Peptide-conjugated fluorescent probe contrast agent for bladder cancer diagnosis

Abstract

A peptide-conjugated fluorescent probe contrast agent and a use thereof in the diagnosis and treatment of bladder cancer. A targeted recognition mechanism of the peptide-conjugated fluorescent probe contrast agent improves the specificity of the contrast agent, and an in situ assembled nanostructure improves the sensitivity of the contrast agent, solves the problem of photobleaching of conventional fluorescent contrast agents, prolongs a stable imaging time window, and meets clinical requirements for bladder cancer diagnosis. In addition, after bladder instillation in Beagle dogs, the peptide-conjugated fluorescent probe exhibits high safety with no systemic drug absorption.

Description

A peptide-coupled fluorescent probe contrast agent for bladder cancer diagnosis

[0001] This application claims priority to a prior application, patent application number 202311748713.1, filed with the State Intellectual Property Office of China on December 19, 2023, entitled “A Polypeptide-Conjugated Fluorescent Probe Contrast Agent for Bladder Cancer Diagnosis.” The entire text of that prior application is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of medical technology, and in particular relates to a polypeptide-coupled fluorescent probe contrast agent for bladder cancer diagnosis and its application in bladder cancer diagnosis and treatment. Background Art

[0003] The incidence of bladder cancer ranks seventh among male malignant tumors and is one of the most common malignant tumors of the urinary system. Bladder cancer can occur at any age, even in children. Its incidence increases with age, and the peak age is 50-70 years old. The incidence of bladder cancer in men is 3-4 times that of women. According to Frost & Sulliven statistics, the number of new cases of bladder cancer worldwide reached 573,000 in 2020, and is expected to reach 651,000 in 2025, with a compound annual growth rate of 2.6%; the number of new cases of bladder cancer in China in 2020 was 86,000, and is expected to reach 101,000 in 2025, with a compound annual growth rate of 3.4%, which is higher than the global average.

[0004] Early detection is the first prerequisite for improving the treatment effect of bladder tumors. The diagnosis of bladder cancer is usually confirmed through urine analysis, urine cytology, flow cytometry, ultrasound and cystoscopy (including biopsy). Standard cystoscopy under white light can easily show a variety of macroscopic tumors including non-invasive papilloma in the bladder. However, when using standard cystoscopy, it is easy to overlook atypical hyperplasia and carcinoma in situ. The current recurrence rate of bladder cancer is 50%-70%, indicating that white light cystoscopy is insufficient in detecting and indicating the resection of bladder lesions.

[0005] Compared with white light cystoscopy, fluorescence cystoscopy can effectively improve the detection rate of non-muscle invasive bladder cancer (NMIBC), especially the detection rate of carcinoma in situ, and make the resection surgery more complete, thereby reducing the tumor recurrence rate and the risk of patients having to undergo cystectomy due to tumor muscle invasion.

[0006] Currently, there is no fluorescent contrast agent for bladder cancer diagnosis in the Chinese domestic market. HAL (5-aminolevulinic acid hexyl ester hydrochloride) is the only contrast agent approved in the world to assist in the diagnosis or surgery of bladder cancer. Compared with white light cystoscopy, HAL combined with blue light cystoscopy can improve the detection rate of primary tumors and tumor recurrences, reduce the recurrence rate, and prolong the time to first recurrence. However, because HAL needs to be converted into protoporphyrin through a biosynthetic pathway in cells to emit a fluorescent signal, its clinical application still has the following problems: (1) Poor specificity and poor photostability, resulting in rapid fluorescence decay, so cystoscopy needs to be completed within 1 hour; (2) The differentiation between carcinoma in situ and non-malignant inflammation is not high, and the false positive rate can reach 30%; (3) If HAL is allowed to stay in the bladder for more than 3 hours, nonspecific imaging of the bladder mucosa will interfere with tumor identification; (4) Hemoglobin in hematuria will significantly reduce the effectiveness of HAL.

[0007] Although there are currently several antibody- or peptide-conjugated fluorescent contrast agents in the early stages of research, this type of contrast agent only improves targeting and does not fundamentally address the bottleneck problem of fluorescent contrast agents being easily photobleached during clinical application. Summary of the Invention

[0008] The present invention provides use of a compound represented by formula I or a salt or solvate thereof in preparing a contrast agent for diagnosing or treating bladder cancer.

[0009] The compound is a polypeptide-coupled fluorescent probe molecule.

[0010] In one embodiment of the present invention, the present invention provides a compound of formula I or a salt or solvate thereof, which is distributed in bladder cancer tumor cells and then excited by near-infrared wavelength light to cause the compound of formula I to fluoresce, thereby highly selectively targeting the tumor cells.

[0011] In one embodiment of the present invention, the present invention provides a compound represented by Formula I or a salt or solvate thereof having binding affinity to XIAP protein.

[0012] In one embodiment of the present invention, the present invention provides a method for optical imaging of bladder cancer biological tissue: the method comprises: (a) contacting the biological tissue with a composition comprising a compound represented by Formula I or a salt or solvate thereof; (b) allowing time for the compound represented by Formula I or a salt or solvate thereof in the composition to distribute in the biological tissue; (c) illuminating the tissue with excitation light of a wavelength absorbable by the compound represented by Formula I; and (d) detecting the fluorescent signal emitted by the compound represented by Formula I.

[0013] The kit comprising the compound represented by formula I or a salt or solvate thereof can be used for fluorescence imaging of bladder cancer cells and bladder cancer tissues.

[0014] When the compound of formula I or its salt or solvate is used as a contrast agent for diagnosing or treating bladder cancer, the preferred route of administration is intravesical instillation. Therefore, in one embodiment of the present invention, the present invention provides the use of a compound of formula I or its salt or solvate in the preparation of a contrast agent for diagnosing or treating bladder cancer for intravesical instillation.

[0015] According to the present invention, the bladder cancer can be non-muscle invasive bladder cancer or muscle invasive bladder cancer. Pathologically, the bladder cancer includes urothelial carcinoma, squamous cell carcinoma, adenocarcinoma, clear cell carcinoma, small cell carcinoma, carcinoid tumors, and other primary or metastatic tumors originating from other tissues and organs. Furthermore, the bladder cancer can be located in any part of the bladder, such as the bladder fundus, bladder neck, and bladder dome.

[0016] In one embodiment of the present invention, the bladder cancer is non-muscle invasive bladder cancer.

[0017] In one embodiment of the present invention, the bladder cancer is bladder urothelial carcinoma.

[0018] The diagnosis includes but is not limited to confirming the patient's disease, diagnosing whether the patient has recurred after treatment, and diagnosing the location, area and size of the patient's tumor in the bladder.

[0019] The diagnosis method includes but is not limited to cystoscopy. In one embodiment of the present invention, the diagnosis is performed by fluorescence cystoscopy, and preferably by using near-infrared fluorescence for cystoscopy.

[0020] Therefore, in another embodiment of the present invention, the present invention provides use of a compound represented by Formula I or a salt or solvate thereof in the preparation of a contrast agent for diagnosing bladder cancer for transvesical instillation for use in conjunction with a fluorescent cystoscope, preferably the fluorescent cystoscope is a near-infrared fluorescent cystoscope.

[0021] The treatment includes but is not limited to bladder tumor transurethral resection, partial cystectomy, intravesical instillation therapy, chemotherapy and immunotherapy. In one embodiment of the present invention, the treatment is bladder tumor transurethral resection or partial cystectomy, preferably the treatment is bladder tumor transurethral resection or partial cystectomy performed under fluorescence cystoscopy, preferably the fluorescence cystoscopy is near-infrared fluorescence cystoscopy.

[0022] Therefore, in another embodiment of the present invention, the present invention provides the use of a compound represented by Formula I or a salt or solvate thereof in the preparation of a contrast agent for transvesical instillation of bladder tumors for use in conjunction with a fluorescent cystoscope for transvesical resection of bladder tumors or partial cystectomy, preferably the fluorescent cystoscope is a near-infrared fluorescent cystoscope.

[0023] The contrast agent may be a lyophilized preparation or a liquid preparation.

[0024] When the contrast agent is a lyophilized preparation, it can be reconstituted with a physiologically acceptable aqueous solution into a liquid preparation before use and then administered intravesically. The lyophilized preparation can be prepared using a lyophilized preparation preparation method known in the art.

[0025] The liquid preparation is prepared by dissolving the compound of formula I or its salt or solvate in a physiologically acceptable aqueous solution.

[0026] The compound represented by Formula I is described in Chinese Patent No. ZL201710891549.8, the entire contents of which are incorporated herein by reference.

[0027] According to the present invention, the salt of the compound of formula I can be a pharmaceutically acceptable salt, which can be prepared by reacting with a non-toxic inorganic or organic acid, or an inorganic or organic base. Inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfuric acid, nitric acid, hydrogen sulfate, boric acid, hemisulfuric acid, and the like; organic acids include trifluoroacetic acid, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid, undecanoic acid, palmitic acid, stearic acid, oleic acid, oxalic acid, malonic acid, adipic acid, lactic acid, malic acid, maleic acid, hippuric acid, tartaric acid, citric acid, succinic acid, ascorbic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, benzoic acid, camphoric acid, camphorsulfonic acid, citric acid, fumaric acid, gluconic acid, galacturonic acid, lauryl sulfate, and various amino acids. Inorganic bases include alkali metal salts, alkaline earth metal salts or optionally substituted ammonium salts, such as hydroxides, carbonates, bicarbonates, etc.; organic bases include trimethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, tromethamine or other basic amino acids, such as lysine, ornithine or arginine, etc.

[0028] The present invention also provides a compound represented by formula I or a salt or solvate thereof for use in imaging in the diagnosis or treatment of bladder cancer.

[0029] In one embodiment of the present invention, the present invention provides a compound of formula I or a salt or solvate thereof for use in intravesical instillation for imaging in the diagnosis or treatment of bladder cancer.

[0030] In another embodiment of the present invention, the present invention provides a compound of formula I or a salt or solvate thereof for use in conjunction with a fluorescent cystoscope for transvesical instillation of bladder cancer for diagnosis, wherein the fluorescent cystoscope is preferably a near-infrared fluorescent cystoscope.

[0031] In another embodiment of the present invention, the present invention provides a compound represented by formula I or a salt or solvate thereof for use in conjunction with a fluorescent cystoscope for transvesical instillation of bladder tumors or partial cystectomy, wherein the fluorescent cystoscope is preferably a near-infrared fluorescent cystoscope.

[0032] The present invention also provides a contrast agent for diagnosing or treating bladder cancer, wherein the contrast agent contains the compound represented by formula I or a salt or solvate thereof.

[0033] In one embodiment of the present invention, the present invention provides a contrast agent for diagnosing or treating bladder cancer by bladder perfusion, wherein the contrast agent contains the compound represented by Formula I or a salt or solvate thereof.

[0034] In another embodiment of the present invention, the present invention provides a contrast agent for diagnosing bladder cancer for use in conjunction with a fluorescent cystoscope for transvesical instillation, wherein the contrast agent contains a compound represented by Formula I or a salt or solvate thereof, and preferably the fluorescent cystoscope is a near-infrared fluorescent cystoscope.

[0035] In another embodiment of the present invention, the present invention provides a contrast agent for transvesical instillation of bladder tumors or partial cystectomy for use in conjunction with a fluorescent cystoscope, wherein the contrast agent contains a compound represented by formula I or a salt or solvate thereof, and preferably the fluorescent cystoscope is a near-infrared fluorescent cystoscope.

[0036] The contrast agent can be a lyophilized preparation or a liquid preparation. When the contrast agent is a lyophilized preparation, it is reconstituted into a liquid preparation with a physiologically acceptable aqueous solution before use. The liquid preparation is prepared by dissolving the compound of formula I or its salt or solvate in a physiologically acceptable aqueous solution. In the present invention, the concentration of the compound of formula I or its salt or solvate in the contrast agent refers to the concentration of the compound of formula I or its salt or solvate in the contrast agent after the lyophilized preparation is reconstituted into a liquid preparation or in the contrast agent originally in the form of a liquid preparation.

[0037] In one embodiment of the present invention, the present invention also provides a kit for imaging in the diagnosis or treatment of bladder cancer, wherein the kit contains the compound represented by formula I or its salt or solvate, or contains the above-mentioned contrast agent.

[0038] As mentioned above, the diagnosis method includes but is not limited to cystoscopy. In one embodiment of the present invention, the diagnosis is performed by fluorescence cystoscopy, and preferably by using near-infrared fluorescence for cystoscopy.

[0039] The treatment includes but is not limited to bladder tumor transurethral resection, partial cystectomy, intravesical instillation therapy, chemotherapy and immunotherapy. In one embodiment of the present invention, the treatment is bladder tumor transurethral resection or partial cystectomy, preferably the treatment is bladder tumor transurethral resection or partial cystectomy performed under fluorescence cystoscopy, preferably the fluorescence cystoscopy is near-infrared fluorescence cystoscopy.

[0040] In one embodiment of the present invention, the concentration of the compound represented by formula I or its salt or solvate in the contrast agent is 0.1-100 μM, preferably 1-90 μM, more preferably 10-80 μM, for example: 12 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 52 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 79 μM, etc.

[0041] In another embodiment of the present invention, the concentration of the compound of Formula I or its salt or solvate in the contrast agent, calculated as the prototype of the compound of Formula I, is 0.001 mg / mL-100 mg / mL, preferably 0.01 mg / mL-10 mg / mL, and more preferably 0.1 mg / mL-5 mg / mL.

[0042] During use, the bladder can be perfused with 10-200 mL of a liquid containing a suitable concentration of a compound of formula I or a salt or solvate thereof (e.g., the aforementioned contrast agent), preferably 10-100 mL, and allowed to remain in the bladder for 5-180 minutes, preferably 20-40 minutes, and then flushed with a physiologically acceptable aqueous solution 1-4 times, for example 3 times.

[0043] The present invention also provides a method for diagnosing bladder cancer, comprising administering a compound of Formula I, or a salt or solvate thereof, or a contrast agent comprising the compound of Formula I, or a salt or solvate thereof, to a patient in need thereof. In one embodiment of the present invention, the compound of Formula I, or a salt or solvate thereof, or a contrast agent comprising the compound of Formula I, or a salt or solvate thereof, is administered by bladder instillation. In one embodiment of the present invention, the diagnostic method is performed using a fluorescent cystoscope, preferably a near-infrared fluorescent cystoscope.

[0044] The present invention also provides a method for treating bladder cancer, comprising administering to a patient in need thereof a compound of Formula I, or a salt or solvate thereof, or a contrast agent comprising the compound of Formula I, or a salt or solvate thereof. In one embodiment of the present invention, the compound of Formula I, or a salt or solvate thereof, or a contrast agent comprising the compound of Formula I, or a salt or solvate thereof, is administered by intravesical instillation. In one embodiment of the present invention, the method of treatment is transurethral resection of bladder tumors or partial cystectomy under the guidance of a fluorescent cystoscope, preferably a near-infrared fluorescent cystoscope.

[0045] The present invention also provides the application or use of the compound represented by formula I or its salt or solvate in the diagnosis or treatment of bladder cancer. The application or use is imaging in the diagnosis or treatment of bladder cancer.

[0046] The present invention also provides a method for using the compound of formula I or its salt or solvate in diagnosing or treating bladder cancer. The method is to use the compound of formula I or its salt or solvate in diagnosing or treating bladder cancer for imaging.

[0047] According to the present invention, when the compound of formula I or its salt or solvate is used in the diagnosis or treatment of bladder cancer, the preferred route of administration is bladder perfusion.

[0048] According to the present invention, the diagnosis includes, but is not limited to, confirming the patient's disease, diagnosing whether the patient has relapsed after treatment, and diagnosing the location, area, and size of the patient's tumor within the bladder. The diagnostic method includes, but is not limited to, cystoscopy. In one embodiment of the present invention, the diagnosis is performed using fluorescent cystoscopy, preferably using near-infrared light.

[0049] The treatment includes but is not limited to bladder tumor transurethral resection, partial cystectomy, bladder instillation therapy, chemotherapy and immunotherapy. In one embodiment of the present invention, the treatment method is bladder tumor transurethral resection or partial cystectomy under the guidance of fluorescent cystoscopy, preferably the fluorescent cystoscopy is a near-infrared fluorescent cystoscopy.

[0050] In one embodiment of the present invention, in the above-mentioned diagnostic or therapeutic methods or applications, the compound of Formula I or its salt or solvate is used at a concentration of 0.1-100 μM, preferably 1-90 μM, more preferably 10-80 μM, for example, 12 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 52 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 79 μM, etc. The compound of Formula I or its salt or solvate can be prepared into a solution within the above-mentioned concentration range using a physiologically acceptable aqueous solution for use.

[0051] Alternatively, the prepared contrast agent is directly used, wherein the concentration of the compound represented by Formula I or its salt or solvate in the contrast agent is 0.1-100 μM, preferably 1-90 μM, more preferably 10-80 μM, for example: 12 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 52 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 79 μM, etc.

[0052] In another embodiment of the present invention, in the above-mentioned diagnostic or therapeutic methods or applications, the compound of Formula I or its salt or solvate is used at a concentration of 0.001 mg / mL to 100 mg / mL, preferably 0.01 mg / mL to 10 mg / mL, and more preferably 0.1 mg / mL to 5 mg / mL, based on the prototype of the compound of Formula I. The compound of Formula I or its salt or solvate can be prepared into a solution in the above-mentioned concentration range using a physiologically acceptable aqueous solution for use.

[0053] Alternatively, the prepared contrast agent is directly used, wherein the concentration of the compound represented by Formula I or its salt or solvate in the contrast agent is 0.001 mg / mL-100 mg / mL, preferably 0.01 mg / mL-10 mg / mL, and more preferably 0.1 mg / mL-5 mg / mL, based on the prototype of the compound represented by Formula I.

[0054] During use, the bladder can be perfused with 10-200 mL of a liquid (e.g., a contrast agent) containing a suitable concentration of a compound of formula I or a salt or solvate thereof, and allowed to remain in the bladder for 5-180 minutes, preferably 20-40 minutes, and then flushed with a physiologically acceptable aqueous solution 1-4 times, for example 3 times.

[0055] According to the present invention, when the compound of formula I or a salt or solvate thereof is used in combination with a fluorescent cystoscope for the diagnosis or treatment of bladder cancer, the cystoscopic operation can be completed within 10 minutes to 6 hours after flushing the bladder, preferably within no more than 4.5 hours.

[0056] According to the present invention, a physiologically acceptable aqueous solution refers to a solution with water as a solvent, including but not limited to water, various physiologically acceptable buffer systems, such as purified water, deionized water, distilled water, physiological saline (such as 0.9% sodium chloride injection), glucose solution for injection, PBS buffer, etc.

[0057] In the present invention, contrast agent has the same meaning as imaging agent or developing agent.

[0058] The polypeptide-coupled fluorescent probe molecules of the present invention can effectively bind to the target specifically through active targeting of molecular recognition, activate downstream signal proteases, and the latter cleave the polypeptide-coupled fluorescent probe molecules at specific sites. The residues after cleavage assemble to form a nanofiber structure, thereby achieving an orderly arrangement of the fluorescent groups and improving the optical stability and fluorescence quantum yield of the fluorescent groups.

[0059] In addition, the targeted recognition cascade activation of proteases by peptide-coupled fluorescent probe molecules improves the specificity of the contrast agent; the in situ assembled nanostructure improves the sensitivity and fluorescence intensity of the contrast agent, overcomes the photobleaching problem of the fluorescent group, extends the stable imaging time window, and has an excellent signal-to-noise ratio, which can meet the clinical needs of bladder cancer diagnosis and intraoperative navigation.

[0060] After the polypeptide-coupled fluorescent probe molecule of the present invention is administered via bladder perfusion to beagle dogs, no systemic drug absorption occurs, and the safety is high.

[0061] In the present invention, "and / or" will be regarded as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0062] "Comprising" and "including" have the same meaning and are intended to be open ended and permit, but not require, the inclusion of additional elements or steps. When the terms "comprising" or "including" are used herein, the terms "consisting of" and / or "consisting essentially of" are also included and disclosed. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Affinity determination of the compound of formula I and the target protein XIAP

[0064] Figure 2 Statistical graph of fluorescence intensity of the compound of formula I, HAL, and the compound of formula II in different bladder cancer cells (T24, RT4, and UM-UC-3) and normal urothelial cells (SV-HUC-1)

[0065] Figure 3 Dose-fluorescence intensity curve of human bladder cancer cell T24 for the uptake of compound I

[0066] Figure 4 Time-fluorescence intensity curves of the compound of formula I and HAL in human bladder cancer cells RT4 and human normal urothelial cells SV-HUC-1

[0067] Figure 5 Imaging photos, signal-to-noise ratio analysis, and histological photos of the compound of Formula I and the compound of Formula II in an orthotopic human bladder cancer model in mice

[0068] Figure 6 Statistical graph of mean fluorescence intensity of compound I and compound II on tumor tissue and non-tumor tissue of bladder cancer patients, as well as AUC graph of specificity and sensitivity analysis

[0069] Figure 7 Imaging and histological photos of the compound of formula I on freshly isolated tumor-bearing organs from bladder cancer patients

[0070] Figure 8 Structural formula of compound I DETAILED DESCRIPTION

[0071] The present invention is further described below with reference to the following examples. It should be noted that the examples are not intended to limit the scope of protection of the present invention, and those skilled in the art will understand that any improvements and variations based on the present invention are within the scope of protection of the present invention.

[0072] The conventional reagents used in the following examples are all commercially available. The biological and pharmacological experimental methods and operations used are all methods known in the art, and the corresponding detection can be carried out with reference to the instructions of the kits used.

[0073] Example 1 Preparation of the compound represented by formula I

[0074] Wang resin with a 0.35 mM modification density was used. The N-terminus of the first amino acid (lysine) was protected with Fmoc, and the C-terminus was fixed to the resin. The N-terminal Fmoc protection was removed using a 20% volume fraction of hexahydropyridine in DMF, and the deprotection was monitored using the ninhydrin test. The carboxyl group of the next amino acid was then activated with a 0.4 M solution of N-methylmorpholine (NMM) and 10 times the amount of benzotriazole-N,N,N",N"-tetramethyluronium hexafluorophosphate (HBTU) in DMF. The activated amino acid was then added to the deprotected resin and reacted for 2 hours. According to this method, all remaining amino acids are linked via a condensation reaction to form a linked polypeptide fixed to the resin; the synthesized polypeptide is removed from the resin using a trifluoroacetic acid solution containing 2.5% water and 2.5% triisopropylsilane, and the amino acid side chain protection is simultaneously removed; the trifluoroacetic acid is removed by rotary evaporation, and the crude polypeptide product is then precipitated with anhydrous ether, washed, and dried; finally, the polypeptide is purified by reverse-phase preparative liquid chromatography; the polypeptide molecule and Cy7-Cl are then reacted in PBS at a molar ratio of 1:1.1 at room temperature for 2 hours. The reaction solution is washed three times with dichloromethane to remove unreacted Cy7-Cl molecules, and the aqueous phase is added to a dialysis bag and dialyzed against deionized water. Finally, it is purified by reverse-phase preparative liquid chromatography to obtain a Cy7-Cl-labeled polypeptide-coupled fluorescent probe molecule, i.e., a molecule having the structure represented by Formula I of the present invention.

[0075] Example 2 Preparation of the Compound of Formula II

[0076] The reference molecular imaging probe compound II was synthesized using the synthesis method in Chinese patent ZL201810648875.0. The Wang resin with a modification density of 0.35 mM was selected to synthesize the polypeptide on the resin, and the Fmoc protection of the amino terminus was removed with a DMF solution of hexahydropyridine. The carboxyl group of the next amino acid was activated with a DMF solution of 4-methylmorpholine (NMM) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and then condensed with the first deprotected amino acid. The above steps were repeated until the condensation of all amino acids was completed; the synthesized polypeptide was removed from the resin with a trifluoroacetic acid solution containing 2.5% water and 2.5% triisopropylsilane, and the side chain protection of the amino acid was removed at the same time; the trifluoroacetic acid was removed by rotary evaporation, and the crude polypeptide product was precipitated with anhydrous ether, washed and dried; finally, the polypeptide was purified by reverse phase preparative liquid chromatography; the IR783 molecule was precipitated at pH The coupling reaction was carried out with the Cys side chain thiol group on the polypeptide in a Tris buffer of 7.5-8.5. After stirring and reacting overnight at room temperature in the dark, the unreacted IR783 was separated by dialysis. The dialysate was then evaporated, concentrated and extracted to obtain the control molecular imaging probe compound II.

[0077] Example 3 Affinity determination of the compound of formula I and the target protein XIAP

[0078] Test instrument: Octet R8 (Sartorius), SSA biosensor (Sartorius, 18-5057)

[0079] Preparation of test solution: Measure 80 mL PBST and 120 mL PBS, mix and sonicate to obtain PBST buffer with a Tween 20 content of 0.02%. Measure 99 mL PBST buffer and 1 mL DMSO, mix and sonicate to obtain a PBST working solution containing 0.02% Tween 20 and 1% DMSO.

[0080] 0.95 mg of the compound of formula I was weighed, 65.65 μL of DMSO was added and sonication was performed to dissolve the compound to obtain a 5 mM stock solution of the compound of formula I.

[0081] To 100 μg of XIAP protein, add 100 μL of H 2 O and 400 μL of PBST buffer, and gently pipette to dissolve and mix the protein to obtain a 200 μg / mL XIAP solution.

[0082] The stock solution of the compound of formula I was diluted with DMSO to obtain a DMSO gradient solution with the concentrations of 25 μM, 10 μM, 5 μM, 2.5 μM, 1 μM, 0.5 μM, and 0.25 μM.

[0083] Take 3 μL of the DMSO gradient solution and add 297 μL of PBST buffer, then sonicate. Take another 300 μL of PBST working solution to obtain a 300 μL gradient solution of Formula I with concentrations of 250 nM, 100 nM, 50 nM, 25 nM, 10 nM, 5 nM, 2.5 nM, and 0 nM.

[0084] Affinity test:

[0085] A gradient solution of the compound of formula I was added to a black 96-well plate, and the program was set up and run to perform affinity determination.

[0086] The results showed that the compound of formula I exhibited high affinity for human XIAP protein (X-linked inhibitor of apoptosis protein), with a Kd value of 55.6 nM ( FIG. 1 ).

[0087] Example 4 Determination of the Selectivity of Formula I Compounds for Bladder Cancer Cells

[0088] Three human bladder cancer cell lines (T24, RT4, UM-UC-3) and one human ureteral epithelial immortalized cell line (SV-HUC-1) were selected and cultured at a rate of 8×10 5Plate 100 cells / well in a 6-well plate and incubate overnight at 37°C in a cell culture incubator. The next day, add the compound of Formula I to a final concentration of 20 μM to the cell culture medium and incubate the cells again at 37°C for 1 hour. After incubation, aspirate the culture supernatant containing the test compound and wash the cells three times with PBS. Discard the PBS, digest the cells with trypsin, and then add complete culture medium for neutralization. After centrifugation, aspirate the supernatant, resuspend the cells in PBS, and analyze them on a flow cytometer.

[0089] Using the same experimental methods and conditions, a control contrast agent HAL (5-aminolevulinic acid hexyl ester hydrochloride) and a control molecular imaging probe compound of formula II were tested in parallel.

[0090] As shown in Figure 2, the mean fluorescence intensities of the three bladder cancer cell lines tested with the Formula I compound were similar, approximately 4.8-6.4 times that of the immortalized epithelial cell line SV-HUC-1. However, the mean fluorescence intensities of HAL on the three bladder cancer cell lines were generally lower, and there was significant variation between the different cell types, with the lowest being UM-UC-3 cells, approximately 1.6 times that of SV-HUC-1. The mean fluorescence intensity of the control molecular imaging probe Formula II on the three bladder cancer cell lines was also lower. Even on the RT4 cells, which had the highest fluorescence intensity, the selectivity was only 2.1 times compared to the SV-HUC-1 group. This demonstrates that the Formula I compound has generally strong recognition and binding ability for bladder cancer cells, as well as high fluorescence intensity, and significantly better tumor cell selectivity than HAL and the control Formula II compound.

[0091] Example 5 Uptake behavior of the compound of formula I in tumor cells

[0092] A human bladder cancer cell line (T24) was selected and 8×10 5 Cells / well were plated in a 6-well plate and incubated overnight at 37°C in a cell culture incubator. The next day, a series of compounds of Formula I were added to the cell culture medium at final concentrations of 0 μM, 1 μM, 3 μM, 27 μM, and 81 μM, and the cells were again incubated at 37°C for 1 hour. After incubation, the culture supernatant containing the test compound was aspirated and the cells were washed three times with PBS. The PBS was aspirated and the cells were digested with trypsin and then neutralized with complete culture medium. After centrifugation, the supernatant was aspirated and the cells were resuspended in PBS and analyzed on a flow cytometer.

[0093] The results are shown in Figure 3. The uptake of the compound of formula I in T24 cells increases linearly with increasing drug concentration, indicating that human bladder cancer cells can take up the compound of formula I and the uptake behavior has a good concentration dependence.

[0094] Example 6 Retention time of the compound of formula I in tumor cells

[0095] A human bladder cancer cell line (RT4) and a human ureteral epithelial immortalized cell line (SV-HUC-1) were selected and cultured at a rate of 8×10 5 Cells / well were plated in a 6-well plate and incubated in a cell culture incubator at 37°C overnight. The next day, a compound of formula I and an equal concentration of the control substance HAL were added to the cell culture medium at a final concentration of 20 μM, and the cells were again incubated in an incubator at 37°C for 1 hour. After the incubation, the culture supernatant containing the test substance was aspirated and the cells were washed 3 times with PBS. The PBS was discarded, the cells were digested with trypsin, and then complete culture medium was added for neutralization. After centrifugation, the supernatant was discarded, the cells were resuspended with PBS, and then detected on a flow cytometer. The cells were detected immediately after the incubation, 2 hours after the incubation, and 4 hours after the incubation.

[0096] The results, as shown in Figure 4, show that the compound of Formula I accumulated in RT4 tumor cells for up to 4 hours, with signal intensity decreasing by only 20% over the 4-hour period. In contrast, the compound of Formula I showed minimal uptake in normal SV-HUC-1 cells and was essentially completely metabolized by 2 hours. The control, HAL, exhibited linear metabolic clearance in RT4 tumor cells, being completely eliminated by 2 hours.

[0097] Example 7: Compound I has high signal-to-noise ratio imaging effect in an orthotopic mouse human bladder cancer model

[0098] First, a mouse model of orthotopic human bladder tumor xenografts was established. 6-8 week old BALB / c nude mice were anesthetized intraperitoneally with avertin and the bladder was emptied by intravenous catheterization with a 24G indwelling needle. The bladder was then flushed with PBS and the bladder mucosa was slightly damaged with a needle tip. Human bladder cancer cells EJ-1, which were in the logarithmic growth phase, were collected and inoculated at a rate of 5×10 4 100 μL of the tumor was injected into the bladder at a concentration of 100 μL and incubated in the bladder for 1 hour. Finally, the mouse urethra was clamped with suture ligation to prevent tumor cells from spilling out of the urethra. After 1 hour, the ligature was removed, and the mouse was allowed to resume movement. Bladder tumor growth was assessed daily by abdominal palpation and observation for gross hematuria.

[0099] In a successfully established orthotopic mouse model of human bladder cancer, 100 μL of a solution of the compound of Formula I and a control compound of Formula II (dissolved in PBS buffer to a final concentration of 50 μM) were instilled into the bladder for 30 minutes. The contrast agent was then removed, and the bladder was flushed three times with PBS buffer. The bladder was then dissected for in vivo fluorescence imaging of the small animal. As shown in Figure 5, the compound of Formula I was able to accurately identify tumor boundaries with a signal-to-noise ratio of up to 4.5, and could simultaneously identify multiple tumor lesions. In contrast, the control compound of Formula II had a signal-to-noise ratio of only 1.2, and its ability to identify tumor boundaries and signal-to-noise ratio were inferior to those of the compound of Formula I.

[0100] Example 8: The compound of formula I has high specificity and high sensitivity in tumor tissue of bladder cancer patients

[0101] All experiments using human specimens were reviewed and approved by the Ethics Review Committee of the Fourth Hospital of Harbin Medical University. After obtaining informed consent from the patients, fresh intact tumor-bearing bladders were removed from patients who were scheduled to undergo radical cystectomy. After the bladder was instilled with sterile saline via a urinary catheter to remove residual blood, 50 mL of a solution of the compound of formula I and the control compound of formula II (dissolved in PBS buffer to a final concentration of 50 μM) was instilled for 30 minutes. The bladder was rinsed three times with sterile saline, the bladder was opened by a longitudinal incision on the upper surface of the bladder, and bladder cancer tissue and normal bladder tissue were cut into pieces and stored in the dark. Subsequently, the tissue (5×5 mm) was fixed with OCT at -20°C in the dark for 12 hours. Finally, the fixed tumor tissue was frozen and sectioned, and then observed and quantified using a laser confocal microscope. Finally, the fluorescent area represented was pathologically analyzed to confirm the detection results of fluorescence imaging. Among them, the compound group of formula I took 240 human bladder cancer tissues and 98 non-tumor tissues (including 60 normal tissues, 20 inflammatory tissues and 18 necrotic tissues), and the compound group of formula II took 27 human bladder cancer tissues and 29 normal tissues.

[0102] As shown in Figure 6, pathological analysis of bladder cancer tissues from the bladder group treated with the compound of Formula I revealed 160 cases of urothelial carcinoma, 20 cases of squamous cell carcinoma, 30 cases of adenocarcinoma, 10 cases of carcinoma in situ (CIS), and 20 cases of other pathological types. The average fluorescence quantitative values ​​for all of these tumor tissues were much higher than those for normal tissue, inflammatory tissue, and necrotic tissue, and the differences were statistically significant. Statistical calculations showed that the specificity of the compound of Formula I for distinguishing bladder cancer from non-cancer tissues was greater than 90%, and its sensitivity was greater than 98%. While the control compound of Formula II also showed differences in the average fluorescence quantitative values ​​for tumor tissue and non-cancer tissue, its specificity for distinguishing bladder cancer from non-cancer tissue was only 62.9%, with a sensitivity of 65.5%.

[0103] Example 9: Compound I can accurately identify tumor tissue in freshly isolated tumor-bearing organs from bladder cancer patients

[0104] All experiments using human specimens were reviewed and approved by the Ethics Review Committee of the Fourth Hospital of Harbin Medical University. After obtaining informed consent from the patients, fresh, intact tumor-bearing bladders were removed from patients scheduled for radical cystectomy. After sterile saline was instilled via catheter to remove residual blood, the bladder was instilled with 50 mL of a solution of the compound of Formula I (dissolved in PBS buffer to a final concentration of 50 μM) for 30 minutes. The bladder was flushed three times with sterile saline, opened through a longitudinal incision on the upper surface of the bladder, and observed using an intraoperative navigation fluorescence imaging system.

[0105] As shown in Figure 7, Brightfield refers to the tumor tissue morphology observed under a white light microscope, fluorescence refers to the distribution of fluorescent signals observed under a fluorescent microscope, and H&E is a histopathological analysis of the fluorescent area. In Area 1 of Figure 7, the tumor tissue boundary visible to the naked eye under a white light microscope can clearly show the tumor tissue boundary imaged by the compound of Formula I under a fluorescent microscope, and histopathological analysis also confirms that the tissue is tumor tissue. In Area 2 of Figure 7, no tumor tissue was found under a white light microscope, but a tiny tumor foci (<3mm) imaged by the compound of Formula I could be clearly found under a fluorescent microscope, and histopathological analysis also confirmed that the tissue was tumor tissue. In Area 3 of Figure 7, it was observed as inflammatory tissue under a white light microscope, and it was found that the tissue was not fluorescently labeled by the compound of Formula I under a fluorescent microscope, and histopathology also confirmed that the tissue was inflammatory tissue. In Area 4 of Figure 7, it was observed as necrotic tissue under a white light microscope, and it was found that the tissue was not fluorescently labeled by the compound of Formula I under a fluorescent microscope, and histopathology also confirmed that the tissue was necrotic tissue. The above results indicate that the compound of formula I can accurately mark tumor boundaries, identify tiny tumor foci (<3 mm), and distinguish tumor tissue from inflammatory and necrotic tissue.

[0106] Example 10: Toxicological Experiments of Compounds of Formula I

[0107] Beagle dogs were administered a compound of Formula I via intravesical instillation or intravenous injection for two weeks (once weekly for a total of three doses). Toxic reactions induced by the compound of Formula I were observed over two weeks and its toxicokinetics were studied. Five groups were included in this study: a vehicle control (intravesical instillation with 0.9% sodium chloride), low- and high-dose intravesical instillation groups (doses of 5 and 25 mg / animal, respectively), and low- and high-dose intravenous instillation groups (doses of 1.2 and 5 mg / kg, respectively). The intravesical instillation volume was 5 mL / animal, and the intravenous instillation volume was 2 mL / kg. Each group consisted of two animals, half male and half female. Key toxicity observations included death and moribundity, general observations, body weight, food intake and weight gain, and clinical pathology (including hematology, coagulation, serum biochemistry, and urinalysis). At the end of the dosing period (D16), all animals in the main experimental groups underwent planned autopsies, and major tissues and organs were harvested for histopathological examination. Blood samples were collected from the intravesical instillation group at various time points before and after the first and last doses (D1 and D15) for toxicokinetic analysis.

[0108] The dosage design of this experiment is shown in the table below:

[0109] The toxicokinetic results are shown in the following table:

[0110] When 5 mg and 25 mg were administered intravesically, no blood drug concentration was detected at any time point before and after the first and last doses.

[0111] Intravenous injection in the range of 1.2 to 5 mg / kg, Beagle dogs were repeatedly intravenously administered with the compound of formula I. The exposure of the compound of formula I in the plasma of male and female animals in each dose group (AUC last and C max ) There is no obvious gender difference; AUC of the compound of formula I in animals last and C max The compound of formula I was administered intravenously for 2 consecutive weeks. There was no significant accumulation of the compound of formula I in the low and high dose groups (measured by AUC last count).

[0112] All animals survived until the planned autopsy. No abnormal changes were found in the general observation, body weight, food intake, serum biochemistry, hematology (only in the intravenous group), coagulation, urinalysis, and gross anatomy of the animals in all dose groups.

[0113] Histopathological results showed that the kidneys, bladders, urethra and ureters of the groups with intravenous injection of 1.2 and 5 mg / kg and intravesical instillation of 5 mg / animal were normal; and the kidneys, bladder, urethra and ureters of the group with intravesical instillation of 25 mg / animal were normal.

[0114] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Use of a compound represented by formula I or a salt or solvate thereof in the preparation of a contrast agent for diagnosing or treating bladder cancer, Preferably, the compound represented by formula I or its salt or solvate is used in the preparation of a contrast agent for diagnosing or treating bladder cancer for transvesical instillation.

2. The use according to claim 1, characterized in that The diagnosis is performed by using a fluorescent cystoscope, preferably a near-infrared fluorescent cystoscope; Preferably, the compound represented by formula I or its salt or solvate is used in the preparation of a contrast agent for diagnosing bladder cancer for transvesical instillation for use in conjunction with a fluorescent cystoscope, and preferably the fluorescent cystoscope is a near-infrared fluorescent cystoscope.

3. The use according to claim 1, characterized in that The treatment is bladder tumor transurethral resection or partial cystectomy, preferably the treatment is bladder tumor transurethral resection or partial cystectomy performed under near-infrared light fluorescence cystoscopy; Preferably, the compound represented by formula I or its salt or solvate is used in the preparation of a contrast agent for transvesical instillation of bladder tumors or partial cystectomy for use in conjunction with a fluorescent cystoscope, and preferably the fluorescent cystoscope is a near-infrared fluorescent cystoscope.

4. A method for diagnosing bladder cancer, characterized in that: administering a compound of formula I or a salt or solvate thereof or a contrast agent comprising the compound of formula I or a salt or solvate thereof to a patient in need thereof, Preferably, the compound of formula I or its salt or solvate or the contrast agent comprising the compound of formula I or its salt or solvate is administered by bladder instillation; Preferably, the diagnostic method is to use a fluorescent cystoscope for diagnosis, and preferably the fluorescent cystoscope is a near-infrared fluorescent cystoscope.

5. A method for treating bladder cancer, characterized in that: administering a compound of formula I or a salt or solvate thereof or a contrast agent comprising the compound of formula I or a salt or solvate thereof to a patient in need thereof, Preferably, the administration method of the compound of formula I or its salt or solvate or the contrast agent comprising the compound of formula I or its salt or solvate is bladder instillation; Preferably, the treatment method is bladder tumor transurethral resection or partial cystectomy under the guidance of a fluorescent cystoscope, and preferably the fluorescent cystoscope is a near-infrared fluorescent cystoscope.

6. The use or method according to any one of claims 1 to 5, characterized in that The contrast agent is a lyophilized preparation or a liquid preparation.

7. The use or method according to claim 6, characterized in that The concentration of the compound represented by formula I or its salt or solvate in the contrast agent is 0.1-100 μM, preferably 1-90 μM, more preferably 10-80 μM; Alternatively, the concentration of the compound of formula I or its salt or solvate in the contrast agent, calculated as the prototype of the compound of formula I, is 0.001 mg / mL-100 mg / mL, preferably 0.01 mg / mL-10 mg / mL, and more preferably 0.1 mg / mL-5 mg / mL.

8. The use or method according to any one of claims 1 to 7, characterized in that The salts of the compounds of formula I are pharmaceutically acceptable salts, which are prepared by reaction with non-toxic inorganic or organic acids, inorganic or organic bases; Preferably, the inorganic acid is selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfuric acid, nitric acid, hydrogen sulfate, boric acid, hemisulfuric acid; Preferably, the organic acid is selected from trifluoroacetic acid, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, heptanoic acid, undecanoic acid, palmitic acid, stearic acid, oleic acid, oxalic acid, malonic acid, adipic acid, lactic acid, malic acid, maleic acid, hippuric acid, tartaric acid, citric acid, succinic acid, ascorbic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, benzoic acid, camphoric acid, camphorsulfonic acid, citric acid, fumaric acid, gluconic acid, galacturonic acid, dodecyl sulfuric acid, amino acids; Preferably, the inorganic base is selected from the hydroxide, carbonate or bicarbonate of an alkali metal or alkaline earth metal or ammonium; Preferably, the organic base is selected from trimethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, tromethamine or a basic amino acid.

9. The use or method of any one of claims 1 to 8, wherein the bladder cancer is non-muscle-invasive bladder cancer or muscle-invasive bladder cancer.

10. The use or method according to any one of claims 1 to 8, wherein the bladder cancer is selected from bladder urothelial carcinoma, bladder squamous cell carcinoma, bladder adenocarcinoma, bladder clear cell carcinoma, bladder small cell carcinoma, and bladder carcinoid.

11. The use or method according to any one of claims 1 to 8, wherein the bladder cancer is a primary cancer or a metastatic tumor derived from other tissues and organs.

12. A kit comprising a compound of formula I or a salt or solvate thereof for use in fluorescence imaging of bladder cancer cells and bladder cancer tissues, 13. An optical imaging method for bladder cancer biological tissue, characterized in that include: (a) contacting a biological tissue with a composition comprising a compound represented by Formula I or a salt or solvate thereof; (b) allowing time for the compound represented by Formula I or a salt or solvate thereof in the composition to distribute in the biological tissue; (c) illuminating the tissue with excitation light of a wavelength absorbable by the compound represented by Formula I; (d) detecting the fluorescent signal emitted by the compound of formula I,

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