Compounds and compositions for tumor detection and surgical guidance

Abstract

Disclosed is a compound having the following structure: TIFF2023537627000024.tif61169X is an anion (e.g., a biologically relevant anion such as chloride, iodide, etc.); Y is NH, NR 10 , or CR 11 R 12 where Z is a heteroatom (e.g., O, S, or Se), and R and R 1 are independently selected from methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and the like, and combinations thereof. In various examples, R and R 1 At least one of the atoms is not an oxygen atom (both cannot be oxygen atoms to form -NO2). 2 and R 3 R is independently selected from methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), etc., and combinations thereof. 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 are independently selected from hydrogen, alkyl groups (e.g., methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), etc.), and combinations thereof. Also disclosed are compositions and methods of using the compounds and compositions. The compounds or compositions can be used to visualize or identify tumors.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 066,072, filed August 14, 2020, the disclosure of which is incorporated herein by reference.

[0002] [Background of disclosure] Surgical resection of cancerous tissue is an important procedure for the treatment of solid tumors. Surgery is one of the most effective methods for treating solid tumors. If all cancerous tissue can be removed during treatment, there is a high possibility of a prolonged disease-free period or a cure. However, surgical outcomes can vary because some cancerous tissue may not be apparent by macroscopic surveillance.

[0003] Although preoperative imaging with MRI, CT, and PET has significantly improved the effectiveness of cancer diagnosis and treatment planning, current intraoperative surgical techniques still rely heavily on the surgeon's experience, skill, thoroughness, and perseverance. Surgeons typically use visual inspection and palpation to identify potentially cancerous tissue, but these two methods are often insufficient. During surgery, surgeons often identify cancerous tissue by visual inspection under white light, often without assistance, so the results depend heavily on the surgeon's experience. Large tumors that are easily visible with the naked eye can be quickly removed, but small lesions that are nearly impossible to detect may be left behind. Because incomplete resection can lead to disease recurrence and overresection can cause surgical complications, a new technique, fluorescence-guided surgery (FGS), is being actively explored to increase the precision, effectiveness, and efficiency of intraoperative procedures. FGS requires a fluorescent probe to enhance the visibility of cancerous tissue and a fluorescent imaging system to detect the fluorescent signal in real time. Conventional nontargeted fluorescent dyes, such as indocyanine green, methylene blue, and fluorescein, are FDA-approved for tracking blood, lymph, and urine flow. However, their passive accumulation in tumors limits their usefulness in cancer imaging. Various fluorescent probes have been designed to highlight cancerous tissue by exploiting the unique physiological properties of cancer (e.g., high receptor expression and high enzyme activity). Fluorophores have been conjugated to targeting ligands or responsive triggers to construct tumor-binding or enzyme-activatable fluorescent probes. Systemically administered fluorescent probes specifically illuminate cancerous tissue (but not normal tissue) through preferential binding or enzyme activation, resulting in high tumor-to-normal tissue contrast. Several promising fluorescent probes are currently undergoing clinical trials for FGS.

[0004] To obtain maximum signal-to-background contrast, most probes are injected intravenously (IV) several hours or even one or several days before imaging to allow clearance of background signal from unbound probes or signal generation from enzyme-activated probes. Therefore, patients must stay in the hospital for hours, sometimes even days, before the actual surgical procedure for contrast injection, which increases their hospital time. Furthermore, IV-administered probes may have limited sensitivity to small tumors (<2 mm) because their poor vasculature may prevent them from reaching such tumors. Systemic agents may not help in this situation, as small tumors are already easily missed by visual observation during the procedure and can be a source of recurrence. Systemic administration of probes also requires large doses, which can cause systemic side effects.

[0005] Typical "always-on" fluorescent conjugated probes with fast on-rates to tumors are not well suited to the "spray-and-see" approach (because excess agent applied to normal tissue must be washed away before imaging). Conversely, low-background enzyme-activated probes eliminate the need for a washout step, but their slow catalytic enzymatic reaction prevents real-time imaging. Tumor cells typically exhibit enhanced glycolysis to maintain their rapid growth and proliferation, and the aerobic environment in solid tumors alters their metabolic pathways to convert glucose to lactate rather than pyruvate. They actively pump out protons to reduce intracellular lactate accumulation, which ultimately significantly reduces extracellular pH in tumors from 7.4 to 6.2–6.9. Tumor acidity correlates with enhanced tumor growth, invasiveness, and metastasis. This tumor-associated acidity has also been exploited to develop numerous intravenously delivered pH-responsive fluorescent probes by conjugating pH-sensitive dyes to tumor-targeting groups (such as antibodies or peptides). Their tumor-specific binding and internalization into acidic endosomes and lysosomes (pH = 4.5-5.0) allow tumors to emit strong fluorescence. However, this type of probe has the same drawbacks as common IV-administered probes. Summary of the Invention

[0006] In one aspect, the disclosure provides compounds that can be used to visualize (e.g., highlight) cancerous tissue during treatment.

[0007] In various examples, the disclosure provides compounds having the following structure: [ka] X is an anion (e.g., a biologically suitable anion, e.g., chloride, iodide, etc.); Y is NH, NR 10 , or CR 11 R 12 Z is a heteroatom (e.g., O, S, or Se). R and R1 are independently selected from methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and the like, and combinations thereof. In various examples, R and R 1 and R cannot both be oxygen atoms (-NO2 cannot be formed). In various examples, R and R 1 R cannot be a hydrogen atom at the same time. 2 and R 3 is independently selected from methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and the like, and combinations thereof. 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently selected from hydrogen, alkyl groups (e.g., methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl)), and the like, and combinations thereof. In various examples, R 4 and R 5 may be the same alkyl group (e.g., a methyl group). 6 and R 7 may be the same alkyl group (for example, a methyl group).

[0008] In one aspect, the present disclosure provides a composition comprising one or more compounds of the present disclosure. The composition may include one or more pharmaceutically acceptable carriers.

[0009] In one aspect, the present disclosure provides a method for using one or more compounds or compositions of the present disclosure. The method of the present disclosure can be used on individuals who have or are suspected of having cancer (e.g., solid tumors). The method can be used to detect, identify, visualize, or image solid tumors. [Brief explanation of the drawings]

[0010] For a fuller understanding of the nature and objects of the present disclosure, reference should be made to the following detailed description taken in conjunction with the drawings accompanying this specification.

[0011] FIG. 1 shows compounds of the present disclosure.

[0012] FIG. 2 shows compounds of the present disclosure.

[0013] FIG. 3 shows the emission maxima and intensity differences at pH 5.0 and 7.5.

[0014] FIG. 4 shows the fluorescence spectra of compounds of the present disclosure.

[0015] Figure 5 shows the cytotoxicity (cell toxicity) data of compounds of the present disclosure. CCK8 MMT assays were performed using 1 μM of each compound, 0.1% DMSO, and RPMI. Cells were incubated for 0.5 or 1 hour, washed with fresh medium, and then incubated for 3 days.

[0016] Figure 6 shows the absorbance and fluorescence spectra of CypH-11 (2 μM in phosphate buffer at pH 5.0). max = 766 nm, and Em max =785 nm

[0017] FIG. 7 shows a comparison of pH-responsive CypH-11 with pH-insensitive Cy7.

[0018] FIG. 8 shows a comparison between CypH-1 and CypH-11.

[0019] FIG. 9 shows the tumor / muscle contrast ratios for CypH-11, CypH-1, and Cy7 at various time points.

[0020] FIG. 10 shows a general synthetic scheme for compounds of the present disclosure.

[0021] FIG. 11 shows the synthesis scheme of CypH-11.

[0022] FIG. 12 shows the chemical structures and characterization of CypH-11 and CypH-1. (A) CypH-1 structure (B) Schematic of CypH-11 fluorescence activation by protonation. (C) Measurement of the pKa values ​​of CypH-11 and CypH-1 (6.0 and 4.7, respectively). Their fluorescence intensities (λ) were measured over a pH range (2.0–11.0) using a plate reader. ex = 725 nm, λ em =785nm). (D) Fluorescence images of CypH-11 and CypH-1 in a 96-well plate at various pH levels.

[0023] FIG. 13 shows the cellular imaging and subcellular localization of CypH-11, CypH-1, and Cy7. (A) OVASAHO cells were incubated with CypH-11, CypH-1, or Cy7 (2 μM each) for 1 hour and then imaged without washing. Scale bar: 50 μm. (B) Colocalization of CypH-11 and CypH-1 with mitochondria and lysosomes. OVASAHO cells were incubated with CypH-11 and CypH-1 (2 μM each) for 1 hour, after which the cells were further stained with organelle trackers (mitochondrial or lysosome trackers) for 10 minutes. Cell images were acquired using the NIR channel (excitation: 690-730 nm, emission: 770-850 nm) and the GFP channel (excitation: 457-487 nm, emission: 502-538 nm). Scale bar: 50 μm. (C) Cell viability of CypH-11 and CypH-1. OVASAHO cells were first treated with CypH-11 or CypH-1 (2 μM each) for 1 h, and after replacing the old medium with fresh cell culture medium, the cells were grown for an additional 72 h. Cell viability was assessed by CCK assay. Each column represents the average of three separate experiments.

[0024] FIG. 14 shows in vivo and ex vivo images of CypH-11, CypH-1, and Cy7 in a subcutaneous OVASAHO / RFP-Luc tumor model. (A) Representative white light and fluorescence composite images of nude mice (10 tumors for CypH-11 and 3 tumors for Cy7) at various time points before and after spraying the surgical area with CypH-11 and Cy7 (2 μM each). RFP images show tumor size and location, and NIR images show the fluorescence changes of CypH-11 and Cy7 after spraying. Scale bar = 1 cm. (B) White light and fluorescence composite images before and after spraying CypH-11 (2 μM, right flank) and CypH-1 (2 μM, left flank). Scale bar = 1 cm. (C) Ex vivo fluorescence image of excised tumor and muscle after spraying with the probe. Scale bar = 1 cm. (D) Tumor-to-normal tissue ratio of fluorescence at various time points after spraying the probe onto the surgical area.

[0025] FIG. 15 shows in vivo imaging of CypH-11 in a subcutaneous SKOV3 / GFP-Luc tumor model. (A) Representative white light and fluorescence composite images of nude mice at various time points before and after spraying with CypH-11 (2 μM, 6 tumors). The GFP image shows the location and size of the tumor, and the CypH-11 image shows the NIR fluorescence generated by CypH-11. Scale bar = 1 cm. (B) Tumor-to-normal tissue ratio of fluorescence intensity at various time points after spraying CypH-11 on the surgical area. N=6 (C) Histological correlation of tumor fluorescence and CypH-11 signals. GFP and DAPI signals were present throughout the tumor, while NIR signal from CypH-11 was present only at the margin. Scale bar = 100 μm.

[0026] FIG. 16 shows in vivo and ex vivo white light and fluorescence composite images of disseminated SKOV3 / RFP-Luc tumors in the peritoneal cavity after IP administration of CypH-11. (A) Three representative mice were imaged 1 hour after IP administration of CypH-11 (200 μL, 2 μM solution). GFP images showed the location and size of disseminated tumors (top row), and CypH-11 images showed the fluorescent signal generated by CypH-11 (bottom row). Scale bar = 1 cm. (B) Ex vivo white light and fluorescence composite images of excised tumor-bearing organs (tumor, spleen, stomach, liver, and intestine). Scale bar = 5 mm. (C) Tissue-to-peritoneal ratio of fluorescence intensity in SKOV3 mice after IP administration (D) Histological correlation between tumor fluorescence and CypH-11 signals. Scale bar = 100 μm.

[0027] FIG. 17 shows the chemical synthesis and spectra of CypH-11. (A) Synthesis scheme of CypH-11 (B) Normalized absorption and emission spectra of CypH-11 in PBS buffer at pH = 5. Ex max = 765nm; Em max =785nm

[0028] FIG. 18 shows the chemical structure and optical properties of Cy7 from GE Healthcare. (A) Cy7 structure (B) Measurement of Cy7 fluorescence intensity (λ) at various pH values ex = 725 nm, λ em = 785 nm). Plate reader used. (C) Fluorescence images of Cy7 in a 96-well plate at various pH values.

[0029] Figure 19 shows OVASAHO cells incubated with CypH-11, CypH-1, and Cy7 (2 μM each) for 1 hour, washed with PBS, and then imaged. Scale bar: 50 μm. Cell images were acquired in the NIR channel (excitation: 690-730 nm, emission: 770-850 nm).

[0030] FIG. 20 shows depth measurements of CypH-11 signal in sprayed and IP injected tumors (40X). (A) Nuclear DAPI staining showed that sprayed CypH-11 could penetrate only 2–3 layers of cells in 15 min. (B) IP-injected CypH-11 was able to reach 6–7 layers of cells in 1 h. Scale bar = 50 μm

[0031] FIG. 21 shows the development of CypH-11 signals in live and dead tissue. (A) In vivo spraying: CypH-11 was first sprayed onto tissues of live animals, and the tissues were then excised 20 minutes later. Good correlation between tumor GFP and NIR signals was observed. (B) Ex vivo spray: Tissue was excised and held for 20 minutes before being sprayed with CypH-11. No CypH-11 signal was observed within the tumor or muscle, indicating that dead tissue cannot generate a CypH-11 signal, likely due to poor uptake of the probe. Scale bar = 5 mm.

[0032] FIG. 22 shows characterization data for CypH-11. (A) 1 H NMR; (B) 13 C NMR; and (C) Mass Spectrometry Disclosure

[0033] Although the claimed subject matter is described through certain examples, other examples (including examples that do not provide all of the advantages and features described herein) are within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of this disclosure.

[0034] A range of values ​​is disclosed herein. The range is set out from a lower limit and an upper limit. Unless otherwise specified, the range includes the lower limit, the upper limit, and all values ​​between the lower and upper limits, including, but not limited to, all values ​​up to the order of the minimum value (either the lower or upper limit) of the range.

[0035] As used herein, unless otherwise stated, the term "group" refers to a chemical entity that is monovalent (i.e., has one end that can be covalently bonded to another chemical species), divalent, or polyvalent (i.e., has two or more ends that can be covalently bonded to other chemical species). The term "group" also includes radicals (e.g., monovalent and polyvalent, e.g., divalent radicals, trivalent radicals, etc.). Illustrative examples of groups include the following: [ka]

[0036] As used herein, unless otherwise specified, the term "alkyl group" refers to a branched or unbranched saturated hydrocarbon group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl, etc. For example, alkyl groups include C1-C 20 and all integer carbons and ranges of carbon numbers therebetween (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 ) The alkyl group can be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, various substituents such as halogens (-F, -Cl, -Br, -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, etc.), aryl groups, alkoxide groups, carboxylate groups, carboxylic acids, ether groups, amine groups, etc., and combinations thereof.

[0037] The present disclosure provides compounds and compositions suitable for visualizing solid tumors. The compounds of the present disclosure or compositions containing the compounds may be used to visualize (e.g., highlight) cancerous tissue during a procedure (e.g., a medical procedure, such as surgery (e.g., tumor removal)). Visualization may be used to minimize unwanted oversights and achieve better overall surgical outcomes. Methods of using the compounds and compositions are also provided.

[0038] In one aspect, the present disclosure provides compounds that may be used to visualize (e.g., highlight) cancerous tissue during treatment.

[0039] In various examples, the disclosure provides compounds having the following structure: [ka] X is an anion (e.g., a biologically suitable anion, e.g., chloride, iodide, etc.); Y is NH, NR 10 , or CR 11 R 12 Z is a heteroatom (e.g., O, S, or Se). R and R 1 are independently selected from methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and the like, and combinations thereof. In various examples, R and R 1 In various examples, R and R cannot both be oxygen atoms (as would form -NO). 1 Both of R cannot be hydrogen atoms. 2 and R 3 is independently selected from methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and the like, and combinations thereof. 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R10 , R 11 , and R 12 are independently selected from hydrogen, alkyl groups (e.g., methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl)), and the like, and combinations thereof. In various examples, R 4 and R 5 may be the same alkyl group (e.g., a methyl group). 6 and R 7 may be the same alkyl group (for example, a methyl group).

[0040] In various examples, the disclosure provides compounds having the following structure: [ka] During the ceremony, X is an anion (e.g., a biologically suitable anion, e.g., chloride, iodide, etc.); Y is NH, NR 10 , or CR 11 R 12 Z is a heteroatom (e.g., O, S, or Se). R and R 1 are independently selected from methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and the like, and combinations thereof. In various examples, R and R 1 In various examples, R and R cannot both be oxygen atoms (as would form -NO). 1 Both of R cannot be hydrogen atoms. 2 and R 3 is independently selected from methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and the like, and combinations thereof. 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R10 , R 11 , and R 12 are independently selected from hydrogen, alkyl groups (e.g., methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl)), and the like, and combinations thereof. Compounds of the present disclosure do not have the following structure: [ka]

[0041] Without intending to be bound by any particular theory, the compounds of the present disclosure are pH-sensitive. The compounds may be non-fluorescent in normal tissue, but may fluoresce when absorbed by acidic cancer tissue. The ability to selectively stain cancer will make surgical procedures more accurate and effective. Medical professionals will be able to accurately identify the location of tumors regardless of size and shape, and accurately perform all necessary procedures in a timely manner.

[0042] The compounds of the present disclosure have desirable pKa values. The compounds can have a pKa in the range of 5.5 to 6.5, including all values ​​and ranges therebetween. Compounds with pKa values ​​less than 5 may not have desirable fluorescence for topical application (e.g., spray application).

[0043] Examples of compounds of the present disclosure include, but are not limited to: [ka] [ka] [ka]

[0044] In one aspect, the present disclosure provides a composition comprising one or more compounds of the present disclosure. The composition may include one or more pharmaceutically acceptable carriers.

[0045] The compositions described herein may include one or more standard pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may be determined in part by the particular composition being administered and the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of the pharmaceutical compositions of the present disclosure. The compound may be suspended free in a pharmaceutically acceptable carrier, or the compound may be encapsulated in liposomes and then suspended in a pharmaceutically acceptable carrier. Examples of carriers include solutions, suspensions, emulsions, solid injectable compositions that are dissolved or suspended in a solvent prior to use, and the like. Injectables can be prepared by dissolving, suspending, or emulsifying one or more active ingredients in a diluent. Examples of diluents include, but are not limited to, distilled water for injection, saline, vegetable oil, alcohol, dimethyl sulfoxide, and combinations thereof. In addition, injectables may contain stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, etc. Injectables may be sterilized in the final formulation process or prepared by sterilization procedures. The compositions of the present disclosure may also be formulated as sterile solid preparations (e.g., by lyophilization) and used after sterilization in sterile water for injection or other sterile diluents or after dissolving in sterile water for injection or other sterile diluents immediately before use. Further examples of pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; celluloses including sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, for example, cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, phosphate buffers, and other non-toxic compatible substances used in pharmaceutical formulations.Further non-limiting examples of pharmaceutically acceptable carriers can be found in "Remington: The Science and Practice of Pharmacy (2005) 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins." Effective formulations include, but are not limited to, oral and nasal formulations, topical formulations, parenteral formulations, and compositions formulated for sustained release. Parenteral administration includes injection, e.g., intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous, etc.

[0046] In various examples, the composition has desirable permeability characteristics and biologically appropriate osmolality. Carriers with desirable permeability characteristics include, but are not limited to, propylene glycol, isopropanol, oleic acid, polyethylene glycol analogs, and the like, and combinations thereof. It is desirable that the composition is non-lethal to cells. Osmolality adjusters may also be used. Examples of osmolality adjusters include, but are not limited to, sugars, such as monosaccharides, for example, glucose, fructose, sorbose, xylose, ribose, and the like, and combinations thereof, disaccharides, for example, sucrose, and the like, sugar alcohols, for example, mannitol, glycerol, inositol, xylitol, adonitol, and the like, and combinations thereof, and amino acids, such as glycine, arginine, and the like, and combinations thereof.

[0047] In various examples, the composition is suitable for local administration. The composition may be sprayed onto a subject with or suspected of having a solid tumor at the location where the subject is suspected of having a solid tumor, or may be used as an oral rinse for oral and / or esophageal cancer. The spray may also be applied to assist in endoscopic / laparoscopic diagnosis in patients with ovarian cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, oral cancer, and other cancers. The composition may be administered (e.g., sprayed) directly from an endoscope, colonoscope, or laparoscope. In various examples, the compound or composition may be administered during all surgical resections or to verify resected tissue.

[0048] In various examples, the composition may comprise 0.5-10 μM (including all 0.01 μM values ​​and ranges therebetween) of a compound of the present disclosure in phosphate buffered saline at a pH of 6.5-7.5 (including all 0.01 pH values ​​and ranges therebetween) and 0.1-1.0% (including all 0.01% by volume values ​​and ranges therebetween) of DMSO. In various examples, the composition may comprise 0.5-10 μM (including all 0.01 μM values ​​and ranges therebetween) of a compound in phosphate buffered saline at a pH of 6.5-7.5 (including all 0.01 pH values ​​and ranges therebetween).

[0049] In one aspect, the present disclosure provides a method for using one or more compounds or compositions of the present disclosure. The method of the present disclosure can be used on individuals who have or are suspected of having cancer (e.g., solid tumors). The method can be used to detect, identify, visualize, or image solid tumors.

[0050] The methods of the present disclosure can be used to determine the presence and / or location of, and / or image, solid tumors, and may be used in combination with other methods used to identify or remove solid tumors.

[0051] A method for determining the presence and / or location of a solid tumor in an individual may include administering a compound or composition of the present disclosure to a region of interest on or within the individual. The region of interest may be an area in the individual that has or is suspected of having a tumor. The compound or composition is exposed to (e.g., irradiated with) electromagnetic radiation (e.g., light having a wavelength within the near-infrared region (NIR) (e.g., 750-1500 nm)). Following irradiation, the region of interest can be imaged or visualized. Imaging or visualization may include measuring or observing a fluorescent signal in the region of interest. After application of the compound or composition, the signal can be detected within a few minutes (e.g., less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 55 seconds, less than 50 seconds, less than 45 seconds, less than 40 seconds, less than 35 seconds, less than 30 seconds, less than 25 seconds, less than 20 seconds, less than 15 seconds, less than 10 seconds, or less than 5 seconds). In various examples, no washout is performed prior to imaging and / or visualization. The fluorescent signal will be expressed in neoplastic tumor tissue.

[0052] The method of the present disclosure may be a method of imaging a solid tumor. The method may include applying or administering a compound or composition of the present disclosure to the solid tumor, exposing the area of ​​interest to electromagnetic radiation, and obtaining an image of the solid tumor. In various examples, no washing is performed before imaging and / or visualization. After applying the compound or composition, a signal may be detected within a few minutes (e.g., less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 55 seconds, less than 50 seconds, less than 45 seconds, less than 40 seconds, less than 35 seconds, less than 30 seconds, less than 25 seconds, less than 20 seconds, less than 15 seconds, less than 10 seconds, or less than 5 seconds). Administration may be by topical administration (e.g., spraying the area of ​​interest) or various non-intravenous delivery methods, such as intraperitoneal delivery (ip).

[0053] Tumor detection, identification, and / or imaging can include measuring a fluorescent signal. Excitation and emission can vary depending on the compound used to generate the fluorescent signal. Measurements can also include measuring background fluorescence. Signals can be measured at various time points (e.g., 1, 3, 5, 7, 10, and 15 minutes). Measurements can also be used to determine tumor-to-normal tissue ratios by calculating the mean fluorescence intensity of the tumor compared to that of normal regions.

[0054] Administration may be by various non-intravenous delivery methods, such as topical administration (e.g., sprayed onto the area of ​​interest) or intraperitoneal delivery (ip). Additionally, the compound or composition may be administered systemically. As used herein, the term "systemic" includes parenteral, topical, oral, spray inhalation, rectal, nasal, and buccal administration. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial administration. In various examples, the compound or composition is applied or administered via topical application (topical application) or local administration. In various examples, the compound or composition is sprayed onto the area of ​​interest. In other examples, the composition is an oral rinse. For example, the method may be a "spray and see" technique.

[0055] The disclosed methods can include determining the tumor boundary (tumor margin) of a tumor (e.g., a solid tumor). For example, a compound or composition is applied to the tumor (e.g., a solid tumor) site, and a fluorescent signal is measured. The excitation and emission can vary depending on the compound used to generate the fluorescent signal. The measurement can also include measuring background fluorescence. The signal can be measured at various time points (e.g., 1, 3, 5, 7, 10, and 15 minutes). The signal can be compared to the fluorescent signal of non-cancerous tissue within / in the region of interest to determine the tumor boundary. The comparison can be used to determine which portions of the region of interest are cancerous and which are non-cancerous. The signal can also be used to determine the tumor boundary to ensure complete resection of the tumor.

[0056] The methods of the present disclosure can be used in a variety of individuals. In various examples, the individual is a human or non-human mammal. Examples of non-human mammals include, but are not limited to, livestock such as cows, pigs, sheep, etc., as well as pet or sport animals such as horses, dogs, cats, etc. Further non-limiting examples of individuals include, but are not limited to, rabbits, rats, mice, etc. The compounds or compositions of the present disclosure may be administered to an individual, for example, in a pharmaceutically acceptable carrier (which facilitates transport of the compound from one organ or part of the body to another organ or part of the body), or may be applied directly to the organ or part of the body of interest.

[0057] A variety of tumors can be identified, imaged, or visualized using the methods of the present disclosure. For example, the tumor may be a solid tumor. Examples of tumors include, but are not limited to, ovarian tumors, skin cancers, pancreatic cancers, genitourinary cancers, colon tumors, bladder tumors, brain tumors, esophageal tumors, cervical tumors, oral tumors, and the like, and combinations thereof.

[0058] The process steps described in the various embodiments and examples disclosed herein are sufficient to produce the disclosed compounds or practice the disclosed methods. Thus, in various embodiments, the process consists essentially of a combination of the process steps disclosed herein. In various other embodiments, the process consists solely of such steps.

[0059] In some embodiments, the present disclosure provides kits, which may include the composition or materials for preparing the composition (e.g., a pharmaceutical carrier and one or more compounds of the present disclosure) and printed materials.

[0060] In various examples, the kit includes a sealed or sealed package containing the pharmaceutical formulation. In various examples, the package includes one or more sealed or sealed vials, bottles, blister (bubble) packs, or any other suitable package for the sale, distribution, or use of the compounds of the present disclosure and compositions comprising the compounds of the present disclosure. The printed material may include printed information. The printed information may be provided on a label, an insert, or may be printed on the packaging material itself. The printed information may include information identifying the amount and type of the compound, other active ingredients, and / or inactive ingredients in the package, as well as instructions for administering the composition, e.g., the number of doses over a certain period of time, and / or information directed to a pharmacist and / or other healthcare provider (e.g., a physician) or a patient. In various examples, the product includes a label describing the contents of the container and providing indications and / or instructions for using the contents of the container. The kit may include a single dose or multiple doses. The kit may further include a device or item necessary for administering the compound or composition. The item or device may be, for example, a spray bottle or an atomizer.

[0061] The following examples are presented to illustrate the present disclosure and are not intended to be limiting in any way.

[0062] Example A. A compound having the following structure: [ka] where X is an anion (e.g., a biologically suitable anion such as chloride, iodide, etc.); Y is NH, NR 10 , or CR 11 R 12 Z is a heteroatom (e.g., O, S, or Se); R and R 1 is independently selected from methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and the like, and combinations thereof; R 2 and R 3 is independently selected from methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and the like, and combinations thereof; R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are independently selected from hydrogen, alkyl groups (e.g., methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), etc.), and combinations thereof, with the proviso that the compound does not have the following structure: [ka] For example, in various instances, R 4 and R 5 may be the same alkyl group (e.g., a methyl group). For example, in various examples, R 6 and R 7 may be the same alkyl group (e.g., a methyl group). In various examples, the compound has the following structure: [ka] [ka] [ka] In various examples, the compound has the following structure: [ka]

[0063] Example B: A composition comprising a compound described in Example A and a pharmaceutically acceptable carrier For example, the composition may have desirable permeability properties. Carriers with desirable permeability properties include, but are not limited to, propylene glycol, isopropanol, oleic acid, polyethylene glycol analogs, and the like, and combinations thereof. It is desirable that the composition be non-lethal to cells. Osmolarity adjusting agents may be used. Examples of osmolarity adjusting agents include, but are not limited to, monosaccharides such as glucose, fructose, sorbose, xylose, ribose, and the like, and combinations thereof; disaccharides such as sucrose, and the like; sugar alcohols such as mannitol, glycerol, inositol, xylitol, adonitol, and the like, and combinations thereof; and amino acids such as glycine, arginine, and the like, and combinations thereof. Stabilizing agents may also be used. Examples of stabilizers are known in the art. In various examples, the composition may comprise 0.5-10 μM (including all 0.01 μM values ​​and ranges therebetween) of the compound in phosphate buffered saline at pH 6.5-7.5 (including all 0.01 pH values ​​and ranges therebetween) and 0.1-1.0% (including all 0.01% by volume values ​​and ranges therebetween) of DMSO. In various examples, the composition may comprise 0.5-10 μM (including all 0.01 μM values ​​and ranges therebetween) of the compound in phosphate buffered saline at pH 6.5-7.5 (including all 0.01 pH values ​​and ranges therebetween). The composition is suitable for topical and / or oral administration (e.g., a sprayable composition).

[0064] Example C. A method for determining the presence and / or location of a solid tumor in an individual in need of treatment, comprising administering a compound according to Example A or a composition according to Example B to an area of ​​interest on or within the individual; exposing the area of ​​interest to electromagnetic radiation (e.g., light having a wavelength in the near-infrared region (NIR)) (e.g., 750-1500 nm); and imaging and / or visualizing the area of ​​interest, thereby determining the presence and / or location of the solid tumor. After application of the compound or composition, a signal can be detected within a few minutes (e.g., less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 55 seconds, less than 50 seconds, less than 45 seconds, less than 40 seconds, less than 35 seconds, less than 30 seconds, less than 25 seconds, less than 20 seconds, less than 15 seconds, less than 10 seconds, or less than 5 seconds). A fluorogenic signal will be generated in neoplastic tumor tissue. Administration may be by various non-intravenous delivery methods, such as topical administration (e.g., sprayed onto the area of ​​interest) or intraperitoneal delivery (ip). Administration may be topical administration. Topical administration may be by spraying. In various examples, topical administration is an oral rinse. In various examples, no rinsing is performed prior to imaging and / or visualization. In various examples, the solid tumor is selected from ovarian tumors, skin cancers, pancreatic cancers, genitourinary cancers, colon tumors, bladder tumors, brain tumors, esophageal tumors, cervical tumors, oral tumors, etc., and combinations thereof. Application or administration to the solid tumor may result in protonation of the compound. In various examples, the electromagnetic radiation is in the near-infrared region.

[0065] Example D. A method of imaging a solid tumor, comprising applying or administering a compound according to Example A or a composition according to Example B to the solid tumor; exposing the area of ​​interest to electromagnetic radiation; and acquiring an image of the solid tumor. After applying the compound or composition, a signal may be detected within minutes (e.g., less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 55 seconds, less than 50 seconds, less than 45 seconds, less than 40 seconds, less than 35 seconds, less than 30 seconds, less than 25 seconds, less than 20 seconds, less than 15 seconds, less than 10 seconds, or less than 5 seconds). Administration may be by topical administration (e.g., spraying onto the area of ​​interest) or various non-intravenous delivery methods, such as intraperitoneal delivery (ip). The application or administration is topical application or local administration. Topical administration may be by spraying. The solid tumor may be selected from ovarian tumors, skin cancers, pancreatic cancers, genitourinary cancers, brain tumors, colon tumors, bladder tumors, esophageal tumors, cervical tumors, oral tumors, etc., and combinations thereof. The electromagnetic radiation may be in the near infrared range.

[0066] Example E. A method for determining the boundaries of a solid tumor, comprising administering a compound according to Example A or a composition according to Example B to an area of ​​interest on / in an individual; exposing the area of ​​interest to electromagnetic radiation (e.g., light having a wavelength in the near-infrared region (NIR)) (e.g., 750-1500 nm); and imaging and / or visualizing the area of ​​interest to determine the boundaries of the solid tumor. After application of the compound or composition, a signal can be detected within a few minutes (e.g., less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 55 seconds, less than 50 seconds, less than 45 seconds, less than 40 seconds, less than 35 seconds, less than 30 seconds, less than 25 seconds, less than 20 seconds, less than 15 seconds, less than 10 seconds, or less than 5 seconds). The signal may be compared to the fluorescent signal of non-cancerous tissue in / to the area of ​​interest to determine the boundaries of the tumor. Administration may be by various non-intravenous delivery methods, such as topical administration (e.g., sprayed onto the area of ​​interest) or intraperitoneal delivery (ip). The application or administration is topical application or administration. Topical administration may be by spraying. The solid tumor may be selected from ovarian tumors, skin cancers, pancreatic cancers, genitourinary cancers, brain tumors, colon tumors, bladder tumors, esophageal tumors, cervical tumors, oral tumors, etc., and combinations thereof. The electromagnetic radiation may be in the near-infrared region.

[0067] Example F. A kit comprising a compound according to Example A or a composition according to Example B. The kit may include the compound (e.g., the compound as a lyophilized powder or film) and a pharmaceutically acceptable carrier. The two components may be mixed and sprayed onto the tissue of interest.

[0068] [Example 1] The following examples illustrate compounds of the present disclosure, as well as toxicity and in vivo data for said compounds.

[0069] 1 and 2 show compounds of the present disclosure.

[0070] FIG. 3 shows the emission maxima and intensity differences at pH 5.0 and 7.5.

[0071] FIG. 4 shows the fluorescence spectra of compounds of the present disclosure.

[0072] Figure 5 shows the cytotoxicity data of compounds of the present disclosure. CCK8 MMT assays were performed using 1 μM of each compound, 0.1% DMSO, and RPMI. Cells were incubated for 0.5 or 1 hour, washed with fresh medium, and then incubated for 3 days.

[0073] Figure 6 shows the absorbance and fluorescence spectra of CypH-11 (2 μM in phosphate buffer at pH 5.0). max = 766 nm, Em max =785nm

[0074] In vivo imaging studies Animals inoculated with RFP-ovsaho cells in the flank were used for imaging studies. Tumor size was controlled at 2 mm to mimic the morphology of metastatic human ovarian cancer. The skin was removed before spraying with the dye (2 μM in saline). The tumor area was sprayed and imaged at various time points using a Cy7 filter set. RFP images were also acquired for tumor co-registration. The contrast ratio was calculated as [tumor signal] / [adjacent muscle signal].

[0075] Following in vitro and cellular validation, we hypothesized that our best candidate, CypH-11, possessing excellent fluorescence properties at different pH levels and in cells, could be an ideal agent for enhancing the detection of small cancerous lesions that would otherwise be imperceptible to surgeons. To validate our hypothesis, we subcutaneously inoculated mice with RFP-positive ovarian cancer cells. When tumors reached approximately 2 mm in size, the skin was removed, and a CypH-11 solution (2 μM in saline) was sprayed onto the tumor area. A fluorescent signal highlighting the tumor rapidly developed within 1 minute after application of the spray. The contrast continued to increase slightly and rapidly reached a plateau (approximately 7 minutes). The tumor signal was approximately 150% higher than that of adjacent muscle tissue, and the CypH-11 signal co-registered well with the RFP signal. Notably, no signal increase was observed in normal tissue, suggesting that this CypH-11 signal enhancement was tumor-specific. In another set of experiments, animals were inoculated with two tumors. Each tumor was sprayed with either the prototype dye CypH-1 or CypH-11 and imaged simultaneously. CypH-11 showed nearly instantaneous signal enhancement, while CypH-1 showed only minimal contrast. This result strongly supports the benefits of our modification.

[0076] To further confirm that the signal enhancement was pH-dependent, Cy7, a commercially available always-on dye with similar absorption and emission properties, was applied to tumors under the exact same conditions. As expected, this pH-independent Cy7 dye provided strong signals in all tissues. Unlike CypH-11, Cy7 did not show any appreciable contrast difference. Due to the fluorescent properties of CypH-11, signal generation could be directly imaged without the need for a washing step. These spray experiments suggested that pH-dependent CypH-11 could be used as an aerosol spray for real-time tumor detection.

[0077] FIG. 7 shows a comparison of pH-responsive CypH-11 and pH-insensitive Cy7.

[0078] FIG. 8 shows a comparison between CypH-1 and CypH-11.

[0079] FIG. 9 shows the tumor / muscle contrast ratios for CypH-11, CypH-1, and Cy7 at various time points.

[0080] [Example 2] The following examples illustrate compounds of the present disclosure, as well as their synthesis and properties.

[0081] Elucidation of characteristics All new CypH dyes were characterized by proton NMR and mass spectrometry to confirm their identity. NMR data were consistent with the structure, and mass spectrometry results gave the expected mass of the dye ±0.5 amu. An HPLC method (see below) was developed and used to assess dye purity. All dyes showed good purity (>95%). Column retention time generally correlated with the dye's lipophilicity, with water-soluble dyes containing sulfate groups eluting earlier (10.6 min for CypH-3, 6, and 9) than CypH-1 (11.9 min), while more lipophilic dyes eluted later. The optical properties, acid dissociation constants, and solubility of the dyes were also determined. Characterization data are summarized in Table 1. Synthetic intermediates were also characterized by proton NMR.

[0082] The HPLC method used for dye purity consisted of a Phenomenex reverse-phase Luna C8(2) column (5 μm, 100A, 250 × 4.6 mm, cat. # 00G-4248-30) using solvents A (50% aqueous methanol + 0.1% TFA) and B (100% methanol + 0.1% TFA). The solvent gradient was 0–3 min (0% B), 3–10 min (100% B), 10–20 min (100% B), and 20–25 min (0% B). The flow rate was 1 mL / min. Detection was performed by a photodiode array at the dye's absorption maximum.

[0083] [Table 1]

[0084] [Table 2]

[0085] Structural optimization The new pH-responsive dyes were modified from a previously published lead probe, CypH-1, a heptamethine cyanine dye that exhibits little fluorescence under neutral and basic conditions (≥7.0) but emits fluorescence under mildly acidic conditions (≤5.0). The excitation and emission maxima of CypH-1 are 760 and 777 nm, respectively. The signal intensity ratio between pH 5 and pH 7.5 was approximately 10. The meso-bridged ring size, lipophilicity, and charge density of CypH-1 were modified to provide better optical properties. In a first round of screening from a library of 10 analogs, dyes with meso-cyclopentane rings were found to have low fluorescence properties at both pH 5 and pH 7.5, while hydrophilic CypH with -CH2CH2SO3- substitutions was found to have good fluorescence properties but poor uptake into the cell membrane. The best compound from this group was CypH-5, which showed an increased pH 5.0 / pH 7.5 fluorescence ratio of 22. Based on this initial structure-property relationship, a second round of 10 novel analogs was designed using CypH-5 as the core, focusing on modifying the electron density on the aniline and indolinium rings. Various alkyl groups, such as methyl, ethyl, propyl, isopropyl, and combinations thereof, were applied to these two positions. The general synthetic route is shown in Figure 10.

[0086] Direct measurement of fluorescence intensity showed that CypH analogs bearing methyl groups on the indolinium ring (CypH-11–20) had higher background fluorescence at pH 7.5. Other CypH analogs bearing longer alkyl chains had much lower background fluorescence. Their fluorescence ratios at pH 5.0 / pH 7.5 were significantly improved from 10–20 to 50–110. Among them, CypH-11, bearing methylisopropylaniline and isopropyl groups on the indolinium ring, gave a 112-fold enhancement in fluorescence signal. CypH-11 had absorption and emission maxima at 766 nm and 785 nm, respectively (Figure 4) and was selected as a lead compound (see below).

[0087] Synthetic details and further characterization of the lead compound, CypH-11

[0088] CypH-11 was synthesized according to the scheme shown in Figure 11 using the following experimental procedures.

[0089] Preparation of 4-(N-isopropyl, N-methyl)aminophenol starting material 4-N-methylaminophenol (1.72 g, 0.01 mol), isopropyl iodide (1.70 g, 0.01 mol), and triethylamine (2.8 mL, 0.02 mol) were stirred in 10 mL of anhydrous chloroform at room temperature overnight. The solution was then concentrated, dissolved in a minimum volume of dichloromethane, and purified by silica gel chromatography (eluting with a gradient of increasing ethyl acetate in hexanes, 25–40% in 5% increments) to give 4-(N-isopropyl,N-methyl)aminophenol (0.926 g, 56% yield). 1 H NMR (in d6-DMSO): 8.60 (s, 1H,-OH), 6.67-6.61 (m, 4H), 3.80 (m, 1H), 2.50 (s, 3H, -CH3), 1.02 (d, J=6.6Hz, 6H, -(CH3)2)

[0090] Synthesis of N-isopropyl-2,3,3-trimethylindolinium iodide (2) 2,3,3-Trimethylindoleine (1) (4 g, 0.025 mol) and 2-iodopropane (14 mL, 0.140 mol) were heated at 140 °C for 72 h. After cooling, the resulting thick oil was washed with diethyl ether to remove excess starting material, and the oil was then placed under high vacuum to remove residual volatiles. The crude material (4.08 g, 49.6%) was analyzed by proton NMR and used in the next reaction. 1 H NMR (CDCl3): 7.86-7.84 (m, 1H), 7.61-7.56 (m, 3H), 5.51 (m, 1H, N-CH), 3.31 (s, 3H, -CH3), 1.92 (d, 6H, J= 6.9 Hz, -(CH3)2), 1.67 (s, 6H, -(CH3)2)

[0091] Synthesis of 2-[2-[2-chloro-3-[2-(1,3-dihydro-1-isopropyl-3,3-dimethyl-2H-indol-2-ylidene)-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-1-isopropyl-3,3-dimethyl-3H-indolium iodide (4) N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline monohydrochloride (3) (0.281 g, 0.78 mmol, Millipore Sigma, St. Louis, MO) and N-isopropyl-2,3,3-trimethylindolinium iodide (0.575 g, 1.75 mmol) were heated to reflux in ethanol (20 mL) containing anhydrous sodium acetate (0.158 g, 1.93 mmol) for 3 h. The reaction mixture was concentrated and purified by silica gel chromatography eluting with increasing amounts of methanol (2–5% in 1% increments) in dichloromethane to give (4) as a green solid (0.25 g, 48%). 1 H NMR (CDCl3): 8.38 (d, J=14.0 Hz, 1H), 7.41-7.37 (m, 3H), 7.26 (m, 1H), 6.43 (d, J=14Hz, 1H), 5.10 (m, 1H), 2.81 (t, J=6.2Hz, 2H), 2.02 (m, 1H), 1.80-1.60 (m, ~12H)

[0092] Synthesis of CypH-11 A solution of 4-(N-isopropyl,N-methyl)aminophenol (0.081 g, 0.491 mmol) in anhydrous N,N-dimethylformamide (5 mL) was stirred at RT, and sodium hydride (0.022 g, 60% in oil, 0.55 mmol) was added, followed by stirring for an additional 15 min to form the sodium phenoxide salt. Dye (2) (0.150 g, 0.225 mmol) was then added, and the mixture was stirred overnight at RT. The DMF was removed in vacuo, and the residue was dissolved in a small amount of dichloromethane and purified by silica gel chromatography eluting with an increasing gradient of methanol in dichloromethane (0-6% in 1% increments) to give CypH-11 as a green solid (51.0 mg, 28.5%). 1 H NMR (500 MHz, CDCl3) δ 7.99 (d, J=14.1 Hz, 2H), 7.33-7.24 (m, 6H), 7.20-7.13 (m, 2H), 6.95 (d, J=9.1 Hz, 2H), 6.82 (d, J=9.1 Hz, 2H), 6.21 (d, J=14.1 Hz, 2H), 4.96-4.87 (m, 2H), 3.94-3.86 (m, 1H), 2.76 (t, J=6.0 Hz, 4H), 2.63 (s, 3H), 2.10-2.00 (m, 2H), 1.66 (d, J=7.0 Hz, 12H), 1.35 (s, 12H), 1.07 (d, J=6.6 Hz, 6H). 13 C NMR (126 MHz, CDCl3) δ 171.81, 165.23, 152.98, 146.18, 142.71, 141.85, 141.05, 128.22, 124.54, 123.07, 122.26, 116.84, 114.98, 112.76, 100.48, 50.97, 48.94, 48.75, 30.63, 28.19, 24.77, 21.22, 19.72, 18.83

[0093] [Example 3] The following examples illustrate compounds, their synthesis and characterization, and methods of the disclosure.

[0094] CypH-11, a near-infrared pH-responsive fluorescent dye, was designed as a sensitive cancer spray to highlight cancerous tissue during surgery, minimizing the surgeon's subjective judgment. CypH-11 (pKa 6.0) emits little fluorescence at neutral pH and fluoresces brightly in acidic environments (a ubiquitous consequence of cancer cell growth). After topical application, CypH-11 was rapidly absorbed, and its fluorescent signal developed in cancerous tissue within 1 minute. The signal-to-background ratios were 1.3 and 1.5 at 1 and 10 minutes, respectively. The fluorescence and near-instant signal development capability enable the "spray-and-see" concept. This fast-acting CypH-11 spray may be a convenient and effective tool for fluorescence-guided surgery, enabling real-time identification of small cancerous lesions for optimal resection without systemic toxicity.

[0095] Design and characterization of pH-responsive fluorescent CypH-11 The acidic pH in the tumor microenvironment caused by enhanced glycolysis is a widely used target for tumor diagnosis and therapeutic development. A pH-responsive fluorescent dye, CypH-1, was previously created by installing a pH-sensitive amino moiety onto a near-infrared (NIR) cyanine fluorophore (Figure 12A). At physiological pH (pH = 7.4), the dimethylaminophenol group is not protonated, and CypH-1 exhibits extremely low fluorescence due to photoinduced electron transfer (PeT) quenching. Under acidic conditions, however, the amino group on CypH-1 is protonated, blocking PeT quenching and resulting in high fluorescence recovery (Figure 12). When tested in a mouse ovarian cancer model by IP injection, CypH-1 exhibited significantly higher fluorescence in small lesions than in normal tissue, but failed to detect ovarian tumors by spray ionization, likely due to its low pKa (pKa = 4.7). Therefore, the pKa of this fluorescent probe was not suitable for optimal imaging by spray ionization. Considering that the pH in tumors is around 6.2–6.9 and that of normal tissues is 7.4, a fluorescent probe with a pKa close to this 6.2–6.9 window would be preferable. Therefore, the 4-(dimethylamino)phenol moiety of CypH-1 was replaced with a more electron-rich 4-(N-isopropyl,N-methyl)aminophenol group, and the methyl group on the indolinium ring was replaced with an electron-donating isopropyl group to provide a more optimized dye, CypH-11 (Figure 12B).

[0096] CypH-11 was synthesized by reacting 4-(N-isopropyl,N-methyl)aminophenol with fluorophore 1 under basic conditions (Figure 17A). The absorption and emission peaks were centered at 765 nm and 785 nm, respectively (Figure 17B). The quantum yield (Φ) of CypH-11 at pH 4.0 was 3.3%. As designed, CypH-11 exhibited a significantly higher pKa value (pKa = 6.0) than CypH-1 (pKa = 4.7). Both CypH-11 and CypH-1 exhibited low fluorescence in neutral and basic solutions, but strong fluorescence in acidic solutions (Figures 12C and 12D). Titration curves and images in various pH solutions confirmed that CypH-11 was more sensitive to pH fluctuations (pH 5.0-7.0) in physiological environments. Cy7, a commercially available pH-insensitive fluorophore with similar excitation and emission wavelengths, was included as a reference in the biological studies. Cy7 fluoresced brightly at all pH values ​​and showed no pH dependence (Figure 18).

[0097] Evaluation of CypH-11 in cancer cells The performance of CypH-11, CypH-1, and Cy7 was evaluated using the ovarian cancer cell line, OVASAHO. OVASAHO cells were incubated with each probe (2 μM) for 1 hour, and cellular fluorescence images were acquired in the presence of dye-containing medium (Figure 13A). Strong intracellular fluorescence and low / moderate fluorescence were observed in CypH-11- and CypH-1-treated wells, whereas Cy7-treated wells showed supersaturated fluorescence. This distinct difference is due to the pH sensitivity of CypH-11 and CypH-1. Both have low fluorescence in cell culture medium (pH = 7.4), but their fluorescence is turned on upon entering the cellular acidic compartment. In contrast, Cy7 showed constant high fluorescence in the physiological pH range. After washing with fresh medium, cellular images were acquired again (Figure 19). Both CypH-11 and CypH-1-treated cells exhibited high fluorescence, suggesting their significant cell permeability and retention, whereas Cy7 exhibited very dim fluorescence, suggesting poor cell permeability or retention. The intracellular distribution of the dyes was investigated by co-staining with mitochondrial, lysosomal, and nuclear trackers (Figure 13B). Both CypH-11 and CypH-1 showed much better overlap with the mitochondrial tracker than with the lysosomal tracker (Pearson's values: 0.70 and 0.90, respectively). Furthermore, cytotoxicity studies with OVASAHO cells showed that treatment with 2 μM CypH-11 or CypH-1 for 1 h did not significantly affect cell viability (91.0 ± 4.6% and 95.5 ± 6.9%, respectively) (Figure 13C).

[0098] Detection of subcutaneous tumors by spray To evaluate the in vivo performance of fluorescent probes using the "spray-and-see" technique, we used the OVASAHO subcutaneous tumor model. For easy signal coregistration, cells were first engineered to express red fluorescent protein (RFP). Two weeks after subcutaneous inoculation of OVASAHO / RFP-Luc cells into both flanks, tumors reached a size of approximately 5 mm. The skin over the tumor area was removed, and then CypH-11 or Cy7 solution (2 μM in PBS) was sprayed once onto the exposed area. Whole-body fluorescence images were acquired continuously at various time points without washing (Figure 14A). Tumor tissues were revealed by their inherent RFP fluorescence. Near-infrared fluorescence generated by CypH-11 was observed immediately (<1 min) in the tumor area but not in adjacent normal areas, reaching a plateau within 10 min. In contrast, Cy7, due to its pH-insensitive "always-on" property, exhibited strong fluorescence throughout the sprayed area.

[0099] CypH-11 and CypH-1 were sprayed on either side of the tumor for control comparison. CypH-11 showed high fluorescence only in the tumor but not in normal tissue, while CypH-1 showed very low fluorescence enhancement in both the tumor and adjacent normal tissue (Figure 14B). The sprayed area was then washed with PBS, and the signal was found to remain within the tumor, suggesting that CypH-11 was absorbed into the tumor tissue and the signal was generated internally (Figure 14B). Similar results were observed for excised tumor and adjacent normal tissue (Figure 14C). The tumor-to-muscle signal ratios of these three dyes were plotted against time (Figure 14D). Immediately after spraying CypH-11, significant levels of fluorescent signal were detected in the tumor, and the signal continued to increase up to 10 minutes. The signal-to-background ratios at 1, 5, and 10 minutes were 1.3, 1.4, and 1.5, respectively. In contrast, the tumor-to-muscle ratios for dark CypH-1 and always-on Cy7 remained near 1.0, suggesting an inability to detect tumors.

[0100] To confirm that OVASAHO tumor staining was not the only incident staining, CypH-11 was further evaluated in a second subcutaneous tumor model, SKOV3 / GFP-Luc. After spraying CypH-11 onto the surgical field, rapid signal development was observed around the tumor (Figure 15A). Compared to the GFP signal, which indicated the precise location of the tumor, the NIR signal was highest at the border of the SKOV3 tumor. Interestingly, the signal pattern differed from that seen in OVASAHO tumors, where the signal was constant across the region. The trend of signal increase in SKOV3 tumors was similar to that seen in OVASAHO tumors (Figure 15B), with signal-to-background ratios reaching 1.3, 1.4, and 1.6 at 1, 5, and 10 minutes, respectively. As before, PBS washing failed to wash away the fluorescent signal, supporting internalization of the sprayed CypH-11 (Figure 15A).

[0101] To evaluate the distribution of CypH-11 in tumors after spraying, tumors were excised and sectioned onto multiple slides at 14 μm thickness. The slides were stained with H&E and DAPI nuclear dye. Under a fluorescent microscope, GFP and DAPI fluorescent signals were uniformly distributed throughout the tumor, while the CypH-11 signal was mainly located in the outer layer of the tumor (Figure 15C). High-magnification images showed that the NIR signal depth was approximately 2–3 cell layers (Figure 20A). This shallow surface penetration may be due to the short, limited contact with the sprayed CypH-11.

[0102] We investigated the use of CypH-11 on harvested tissue. If successful, CypH-11 could be used for postoperative tissue evaluation. When CypH-11 was sprayed onto tissue from live animals, the signal developed rapidly and remained within the tumor (Figure 21A). The signal in excised tissue was detectable for weeks to months after storage. Conversely, when tumor and muscle tissues were first harvested and then CypH-11 was sprayed onto these 20-minute-old "dead" tissues, no NIR signal was detectable (Figure 21B), suggesting that only live tissues can absorb and convert topically applied fluorescent CypH-11.

[0103] Detection of Microdisseminated Ovarian Tumors with Intraperitoneally Delivered CypH-11 Following the promising "spray-and-see" application of CypH-11, we were also interested in whether this fast-response fluorescent dye could be used for the rapid detection of small peritoneally disseminated ovarian tumors. To mimic peritoneal disseminated ovarian cancer, SKOV3 / GFP-Luc cells were directly injected into the peritoneal cavity of mice, and tumor growth was followed by monitoring D-luciferin-induced bioluminescence. It took approximately two weeks to reach a strong bioluminescence signal, indicating tumor growth. CypH-11 (2 μM, 200 μL in PBS) was administered intraperitoneally. One hour later, the peritoneal cavity was surgically exposed, and brightfield and near-infrared (NIR) fluorescence images were immediately acquired without washing. The GFP signal indicated tumor location, while the near-infrared fluorescence was generated by CypH-11 (Figure 16A). Due to the fluorescent properties of CypH-11, background signals were very low in normal tissues and organs, eliminating the need for a washing step. Excellent overlap between the CypH-11-generated fluorescence and the tumor GFP signal was observed. Following whole-body imaging, which only allowed for the identification of large, superficial tumors (>3 mm) in the peritoneal cavity, tissues and major organs (spleen, stomach, liver, and intestine) were collected to identify small and barrier tumors. Zoom-in views also demonstrated favorable correlation between tumor and CypH-11 signal (Figure 16B). All tumors of different sizes were highlighted, with signals 3-4 times higher than the peritoneal, liver, and intestinal background (Figure 16C). More importantly, tumors as small as 1 mm, which posed great challenges for surgeons to remove, were clearly detected. Histological analysis also showed that CypH-11 was primarily located in the outer layer of the tumor, but the signal descended to 6-7 layers of cells within one hour (Figures 16D and 20B). The deeper CypH-11 penetration observed here compared to its application by spray could likely be attributed to longer contact with a larger volume of CypH-11 solution.

[0104] Consideration

[0105] FGS is a promising technology due to its real-time visualization capabilities. Under excitation light, tumor-specific fluorescent probes allow surgeons to "see" fluorescent tumors through a video camera. Topically sprayable probes can be extremely useful during surgical procedures, especially for identifying small tumors and confirming tumor margins. If necessary, probes can be sprayed on suspicious areas to highlight the presence of cancerous tissue that should be removed or normal tissue that should be avoided, thereby improving safety. Recently, at least two topical agents, β-galactosidase-sensitive SPiDER-βGal and γ-glutamyl transpeptidase-sensitive gGL-HMRG, have been reported for tumor detection, but their application is limited to tumors expressing the target enzyme. To provide a universal "spray-and-see" probe for real-time tumor visualization, the probe should target common cancer hallmarks, and signal transduction should be tumor-specific and immediate. Because tumor acidosis is a ubiquitous consequence of cancer cell proliferation and growth and the protonation reaction is instantaneous, the tumor pH-sensitive fluorogenic CypH-11 was designed to image cancer tissue without the need to wash away excess dye.

[0106] CypH-11 was derived from a previously developed NIR cyanine dye, CypH-1. CypH-1 is pH-responsive, but its pKa was not optimized for the pH of the tumor environment. Without intending to be bound by any particular theory, a dye with a pKa close to the pH of the tumor environment would be an improved dye for tumor detection. The introduction of electron-donating groups increased the pKa of CypH-11 to 6.0. Under basic conditions, fluorescence is quenched by the PeT effect between the lone electron pair on the isopropyl-methylamino group and the cyanine backbone of CypH-11 (Figure 12B). Under acidic conditions, the amino group is protonated, masking the lone electron pair and resulting in a strong fluorescent signal. Measurement of the probe fluorescence output in solution showed that the normalized fluorescence of CypH-11 at pH 6.0–6.5 was approximately 3.4-fold higher than that of CypH-1 (Figures 12C and 12D).

[0107] Cellular imaging experiments confirmed the advantages of the fluorogenic properties (Figure 13). Both CypH-11 and CypH-1 gave very low background signals in neutral medium (pH = 7.4), allowing their distribution to intracellular acidic compartments to be clearly visualized (without a wash step), whereas the highly fluorescent, pH-insensitive Cy7 over-enhanced the tumor both in cell culture and in vivo. CypH-1, likely due to its low pKa, showed minimal signal enhancement when sprayed onto the tumor area, resulting in ineffective fluorescence activation. In contrast, CypH-11 highlighted the tumor and revealed the tumor border with minimal background signal (Figures 14 and 15). Because the protonation process is a nearly instantaneous reaction, the fluorescence activation of CypH-11 in the tumor was rapid (<1 min), requiring virtually no waiting time. The ability to immediately visualize the fluorescence activation in situ is a key feature of the spray formulation.

[0108] Previously, we demonstrated that IP-administered polymer-based protease-activated probes could better detect small ovarian tumors compared with IV-administered ones, demonstrating that IP-administered CypH-1 is effective for detecting small tumors. In this study, we demonstrated that IP-injected CypH-11 can label very small ovarian tumors (<1 mm) within an hour, eliminating the need for a washout step before imaging (Figure 16). Based on this rapid response rate and tumor selectivity, IP-delivered CypH-11 may be readily translated into clinical practice for optimal cell ablation.

[0109] CypH-11 fluorescent signal generation in tumors is due to direct contact with tumor tissue. Naturally, due to the local spray delivery and limited probe solution, the NIR signal was limited to the top layer of cells (Figure 20). Because acidic pH is a universal cancer marker, the pH-sensitive spray technique may be useful for many types of superficial tumors, such as ovarian, cervical, and colon cancer. A study of stage III or IV ovarian cancer patients treated with maximal cytoresection (no gross residual disease) demonstrated that each 10% increase in optimal cytoresection was associated with a 5.5% increase in median survival. Median survival times longer than 13 months were reported in patients with no residual tumor after optimal cytoresection compared with patients with residual tumor, suggesting that complete surgical cytoresection is the most important prognostic indicator of survival. Unfortunately, current surgical procedures are insufficient, failing to remove all tumor, microscopic residual tumors, and undetected tumor nodules 40% of the time. A spray like CypH-11 that improves a surgeon's ability to visualize and remove diseased tissue during surgery could have a major impact.

[0110] conclusion

[0111] CypH-11 is a simple pH-sensitive fluorophore that exhibits negligible fluorescence at neutral pH but rapidly becomes brightly fluorescent under mildly acidic conditions. Its pKa value (6.0) is suitable for detecting tumor-associated pH changes. Its imaging potential as a spray agent for tumor detection and tumor boundary determination was confirmed using a subcutaneous tumor model. Its ability to detect small ovarian tumors was further demonstrated by IP administration of CypH-11 in a disseminated tumor model.

[0112] Materials and Methods

[0113] General Information for Chemical Synthesis All chemicals and solvents used for synthesis were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Waltham, MA). 4-(N-isopropyl, N-methyl)aminophenol and compound 1 used in the synthesis of CypH-11 were synthesized according to previously reported procedures with necessary modifications. Compound CypH-1 was synthesized as previously reported, and Cy7 was purchased from GE Healthcare (Chicago, IL). Both compounds were used to compare their imaging capabilities with CypH-11. Compounds and intermediates were isolated and purified by silica gel flash chromatography. 1 H and 13 C NMR spectra were collected on a Bruker Ascend-500 spectrometer, and high-resolution mass spectrometry (HRMS) was collected on a PE Sciex API 100 mass spectrometer.

[0114] Synthesis and characterization of CypH-11 To a solution of 4-(N-isopropyl,N-methyl)aminophenol (81 mg, 0.491 mmol) in anhydrous N,N-dimethylformamide (DMF, 5 mL) was added sodium hydride (NaH, 22 mg, 60% in oil, 0.55 mmol). The reaction was stirred for 15 minutes to form the sodium phenoxide salt. Compound 1 (150 mg, 0.225 mmol) was then added, and the mixture was stirred at room temperature overnight. Upon completion, the DMF solvent was removed under vacuum. The residue was purified using silica gel chromatography using an increasing gradient of methanol (0-6%) in dichloromethane. The desired CypH-11 compound was obtained as a green solid (0.051 mg, 28.5%). 1 H NMR (500 MHz, CDCl3) δ 7.99 (d, J=14.1 Hz, 2H), 7.33-7.24 (m, 6H), 7.20-7.13 (m, 2H), 6.95 (d, J=9.1 Hz, 2H), 6.82 (d, J=9.1 Hz, 2H), 6.21 (d, J=14.1 Hz, 2H), 4.96-4.87 (m, 2H), 3.94-3.86 (m, 1H), 2.76 (t, J=6.0 Hz, 4H), 2.63 (s, 3H), 2.10-2.00 (m, 2H), 1.66 (d, J=7.0 Hz, 12H), 1.35 (s, 12H), 1.07 (d, J=6.6 Hz, 6H). 13 C NMR (126 MHz, CDCl3) δ 171.81, 165.23, 152.98, 146.18, 142.71, 141.85, 141.05, 128.22, 124.54, 123.07, 122.26, 116.84, 114.98, 112.76, 100.48, 50.97, 48.94, 48.75, 30.63, 28.19, 24.77, 21.22, 19.72, 18.83. 46 H 58 N3O] + For: Expected m / z=668.4580 [M]+; Measured m / z=668.4557 [M]+; 3.4 ppm error

[0115] spectroscopic analysis Stock solutions of CypH-11, CypH-1, and Cy7 (1 mM in DMSO) were stored in a freezer at -30°C and used in the following experiments. For pKa measurements, each compound was diluted with 20 mM phosphate buffer solution (PBS, pH 2.0-11.0) to a final concentration of 2 μM. Fluorescence intensity (λ ex = 725 nm, and λ em The fluorescence intensity (λ = 785 nm) was measured using a plate reader (Tecan Infinite M1000 Pro), and fluorescence images were recorded using a fluorescence imaging system (Bruker In-vivo F Pro). For quantum yield measurements, indocyanine green (ICG, Φ = 1.2%, in water) was used as a standard compound. Each compound (0.4, 0.8, 1.2, 1.6, and 2.0 μM) in PBS solution (pH = 4.0 and pH = 7.4) was measured using a Cary 60 UV-Vis spectrophotometer and a Cary Eclipse fluorescence spectrophotometer (Agilent). The relative quantum yield was calculated by comparing the fluorescence-to-absorbance slope with that of ICG.

[0116] Cell lines and culture The ovarian cancer OVASAHO cell line was purchased from the JCRB Cell Bank (Osaka, Japan). OVASAHO / RFP-Luc cells were transduced with FLus-F2A-RFP-IRES-Puro lentivirus (Biosettia, San Diego, CA) and selected with puromycin. SKOV3 / GFP-Luc cells were purchased from Cell Biolabs (San Diego, CA). Both OVASAHO and OVASAHO / RFP-Luc cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C under 5% CO. SKOV3 / GFP-Luc cells were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C under 5% CO.

[0117] In vitro fluorescence microscopy OVASAHO cells were used to compare the cellular performance and cellular distribution of CypH-11, CypH-1, and Cy7. OVASAHO cells (1.0 × 10 4 ) were seeded onto a 96-well black plate (Corning, NY) and incubated in supplemented medium for 24 hours. Compounds (2 μM) were added, and the cells were incubated for 1 hour. Before PBS washing, cell fluorescence images were captured with a fluorescence microscope (Cy7 filter, excitation: 690-730 nm, emission: 770-850 nm). After PBS washing (3x), cell images were captured again. For colocalization experiments, OVASAHO cells (5.0 × 10 3 ) were incubated in a 96-well plate with a clear, flat bottom (ibiTreat, 180 μm coverslips, ibidi). After treatment with CypH-11 (2 μM) or CypH-1 (2 μM) for 1 hour, the cells were washed with medium (3x). The cells were stained with Mitoview Green (Biotium, Parkway Fremont, CA) for 15 minutes or Lysoview 488 (Biotium) for 30 minutes, and then stained with DAPI (Invitrogen). After washing with medium (3x), the cells were stained with DAPI (Invitrogen) for an additional 10 minutes. After replacing the medium with fresh cell culture medium, fluorescent images were captured using a fluorescence microscope (EVOS). CypH-11 and CypH-1 images were obtained using a Cy7 filter, a DAPI image using a DAPI filter (excitation: 352-402 nm, emission: 417-477 nm), and a Mitoview Green & Lysoview 488 image using a GFP filter (excitation: 457-487 nm, emission: 502-538 nm).

[0118] Cell viability The cell viability of CypH-11 and CypH-1 was assessed using the Cell Counting Kit-8 (CCK-8) from Dojindo (Rockville, MD). OVASAHO cells (5.0 × 10 3) were seeded into 96-well plates and cultured for 24 hours. The cells were then treated with CypH-11 (2 μM) and CypH-1 (2 μM) for 1 hour. After replacing the medium with fresh cell culture medium, the cells were incubated for an additional 72 hours. Cell viability was assessed by treating with CCK-8 solution for 3 hours and reading the absorbance at 450 nm using a plate reader.

[0119] Subcutaneous and peritoneal implant tumor models All animal procedures were performed in accordance with the approved animal protocols and guidelines of the Animal Care and Use Committee of Weill Cornell Medical College. Mice (female, SCID Hairless Outbred Mice) were purchased from Charles River (Wilmington, MA). To establish subcutaneous implants, OVASAHO / RFP-Luc cells (5.0 × 10 6 ) or SKOV3 / GFP-Luc cells (5.0 × 10 6 A suspension of SKOV3 / GFP-Luc cells (5.0 × 10 cells) suspended in 200 μL of PBS was inoculated into each flank of female nude mice (bilaterally). After 2 weeks, the tumor implants reached a size of approximately 5 mm and were used for spray experiments. To establish peritoneal implants, SKOV3 / GFP-Luc cells (5.0 × 10 cells) suspended in 200 μL of PBS were inoculated into each flank of the mice. 6 ) was injected intraperitoneally into female nude mice. Two weeks later, tumor growth was examined using an in vivo bioluminescence imaging system followed by peritoneal injection of D-luciferin potassium solution (200 μL, 15 mg / mL) for 10 minutes. Mice bearing multiple peritoneal implants with a diameter of 5 mm were generally used for the experiments.

[0120] In vivo fluorescence imaging of subcutaneous tumors We compared the imaging capabilities of CypH-11, CypH-1, and Cy7 using two subcutaneous tumor models (OVASAHO / RFP-Luc and SKOV3 / GFP-Luc). Mice with subcutaneous implants were anesthetized using 2% isoflurane in an induction chamber and maintained with 1.5–2.0% isoflurane via a nose cone. Sterile surgical tools were used to remove the skin surrounding the tumor. Images of the mice were captured using an IVIS Spectrum CT System from PerkinElmer (Waltham, MA). OVASAHO / RFP-Lu and SKOV3 / GFP-Luc tumors were captured under the RFP channel (excitation: 520–550 nm, emission: 570–590 nm) and the GFP channel (excitation: 450–480 nm, emission: 510–530 nm), respectively. The Cy7 channel (excitation: 730-760 nm, emission: 790-810 nm) was applied to evaluate the fluorescence generated by CypH-11, CypH-1, and Cy7. After skin removal, the tumor area and background fluorescence under the Cy7 channel were first measured. Solutions of CypH-11, CypH-1, and Cy7 (2 μM each) were sprayed onto the surgical area, and the fluorescence of the Cy7 channel was continuously measured at each time point (1, 3, 5, 7, 10, and 15 min). Ten, five, and three OASAHO tumors were used to evaluate CypH-11, CypH-1, and Cy7, respectively. Six SKOV3 tumors were used to evaluate CypH-11. To evaluate the tumor-to-normal tissue ratio, the entire tumor area and adjacent open skin areas were sketched, and their fluorescence intensities were acquired by IVIS software. The tumor-to-normal tissue ratio was calculated by dividing the mean fluorescence intensity of the tumor by the mean fluorescence intensity of the normal region. Tumor-bearing mice were euthanized by carbon dioxide inhalation or high-dose isoflurane (5%). The subcutaneous tumor and adjacent muscle were then extracted and sprayed with CypH-11 (2 μM). Images were then captured under the GFP / RFP / Cy7 channels.

[0121] In vivo fluorescence imaging of disseminated peritoneal tumors To further evaluate the imaging ability of CypH-11 to highlight disseminated micrometastases within the peritoneal cavity, SKOV3 / GFP-Luc tumors were implanted and allowed to grow and disseminate within the peritoneal cavity of mice (similar to that of ovarian cancer patients). Tumor-bearing mice were intraperitoneally injected with CypH-11 solution (200 μL, 2 μM) in PBS. After 1 h, the mice were anesthetized using 2% isoflurane in an induction chamber, and anesthesia was maintained via a nose cone using 1.5%–2% isoflurane. Sterile surgical tools were used to open the abdominal cavity. Fluorescent images were captured throughout the cavity under both the GFP and Cy7 channels. After imaging, the mice were euthanized with a high dose of isoflurane (5%), and the disseminated tumors and major organs of interest (i.e., heart, liver, lungs, kidneys, spleen, stomach, and intestines) were harvested. The harvested organs were placed on a glass plate and imaged under both the GFP and Cy7 channels. Regions of interest (ROIs) within tumor nodules and adjacent normal areas in the abdominal cavity (n = 6, mean area = 0.28 ± 0.1 cm) were defined. 2 ) was plotted and tumor-to-normal tissue ratios were calculated.

[0122] histology To obtain information on the distribution of CypH-11 in tumors after administration by spray and ip injection, tumors were excised and analyzed. First, tumors were embedded in molds using optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura Finetek, Torrance, CA) on dry ice for 20 minutes. The frozen tissue was sectioned to the desired thickness (14 μm) using a cryotome. The slides were stored at -80°C until further use. The slides were first imaged using a fluorescence microscope (EVOS, Thermofisher Scientific, Waltham, MA), then stained with hematoxylin and eosin Y solution (H&E), and their histological changes were evaluated under a light microscope.

[0123] statistical analysis Cytotoxicity, fluorescence ratio, and histological analysis were subjected to unpaired t-tests. All p-values ​​were two-sided, and p-values ​​<0.05 were considered significant. Plotted values ​​are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism (GraphPad Software Inc, San Diego, CA).

[0124] Although the present disclosure has been described with reference to one or more particular embodiments and / or examples, it will be understood that other embodiments and / or examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

1. A compound having the following structure: 【Chemistry 1】 (In the formula, X is an anion; Y is CR 11 R 12 and Z is O; R and R 1 are independently selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, n-butyl, t-butyl, and combinations thereof; R 2 and R 3 are independently selected from ethyl, propyl, isopropyl, butyl, isobutyl, n-butyl, t-butyl, and combinations thereof; R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R and R 12 is hydrogen).

2. 2. The compound of claim 1, wherein the compound has the following structure: 【Chemistry 4】 【Transformation 5】

3. 3. The compound of claim 2, wherein the compound has the following structure: 【Transformation 6】

4. A composition comprising a compound according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

5. The composition according to claim 4, wherein the concentration of the compound is 0.5 to 10 μM.

6. The composition of claim 4, wherein the composition is a sprayable composition or an oral rinse.

7. A composition comprising a compound according to any one of claims 1 to 3 for use in determining the presence and / or location of a solid tumor in an individual in need of treatment.

8. 1. A composition for use in a method for determining the presence and / or location of a solid tumor in an individual in need of treatment, comprising: administering or applying said composition to the area of ​​interest on / in the individual; exposing the area of ​​interest to near-infrared electromagnetic radiation; and imaging and / or visualizing the region of interest; The composition of claim 7 used in a method comprising:

9. 9. The composition of claim 8, wherein a signal is generated from the exposure, and the signal is detected in less than 5 minutes.

10. 10. The composition of claim 9, wherein the signal is detected in less than 1 minute.

11. The composition of claim 8, wherein the administration / application is topical administration / application.

12. The composition of claim 11 , wherein the topical administration / application is a spray or oral rinse.

13. 9. The composition of claim 8, wherein the solid tumor is selected from ovarian tumor, skin cancer, pancreatic cancer, genitourinary cancer, colon tumor, bladder tumor, brain tumor, esophageal tumor, cervical tumor, oral tumor, and combinations thereof.

14. The composition of claim 8 , wherein said administration / application to said solid tumor results in protonation of said compound.

15. A composition comprising a compound according to any one of claims 1 to 3, for use in imaging solid tumors.

16. 1. A composition for use in a method of imaging a solid tumor, comprising: applying or administering the composition to a solid tumor; exposing the region of interest to near-infrared electromagnetic radiation; and obtaining an image of said solid tumor; 16. The composition of claim 15 used in a method comprising:

17. The composition described in claim 16, wherein a signal is generated from the exposure after application or administration of the composition, and the signal is detected in less than 5 minutes.

18. 18. The composition of claim 17, wherein the signal is detected in less than 1 minute.

19. The composition of claim 16, wherein the administration / application is topical administration / application.

20. 20. The composition of claim 19, wherein the topical administration / application is a spray or oral rinse.

21. 17. The composition of claim 16, wherein the solid tumor is selected from ovarian tumor, skin cancer, pancreatic cancer, genitourinary cancer, brain tumor, colon tumor, bladder tumor, esophageal tumor, cervical tumor, oral tumor, and combinations thereof.

22. A composition comprising a compound according to any one of claims 1 to 3, for use in determining the boundaries of a solid tumor.

23. 1. A composition for use in a method for determining the borders of a solid tumor, comprising: applying or administering said composition to a solid tumor; exposing the area of ​​interest to near-infrared electromagnetic radiation; Imaging and / or visualizing the region of interest; and determining the boundaries of said solid tumor; 23. The composition of claim 22 for use in a method comprising:

24. 24. The composition of claim 23, wherein the application / administration is a spray or oral rinse.

25. 24. The composition of claim 23, wherein a signal is produced from the exposure, and the signal is detected in less than 5 minutes.

26. 26. The composition of claim 25, wherein the signal is detected in less than 1 minute.

27. 24. The composition of claim 23, wherein the solid tumor is selected from ovarian tumor, skin cancer, pancreatic cancer, genitourinary cancer, brain tumor, colon tumor, bladder tumor, esophageal tumor, cervical tumor, oral tumor, and combinations thereof.

28. A kit comprising a compound according to any one of claims 1 to 3.

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