- Email: [email protected]
Intraoperative Near-Infrared Fluorescence Imaging of Thymus in Preclinical Models
Intraoperative Near-Infrared Fluorescence Imaging of Thymus in Preclinical Models Hideyuki Wada, MD, Hoon Hyun, PhD, Homan Kang, PhD, Julien Gravier, PhD, Maged Henary, PhD, Mark W. Bordo, BS, Hak Soo Choi, PhD, and John V. Frangioni, MD, PhD Division of Plastic and Reconstructive Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts; Department of Gastroenterological Surgery II, Hokkaido University Graduate School of Medicine, Sapporo, Japan; Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts; Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju, Republic of Korea; Department of Chemistry, Georgia State University, Atlanta, Georgia; Curadel, LLC, Marlborough, Massachusetts; and Department of Radiology, Beth Israel Deaconess Medical Center, Boston, Massachusetts
Background. There are currently no thymus-specific contrast agents for biomedical imaging. Thus, finding ectopic thymic tissue during certain operations is extremely difficult. The purpose of the present study was to determine if near-infrared (NIR) fluorescence imaging could provide high sensitivity, real-time identification of thymic tissue during the operation. Methods. After initial in vivo screening of a 315compound NIR fluorophore library for thymic uptake, methylene blue and five different 700-nm emitting candidate molecules were injected into CD-1 mice for quantitation of the signal-to-background ratio as a function of kinetics and dosing. Results were confirmed in 35kg Yorkshire pigs. Dual-channel NIR imaging was also performed using a variety of 800-nm emitting NIR fluorophores targeted to various tissues in the mediastinum and neck.
Results. The compound Oxazine 170 demonstrated the highest signal-to-background ratio (‡3) for thymic tissue relative to mediastinal fat, heart, lung, muscle, thyroid gland, and parathyroid gland, with peak signal-to-background ratio occurring 4 h after 1 intravenous injection of a human equivalent dose of approximately 7 mg. Simultaneous dual-channel NIR imaging permitted unambiguous identification of the thymus from surrounding tissues, such as endocrine glands and lymph nodes. Conclusions. In mouse and pig, NIR fluorescence imaging using Oxazine 170 permits high sensitivity, real-time identification of thymic tissue for surgical procedures requiring its resection or avoidance. The performance of Oxazine 170 for imaging human thymic tissue is currently not known.
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on the surgical field, where it exhibits high uptake in the target of interest. To date, NIR fluorescence imaging with various contrast agents has been studied in more than 1,000 patients worldwide, in a wide variety of operations. Yet, no studies have been performed for thymus imaging because no thymus-specific contrast agent has been described. In this study, we applied a chemical library screening approach to discover molecules with high uptake in the thymus and conducted preclinical
he thymus plays a critical role in the development of the immune system, especially T-cell maturation [1–3]. Although thymic tissue is typically located in the anterior mediastinum, ectopic thymic tissue can appear anywhere in the mediastinum and neck, from the level of the thyroid to the diaphragm [4–6]. Despite improved surgical approaches [7], the inability to highlight thymic tissue in the context of surrounding fat and muscle and to locate ectopic thymus results in an unsatisfactory outcome in certain operations, such as those for myasthenia gravis [8–10]. Near-infrared (NIR) fluorescence is a relatively new technology that provides surgeons with high-resolution, high-sensitivity optical imaging in real time. Although NIR light between 700 and 900 nm is invisible to the human eye, special imaging systems can be used to see the light and guide the surgeon. NIR requires the intravenous injection of a fluorescent contrast agent (ie, a chemical or drug) with specificity for a particular target Accepted for publication Sept 8, 2016. Address correspondence to Dr Frangioni, Curadel, LLC, 257 Simarano Dr, Marlborough, MA 01752; email: [email protected].
Ó 2016 by The Society of Thoracic Surgeons Published by Elsevier
(Ann Thorac Surg 2016;-:-–-) Ó 2016 by The Society of Thoracic Surgeons
Dr Frangioni discloses a financial relationship with Curadel, LLC, and Beth Israel Deaconess Medical Center.
The Supplemental Figures can be viewed in the online version of this article [http://dx.doi.org/10.1016/ j.athoracsur.2016.09.050] on http://www.annalsthoracic surgery.org.
0003-4975/$36.00 http://dx.doi.org/10.1016/j.athoracsur.2016.09.050
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Abbreviations and Acronyms Co D Fa FLARE
= = = =
He LN MB Me Ne NIR OX QY ROI SBR TG Th Tr
= = = = = = = = = = = = =
cortex distribution coefficient fat tissues Fluorescence-Assisted Resection and Exploration heart lymph nodes methylene blue medulla nerve near-infrared oxazine quantum yield region of interest signal-to-background ratio thyroid gland thymus trachea
experiments in small and large animals to quantitate contrast agent performance.
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distribution (D) coefficient (logD at pH 7.4) and threedimensional minimized structures were performed using Marvin and JChem calculator plug-ins (ChemAxon, Budapest, Hungary).
Animal Models Male CD-1 mice (n ¼ 57) including three 10- to 12-monthold mice averaging 22 g (Charles River Laboratories, Wilmington, MA) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneally (Webster Veterinary, Fort Devens, MA) and a median sternotomy was performed to open the mediastinum and thorax. Female Yorkshire pigs (n ¼ 6) averaging 34.8 kg (E.M. Parsons and Sons, Hadley, MA) were induced with 4.4 mg/kg intramuscular Telazol (Fort Dodge Laboratories, Fort Dodge, IA) and intubated. Anesthesia was maintained with 2% isoflurane (Baxter Healthcare, Deerfield, IL). After anesthesia, a 14-gauge central venous catheter was inserted into the external jugular vein, and saline was administered as needed. A median sternotomy was performed for satisfactory intraoperative imaging. Electrocardiogram, heart rate, pulse oximetry, and body temperature were monitored during the experiment.
NIR Fluorescence Imaging System Materials and Methods The animals used in this study were housed in an facility certified by the Association for Assessment and Accreditation of Laboratory Animal Care and were studied under the supervision of the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee in accordance with approved institutional protocols #024-2013 for rodents and #034-2013 for pigs. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals.
NIR Fluorescent Contrast Agents A 315-compound chemical library consisting of polymethine indocyanine, phenoxazine, and phenothiazine derivatives was screened for thymic uptake using CD-1 mice. Methylene blue (MB; 10 mg/mL; 31.3 mmol/L; Taylor Pharmaceuticals, Decatur, IL), was used as a control. Oxazine (OX) 170 and OX750 were purchased from Sigma-Aldrich (St. Louis, MO). OX18, OX27, and OX89 were synthesized in our laboratory. The 800-nm NIR fluorophores for dual imaging were T800-F for thyroid and parathyroid imaging [11] and ZW800-3C for mediastinal lymph nodes [12]. All NIR fluorophores, except MB, were prepared as 10 mmol/L stock solutions in dimethylsulfoxide.
Measurement of Optical Properties Optical properties were measured in phosphate-buffered saline (pH 7.4) with 5% bovine serum albumin. Quantum yield (QY) of 700 nm and 800 NIR fluorophores was measured using OX725 (Sigma-Aldrich) in ethylene glycol (QY ¼ 19%) and indocyanine green in dimethylsulfoxide (QY ¼ 13%), respectively, under conditions of matched absorbance [13, 14]. In silico calculations of
The dual-NIR channel Fluorescence-Assisted Resection and Exploration (FLARE) (Curadel, LLC, Marlborough, MA) imaging system has been described in detail previously [15, 16]. Color image and 2 independent channels (700 nm and 800 nm) of NIR fluorescence images were acquired simultaneously with custom software at rates up to 15 Hz over a 15-cm diameter field of view. The 700-nm NIR fluorescence and 800-nm fluorescence were pseudocolored red and lime green, respectively, in merged images.
Intraoperative NIR Imaging of the Thymus in Mice and Pigs OX170, OX750, OX98, OX170, and OX750 (100 nmol) were injected intravenously into CD-1 mice 4 hours before imaging (n ¼ 3 for each fluorophore). As a control, 100 nmol of MB was injected intravenously 4 hours before imaging (n ¼ 3). For age-related studies, 100 nmol of OX170 was also injected into young (4-week-old) CD-1 mice (n ¼ 3) and adult (10-to 12-month old) CD-1 mice (n ¼ 3). For kinetic studies, 100 nmol of OX170 was quantified over 8 hours (n ¼ 3 mice per each time point). For dose optimization, images were acquired at 4 hours (n ¼ 3 per each dose). Each NIR agent was diluted into 100 mL of saline containing 5% bovine serum albumin before injection. For large animal studies, 10 mmol of OX170 was injected intravenously into pigs, and thymic tissue in the anterior mediastinum and neck was observed over 8 hours (n ¼ 3). Dual-NIR imaging of the thymus along with surrounding tissues and glands, such as anterior mediastinal lymph nodes, thyroid gland, and parathyroid glands, was performed by injecting 5 mmol of T800-F for thyroid and parathyroid imaging, and 1 mmol of ZW800-3C for lymph node imaging. Dual-NIR images were acquired at 4 hours after the intravenous injection of OX170 and T800-F or
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WADA ET AL NIR FLUORESCENCE IMAGING OF THYMUS
Fig 1. Chemical structure of near-infrared (NIR) fluorophores. Chemical formula, molecular weight (MW), and distribution coefficient (logD) at pH 7.4 were calculated using MarvinSketch (ChemAxon, Budapest, Hungary) for (A) 700-nm NIR fluorophores and (B) 800 nm NIR fluorophores (MB ¼ methylene blue; OX ¼ oxazine.)
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Channel 2 (800 nm) 445.57 770.12 4.37 –1.25 110,000 309,000 758 774 771 789 18.9 16.1 20,790 49,749
ZW800-3C. Each NIR agent was diluted into 10 mL of saline containing 5% bovine serum albumin before injection.
Histologic Analysis and NIR Microscopy For histologic evaluation, thymus resected from young and old mice injected with OX170 was embedded in Tissue-Tek optimal cutting temperature compound (Fisher Scientific, Pittsburgh, PA) and flash frozen in liquid nitrogen. Cryosectioned (10 mm) tissue was observed using a NIR fluorescence microscope. Consecutive sections were stained with hematoxylin and eosin.
469.92 2.34 44,000 676 684 5.0 2,200
OX ¼ oxazine. MB ¼ methylene blue; FLARE ¼ fluorescence-assisted resection and exploration; D ¼ distribution coefficient;
Fluorophores were measured in 100% serum with 50 mmol/L (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at pH 7.4.
Quantitation and Statistical Analysis
a
431.87 1.65 69,000 630 655 24.5 16,905 282.36 0.41 184,000 633 655 8.4 15,456 FLARE imaging channel Molecular weight, Da LogD, pH 7.4 Extinction coefficient, M–1 cm–1 Absorbance maximum, nm Emission maximum, nm Quantum yield, % Molecular brightness (M–1 cm–1)
Channel 1 (700 nm) 319.82 318.39 –0.62 0.6 49,500 29,000 667 635 686 678 3.8 6.0 1,881 1,740
456.53 1.38 38,000 645 700 7.1 2,698
OX170 Property
MB
OX18
OX27
OX89
OX750
T800-F
ZW800-3C
WADA ET AL NIR FLUORESCENCE IMAGING OF THYMUS
Table 1. Optical Properties of Near-Infrared Fluorophoresa
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The fluorescent intensity of a region of interest over various tissues was quantified using custom FLARE software. Signal-to-background ratio (SBR) is fluorescent intensity of the region of interest/background intensity. The muscles, heart, lung, fat tissues, nerve, and thyroid gland were used as background for this calculation to yield thymus/muscle, thymus/heart, thymus/lung, thymus/fat tissue, thymus/nerve, and thymus/thyroid gland SBRs. Results are presented as mean (n 3) SD. Statistical analysis was performed using a one-way analysis of variance between multiple groups. A p value of less than 0.05 was considered significant.
Results Optical Properties of NIR Fluorescent Contrast Agents Chemical structures and optical spectra for 700-nm (MB, OX18, OX27, OX89, OX170, and OX750) and 800-nm NIR fluorophores (T800-F and ZW800-3C) are shown in Figure 1 and Supplemental Figure 1, respectively. Table 1 details the optical properties of each agent under bloodlike conditions. Although the extinction coefficient for OX170 is similar to the value from MB, OX170 exhibits more than sixfold higher QY compared with MB (24.5% vs 3.8%), resulting in a total molecular brightness ninefold greater. OX170 and 800-nm emitting fluorophores (ie, T800-F and ZW800-3C) have ideal separation in their absorbance and fluorescence spectra, which matches the 2 channels (700 nm and 800 nm) of the FLARE imaging system and thus enables simultaneous dual-channel NIR fluorescence imaging.
In Vivo Screening in Mice to Identify Thymus-Targeted NIR Fluorophores Five different OX derivatives, OX18, OX27, OX89, OX170, and OX750 (Fig 1A), were selected from our initial 315-compound chemical library screen. Thymus uptake was then compared with a control fluorophore, MB, which is the only 700-nm NIR fluorophore currently available clinically. MB showed virtually no uptake in thymus, likely because of its hydrophilicity (logD at pH 7.4 ¼ –0.62; Fig 2A). MB also has rather poor optical properties (ie, low molecular brightness; see below) and chemical instability (ie, reduction to leuco-MB in blood).
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Fig 2. In vivo screening near-infrared (NIR) fluorophores targeting the thymus in mice: (A) 100 nmol injected intravenously into CD-1 mice (n ¼ 3 mice per fluorophore) 4 hours before imaging of the thymus (arrowheads). (He ¼ heart; Lu ¼ lung; Th ¼ thymus.) Scale bars ¼ 5 mm. All NIR fluorescence images for all figures have identical exposure times and normalizations. (B) Signal-to-background ratio (SBR) of thymus (Th) relative to muscle (Mu), heart (He), and lung (Lu) was compared using various NIR fluorophores. Data are shown as mean SD (range bars). (MB ¼ methylene blue; OX ¼ oxazine.) *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
OX27 and OX89 also failed to show thymus uptake and were deemed false positives from the initial screen. However, OX18, OX170, and OX750 showed unusually high uptake in the thymus, confirming initial screening results. Uptake in nearby surrounding tissue varied
considerably among the compounds (Fig 2A), with OX170 having the lowest uptake in muscle and heart and thus the highest SBR in thymus (Fig 2B). All compounds showed relatively high uptake in the lung. These results led us to select OX170 for further study.
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Fig 3. Intraoperative near-infrared (NIR) fluorescence and histologic analysis in young and adult mice: (A) 100 nmol of oxazine (OX) 170 injected intravenously into young mice (4 weeks old) and adult mice (10 to 12 months old; n ¼ 3 mice per group) 4 hours before imaging of the thymus (arrowheads; scale bars ¼ 5 mm). (He ¼ heart; Lu ¼ lung; Th ¼ thymus.) (B) Histologic analysis: Consecutive thymus sections from young mice and adult mice stained with hematoxylin and eosin or unstained and observed using a NIR fluorescence microscope (scale bars ¼ 300 mm). (Co ¼ cortex; Me ¼ medulla.)
Comparison of NIR Fluorescence Imaging and Histologic Analysis of Thymus Between Young and Adult Mice Because thymus shows age-related involution and atrophic thymus can be present even in young women [17, 18], we explored whether OX170 could visualize the thymus in young mice (4 weeks old) and adult mice (10 to 12 months old). Although there was a clear shrinkage of thymus size in adult mice, it still showed high fluorescence signal and high SBR relative to surrounding tissue (Fig 3A). Histologically, thymic tissue in both young and adult mice showed NIR fluorescent signal in nonfat cells (Fig 3B). The cortex had a higher signal than the medulla in both young and adult thymus.
Kinetics and Dose Optimization of OX170 in Mice To determine optimal imaging time after intravenous injection, images were acquired over 8 hours after an intravenous injection of 100 nmol OX170 into CD-1 mice (Fig 4A). NIR fluorescence in the thymus rose quickly and was highest from 1 to 4 hours postinjection, while the
background signal decreased continuously over time. The highest SBR relative to muscle, heart, and lung was achieved at 4 hours postinjection; therefore, the dose optimization study was performed at 4 hours postinjection (Fig 4B). Although the thymus signal increased proportionally to the dose, the signal in the surrounding background tissue also increased, such that SBR was similar at doses between 100 and 200 nmol. We concluded that a dose of 100 nmol OX170 was optimal in mice. No adverse reactions in mice were encountered during the observation period.
Intraoperative NIR Fluorescence Thymus Imaging in Pigs To confirm that the results in small animals were not species dependent, thymus imaging was performed in pigs using a body surface area–scaled dose of 10 mmol. OX170 was injected intravenously, and NIR imaging was performed over 8 hours postinjection (Fig 5A). The thymus was highlighted with high contrast relative to
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Fig 5. Intraoperative thymus imaging in pigs: (A) 10 mmol of oxazine (OX) 170 injected intravenously into 35-kg Yorkshire pigs (n ¼ 3 pigs) and imaged at 8 hours. Thymic tissue (arrowheads) in the mediastinum (first row), magnified thymus in the mediastinum (second row), and thymus in the neck, with dashed arrow highlighting the thyroid gland (3rd row). Shown are a color image, 700-nm near-infrared (NIR) fluorescence, and a merged image of the two. For merge, channel 1 (700 nm) is pseudocolored in red. Scale bars ¼ 1 cm. (B) Quantitative kinetics of signal-tobackground ratios (SBR) for thymus and surrounding organs/tissues. Data are shown as mean SD (error bars). (Fa ¼ fat tissues; He, ¼ heart; LN ¼lymph nodes; Ne ¼ nerve; TG ¼ thyroid gland; Th ¼ thymus; Tr ¼ trachea.)
Fig 4. Kinetics and dose-response of oxazine (OX) 170 uptake in mice: (A) Quantitative kinetics: 100 nmol of OX170 injected intravenously into CD-1 mice and signal-to-background ratios (SBR) for thymus (Th)/heart (He), Th/muscle (Mu), and Th/lung (Lu) measured over time (n ¼ 3 mice per time point). Data are shown as mean SD (range bars) (B) Dose-response curve: SBRs for Th/He, Th/Mu, and Th/Lu measured 4 hours after injection into CD-1 mice. Scale bars ¼ 5 mm. Arrowheads indicate thymus. *p < 0.05, **p < 0.01, ***p < 0.001, and ****P < 0.0001.
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Fig 6. Simultaneous dual near-infrared (NIR) channel imaging of thymus and surrounding organs: 10 mmol of 700-nm emitting OX170 injected into 35 kg Yorkshire pigs 4 h before imaging. At 10 minutes after the injection of OX170, 5 mmol of 800-nm emitting T800-F targeted to both thyroid and parathyroid (first and second rows; n ¼ 1 each) or 1 mmol of ZW800-3C targeted to mediastinal lymph nodes (third row; n ¼ 1) were injected. Shown are a color image, 700-nm NIR fluorescence, 800-nm NIR fluorescence, and a merged image of the three. For the merge image, channel 1 (700 nm) is pseudocolored in red and channel 2 (800 nm) in lime green. The white arrowheads indicate thymic tissue. The red dashed arrows indicate parathyroid glands (first and second rows). The white dashed arrow indicate thyroid gland (first row). Red arrowheads indicate mediastinal lymph nodes (third row). Scale bars ¼ 1 cm. (LN ¼ lymph nodes; PG ¼ parathyroid gland; TG ¼ thyroid gland; Th ¼ thymus.)
surrounding background tissues, including heart, fat, nerve, and lymph nodes in the mediastinum and neck. Because OX170 also stained the thyroid, the contrast between thymus and thyroid was poor when only a single NIR channel was used for imaging (Fig 5B). Overall, OX170 showed relatively high SBRs for up to 8 hours, although the peak SBR occurred between 2 and 6 hours postinjection. No adverse reactions in the pigs were encountered during the 8-hour observation.
Simultaneous Dual-NIR Imaging of the Anterior Mediastinum Because the proximity of the thymus to surrounding tissue and glands sometimes makes it difficult to discriminate thymus from nonthymus, we explored the use of dual-NIR imaging to improve identification in the anterior mediastinum. The FLARE imaging system has 2 independent channels of NIR fluorescence, 1 centered at 700 nm and 1 at 800 nm. With the assistance of 800-nm NIR fluorophores previously described by our group [11, 19], we were able to unambiguously discriminate thymus from thyroid gland, parathyroid glands, and mediastinal lymph nodes (Fig 6). In particular, pseudocolored merged images of color video, 700-nm NIR fluorescence, and 800-nm NIR fluorescence provided a rapid visual coding, such that red pseudocolor was true positive
for the presence of thymus, green pseudocolor was true negative, and yellow pseudocolor was false positive. Resolution of this discrimination was less than 1 mm at a field of view of 15 cm and improved to less than 100 mm when zoomed in to a field of view of 2 cm.
Comment In this preclinical study, we demonstrated that the 700nm NIR fluorophore OX170 successfully highlighted all thymic tissue in mice and pigs, with a relatively high SBR relative to surrounding tissue except lung. We do not know the molecular mechanism by which OX170 is taken up by the cortex of thymic tissue and the lung. Although future studies will focus on modifying the chemical structure of OX170 to improve the thymus-tolung ratio and to induce a bathochromic shift, it is not clear that this is an imperative because lung is so easily identified visually during anterior mediastinal procedures. OX170 appears to be species independent, providing thymus imaging over 2 to 8 hours after a single intravenous injection. Of note, the human equivalent dose of OX170 is only approximately 7 mg, which is consistent with dosing of the United States Food and Drug Administration–approved NIR fluorophore indocyanine
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green for various procedures. At least in animals, OX170 can be used during dual imaging to unambiguously differentiate thymic tissue from other tissue in the neck and anterior mediastinum. Although we show data for the open surgical version of the FLARE imaging system, equal performance is seen with our new minimally invasive thoracoscopic version [20]. It is tempting to speculate from our large animal results how OX170 and similar molecules might someday prove useful clinically. Although thymectomy is considered an effective treatment for achieving complete stable remission of nonthymomatous myasthenia gravis, its success rate ranges from only 21% to 63% [17, 21–26]. Persistent ectopic thymus remains an important predictor of a poor response [18]. NIR fluorescence imaging of the thoracic cavity could provide surgeons with improved detectability of ectopic thymic tissue and thus the possibility of improved outcome. And, as shown above, a thymus-specific contrast agent can be combined with a second NIR fluorophore emitting at a different wavelength to simultaneously identify critical structures in the surgical field. Nevertheless, our preclinical study has important limitations and should not yet be extrapolated to human use. First, we used mice and pigs, which have subtle differences in their thymic anatomy compared with humans. Second, we did not observe ectopic tissue in these animals, although pigs have rather diffuse thymic tissue that does appear to mimic ectopic deposits (Figs 5 and 6). And, finally, although no obvious changes in vital signs or heart rhythm were encountered during the 8-hour monitoring period, we could have missed subacute toxicity. Future studies will have to address these shortcomings. In particular, initial human clinical trials will need to focus first on the safety of OX170, because it is not approved by regulatory agencies for human use, and the lethal dose that kills 50% of a test sample has not yet been reported. Then, uptake in human thymic tissue can be confirmed before dose optimization and pharmacokinetic studies. The authors wish to thank Rita G. Laurence, David J. Burrington, Jr, Eugenia Trabucchi, Sylvain Gioux, Frank Kettenring, Florin Neacsu, and Joseph Angelo for their assistance. This work was supported by National Institutes of Health grants R01-CA-115296 and R01-EB-011523.
References 1. Nasseri F, Eftekhari F. Clinical and radiologic review of the normal and abnormal thymus: pearls and pitfalls. Radiographics 2010;30:413–28. 2. Bertho JM, Demarquay C, Moulian N, Van Der Meeren A, Berrih-Aknin S, Gourmelon P. Phenotypic and immunohistological analyses of the human adult thymus: evidence for an active thymus during adult life. Cell Immunol 1997;179: 30–40. 3. Taub DD, Longo DL. Insights into thymic aging and regeneration. Immunol Rev 2005;205:72–93. 4. Masaoka A, Nagaoka Y, Kotake Y. Distribution of thymic tissue at the anterior mediastinum. Current procedures in thymectomy. J Thorac Cardiovasc Surg 1975;70:747–54.
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5. Fukai I, Funato Y, Mizuno T, Hashimoto T, Masaoka A. Distribution of thymic tissue in the mediastinal adipose tissue. J Thorac Cardiovasc Surg 1991;101:1099–102. 6. Ashour M. Prevalence of ectopic thymic tissue in myasthenia gravis and its clinical significance. J Thorac Cardiovasc Surg 1995;109:632–5. 7. Sonett JR, Jaretzki A. Thymectomy for nonthymomatous myasthenia gravis: a critical analysis. Ann N Y Acad Sci 2008;1132:315–28. 8. Takahashi K, Al-Janabi NJ. Computed tomography and magnetic resonance imaging of mediastinal tumors. J Magn Reson Imaging 2010;32:1325–39. 9. Ackman JB, Wu CC. MRI of the thymus. AJR Am J Roentgenol 2011;197:W15–20. 10. Priola AM, Priola SM. Imaging of thymus in myasthenia gravis: from thymic hyperplasia to thymic tumor. Clin Radiol 2014;69:e230–45. 11. Hyun H, Park MH, Owens EA, et al. Structure-inherent targeting of near-infrared fluorophores for parathyroid and thyroid gland imaging. Nat Med 2015;21:192–7. 12. Wada H, Hyun H, Vargas C, et al. Sentinel lymph node mapping of liver. Ann Surg Oncol 2015;22(Suppl 3): S1147–55. 13. Choi HS, Ashitate Y, Lee JH, et al. Rapid translocation of nanoparticles from the lung airspaces to the body. Nat Biotechnol 2010;28:1300–3. 14. Ashitate Y, Tanaka E, Stockdale A, Choi HS, Frangioni JV. Near-infrared fluorescence imaging of thoracic duct anatomy and function in open surgery and video-assisted thoracic surgery. J Thorac Cardiovasc Surg 2011;142:31–8; e1–2. 15. Troyan SL, Kianzad V, Gibbs-Strauss SL, et al. The FLARE intraoperative near-infrared fluorescence imaging system: a first-in-human clinical trial in breast cancer sentinel lymph node mapping. Ann Surg Oncol 2009;16: 2943–52. 16. Gioux S, Choi HS, Frangioni JV. Image-guided surgery using invisible near-infrared light: fundamentals of clinical translation. Mol Imaging 2010;9:237–55. 17. Tomulescu V, Sgarbura O, Stanescu C, et al. Ten-year results of thoracoscopic unilateral extended thymectomy performed in nonthymomatous myasthenia gravis. Ann Surg 2011;254: 761–5; discussion 765–6. 18. Ambrogi V, Mineo TC. Active ectopic thymus predicts poor outcome after thymectomy in class III myasthenia gravis. J Thorac Cardiovasc Surg 2012;143:601–6. 19. Wada H, Hyun H, Vargas C, et al. Pancreas-targeted NIR fluorophores for dual-channel image-guided abdominal surgery. Theranostics 2015;5:1–11. 20. Venugopal V, Park M, Ashitate Y, et al. Design and characterization of an optimized simultaneous color and nearinfrared fluorescence rigid endoscopic imaging system. J Biomed Opt 2013;18:126018. 21. Jaretzki A, Penn AS, Younger DS, et al. “Maximal” thymectomy for myasthenia gravis. Results. J Thorac Cardiovasc Surg 1988;95:747–57. 22. Cheng C, Liu Z, Xu F, et al. Clinical outcome of juvenile myasthenia gravis after extended transsternal thymectomy in a Chinese cohort. Ann Thorac Surg 2013;95:1035–41. 23. Prokakis C, Koletsis E, Salakou S, et al. Modified maximal thymectomy for myasthenia gravis: effect of maximal resection on late neurologic outcome and predictors of disease remission. Ann Thorac Surg 2009;88:1638–45. 24. Shrager JB, Nathan D, Brinster CJ, et al. Outcomes after 151 extended transcervical thymectomies for myasthenia gravis. Ann Thorac Surg 2006;82:1863–9. 25. Budde JM, Morris CD, Gal AA, Mansour KA, Miller JI. Predictors of outcome in thymectomy for myasthenia gravis. Ann Thorac Surg 2001;72:197–202. 26. Ruffini E, Guerrera F, Filosso PL, et al. Extended transcervical thymectomy with partial upper sternotomy: results in non-thymomatous patients with myasthenia gravis. Eur J Cardiothorac Surg 2015;48:448–54.
Background. There are currently no thymus-specific contrast agents for biomedical imaging. Thus, finding ectopic thymic tissue during certain operations is extremely difficult. The purpose of the present study was to determine if near-infrared (NIR) fluorescence imaging could provide high sensitivity, real-time identification of thymic tissue during the operation. Methods. After initial in vivo screening of a 315compound NIR fluorophore library for thymic uptake, methylene blue and five different 700-nm emitting candidate molecules were injected into CD-1 mice for quantitation of the signal-to-background ratio as a function of kinetics and dosing. Results were confirmed in 35kg Yorkshire pigs. Dual-channel NIR imaging was also performed using a variety of 800-nm emitting NIR fluorophores targeted to various tissues in the mediastinum and neck.
Results. The compound Oxazine 170 demonstrated the highest signal-to-background ratio (‡3) for thymic tissue relative to mediastinal fat, heart, lung, muscle, thyroid gland, and parathyroid gland, with peak signal-to-background ratio occurring 4 h after 1 intravenous injection of a human equivalent dose of approximately 7 mg. Simultaneous dual-channel NIR imaging permitted unambiguous identification of the thymus from surrounding tissues, such as endocrine glands and lymph nodes. Conclusions. In mouse and pig, NIR fluorescence imaging using Oxazine 170 permits high sensitivity, real-time identification of thymic tissue for surgical procedures requiring its resection or avoidance. The performance of Oxazine 170 for imaging human thymic tissue is currently not known.
T
on the surgical field, where it exhibits high uptake in the target of interest. To date, NIR fluorescence imaging with various contrast agents has been studied in more than 1,000 patients worldwide, in a wide variety of operations. Yet, no studies have been performed for thymus imaging because no thymus-specific contrast agent has been described. In this study, we applied a chemical library screening approach to discover molecules with high uptake in the thymus and conducted preclinical
he thymus plays a critical role in the development of the immune system, especially T-cell maturation [1–3]. Although thymic tissue is typically located in the anterior mediastinum, ectopic thymic tissue can appear anywhere in the mediastinum and neck, from the level of the thyroid to the diaphragm [4–6]. Despite improved surgical approaches [7], the inability to highlight thymic tissue in the context of surrounding fat and muscle and to locate ectopic thymus results in an unsatisfactory outcome in certain operations, such as those for myasthenia gravis [8–10]. Near-infrared (NIR) fluorescence is a relatively new technology that provides surgeons with high-resolution, high-sensitivity optical imaging in real time. Although NIR light between 700 and 900 nm is invisible to the human eye, special imaging systems can be used to see the light and guide the surgeon. NIR requires the intravenous injection of a fluorescent contrast agent (ie, a chemical or drug) with specificity for a particular target Accepted for publication Sept 8, 2016. Address correspondence to Dr Frangioni, Curadel, LLC, 257 Simarano Dr, Marlborough, MA 01752; email: [email protected].
Ó 2016 by The Society of Thoracic Surgeons Published by Elsevier
(Ann Thorac Surg 2016;-:-–-) Ó 2016 by The Society of Thoracic Surgeons
Dr Frangioni discloses a financial relationship with Curadel, LLC, and Beth Israel Deaconess Medical Center.
The Supplemental Figures can be viewed in the online version of this article [http://dx.doi.org/10.1016/ j.athoracsur.2016.09.050] on http://www.annalsthoracic surgery.org.
0003-4975/$36.00 http://dx.doi.org/10.1016/j.athoracsur.2016.09.050
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Abbreviations and Acronyms Co D Fa FLARE
= = = =
He LN MB Me Ne NIR OX QY ROI SBR TG Th Tr
= = = = = = = = = = = = =
cortex distribution coefficient fat tissues Fluorescence-Assisted Resection and Exploration heart lymph nodes methylene blue medulla nerve near-infrared oxazine quantum yield region of interest signal-to-background ratio thyroid gland thymus trachea
experiments in small and large animals to quantitate contrast agent performance.
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distribution (D) coefficient (logD at pH 7.4) and threedimensional minimized structures were performed using Marvin and JChem calculator plug-ins (ChemAxon, Budapest, Hungary).
Animal Models Male CD-1 mice (n ¼ 57) including three 10- to 12-monthold mice averaging 22 g (Charles River Laboratories, Wilmington, MA) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneally (Webster Veterinary, Fort Devens, MA) and a median sternotomy was performed to open the mediastinum and thorax. Female Yorkshire pigs (n ¼ 6) averaging 34.8 kg (E.M. Parsons and Sons, Hadley, MA) were induced with 4.4 mg/kg intramuscular Telazol (Fort Dodge Laboratories, Fort Dodge, IA) and intubated. Anesthesia was maintained with 2% isoflurane (Baxter Healthcare, Deerfield, IL). After anesthesia, a 14-gauge central venous catheter was inserted into the external jugular vein, and saline was administered as needed. A median sternotomy was performed for satisfactory intraoperative imaging. Electrocardiogram, heart rate, pulse oximetry, and body temperature were monitored during the experiment.
NIR Fluorescence Imaging System Materials and Methods The animals used in this study were housed in an facility certified by the Association for Assessment and Accreditation of Laboratory Animal Care and were studied under the supervision of the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee in accordance with approved institutional protocols #024-2013 for rodents and #034-2013 for pigs. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals.
NIR Fluorescent Contrast Agents A 315-compound chemical library consisting of polymethine indocyanine, phenoxazine, and phenothiazine derivatives was screened for thymic uptake using CD-1 mice. Methylene blue (MB; 10 mg/mL; 31.3 mmol/L; Taylor Pharmaceuticals, Decatur, IL), was used as a control. Oxazine (OX) 170 and OX750 were purchased from Sigma-Aldrich (St. Louis, MO). OX18, OX27, and OX89 were synthesized in our laboratory. The 800-nm NIR fluorophores for dual imaging were T800-F for thyroid and parathyroid imaging [11] and ZW800-3C for mediastinal lymph nodes [12]. All NIR fluorophores, except MB, were prepared as 10 mmol/L stock solutions in dimethylsulfoxide.
Measurement of Optical Properties Optical properties were measured in phosphate-buffered saline (pH 7.4) with 5% bovine serum albumin. Quantum yield (QY) of 700 nm and 800 NIR fluorophores was measured using OX725 (Sigma-Aldrich) in ethylene glycol (QY ¼ 19%) and indocyanine green in dimethylsulfoxide (QY ¼ 13%), respectively, under conditions of matched absorbance [13, 14]. In silico calculations of
The dual-NIR channel Fluorescence-Assisted Resection and Exploration (FLARE) (Curadel, LLC, Marlborough, MA) imaging system has been described in detail previously [15, 16]. Color image and 2 independent channels (700 nm and 800 nm) of NIR fluorescence images were acquired simultaneously with custom software at rates up to 15 Hz over a 15-cm diameter field of view. The 700-nm NIR fluorescence and 800-nm fluorescence were pseudocolored red and lime green, respectively, in merged images.
Intraoperative NIR Imaging of the Thymus in Mice and Pigs OX170, OX750, OX98, OX170, and OX750 (100 nmol) were injected intravenously into CD-1 mice 4 hours before imaging (n ¼ 3 for each fluorophore). As a control, 100 nmol of MB was injected intravenously 4 hours before imaging (n ¼ 3). For age-related studies, 100 nmol of OX170 was also injected into young (4-week-old) CD-1 mice (n ¼ 3) and adult (10-to 12-month old) CD-1 mice (n ¼ 3). For kinetic studies, 100 nmol of OX170 was quantified over 8 hours (n ¼ 3 mice per each time point). For dose optimization, images were acquired at 4 hours (n ¼ 3 per each dose). Each NIR agent was diluted into 100 mL of saline containing 5% bovine serum albumin before injection. For large animal studies, 10 mmol of OX170 was injected intravenously into pigs, and thymic tissue in the anterior mediastinum and neck was observed over 8 hours (n ¼ 3). Dual-NIR imaging of the thymus along with surrounding tissues and glands, such as anterior mediastinal lymph nodes, thyroid gland, and parathyroid glands, was performed by injecting 5 mmol of T800-F for thyroid and parathyroid imaging, and 1 mmol of ZW800-3C for lymph node imaging. Dual-NIR images were acquired at 4 hours after the intravenous injection of OX170 and T800-F or
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Fig 1. Chemical structure of near-infrared (NIR) fluorophores. Chemical formula, molecular weight (MW), and distribution coefficient (logD) at pH 7.4 were calculated using MarvinSketch (ChemAxon, Budapest, Hungary) for (A) 700-nm NIR fluorophores and (B) 800 nm NIR fluorophores (MB ¼ methylene blue; OX ¼ oxazine.)
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Channel 2 (800 nm) 445.57 770.12 4.37 –1.25 110,000 309,000 758 774 771 789 18.9 16.1 20,790 49,749
ZW800-3C. Each NIR agent was diluted into 10 mL of saline containing 5% bovine serum albumin before injection.
Histologic Analysis and NIR Microscopy For histologic evaluation, thymus resected from young and old mice injected with OX170 was embedded in Tissue-Tek optimal cutting temperature compound (Fisher Scientific, Pittsburgh, PA) and flash frozen in liquid nitrogen. Cryosectioned (10 mm) tissue was observed using a NIR fluorescence microscope. Consecutive sections were stained with hematoxylin and eosin.
469.92 2.34 44,000 676 684 5.0 2,200
OX ¼ oxazine. MB ¼ methylene blue; FLARE ¼ fluorescence-assisted resection and exploration; D ¼ distribution coefficient;
Fluorophores were measured in 100% serum with 50 mmol/L (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at pH 7.4.
Quantitation and Statistical Analysis
a
431.87 1.65 69,000 630 655 24.5 16,905 282.36 0.41 184,000 633 655 8.4 15,456 FLARE imaging channel Molecular weight, Da LogD, pH 7.4 Extinction coefficient, M–1 cm–1 Absorbance maximum, nm Emission maximum, nm Quantum yield, % Molecular brightness (M–1 cm–1)
Channel 1 (700 nm) 319.82 318.39 –0.62 0.6 49,500 29,000 667 635 686 678 3.8 6.0 1,881 1,740
456.53 1.38 38,000 645 700 7.1 2,698
OX170 Property
MB
OX18
OX27
OX89
OX750
T800-F
ZW800-3C
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Table 1. Optical Properties of Near-Infrared Fluorophoresa
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The fluorescent intensity of a region of interest over various tissues was quantified using custom FLARE software. Signal-to-background ratio (SBR) is fluorescent intensity of the region of interest/background intensity. The muscles, heart, lung, fat tissues, nerve, and thyroid gland were used as background for this calculation to yield thymus/muscle, thymus/heart, thymus/lung, thymus/fat tissue, thymus/nerve, and thymus/thyroid gland SBRs. Results are presented as mean (n 3) SD. Statistical analysis was performed using a one-way analysis of variance between multiple groups. A p value of less than 0.05 was considered significant.
Results Optical Properties of NIR Fluorescent Contrast Agents Chemical structures and optical spectra for 700-nm (MB, OX18, OX27, OX89, OX170, and OX750) and 800-nm NIR fluorophores (T800-F and ZW800-3C) are shown in Figure 1 and Supplemental Figure 1, respectively. Table 1 details the optical properties of each agent under bloodlike conditions. Although the extinction coefficient for OX170 is similar to the value from MB, OX170 exhibits more than sixfold higher QY compared with MB (24.5% vs 3.8%), resulting in a total molecular brightness ninefold greater. OX170 and 800-nm emitting fluorophores (ie, T800-F and ZW800-3C) have ideal separation in their absorbance and fluorescence spectra, which matches the 2 channels (700 nm and 800 nm) of the FLARE imaging system and thus enables simultaneous dual-channel NIR fluorescence imaging.
In Vivo Screening in Mice to Identify Thymus-Targeted NIR Fluorophores Five different OX derivatives, OX18, OX27, OX89, OX170, and OX750 (Fig 1A), were selected from our initial 315-compound chemical library screen. Thymus uptake was then compared with a control fluorophore, MB, which is the only 700-nm NIR fluorophore currently available clinically. MB showed virtually no uptake in thymus, likely because of its hydrophilicity (logD at pH 7.4 ¼ –0.62; Fig 2A). MB also has rather poor optical properties (ie, low molecular brightness; see below) and chemical instability (ie, reduction to leuco-MB in blood).
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Fig 2. In vivo screening near-infrared (NIR) fluorophores targeting the thymus in mice: (A) 100 nmol injected intravenously into CD-1 mice (n ¼ 3 mice per fluorophore) 4 hours before imaging of the thymus (arrowheads). (He ¼ heart; Lu ¼ lung; Th ¼ thymus.) Scale bars ¼ 5 mm. All NIR fluorescence images for all figures have identical exposure times and normalizations. (B) Signal-to-background ratio (SBR) of thymus (Th) relative to muscle (Mu), heart (He), and lung (Lu) was compared using various NIR fluorophores. Data are shown as mean SD (range bars). (MB ¼ methylene blue; OX ¼ oxazine.) *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
OX27 and OX89 also failed to show thymus uptake and were deemed false positives from the initial screen. However, OX18, OX170, and OX750 showed unusually high uptake in the thymus, confirming initial screening results. Uptake in nearby surrounding tissue varied
considerably among the compounds (Fig 2A), with OX170 having the lowest uptake in muscle and heart and thus the highest SBR in thymus (Fig 2B). All compounds showed relatively high uptake in the lung. These results led us to select OX170 for further study.
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Fig 3. Intraoperative near-infrared (NIR) fluorescence and histologic analysis in young and adult mice: (A) 100 nmol of oxazine (OX) 170 injected intravenously into young mice (4 weeks old) and adult mice (10 to 12 months old; n ¼ 3 mice per group) 4 hours before imaging of the thymus (arrowheads; scale bars ¼ 5 mm). (He ¼ heart; Lu ¼ lung; Th ¼ thymus.) (B) Histologic analysis: Consecutive thymus sections from young mice and adult mice stained with hematoxylin and eosin or unstained and observed using a NIR fluorescence microscope (scale bars ¼ 300 mm). (Co ¼ cortex; Me ¼ medulla.)
Comparison of NIR Fluorescence Imaging and Histologic Analysis of Thymus Between Young and Adult Mice Because thymus shows age-related involution and atrophic thymus can be present even in young women [17, 18], we explored whether OX170 could visualize the thymus in young mice (4 weeks old) and adult mice (10 to 12 months old). Although there was a clear shrinkage of thymus size in adult mice, it still showed high fluorescence signal and high SBR relative to surrounding tissue (Fig 3A). Histologically, thymic tissue in both young and adult mice showed NIR fluorescent signal in nonfat cells (Fig 3B). The cortex had a higher signal than the medulla in both young and adult thymus.
Kinetics and Dose Optimization of OX170 in Mice To determine optimal imaging time after intravenous injection, images were acquired over 8 hours after an intravenous injection of 100 nmol OX170 into CD-1 mice (Fig 4A). NIR fluorescence in the thymus rose quickly and was highest from 1 to 4 hours postinjection, while the
background signal decreased continuously over time. The highest SBR relative to muscle, heart, and lung was achieved at 4 hours postinjection; therefore, the dose optimization study was performed at 4 hours postinjection (Fig 4B). Although the thymus signal increased proportionally to the dose, the signal in the surrounding background tissue also increased, such that SBR was similar at doses between 100 and 200 nmol. We concluded that a dose of 100 nmol OX170 was optimal in mice. No adverse reactions in mice were encountered during the observation period.
Intraoperative NIR Fluorescence Thymus Imaging in Pigs To confirm that the results in small animals were not species dependent, thymus imaging was performed in pigs using a body surface area–scaled dose of 10 mmol. OX170 was injected intravenously, and NIR imaging was performed over 8 hours postinjection (Fig 5A). The thymus was highlighted with high contrast relative to
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Fig 5. Intraoperative thymus imaging in pigs: (A) 10 mmol of oxazine (OX) 170 injected intravenously into 35-kg Yorkshire pigs (n ¼ 3 pigs) and imaged at 8 hours. Thymic tissue (arrowheads) in the mediastinum (first row), magnified thymus in the mediastinum (second row), and thymus in the neck, with dashed arrow highlighting the thyroid gland (3rd row). Shown are a color image, 700-nm near-infrared (NIR) fluorescence, and a merged image of the two. For merge, channel 1 (700 nm) is pseudocolored in red. Scale bars ¼ 1 cm. (B) Quantitative kinetics of signal-tobackground ratios (SBR) for thymus and surrounding organs/tissues. Data are shown as mean SD (error bars). (Fa ¼ fat tissues; He, ¼ heart; LN ¼lymph nodes; Ne ¼ nerve; TG ¼ thyroid gland; Th ¼ thymus; Tr ¼ trachea.)
Fig 4. Kinetics and dose-response of oxazine (OX) 170 uptake in mice: (A) Quantitative kinetics: 100 nmol of OX170 injected intravenously into CD-1 mice and signal-to-background ratios (SBR) for thymus (Th)/heart (He), Th/muscle (Mu), and Th/lung (Lu) measured over time (n ¼ 3 mice per time point). Data are shown as mean SD (range bars) (B) Dose-response curve: SBRs for Th/He, Th/Mu, and Th/Lu measured 4 hours after injection into CD-1 mice. Scale bars ¼ 5 mm. Arrowheads indicate thymus. *p < 0.05, **p < 0.01, ***p < 0.001, and ****P < 0.0001.
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Fig 6. Simultaneous dual near-infrared (NIR) channel imaging of thymus and surrounding organs: 10 mmol of 700-nm emitting OX170 injected into 35 kg Yorkshire pigs 4 h before imaging. At 10 minutes after the injection of OX170, 5 mmol of 800-nm emitting T800-F targeted to both thyroid and parathyroid (first and second rows; n ¼ 1 each) or 1 mmol of ZW800-3C targeted to mediastinal lymph nodes (third row; n ¼ 1) were injected. Shown are a color image, 700-nm NIR fluorescence, 800-nm NIR fluorescence, and a merged image of the three. For the merge image, channel 1 (700 nm) is pseudocolored in red and channel 2 (800 nm) in lime green. The white arrowheads indicate thymic tissue. The red dashed arrows indicate parathyroid glands (first and second rows). The white dashed arrow indicate thyroid gland (first row). Red arrowheads indicate mediastinal lymph nodes (third row). Scale bars ¼ 1 cm. (LN ¼ lymph nodes; PG ¼ parathyroid gland; TG ¼ thyroid gland; Th ¼ thymus.)
surrounding background tissues, including heart, fat, nerve, and lymph nodes in the mediastinum and neck. Because OX170 also stained the thyroid, the contrast between thymus and thyroid was poor when only a single NIR channel was used for imaging (Fig 5B). Overall, OX170 showed relatively high SBRs for up to 8 hours, although the peak SBR occurred between 2 and 6 hours postinjection. No adverse reactions in the pigs were encountered during the 8-hour observation.
Simultaneous Dual-NIR Imaging of the Anterior Mediastinum Because the proximity of the thymus to surrounding tissue and glands sometimes makes it difficult to discriminate thymus from nonthymus, we explored the use of dual-NIR imaging to improve identification in the anterior mediastinum. The FLARE imaging system has 2 independent channels of NIR fluorescence, 1 centered at 700 nm and 1 at 800 nm. With the assistance of 800-nm NIR fluorophores previously described by our group [11, 19], we were able to unambiguously discriminate thymus from thyroid gland, parathyroid glands, and mediastinal lymph nodes (Fig 6). In particular, pseudocolored merged images of color video, 700-nm NIR fluorescence, and 800-nm NIR fluorescence provided a rapid visual coding, such that red pseudocolor was true positive
for the presence of thymus, green pseudocolor was true negative, and yellow pseudocolor was false positive. Resolution of this discrimination was less than 1 mm at a field of view of 15 cm and improved to less than 100 mm when zoomed in to a field of view of 2 cm.
Comment In this preclinical study, we demonstrated that the 700nm NIR fluorophore OX170 successfully highlighted all thymic tissue in mice and pigs, with a relatively high SBR relative to surrounding tissue except lung. We do not know the molecular mechanism by which OX170 is taken up by the cortex of thymic tissue and the lung. Although future studies will focus on modifying the chemical structure of OX170 to improve the thymus-tolung ratio and to induce a bathochromic shift, it is not clear that this is an imperative because lung is so easily identified visually during anterior mediastinal procedures. OX170 appears to be species independent, providing thymus imaging over 2 to 8 hours after a single intravenous injection. Of note, the human equivalent dose of OX170 is only approximately 7 mg, which is consistent with dosing of the United States Food and Drug Administration–approved NIR fluorophore indocyanine
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green for various procedures. At least in animals, OX170 can be used during dual imaging to unambiguously differentiate thymic tissue from other tissue in the neck and anterior mediastinum. Although we show data for the open surgical version of the FLARE imaging system, equal performance is seen with our new minimally invasive thoracoscopic version [20]. It is tempting to speculate from our large animal results how OX170 and similar molecules might someday prove useful clinically. Although thymectomy is considered an effective treatment for achieving complete stable remission of nonthymomatous myasthenia gravis, its success rate ranges from only 21% to 63% [17, 21–26]. Persistent ectopic thymus remains an important predictor of a poor response [18]. NIR fluorescence imaging of the thoracic cavity could provide surgeons with improved detectability of ectopic thymic tissue and thus the possibility of improved outcome. And, as shown above, a thymus-specific contrast agent can be combined with a second NIR fluorophore emitting at a different wavelength to simultaneously identify critical structures in the surgical field. Nevertheless, our preclinical study has important limitations and should not yet be extrapolated to human use. First, we used mice and pigs, which have subtle differences in their thymic anatomy compared with humans. Second, we did not observe ectopic tissue in these animals, although pigs have rather diffuse thymic tissue that does appear to mimic ectopic deposits (Figs 5 and 6). And, finally, although no obvious changes in vital signs or heart rhythm were encountered during the 8-hour monitoring period, we could have missed subacute toxicity. Future studies will have to address these shortcomings. In particular, initial human clinical trials will need to focus first on the safety of OX170, because it is not approved by regulatory agencies for human use, and the lethal dose that kills 50% of a test sample has not yet been reported. Then, uptake in human thymic tissue can be confirmed before dose optimization and pharmacokinetic studies. The authors wish to thank Rita G. Laurence, David J. Burrington, Jr, Eugenia Trabucchi, Sylvain Gioux, Frank Kettenring, Florin Neacsu, and Joseph Angelo for their assistance. This work was supported by National Institutes of Health grants R01-CA-115296 and R01-EB-011523.
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