CN110041400B

CN110041400B - Polypeptide and application thereof in early diagnosis of colorectal cancer

Info

Publication number: CN110041400B
Application number: CN201810026559.XA
Authority: CN
Inventors: 王琰; 陈晓光; 杨潇骁; 曹之宪; 李燕; 温宝莹; 冯茹; 张森; 黄敏
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2018-01-15
Filing date: 2018-01-15
Publication date: 2022-07-29
Anticipated expiration: 2038-01-15
Also published as: CN110041400A

Abstract

The invention discloses a polypeptide and application thereof in early diagnosis of colorectal cancer, and particularly relates to a novel polypeptide compound specifically combined with colorectal cancer, pharmaceutically acceptable salts thereof and a preparation method thereof. The invention relates to a polypeptide compound and pharmaceutically acceptable salts thereof. The kit has good targeting and detection sensitivity for the colorectal cancer, has the characteristic of fast elimination of in vivo metabolism, is expected to be applied to diagnosis of the colorectal cancer, improves the accuracy of early diagnosis of the colorectal cancer, and reduces the incidence of the colorectal cancer.

Description

Polypeptide and application thereof in early diagnosis of colorectal cancer

Technical Field

The invention relates to a novel polypeptide compound capable of being specifically combined with colorectal cancer, pharmaceutically acceptable salts thereof and a preparation method thereof.

Background

Worldwide, Colorectal cancer (CRC) is a serious health-threatening disease in humans, and is long-standing in the third place of common malignancies; in our country, the incidence of colorectal cancer has also been on the rise in recent years. The fact that the national cancer center released the current cancer data of residents in China for the first time in 2015 shows that colorectal cancer is the second most likely among cancer patients diagnosed with cancer and still alive within 5 years. The main reasons are as follows:

(1) The lack of effective early diagnostic measures and post-diagnosis treatments has led to high morbidity and mortality rates (Cheng hong, 2008; Liu Cheng Ying, 2012; Deschoolmester V, 2010; Rongshou Zheng, 2016). Therefore, the internationally accepted principle is that the primary focus of the tiny tumor can be found as early as possible, which becomes the key for treating the tumor, and the five-year survival rate of the patient after the operation can be greatly increased.

(2) Tumor metastasis is also a leading cause of death in cancer patients. The micrometastases of lymph nodes and distant metastases of primary foci are difficult to detect imagewise and lack good diagnostic methods.

In recent years, new colorectal cancer diagnosis technologies emerge endlessly, such as discovery and application of various novel tumor markers, application of various endoscopes, application of ultrasonic waves, X-ray, CT and MRI, and the like. Although these new techniques and means greatly help the early detection of tumors, they still suffer from insufficient specificity and sensitivity, and their early prediction ability is limited.

Tumor angiogenesis is taken as a target, and the design of a high-specificity molecular probe or a targeted drug becomes one of the important directions of tumor diagnosis and treatment research. In recent decades, the application of polypeptide drugs in tumor diagnosis and treatment has attracted high importance to the scientific community (Kerbel RS, 2006; Trejtnar F, 2008; Jemal a, 2010; Fani M, 2012).

There are two main types of molecular targeted therapies: monoclonal antibodies and targeting polypeptides (Homing peptides). Targeting polypeptide (Homing peptide) is a carrier which is considered to be ideal at present for tumor diagnosis and targeting therapy, and has the following characteristics: the plasma is cleared quickly, and the affinity and specificity to tumor tissues are high; good tissue penetrability and can be taken up by tumor cells; and thirdly, the monoclonal antibody is easy to chemically synthesize and has low immunogenicity, so that the defects of the monoclonal antibody can be reduced or avoided.

The compound of the formula I is a novel polypeptide which can be combined with early colorectal cancer targeting. Fluorescein FITC labeled compound 1A is injected into Balb/c in-situ colorectal cancer tumor-bearing mice through tail veins, after 1 hour, the injection is fully carried out, tissues in the bodies are taken out, frozen sections are made and are directly used for observation, and the other tissues except tumor tissues have no obvious green fluorescence. In vitro immunofluorescent staining also showed that compound 1A co-localized with CD31 in both mouse and human colorectal tumor tissues, and no fluorescence was seen in normal colorectal tissues.

The invention relates to a novel polypeptide compound specifically combined with colorectal cancer, pharmaceutically acceptable salt thereof and a preparation method thereof, and the novel polypeptide has good targeting property and detection sensitivity on the colorectal cancer.

Disclosure of Invention

The invention aims to provide a polypeptide compound shown in a formula I, pharmaceutically acceptable salts thereof, a pharmaceutical composition and application thereof.

In order to solve the technical problem, the invention provides the following technical scheme:

the first aspect of the technical scheme of the invention provides polypeptide compounds shown in formula I and pharmaceutically acceptable salts thereof, which are characterized in that the general chemical structures of the polypeptide compounds are shown in formula I:

wherein the content of the first and second substances,

x is fluorine or a radioisotope thereof ¹⁸ F。

A compound according to any one of the first aspect of the invention, said compound having the structure:

in a second aspect of the present invention there is provided a diagnostic reagent composition comprising a diagnostically effective amount of a polypeptide compound according to any one of the first aspect of the present invention and pharmaceutically acceptable salts thereof, and optionally one or more pharmaceutically acceptable carriers or excipients.

For administration purposes, the diagnostic reagent compositions of the present invention may be administered by any known method of administration.

The dosage of the polypeptide compound and the pharmaceutically acceptable salt thereof of the present invention to be administered may vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration and the dosage form, and the like. Generally, the suitable dosage of the polypeptide compounds and pharmaceutically acceptable salts thereof of the present invention is intravenous administration in the range of 0.001-2.5mg/Kg body weight, preferably 0.001-2mg/Kg body weight, more preferably 0.001-1.5mg/Kg body weight, and most preferably 0.001-1mg/Kg body weight; the above-described dosage may be administered in one dosage unit or divided into several dosage units, depending on the clinical experience of the physician and the dosage regimen including the use of other therapeutic means.

The diagnostic reagent composition of the present invention can be used alone or in combination with other therapeutic agents or symptomatic agents. When the diagnostic reagent composition of the present invention is synergistically effective with other therapeutic agents, its dosage should be adjusted according to the actual situation.

In a third aspect of the present invention, there is provided a polypeptide compound of the first aspect of the present invention and a pharmaceutically acceptable salt thereof, or a diagnostic reagent composition of the second aspect of the present invention for use in early diagnosis and targeted therapy of colorectal cancer.

Advantageous technical effects

All the compounds in the invention have novel chemical structures, and the novel polypeptide compound in the formula I disclosed by the invention has the characteristic of specific binding with colorectal tumors, and is expected to be used for early diagnosis of clinical colorectal cancer. The synthetic route of the compound shown in the formula I has the advantages of easily obtained raw materials, simple and convenient operation, mild reaction conditions, short reaction time, solvent saving, pollution reduction and the like.

Drawings

FIG. 1 imaging results of FITC-Compound 1A after injection into tumor mice

FIG. 2 immunofluorescence staining of mouse colorectal tissue with Compound 1A

FIG. 3 immunofluorescence staining of human colorectal tissue for Compound 1A

FIG. 4 internal standard TH-7 formula

FIG. 5 mean plasma concentration-time plot of compound 1A administered intravenously to the tail of mice

(A) Normal mice (n ═ 5); (B) tumor mouse (n ═ 3)

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.

For all of the following examples, standard procedures and purification methods known to those skilled in the art may be used. Unless otherwise indicated, all temperatures are expressed in degrees Celsius. The structure of the compounds is determined by High Resolution Mass Spectrometry (HRMS) and/or nuclear magnetic resonance spectroscopy (NMR).

Preparation examples section

The structure of the compound is shown by nuclear magnetic resonance hydrogen spectrum ( ¹ H-NMR) and Mass Spectrometry (MS). The hydrogen and carbon spectral shifts (δ) for nuclear magnetic resonance are given in parts per million (ppm). NMR Hydrogen spectra were measured using a Mercury-400 NMR spectrometer, deuterated chloroform (CDCl) ₃ ) Or heavy water (D) ₂ O) or deuterated dimethyl sulfoxide (DMSO-d6) or deuterated methanol (CD) ₃ OD) as solvent and Tetramethylsilane (TMS) as internal standard.

Mass spectra were determined using a Thermo active Plus (ESI/Orbitrap) LC MS.

The balance used was an electronic balance of the Sartorius-BSA type Germany.

Perkin-Elmer 343 plus polarimeter;

eye rotary evaporator;

vacuum diaphragm pump;

the anhydrous reagent is a commercially available analytical Pure reagent prepared by removing water through Pure Solv. solvent purification system, and other reagents are commercially available analytical Pure reagents.

When the Chinese or English abbreviation amino acid is used in the specification of the invention, the general amino acid name and English abbreviation form in the field are adopted, if the independent amino acid name and English abbreviation of the amino acid are not explicitly described, the L-type amino acid is represented, for example, serine or serine amide (Ser) represents that the L-type serine or L-type seryl is represented; the corresponding D-form amino acid is added before Chinese character "D-" or English abbreviation "(D)", such as D-serine or D-serine amide.

When the three characters are used for abbreviation of amino acid, the invention adopts the common amino acid name and English abbreviation form in the field, when the right side is '-OH', the amino acid is in the form of free carboxylic acid, and when the left side is 'H-' the amino acid is in the form of free amino, for example, 'H-Ser-OH' indicates that the amino acid is L-serine with free amino and carboxyl.

Forward preparative chromatographic conditions: 300-400 mesh silica gel, the silica gel amount is 50-80 times of the sample amount, and the detection wavelength is 254 nm;

wherein, the abbreviations for the chemical reagents and solvents are given in the following forms,

TFA is trifluoroacetic acid, i.e. trifluoroacetic acid;

DMF is N, N-Dimethylformamide, namely N, N-Dimethylformamide;

THF is tetrahydrofuran, i.e. tetrahydrofuran;

NMP is l-Methyl-2-pyrrolidone, 1-Methyl pyrrolidone;

DCM is dichlormethane, i.e. Dichloromethane;

EtOAc is ethyl acetate;

TEA is triethylamine;

DCC is dicyclohexylcarbodiimide;

MsCl is methanesulfonyl chloride;

DIEA is N, N-diisopropylethylamine

Preparation example

Synthesis of intermediates

Preparation example 1.Synthesis of 4- (2-azidoethyl) -1-fluorobenzene (1)

Dissolving 1.0g of p- (2-bromoethyl) fluorobenzene in 10ml of dry DMSO, adding 0.36g of sodium azide, heating in an argon protective oil bath at 45 ℃ for 5 hours, reacting at room temperature overnight, diluting the reaction solution with 50ml of EtOAc, and adding saturated NaHCO ₃ The aqueous solution and saturated aqueous NaCl solution were washed 3 times, and the organic layer was washed with Na ₂ SO ₄ Drying, filtration and concentration of the filtrate gave 770mg of a yellow clear oil, 93% yield.

And (3) product analysis: ESI-MS calculated value 166.07[ M + H ]] ⁺ Measured value: 166.07. H-NMR nuclear magnetism ¹ H NMR(400MHz,Chloroform-d)δ7.19(dd,J＝8.3,5.4Hz,2H),7.02(t,J＝8.5Hz,2H),3.49(t,J＝7.2Hz,2H),2.87(t,J＝7.2Hz,2H).

Preparation example 2.Synthesis of 4- (2-azidoethyl) -phenol (2)

1.37g 4-aminoethyl-phenol (tyramine) was dissolved in 50ml anhydrous methanol, 2.50g imidazole-1-sulfonyl azide, 2.34g K was added ₂ CO ₃ And 0.25mg copper sulfate pentahydrate, stirring at room temperature under argon atmosphere for 8 hours, adding concentrated hydrochloric acid 50ml, extracting with 50ml EtOAc 3 times, washing with saturated NaCl aqueous solution 3 times, and adding Na to organic layer ₂ SO ₄ Drying, filtering, concentrating the filtrate, and separating by silica gel medium-pressure column chromatography to obtain 1.5g of yellow transparent oily substance with a yield of 92%. TLC Rf 0.8 (petroleum ether: ethyl acetate 1:1) nuclear magnetic resonance ¹ H NMR(400MHz,Chloroform-d)δ7.09(m,2H),6.79(m,2H),3.46(t,J＝7.2Hz,2H),2.82(t,J＝7.2Hz,2H).

Preparation example 3.Synthesis of 4- (2-azidoethyl) phenylmethanesulfonate (3)

1.63g of intermediate 2 synthesized in production example 2 was dissolved in 20ml of anhydrous dichloromethane, 1.5ml of triethylamine was added, 1.27g of methanesulfonyl chloride was added dropwise at 0 ℃ in an ice water bath, the mixture was stirred for 1 hour and then reacted at room temperature for 2 hours, 50ml of dichloromethane was added, the mixture was washed three times with 50ml of a saturated aqueous sodium bicarbonate solution, 3 times with a saturated aqueous salt solution, and the organic layer was dried over anhydrous sodium sulfate and then filtered, followed by concentration to obtain 2.216 (92.2%) as a pale yellow oil. Nuclear magnetic analysis: ¹ H NMR(400MHz,Chloroform-d)δ7.41-7.27(m,2H),7.27-7.19(m,2H),3.53(s,3H),2.60(tt,J＝5.1,1.1Hz,2H),1.70(t,J＝5.1Hz,2H),.

preparation example 4.4- (2-Azidoethyl) -1- ¹⁸ Synthesis of fluorobenzene (4)

0.2g of intermediate 3 from preparation 3 are dissolved in 5ml of anhydrous acetonitrile and the CTI RDS111 accelerator is passed through ¹⁸ O(p,n) ¹⁸ F reaction to produce ¹⁸ F ^- Is pneumatically driven to ¹⁸ F ^- From the target system to the synthesizer, ¹⁸ F ^- captured by QMA anion column. On the anion column QMA ¹⁸ F ^- Eluted by acetonitrile solution of potassium carbonate and cryptate K222 into a reaction tube, and heated acetonitrile is subjected to azeotropic dehydration under the condition of introducing nitrogen. Cooling, adding intermediate 3 dissolved in anhydrous acetonitrile into the residue, heating ¹⁸ F ^- Nucleophilic substitution reaction with intermediate to generate ¹⁸ F is substituted azide ethyl fluorobenzene 4, acetonitrile in the product is evaporated, and the product is subjected to solid phase extraction and desalination by C18 and then is concentrated to obtain ¹⁸ Fluorine labels intermediate 4. TLC radiochemical purity>95 percent and the specific activity is 1.3-5 Ci/mmol.

Preparation example 5.NH ₂ Synthesis of-Cys (Trt) - (2-Cl-Trt Resin) (5)

Taking 2.0g of Resin (2-Cl-Trt Resin, the degree of substitution is 0.7mmol/g), swelling in dichloromethane for 10min, adding 2.46g of Fmoc-Cys (Trt) -OH and 15ml of a DMF solution of 2.92ml of DIEA, shaking for 2 hours at room temperature, washing the Resin with DMF for 3 times and washing with dichloromethane for 2 times.

② adding 20 percent piperidine/DMF solution 15ml into the resin, shaking for 15min at room temperature, repeating for 2 times, washing the resin with DMF for 3 times, washing with dichloromethane for 2 times, and obtaining the target resin intermediate.

Preparation example 6.NH ₂ Synthesis of (6) -His (Trt) -Cys (Trt) - (2-Cl-Trt Resin)

Dissolving 2.6g Fmoc-His (Trt) -OH, 1.6g HBTU, 0.72gCl-HOBt and 1.46ml DIEA in 15ml DMF, adding to the resin, shaking at room temperature for 1.5 h, washing the resin with DMF 3 times and dichloromethane 2 times.

② adding 20 percent piperidine/DMF solution 15ml into the resin, shaking for 15min at room temperature, repeating for 2 times, washing the resin with DMF for 3 times, and washing with dichloromethane for 2 times to obtain the target resin intermediate.

Preparation example 7.NH ₂ Synthesis of (7) -Ser (tBu) -His (Trt) -Cys (Trt) - (2-Cl-Trt Resin)

1.6g of Fmoc-Ser (tBu) -OH, 1.6g of HBTU, 0.72gCl-HOBt and 1.46ml of DIEA were dissolved in 15ml of DMF, added to the resin, shaken at room temperature for 1.5 hours, and the resin was washed with DMF 3 times and dichloromethane 2 times.

Preparation example 8.NH ₂ Synthesis of-Phe-Ser (tBu) -His (Trt) -Cys (Trt) - (2-Cl-Trt Resin) (8)

Firstly, 1.62g of Fmoc-Phe-OH, 1.6g of HBTU, 0.72gCl-HOBt and 1.46ml of DIEA are dissolved in 15ml of DMF, added to the resin, shaken at room temperature for 1.5 hours, and the resin is washed with DMF for 3 times and dichloromethane for 2 times.

Preparation example 9.NH ₂ Synthesis of-Pro-Phe-Ser (tBu) -His (Trt) -Cys (Trt) - (2-Cl-Trt Resin) (9)

1.42g of Fmoc-Pro-OH, 1.6g of HBTU, 0.72gCl-HOBt and 1.46ml of DIEA were dissolved in 15ml of DMF, added to the resin, shaken at room temperature for 1.5 hours, and the resin was washed with DMF 3 times and dichloromethane 2 times.

Preparation example 10 NH ₂ Synthesis of Ser (tBu) -Pro-Phe-Ser (tBu) -His (Trt) -Cys (Trt) - (2-Cl-Trt Resin) (10)

1.6g of Fmoc-Ser (tBu) -OH, 1.6g of HBTU, 0.72gCl-HOBt and 1.46ml of DIEA were dissolved in 15ml of DMF, added to the resin, shaken at room temperature for 1.5 hours, the resin was washed with DMF 3 times and dichloromethane 2 times.

Preparation example 11.NH ₂ Synthesis of-Pro-Ser (tBu) -Pro-Phe-Ser (tBu) -His (Trt) -Cys (Trt) - (2-Cl-Trt Resin) (11)

Dissolving 1.42g of Fmoc-Pro-OH, 1.6g of HBTU, 0.72gCl-HOBt and 1.46ml of DIEA in 15ml of DMF, adding the solution into the resin, shaking the solution at room temperature for 1.5 hours, washing the resin with DMF for 3 times and washing the resin with dichloromethane for 2 times.

Preparation example 12.NH ₂ Synthesis of-Thr (tBu) -Pro-Ser (tBu) -Pro-Phe-Ser (tBu) -His (Trt) -Cys (Trt) - (2-Cl-Trt Resin) (12)

Dissolving 1.3g of Fmoc-Pro-OH, 1.6g of HBTU, 0.72gCl-HOBt and 1.46ml of DIEA in 15ml of DMF, adding the solution into resin, shaking at room temperature for 1.5 hours, washing the resin with DMF for 3 times and dichloromethane for 2 times.

② adding 20 percent piperidine/DMF solution 15ml into the resin, shaking for 15min at room temperature, repeating for 2 times, washing the resin with DMF for 3 times, washing with dichloromethane for 2 times, and obtaining the target resin intermediate 10.

Preparation example 13.

Synthesis of (2)

Firstly, 1.46g S-2- (N-fluorenylmethoxycarbonylamino) -5-alkynyl hexanoic acid, 1.6g HBTU, 0.72gCl-HOBt and 1.46ml DIEA are dissolved in 15ml DMF, added into resin and shaken for 1.5 hours at room temperature, and the resin is washed 3 times by DMF and 2 times by dichloromethane.

② adding 20 percent piperidine/DMF solution 15ml into the resin, shaking for 15min at room temperature, repeating for 2 times, washing the resin with DMF for 3 times, washing with dichloromethane for 2 times, obtaining the target resin intermediate 11.

Preparation example 14.

Synthesis of (2)

0.46g of 3-mercaptopropionic acid, 1.6g of HBTU, 0.72gCl-HOBt and 1.46ml of DIEA were dissolved in 15ml of DMF, and added to the resin, and shaken at room temperature for 1.5 hours, and the resin was washed with DMF 3 times and dichloromethane 2 times to obtain the objective resin intermediate 12.

Preparation example 15.

Cyclic peptides

Synthesis of (2)

Preparing TFA, TESI and H solution ₂ 20ml of O (95:2.5:2.5) was added to 1.2g of the resin intermediate 12, and reacted at room temperature for 2 hours, followed by collecting the filtrate, concentration and ether precipitation to obtain 410mg of a white solid. And purifying by HPLC to obtain reduced polypeptide solution.

Adjusting the pH of the acetonitrile/water solution of the purified reduced polypeptide to 8-9 by using ammonia water, introducing air at room temperature for oxidation reaction for 24 hours, and carrying out mass spectrum detection reaction completely.After filtration, concentration and purification by HPLC, the target product peaks were pooled and lyophilized to yield 150mg of target intermediate 12. HRMS calculated 1070.4076; found 1070.4021[ M + H] ⁺

EXAMPLE 1 Synthesis of preferred Compound 1A

Intermediate 15(23.4mg) prepared in preparation example 15 was taken, sodium ascorbate (7mg) and CuI (7.6mg) were added, and dissolved in 1.5ml of water; dissolving compound intermediate 3(66mg) in 0.5ml acetonitrile, mixing the two solutions, adding DIEA (70ul), and stirring at high speed under the protection of argon; the reaction was completed for 10 min. Filtration, HPLC separation, purification, and lyophilization gave 5mg of a white powder. HRMS calculated 1235.4778, found 1235.4738[ M + H ] ] ⁺ 。

EXAMPLE 2 Synthesis of preferred Compound 2A

Intermediate 15(30mg) prepared in preparation example 15 was taken, and sodium ascorbate (8mg) and CuI (8.6mg) were added and dissolved in 2ml of water; dissolving compound intermediate 4(80mg) in 0.5ml acetonitrile, mixing the two solutions, adding DIEA (70ul), and stirring at high speed under the protection of argon; the reaction was completed for 10 min. Filtration, HPLC separation, purification, and lyophilization gave 6.2mg of a white powder. HRMS calculated 1234.4803, found 1234.4828[ M + H ]] ⁺ Purity of radioactivity>95 percent and the specific activity is 0.2-0.8 Ci/mmol.

Pharmacological experiments

The invention designs, synthesizes and screens out a novel polypeptide which is specifically combined with colorectal tumor tissues, namely, the compound of the formula I, and the combination specificity of the compound of the formula I to colorectal tumors can be judged by adopting the following scheme:

EXAMPLE 1 (I) in vivo targeting of murine colorectal tumors

To verify the targeting properties of the compound of formula i, FITC-labeled compound 1A was administered by tail vein injection to colorectal cancer-bearing mice in situ, and the distribution of FITC-compound 1A in each tissue of the tumor-bearing mice was observed in combination with immunohistochemical method.

In the experiment, a Balb/c mouse tumor model of the cell line Colon 26 was first established:

healthy Balb/c mice, male, 7-8 weeks old, weight 18-22 g. Culturing Balb/c mouse colorectal cancer cells Colon 26 in large scale, collecting after the cell density reaches 80%, digesting with 0.25% pancreatin containing 0.01% EDTA, centrifuging at 1,100rpm for 4min, removing supernatant, adding serum-free RPMI-1640 culture solution for heavy suspension precipitation, adjusting cell density by cell counting, and injecting 0.2mL colorectal cancer cells Colon 26 suspension via anus, about 2 × 10 ⁶ After about 10 days, the anus can be seen with the naked eyes, the tumor formation rate reaches over 95 percent, the tumor grows by about 1 multiplied by 1cm, and the test can be carried out.

FITC-compound 1A (1mg/ml) is injected into tail vein by 0.3ml, after 1h, 20% urethane is anesthetized, PBS is used for full perfusion, then 4% PFA is used for perfusion, brain, heart, lung, liver, spleen, kidney, stomach, small intestine, colon and tumor tissues are taken, after gradient sucrose dehydration, frozen section embedding medium OCT is added, liquid nitrogen is used for cooling and molding, and after being cut into thin slices with thickness of 5 mu m by a freezing microtome, FITC-compound 1A (figure 1) is directly observed for fluorescent staining.

FITC-compound 1A can be specifically targeted to colorectal tumors by tail vein injection into tumor mice (n ═ 9). The imaging result shows that the compound 1A can identify the tumor in the tumor mouse, and the other tissues except the tumor tissue have no obvious green fluorescent staining. Shows that the compound 1A has the characteristic of specifically targeting colorectal tumors.

EXAMPLE 2 (II) in vitro targeting of murine colorectal tumors

In order to investigate the early diagnosis capacity of the compound shown in the formula I for colorectal cancer, namely the targeting rate aiming at colorectal tumors and the false positive condition aiming at normal colorectal tissues, the compound is adopted for obtaining mouse tumors in different growth periods, and the targeting specificity of the compound shown in the formula I on the tumor tissues of in-situ colorectal cancer mice is evaluated in vitro by using an immunofluorescence staining method.

In the experiment, a Balb/c mouse tumor model of the cell line Colon 26 is established, mouse colorectal tumor tissues and normal tissues are collected in different growth stages of tumors, and the diameter of the mouse tumor tissues is recorded within the range of 1-15 mm. Frozen sections (5 mu m) of colorectal tumor tissues and normal tissues of mice are prepared after OCT embedding, and are washed 3 times with PBS after being fixed for 10min by 95% ethanol and 5 min/time.

After blocking mouse tissue cryosections with 10% goat serum for 1h, 100. mu.L of FITC-Compound 1A at a concentration of 2.5. mu.g/mL was added, along with 100. mu.L of rat anti-mouse CD31 monoclonal antibody (goat serum 1:20 dilution), and incubated overnight at 4 ℃. PBS wash 3 times, 5 min/time, add 100 u L Alexa Fluor 568 goat anti-rat IgG (H + L) (10% goat serum 1:100 dilution), 37 degrees 1H incubation. PBS washing 3 times, 5 min/time, using containing DAPI and fluorescence quenching agent mounting plate. The green fluorescence intensity was observed under a fluorescence inverted microscope and photographed (FIG. 2).

Immunofluorescent staining results showed that compound 1A co-localized with CD31 in mouse tumor tissue, but did not show fluorescence in normal colorectal tissue and did not bind to normal tissue vessels. The compound 1A is shown to have the specificity of targeting mouse colorectal tumors, but has no binding specificity to normal mouse colorectal tissues.

Experimental example 3 (III) in vitro targeting of human colorectal tumors

In a specific test for investigating the compound of the formula I in a target clinical human early colorectal cancer specimen, in order to deeply evaluate the specific targeting property of the compound of the formula I, the early diagnosis capability of the compound of the formula I for colorectal cancer is evaluated, and the property that the compound of the formula I is highly specifically combined with colorectal cancer tumor tissue is verified by combining histopathology and immunofluorescence identification.

Human colorectal tissues including tissues of different stages of tumors and paracancerous tissues, normal mucosa, adenoma and polyp are collected, frozen sections (5 mu m) are prepared after OCT embedding, PBS is used for 3 times after 95% ethanol is fixed for 10min, 5min is carried out for each time, after 10% goat serum of the human tissue frozen sections is sealed for 1h, 100 mu L FITC-compound 1A (10% goat serum preparation) with the concentration of 2.5 mu g/mL is added, and 100 mu L mouse anti-human CD31 monoclonal antibody (10% goat serum 1:50 dilution) is added at the same time, and the mixture is incubated overnight at 4 ℃. PBS wash 3 times, 5 min/time, add 100 u L Alexa Fluor 568 goat anti-mouse IgG (H + L) (10% goat serum 1:100 dilution), 37 degrees 1H incubation. The plates were washed 3 times with PBS 5 min/time, mounted with mounting medium containing DAPI and an anti-fluorescence quencher, and observed by fluorescence inverted microscopy (FIG. 3).

Immunofluorescent staining results showed that compound 1A co-localized with CD31 in human tumor tissue and showed no fluorescence in normal colorectal tissue as well as in inflammatory polyp specimens. The immunofluorescence identification result of human colorectal tissues also shows that the compound 1A has specificity of targeting human colorectal tumors, and has no specific targeting on normal colorectal tissues and inflammatory polyps.

EXAMPLE 4 (IV) in vivo plasma kinetics of Polypeptides

In order to make the present invention more useful for clinical diagnosis, in vivo pharmacokinetic tests of the compounds of formula I were performed. In the test, 25mg/kg was administered in saline: compound 1A was prepared at 2.5mg/mL in PB buffer (V: V) ═ 1:1, and approximately 0.2mL was administered to the tail vein of mice. Blood was drawn from the mouse orbit into centrifuge tubes with heparin sodium at various time points (0,2,5,10,15,20,30,45,60,90,120,180 and 240min) after dosing. Centrifuging at 5,000g for 5min, collecting supernatant as plasma sample, and storing at-80 deg.C. Taking 10 μ L of plasma, adding internal standard TH-7 (molecular formula C) ₃₅ H ₄₉ N ₉ O ₁₁ Molecular weight 771.36, structural formula shown in figure 4) solution (10 μ g/mL prepared from methanol) 5 μ L, adding acetonitrile 40 μ L, vortex mixing for 1min, centrifuging at 14,000rpm for 5min, collecting supernatant, blow drying at 40 deg.C under nitrogen flow, re-dissolving residue with mobile phase 100 μ L, filtering with 0.22 μm filter membrane, and analyzing by LC-MS/MS 8050 sample injection.

Compound 1A reference substance is precisely weighed, dissolved in methanol and then diluted in proportion to obtain a series of reference substance solutions with different concentrations, and 0.1, 0.5, 1.0, 5.0, 10,50,100, 400 and 800 μ g/mL are used for establishing a standard curve. The internal standard polypeptide TH-7 was precisely weighed and prepared into an internal standard solution of 10. mu.g/mL with methanol.

Taking 10 mu L of Bal b/c mouse blank plasma, adding 1 mu L of compound 1A series standard solution, adding 5 mu L of internal standard (10 mu g/mL TH-7 solution), preparing a compound 1A plasma sample solution with the plasma concentration of 10,50,100,500,1000,5000,10000,40000 and 80000ng/mL, operating according to a plasma sample processing method, and recording peak areas of the compound 1A and the internal standard. And (3) taking the concentration as an abscissa and the peak area ratio of the compound 1A to the internal standard TH-7 as an ordinate to draw a standard curve.

LC-MS/MS 8050 analysis conditions

A chromatographic column: alltima HP C18-HL (50 mm. times.2.1 mm, 3 μm).

Liquid phase conditions: mobile phase 0.1% formic acid water (a) -0.1% formic acid methanol (B): 0min,85: 15; 1.5min,85: 15; 3.0min,40: 60; 4.00min,40: 60; 4.01min,85: 15; flow rate: 0.3 mL/min ^-1 (ii) a Column temperature: 40 ℃; sample introduction amount: 2 μ L.

Mass spectrum conditions: capillary voltage (CE): compound 1A is-55.0 eV; IS IS-42.0 eV; atomizing gas (Nebulizing): 3L/min; drying Gas (Drying Gas): 10.0L/min; interface (Interface): -3.0 kv; CID gas flow (CID gas): 270 kPa; the detection mode is positive ion mode detection, and the detected ions are [ M +2H ] ] ²⁺ (ii) a The scanning mode is as follows: multiple reaction detection mode, ion source: an ESI source; the ion reaction pairs used for quantitative analysis were: compound 1A was 618.50 → 70.45, internal standard 386.80 → 70.50.

Injecting the compound 1A into Balb/c normal mice and tumor model mice by tail vein injection to study plasma pharmacokinetics in vivo, taking blood at different time, removing protein in plasma by acetonitrile precipitation, quantifying the compound 1A in plasma by LC-MS/MS method, and taking time as abscissa and blood concentration as ordinate, GraphPad

6 the dosing profile for compound 1A is prepared, as shown in FIG. 5. Plasma drug concentrations were analyzed using DAS3.0 pharmacokinetic software, and the pharmacokinetic parameters are shown in table 1.

TABLE 1 mouse caudal vein administration of Compound 1A pharmacokinetic parameters

After intravenous administration in normal mice, the plasma concentration of compound 1A decreased with time, with the maximum plasma concentration being at the first blood draw point (2min), after which the plasma concentration decreased rapidly, with a plasma half-life of 24.77min, which was undetectable after 360min and was below the detection limit of the assay. The pharmacokinetic parameters apparent volume of distribution Vz and total clearance CLt were greater, being about 2270mL/kg and 64mL/min/kg, respectively. Compound 1A has better plasma stability and exists mainly as proto-drug in blood.

After tail vein administration, the plasma pharmacokinetic profile of compound 1A in tumor model mice showed significant differences compared to normal mice. Plasma exposure levels of compound 1A were lower in tumor model mice than in normal mice. The compound 1A has tumor targeting property, can prolong the in vivo retention time, and has the plasma half-life prolonged to 39.39 min. Compound 1A binds to the target, accelerating its translocation from the blood to the target, with a lower area under the curve, AUC, in plasma drug in tumor model mice; the plasma clearance CLt and apparent volume of distribution Vz were greater, approximately 2-fold that of normal mice.

Claims

1. A polypeptide compound and pharmaceutically acceptable salts thereof, wherein the structural formula of the polypeptide compound is shown as 1A or 2A:

2. a diagnostic reagent composition comprising a diagnostically effective amount of the polypeptide compound of claim 1 and pharmaceutically acceptable salts thereof, and optionally one or more pharmaceutically acceptable carriers or excipients.

3. Use of the polypeptide compound of claim 1 and pharmaceutically acceptable salts thereof for the preparation of an early diagnostic reagent for colorectal cancer.

4. The use of the polypeptide compounds of claim 1 and pharmaceutically acceptable salts thereof as pharmaceutical carriers for the manufacture of a medicament for the targeted treatment of colorectal cancer.