Stable liquid phantom for near-infrared fluorescence validation

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 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. [Background technology]

[0002] Near-infrared fluorescence imaging devices are used in clinical settings to provide images of fluorescence in tissues, typically derived from exogenous contrast agents administered to the patient before or during the imaging session. Performance validation of such near-infrared fluorescence detection systems is crucial to ensure reproducible quantitative evaluations. Imaging devices need to be tested to ensure they are working correctly to avoid defects that may go unnoticed by the user, such as reduced excitation light intensity or mechanical problems in the detection optics. This is particularly important in medical applications, where the performance of the imaging device may affect diagnostic and therapeutic (i.e., surgical) outcomes. However, despite recent advances in fluorescence imaging, the availability of appropriate validation systems and criteria to evaluate the sensitivity of imaging systems remains unmet.

[0003] Testing of the imaging device may be performed using specific metrology equipment, which, however, cannot simultaneously verify the illumination and acquisition units of the imaging device.

[0004] Another possibility is the use of a curable polyurethane matrix or composite phantoms embedded with different concentrations of quantum dots (small particles produced in semiconductor processes), as described, for example, in US Pat. No. 9,167,240, which relates to a composite of methods and 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 produce and control, and quantum dots have a very high absorption of visible light (especially much higher than the fluorescent agents typically used in medical applications), so they can only be used to validate the performance of imaging devices in lighting-controlled environments.

[0005] Alternatively, tissue-mimicking phantoms have been designed by combining materials with absorption and scattering properties similar to those of human tissue (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 No. 2006-056580 and Grand et al., J. Biomed. Opt. 2006, 11(1): 014007, which disclose tissue-like solid phantoms for validation of fluorescent imaging systems. However, this kind of phantom is relatively complex to prepare, and large-scale production can be tedious as well as quite costly, considering the variety of materials required (polymers, scattering agents, absorbers, dyes, buffers, excipients).

[0006] A simpler alternative to these systems is represented by the use of liquid phantoms composed of an organic dye in an appropriate buffer. Liquid phantoms are more user-friendly and customizable than solid phantoms, since the dye solution can be filled into disposable devices commonly found in laboratories and hospitals, such as multi-well plates, vials or capillary tubes. An example of a commonly used liquid phantom is the Fluorescein NIST-traceable standard (code: F36915) sold by ThermoFisher. However, fluorescein emits in the visible electromagnetic spectrum (515 nm), and there are no NIST-traceable standards available for the control, validation and calibration of near-infrared fluorescence imaging devices operating at wavelengths above 650 nm.

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

[0008] However, these test devices require manual intervention (e.g., on-site preparation), are operator-dependent, and prone to error. Moreover, most organic dyes have a relatively low stability in solution, and ordinary users lack the appropriate equipment, know-how, procedures, and analytical methods to control the quality (i.e., purity, concentration, identity, etc.) of the dye solution after preparation and during storage. Furthermore, the above references mention the use of a high molecular weight molecule in which the NIR dye is conjugated with an antibody (bevacizumab-800CW), so even in this case, it may be very complicated and costly to prepare such phantoms on a large scale for routine system performance validation. Moreover, although bevacizumab-800CW is dissolved in 2% Intralipid®, a lipid emulsion consisting of phospholipids and fatty acids that mimics human tissue, the exact concentration of the dye cannot be adequately controlled due to optical interference with standard absorbance measurements. Finally, Ter Weele et al., Eur. J. Pharm. Biopharm. 104 (2016), 226-234, reported that bevacizumab-800CW is stable when formulated in isotonic phosphate-buffered sodium chloride solution at pH 7, but the presence of other components in the formulation reduces its stability, so the long-term stability of the formulation in 2% Intralipid® is not guaranteed, introducing potential bias during routine system performance validation. Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, stable and convenient liquid phantoms based on organic near-infrared dyes are required for routine performance evaluation of fluorescence systems for optical imaging operating at wavelengths in the near-infrared spectrum. [Means for solving the problem]

[0010] Summary of the invention The present invention relates to the use of a formulation comprising an organic near-infrared dye dissolved in Good's buffer as a suitable phantom for evaluating, validating, and calibrating near-infrared fluorescence imaging systems. The solution may optionally include at least one additive. In particular, the near infrared dye is a compound of formula (I) as shown in the detailed description below.

[0011] The formulation may be supplied in a final container for storage that does not interfere with the near-infrared imaging procedure and does not require additional manipulation by the end user, such as dilution, division or quality verification.

[0012] Thus, another aspect of the present invention relates to a kit for verifying the performance of a near-infrared fluorescent device, comprising a set of containers containing different concentrations of the formulation of the present invention (i.e., different dilutions of the near-infrared dye in Good's buffer), allowing for testing of the fluorescent device at multiple concentrations simultaneously.

[0013] A further aspect of the invention relates to the use of such a stable formulation for the performance validation of a near-infrared fluorescence imaging system consisting of at least an illumination unit and an acquisition unit. Furthermore, the invention relates to the validation of a near-infrared fluorescence imaging system intended for biomedical imaging applications, the imaging being microscopic imaging of organic and inorganic materials, cells and subcellular 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 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 sonofluorescence.

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

[0016] [Figure 1] FIG. 1 shows a linear regression plot of dye concentration (nM, x-axis) versus fluorescence intensity (mean radiant efficiency, y-axis) obtained from data collected from Example 4 (R2=0.999). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description of the Invention The first aspect of the present invention is a compound of formula (I): [ka] (I) [In the formula, R7 is selected from hydrogen, chlorine, -SO3H, -COOH, -CONH-Y, phenyl optionally substituted with -alkyl-COOH or -alkyl-CONH-Y groups, and -O-phenyl, where Y is -SO3H or a divalent alkyl substituted with at least two hydroxyl groups; R1, R2, R3 and R4 are each independently selected from hydrogen, -SO3H, -COOH and -CONHY, where Y is -SO3H or a divalent alkyl substituted with at least two hydroxyl groups; 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 a divalent alkyl optionally substituted with a group selected from -SO3H, -COOH, and -CONH2. The dye is represented by Use of the formulation, dissolved in Good's buffer, optionally containing at least one additive, as a phantom to verify the performance of a fluorescent imaging device.

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

[0019] Furthermore, it has been found that dissolving the dye of formula (I) in a suitable Good's buffer provides the formulation with a desirable long shelf life, thus allowing centralized production, storage, and remote transportation to the testing site. The components of the formulation of the present invention are relatively inexpensive, and the manufacturing process is reproducible and suitable for large-scale supply. Standard analytical procedures can be applied to control the quality of the formulation prior to release.

[0020] In a preferred embodiment, the near infrared dye is a compound represented by the above formula (I) in which R2 and R3 are hydrogen, i.e., formula (Ia): [ka] (Ia) [In the formula, R1 and R4 are each independently selected from hydrogen, -SO3H, -COOH, and -CONHY, where Y is -SO3H or a divalent alkyl substituted with at least two hydroxyl groups, and R5, R6, and R7 are as defined above. It is a compound represented by the formula:

[0021] More preferably, the near infrared dye is a compound represented by formula (Ia) in which 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 -SO3H groups.

[0022] In a further 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 the above formula (I) in which R1 together with R2 and R3 together with R4 form a benzo group, i.e., formula (Ib): [ka] (Ib) [In the formula, R5, R6 and R7 are defined as above, and each R8 is independently hydrogen or -SO3H.] It is a compound represented by the formula:

[0024] Preferred compounds of formula (Ib) are represented, for example, by 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, has minimal salt effects, is chemically stable, and is optically transparent. In order to conveniently use the formulation of the present invention as a phantom for verifying the performance of a fluorescence imaging device, the buffer should not interfere with the absorption and emission properties of the dye and should produce an electromagnetic spectrum in the ultraviolet and visible range equivalent to that of the same dye dissolved in distilled water.

[0026] In a preferred embodiment, the suitable buffer is a zwitterionic biological buffer comprising a divalent C1-C4 alkyl substituted with -SO3H or -COOH groups.

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

[0028] The chemical structures and pKa at 20° C. of preferred Good's buffers are shown in Table I below.

[0029] Table I - Chemical structures and pKa at 20°C of preferred Good's buffers [Table 1]

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

[0031] The near infrared dyes represented by formula (I) can be readily dissolved in an aqueous solution containing a Good's buffer at a concentration compatible with the sensitivity of common fluorescence detection systems. In particular, such a concentration is 1 mg / mL or less. For example, such a concentration can be in the range of 1-1000 μg / mL solution for low sensitivity fluorescence detection systems and in the range of 1-1000 ng / mL solution for high sensitivity fluorescence detection systems. Furthermore, for very sensitive fluorescence detection systems, the concentration range can be 1-1000 pg / mL solution.

[0032] In a preferred embodiment of the present invention, the near infrared dye of formula (I) is dissolved in aqueous Good's buffer at a concentration comprised between 1 nM and 100 nM.

[0033] The concentration of the Good's buffer ranges from 1 mM to 100 mM, and more preferably between 5 mM and 50 mM.

[0034] In a further embodiment of the present invention, the near infrared dye of formula (I) is dissolved in Good's buffer at a pH value between 6 and 8, more preferably between 6.5 and 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 antibacterial substances. Suitable organic solvents include, for example, ethanol, methanol, dimethyl sulfoxide, formamide, dimethylformamide, N-methylformamide. Suitable surfactants include, for example, polysorbates such as Tween 20 and Tween 80, polyethylene glycols of different size distributions (for example, PEG40, PEG100, PEG300, PEG400), sodium stearate, sodium lauryl sulfate, Triton X-100, and NP-40. Suitable antibacterial substances include, for example, sodium azide and benzyl alcohol.

[0036] In a further aspect, the present invention provides the use of the formulation as defined above, provided as a concentrate in a container closure system, the container closure system being suitable for containing the solution without risk of leakage or evaporation.For example, the container closure system is selected from a bottle, a tube, a vial, a container, a storage bag, etc.

[0037] Another aspect of the invention relates to a validation kit comprising the stable formulation as defined above contained in a primary package suitable for fluorescence detection. The primary package is a suitable container suitable for storing the solution. For example, the primary package can be a tube, a vial, an ampoule, a syringe, a cuvette, a multi-well plate with a suitable lid, etc.

[0038] In a further embodiment, the validation kit described above comprises a set of multiple primary packages, e.g. multiple vials or tubes, each pre-filled with a different dilution of the formulation of the invention in an aqueous solution of an organic buffer compound as defined above, allowing for simultaneous testing of the fluorescent device at multiple concentrations.

[0039] Preferably, the validation kit comprises a set of four primary packages (e.g., tubes) containing the formulations 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 fluorescent dilutions by using color-coded caps with different colors for each dilution.

[0040] In a further embodiment, the primary packaging is contained within a secondary packaging suitable for preserving the quality of the product over time and limiting exposure of the primary packaging to light. For example, the secondary packaging is selected from a cardboard box, a foil pouch, an envelope, a sleeve, a canister, a zip lock storage bag, and optionally includes instructions.

[0041] In another aspect, the present invention provides a method of calibrating a fluorescence imaging device, comprising the steps of: a) exposing the validation kit defined above to an appropriate excitation source of a fluorescence system; b) collecting the fluorescent emission with an appropriate detection system; c) Recording the fluorescence data with a suitable computerized system.

[0042] definition For the purposes of this description, unless otherwise specified, the following terms and phrases as used herein are intended to have the following meanings:

[0043] The term "alkyl" refers to a straight or branched chain aliphatic hydrocarbon radical group having 1 to 6 carbon atoms in the chain. For example, "C1-C4 alkyl" includes within its meaning straight or branched chains 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, a straight or branched chain alkyl is a monovalent radical group. In some cases, it may be a "divalent" or "polyvalent" radical group, such as methylene, ethylene, isopropylene groups, in which two or more hydrogen atoms have been removed from the above hydrocarbon radical groups and replaced.

[0044] The expressions "Good's buffer" or "biological buffer" or "buffer" as used herein refer to water-soluble organic substances that maintain a constant pH over a given optimum range (usually a pH of 6 to 8) by neutralizing the effects of hydrogen ions. Preferably, they have a pKa value of 6 to 10 and are zwitterionic molecules that are derivatives of aminoethane or aminopropane, optionally substituted with sulfonic and / or carboxylic acids. Examples of Good's buffers are MES (2-morpholin-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), Bis-Tris Propane (1,3-bis[tris(hydroxymethyl)methylamino]propane), BES (2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid), MOPS (3-(morpholin-4-yl)propane-1-sulfonic acid), TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid), HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), DIPSO (3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid), MOBS (4-(N-morpholino)butanesulfonic acid) acid), TAPSO (3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-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)), Tricine ({[1,3-dihydroxy-2-(hydroxymethyl)propan-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 buffer may 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 buffers used in the present invention are characterized by a solubility in water ranging from about 0.05 M to about 4 M at 20° C. Preferably, they have a solubility in water of at least 0.1 M.

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

[0047] The term "zwitterionic compound" refers to a molecule that contains equal numbers of positively and negatively charged functional groups. It typically refers to a dipolar ion that contains both an acid (e.g., carboxylic acid or -SO3H) and a base (e.g., amine) moiety, such as an amino acid derivative.

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

[0049] Experimental Part The invention and its specific embodiments described in the following parts are merely exemplary and should not be considered as limiting the invention; they show how the invention can be put into practice and are intended to illustrate 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 Tween 20 were purchased from SIGMA. Other reagents were purchased from Merck KGaA and were of at least analytical grade. MilliQ water provided by a MilliQ apparatus (Merck Millipore) was used for preparation of buffers.

[0052] Stability studies were performed 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-morpholin-4-ylethanesulfonic acid (CAS number: 4432-31-9) MOPS 3-(morpholin-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]amino}acetic acid (CAS number: 5704-04-1) Tween 20 Polyethylene glycol sorbitan monolaurate

[0055] Example 1: Preparation of organic buffer Aqueous solutions containing organic buffer compounds suitable for dissolving the near infrared dyes of formula (I) were prepared, for example, as reported for some representative buffer compounds by the following procedures: a) 50mM HEPES(pH7.4): 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. The buffer was then made up to 500 mL with water, filtered through a 0.22 μm membrane under sterile conditions, and stored at +2-8 °C for up to 3 months. b) 50mM MOPS(pH7.0):5.23 g of MOPS was dissolved in 450 mL of water and the same procedure as in example a) was followed to obtain the desired pH and volume. c) 50mM TRICINE(pH8.0): 4.48 g of TRICINE was dissolved in 450 mL of water and the same procedure as in example a) was followed to obtain the desired pH and volume. d) 50mM MES (pH 6.2): 4.88 g of MES was dissolved in 450 mL of water and the same procedure as in example a) was followed to obtain the desired pH and volume. e) 50mM PBS(pH7.4): To prepare 100 mL of 50 mM PBS solution for the 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 +2-8 °C for up to 3 months.

[0056] Example 2: Preparation of stock solution (IRDye 800CW dissolved in 50 mM HEPES pH 7.4) A stock solution of IRDye 800CW was prepared by dissolving IRDye 800CW carboxylate in 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 exact concentration of the stock solution is determined using the Lambert-Beer formula: A = εcl [where A is the measured absorbance, c is the molar concentration, l is the path length, and ε is the molar extinction coefficient of the dye (i.e., for the IRDye 800CW, ε is 240,000 M -1 cm -1 ). The concentration was determined by UV / VIS at 774 nm using a 500 .mu.m.times.10 ...

[0058] Determination of the concentration of IRDye 800CW carboxylate in a stock solution prepared as above and diluted 1:2 with HEPES buffer revealed a concentration of 4.27±0.05 μM (average of triplicate determinations).

[0059] Example 3: Preparation of validation kit (standard solution) From the stock solution prepared as described in Example 2, three different dilutions were performed with HEPES buffer, obtaining 70 mL of standard solutions with concentrations of 32 nM, 8 nM and 2 nM by diluting 225 μL, 56.2 μL and 14 μL of the stock solution in a volumetric flask, respectively, with HEPES buffer to a final volume of 30 mL.

[0060] For each IRDye 800CW standard (32, 8, 2 nM), 30 clear plastic vials were filled with 1.6 mL of standard solution 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 kit (32, 8, 2, 0 nM vial) was placed in an individual aluminum foil envelope and labeled. The kits were stored at +2-8°C.

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

[0063] Signal intensity was calculated by placing an area of ​​interest in each of the four vials of the kit. To assess linearity, the fluorescence intensity values ​​were plotted against concentration. An example of a linearity plot of dye concentration versus fluorescence intensity obtained using the validation kit described in Example 3 is shown in Figure 1. The validation kit revealed 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 carried out to evaluate the effect of various buffers, excipients or preservatives, and storage conditions on the dye formulation. In particular, the stability of the formulation after storage in a refrigerator at +2-8°C was first investigated.

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

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

[0067] The residual absorbance results 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 which case the dye buffer formulation was less stable, with less than 80% remaining absorbance after 4 weeks at +2-8°C due to decomposition of the chromophore species.

[0069] Table II: Dyes in various organic buffers and PBS IRDye 800CW (3μM) Residual Absorbance Percentage [Table 2]

[0070] Table III: Dyes in various organic buffers S0456 (3μM) Residual Absorbance Percentage [Table 3]

[0071] The stability of the above solution confirmed over a period of 4 weeks (approximately 1 month) showed a residual absorbance of approximately 95% or more.

[0072] Additionally, 3 μM solutions of IRDye 800BK dye in MOPS and BES buffers at different concentrations (10 and 50 mM) and different pH conditions were refrigerated at +2–8 °C to confirm stability.

[0073] The results for percentage remaining absorbance are shown in Tables IV and V, respectively. These data indicate that small variations in the concentration and pH of the buffer compound do not affect the stability of the phantom formulations.

[0074] Table IV: At different pH and different buffer concentrations MOPS Dye in Buffer IRDye 800BK (3μM) Residual Absorbance Percentage [Table 4]

[0075] Table V: At different pH and different buffer concentrations BES Dye in Buffer IRDye 800BK (3μM) Residual Absorbance Percentage [Table 5]

[0076] The stability of the above solution observed over a 4 week period (approximately 1 month) showed approximately 90% remaining absorbance.

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

[0078] Further stability studies were performed by storing representative formulations of the invention containing 3 μM IRDye 800CW in 50 mM HEPES or TRICINE buffer containing 0.04% Tween 20 or 0.02% sodium azide, and 3 μM IRDye 800BK in 10 mM HEPES, MOPS or BES buffer containing 10% EtOH at +2-8° C. in a refrigerator. The preparation of the above buffers is described below:

[0079] 50mM HEPES(pH7.4)+0.04% Tween20: To prepare 100 mL of buffer, 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. The buffer was then made up to a volume of 100 mL with water 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, 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. The buffer was then made up to a volume of 100 mL with water and filtered through a 0.22 μm membrane under sterile conditions.

[0081] 10mM HEPES (pH 7.0) + 10% EtOH:To prepare 100 mL of buffer, 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. Then, 10 mL of 100% ethanol was added. The buffer was then made up to a volume of 100 mL with water and filtered through a 0.22 μm membrane under sterile conditions.

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

[0083] The results of the stability studies, shown in Tables VI and VII in terms of percentage absorbance remaining, demonstrate that the presence of excipients does not significantly affect the stability of the formulation.

[0084] Table VI: Dyes in various biological buffers in the presence of Tween 20 or sodium azide IRDye 800BK (3μM) Residual Absorbance Percentage [Table 6]

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

[0086] Example 7: Stability of phantom solutions 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 at 25 °C in a dark incubator for 2 weeks.

[0087] For representative embodiments of the present invention, the results of experiments showing that the formulations can be stored at 25° C. (eg, on a work bench) for at least two weeks without significant deterioration are shown below in Table VIII.

[0088] Table VIII: Dyes in various organic buffers after storage at 25°C. IRDye 800BK (3μM) Residual Absorbance Percentage [Table 8]

[0089] Example 8: Stability of phantom solutions at +2-8°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 stored refrigerated at +2-8 °C for at least 6 months. Samples of for long-term stability studies at +2-8 °C.

[0090] The results of the experiment are shown below in Table IX, which shows that for several exemplary embodiments of the present invention, the formulations can be stored at 2-8° C. for at least 6 months without significant degradation.

[0091] Table IX: Dye in HEPES buffer after storage at +2-8°C for at least 6 months IRDye 800BK (3μM) Residual Absorbance Percentage [Table 9]

[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. Formula (I): 【Chemistry 1】 (I) [In the formula, R7 is hydrogen, chlorine, -SO 3 H, -COOH, -CONH-Y, phenyl optionally substituted with -alkyl-COOH or -alkyl-CONH-Y groups, and -O-phenyl, where Y is -SO 3 H or a divalent alkyl substituted with at least two hydroxyl groups; R1, R2, R3 and R4 each independently represent hydrogen, -SO 3 H, -COOH and -CONHY, where Y is -SO 3 H or a divalent alkyl substituted with at least two hydroxyl groups, or R1 together with R2, and R3 together with R4, each of which is at least one -SO 3 forming a benzo group optionally substituted with H groups; and R5 and R6 each independently represent -SO 3 H, -COOH and -CONH 2 is a divalent alkyl optionally substituted with a group selected from The dye is represented by A formulation, dissolved in Good's buffer, optionally containing at least one additive, for use as a phantom for verifying the performance of a fluorescent imaging device.

2. The dye is represented by formula (Ia): 【Chemistry 2】 (Ia) [In the formula, R1 and R4 each independently represent 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 are as defined above. The formulation according to claim 1, wherein the compound is represented by the formula:

3. R1 and R4 are -SO 3 H group, and R5 and R6 are each independently -SO 3 R7 is a divalent alkyl optionally substituted with H or -COOH, and R7 is chlorine, or -SO 3 3. The formulation of claim 2, which is a compound of formula (Ia), which is -O-phenyl optionally substituted with an H group.

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

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

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

7. Good buffer, -SO 3 Divalent C substituted with H and / or -COOH groups 1 -C 4 2. The formulation of claim 1, which is an alkyl-containing zwitterionic biological buffer.

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

9. 9. The formulation of claim 8, wherein the buffer is MOPS, BES, HEPES or TRICINE.

10. 2. The formulation of claim 1, wherein the optional additive is selected from a surfactant, an organic solvent and an antimicrobial compound.

11. 11. The formulation of claim 10, wherein the surfactant is selected from Tween 20, Tween 80, PEG 40, PEG 100, PEG 300, PEG 400, PEG 4000, sodium stearate, sodium lauryl sulfate, Triton X-100 and NP-40.

12. 11. The formulation of claim 10, wherein the organic solvent is selected from ethanol, methanol, dimethylsulfoxide, formamide, dimethylformamide and N-methylformamide.

13. 11. The formulation of claim 10, wherein the antibacterial compound is selected from sodium azide and benzyl alcohol.

14. 13. A validation kit for calibrating a fluorescence imaging device comprising the formulation defined in claim 1 contained within a primary package for fluorescence detection selected from a tube, a vial, an ampoule, a syringe, a cuvette, a multi-well plate with a suitable lid.

15. Steps below: a) exposing the verification kit defined in claim 14 to a suitable excitation source of a fluorescence system; b) collecting the fluorescent emission with an appropriate detection system; c) recording the fluorescence data with an appropriate computerized system; A method for calibrating a fluorescence imaging device, comprising:

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