Stable liquid phantom for near-infrared fluorescence verification

Abstract

The present invention relates to the field of optical imaging. More specifically, the present invention relates to the use of a formulation of an organic dye with near-infrared emission dissolved in Good's buffer as a suitable phantom for evaluating, validating and calibrating near-infrared fluorescence imaging systems.

Description

Technical Field

[0001] The present invention relates to the field of optical imaging. More specifically, the present invention relates to the use of a formulation in which an organic dye having near-infrared radiation is dissolved in a good buffer as an appropriate phantom for evaluating, verifying, and calibrating a near-infrared fluorescence imaging system.

Background Art

[0002] Near-infrared fluorescence imaging devices are used in clinical settings and typically provide fluorescence images in tissues derived from an exogenous contrast agent administered to a patient before or during an imaging session. Performance verification of such a near-infrared fluorescence detection system is very important to ensure a reproducible quantitative evaluation. The imaging device needs to be tested to confirm that it is operating correctly to avoid defects that the user may not notice, such as a decrease in excitation light intensity or mechanical problems in the detection optical system. This is particularly important in medical applications where the performance of the imaging device can affect the outcome of diagnosis and treatment (i.e., surgery). However, despite recent advances in fluorescence imaging, the availability of appropriate verification systems and criteria for evaluating the sensitivity of imaging systems remains unsatisfied.

[0003] Testing of the imaging device may be performed using specific measuring instruments. However, this cannot simultaneously verify the illumination unit and the acquisition unit of the imaging device.

[0004] Another possibility is the use of a curable polyurethane matrix or composite phantom embedded with quantum dots (small particles manufactured by semiconductor processes) of different concentrations, as described, for example, in U.S. Patent No. 9,167,240 relating to a method and composite of solid phantoms for the validation of fluorescence imaging and tomography devices, and in Gorpas et al., J. Biomed. Opt. 2017, 22(1): 016009, which describes the use of composite solid phantoms for the validation and standardization of fluorescence imaging devices. However, composite phantoms are complex to manufacture and control, and quantum dots have very high visible light absorption (especially much higher than fluorescent agents commonly used in medical applications), meaning they can only be used to validate the performance of imaging devices in environments with controlled illumination.

[0005] Alternatively, tissue-mimicking phantoms are designed by combining materials with absorption and scattering properties similar to human tissues (e.g., hemoglobin and intralipid) with organic fluorescent dyes such as indocyanine green (ICG). Examples of such phantoms are described in U.S. Patent Application Publication 2006-056580 and Grand et al., J. Biomed. Opt. 2006, 11(1): 014007, which disclose tissue-like solid phantoms for validation of fluorescence imaging systems. However, because the preparation of these types of phantoms is relatively complex, large-scale production can be not only cumbersome but also quite costly, considering the various materials required (polymers, scattering agents, absorbing agents, dyes, buffers, and excipients).

[0006] A simpler alternative to these systems is the use of liquid phantoms, which consist of organic dyes in a suitable buffer. Liquid phantoms are more user-friendly and customizable than solid phantoms because the dye solution can be filled into disposable instruments commonly found in laboratories and hospitals, such as multiwell plates, vials, or capillary tubes. An example of a commonly used liquid phantom is the fluorescein NIST traceable standard solution (code: F36915) sold by ThermoFisher. However, fluorescein emits in the visible electromagnetic spectrum (515 nm), and there are no NIST traceable standards available for controlling, verifying, and calibrating near-infrared fluorescence imaging devices that operate at wavelengths above 650 nm.

[0007] Further examples of such phantom solutions have been reported by Koller et al., Nat. Commun. 2018, 9(1): 3739 and Hoogstins et al., Mol. Imaging. Biol. 2019, 21(1): 11-18, disclosing the use of a liquid phantom containing a near-infrared dye for the validation of intraoperative fluorescence imaging devices, and a test device (holder) named CalibrationDisk (SurgVision) filled with a vial containing the near-infrared dye solution before the device validation procedure.

[0008] However, these testing devices require manual intervention (such as on-site preparation), are operator-dependent, and are prone to errors. Furthermore, the stability of most organic dyes in solution is relatively low, and the average user lacks the appropriate equipment, know-how, procedures, and analytical methods to control the quality of dye solutions (i.e., purity, concentration, identity, etc.) after preparation and during storage. Moreover, the above references mention the use of high molecular weight molecules (bevacizumab-800CW) in which NIR dyes are bound to antibodies, and even in this case, preparing such phantoms on a large scale for routine system performance verification can be extremely complex and costly. Furthermore, although bevacizumab-800CW is dissolved in 2% Intralipid®, a lipid emulsion consisting of phospholipids and fatty acids that mimic human tissue, accurate control of the dye concentration cannot be properly achieved due to optical interference with standard absorbance measurements. Finally, according to Ter Weele et al., Eur. J. Pharm. Biopharm. 104(2016), 226-234, bevacizumab-800CW is stable when formulated in an isotonic sodium phosphate-buffered chloride solution at pH 7, but its stability decreases when other components are included in the formulation. Therefore, the long-term stability of the formulation in 2% Intralipid® is not guaranteed, and potential bias is introduced during routine system performance validation. [Overview of the project] [Problems that the invention aims to solve]

[0009] Therefore, a stable and convenient liquid phantom based on organic near-infrared dyes is needed for the routine performance evaluation of fluorescence systems for optical imaging operating at near-infrared wavelengths. [Means for solving the problem]

[0010] Outline of the invention The present invention relates to the use of a formulation containing an organic near-infrared dye dissolved in Good's buffer as a suitable phantom for evaluating, verifying, and calibrating near-infrared fluorescence imaging systems. The aforementioned solution may optionally contain at least one additive. In particular, near-infrared dyes are compounds of formula (I), as shown in the detailed explanation below.

[0011] The formulation may be supplied in a final storage container that does not interfere with near-infrared imaging procedures and does not require additional end-user operations such as dilution, splitting, or quality verification.

[0012] Accordingly, another aspect of the present invention relates to a kit for verifying the performance of a near-infrared fluorescence apparatus, comprising a set of containers for containing formulations of the present invention at different concentrations (i.e., different dilutions of the near-infrared dye in Good buffer), enabling simultaneous testing of the fluorescence apparatus at multiple concentrations.

[0013] Further aspects of the present invention relate to the use of such a stable formulation for performance verification of a near-infrared fluorescence imaging system comprising at least an illumination unit and an acquisition unit. Furthermore, the present invention relates to the verification of a near-infrared fluorescence imaging system intended for biomedical imaging applications, where the imaging is microscopic imaging of organic and inorganic substances, cells and intracellular structures, or tomographic imaging of tissues and organs. The near-infrared imaging system is either a preclinical imaging system or a clinical imaging system.

[0014] The formulations of the present invention can be used to verify the performance of near-infrared imaging devices prior to biomedical imaging procedures such as fluorescence endoscopy, fluorescence minimally invasive surgery or laparoscopy, fluorescence robotic surgery, open-field surgery, laser-guided surgery, photodynamic therapy, fluorescence lifetime imaging, or photoacoustic or ultrasonic fluorescence.

[0015] In yet another aspect, the present invention relates to a method for performing a fluorescence verification procedure of a near-infrared fluorescence imaging system using such a stable formulation. [Brief explanation of the drawing]

[0016] [Figure 1] Figure 1 shows a linear regression plot (R²=0.999) representing dye concentration (nM, x-axis) versus fluorescence intensity (mean radiative efficiency, y-axis) obtained from data collected from Example 4. [Modes for carrying out the invention]

[0017] Detailed description of the invention A first aspect of the present invention is formula (I): [ka] (I) [In the formula, R7 is selected from hydrogen, chlorine, phenyl and -O-phenyl optionally substituted with -SO3H,-COOH,-CONH-Y,-alkyl-COOH or -alkyl-CONH-Y groups, where Y is a divalent alkyl group substituted with -SO3H or at least two hydroxyl groups; R1, R2, R3, and R4 are each independently selected from hydrogen, -SO3H, -COOH, and -CONHY, where Y is a divalent alkyl substituted with -SO3H or at least two hydroxyl groups, or R1 together with R2, and R3 together with R4, each form a benzo group optionally substituted with at least one -SO3H group; and R5 and R6 are each independently divalent alkyl groups optionally substituted with a group selected from -SO3H, -COOH, and -CONH2. It contains the pigment shown, The formulation is used as a phantom to verify the performance of a fluorescence imaging device, and is dissolved in a Good buffer containing at least one additive as an option.

[0018] Near-infrared dyes useful in the present invention typically have a maximum absorbance between 750 nm and 850 nm and a maximum fluorescence emission between 770 nm and 900 nm in an aqueous medium. Thus, the near-infrared spectrum of the dye is compatible with most near-infrared imaging systems.

[0019] Furthermore, it has been found that when the dye of formula (I) is dissolved in a suitable good buffer, a desirable long-term storage period of the formulation is obtained, thus enabling intensive production, storage, and remote transportation to the test site. The components of the formulation of the present invention are relatively inexpensive, the manufacturing process is reproducible, and it is suitable for large-scale supply. Standard analytical procedures can be applied to control the quality of the formulation before sale.

[0020] In a preferred embodiment, the near-infrared dye is a compound represented by the above formula (I) wherein R2 and R3 are hydrogen, that is, formula (Ia):

Chemical formula

[0021] More preferably, the near-infrared dye is a compound represented by formula (Ia) wherein R1 and R4 are -SO3H groups, R5 and R6 are each independently a divalent alkyl optionally substituted with -SO3H or -COOH, and R7 is chlorine or -O-phenyl optionally substituted with a -SO3H group.

[0022] In a more preferred embodiment, the near-infrared dye is a compound selected from sulfo-Cy7 (CAS Nr.: 2104632-29-1), S0456 (CAS Nr.: 1252007-83-2), IRDye800CW (CAS Nr.: 1088919-86-1), and IRDye800BK (CAS Nr.: 748120-01-6).

[0023] In another preferred embodiment, the near-infrared dye is a compound represented by formula (I) above, where R1 together with R2 and R3 together with R4, respectively, forming a benzo group, i.e., formula (Ib): [ka] (Ib) [In the formula, R5, R6, and R7 are as defined above, and R8 is independently either hydrogen or -SO3H.] It is a compound represented by [the formula shown].

[0024] The compound represented by the preferred formula (Ib) is, for example, IR-820 (CAS Nr.: 172616-80-7) and its derivatives.

[0025] Preferably, the buffer used in the formulation of the present invention is highly water-soluble, minimally affected by salt, chemically stable, and optically transparent. For the formulation of the present invention to be conveniently used as a phantom for verifying the performance of a fluorescence imaging device, the buffer needs to not interfere with the absorption and emission properties of the dye and to generate an electromagnetic spectrum in the ultraviolet-visible region equivalent to that of the same dye dissolved in distilled water.

[0026] In preferred embodiments, suitable buffers are amphoteric biological buffers containing divalent C1-C4 alkyl groups substituted with -SO3H or -COOH groups.

[0027] More preferably, the Good buffer is selected from the group consisting of MOPS (3-(morpholine-4-yl)propane-1-sulfonic acid), MES (2-morpholine-4-ylethanesulfonic acid), TRICINE ({[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino}acetic acid), HEPES (2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid), BES (2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid), TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino]ethanesulfonic acid), TAPSO (3-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino]-2-hydroxypropane-1-sulfonic acid), PIPES (1,4-piperazinediethanesulfonic acid), and the like. The most preferred buffering compound is selected from MOPS, BES, HEPES, and TRICINE.

[0028] Table I below shows the chemical structure and pKa at 20°C of preferred Good buffers.

[0029] Table I - Chemical structure and pKa of preferred good buffers at 20°C [Table 1]

[0030] The formulation defined above has been proven to be stable for at least one month when stored at 2-8°C and for at least two weeks when stored on a workbench at 25°C. Furthermore, it can be easily prepared from a stock solution that can be stored in a refrigerator.

[0031] The near-infrared dye represented by formula (I) can be readily dissolved in aqueous solutions containing Good's buffer at concentrations compatible with the sensitivity of typical fluorescence detection systems. In particular, such concentrations are less than 1 mg / mL. For example, such concentrations fall within the range of 1 to 1000 μg / mL solutions for low-sensitivity fluorescence detection systems and within the range of 1 to 1000 ng / mL solutions for high-sensitivity fluorescence detection systems. Furthermore, for very high-sensitivity fluorescence detection systems, the concentration range can be 1 to 1000 pg / mL solutions.

[0032] In a preferred embodiment of the present invention, the near-infrared dye represented by formula (I) is dissolved in an aqueous Good buffer at a concentration in the range of 1 nM to 100 nM.

[0033] The concentration of Good buffer is in the range of 1 mM to 100 mM, more preferably between 5 mM and 50 mM.

[0034] In a further embodiment of the present invention, the near-infrared dye represented by formula (I) is dissolved in Good's buffer at a pH of 6 to 8, more preferably 6.5 to 7.5.

[0035] In another embodiment, the formulation of the present invention further comprises at least one additive. Suitable additives include organic solvents, surfactants, and antimicrobial substances. Suitable organic solvents include, for example, ethanol, methanol, dimethyl sulfoxide, formamide, dimethylformamide, and N-methylformamide. Suitable surfactants include, for example, polysorbates such as Tween 20 and Tween 80, polyethylene glycols of different size distributions (e.g., PEG40, PEG100, PEG300, PEG400), sodium stearate, sodium lauryl sulfate, Triton X-100, and NP-40. Suitable antimicrobial substances include, for example, sodium azide and benzyl alcohol.

[0036] In a further embodiment, the present invention provides the use of the formulation defined above, provided as a stock solution in a container stopper system. The container stopper system is suitable for containing the solution without the risk of leakage or evaporation. For example, the container stopper system can be selected from bottles, tubes, vials, containers, storage bags, and the like.

[0037] Another aspect of the present invention relates to a verification kit comprising a stable formulation as defined above, contained within a primary packaging suitable for fluorescence detection. The primary packaging is a suitable container suitable for storing the solution. For example, the primary packaging may be a tube, vial, ampoule, syringe, cuvette, or multiwell plate with a suitable lid.

[0038] In a further embodiment, the verification kit includes a set of multiple primary packages, for example, multiple vials or tubes, each of which is pre-filled with different dilutions of the formulation of the present invention in an aqueous solution of the organic buffer compound defined above, allowing the fluorescence device to be tested simultaneously at multiple concentrations.

[0039] Preferably, the validation kit includes a set of four primary packages (e.g., tubes) containing the formulation defined above, with the near-infrared dye present at selected concentrations such as 0 nM, 2 nM, 8 nM, and 32 nM, respectively. Optionally, such primary packages can be identified and associated with different concentrations of fluorescence dilutions by using color-coded caps of different colors for each dilution.

[0040] In further embodiments, the primary packaging is contained within secondary packaging suitable for preserving the product's quality over a long period and limiting the primary packaging's exposure to light. For example, the secondary packaging may be selected from cardboard boxes, aluminum pouches, envelopes, sleeves, canisters, or resealable bags. The secondary packaging may optionally include instructions.

[0041] In another embodiment, the present invention provides a method for calibrating a fluorescence imaging apparatus, comprising the following steps: a) Expose the validation kit defined above to an appropriate excitation source for the fluorescence system; b) Collect fluorescence emission using an appropriate detection system; c) Record fluorescence data using an appropriate computerized system.

[0042] definition In this description, unless otherwise specified, the following terms and phrases used herein are intended to have the meanings set forth below.

[0043] The term "alkyl" refers to a linear or branched aliphatic hydrocarbon radical group having 1 to 6 carbon atoms in the chain. For example, "C1-C4 alkyl" includes linear or branched groups containing 1 to 4 carbon atoms. Representative and preferred alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, pentyl, and hexyl. Unless otherwise specified, linear or branched alkyl groups are monovalent radical groups. In some cases, they may be "divalent" or "polyvalent" radical groups, such as methylene, ethylene, and isopropylene groups, which are obtained by removing and substituting two or more hydrogen atoms from the above hydrocarbon radical groups.

[0044] As used herein, the terms “Good buffer,” “biological buffer,” or “buffer” refer to water-soluble organic substances that maintain a constant pH over a predetermined optimal range (typically pH 6–8) by neutralizing the effects of hydrogen ions. Preferably, they are amphoteric molecules having a pKa value of 6–10 and being derivatives of aminoethane or aminopropane, optionally substituted with sulfonic acids and / or carboxylic acids. Examples of Good buffers include MES (2-morpholine-4-ylethanesulfonic acid), Bis-Tris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol), ADA (2,2',2''-nitrilotriacetic acid), PIPES (1,4-piperazinediethanesulfonic acid), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), and Bis-Tris Propan (1,3-bis[tris(hydroxymethyl)methylamino]propane), BES (2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid), MOPS (3-(morpholine-4-yl)propane-1-sulfonic acid), TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino]ethanesulfonic acid), HEPES (2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid), DIPSO (3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid), MOBS (4-(N-morpholino)butanesulfonic acid) (N-acid), TAPSO (3-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino]-2-hydroxypropane-1-sulfonic acid), HEPPSO (N-(hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid), POPSO (piperazine-N,N'-bis(2-hydroxypropanesulfonic acid)), EPPS (N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid)), Tricinel ({[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino}acetic acid), Gly-Gly (glycyl-glycine), Bicine (N,N-bis(2-hydroxyethyl)glycine), HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)), TAPS ([(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid), AMPD (2-amino-2-methyl-1,3-propanediol), TABS (N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid), AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid), CHES (2-(cyclohexylamino)ethanesulfonic acid), CAPSO The buffers can be selected from the group consisting of 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid, CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), and CABS (4-(cyclohexylamino)-1-butanesulfonic acid) buffers, i.e., biological buffers commonly known as Good's buffers.

[0045] The buffering agents used in the present invention are characterized by having a solubility in water at 20°C in the range of about 0.05 M to about 4 M. Preferably, they have a solubility of at least 0.1 M in water.

[0046] The term "buffer solution" refers to an aqueous solution containing a biological buffer.

[0047] The term "amphoterionic compound" refers to a molecule containing an equal number of positively and negatively charged functional groups. This typically represents a bipolar ion containing both an acidic (e.g., carboxylic acid or -SO3H) and a basic (e.g., amine) component, such as amino acid derivatives.

[0048] The terms "low sensitivity" or "high sensitivity" for fluorescence detection systems refer to the system's detection limit, i.e., the lowest fluorescence signal that can be distinguished from a blank. [Examples]

[0049] Experiment Part The invention and its specific embodiments described in the following parts are merely illustrative and should not be considered limiting to the invention; they illustrate how the invention may be carried out and are intended to be illustrative without limiting the scope of the invention.

[0050] Materials and equipment IRDye 800CW carboxylate was purchased from LI-COR Inc (Lincoln, Nebraska, USA; code 929-09406, lot C80209-01). S0456 was purchased from Few Chemicals GmbH (Bitterfeld-Wolfen, Germany; code 420456, lot 5114017). IRDye 800BK was synthesized as described in EP 1113822B1. The purity of IRDye 800BK sodium salt was 99.6% at 776 nm (maximum absorbance).

[0051] HEPES, MOPS, MES, BES, TRICINE, sodium azide, and Tween20 were purchased from SIGMA. Other reagents were purchased from Merck KGaA and were at least analytical grade. MilliQ water provided by the MilliQ instrument (Merck Millipore) was used to prepare the buffer.

[0052] Stability studies were conducted using a New Brunswick Scientific Innova 4230 Incubator Shaker (Marshall Scientific LLC). Absorbance, excitation, and emission values ​​were evaluated using a SPECORD 200 PLUS spectrophotometer (Analytik Jena GmbH).

[0053] Fluorescence imaging studies were performed using the preclinical fluorescence imaging system IVIS Spectrum (Perkin Elmer).

[0054] List of abbreviations BES 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid (CAS number: 10191-18-1) EtOH Ethanol HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (CAS number: 7365-45-9) MES 2-Morpholine-4-ylethanesulfonic acid (CAS number: 4432-31-9) MOPS 3-(morpholine-4-yl)propane-1-sulfonic acid (CAS number: 1132-61-2) PIPES 1,4-Piperazinediethanesulfonic acid (CAS number: 5625-37-6) PBS (phosphate-buffered saline) TAPSO 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid (CAS number: 68399-81-5) TES 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (CAS number: 7365-44-8) TRICINE {[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]aminoacetic acid (CAS number: 5704-04-1) Tween20 Polyethylene Glycol Sorbitan Monolaurate

[0055] Example 1: Preparation of organic buffer solution Aqueous solutions containing organic buffer compounds suitable for dissolving the near-infrared dye represented by formula (I) were prepared, for example, as reported for several representative buffer compounds using the following procedure: a) 50mM HEPES (pH 7.4): To prepare 500 mL of 50 mM buffer, 5.96 g of HEPES was dissolved in 450 mL of water. The pH of the solution was adjusted to 7.4 by adding 0.1 M HCl. Next, the buffer was diluted with water to 500 mL, filtered through a 0.22 μm membrane under sterile conditions, and stored at +2 to 8°C for up to 3 months. b) 50 mM MOPS (pH 7.0):5.23 g of MOPS was dissolved in 450 mL of water, and the desired pH and volume were obtained by following the same procedure as in Example a). c) 50mM Tricinene (pH 8.0): 4.48 g of TRICINE was dissolved in 450 mL of water, and the desired pH and volume were obtained by following the same procedure as in Example a). d) 50 mM MES (pH 6.2): 4.88 g of MES was dissolved in 450 mL of water, and the desired pH and volume were obtained by following the same procedure as in Example a). e) 50mM PBS (pH 7.4): To prepare 100 mL of 50 mM PBS solution for comparative experiments, 0.72 g of Na2HPO4, 4 g of NaCl, and 0.1 g of KCl were dissolved in 100 mL of water. The solution was filtered through a 0.22 μm membrane under sterile conditions and stored at +2 to 8°C for up to 3 months.

[0056] Example 2: Preparation of stock solution (IRDye 800CW dissolved in 50mM HEPES pH 7.4) The stock solution of IRDye 800CW was prepared by dissolving IRDye 800CW carboxylate in a 50 mM HEPES buffer at pH 7.4, prepared as described in Example 1, a). For example, 20 nmol of IRDye 800CW was dissolved in 3 mL of 50 mM HEPES solution.

[0057] The precise concentration of the stock solution is given by the Lambert-Beer formula: A = εcl [Here, A is the measured absorbance, c is the molar concentration, l is the optical path length, and ε is the molar extinction coefficient of the dye (i.e., for IRDye 800CW, ε is 240,000 M)] -1 cm -1 (That is.) This was determined by UV / VIS at 774nm.

[0058] The stock solution, prepared as described above and diluted 1:2 with HEPES buffer, was found to contain 4.27 ± 0.05 μM of IRDye 800CW carboxylate.

[0059] Example 3: Preparation of a verification kit (standard solution) As described in Example 2, the stock solution was prepared, and three different dilutions were performed with HEPES buffer. 225 μL, 56.2 μL, and 14 μL of the stock solution in the volumetric flask were each diluted with HEPES buffer to a final volume of 30 mL, yielding 70 mL of standard solutions with concentrations of 32 nM, 8 nM, and 2 nM.

[0060] For each IRDye 800CW standard solution (32, 8, and 2 nM), 1.6 mL of the standard solution was filled into 30 clear plastic vials and capped with color-coded screw caps (green for 32 nM, orange for 8 nM, and yellow for 2 nM). In addition, 30 vials labeled "blank" (0 nM) were filled with HEPES buffer using the same procedure and capped with clear screw caps.

[0061] Each of the above kits (32, 8, 2, and 0 nM vials) was placed in an individual aluminum foil envelope and labeled. The kits were stored at +2 to 8°C.

[0062] Example 4: Fluorescence imaging verification test A validation kit consisting of three vials (i.e., 32 nM, 8 nM, and 2 nM) containing standard solutions prepared as described in Example 3 and stored in an aluminum foil envelope at +2 to 8°C, and one vial (0 nM, blank) containing HEPES buffer, was removed from the refrigerator and equilibrated at room temperature for 30 to 60 minutes. The vials were then removed from the envelope and placed in the collection chamber of the IVIS Spectrum preclinical imaging system. Fluorescence imaging was performed using predefined acquisition settings with excitation at 745 ± 15 nm and detection at 800 ± 10 nm. At the end of the imaging session, a fluorescence image of the phantom kit was acquired.

[0063] The signal intensity was calculated by placing the target region in each of the four vials of the kit. To evaluate linearity, the fluorescence intensity values ​​were plotted against concentration. Figure 1 shows an example of a linearity plot of dye concentration versus fluorescence intensity obtained using the validation kit described in Example 3. The validation kit demonstrated high detection linearity of the tested imaging system in the selected concentration range (2–32 nM).

[0064] Example 5: Stability of phantom solution at +2 to 8°C Several stability studies were conducted to evaluate the effects of various additives, such as buffers, excipients, or preservatives, and storage conditions on the dye formulations. In particular, the stability of the formulations after storage in a refrigerator at +2 to 8°C was investigated first.

[0065] Stability was measured as a decrease in absorbance at the maximum wavelength of the dye, obtained using a UV / VIS spectrophotometer, indicating a decrease in the dye monomer content in the solution. All results are reported as a residual percentage relative to baseline (T=0), with the percentage at time=0 being 100%. The decrease in absorbance is associated with a decrease in the dye concentration in the formulation buffer, indicating decreased stability and degradation of the major chromophore species.

[0066] In the table below, the residual absorbances of various dyes, namely IRDye 800CW and S0456, were recorded after storing the formulations in several representative organic buffers at +2 to 8°C. Specifically, the dyes IRDye 800CW and S0456 were dissolved at a concentration of 3 μM in 50 mM buffer MES, HEPES, MOPS, or TRICINE, and the solutions were refrigerated for 4 weeks to confirm their stability.

[0067] The results for residual absorbance are shown in Tables II and III, respectively.

[0068] Table II also shows the results of a comparative experiment in which IRDye 800CW was dissolved at a concentration of 3 μM in 50 mM inorganic buffer PBS (phosphate-buffered saline). In this case, the stability of the dye buffer formulation was low, and after 4 weeks at +2 to 8°C, the residual absorbance was less than 80% due to the decomposition of the chromophore.

[0069] Table II: Various Organic Buffers and Dyes in PBS IRDye 800CW Percentage of residual absorbance at (3 μM) [Table 2]

[0070] Table III: Dyes in various organic buffers S0456 Percentage of residual absorbance at (3 μM) [Table 3]

[0071] The stability of the above solution observed over four weeks (approximately one month) showed a residual absorbance of over 95%.

[0072] Furthermore, the stability of 3 μM solutions of IRDye 800BK dye dissolved in MOPS and BES buffers at different concentrations (10 and 50 mM) and different pH conditions was confirmed by refrigeration at +2 to 8°C.

[0073] The results for residual absorbance percentages are shown in Tables IV and V, respectively. These data demonstrate that slight variations in buffer compound concentration and pH do not affect the stability of the phantom formulation.

[0074] Table IV: At various pH levels and buffer concentrations MOPS Dyes in buffering agent IRDye 800BK Percentage of residual absorbance at (3 μM) [Table 4]

[0075] Table V: At various pH levels and buffer concentrations BES Dyes in buffering agent IRDye 800BK Percentage of residual absorbance at (3 μM) [Table 5]

[0076] The stability of the above solution observed after 4 weeks (approximately 1 month) showed a residual absorbance of approximately 90%.

[0077] Example 6: Stability of phantom solution at +2 to 8°C in the presence of additives The effect of the presence of additives on the stability of the formulation of the present invention has also been investigated.

[0078] Further stability studies were conducted by storing representative formulations of the present invention, containing 3 μM IRDye 800CW dissolved in 50 mM HEPES or TRICINE buffer containing 0.04% Tween20 or 0.02% sodium azide, and formulations containing 3 μM IRDye 800BK dissolved in 10 mM HEPES, MOPS, or BES buffer containing 10% EtOH, in a refrigerator at +2 to 8°C. The preparation of the above buffers is described below:

[0079] 50mM HEPES(pH7.4)+0.04% Tween20: To prepare 100 mL of buffer solution, 1.19 g of HEPES was dissolved in 80 mL of water; 40 μL of Tween 20 was added, and the pH was adjusted to 7.4 with 0.1 M HCl. Next, the buffer solution was diluted with water to a volume of 100 mL and filtered through a 0.22 μm membrane under sterile conditions.

[0080] 50 mM HEPES (pH 7.4) + 0.02% sodium azide: To prepare 100 mL of buffer solution, 1.19 g of HEPES and 20 mg of sodium azide were dissolved in 80 mL of water, and the pH was adjusted to 7.4 with 0.1 M HCl. Next, the buffer solution was diluted with water to a volume of 100 mL and filtered through a 0.22 μm membrane under sterile conditions.

[0081] 10 mM HEPES (pH 7.0) + 10% EtOH:To prepare 100 mL of buffer solution, 0.24 g of HEPES was dissolved in 80 mL of water and the pH was adjusted to 7.0 with 0.1 M HCl. Next, 10 mL of 100% ethanol was added. Then, the buffer solution was diluted with water to a volume of 100 mL and filtered through a 0.22 μm membrane under sterile conditions.

[0082] Similar solutions were prepared for MOPS, BES, and TRICINE buffers using the same procedure.

[0083] The results of the stability studies shown in Tables VI and VII regarding residual absorbance percentage demonstrate that the presence of the additive does not significantly affect the stability of this formulation.

[0084] Table VI: Dyes in various biological buffers in the presence of Tween20 or sodium azide IRDye 800BK Percentage of residual absorbance at (3 μM) [Table 6]

[0085] Table VII: Dyes in various biological buffers in the presence of 10% EtOH IRDye 800BK Percentage of residual absorbance at (3 μM) [Table 7]

[0086] Example 7: Stability of phantom solution at 25°C The stability of the formulations under stress conditions was also investigated. In particular, samples of 3 μM IRDye 800CW dissolved in HEPES or MOPS buffer (all 50 mM) were stored for two weeks at 25°C in a dark incubator.

[0087] Table VIII below shows experimental results demonstrating that a typical embodiment of the present invention allows the formulation to be stored at 25°C (for example, on a workbench) for at least two weeks without significant degradation.

[0088] Table VIII: Pigments in various organic buffers after storage at 25°C IRDye 800BK Percentage of residual absorbance at (3 μM) [Table 8]

[0089] Example 8: Stability of phantom solution at +2 to 8°C during long-term storage The stability of the formulations under long-term storage conditions was also investigated. In particular, samples of 3 μM IRDye 800CW and 3 μM IRDye 800BK dissolved in HEPES buffer were protected from light exposure and refrigerated at +2 to 8°C for at least 6 months. Samples were used for long-term stability studies at 2 to 8°C.

[0090] The experimental results are shown in Table IX below. Here, it is shown that, in some representative embodiments of the present invention, the formulation can be stored at 2-8°C for at least 6 months without significant degradation.

[0091] Table IX: Dyes in HEPES buffer after storage at +2 to 8°C for at least 6 months. IRDye 800BK Percentage of residual absorbance at (3 μM) [Table 9] Furthermore, the present invention includes the following embodiments. [Aspect 1] Equation (I): [ka] (I) [In the formula, R7 is hydrogen, chlorine, -SO 3 Selected from phenyl and -O-phenyl optionally substituted with H,-COOH,-CONH-Y,-alkyl-COOH, or -alkyl-CONH-Y groups, where Y is -SO 3 It is a divalent alkyl group substituted with H or at least two hydroxyl groups; R1, R2, R3, and R4 are each independently hydrogen, -SO4. 3 H is selected from -COOH and -CONHY, where Y is -SO 3 It is a divalent alkyl group substituted with H or at least two hydroxyl groups, or R1 together with R2, and R3 together with R4, each with at least one -SO 3 Forms a benzo group optionally substituted with an H group; and R5 and R6 are independent of each other, -SO 3 H, -COOH, and -CONH 2 [A divalent alkyl group optionally substituted with a group selected from the following.] It contains the pigment shown, Use of a formulation dissolved in a Good buffer containing at least one additive as a phantom for verifying the performance of a fluorescence imaging device. [Aspect 2] The pigment is given by formula (Ia):

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[0092] References: 1. US 9,167,240 2. Gorpas et al.、J.Biomed.Opt.2017、22(1):016009 3. US2006-056580 4. De Grand et al.、J.Biomed.Opt.2006、11(1):014007 5. Koller et al.、Nat.Commun.2018、9(1):3739 6. Hoogstins at al.、Mol.Imaging.Biol.2019、21(1):11-18 7. Ter Weele et al.、Eur.J.Pharm.Biopharm.2016、104:226-34 8. EP1113822

Claims

1. Equation (I): 【Chemistry 1】 (I) [In the formula, R7 is hydrogen, chlorine, -SO 3 Selected from phenyl and -O-phenyl substituted with H,-COOH,-CONH-Y,-alkyl-COOH, or-alkyl-CONH-Y groups, where Y is -SO 3 It is a divalent alkyl group substituted with H or at least two hydroxyl groups; R1, R2, R3, and R4 are each independently hydrogen, -SO4. 3 H is selected from -COOH and -CONHY, where Y is -SO 3 It is a divalent alkyl group substituted with H or at least two hydroxyl groups, or R1 together with R2, and R3 together with R4, each with at least one -SO 3 Forms a benzo group substituted with an H group; and R5 and R6 are independent of each other, -SO 3 H, -COOH, and -CONH 2 [A divalent alkyl group substituted with a group selected from the following] It contains the pigment shown, A formulation dissolved in Good buffer for use as a phantom to verify the performance of a fluorescence imaging device.

2. The pigment is given by formula (Ia): 【Chemistry 2】 (Ia) [In the formula, R1 and R4 are each independently selected from hydrogen, -SO 3 H, -COOH and -CONHY, where Y is -SO 3 H or a divalent alkyl substituted with at least two hydroxyl groups, and R5, R6 and R7 have the same meanings as described above] The formulation according to claim 1, wherein the compound is represented by [formula].

3. R1 and R4 are -SO 3 It is an H group, and R5 and R6 are independently -SO 3 It is a divalent alkyl group substituted with H or -COOH, and R7 is chlorine or -SO 3 The formulation according to claim 2, wherein the compound is represented by formula (Ia), which is a -O-phenyl substituted with an H group.

4. The formulation according to claim 3, wherein the dye is a compound selected from sulfo-Cy7, S0456, IRDye 800CW, and IRDye 800BK.

5. The pigment is given by formula (Ib): 【Transformation 3】 (Ib) [In the formula, R5, R6, and R7 are as defined in claim 1, and R8 is independently hydrogen or -SO4. 3 [H is] The formulation according to claim 1, wherein the compound is represented by [formula].

6. The formulation according to claim 5, wherein the dye is IR-820 or a derivative thereof.

7. Good cushioning agent, -SO 3 Divalent carbon substituted with H and / or -COOH groups 1 -C 4 The formulation according to claim 1, which is an alkyl-containing amphoteric biological buffer.

8. The formulation according to claim 7, wherein the good buffer is selected from the group consisting of MOPS, MES, TRICINE, HEPES, BES, TES, TAPSO, and PIPES.

9. The formulation according to claim 8, wherein the buffering agent is MOPS, BES, HEPES, or TRICINE.

10. The formulation according to claim 1, wherein the Good buffer comprises at least one additive selected from surfactants, organic solvents, and antimicrobial compounds.

11. The formulation according to claim 10, wherein the surfactant is selected from Tween20, Tween80, PEG40, PEG100, PEG300, PEG400, PEG4000, sodium stearate, sodium lauryl sulfate, Triton X-100, and NP-40.

12. The formulation according to claim 10, wherein the organic solvent is selected from ethanol, methanol, dimethyl sulfoxide, formamide, dimethylformamide, and N-methylformamide.

13. The formulation according to claim 10, wherein the antimicrobial compound is selected from sodium azide and benzyl alcohol.

14. A validation kit for calibrating a fluorescence imaging device, comprising a formulation as defined in claim 1, contained within a primary packaging for fluorescence detection selected from tubes, vials, ampoules, syringes, cuvettes, and multiwell plates with appropriate lids.

15. The following steps: a) Expose the verification kit defined in claim 14 to a suitable excitation source of the fluorescence system; b) Collect fluorescence emission using an appropriate detection system; c) Record fluorescence data using an appropriate computerized system; A method for calibrating a fluorescence imaging device that includes [specific component / tool].

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