WO2016078207A1 - Fluorescent conjugate compound and uses thereof - Google Patents

Abstract

A fluorescent conjugate compound and uses thereof. The fluorescent conjugate compound consists of free fatty acids marked by fluorescent molecules, is useful for detecting precancerous lesion cells or cancer cells, and also is useful for the assessment of human tumor and precancerous diseases, pathologic biopsy, surgical resection range determination, as well as the preparation of drug or tracer reagents for detecting tumor or precancerous disease.

Description

Fluorescent conjugated compound and application thereof Technical field

The invention relates to a fluorescent conjugated compound and an application thereof, in particular to a fluorescent molecule labeled fatty acid and application thereof, and belongs to the technical field of medicine.

Background technique

Tumors are new organisms that form abnormal growth of cells in the body. This proliferation is an unregulated malignant proliferation that eventually forms a mass. The normal cells of the body proliferate and differentiate when they are damaged to repair the damage, while the tumor cells lose the ability to differentiate and mature and infinitely proliferate and squeeze the surrounding normal tissues and carry out distant metastasis.

The survival of a tumor patient is closely related to the stage of the tumor at the time of diagnosis. If the tumor can be detected early, the tumor can be controlled or cured. The clinical stage of the tumor is based on the size of the primary tumor and the degree of dissemination to describe the severity and extent of the malignant tumor. The tumor in the first stage does not invade the surrounding tissue, and the tumor in the fourth stage has been excluded from the tumor at the primary site. Transfer to other parts of the tissue and proliferate at the metastatic site to form new tumors. However, the current patients do not have early treatment, early detection of tumors still have defects, which is the main cause of death of cancer patients.

At present, the main treatments for tumors are radiotherapy and chemotherapy, biological immunotherapy, surgery, etc., which are occupied by surgery in most solid tumors such as colon cancer, lung cancer, breast cancer, prostate cancer, cervical cancer, esophageal cancer, bladder cancer and melanoma. important position. However, due to incomplete resection, the tumor recurrence rate and metastasis rate were still high. For example, the recurrence rate after radical resection of colorectal cancer was 24.1%, and 34.3% of these relapse cases occurred within 1 year after surgery. Therefore, accurate localization of tumors has important guiding significance both in routine examination and in surgery, and is closely related to the prognosis and survival rate of patients.

At present, there are mainly two factors that affect the detection of tumors:

1. Biological characteristics of tumor development: The occurrence and development of most tumors is a complex process with multiple factors and multiple stages. From carcinogenic factors to normal cells to the formation of clinically detectable tumors often need to go through a long incubation period, which is roughly divided into the excitation phase and the promotion phase. The cancer is stimulated by carcinogenic factors that irreversibly transform normal cells into latent cells. The initial step of tumor cells, the promotion process is the process of transforming latent tumor cells into cancer cells. At this time, the cancer cells undergo malignant transformation and gradually exhibit a malignant phenotype. The mutant cells are given new characteristics, unlike normal cells after they mature. Proliferate and only update damaged cells in the body. The proliferation of cancer cells can occur in early childhood. It generally does not differentiate into mature, biologically functional cells. When entering the uncontrolled proliferation stage, it invades and destroys adjacent tissues. It will eventually metastasize or spread to other parts of the body. However, when a number of cancer cell proliferation reaches ^0910, the clinical detection of possible, many patients have entered at this time advanced, and when the number of cancer cells reaches ^12:10, it has spread.

In view of the complexity and uncontrollability of the above-mentioned tumor development process, it is difficult to know the stage and extent of disease development, and for the latent tumor, the structure and function of the tissue have not changed significantly, relying only on the clinical experience of the physician and the macroscopic morphology of the tumor, Judging from serum tumor markers, it is difficult to find early colon cancer, lung cancer, breast cancer, prostate cancer, cervical cancer, esophageal cancer, bladder cancer, melanoma, leukemia, lymphoma and the like.

2, the current diagnosis and treatment of defects: the current early detection of tumors mainly rely on tumor markers, imaging and pathology examinations. Tumor molecular marker examination and imaging examination are not specific to the discovery of tumors. Histopathological biopsy is invasive, and sometimes it requires surgical resection of the tissue for analysis, which may cause the spread of cancer cells. For example, the detection of skin cancer melanoma is usually performed by physical examination of selected cancerous skin for biopsy, which depends to a large extent on the experience of the physician, because cancerous tissue may exceed the scope of its physical judgment, easily missed diagnosis and endanger the life of the patient. On the contrary, some skin biopsy is not cancer, and patients will suffer unnecessary damage. For example, when there is melanin on the cheek, the biopsy sample from the patient's face will affect the appearance.

For patients with confirmed cancer, if the tumor infiltration depth stays at 1-2, a good therapeutic effect can usually be achieved by radical tumor surgery. Before the operation, routine anatomical imaging such as X-ray, CT, MRI, etc., is used to initially locate the tumor to guide the operation. However, during the operation, these pictures can not guide the surgeon to locate the tumor in real time. The doctor only determines the tumor based on his own senses. The location and depth. To finalize the pathological grade of the tumor, the surgeon takes the biopsy tissue at the site of the lesion before the surgery is completed, and then sends a quick frozen section to confirm that all cancerous tissue has been cleared. However, this process often requires a trained pathologist to quickly and accurately analyze the tissue sample, while the patient is still in an open or other surgical state before the outcome of the test, and if the test results indicate that the cancer tissue is metastasized Surgeons will continue to use their senses to remove potentially residual tumors. Although after secondary clearance, there are still 15%-25% of cancer tissue residues remaining. And compared with patients who have completely removed tumor tissue, these patients often need to experience more expensive and painful treatments to control the cancer remaining at the time of surgery and have a higher risk of death.

In recent years, functional imaging has attracted the attention of more and more medical researchers. Functional imaging provides a good opportunity for the detection of benign and malignant tumors. It uses optical, ultrasound, nuclear medicine, magnetic resonance imaging and other imaging methods to target specific targets. Imaging is performed to reveal abnormalities in cell function and gene molecular level in tumors, especially during early tumorigenesis. For example, the application of PET-CT in judging human benign and malignant tumors is the best practice, but PET-CT has a blind spot in the discovery of gastrointestinal tumors, and compared with endoscopy in the digestive tract. Tumor discovery does not have any advantage. Moreover, PET-CT radiation is relatively large, requiring expensive and cumbersome testing equipment and expensive to inspect, and it is difficult for ordinary patients to bear. In addition, PET-CT cannot be used to locate cancer in a doctor's physical examination room, an operation room, or an operating room operation.

Laser confocal microendoscopy is a newly developed endoscope in recent years. It is a product of the integration of traditional electron endoscopy and confocal laser microscopy. Its preparation and operation are almost the same as general endoscopy. Special fluorescent agents such as intravenous sodium fluorescein and topical acridine yellow solution are used during the examination. It has been applied to the examination of gastroscope and colonoscopy in China, and has a fast and accurate advantage for the tracking of smaller lesions and early gastrointestinal tumors. However, the most commonly used fluorescent contrast agents, sodium fluorescein and acridine hydrochloride, are imaged by fluorescein dyed into peripheral blood cells, which cannot enter the cells and can appear after the patient uses them. Sexual allergic reaction, acridine yellow can enter the cell to stain the nucleus, the imaging is clear, but because of its carcinogenicity, it has been banned for the FDA, which requires the development of high specificity and safe and reliable biological agents to improve the confocal microscopy The inspection effect of the mirror.

On the other hand, the absorption and metabolism of free fatty acids is an important physiological process of the body, and almost all tissues such as the intestine, heart, fat, kidney and liver have long-chain fatty acid transporters. The abnormal function of fatty acid transporters is an important cause of the occurrence of various diseases, so studying fatty acid absorption and metabolism is an important scientific problem. At present, the study of fatty acid absorption and metabolism mainly uses radioactive element-labeled fatty acid molecular tracer technology, which requires special detection instruments, and the cost is high. The biggest point is the radioactive contamination of the environment, which is generally not directly applicable to the human body.

There is no fluorescent conjugated compound in the prior art which is composed of a fluorescent molecule labeled free fatty acid, and the human cancer and precancerous diseases are realized according to the difference in absorption or metabolism of free fatty acids in the fluorescent conjugated compound by normal cells and tumor cells. Application of disease assessment, pathological biopsy, and scope of surgical resection.

Summary of the invention

The object of the present invention is to provide a fluorescent conjugated compound which is composed of a fluorescent molecule labeled free fatty acid and which can be used for absorption or metabolism of free fatty acids in the fluorescent conjugated compound according to normal cells and tumor cells. Difference, fluorescence imaging by fluorescence microscopy, laser confocal microscopy, fluorescence endoscopy or laser confocal microscopy, which directly reflects the fluorescence signal intensity of the site to be tested, thereby distinguishing tumor cells from normal cells, and then discovering Precancerous lesions or cancer cells, to achieve disease assessment, pathological biopsy, and surgical resection of human tumors and precancerous diseases.

The technical scheme of the present invention is as follows: a fluorescent conjugated compound is composed of a fluorescent molecule labeled with a free fatty acid, It is applied to detect precancerous lesion cells or cancer cells; the fluorescent molecule is selected from the group consisting of sodium fluorescein, acridine yellow, rhodamine, fluoroboron fluorescens dye, fluorescent dye NBD, dansyl, coumarin dye, cyanine dye Any one of the free fatty acids is a long-chain saturated or monounsaturated fatty acid; the label means that the fluorescent molecule and the free fatty acid pass through 2 to 30 by a chemical reaction from C, N, O, P, S, Si. Any one or more of a linear, cyclic, benzene ring or a combination of the above-mentioned linking groups; the emission wavelength of the fluorescent molecule is from 400 nm to 1200 nm.

Preferably, the fluorescent conjugated compound is composed of sodium fluorescein C ₂ 0H ₁₀ Na ₂ O ₅ labeled octadecenoic acid C ₁₈ H ₃₄ O ₂ , and sodium fluorescein passes through the CO bond at the carbon 9 position of octadecenoic acid. It is linked with octadecenoic acid and its preparation method is as follows:

(1) Under the protection of nitrogen, the molar ratio of sodium fluorescein to mercaptopropionic acid is 1:0.1-0.15, stirred at 45-55 ° C until a solution is formed, and ruthenium chloride with a molar mass of 0.2 fluorescein sodium is added to the solution. (IV) tetrahydrofuran complex (1:2), toluene, then refluxed at 125-135 ° C for 15-18 h, the reaction is completed, distillation under reduced pressure, toluene is removed, to obtain an intermediate product;

(2) Under the protection of nitrogen, octadecenoic acid, an intermediate product, azobisisobutyronitrile, and toluene were sequentially added to the dried flask, and the reaction was carried out at 75 to 85 ° C for 45 to 50 hours. The reaction was completed, and the product was precipitated in ethanol. The aqueous solution is centrifuged and dried under vacuum at 50 ° C to obtain sodium oleic acid labeled with fluorescein; the molar ratio of the mixture of octadecenoic acid, intermediate product and azobisisobutyronitrile is 1:4 ~6: 0.2 to 0.5.

The amount of toluene in the step (1) is: the amount of toluene per 1 mol of sodium fluorescein in the reaction system is from 180 to 200 ml.

The amount of toluene in the step (2) is: the amount of toluene per 1 mol of sodium fluorescein-SH in the reaction system is from 250 to 300 ml.

The fluorescent conjugated compounds of the present invention can also be prepared as a drug, stain or tracer for detecting human tumors and precancerous diseases.

The human tumor includes colon cancer, lung cancer, breast cancer, prostate cancer, cervical cancer, esophageal cancer, bladder cancer, melanoma, leukemia, lymphoma, and the like; the precancerous diseases include Barrett's esophagus and adenoma. Sexual polyps, inflammatory bowel disease.

The medicament comprises a medically approved tablet, capsule, pill, powder, ointment, suppository or the solution of the fluorescent conjugated compound in a water soluble propylene glycol or sodium chloride solution to form a sterile solution or suspension.

The content of the fluorescent conjugated compound in the drug for detecting human tumors and precancerous diseases is 0.05 to 0.1%.

The fluorescent conjugated compound of the present invention can be actively transported into cells and accumulated in cells without being metabolized, due to differences in absorption or metabolism of free fatty acids in fluorescent conjugated compounds by normal cells and tumor cells, by fluorescence microscopy, laser confocal Fluorescence imaging by microscopy, fluorescence endoscopy or laser confocal microscopy can visually reflect the fluorescence signal intensity of the site to be tested, and distinguish tumor cells from normal cells to detect precancerous cells or cancer cells.

The fluorescence intensity of the fluorescent conjugated compound of the present invention is about 1.5 to 9.8 times that of the tumor tissue (average 3.4 times). In the fluorescence imaging image, the tumor growth and infiltration range are shown in the dark region.

The fluorescent conjugated compound may be administered in a living body by topical administration or intravenous administration or orally; preferably, the living body is administered topically, and the imaging effect is better.

The fluorescent conjugated compound of the present invention is applied to a living body to be detected by fluorescence microscopy, laser confocal microscopy, fluorescence endoscopy or laser confocal microendoscopic imaging, preferably laser confocal microendoscopy.

The invention provides a fluorescent label which can be detected by a fluorescence microscope and/or an endoscopic device based on the metabolic characteristics of the tumor tissue, and visually displays the metabolism of the specific biological substance in the cell in the form of an image, thereby realizing Timely and rapid diagnosis of diseases and abnormal tumors with abnormal cell biological metabolic activity, and can specifically guide targeted biopsy, and the detection results are accurate and reliable. The fluorescent conjugated compound of the invention has stable biological properties, and the application is safe and fast, and can be directly applied to the human body. The invention establishes a specific and safe endoscopic fluorescent molecular imaging method.

The fluorescent conjugated compound of the present invention and its use have the following advantages:

(1) Fluorescent conjugated compounds are highly sensitive to the discovery of tumor tissues: pre-cancerous cells or tumor cells do not (lower) absorb this fluorescence after intravenous, inhalation or local perfusion of the fluorescent conjugated compound to the patient. The compound can absorb more in normal cells, and a significant concentration difference is formed between the diseased tissue and the normal tissue after a certain period of time. Under a certain wavelength of laser irradiation, the normal tissue emits a strong fluorescence with a specific wavelength. The diseased tissue has no such absorption peak or weak absorption peak, and the fluorophore that absorbs metabolism is not easily catalyzed by other enzymes in the body and accumulates in the cells. This property of the fluorescent conjugated compound increases its sensitivity to recognize tumor tissues. And distinguishing tumors from normal tissues; this method enables tumor tissue to be found at the cellular level compared to existing tumor tracing methods;

(2) Fluorescent conjugated compounds have high stability in vivo: fluorescein can directly bind to small molecule compounds through chemical bonds such as alkyl groups, amide groups, and sulfonamide groups. These chemical bonds are relatively stable in the reaction system in vivo. At the same time, although the linking group separates fluorescein and small molecule compounds, it is small molecule The compound is not affected by fluorescein, and can be absorbed and involved in the metabolism of the body through its specific transporter system like a small molecule compound of a normal organism, and has the characteristics of no toxic side effects;

(3) Fluorescent conjugated compounds provide clear, intuitive and accurate localization of tumor sites: fluorescent conjugated compounds enter the cells by active transport, in addition to clearly showing the contours and arrangement of cells, and even clearly distinguishing cytoplasm and nucleus, imaging quality is beyond current All fluorescent dyes used in clinical practice, combined with fluorescence microscopy, laser confocal microscopy, fluorescent endoscopy, laser confocal endoscopy and other detection equipment to visually display the distribution of fluorescent markers;

(4) Fluorescent marker direct intestinal staining, the method is fast: the previous fluorescent contrast agent such as sodium fluorescein is injected into the body by intravenous injection, and its preliminary preparation and drug action time is long, and the fluorescently labeled biological small molecule can be directly localized. Spray dyeing, direct spraying on the target at the same time of inspection, which is actively transported to the intracellular cells by fluorescence imaging, reflecting the disease state, and the dyeing method is convenient and quick;

(5) Low immunogenicity and toxicity of fluorescent conjugated compounds: fluorescently labeled small molecular compounds are mostly fatty acids or their derivatives that are normally absorbed by the human body, and have no toxic and side effects on the human body. The immune and allergic reactions caused by the body are only possible. Is a fluorescein substance that acts as a label, so fluorescein which has been used for non-toxic side effects of the human body is preferred;

(6) Fluorescent conjugated compounds are simple and easy to use, and can be widely used in clinical applications: the difference in absorption and metabolism of fluorescent conjugated compounds by tumor cells and normal cells can more accurately distinguish tumors from non-tumor tissues at the cellular level. The method is simple and convenient, and can not only guide the endoscopic biopsy, but also can be widely used in clinical preparation of drugs or tracer reagents for detecting human tumors and precancerous diseases, and the fluorescent conjugated compounds can be made into tablets. Capsules, pills, powder forms for convenient oral administration or dissolution in a sterile solution or suspension formed in a water-soluble propylene glycol or sodium chloride solution for parenteral injection, can be used in the form of ointment or as a suppository for rectal administration Medication.

DRAWINGS

The invention is further illustrated by the following figures, but the contents of the drawings do not constitute any limitation of the invention.

Figure 1 is a bar graph comparing the absorption competition of Fluorescein sodium-FA and stearic acid C18:0 in different intestinal cancer cell lines measured by flow cytometry in Example 1.

Figure 2 is a comparison of the in vivo absorption of Fluorescein sodium-FA in the colonic mucosa of normal mice by fluorescence microscopy in Example 1, from left to right: HE staining, Fluorescein sodium-FA fluorescence (cytoplasmic staining), DAPI fluorescence Figure (stained cell nucleus), Fluorescein sodium-FA and DAPI synthesis map.

Figure 3 is a comparison of absorption spectra of Fluorescein sodium-FA in normal mice for 30 min after fluorescence microscopy in Example 1. From left to right: HE staining, Fluorescein sodium-FA fluorescence, DAPI fluorescence, Fluorescein Sodium-FA and DAPI synthesis map.

4 is an imaging diagram of HE staining and fluorescence microscopy after in vivo absorption of Fluorescein sodium-FA by a mouse chemically induced colon cancer model in Example 1, wherein Fig. 1 is a mouse chemically induced colon cancer at a magnification of 40X, Fig. 2. HE staining of the inflamed site and normal colonic mucosa of the mouse at a magnification of 200X and contrast imaging under fluorescence microscopy (from left to right: HE staining, Fluorescein sodium-FA fluorescence, DAPI fluorescence, Fluorescein sodium-FA) Fig. 3 is a comparison of HE staining and imaging contrast under fluorescence microscope in mouse adenocarcinoma at magnification of 200X (from left to right: HE staining, Fluorescein sodium-FA fluorescence, DAPI fluorescence, Fluorescein sodium-FA and DAPI synthesis map).

Figure 5 shows different fluorescent dyes under laser confocal endoscopy (left: Fluorescein sodium-FA, middle panel: control stain NBD-labeled deoxyglucose (2-NBDG), right panel: control stain acridine yellow Fluorescence imaging contrast in normal colonic mucosa of mice.

Figure 6 is a graph showing the different periods of Fluorescein sodium-FA in a mouse chemically induced colon cancer model under laser confocal microscopy in Example 1 (left panel: normal colon, middle panel: low-grade intraepithelial neoplasia, right panel) : Adenocarcinoma) Comparison of absorption of colonic mucosa.

Figure 7 is a comparison of absorption of Fluorescein sodium-FA in normal human colon mucosa (left), colon adenoma after human ESD (middle), and adenocarcinoma tumor specimen (right) under laser confocal microscopy in Example 1. Figure.

detailed description

The invention is further illustrated by the following examples, which are intended to illustrate the invention and not to limit the scope of the invention.

Example 1 generates fluorescence by a fluorescent molecule (sodium fluorescein, emission wavelength 525 nm, green fluorescent dye) and free fatty acid (octadecenoic acid: carbon 18 monounsaturated fatty acid) by CO linkage at the carbon 9 position of oleic acid The conjugated compound (denoted as Fluorescein sodium-FA, the same below), the resulting Fluorescein sodium-FA has fluorescence similar to the original fluorescein molecule, and the preparation method is as follows:

(1) Under the protection of nitrogen, the molar ratio of sodium fluorescein to mercaptopropionic acid is 1:0.13, 0.1 mol of sodium fluorescein and 0.13 mol of mercaptopropionic acid are stirred at 50 ° C until a solution is formed, and fluorescence is added to the solution. Sodium ruthenium (IV) tetrahydrofuran complex (1:2) with a molar mass of 0.2 ‰ (0.02 mmol), 20 ml of toluene, connected to a water separator, and refluxed at 130 ° C for 16 h, the reaction is finished, minus Press distillation to remove toluene to obtain an intermediate product;

(2) Under the protection of nitrogen, 0.1 mol of oleic acid was sequentially added to the dried flask at a molar ratio of octadecenoic acid, an intermediate product, and an azobisisobutyronitrile mixture: 1:5:0.3. 0.5 mol of intermediate product, 0.03 mol of azobisisobutyronitrile, and 150 ml of toluene were reacted at 80 ° C for 48 h. At the end of the reaction, the product was precipitated in an aqueous ethanol solution, centrifuged, and dried under vacuum at 50 ° C to obtain sodium fluorescein. Labeled oleic acid Fuorescein sodium-FA.

The following experiment was carried out by taking the obtained Fluorescein sodium-FA as an example to illustrate the application effect.

1. The competition experiment of absorption of Fluorescein sodium-FA and stearic acid C18:0 in different intestinal cancer cell lines, the steps are as follows:

(1) Different intestinal cancer cell lines HT29, SW480, HCT116 and LS174T grown in the logarithmic growth phase of the six-well plate were selected, and the flow point tube was marked for 30 min at the time point, and each group had 3 duplicate wells;

(2) Premixed with HBSS buffer and 0.1% fatty acid-free calf albumin (BSA) solution to prepare Fluorescein sodium-FA at a final concentration of 1 uM, and 0.15 uM, 1.5 uM, 15 uM in 0.1% BSA solution, respectively. a stearic acid mixture, the above two liquids are mixed in equal volume;

(3) Add 1 ml of the above mixture to different intestinal cancer cell culture plates, incubate for 30 min in a 37-degree incubator; add 30 ml of pre-cooled PBS to the inside after 30 min, and quickly insert into the ice box;

(4) Digested cell strain, resuspended with HBSS, respectively, the 4 degree centrifuge should be pre-cooled to 4 degrees, then centrifuged, 2000 rpm / separation of the heart for 5 minutes; after centrifugation, the upper cell cytometry to detect the absorption of fluorescein strength.

The experimental results are shown in Figure 1. The results show that stearic acid C18:0 can inhibit the absorption of Fluorescein sodium-FA well. Both have a common long-chain fatty acid transporter, indicating that Fluorescein sodium-FA is similar to natural fatty acids. Things.

2. Absorption experiments of Fluorescein sodium-FA in normal mouse colonic mucosa and death for 30 min, the steps are as follows:

(1) The mice were fasted for 1 day to ensure the cleansing of the intestine of the mice. The anesthetized Balb/C mice were intraperitoneally injected with pentobarbital 0.01 g/ml at a dose of 6-7 ul/g (the dead mice were treated for 30 minutes after the neck was broken). Open the intestine directly);

(2) After successful anesthesia in the mice, open the abdominal cavity of the mouse, find the ileocecal valve, and intermittently separate 3 colons of about 1 cm in length, and ligature the ends of the separated intestines with surgical sutures. The degree of ligation cannot be passed by fluorescent dyes. The ligature is not affected at the same time. 100 ul of 200 uM Fluorescein sodium-FA mixture was pipetted with a 1 ml syringe and injected into the ligated intestine. The stain of the control group was 0.02% acridine yellow and 500 uM of dye NBD-labeled deoxyglucose 2-NBDG;

(3) After incubating for 10 min at room temperature, remove the sutures on both sides, cut the intestinal wall and rinse the infected site with PBS twice for 3 min each time. Then the endoscopic surgeon observed the colon with laser confocal microendoscopy. Confocal imaging images to determine the tumor or normal site, using a 29g syringe that sucked Indian ink to accurately locate the examination site that was judged by the endoscopist, marking the part number;

(4) Specimens found by endoscopic confocal endoscopy and labeled, frozen sections, observed under a fluorescence microscope and observed under a microscope after HE staining, and the resulting HE stained slides were diagnosed by an experienced pathologist. The results of "gold standard" were judged to be normal and tumor tissues; the results of HE staining and laser confocal microscopy and fluorescence microscopy were used to determine the absorption of Fluorescein sodium-FA by colon cancer cells.

The experimental results are shown in Figures 2, 3 and 5. Figure 2 shows the normal mouse colon. The fluorescence intensity is high due to the absorption of a large amount of Fluorescein sodium-FA. Figure 3 shows the colon of dead mice, which does not absorb Fluorescein sodium- FA, the fluorescence intensity is extremely weak. It is indicated that Fluorescein sodium-FA is actively transported into cells and can only be imaged in active tissue cells. Figure 5: Under confocal microendoscopy, Fluorescein sodium-FA was absorbed into the cytoplasm by the normal colonic mucosa, clearly showing the outline of the nucleus and gland. The control stain 2-NBDG was hardly absorbed by the normal mucosa. The control stain acridine yellow can enter the cytoplasm and nucleus, and the nuclear boundary is confused, which interferes with cell morphology.

3, mouse chemically induced colon cancer absorption of Fluorescein sodium-FA, the steps are as follows:

(1) Male Balb/C mice were selected for 6-8 weeks. After the mice were acclimated to the environment, the first day of intraperitoneal injection of AOM12.5mg/kg, one week rest, fed with 3% DSS (dextran sulfate sodium) The aqueous solution was 7 days (normal food feeding) and then rested for 14 days, thus repeating 3 cycles;

(2) Pentobarbital 0.01g / ml intraperitoneal injection of Balb / C mice dose of 6-7ul / g, about 5min, open the mouse abdominal cavity, find the distal colon near the rectum, gently separated with tweezers The 1cm long intestine segment ensures that there is no content in the intestine, and then the separated intestine is closed with two surgical sutures. It is suitable for puncture. The puncture of the colon serosa is performed with a 1ml syringe, and 100ul of 200uM Fluorescein sodium is injected into the closed intestine. -FA, after 10min, loosen the sutures at both ends, cut the intestines, rinse the residual fluorescein with PBS, observe under the laser confocal endoscope, and accurately locate the examination site judged by the endoscopist. number;

(3) Specimens that were found by endoscopic confocal endoscopy and labeled, frozen sections, observed under a fluorescence microscope and observed under a microscope after HE staining, and the resulting HE stained slides were diagnosed by an experienced pathologist. To obtain the "gold standard" results to judge normal and tumor tissue; comprehensive HE staining results and laser confocal microscopy endoscopy and fluorescence microscopy results to determine colon cancer tumor cells against Fluorescein Absorption of sodium-FA.

The results are shown in Figures 4 and 6. Figure 4 shows that under fluorescence microscopy, Fluorescein sodium-FA has significant differences in the absorption of normal and adenocarcinoma in the same induced cancer mice. Fluorescence microscopy absorbs more fluorescent substances in the normal part, and the fluorescence intensity is large, while the refractive area of the adenocarcinoma site absorbs very little Fluorescein sodium-FA, and the fluorescence intensity is very weak. As shown in Figure 6, the fluorescence intensity of Fluorescein sodium-FA in normal, low-grade intraepithelial neoplasia and adenocarcinoma was gradually reduced in confocal microendoscopy.

4, human colon adenoma - colon cancer in vitro specimens of Fluorescein sodium-FA absorption experiments, the steps are as follows:

(1) After the patient's ESD, immediately take a colon adenoma or colon cancer specimen into a six-well plate wrapped in foil paper, and dilute 200uM Fluorescein sodium-FA until the specimen is completely covered and placed in a 37-degree thermostat;

(2) After staining for 10 minutes, the scorpion was pulled out of the tissue, washed twice with PBS, and the surface residual water was wiped dry with a cotton swab, and observed under confocal endoscopy;

(3) Specimens that were found by endoscopic confocal endoscopy and labeled, frozen sections, observed under a fluorescence microscope and observed under a microscope after HE staining, and the resulting HE stained slides were diagnosed by an experienced pathologist. The results of "gold standard" were judged to be normal and tumor tissues; the results of HE staining and laser confocal microscopy and fluorescence microscopy were used to determine the absorption of Fluorescein sodium-FA by colon cancer cells.

The experimental results are shown in Fig. 7. Under confocal microendoscopy, the absorption intensity of Fluorescein sodium-FA in normal human colon, adenoma, and adenocarcinoma tissues gradually decreased.

Example 2 The fluorescent conjugated compound Fluorescein sodium-FA prepared in Example 1 was prepared into an oral powder and administered orally to the human body. After oral administration, the fluorescent conjugated compound was actively transported into human cells and accumulated in the cells. Not being metabolized.

After 10 minutes of oral administration, the fluorescence endoscopy was used to detect the absorption fluorescence of normal cells and tumor cells by fluorescein-labeled free fatty acids. The absorption fluorescence intensity of normal tissue was 3.4 times that of tumor tissue. In the obtained fluorescence imaging image, the dark region form shows the range of tumor growth and infiltration, and the tumor cells and the normal cells are distinguished, thereby finding precancerous cells or cancer cells.

Example 3 The staining agent prepared by the fluorescent conjugated compound Fluorescein sodium-FA prepared in Example 1 was directly sprayed through the site to be observed (in colorectal cancer), and under the confocal endoscope, Fluorescein sodium-FA was sprayed 1 After a minute, the cell alignment and atypia can be displayed. Compared with the traditional sodium fluorescein, intravenous injection into the human body, direct injection of Fluorescein sodium-FA can significantly reduce the patient's use. The side effects, while direct spray effect, quickly accelerated the work efficiency of endoscopists, suitable for intense, busy clinical work, previously studied for clinical direct spray dyed acridine yellow, because its carcinogenicity has been banned by the FDA. Direct injection of Fluorescein sodium-FA can specifically display the tumor site through a "dark zone" (absorbing less fatty acids), and simultaneously enter the cell by means of confocal endoscopy and its active transport, visually reflecting the type of lesion at the cell level, Degree and staging, equivalent to "in vivo immunohistochemistry", and because fatty acid absorption decreases in the early stage of colorectal cancer, it can be used for early colorectal cancer screening, and for precancerous lesions and inflammation-related tumor detection Importantly, there is no dye in the field of clinical application and scientific research at home and abroad to achieve this effect, and other epithelial tumors such as gastric cancer, cervical cancer, bladder cancer, etc. are also applicable.

Example 4

(1) Under the protection of nitrogen, the molar ratio of sodium fluorescein to mercaptopropionic acid is 1:0.1, 0.1 mol of sodium fluorescein and 0.1 mol of mercaptopropionic acid are stirred at 45 ° C until a solution is formed, and fluorescence is added to the solution.摩尔(IV) tetrahydrofuran complex (1:2) with a molar mass of 0.2‰(0.02mmol), 18ml of toluene, connected to the water separator, and refluxed at 125°C for 18h, the reaction is finished, minus Press distillation to remove toluene to obtain an intermediate product;

(2) Under nitrogen protection, 0.1 mol of oleic acid was sequentially added to the dried flask at a molar ratio of octadecenoic acid, an intermediate product, and an azobisisobutyronitrile mixture: 1:4:0.4. 0.4 mol of intermediate product, 0.04 mol of azobisisobutyronitrile, and 120 ml of toluene were reacted at 75 ° C for 50 h. At the end of the reaction, the product was precipitated in an aqueous ethanol solution, centrifuged, and dried under vacuum at 50 ° C to obtain sodium fluorescein. Labeled oleic acid Fuorescein sodium-FA.

Example 5

(1) Under the protection of nitrogen, the molar ratio of sodium fluorescein to mercaptopropionic acid is 1:0.15, 0.1 mol of sodium fluorescein and 0.15 mol of mercaptopropionic acid are stirred at 45 ° C until a solution is formed, and fluorescence is added to the solution. Sodium hydride (IV) tetrahydrofuran complex (1:2) with a molar mass of 0.2 ‰ (0.02 mmol), 18 ml of toluene, connected to a water separator, and refluxed at 135 ° C for 15 h, the reaction is finished, minus Press distillation to remove toluene to obtain an intermediate product;

(2) Under the protection of nitrogen, 0.1 mol of oleic acid was sequentially added to the dried flask at a molar ratio of octadecenoic acid, an intermediate product, and an azobisisobutyronitrile mixture: 1:6:0.2. 0.6 mol of intermediate product, 0.02 mol of azobisisobutyronitrile, and 180 ml of toluene, reacted at 85 ° C for 50 h, and the product was precipitated in an aqueous ethanol solution after centrifugation, and dried under vacuum at 50 ° C to obtain sodium fluorescein. Labeled oleic acid Fuorescein sodium-FA.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than the present invention. The invention is described in detail with reference to the preferred embodiments, and those skilled in the art should understand that the invention may be modified or substituted without departing from the spirit of the invention. And scope.

Claims (9)

A fluorescent conjugated compound, characterized in that the fluorescent conjugated compound is composed of a fluorescent molecule labeled free fatty acid, and is used for detecting precancerous lesion cells or cancer cells; the fluorescent molecule is selected from the group consisting of sodium fluorescein, acridine yellow, and rodin Any one of a bright, fluoroboron fluorescent dye, a fluorescent dye NBD, a dansyl, a coumarin dye, a cyanine dye; the free fatty acid is a long-chain saturated or monounsaturated fatty acid; and the label refers to a chemical reaction The fluorescent molecule and the free fatty acid are linked by 2 to 30 linking groups consisting of a linear, cyclic, benzene ring or a combination thereof of any one or more of C, N, O, P, S, Si; The fluorescent molecule has an emission wavelength of 400 nm to 1200 nm. The fluorescent conjugated compound according to claim 1, wherein the fluorescent conjugated compound is composed of sodium fluorescein-labeled octadecanoic acid, and sodium fluorescein is passed through a CO bond at the carbon 9 position of oleic acid. The oleic acid linkage is prepared as follows: (1) Under the protection of nitrogen, the molar ratio of sodium fluorescein to mercaptopropionic acid is 1:0.1-0.15, stirred at 45-55 ° C until a solution is formed, and ruthenium chloride with a molar mass of 0.2 fluorescein sodium is added to the solution. (IV) tetrahydrofuran complex (1:2), toluene, then refluxed at 125-135 ° C for 15-18 h, the reaction is completed, distillation under reduced pressure, toluene is removed to obtain an intermediate product; under nitrogen protection, drying The flask was sequentially charged with oleic acid, an intermediate product, azobisisobutyronitrile, and toluene, and reacted at 75 to 85 ° C for 45 to 50 hours. At the end of the reaction, the product was precipitated in an aqueous ethanol solution, centrifuged, and vacuum dried at 50 ° C. That is, sodium oleic acid labeled with sodium fluorescein is prepared; the molar ratio of the mixture of octadecenoic acid, intermediate product and azobisisobutyronitrile is 1:4-6:0.2-0.5. The fluorescent conjugated compound according to claim 2, wherein the amount of toluene in the step (1) is from 180 to 200 ml per 1 mol of sodium fluorescein in the reaction system. The fluorescent conjugated compound according to claim 2, wherein the amount of toluene in the step (2) is from 250 to 300 ml per 1 mol of sodium fluorescein-SH in the reaction system. The fluorescent conjugated compound according to claim 1 or 2, wherein the fluorescent conjugated compound is prepared as a drug, a stain or a tracer for detecting a human tumor and a precancerous disease. The fluorescent conjugated compound according to claim 5, wherein the human tumor comprises colon cancer, lung cancer, breast cancer, prostate cancer, cervical cancer, esophageal cancer, bladder cancer, melanoma, leukemia, lymphoma. Malignant tumors; the precancerous diseases include Barrett's esophagus, adenomatous polyps, and inflammatory bowel disease. The fluorescent conjugated compound according to claim 5, wherein the drug comprises a medically approved tablet, capsule, pill, powder, ointment, suppository or dissolving the fluorescent conjugated compound in water-soluble propylene glycol or A sterile solution or suspension is formed in the sodium chloride solution. The fluorescent conjugated compound according to claim 5, wherein the fluorescent conjugated compound is contained in a drug for detecting a human tumor and a precancerous disease in an amount of 0.05 to 0.1%. The fluorescent conjugated compound according to claim 1, wherein the fluorescent conjugated compound absorbs the fluorescence intensity of the normal tissue of the living body by 1.5 to 9.8 times the fluorescence intensity of the tumor-absorbing tissue.

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