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-11746086-B2
Publication Date
2023-09-05
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, as well as methods of making and using them. The disclosed conjugates are neutral zwitterionic molecules that exhibit little or no aggregation, specifically showing high resistance to aggregation when conjugated to targeting agents such as antibodies or receptor ligands.
The problem being solved is the formation of non-emissive dye aggregates, especially H-aggregates, which cause a blue-shifted absorption band and reduced fluorescence intensity upon fluorophore bioconjugation. Conventional methods like persulfonation, while successful for visible-range fluorophores, have limited success in preventing aggregation in near-infrared (NIR) fluorophores, which are inherently less emissive. Therefore, there is a pressing need for NIR fluorophores that avoid aggregation to maintain brightness, target binding, in vivo stability, and pharmacokinetics.
The disclosed conjugates combine short polyethylene glycol chains on indolenine nitrogens and a substituted quaternary amine alkyl ether at the C4' position, resulting in net-neutral zwitterionic dyes with highly aggregation-resistant properties. These conjugates are synthesizable via concise sequences, efficiently label monoclonal antibodies at neutral pH, exhibit no evidence of H-aggregation even at high labeling densities, and demonstrate exceptionally bright in vivo signals compared to conventional heptamethine cyanines like IRDye®-800CW. Additionally, these conjugates show superior tumor uptake, brightness, and reduced liver uptake in vivo.
Claims Coverage
The patent includes multiple independent claims covering novel conjugates, conjugate precursors, pharmaceutical compositions, and methods of tumor imaging, each with distinctive inventive features.
Novel conjugate and conjugate precursor structures with defined substituents
The invention covers conjugates or conjugate precursors according to Formula I, characterized by specific groups including R2 as H, halo, optionally substituted alkyl or aryl; R3 to R16, sulfonate-containing groups at R11 and R12; and defined parameters m, n, p, q. These structures feature a targeting agent attached via Ra to Rc.
Use of targeting agents including antibodies such as panitumumab or trastuzumab
Certain claims specify that the targeting agent connected to the conjugate is an antibody, including monoclonal antibodies panitumumab or trastuzumab, capable of recognizing and binding to tumor biomarkers, which enhances targeted imaging functionality.
Symmetrical substitution pattern on the conjugate structure
Some claims require symmetrical substitution where pairs of R groups (e.g., R3 and R4, R5 and R8, R6 and R9, etc.) are the same, contributing to the molecular stability and aggregation resistance.
Degree of labeling and multiplexed conjugate structures
Claims include conjugates with multiple heptamethine cyanine moieties per targeting agent, with degrees of labeling (DOL) ranging from 1 to 8, allowing enhanced fluorescence without aggregation, including structures according to Formula II and IIA.
Pharmaceutical composition containing the conjugate with a pharmaceutically acceptable carrier
The invention encompasses pharmaceutical compositions comprising at least one of the disclosed conjugates and a pharmaceutically acceptable carrier suitable for various administration routes.
Methods of tumor imaging using the conjugates with NIR fluorescence detection
The patent claims methods for tumor imaging by contacting biological samples or subjects with the conjugate, irradiating with light of selected wavelength (650-900 nm) and intensity, and detecting fluorescence to indicate target presence, including ex vivo and in vivo applications, with image-guided tumor excision.
Overall, the claims comprehensively cover the composition, structural variants, pharmaceutical formulations, and imaging methods involving aggregation-resistant heptamethine cyanine conjugates with targeting agents, enabling effective NIR fluorescence tumor detection and imaging with improved performance over prior art.
Stated Advantages
High resistance to aggregation, resulting in little or no H-aggregation even at high labeling densities.
Exceptionally bright in vivo fluorescence signals compared to conventional heptamethine cyanines such as IRDye®-800CW.
Superior tumor uptake and brightness in vivo.
Reduced liver uptake, which may improve imaging especially in hepatobiliary regions.
Efficient and concise synthesis and labeling at neutral pH.
Documented Applications
In vivo tumor imaging by administering the conjugate to a subject followed by near-infrared irradiation to visualize tumors.
Ex vivo and in vitro imaging of biological samples containing or suspected of containing a target by contacting with the conjugate and detecting fluorescence.
Fluorescence-guided surgical excision of tumors using the conjugate to localize tumor cells.
Use of the conjugate precursors for customized conjugation to targeting agents to develop tailored imaging agents.
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