Sprayable neurospecific near-infrared fluorescent contrast agent composition for dual-channel imaging and use thereof

Abstract

The present invention relates to a contrast agent composition capable of specifically staining nerve tissue, in which the composition has affinity for myelin and can specifically stain nerve tissue rather than other tissues, has a wavelength in the near-infrared (NIR) region of 700 nm, and can stably emit fluorescence in vivo, and thus can be effectively used for real-time fluorescence imaging to differentiate from other tissues.

Description

Composition of a near-infrared fluorescent contrast agent for nebulizable neurospecific dual-channel imaging and use thereof

[0001] The present invention relates to a near-infrared fluorescent contrast agent composition for neurospecific imaging and its use.

[0002] Imaging technology using near-infrared fluorescence holds great potential in medicine, particularly in diagnosis and image-guided surgery. In modern medicine, precise imaging of neural tissue is crucial for the diagnosis and treatment of various neurological disorders. Accurately identifying and protecting neural tissue during surgery is essential to minimize nerve damage and improve patient outcomes. Recurrent laryngeal nerve damage occurs in up to 50% of cases after esophagectomy and thyroidectomy, which can lead to hoarseness, aspiration, and dyspnea. Furthermore, cavernous nerve damage has been reported in up to 70% of cases after prostatectomy, which can lead to sexual and urinary dysfunction. Therefore, accurate identification and preservation of nerves during cancer surgery is a critical issue, and various imaging techniques and contrast agents have been developed to address this issue.

[0003] Various methods, such as electromyography, ultrasound, and optical coherence tomography, have been used to identify nerves during surgery. However, these methods have limitations in wide-ranging imaging and resolution, making it difficult to provide real-time visual feedback. Therefore, real-time fluorescent imaging contrast agents using near-infrared wavelengths are being utilized effectively. Conventional contrast agents mainly absorb specific wavelengths in the visible or near-infrared region and have been used to image various biological tissues, including nerve tissue. However, these contrast agents have low specificity for nerve tissue, making it difficult to distinguish nerve tissue from other surrounding tissues. In addition, most existing contrast agents have specific absorption characteristics for tumor tissue, limiting their ability to simultaneously distinguish nerve tissue and tumor tissue.

[0004] In particular, nerve damage during cancer surgery, among various surgical procedures, results in significant morbidity and mortality. Despite advances in surgical techniques and equipment, nerves are currently identified by their visible shape and anatomical location, and surgery is performed without image guidance. Branching, trifurcation, medial or anterior displacement, and non-recurrent anatomical variations are also risk factors for nerve damage. Visual identification of nerves during complex cancer surgery is crucial to minimize the high morbidity and mortality associated with nerve damage. However, currently available intravenous nerve-targeting compounds do not provide optimal conditions due to high tissue absorption and scattering at UV and visible wavelengths, as well as short nerve retention times after systemic circulation. Furthermore, these compounds are inherently highly lipophilic and charged, resulting in high background binding to adipose tissue, significantly reducing the signal-to-background ratio (SBR). The relatively high intrinsic tissue autofluorescence in the visible range also limits the in vivo use of these fluorophores.

[0005] Accordingly, the inventors of the present invention aim to provide a contrast agent that absorbs at wavelengths in the 700 nanometer (nm) range in the near-infrared (NIR) region. When used in conjunction with a tumor-specific contrast agent, this agent offers the potential to clearly distinguish tumor tissue from neural tissue. Specifically, if such a contrast agent exhibits high affinity for myelin, allowing for more precise targeting of neural tissue, it offers the potential to overcome the limitations of existing technologies.

[0006] An object of the present invention is to provide a neurospecific contrast agent composition.

[0007] Another object of the present invention is to provide an imaging method comprising a step of administering the contrast agent composition to a subject.

[0008] Another object of the present invention is to provide a dual channel imaging approach using a sprayable formulation.

[0009] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0010] To solve the above problem, the present invention provides a neurospecific contrast agent composition comprising a compound represented by the following [chemical formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient:

[0011] [Chemical Formula 1]

[0012]

[0013] In the above [chemical formula 1],

[0014] R 1 and R 2 are the same or different from each other, and are each independently selected from hydrogen, alkoxy, C1 to C6 alkyl and C1 to C6 alkenyl, or are combined with each other to form an aromatic ring,

[0015] R 3 is C1 to C6 alkyl, C1 to C6 alkylsulfonyl, phenylpropyl and -(CH2) n N + (R 4 ) is one selected from the group consisting of 3,

[0016] n is an integer from 0 to 4,

[0017] R 4 is an alkyl group of C1 to C6,

[0018] X is a halogen element.

[0019] According to one side, R in the above [chemical formula 1] 1 and R 2 is hydrogen, and R 3 can be methyl.

[0020] According to one side, in the above [chemical formula 1], X can be I.

[0021] According to one aspect, the compound represented by the above [chemical formula 1] or a pharmaceutically acceptable salt thereof may specifically bind to myelin.

[0022] According to one aspect, the contrast agent may absorb light in the near-infrared (NIR) region with a wavelength of 700 nm to 800 nm. According to another embodiment of the present invention, an imaging method is provided, comprising the following steps:

[0023] (1) A step of treating a subject with a contrast agent composition containing a compound represented by the following [Chemical Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient; and

[0024] (2) A step of detecting a fluorescent signal emitted from the above object.

[0025] [Chemical Formula 1]

[0026]

[0027] In the above [chemical formula 1],

[0028] R 1 and R 2 are the same or different from each other, and are each independently selected from hydrogen, alkoxy, C1 to C6 alkyl and C1 to C6 alkenyl, or are combined with each other to form an aromatic ring,

[0029] R 3 is C1 to C6 alkyl, C1 to C6 alkylsulfonyl, phenylpropyl and -(CH2) n N + (R 4 ) is one selected from the group consisting of 3,

[0030] n is an integer from 0 to 4,

[0031] R 4 is an alkyl group of C1 to C6,

[0032] X is a halogen element.

[0033] According to one aspect, the imaging method can be achieved by additionally processing a second contrast agent composition having an absorption wavelength of 800 nm or more in addition to a contrast agent composition containing a compound represented by [chemical formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient.

[0034] According to one aspect, in the imaging method, the contrast agent composition including a compound represented by [chemical formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient may specifically stain nerve tissue, and the second contrast agent composition may specifically stain tumor tissue.

[0035] According to one aspect, in the above imaging method, the processing in step (1) may be performed by a dyed gauze method.

[0036] A neurospecific contrast agent according to one embodiment of the present invention has a complex chemical structure containing sufficient double bonds, thereby absorbing light in the near-infrared (NIR) region of 700 nanometers and emitting light of a different wavelength (generally a longer wavelength) than the absorbed light. Furthermore, it is myelin-friendly, enabling nerve tissue-specific staining. This allows it to be used in vivo with a second contrast agent that has a different absorption wavelength and is specific for tissues other than nerves to distinguish nerve tissue from other tissues.

[0037] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0038] Figure 1 shows the chemical structure and in vivo imaging of a neural targeting fluorescent contrast agent.

[0039] Figure 2 shows an experiment to optimize the absorption capacity of NTFP-700 for direct administration in mice.

[0040] Figure 3 shows an experiment to optimize the absorption time of NTFP-700 for direct administration in mice.

[0041] Figure 4 shows the optimization experiment of the washing protocol for NTFP-700 absorption for direct administration in mice.

[0042] Figure 5 shows a comparative experiment of the direct administration method and the dyed gauze method for sciatic nerve dyeing.

[0043] Figure 6 shows intraoperative dual-channel fluorescence imaging in a mouse tumor model.

[0044] Figure 7 shows a precision tumor surgical procedure using intraoperative dual-channel fluorescence imaging guidance in a rabbit tumor model.

[0045] Figure 8 shows histological images of rabbit nerves stained with H&E, NeuroTrace, FluoroMyelin, and NTFP-700 stains.

[0046] The present inventors provide a near-infrared fluorescent contrast agent that absorbs at a wavelength of 700 nm and has high affinity for myelin, and aim to provide a nerve-specific imaging method using this agent. Furthermore, a novel imaging method utilizing dyed gauze to selectively induce contrast agent absorption into nerve tissue is included. These technical features suggest its potential as a precise surgical guide and diagnostic tool capable of effectively distinguishing nerve tissue from tumor tissue.

[0047] This can be achieved by visualizing nerve and cancer tissues in two separate near-infrared (NIR) channels (800 nm for cancer and 700 nm for nerve, or vice versa). The superiority of the present invention's contrast agent lies in its nebulizable formulation, low signal-to-background ratio (SBR), and ability to overcome the challenges of tissue autofluorescence, which may provide improved intraoperative imaging guidance compared to current intravenously injectable nerve-targeting compounds.

[0048] Accordingly, the present invention provides a compound represented by the following [chemical formula 1] or a pharmaceutically acceptable salt thereof.

[0049] The present invention provides a neurospecific contrast agent composition comprising a compound represented by the following [chemical formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient:

[0050] [Chemical Formula 1]

[0051]

[0052] In the above [chemical formula 1],

[0053] R 1 and R 2 are the same or different from each other, and are each independently selected from hydrogen, alkoxy, C1 to C6 alkyl and C1 to C6 alkenyl, or are combined with each other to form an aromatic ring,

[0054] R 3 is C1 to C6 alkyl, C1 to C6 alkylsulfonyl, phenylpropyl and -(CH2) n N + (R 4 ) is one selected from the group consisting of 3,

[0055] n is an integer from 0 to 4,

[0056] R 4 is an alkyl group of C1 to C6,

[0057] X is a halogen element.

[0058] In the present invention, the term “alkyl” refers to a group derived from a straight or branched chain saturated aliphatic hydrocarbon having a specific number of carbon atoms and at least one valence. Examples of such alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, 2-butyl, 3-butyl, pentyl, n-hexyl, and the like.

[0059] In the present invention, the term “alkenyl” refers to a group having one C=C double bond in the alkyl group. Examples of such alkenyl groups include, but are not limited to, an ethenyl group, a propenyl group, a butenyl group, and the like.

[0060] In the present invention, the term “alkylsulfonyl” means a hydrogen at the terminal of the alkyl group is -SO3 - It means that it has been replaced with .

[0061] In the present invention, the term “halogen” refers to elements belonging to group 17 of the periodic table, such as fluorine (F), chloride (Cl), bromine (Br), or iodine (I).

[0062] In the present invention, the term “alkoxy” means an atomic group CnH2n+1O- formed by bonding an oxygen atom to an alkyl group, and examples of such alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, or phthaloxy.

[0063] In the present invention, the term “pharmaceutically acceptable salt” means a formulation of a compound that does not cause serious irritation to an organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The pharmaceutically acceptable salt can be obtained by reacting the compound of the present invention with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., a sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, etc., an organic carboxylic acid such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, etc. In addition, the compound of the present invention can be obtained by reacting it with a base to form a salt such as an alkali metal salt such as an ammonium salt, a sodium or potassium salt, an alkaline earth metal salt such as a calcium or magnesium salt, a salt of an organic base such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, and a salt of an amino acid such as arginine or lysine.

[0064] In the present invention, "including as an active ingredient" means that the ingredient is included in an amount necessary or sufficient to realize a desired biological effect. In actual application, the amount included as an active ingredient can be determined by considering the amount for treating the target disease and not causing other toxicity, and may vary depending on various factors such as the disease or condition being treated, the form of the composition being administered, the size of the subject, or the severity of the disease or condition. A person of ordinary skill in the art to which the present invention pertains can empirically determine the effective amount of an individual composition without undue experimentation.

[0065] In the present invention, the term "contrast agent" refers to a substance administered into the body to strongly and specifically contrast or image cancer cells, etc. in the body, and is currently widely used in the medical and diagnostic fields to enhance images of tissues and cells. The term "contrast agent" in the present invention is not limited to the scope of contrast agents for magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET), and is used to mean imaging agents for ultrasound image analysis, imaging agents for fluorescence image analysis, etc.

[0066] The present invention also provides an imaging method comprising a step of treating a subject with the contrast agent composition.

[0067] In the present invention, the term "imaging," also known as "imaging," refers to all methods for visualizing a target object. In the present invention, imaging may preferably be optical imaging using light.

[0068] The above “imaging” may be, but is not limited to, one or more selected from the group consisting of fluorescence, bioluminescence, magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), single photon emission computed tomography (SPECT), and combinations thereof.

[0069] The imaging in the present invention may preferably be dual-channel imaging or dual contrast agent imaging, which may mean the simultaneous use of two or more contrast agents having different and distinguishable wavelengths. Preferably, the contrast agent composition of the present invention may have a wavelength of 700 nm or more and less than 800 nm, and may specifically stain neural tissue, and the second contrast agent composition may have a wavelength of 800 nm or more.

[0070] In the present invention, the term “subject” or “individual” is not limited to any mammal such as a livestock or human that requires imaging to distinguish neural tissue from other tissues, but may preferably be a human.

[0071] The contrast agent composition according to the present invention can be administered via various routes, including oral, transdermal, subcutaneous, intravenous, or intramuscular. The dosage of the active ingredient can be appropriately selected based on various factors, such as the route of administration, the patient's age, sex, weight, and severity of the condition. Furthermore, the composition of the present invention can be administered in combination with known compounds capable of enhancing the desired effect.

[0072] The contrast agent composition according to the present invention can preferably be administered using the "stained gauze method." The stained gauze method refers to a method in which commercially available gauze is soaked in the contrast agent composition, sufficiently soaked, and then the target neural tissue requiring imaging is wrapped with the gauze to stain it.

[0073] Another preferred method of administering the contrast agent composition according to the present invention is spraying. The contrast agent composition of the present invention may be formulated into a formulation suitable for spraying.

[0074] The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0075] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0076] When describing components of an embodiment, terms such as first, second, A, B, (a), (b), etc. may be used. These terms are only intended to distinguish the components from other components, and the nature, order, or sequence of the components are not limited by the terms. When a component is described as being "connected," "coupled," or "connected" to another component, it should be understood that the component may be directly connected or connected to the other component, but that another component may also be "connected," "coupled," or "connected" between each component.

[0077]

[0078] Hereinafter, embodiments are described in detail with reference to the attached drawings. However, the embodiments may be modified in various ways, and the scope of the patent application is not limited or restricted by these embodiments. It should be understood that all modifications, equivalents, or alternatives to the embodiments are included within the scope of the patent application.

[0079] In addition, when describing with reference to the attached drawings, identical components will be assigned the same reference numerals regardless of the drawing numbers, and redundant descriptions thereof will be omitted. When describing embodiments, if a detailed description of a related known technology is judged to unnecessarily obscure the gist of the embodiment, the detailed description will be omitted.

[0080]

[0081] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0082]

[0083] Manufacturing Example 1. Experimental Method

[0084] The injectable near-infrared fluorescent contrast agent compounds for neuroimaging of the present invention were synthesized according to the following reaction scheme. Various hydrophobic pentamethine cyanine intermediates were synthesized as follows. 2,3,3-Trimethylindolene (1), 4,5-dimethoxy-2,3,3-trimethyl-3H-indole (2), and 1,1,2-trimethyl-1H-benzo[e]indole (3) were reacted with their respective alkyl halides in boiling acetonitrile to obtain heterocyclic derivatives (4 to 9). Each salt (4 to 9) was then condensed with Vilsmeier-Haack reagent to form the final pentamethine cyanines. The reaction mixture was purified by open column chromatography or solvent precipitation procedures (using diethyl ether or methyl tert-butyl ether) to obtain analytical purity (≥95% by LC-ELSD-MS).

[0085]

[0086] Manufacturing Example 1-1. 1,3,3-Trimethyl-2-((1E,3E,5E)-5-(1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indolium iodide (10 in the above figure, hereinafter NTFP-700)

[0087] Yield 64%; 1H NMR (400 MHz, MeOD-d4)δ: 1.71 (s, 12H), 3.63 (s, 6H), 6.28 (d,J= 16 Hz, 2H), 6.65 (t,J= 12 Hz, 1H), 7.24 (t,J= 8 Hz, 2H), 7.29 (d,J= 8 Hz, 2H), 7.39 (t,J= 8 Hz, 2H), 7.48 ( d,J= 8 Hz, 2H), 8.25 (t,J= 12 Hz, 2H); 13 C NMR (100 MHz, MeOD-d4) δ27.90, 31.71, 50.50, 104.44, 111.83, 123.31, 126.20, 129.71, 142.56, 144.29, 155.52, 175.28. TOF HRMS m / z (M + ) calculated for [C 27 H 31 N2] + 383.2487, found 383.2474.

[0088]

[0089] Manufacturing Example 1-2. 1-Butyl-2-((1E,3E,5E)-5-(1-butyl-3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indolium iodide (11)

[0090] Yield: 48%, MP > 260°C, 1 H NMR (400 MHz, DMSO-d6)δ: 0.91 (t,J= 8 Hz, 6H), 1.37 (q,J= 8 Hz, 4H), 1.66 (s, 16H), 4.08 (s, 4H), 6.30 (d,J= 16 Hz, 2H), 6.59 (t,J= 12 Hz, 1H), 7.23 (s, 2H), 7.38 (s, 4H), 7.60 (d,J= 8 Hz, 2H), 8.33 (t,J= 12 Hz, 2H). 13C NMR (100 MHz, DMSO-d6)δ:13.39, 19.11, 26.80, 28.73, 42.84, 48.52, 102.81, 110.74, 122.09, 124.31, 125.25, 128.07, 140.75, 141.65, 153.65, 172.26. TOF HRMS m / z (M + ) calculated for [C 33 H 43 N2] + 467.7074 found 468.4592.

[0091]

[0092] Manufacturing Example 1-3. 2-((1E,3E,5E)-5-(3,3-dimethyl-1-(3-phenylpropyl)indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-1-(3-phenylpropyl)-3H-indolium iodide (12)

[0093] Yield 49%, MP 185-187°C, 1 H NMR (400 MHz, DMSO-d6)δ1.68 (s, 12H), 2.01 (t,J=8 Hz, 4H), 2.74 (t,J=8 Hz, 4H), 4.15 (t,J=8 Hz, 4H), 6.075 (d,J=12 Hz, 2H), 6.46 (t,J= 12 Hz, 1H0, 7.40-7.24 (m,16H), 7.62 (d,J= 8 Hz, 2H), 8.33 (t,J= 12 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6) δ27.60, 29.16, 32.58, 43.55, 49.40, 103.58, 111.47, 122.93, 125.19, 125.19, 125.97, 128.71, 128.91, 141.43, 141.43, 142.46, 154.54, 173.11. TOF HRMS m / z (M + ) calculated for [C 43 H 47 N2] + 591.3734, found 591.2740.

[0094]

[0095] Manufacturing Example 1-4. 5-Methoxy-2-((1E,3E,5E)-5-(5-methoxy-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indolium iodide (13)

[0096] Yield 75%, MP 228-230 °C, 1 H NMR (400 MHz, DMSO-d6): δ1.66 (s, 12H), 3.56 (s, 3H), 3.81 (s, 6H), 6.165 (d,J= 12 Hz, 2H), 6.46 (t,J= 12 Hz, 1H), 6.95 (d,J= 8 Hz, 2H), 7.30-7.28 (m, 4H), 8.23 ​​(t,J= 12 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6): δ26.48, 30.62, 48.45, 55.29, 102.06, 108.43, 111.02, 112.84, 123.73, 135.80, 142.15, 151.94, 156.98, 171.55. TOF HRMS m / z (M + ) calculated for C 29 H 35 N2O2443.2699 found 443.2692.

[0097]

[0098] Manufacturing Example 1-5. 3-Ethyl-2-((1E,3E,5E)-5-(3-ethyl-1,1-dimethyl-1H-benzo[e]indole-2(3H)-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-1H-benzo[e]indolium iodide (14)

[0099] Yield 79%, MP 266-268°C, 1H NMR (400 MHz, DMSO-d6): δ1.333 (t,J= 8 Hz, 6H), 1.962 (s, 12H), 4.297 (t,J= 8 Hz, 4H), 6.37 (d,J= 12 Hz, 2H), 6.637 (t,J= 12 Hz, 1H), 7.51 (t,J= 8 Hz, 2H), 6.68 (t,J= 8 Hz, 2H), 7.74 (d,J= 8 Hz, 2H), 8.08 (t,J= 8 Hz, 4H), 8.25 (d,J= 8 Hz, 2H), 8.46 (t,J= 12 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6) δ11.96, 26.17, 48.04, 50.19, 102.05, 110.90, 121.58, 124.20, 125.07, 127.11, 127.19, 129.39, 129.83, 130.76, 132.72, 138.77, 152.54, 172.71.TOF HRMS m / z (M + ) calculated for C 37 H 39 N2511.3113, found 511.3098.

[0100]

[0101] Manufacturing Example 1-6. 4-(2-((1E,3E,5E)-5-(1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indole-2(3H)-ylidene)penta-1,3-dienyl)-1,1-dimethyl-1H-benzo[e]indoleium-3-yl)butane-1-sulfonate (15)

[0102] Yield 76%, MP > 250°C, 1 H NMR (400 MHz, DMSO-d6):δ 1.80 (m,J= 12 Hz, 9H), 2.00 (s, 12H), 4.2 (q, 4H), 6.4 (d,J= 16 Hz, 2H), 6.7 (t,J= 12 Hz, 1H), 7.50 (t,J= 8 Hz, 2H), 7.70 (t,J= 4 Hz, 2H), 8.10 (q, 4H), 8.35 (d,J= 8 Hz, 2H), 8.50 (t,J= 12 Hz, 2H).

[0103]

[0104] Experimental Example 1. Experimental Method

[0105] Study design

[0106] This study was designed to prevent unnecessary nerve damage during surgery by directly administering a nerve-targeting fluorescent agent during cancer surgery. In addition to cRGD-ZW800-PEG, a tumor-targeting fluorescent contrast agent with a wavelength of 800 nm developed in a previous study, NTFP-700, a fluorescent contrast agent with a neural targeting capability at a different wavelength channel of 700 nm, was developed to visualize tumors and nerves simultaneously during surgery.

[0107] To optimize the dosage, incubation time, and number of washes of the novel neuro-targeting fluorophore, we utilized mouse sciatic nerves (n=4) and brachial plexus (n=4). Furthermore, we present a dyed gauze method, which can be easily applied during clinical surgery, even in locations where visualization is difficult due to steep angles caused by cancer or anatomical structures. Using mouse sciatic nerves, the effectiveness of direct administration and dyed gauze methods, depending on the angle, was evaluated.

[0108] Furthermore, to evaluate the applicability during actual cancer surgery, we utilized mouse (n=4 for each method) and rabbit cancer models (n=4). Tumors were visualized using cRGD-ZW800-PEG (50 nmol for mice, 0.1 mg / kg for rabbits) at a dose optimized in a previous study, and nerves were visualized using an optimal dyed gauze method. All fluorescence images were captured using a FIAT-L imaging system (Nawoo Vision), allowing simultaneous visualization of tumors and nerves in real time during tumor surgery.

[0109]

[0110] animal testing

[0111] All surgical procedures, including animal care and handling, were approved by the Institutional Animal Care and Use Committee of Korea University (KOREA-2023-0189). Five-week-old C57BL / 6 mice (20–25 g) were purchased from Orient Biotech (Seoul, S. Korea), and female New Zealand white rabbits weighing 2.0–2.5 kg were purchased from Koatech (Seoul, S. Korea). All mice (five per cage) and rabbits (one per cage) used in each experiment were housed in cages with food and water ad libitum for 1–2 weeks according to humane animal care protocols.

[0112] A mouse cancer model was established by injecting LLC cells (2×10^5 cells / ml) into the thighs of mice. Furthermore, based on the VX2 lung cancer rabbit model established in a previous study, a VX2 rabbit cancer model was established by injecting a mixture of VX2 carcinoma cells and Matrigel into the thighs of rabbits. The sciatic nerves of mice and rabbits were exposed for tumor surgery and imaging.

[0113]

[0114] Comparison of the neural specificity of fluorescent substances

[0115] The neural specificity of NTFP-700, developed in this study, was compared using oxazine 4 (Oxazine 4, TCI, America), oxazine 1 (Oxazine 1, Santa Cruz Biotechnology), and indocyanine green, which are known to visualize nerves. After exposing the sciatic nerve of a mouse, 100 μL of each fluorophore was directly applied to the nerve and incubated for 1 minute. After removing the fluorophore by absorption with gauze, images were acquired using the Invivo Imaging System (Davinch-K, Korea).

[0116]

[0117] Optimization of direct fluorescent agent administration protocol

[0118] To perform a dose optimization study for direct administration of NTFP-700, a newly developed nerve-targeting fluorescent contrast agent, the sciatic nerve and brachial plexus of mice were exposed (n=4). For dose optimization, 1 μM-100 μM NTFP-700 was evenly applied as drops to the nerve and surrounding fat and muscle tissue. After incubating the nerve for 1 minute, the contrast agent was absorbed and removed with a clean gauze pad. Then, 200 μL of phosphate-buffered saline (PBS) was applied over the nerve and absorbed with gauze. This process was repeated five times. After applying 25 μM NTFP-700 to the sciatic nerve and brachial plexus of mice, the contrast agent was incubated for 1, 3, or 5 minutes, and washed five times with 200 μL of PBS to determine the optimal incubation time for direct administration (n=4). To remove nonspecific staining from non-nerve areas, 25 μM NTFP-700 was incubated for 1 minute, followed by 1 to 10 washes with PBS, with images collected after each wash. Washing was performed by briefly flushing the nerve and surrounding tissue, followed by removal of the PBS with a clean gauze. The optimal NTFP-700 protocol by concentration, incubation time, and number of washes was determined based on the nerve-to-background tissue ratio.

[0119]

[0120] Comparison of optimized direct administration of fluorophores and dyed gauze methods in the mouse sciatic nerve

[0121] To compare the direct administration method and the dyed gauze method according to the angle of the nerve, the established optimal conditions for NTFP-700 were used. Direct administration of 100 μL of 25 μM NTFP-700 was used, along with the dyed gauze method, which involves saturating the fluorophore. The two different methods were applied to nerves positioned at flat and steep angles, respectively, to evaluate the utility of the methods depending on the angle of the nerve.

[0122]

[0123] Simultaneous visualization of nerves and tumors via dual-channel fluorescence imaging in mouse cancer models.

[0124] Lewis lung cancer cells were used as a mouse cancer model. LLC (5 × 10^5) cell lines were injected into the sciatic nerve region of the mouse, and the cancer model was established after 1-2 weeks. The tumor-targeting fluorescent contrast agent cRGD-ZW800-PEG (50 nmol) was administered intravenously 4 hours before surgery, and the sciatic nerve region was exposed. NTFP-700 was applied to the sciatic nerve using an optimized direct administration method and a dyed gauze technique. The nerve (700 nm) and tumor (800 nm) were visualized simultaneously using a near-infrared (NIR) dual fluorescence imaging system.

[0125]

[0126] Simultaneous visualization of nerves and tumors via dual-channel fluorescence imaging in a rabbit cancer model.

[0127] The VX2 rabbit tumor model was developed based on a previous study using VX2 as a model. Briefly, fresh VX2 tissue was harvested using a surgical blade for tumor implantation, washed with phosphate-buffered saline (PBS), and the surrounding necrotic tumor tissue was removed. The tissue was then minced into small pieces and filtered using a 100-μm cell strainer to obtain a VX2 suspension. The suspension was centrifuged at 1200 rpm for 3 min and resuspended in PBS at a concentration of 1 × 10^7 cells / mL. The VX2 tumor cells were mixed with 100 μL Matrigel and prepared using a 1 mL syringe with a 23-gauge needle. The mixture of VX2 tumor cells and Matrigel was injected into the subsciatic muscle of rabbits, and the rabbit tumor model was established 2 weeks later.

[0128] Similarly, the tumor-targeting fluorescent contrast agent cRGD-ZW800-PEG (50 nmol) was administered intravenously 4 hours before surgery, exposing the sciatic nerve area. NTFP-700 was applied to the sciatic nerve using an optimized direct administration method and a dyed gauze technique. A near-infrared (NIR) dual fluorescence imaging system was used to simultaneously visualize the nerve (700 nm) and the tumor (800 nm), and the tumor was removed through real-time image-guided surgery.

[0129]

[0130] Histological confirmation in rabbit sciatic nerve tissue

[0131] Rabbit sciatic nerve tissue was excised after in vivo nerve staining using an optimized dyed gauze staining technique, mounted in optimal cutting temperature (OCT) compound, and flash frozen in liquid nitrogen. Frozen samples were sectioned into cryosections (10 μm per slice). One slide was stained with hematoxylin and eosin (H&E), and serial sections were used for fluorescence microscopy. Sections were then stained with NeuroTrace fluorescent Nissl stain, FluoroMyelin fluorescent myelin stain, and NTFP-700, all provided by ThermoFisher Scientific (Waltham, MA). Fluorescence and color microscopic images were acquired at 2.5x and 10x magnification using an LSM 900 confocal laser scanning microscope (Zeiss, Thornwood, NY).

[0132]

[0133] Statistical analysis

[0134] Statistical analyses were performed using repeated measures or one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Statistical significance was set at P <0.05: * P <0.05, ** P <0.01, *** P <0.001, and **** P <0.0001. Results are expressed as the mean ± SD for all image analyses using the Davinch Invivo Imaging System (Davinch-K), the FIAT-L Imaging System (Nawoo Vision), and the NIR fluorescence microscope. Statistical analyses and curve fitting were performed using Microsoft Excel and Prism version 10 software (GraphPad).

[0135]

[0136] Experimental Example 2. Chemical Structure of Contrast Agents and In Vivo Imaging

[0137] The chemical structures and molecular weights of the neurospecific contrast agents of the present invention, NTFP-700, oxazine 4, oxazine 1, and indocyanine green (ICG), are shown in Fig. 1A. In addition, the fluorescence of NTFP-700, Ox4, Ox1, and ICG directly injected into the mouse sciatic nerve in vivo is shown in Fig. 1B. NTFP-700, Oxazine 4, and Oxazine 1 were imaged through the 700 nm channel, respectively, and ICG was imaged through the 800 nm near-infrared (NIR) channel. It was confirmed that NTFP-700, the contrast agent of the present invention, is suitable for visualizing nerves with a wavelength of 700 nm, and it was confirmed that it could stain nerves specifically compared to the control group.

[0138]

[0139] Experimental Example 3. Optimization of NTFP-700 absorption capacity for direct administration in mice.

[0140] Stained images of the sciatic nerve (SN) and brachial plexus (BP) after each experimental dose (1 μM to 100 μM) are shown in Fig. 2A. White arrows indicate nerve bundles in the mouse. The fluorescence intensities were quantified to calculate the signal-to-background ratio (SBR) of the sciatic nerve (SN) and brachial plexus (BP) and are shown in Fig. 2B (mean ± SD). The images are representative of four independent experiments. **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05.

[0141] As can be seen in Figure 2B, the dose that showed the highest SBR (signal-to-background ratio) of NTFP-700 was 25 mM, which was confirmed to be the optimal concentration for direct administration.

[0142]

[0143] Experimental Example 4. Optimization of NTFP-700 absorption time for direct administration in mice.

[0144] Figure 3A shows images of the sciatic nerve (SN) and brachial plexus (BP) after incubation for each experimental time (1, 3, and 5 min), with white arrows indicating the nerve bundles of the mouse. Figure 3B shows the fluorescence intensity quantified to calculate the signal-to-background ratio (SBR) of the sciatic nerve (SN) and brachial plexus (BP) (mean ± SD). The images are representative of four independent experiments. **** p < 0.0001, *** p < 0.001.

[0145] The optimal incubation time for direct administration of NTFP-700 was 1 minute, which was confirmed to exhibit the highest SBR, demonstrating that the neurospecific contrast agent of the present invention can be usefully utilized in situations requiring medical techniques due to its short incubation time.

[0146]

[0147] Experimental Example 5. Optimization of the Washout Protocol for NTFP-700 Absorption for Direct Administration in Mice

[0148] Figure 4A shows images of the sciatic nerve (SN) and brachial plexus (BP) after each washing time (0 to 10 min). White arrows indicate nerve bundles in the mouse. Figure 4B shows the quantification of fluorescence intensity to calculate the signal-to-background ratio (SBR) of the sciatic nerve (SN) and brachial plexus (BP). The images are representative of four independent experiments. *** p < 0.001, ** p < 0.01.

[0149] The optimal number of washes for direct administration of NTFP-700 was one, which resulted in the highest SBR. A small number of washes, as described above, demonstrates the usefulness of the contrast agent of the present invention in situations requiring medical expertise.

[0150]

[0151] Experimental Example 6. Comparison of the Direct Injection Method and the Stained Gauze Method for Sciatic Nerve Staining

[0152] Schematic diagrams of the direct administration method and the dyed gauze method are shown in Figures 5A and C, respectively. Figures 5B and D show images of the sciatic nerve (SN) stained with NTFP-700 using the direct administration method and the dyed gauze method, respectively, taken at various angles. The red arrow indicates the nerve imaged at a steep angle due to its anatomical structure.

[0153] Both the direct injection method and the dyed gauze technique were effective in visualizing nerves at flat angles. However, at steep angles, the dyed gauze technique demonstrated more effective staining than the direct injection method, preventing contrast agent runoff.

[0154]

[0155] Experimental Example 7. Intraoperative dual-channel fluorescence imaging in a mouse tumor model.

[0156] Figures 6A and B show intraoperative simultaneous dual-channel imaging of tumor (cRGD-ZW800-PEG, 800 nm NIR) and nerve (NTFP-700 directly injected (A) and dyed gauze (B), 700 nm NIR) in a mouse tumor model. The white dotted line indicates the tumor, the red arrow indicates the nerve imaged at a steep angle due to the tumor, and the yellow arrow indicates the nerve imaged at a flat angle. Figure 6C shows the tumor-to-background and nerve-to-background ratios calculated by quantifying the fluorescence intensity (mean ± SD). The images are representative of three independent experiments.

[0157] In a mouse tumor model, both direct administration and dyed gauze techniques could be successfully utilized to simultaneously distinguish nerves and tumors using two different colors during cancer surgery using dual-channel NIR imaging.

[0158]

[0159] Experimental Example 8. Confirmation of precise tumor surgery using intraoperative dual-channel fluorescence imaging guidance in a rabbit tumor model.

[0160] Figure 7A illustrates a schematic of dual-channel fluorescence-guided surgery using cRGD-ZW800-PEG and NTFP-700 in a rabbit tumor model. cRGD-ZW800-PEG was administered intravenously 4 hours before surgery, and NTFP-700 was administered intraoperatively using a dyed gauze technique. Figure 7B shows representative intraoperative dual-channel imaging results of the tumor (green) and nerve (blue). The white dotted line indicates the tumor, and the yellow arrow indicates the nerve.

[0161] In a rabbit model of subcutaneous tumors, dual-channel NIR imaging successfully distinguished nerves and tumors simultaneously using two different colors during cancer surgery. This method enabled successful tumor removal while preserving nerves.

[0162]

[0163] Experimental Example 9. Histological images of rabbit nerves stained with H&E, NeuroTrace, FluoroMyelin, and NTFP-700.

[0164] Figure 8 shows representative H&E staining and fluorescence microscopy images of a transected rabbit sciatic nerve. The white squares in the first row indicate the locations of images magnified 20x (second row). Scale bars = 200 μm and 50 μm, respectively. All fluorescence images had the same exposure time and normalization conditions.

[0165] The fluorescence signal of NTFP-700 was consistent with myelin-specific fluorescence staining, confirming its specificity for nerve tissue. This result confirms that NTFP-700, the neurospecific contrast agent of the present invention, targets myelin and can produce nerve tissue-specific staining results.

[0166]

[0167] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0168] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

[0169] This invention was carried out with the support of the Korea Health Technology Research and Development Project (Project No. RS-2024-00436472) of the Korea Health Industry Development Institute (KHIDI), and was completed with the support of the Ministry of Health and Welfare.

Claims

1. A neurospecific contrast agent composition comprising a compound represented by the following [chemical formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] In the above [chemical formula 1], R 1 and R 2 are the same or different from each other, and are each independently selected from hydrogen, alkoxy, C1 to C6 alkyl and C1 to C6 alkenyl, or are combined with each other to form an aromatic ring, R 3 is C1 to C6 alkyl, C1 to C6 alkylsulfonyl, phenylpropyl and -(CH2) n N + (R 4 ) is one selected from the group consisting of 3, n is an integer from 0 to 4, R 4 is an alkyl group of C1 to C6, X is a halogen element.

2. In paragraph 1, In the above [chemical formula 1], R 1 and R 2 is hydrogen, R 3 A methyl, neurospecific contrast agent composition.

3. In paragraph 1, A neurospecific contrast agent composition, wherein in the above [chemical formula 1], X is I.

4. In paragraph 1, A neurospecific contrast agent composition, wherein the compound represented by the above [chemical formula 1] or a pharmaceutically acceptable salt thereof specifically binds to myelin.

5. In paragraph 1, A neurospecific contrast agent composition, characterized in that the contrast agent absorbs light in the near-infrared (NIR) region with a wavelength of 700 nm to 800 nm.

6. A method of imaging comprising the following steps: (1) A step of treating a subject with a contrast agent composition containing a compound represented by the following [Chemical Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient; and (2) A step of detecting a fluorescent signal emitted from the above object. [Chemical Formula 1] In the above [chemical formula 1], R 1 and R 2 are the same or different from each other, and are each independently selected from hydrogen, alkoxy, C1 to C6 alkyl and C1 to C6 alkenyl, or are combined with each other to form an aromatic ring, R 3 is C1 to C6 alkyl, C1 to C6 alkylsulfonyl, phenylpropyl and -(CH2) n N + (R 4 ) is one selected from the group consisting of 3, n is an integer from 0 to 4, R 4 is an alkyl group of C1 to C6, X is a halogen element.

7. In paragraph 6, The imaging method is an imaging method that is performed by additionally processing a second contrast agent composition having an absorption wavelength of 800 nm or more in addition to a contrast agent composition containing a compound represented by [Chemical Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient.

8. In paragraph 7, In the imaging method, the contrast agent composition comprising a compound represented by [chemical formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient is capable of specifically staining neural tissue, and the second contrast agent composition is capable of specifically staining tumor tissue.

9. In paragraph 6, In the above imaging method, the processing in step (1) is performed by a dyed gauze method or a spraying method.

Images