Non-aggregating heptamethine cyanine fluorophores for in vivo imaging
Inventors
Schnermann, Martin John • Luciano, Michael Philip • Nani, Roger Rauhauser
Assignees
US Department of Health and Human Services
Publication Number
US-10961193-B2
Publication Date
2021-03-30
Expiration Date
2039-02-15
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Abstract
Heptamethine cyanine fluorophore conjugates and conjugate precursors are disclosed. Methods of using the conjugates and conjugate precursors are also disclosed. The disclosed conjugates are neutral zwitterionic molecules and exhibit little or no aggregation.
Core Innovation
This disclosure concerns heptamethine cyanine fluorophore conjugates and conjugate precursors, which are neutral zwitterionic molecules exhibiting little or no aggregation. The conjugates are highly resistant to aggregation when conjugated to a targeting agent such as a biomolecule including antibodies or receptor ligands. The disclosed conjugates also exhibit exceptionally bright in vivo signals compared to conventional heptamethine cyanines.
The problem being solved is that fluorophore conjugation often alters the properties of both the fluorophore and the molecule it is attached to, impacting brightness, target binding, in vivo stability, and pharmacokinetics largely due to dye aggregation. Conventional strategies for avoiding aggregation, such as sulfonation, have limited success in near-infrared fluorophores which are intrinsically less emissive and still prone to forming non-emissive aggregates even at moderate labeling densities.
Claims Coverage
The patent contains two independent claims covering the conjugates/conjugate precursors by chemical structure and the methods of imaging using these conjugates.
Non-aggregating heptamethine cyanine conjugates or conjugate precursors
A conjugate or conjugate precursor, or stereoisomer thereof, defined by Formula IA featuring neutral zwitterionic molecules exhibiting reduced or no aggregation when conjugated to targeting agents such as antibodies. Specific structural features include p=2, 3, or 4; Ra as a linking group to targeting agents; and embodiments where substituents Rb, R3, R4, and R13-R16 can be methyl groups. The conjugates may have degrees of labeling from 1 to 8, with multiple heptamethine cyanine moieties attached to one targeting agent.
Pharmaceutical composition comprising the conjugate
Pharmaceutical formulations comprising the disclosed conjugate and a pharmaceutically acceptable carrier suitable for various modes of administration including parenteral, oral, topical, and more.
Method of imaging a biological sample or subject using the conjugate
A method including contacting a biological sample with a disclosed conjugate containing a targeting agent capable of specific binding to a target, irradiating the sample with near-infrared light to induce fluorescence, and detecting the fluorescence to confirm presence of the target. The method includes in vivo applications where the conjugate is administered to a subject and targeted irradiation and imaging occur at a target region such as a tumor, optionally followed by excision of fluorescent tumor cells.
The independent claims cover novel non-aggregating heptamethine cyanine conjugates/conjugate precursors with specific chemical structures, pharmaceutical compositions comprising them, and methods of imaging using these conjugates with targeted fluorescence detection, especially for tumor localization. The inventive features focus on the molecular structure conferring aggregation resistance, enabling high brightness and improved in vivo imaging performance.
Stated Advantages
The conjugates exhibit highly resistance to aggregation even at high labeling densities, avoiding problems like H-aggregation that reduce fluorescence.
They produce exceptionally bright in vivo fluorescence signals compared to conventional heptamethine cyanines such as IRDye®-800CW.
The disclosed conjugates show superior tumor uptake and brightness in vivo imaging models.
Reduced liver uptake of the conjugates compared to conventional dyes, improving imaging specificity and reducing background.
Conjugates can be prepared by a concise, scalable synthetic sequence and labeled efficiently at neutral pH.
Documented Applications
In vivo fluorescence imaging for visualization and localization of tumors in subjects using conjugates comprising targeting agents such as antibodies.
Ex vivo or in vitro imaging of biological samples to detect targets such as tumor antigens by fluorescence after conjugate binding and irradiation.
Fluorescence-guided surgery where fluorescent signals from the conjugates guide excision of tumor tissue.
Customized conjugation of conjugates precursors with selected targeting agents suitable for diagnostic or therapeutic research and clinical applications.
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