Biotinylated gefitinib probe as well as preparation method and application thereof

Abstract

The invention discloses a biotinylated gefitinib probe as well as a preparation method and application thereof, the biotinylated gefitinib probe is better in stability and high in sensitivity and accuracy, can be stored for a long time, and can be used in experiments needing long-time storage and multiple analysis; the kit can also be used for detecting the binding condition of gefitinib and EGFR under various conditions so as to treat or diagnose cancers; the EGFR mutation type is determined in combination with gene sequencing, the feasibility and effectiveness of the gefitinib for treating the EGFR mutation type can be evaluated, and medication is guided. Therefore, the biotinylated gefitinib probe is expected to become an indispensable tool in precision medical treatment, and more patients are benefited from gefitinib treatment.

Description

Technical Field

[0001] The present invention relates to the field of cancer diagnosis and treatment in the field of biotechnology, and in particular to a biotinylated gefitinib probe and a preparation method and application thereof. Background Art

[0002] Epidermal growth factor receptor (EGFR) plays a key role in cell proliferation, differentiation, migration and survival. EGFR is overexpressed or mutated on the surface of many tumor cells, resulting in abnormally increased tyrosine kinase activity and continuous activation of downstream signaling pathways, such as RAS-RAF-MEK-ERK and PI3K-AKT-mTOR, promoting the growth and survival of tumor cells. Gefitinib is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that can competitively bind to the ATP binding site of EGFR, thereby inhibiting the autophosphorylation of EGFR tyrosine kinase, blocking the activation of downstream signaling pathways, and playing a variety of anti-tumor effects such as inhibiting tumor cell proliferation, inducing cell apoptosis, inhibiting angiogenesis and invasion and metastasis. In addition, gefitinib can also be used in combination with other treatments, such as in combination with chemotherapy drugs to synergistically inhibit tumor growth through different mechanisms of action; or in combination with radiotherapy to enhance the killing effect of radiotherapy on tumor cells.

[0003] Gefitinib is mainly used to treat advanced or metastatic non-small cell lung cancer with EGFR mutations. Gefitinib shows good therapeutic effects on patients with non-small cell lung cancer carrying EGFR sensitive mutations (such as exon 19 deletion mutation, exon 21 L858R point mutation, etc.). Clinically, the standard of gefitinib use is mainly based on the patient's EGFR gene test results. EGFR gene testing cannot quantitatively detect the level of EGFR tyrosine kinase inhibition, so it is impossible to accurately evaluate the effectiveness of gefitinib drug treatment in patients with non-small cell lung cancer. The design of gefitinib probes can accurately detect the binding of probes to tumor cell EGFR in vitro, infer the level of EGFR tyrosine kinase inhibition, and then predict the feasibility and effectiveness of gefitinib treatment in patients with non-small cell lung cancer. At present, patent CN104212440A discloses a class of quinazoline fluorescent probes and their preparation and application, specifically connecting the fluorescent chromophore to gefitinib to prepare a fluorescent probe. Due to the targeting effect of gefitinib, the fluorescent probe can be used to explore the binding of the probe to EGFR, and is applied to the relative quantitative detection of the biomarker EGFR in cells, providing more direct information for cancer diagnosis. At the same time, the probe can effectively inhibit the activity of EGFR tyrosine kinase and has cancer treatment function. However, since the optimal luminescence and preservation pH of fluorescein are usually between 7.0 and 9.0, tumor tissues are often acidic due to the presence of the Warburg effect, that is, tumor cells still mainly rely on glycolysis to obtain energy under aerobic conditions, produce a large amount of lactic acid, and cause the pH value to drop, which is not conducive to the use of fluorescein labeled probes in actual detection; and when the probe is detected using excitation light, since the probe itself contains a fluorescent marker, it will cause background interference during the detection process, resulting in inaccuracy of the detection results. Therefore, it is of great significance to provide a gefitinib probe with higher stability, sensitivity and accuracy, and suitable for detection in a variety of situations. Summary of the invention

[0004] The purpose of the present invention is to overcome the above defects and shortcomings in the prior art and provide a biotinylated gefitinib probe.

[0005] The second object of the present invention is to provide a method for preparing the above-mentioned biotinylated gefitinib probe.

[0006] The third object of the present invention is to provide the use of the above probe in preparing products for cancer treatment or diagnosis.

[0007] The fourth object of the present invention is to provide the use of the above probe in the preparation of a product for guiding cancer patients to take gefitinib.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a biotinylated gefitinib probe, the structural formula of which is shown in formula (I):

[0010]

[0011] Gefitinib is mainly used for the treatment of advanced or metastatic non-small cell lung cancer with EGFR mutations. Gefitinib has shown good therapeutic effects on patients with non-small cell lung cancer carrying EGFR sensitive mutations (such as exon 19 deletion mutation, exon 21 L858R point mutation, etc.). Biotin has a strong specificity and high affinity interaction with avidin. When the biotin-labeled gefitinib probe is labeled with fluorescently labeled streptavidin, even if the mutation type that can bind to gefitinib is low, it can be amplified through the streptavidin-biotin reaction, increasing the sensitivity of the detection. The biotinylated gefitinib probe itself does not contain fluorescence, and will not cause background interference during the detection process, with higher sensitivity and accuracy. At the same time, streptavidin can be combined with a variety of markers (such as enzymes, fluorescent dyes, etc.), not only suitable for various types of experiments (such as immunohistochemistry, Western blot, ELISA, etc.) but also suitable for various detection systems (such as HRP, AP and fluorescence detection systems, etc.), to determine whether the gefitinib target position and EGFR are co-localized, and improve the detection accuracy. Therefore, the biotinylated gefitinib probe can be used in experiments that require long-term storage and multiple analyses because of its better stability, high sensitivity and accuracy, and long-term storage; it can be used to detect the binding of gefitinib to EGFR in various situations and diagnose cancer; combined with gene sequencing to determine the type of EGFR mutation, it can also evaluate the feasibility and effectiveness of gefitinib treatment of the EGFR mutation type and guide medication.

[0012] The present invention also provides a method for preparing the above-mentioned biotinylated gefitinib probe, comprising the following steps:

[0013] S1. subjecting gefitinib to methoxy demethylation reaction to obtain a raw material; the raw material is

[0014]

[0015] S2. The raw material and biotin are subjected to an esterification reaction to obtain the biotinylated gefitinib probe represented by formula (I).

[0016] The present invention firstly performs a methoxy demethylation reaction on gefitinib to convert the methoxy group (-OCH3) on the benzene ring into a hydroxyl group (-OH), and then performs an esterification reaction on the reaction product and biotin to prepare a biotinylated gefitinib probe.

[0017] Furthermore, the methoxy demethylation reaction in step S1 is carried out in an organic solvent using aluminum chloride as a demethylation reagent, and the pH is adjusted to be alkaline to obtain the product through purification.

[0018] Furthermore, the organic solvent is dichloroethane, acetonitrile or dichloromethane.

[0019] Furthermore, the reaction temperature is 58-62°C.

[0020] Furthermore, the purification is dichloromethane / methanol column purification.

[0021] Furthermore, in step S2, the mass ratio of the raw material to biotin is 40-45:28-30.

[0022] Preferably, in step S2, the mass ratio of the raw material to biotin is 432:293.

[0023] Furthermore, the esterification reaction in step S2 is carried out by reacting carbodiimide with 1-hydroxybenzotriazole in an organic solvent.

[0024] Furthermore, the organic solvent is N,N-dimethylformamide (DMF).

[0025] Clinically, the standard of gefitinib use is mainly based on the patient's EGFR gene test results. Current EGFR gene testing focuses on known exon 19 deletion mutations and exon 21 L858R point mutations. However, there are many other rare mutation types in the EGFR gene, and the sensitivity of these rare mutations to gefitinib cannot be effectively evaluated through genetic testing, resulting in the inability to fully evaluate the therapeutic effectiveness of gefitinib. In actual treatment, there are still patients with other EGFR mutations (non-exon 19 deletion mutations or exon 21 L858R point mutations) who can benefit from gefitinib treatment. There are differences in the effectiveness of gefitinib treatment in different patients. The fundamental reason is the difference in the ability of gefitinib to bind to the targeted EGFR. Therefore, the binding ability of gefitinib and EGFR is the key to evaluating the effectiveness of treatment. Biotinylated gefitinib probes can be used to detect the activity state of EGFR at the protein level while performing gene sequencing to determine the type of EGFR mutation, comprehensively judge whether the patient is suitable for gefitinib treatment and predict the treatment effect, and provide patients with more accurate treatment plans. Therefore, biotinylated gefitinib probes are expected to become an indispensable tool in precision medicine, allowing more patients to benefit from gefitinib treatment.

[0026] Therefore, the present invention provides the use of the above-mentioned biotinylated gefitinib probe in preparing a product for cancer treatment or diagnosis.

[0027] The present invention also provides the use of the biotinylated gefitinib probe in preparing a product for guiding cancer patients to take gefitinib.

[0028] Furthermore, the product is used to determine whether a patient should take gefitinib by detecting the ability of the probe to target the epidermal growth factor receptor.

[0029] Furthermore, the cancer is a cancer caused by overexpression or mutation of epidermal growth factor receptor.

[0030] Preferably, the cancer is non-small cell lung cancer.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a biotinylated gefitinib probe and a preparation method and application thereof. The probe of the present invention can be used in experiments requiring long-term storage and multiple analyses because of its better stability, high sensitivity and accuracy, and ability to be stored for a long time. It can be used to detect the binding of gefitinib to EGFR in various situations, diagnose cancer, determine the EGFR mutation type in combination with gene sequencing, and also evaluate the feasibility and effectiveness of gefitinib treatment of the EGFR mutation type, and guide medication. Therefore, the biotinylated gefitinib probe is expected to become an indispensable tool in precision medicine, allowing more patients to benefit from gefitinib treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Mass spectra of biotinylated gefitinib probe.

[0034] Figure 2 H spectrum of biotinylated gefitinib probe.

[0035] Figure 3 To detect the binding ability of biotinylated gefitinib probe to EGFR. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0038] Example 1 Preparation of biotinylated gefitinib probe

[0039] The preparation protocol of biotinylated gefitinib probe is as follows:

[0040] (1) 500 mg of gefitinib and 10 ml of dichloromethane (DCM) were added to a 100 ml single-necked bottle at room temperature, and 1.2 g of aluminum chloride was added. The mixture was placed at 60° C. overnight, quenched with water, and sodium carbonate was added to adjust the base. The mixture was filtered, and the filter cake was mixed with silica gel and purified by column chromatography. The mixture was filtered with dichloromethane / methanol to obtain 420 mg of the raw material product.

[0041]

[0042] (2) 432 mg of the raw material product, 293 mg of biotin, 228 mg of EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), and 156 mg of 1-hydroxybenzotriazole (HOBT) were added to 10 ml of dimethylformamide (DMF), reacted at room temperature overnight, and dried by spinning to obtain 480 mg of biotinylated gefitinib probe.

[0043]

[0044] The mass spectrometry results of the biotinylated gefitinib probe are shown in Figure 1 As shown; H spectrum detection results are as follows Figure 2 As shown, it was shown that the biotinylated gefitinib probe was successfully synthesized.

[0045] Example 2 Detection of the binding ability of biotinylated gefitinib probe to EGFR

[0046] 1. Experimental Methods

[0047] This detection scheme uses immunofluorescence method, and about 10 5 EGFR exon 19 deletion mutation and EGFR wild-type cells were co-incubated with biotinylated gefitinib probe overnight after the cells adhered to the wall, and washed three times with PBS on the next day; fixed with 4% paraformaldehyde for 30 minutes, washed with PBS three times, 5 minutes each time, and blocked with 5% BSA for 30 minutes; diluted EGFR primary antibody and biotin primary antibody (sc-53179, Santa Cruz Biotechnology) were added in sequence, and incubated at 4°C overnight; after overnight, washed with PBS three times, 5 minutes each time, diluted FITC-conjugated secondary antibody and Texas Red fluorescent secondary antibody were added in sequence, incubated at 37°C for 1 hour, washed with PBS three times, 5 minutes each time; DAPI (4',6-diamidino-2-phenylindole, blue fluorescent dye) was added dropwise for incubation for 5 minutes, and the slides were sealed with sealing solution containing anti-fluorescence quencher, and then the images were observed and collected under a fluorescence microscope.

[0048] The results are as follows Figure 3As shown, in cells with EGFR exon 19 deletion mutation, EGFR and biotinylated gefitinib probe co-localized, indicating that EGFR and biotinylated gefitinib probe were closely bound together, while in EGFR wild-type cells, EGFR and biotinylated gefitinib probe were not observed, indicating that gefitinib is effective in treating EGFR exon 19 deletion mutation cells.

[0049] Example 3 Detection of the binding ability of biotinylated gefitinib probe to different EGFR mutation types

[0050] 1. Experimental Methods

[0051] This detection scheme uses immunofluorescence method, and 10 5 EGFR exon 19 deletion mutation, EGFR L858R combined with T790M mutation and EGFR wild-type cells were co-incubated with biotinylated gefitinib probe overnight after the cells adhered to the wall, and washed three times with PBS on the next day; fixed with 4% paraformaldehyde for 30 minutes, washed with PBS three times, 5 minutes each time, and blocked with 5% BSA for 30 minutes; diluted EGFR primary antibody and biotin primary antibody were added in sequence, and incubated at 4°C overnight; after overnight, washed with PBS three times, 5 minutes each time, diluted FITC-conjugated secondary antibody and Texas Red fluorescent secondary antibody were added in sequence, incubated at 37°C for 1 hour, washed with PBS three times, 5 minutes each time; DAPI (4',6-diamidino-2-phenylindole, blue fluorescent dye) was added dropwise for incubation for 5 minutes, and the slides were sealed with sealing solution containing anti-fluorescence quencher, and then the images were observed and collected under a fluorescence microscope.

[0052] The results are as follows Figure 3 As shown in the figure, in cells with EGFR exon 19 deletion mutation, EGFR and biotinylated gefitinib probe co-localize; in T790M mutation cells with gefitinib resistance, the binding between EGFR and biotinylated gefitinib probe is weakened; in EGFR wild-type cells, no binding between EGFR and biotinylated gefitinib probe is observed. This indicates that gefitinib is effective in treating cells with EGFR exon 19 deletion mutation, and EGFR and biotinylated gefitinib probe are tightly bound together; while the therapeutic effect of gefitinib on cells with EGFR L858R and T790M mutation is significantly reduced.

[0053] This experiment verifies the ability of the biotinylated gefitinib probe provided by the present invention to bind to EGFR. The biotinylated gefitinib probe provided by the present invention can distinguish the targeting ability of gefitinib to different EGFR mutations, thereby evaluating the feasibility and effectiveness of gefitinib in treating different EGFR mutations and guiding patients' medication. The probe is expected to become an indispensable tool in precision medicine, allowing more patients to benefit from gefitinib treatment.

Claims

1. A biotinylated gefitinib probe, characterized in that: Its structural formula is shown in formula (I).

2. The method for preparing the biotinylated gefitinib probe according to claim 1, characterized in that: The following steps are involved: S1. subjecting gefitinib to methoxy demethylation reaction to obtain a raw material; the raw material is S2. The raw material and biotin are subjected to an esterification reaction to obtain the biotinylated gefitinib probe represented by formula (I).

3. The preparation method according to claim 2, characterized in that: The methoxy demethylation reaction in step S1 is carried out in an organic solvent using aluminum chloride as a demethylation reagent, and the pH is adjusted to be alkaline to obtain the product through purification.

4. The preparation method according to claim 2, characterized in that: The mass ratio of the raw material to biotin in step S2 is 40-45:28-30.

5. The preparation method according to claim 2, characterized in that: The esterification reaction in step S2 is carried out by reacting carbodiimide with 1-hydroxybenzotriazole in an organic solvent.

6. Use of the biotinylated gefitinib probe according to claim 1 in the preparation of products for cancer treatment or diagnosis.

7. Use of the biotinylated gefitinib probe according to claim 1 in preparing a product for guiding cancer patients to take gefitinib.

8. The use according to claim 7, characterized in that: The product is used to determine the use of gefitinib in patients by detecting the ability of the probe to target the epidermal growth factor receptor.

9. The use according to any one of claims 6 or 7, characterized in that: The cancer is a cancer caused by overexpression or mutation of epidermal growth factor receptor.

10. The use according to claim 9, characterized in that: The cancer is non-small cell lung cancer.

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