ObjectiveFluorophore-labeled contrast imaging agents are moving toward clinical use for a number of applications. The near-infrared dye IRDye 800CW is frequently used in its N-hydroxysuccinamide (NHS) ester form for labeling these agents. Following conjugation or breakdown of a labeled ligand, excess NHS ester is converted to the carboxylate form. To prepare for clinical use as a near-infrared fluorophore, a toxicity study was conducted on IRDye 800CW carboxylate.MethodsMale and female Sprague–Dawley rats were given a single intravenous or intradermal administration of IRDye 800CW carboxylate; Indocyanine Green was used as a comparative control. Animals were injected with varying doses of the test and control articles and observed for up to 14 days. Clinical chemistry, hematological, and pharmacokinetic analyses were performed on subgroups of animals. Organs were analyzed for content of the test article. Tissues were analyzed microscopically for pathological changes.ResultsBased on hematologic, clinical chemistry, and histopathologic evaluation, single administration of IRDye 800CW carboxylate intravenously at dose levels of 1, 5, and 20 mg/kg or 20 mg/kg intradermally produced no pathological evidence of toxicity.ConclusionA dose of 20 mg/kg was identified as the no observed adverse effect level following IV or ID routes of administration of IRDye 800CW.Electronic supplementary materialThe online version of this article (doi:10.1007/s11307-010-0317-x) contains supplementary material, which is available to authorized users.
Purpose ABY-029, a synthetic Affibody peptide, Z03115-Cys, labeled with a near-infrared fluorophore, IRDye® 800CW, targeting epidermal growth factor receptor has been produced under Good Manufacturing Practices for an FDA approved first-in-use human study during surgical resection of glioma, as well as other tumors. Here, the pharmacology, phototoxicity, receptor activity and biodistribution studies of ABY-029 were completed in rats, prior to the intended human use. Procedures Male and female Sprague-Dawley rats were administered a single intravenous dose of varying concentrations (0, 245, 2449, and 24,490 µg/kg corresponding to 10X, 100X, and 1000X an equivalent human microdose level) of ABY-029 and observed for up to 14 days. Histopathological assessment of organs and tissues, clinical chemistry and hematology were performed. In addition, pharmacokinetic clearance, and biodistribution of ABY-029 were studied in sub-groups of the animals. Phototoxicity and ABY-029 binding to human and rat EGFR were assessed in cell culture and on immobilized receptors, respectively. Results Histopathological assessment, and hematological and clinical chemistry analysis demonstrated that single-dose ABY-029 produced no pathological evidence of toxicity at any dose level. No phototoxicity was observed in EGFR positive and negative glioma cell lines. Binding strength and pharmacokinetics of the anti-EGFR Affibody molecules were retained after labeling with the dye. Conclusion Based on the successful safety profile of ABY-029, the 1000X human microdose 24.5 mg/kg was identified as the no observed adverse effect level (NOAEL) following intravenous administration. Conserved binding strength and no observed light toxicity also demonstrated ABY-029 safety for human use.
Fluorescence in the near-infrared (NIR) spectral region is suitable for in vivo imaging due to its reduced background and high penetration capability compared to visible fluorescence. SNAPf is a fast-labeling variant of SNAP-tag that reacts with a fluorescent dye-conjugated benzylguanine (BG) substrate, leading to covalent attachment of the fluorescent dye to the SNAPf. This property makes SNAPf a valuable tool for fluorescence imaging. The NIR fluorescent substrate BG-800, a conjugate between BG and IRDye 800CW, was synthesized and characterized in this study. HEK293, MDA-MB-231 and SK-OV-3 cells stably expressing SNAPf-Beta-2 adrenergic receptor (SNAPf-ADRβ2) fusion protein were created. The ADRβ2 portion of the protein directs the localization of the protein to the cell membrane. The expression of SNAPf-ADRβ2 in the stable cell lines was confirmed by the reaction between BG-800 substrate and cell lysates. Microscopic examination confirmed that SNAPf-ADRβ2 was localized on the cell membrane. The signal intensity of the labeled cells was dependent on the BG-800 concentration. In vivo imaging study showed that BG-800 could be used to visualize xenograph tumors expressing SNAPf-ADRβ2. However, the background signal was relatively high, which may be a reflection of non-specific accumulation of BG-800 in the skin. To address the background issue, quenched substrates that only fluoresce upon reaction with SNAP-tag were synthesized and characterized. Although the fluorescence was successfully quenched, in vivo imaging with the quenched substrate CBG-800-PEG-QC1 failed to visualize the SNAPf-ADRβ2 expressing tumor, possibly due to the reduced reaction rate. Further improvement is needed to apply this system for in vivo imaging.
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