CN118812636B - A polypeptide compound, a pharmaceutical composition containing the same, and its applications - Google Patents

Abstract

The invention discloses a polypeptide compound, a pharmaceutical composition containing the same and application thereof. Specifically discloses a polypeptide compound shown as a formula I or pharmaceutically acceptable salt thereof. The polypeptide compound has good in-vivo and in-vitro stability, high imaging resolution and good specificity, and can be used as a positron emission computed tomography tumor molecule image probe.

Description

Polypeptide compound, pharmaceutical composition containing same and application of pharmaceutical composition

Technical Field

The invention relates to a polypeptide compound, a pharmaceutical composition containing the same and application thereof.

Background

Carbonic anhydrase IX (CA IX) is one of the isoforms of the carbonic anhydrase family, is a novel tumor-associated antigen, can be used as a biomarker for tumor cell specificity, and meanwhile, CA IX is closely related to the hypoxia of tumors, can be used as a marker for tumor hypoxia, and has potential roles in regulating cell proliferation and tumor formation. CA IX expression is regulated by hypoxia, and CA IX is not expressed or expressed very low in normal tissues, but is overexpressed (Niemela AM,et al.Cancer Epidemiol Biomarkers Prev,2007,16:1760-1766.WOELBER L,KRESS K,KERSTEN J F,et al.Carbonic anhydrase IX in tumor tissue and sera of patients with primary cervical cancer[J].Bmc Cancer,2011,11:1-10.).CA IX in tumor (such as colon cancer, renal cell carcinoma, cervical cancer, breast cancer, lung cancer, pancreatic cancer, ovarian cancer and the like) tissues to be used as a prognosis factor of human tumors, and the higher the expression is, the worse the prognosis is in cancers such as cervical cancer, lung cancer, breast cancer and the like. Whereas in advanced renal cell carcinoma, lower CA IX expression levels are indicative of poorer survival (ILIE M,MAZURE N M,HOFMAN V,et al.High levels of carbonic anhydrase IX in tumour tissue and plasma are biomarkers of poor prognostic in patients with non-small cell lung cancer[J].British Journal of Cancer,2010,102(11):1627-35.KON-NO H,ISHII G,NAGAI K,et al.Carbonic anhydrase IX expression is associated with tumor progression and a poor prognosis of lung adenocarcinoma[J].Lung Cancer,2006,54(3):409-18.).

The CA IX has close relation with tumorigenesis, development, metastasis and treatment effects, and is a tumor imaging target with very good prospect. Therefore, the radioactive image probe with specificity to CA IX is researched and prepared, early diagnosis, accurate stage separation, recurrence and metastasis of tumors are realized through nuclear medicine imaging noninvasively and conveniently, a tumor treatment scheme is assisted to be determined, and the radioactive image probe has important practical significance and application value for improving the effect of tumor treatment and reducing the death rate.

The existing radiopharmaceuticals reported for the CA IX target are few, the reported method for labeling the probes is not ideal, and the main problems are low reaction product yield and long reaction time (Mercier F,Paris J,Kaisin G et al Bioconjugate Chem.2011,22,108–114General Method for Labeling siRNA by Click Chemistry with Fluorine-18for the Purpose of PET Imaging;Inkster JA,Adam MJ,Storr T et al Nucleosides Nucleotides Nucleic Acids.2009Nov;28(11):1131-43Labeling of an antisense oligonucleotide with[(18)F]FPy5yne), in-vivo and in-vitro stability. Moreover, the prior literature reports that ¹²⁵ I marked compound similar to the structure of the product has 50% decomposition (Askoxylakis V,Garcia-Boy R,Rana S,et al.A new peptide ligand for targeting human carbonic anhydrase IX,identified through the phage display technology[J].PLoS One,2010,5:e15962.); after being incubated in serum for 1.5 hours, and ¹⁸ F marked compound similar to the structure of the product has (Jia L,Li X,Cheng D,Zhang L.Fluorine-18click radiosynthesis and microPET/CT evaluation of a small peptide-a potential PET probe for carbonic anhydrase IX.Bioorg Med Chem.2019;27:785-789. defects of high organ part uptake, insufficient in vivo stability and the like, namely a ¹⁸ F marked polypeptide compound, and a preparation method and application thereof, and the patent number is CN2015153512. X. Thus, there is currently a lack of a method for stabilizing a labeled polypeptide and a specific molecular imaging probe targeting CA IX.

Disclosure of Invention

The invention aims to overcome the defect of single structure of the existing polypeptide compound. Therefore, the invention provides a polypeptide compound, a pharmaceutical composition containing the polypeptide compound and application of the pharmaceutical composition. The polypeptide compound has good in-vivo and in-vitro stability, high imaging resolution and good specificity, and can be used as a positron emission computed tomography (PET) tumor molecule image probe.

The invention provides a polypeptide compound shown in a formula I or pharmaceutically acceptable salt thereof:

Wherein R is a group containing ⁶⁸ Ga;

L is a C ₁～C₁₀ linear alkylene group, a C ₂～C₁₀ branched alkylene group, a fragment shown in formula 2, a fragment shown in formula 3, a fragment shown in formula 4, or a fragment shown in formula 5;

n ₂ is an integer of 1-10, n ₃ is an integer of 2-10, and the A end of the fragment shown in formula 2, the fragment shown in formula 3, the fragment shown in formula 4 and the fragment shown in formula 5 is connected with R;

NHVPLSPy is the polypeptide Asn-His-Val-Pro-Leu-Ser-Pro- (D-Tyr); (Asn is the N-terminus of the polypeptide)

To an alpha-amino group in Asn.

In one embodiment, in the polypeptide compound of formula I or a pharmaceutically acceptable salt thereof, certain groups are defined as follows, and the remaining groups are defined in any of the other embodiments (hereinafter referred to as "in one embodiment"):

The R consists of ⁶⁸ Ga-containing ions and a chelating group, and the ⁶⁸ Ga-containing ions are chelated with the chelating group.

In one embodiment, the ⁶⁸ Ga-containing ion is ⁶⁸Ga³⁺.

In one embodiment, the chelating group is

In one embodiment, R is:

in one embodiment, L is a C ₁～C₁₀ linear alkylene group.

In one embodiment, the C ₁～C₁₀ linear alkylene is a fragment of formula 1, wherein n ₁ is 0 to 9, preferably 0 to 3, for example n ₁ =0;

in one embodiment, the C ₁～C₁₀ linear alkylene is methylene.

In one embodiment, the polypeptide compound shown in formula I is a compound formed by chelating a compound a with the ⁶⁸ Ga-containing ion (e.g., ⁶⁸Ga³⁺), wherein the structure of the compound a is as follows:

in one embodiment, the polypeptide compound of formula I has the structure:

Wherein M is ⁶⁸ Ga-containing ion, e.g., ⁶⁸Ga³⁺. In one embodiment, the polypeptide compound of formula I has the structure:

Wherein M is ⁶⁸ Ga-containing ion, e.g., ⁶⁸Ga³⁺.

In one embodiment, the polypeptide compound of formula I is

The invention also provides a preparation method of the polypeptide compound shown in the formula I, which comprises the following steps of reacting ⁶⁸ Ga-containing ions with the compound shown in the formula II in a solvent to obtain the polypeptide compound shown in the formula I;

Wherein R' is a chelating group.

In one embodiment, the chelating group is

In the preparation method, the solvent is conventional in the art, and is preferably one or more selected from the group consisting of water, physiological saline, phosphate buffer solution, acetate buffer solution, t-butanol, DMF (N, N-dimethylformamide) and DMSO (dimethyl sulfoxide). The pH of the solvent is conventional in the art, preferably 3 to 6, more preferably 3.5 to 5.5.

In the preparation method, the concentration of the polypeptide compound shown in the formula II in the reaction system is conventional in the art, preferably 1-200 mg/L, and more preferably 15-100 mg/L.

In the preparation method, the concentration of ⁶⁸ Ga-containing ions in a reaction system is conventional in the art, preferably 0.1 mCi/mL-50 mCi/mL, more preferably 1 mCi/mL-20 mCi/mL, and most preferably 2 mCi/mL-10 mCi/mL.

In the preparation method, the reaction time is conventional in the art, preferably 1 to 30min, more preferably 5 to 20min, and most preferably 8 to 15min.

In the preparation method, the reaction temperature is conventional in the art, and the temperature can be appropriately adjusted according to the stability of the compound shown in the formula II and the boiling point of the reaction solvent system adopted, preferably 40-200 ℃, and more preferably 80-150 ℃.

In one embodiment, the preparation method may further comprise the step of further isolating and purifying the polypeptide compound of formula I, as is conventional in the art, preferably by Sep-Pak C18 column.

In one embodiment, the preparation method further comprises the step of filtering the separated and purified polypeptide compound shown in the formula I through a sterile filter membrane and diluting the polypeptide compound with normal saline or phosphate buffer.

The invention also provides a pharmaceutical composition which comprises the polypeptide compound shown in the formula I or pharmaceutically acceptable salt thereof and pharmaceutical excipients.

In one embodiment, the pharmaceutical composition is a pharmaceutical composition for diagnosing a tumor.

In one embodiment, the tumor is colon cancer, rectal cancer, renal cancer, cervical cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer, preferably colon cancer or renal cancer.

In one embodiment, the tumor is a CA IX-expressing tumor.

In one embodiment, the colon cancer is a CA IX-expressed colon cancer.

In one embodiment, the kidney cancer is CA IX-expressed kidney cancer.

The invention also provides application of the polypeptide compound shown in the formula I or pharmaceutically acceptable salt thereof in preparing a medicament for diagnosing tumors.

In one embodiment, the tumor is colon cancer, rectal cancer, renal cancer, cervical cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer, preferably colon cancer or renal cancer.

In one embodiment, the tumor is a CA IX-expressing tumor.

In one embodiment, the colon cancer is a CA IX-expressed colon cancer.

In one embodiment, the kidney cancer is CA IX-expressed kidney cancer.

The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

1. The present invention develops novel high affinity polypeptides targeting carbonic anhydrase IX (CA IX) useful for targeting CA IX. The CA IX receptor is utilized to have high expression in various tumor cells such as colorectal cancer, renal cell carcinoma, liver cancer, breast cancer, cervical cancer and the like, and is based on the principle of specific binding of the polypeptide compound shown as the formula I and the CA IX, so that the polypeptide compound is expected to be applied to early diagnosis, prognosis evaluation and curative effect detection of various tumors such as colorectal cancer, renal cell carcinoma, liver cancer, breast cancer, cervical cancer and the like.

2. The preparation method of the radiolabeled polypeptide is simple, has high radiochemical yield and short reaction time, is easy for commercial batch production, and is more suitable for clinical application of radiopharmaceuticals. The prepared ⁶⁸ Ga marked compound has the radiochemical purity of more than 99 percent.

3. The radioactive molecular image probe obtained by the invention has stable structure, no decomposition in vitro and no gallium removal [ ⁶⁸ Ga ] in vivo.

4. The radiolabeled polypeptide obtained by the invention has high imaging resolution, can specifically identify tumor tissues, has higher tumor uptake and higher tumor-to-muscle uptake ratio, has excellent imaging effect on colorectal cancer and renal cell carcinoma, and has wide application range.

5. The hydrochloric acid, sodium chloride and the like used in the invention are all commercial reagents, and the raw materials are cheap and easy to obtain.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Preparation example 1Fmoc solid phase Synthesis of precursors

Synthetic raw materials and related reagents:

1. protecting amino acid raw material

Fmoc-Asn(Trt)-OH,Fmoc-His(Trt)-OH,Fmoc-Va1-OH,Fmoc-Pro-OH,Fmoc-Leu-OH,Fmoc-Ser(tbu)-OH,Fmoc-D-Tyr(tbu)-OH.

2. Condensing reagent

HBTU,DIEA

3. Solvent(s)

DMF, DCM, methanol, acetonitrile

4. Resin composition

2-Chlorotrityl Chloride resin with a degree of substitution of 1.1 mmol/g

5. Deprotection reagent

Piperidine compounds

6. Detection reagent:

Phenol reagent, pyridine reagent, ninhydrin reagent

7. Cutting reagent

TFA, TIS, EDT, anhydrous diethyl ether

8. Nitrogen gas

9. Precision electronic balance

Instrument apparatus:

1. Twelve-channel semiautomatic polypeptide synthesizer

2. High performance liquid chromatograph

3. Freeze dryer

4. Centrifugal machine

The synthesis process comprises the following steps:

1. Swelling of resin

2-Chlorotrityl Chloride Resin were placed in a reaction tube, DMF (15 m 1/g) was added thereto, and the mixture was shaken for 60 minutes to give a reaction solution 1.

2. With the first amino acid

The solvent of reaction 1 was filtered off with suction through a sand core, 3-fold molar excess of Fmoc-D-Tyr (tbu) -OH (first amino acid at C-terminus) was added, followed by 10-fold molar excess of DIEA, and finally DMF was added for dissolution and shaking for 30min. Methanol seal head for 30min. Reaction solution 2 was obtained.

3. Deprotection of

DMF from reaction solution 2 was removed, 20% piperidine DMF solution (15 ml/g) was added for 5min, and then 20% piperidine DMF solution (15 ml/g) was added for 15min. Reaction liquid 3 was obtained.

4. Detection of

Pumping out piperidine solution of the reaction liquid 3, taking more than ten resin particles, washing with ethanol for three times, adding ninhydrin, KCN and phenol solution into the resin particles, heating the mixture at 105-110 ℃ for 5min, and turning deep blue into positive reaction.

5. Washing

DMF (10 ml/g) was taken twice, methanol (10 ml/g) was taken twice, and DMF (10 ml/g) was taken twice to give product 5.

6. Condensation

To the product 5 was added 3-fold molar excess of Fmoc protected amino acid, 3-fold molar excess of HBTU, followed by 10-fold molar excess of DIEA, and finally DMF was added for dissolution and shaking for 45min.

7. Detection of

Taking more than ten pieces of resin, washing with ethanol for three times, adding ninhydrin, pyridine and phenol solution into the resin, heating the mixture at 105-110 ℃ for 5min, and taking colorless negative reaction.

8. Washing

DMF (10 m 1/g) was taken once, methanol (10 ml/g) was taken twice, and DMF (10 ml/g) was taken twice.

9. Repeating the three to eight steps, and linking from right to left in the sequence Asn-His-Val-Pro-Leu-Ser-Pro- (D-Tyr) until the last amino acid is linked.

10. Finally, 3 times molar excess DOTA-tris (tBu ester), 3 times molar excess HBTU and 10 times molar excess DIEA were added, dissolved in DMF and shaken for 45min.

11. Washing the resin according to the following method, and draining

DMF (10 ml/g) was taken twice, DCM (10 ml/g) three times, methanol (10 ml/g) four times and dried for 10min.

12. Cutting

A cutting fluid (10/g) was prepared with 94.5% TFA, 2.5% water, 2.5% EDT, and 1% TIS.

The cutting time is 180min. Obtaining the lysate.

13. Blow-drying and washing

Drying the lysate with nitrogen as much as possible, precipitating diethyl ether, centrifuging to remove supernatant, washing precipitate with diethyl ether for six times, and volatilizing at normal temperature. Crude product is obtained.

14. Purifying and preparing.

1) Taking a small amount of crude product, and dissolving by H ₂ O/ACN.

2) And taking a small amount of sample, and analyzing and judging the peak time corresponding to the target peak on an HPLC analysis instrument.

3) The preparation system using C18 reverse phase chromatography comprises wavelength of 220nm, flow rate of 15m1/min, sample injection amount of 20mL, column temperature of 25deg.C

Mobile phase A, 0.1% aqueous TFA, mobile phase B, 0.1% acetonitrile TFA, and target peak solution were collected.

4) A few target peak solutions were taken with a 1.5ml centrifuge tube for mass spectrometry confirmation and purity detection.

15. Lyophilizing the qualified target peak solution to obtain the final product To an alpha-amino group in Asn.

16. Authentication

Taking a small amount of finished products respectively, and performing molecular weight identification of MS and purity identification of HPLC analysis.

MS identification, predicted molecular weight 1312.04, ESI as ion source, run time 1min. The result was 438.62[ M+3H ] ³⁺,657.41[M+2H]²⁺,1313.45[M+H]⁺.

HPLC identification was calculated as percentage type, wavelength detected was 220 nm, run time was 20min. The flow rate was 1ml/min and the loading volume was 10. Mu.l. Buffer A is water containing 0.1% trifluoroacetic acid, buffer B is acetonitrile containing 0.1% trifluoroacetic acid, and the linear gradient is 83% -58% buffer B within 20min. The model of the chromatographic column is Kromasil 100-5C18,4.6mm× 250mm,5micron Column.

TABLE 1HPLC identification result

17. Packaging the product in a sealed manner, and preserving at-20 ℃.

Example 1

To the reactor was added 100. Mu.L of B1-1 solution (1 mg/mL in physiological saline as the solvent), ⁶⁸GaCl₃ mL of solution (2 mCi/mL in radioactive concentration and 0.1mol/L in hydrochloric acid solution) and the pH was adjusted to 5.0,110 ℃with 1mol/L of sodium acetate aqueous solution for reaction for 10min. After the reaction was completed, the reaction solution was transferred to a C18 column (Light C18 column from Waters Co., ltd., U.S.A.), the C18 column was rinsed with 10mL of water, the C18 column was slowly rinsed with 1mL of ethanol/water (1:4, volume ratio) solution, and the eluent was collected to obtain a solution containing the A1-1 product. The radiochemical purity of the obtained labeled product A1-1 is more than 99%.

Example 2

Referring to the method of preparation example 1, DOTA-tris (tBu ester) as a raw material in step 10 was replaced with DOTA-GA (tBu) ₄, and the other steps were the same as those of preparation example 1, thereby obtaining compound B1-2.

The labeled product A1-2 was obtained by the method of example 1.

Example 3

The procedure of preparation example 1, steps 1-9, was followed to obtain a polypeptide chain Asn-His-Val-Pro-Leu-Ser-Pro- (D-Tyr). And then repeating the operation of the step 3-8, and connecting Lys at the left side of the polypeptide chain Asn-His-Val-Pro-Leu-Ser-Pro- (D-Tyr) to prepare the polypeptide chain Lys-Asn-His-Val-Pro-Leu-Ser-Pro- (D-Tyr), wherein the raw material of Lys is Fmoc-Lys-OH. Compounds B1 to 3 were obtained by the method of steps 10 to 15 in preparation example 1.

The labeled products A1-3 were obtained by the method of example 1.

Example 4

The procedure of preparation example 1, steps 1-9, was followed to obtain a polypeptide chain Asn-His-Val-Pro-Leu-Ser-Pro- (D-Tyr). And then repeating the operation of the step 3-8, and connecting the Amine-PEG-Acid (the raw material is the Amine-PEG-Acid protected by the Fmoc) with the monomer number of the PEG being 3) on the left side of the polypeptide chain Asn-His-Val-Pro-Leu-Ser-Pro- (D-Tyr). Referring again to the method of steps 10 to 15 in preparation example 1, compounds B1 to 4 were produced.

The labeling products A1 to 4 were obtained by the method of example 1.

Example 5

The procedure of preparation example 1, steps 1-9, was followed to obtain a polypeptide chain Asn-His-Val-Pro-Leu-Ser-Pro- (D-Tyr). Then repeating the steps 3-8, and connecting 5 sarcosines on the left side of the polypeptide chain Asn-His-Val-Pro-Leu-Ser-Pro- (D-Tyr), wherein the raw material is Fmoc-sarcosine (Fmoc-protected sarcosines). Referring again to the method of step 10-15 in preparation example 1, compounds B1-5 were produced.

The method of example 1 was followed to obtain labeled products A1-5.

Effect example 1 in vitro stability

Stability test of the labeled products A1-1, A1-2, A1-3, A1-4 and A1-5 of examples 1-5 in PBS the solutions containing the products A1-1, A1-2, A1-3, A1-4 and A1-5 prepared in examples 1-5 were filtered through sterile filtration membranes, respectively, and diluted with physiological saline to obtain dilutions.

Mu.L of the dilutions (total radioactivity 50. Mu. Ci) were each taken, placed in 1mL of PBS (0.01 mol/L, pH=7.4), incubated at 37℃for 4 hours, and their radiochemical purity was determined by TLC to observe their in vitro stability.

The TLC analysis showed that the labeled products A1-1, A1-2, A1-3, A1-4 and A1-5 of examples 1-5 were not generated as radioactive impurities, indicating good stability in PBS.

The radiochemical purity of the labeled product A1-1 of example 1 was 98.5%.

Effect example 2 stability in serum

Stability test of labeled products A1-1, A1-2, A1-3, A1-4 and A1-5 of examples 1-5 in serum the solutions containing the products A1-1, A1-2, A1-3, A1-4 and A1-5 prepared in examples 1-5 were filtered through sterile filtration membranes, respectively, and diluted with physiological saline to obtain dilutions.

Mu.L of the dilutions (total radioactivity 50. Mu. Ci) were each taken, placed in 1mL of calf serum, incubated at 37℃for 4 hours, and their radiochemical purity was determined by TLC to observe their stability in serum.

The TLC analysis showed that the labeled products A1-1, A1-2, A1-3, A1-4 and A1-5 of examples 1-5 were not generated as radioactive impurities, indicating good stability in calf serum.

The radiochemical purity of the labeled product A1-1 of example 1 was 97.6%.

Effect example 3 in vivo stability

In vivo stability of the labeled products A1-1, A1-2, A1-3, A1-4 and A1-5 of examples 1-5 in mice the solutions containing the products A1-1, A1-2, A1-3, A1-4 and A1-5 prepared in examples 1-5 were filtered through sterile filtration membranes, respectively, and diluted with physiological saline to obtain dilutions.

200. Mu.L of the dilution (total radioactivity 100. Mu. Ci) was injected into mice via tail vein. Urine excreted by the mice was collected by reflex urination 2 hours after injection. The radiochemical purity of the labeled products A1-1, A1-2, A1-3, A1-4 and A1-5 in urine was determined by TLC to observe the in vivo stability.

The labeled products were injected into mice for 2 hours, and the results of urine collected by TLC analysis showed that the labeled products A1-1, A1-2, A1-3, A1-4 and A1-5 of examples 1-5 were not generated with radioactive degradation impurities, indicating good stability in mice.

The labeled product A1-1 of example 1 was injected into mice for 2 hours, and the results of TLC analysis of the collected urine showed that the radiochemical purity of the labeled product A1-1 of example 1 in urine was 100%.

Effect example 4 determination of lipophilicity (Log P)

Determination of the lipophilicity (Log P) of the labeling products A1-1, A1-2, A1-3, A1-4 and A1-5 of examples 1-5 the lipophilicity of the labeling compounds was determined in a system consisting of n-octanol and a water-soluble buffer solution (PBS). This index is an important factor in measuring the absorption, distribution, metabolism and elimination of drugs in the living body. Log P is the lipid partition coefficient of a compound and reflects the degree of lipophilicity of a compound. The measurement and calculation formula is as follows:

The solutions containing the products A1-1, A1-2, A1-3, A1-4 and A1-5 prepared in examples 1-5 were filtered through sterile filters, respectively, and diluted with physiological saline to obtain dilutions.

Mu.L of the dilutions (total radioactivity 10. Mu. Ci) were each taken in a 2mL vial (containing 1mL n-octanol and 1mL PBS), sealed, vortexed well for 2min at room temperature, and centrifuged at high speed for 3min to two-phase equilibrium. 100. Mu.L of each of the organic phase and the aqueous phase was sampled by a pipette and placed in two gamma-counting tubes, and the counts were measured by a gamma counter. The values of Log P were calculated from 6 replicates measured 6 replicates. The lipophilic data show that the labeled products A1-1, A1-2, A1-3, A1-4 and A1-5 of examples 1-5 are more hydrophilic.

Log p= -2.1 of the labeled product A1-1 of example 1.

Effect example 5 application in PET/CT imaging of kidney cancer 786-O tumor mice

Use of the marker products A1-1, A1-2, A1-3, A1-4 and A1-5 of examples 1-5 in PET/CT imaging of kidney cancer 786-O tumor mice:

Modeling procedure kidney cancer 786-O tumor cells were cultured, cell suspensions were prepared at a concentration of 1X 10≡7 cells/100 ul with 0.01mol/L PBS (pH=7.4), and after the cell suspensions were prepared, they were formulated with matrigel at a ratio of 2:1, and immediately after the formulation, injections were made, i.e., 150ul of each mouse was subcutaneously injected.

The solutions containing the products A1-1, A1-2, A1-3, A1-4 and A1-5 prepared in examples 1-5 were filtered through sterile filters, respectively, and diluted with physiological saline to obtain dilutions.

200. Mu.L of the dilutions (total radioactivity 100. Mu. Ci) were injected into mice via tail vein, respectively. Tumor mice were anesthetized with isoflurane after 1 hour. The mice were anesthetized, positioned for CT scan, and then PET scan. In the whole scanning process, 2L/min oxygen flow is ensured, and 1.5% isoflurane is mixed. Inveon Acquisition Workplace (IAW) control the whole scanning process. The image was reconstructed using the OSEM3D (Three-Dimensional Ordered Subsets Expectation Maximum) algorithm.

PET-CT imaging shows that PET probes A1-1, A1-2, A1-3, A1-4 and A1-5 are mainly metabolized by kidney in tumor mice, and the in vivo clearance speed is high. 786-O tumors were specific for uptake by PET probes A1-1, A1-2, A1-3, A1-4 and A1-5, with tumor uptake clearly visible. A1-1, A1-2, A1-3, A1-4 and A1-5 have good stability in mice and have no phenomenon of removing [ ⁶⁸ Ga ] gallium. Therefore, A1-1, A1-2, A1-3, A1-4 and A1-5 are PET probes targeting CA IX with great clinical application prospect, and can be used for early diagnosis of CAIX high-expression tumor.

The Standard Uptake Value (SUV) of the tumor 1.0 after 1 hour of PET probe A1-1 injection. The uptake ratio of tumor to muscle is 8.5 in 1h, the uptake ratio of tumor to brain is 12.5 in 1h, the uptake ratio of tumor to heart is 7.6 in 1h, and the uptake ratio of tumor to lung is 7.9, which are higher than those of a ¹⁸ F labeled polypeptide compound disclosed in document (Askoxylakis V,Garcia-Boy R,Rana S,et al.A new peptide ligand for targeting human carbonic anhydrase IX,identified through the phage display technology[J].PLoS One,2010,5:e15962.;Jia L,Li X,Cheng D,Zhang L.Fluorine-18click radiosynthesis and microPET/CT evaluation of a small peptide-a potential PET probe for carbonic anhydrase IX.Bioorg Med Chem.2019;27:785-789.;, a preparation method and application thereof, and ¹²⁵ I and ¹⁸ F labeled compounds similar to the structure of the product disclosed in the invention and reported in the patent number CN2015153512. X).

Effect example 6 application in PET/CT imaging of kidney cancer 786-O tumor mice

Use of the marker products A1-1, A1-2, A1-3, A1-4 and A1-5 of examples 1-5 in PET/CT imaging of kidney cancer 786-O tumor mice:

The solutions containing the products A1-1, A1-2, A1-3, A1-4 and A1-5 prepared in examples 1-5 were filtered through sterile filters and diluted with physiological saline to obtain dilutions.

200. Mu.L of the dilutions (total radioactivity 100. Mu. Ci) were injected into mice via tail vein, respectively. Tumor mice were anesthetized with isoflurane after 2 hours. The mice were anesthetized, positioned for CT scan, and then PET scan. In the whole scanning process, 2L/min oxygen flow is ensured, and 1.5% isoflurane is mixed. Inveon Acquisition Workplace (IAW) control the whole scanning process. The image was reconstructed using the OSEM3D (Three-Dimensional Ordered Subsets Expectation Maximum) algorithm.

PET-CT imaging shows that PET probes A1-1, A1-2, A1-3, A1-4 and A1-5 are mainly metabolized in tumor mice through kidneys, livers and intestinal tracts, and the in vivo clearance speed is high. 786-O tumors were specific for uptake by PET probes A1-1, A1-2, A1-3, A1-4 and A1-5, with tumor uptake clearly visible. The marked products A1-1, A1-2, A1-3, A1-4 and A1-5 have good stability in mice, and no phenomenon of removing [ ⁶⁸ Ga ] gallium is found. Therefore, the marked products A1-1, A1-2, A1-3, A1-4 and A1-5 are PET probes targeting CAIX with great clinical application prospect, and can be used for early diagnosis of CA IX high-expression tumor.

The Standard Uptake Value (SUV) of the tumor after 2 hours of injection of PET probe A1-1 is 0.8, the uptake ratio of tumor to muscle is 25.0 at 2 hours, the uptake ratio of tumor to brain is 21.6 at 2 hours, the uptake ratio of tumor to heart is 15.4 at 2 hours, and the uptake ratio of tumor to lung is 11.8, which are higher than ¹²⁵ I and ¹⁸ F labeled structurally similar compounds as the products of the invention reported in the literature.

Effect example 7 application in PET/CT imaging of colon cancer HT29 tumor-bearing mice

Use of the marker products A1-1, A1-2, A1-3, A1-4 and A1-5 of examples 1-5 in PET/CT imaging of colon cancer HT29 tumor bearing mice:

The solutions containing the products A1-1, A1-2, A1-3, A1-4 and A1-5 prepared in examples 1-5 were filtered through sterile filters, respectively, and diluted with physiological saline to obtain dilutions.

200. Mu.L of the dilutions (60. Mu. Ci total radioactivity) were injected into mice via tail vein, respectively, and tumor mice were anesthetized with isoflurane. The mice were anesthetized, positioned for CT scan, and then PET scan. In the whole scanning process, 2L/min oxygen flow is ensured, and 1.5% isoflurane is mixed. Inveon Acquisition Workplace (IAW) control the whole scanning process. The image was reconstructed using the OSEM3D (Three-Dimensional Ordered Subsets Expectation Maximum) algorithm.

PET-CT imaging shows that PET probes A1-1, A1-2, A1-3, A1-4 and A1-5 are mainly metabolized by kidney in tumor mice, and the in vivo clearance speed is high. HT29 tumors have specific uptake of PET probes A1-1, A1-2, A1-3, A1-4 and A1-5, and tumor uptake is clearly visible. The marked products A1-1, A1-2, A1-3, A1-4 and A1-5 have good stability in mice, and no phenomenon of removing [ ⁶⁸ Ga ] gallium is found. Therefore, the marked products A1-1, A1-2, A1-3, A1-4 and A1-5 are PET probes targeting CAIX with great clinical application prospect, and can be used for early diagnosis of CA IX high-expression tumor.

The Standard Uptake Value (SUV) of the tumor after 1 hour of injection of PET probe A1-1 is 0.7, the uptake ratio of tumor to muscle is 7.3 at 1h, the uptake ratio of tumor to brain is 11.6 at 1h, the uptake ratio of tumor to heart is 6.0 at 1h, the uptake ratio of tumor to lung is 5.3, which are higher than ¹²⁵ I and ¹⁸ F labeled structurally similar compounds as the products of the invention reported in the literature.

Effect example 8 application in PET/CT imaging of colon cancer HT29 tumor-bearing mice

Use of the markers A1-1, A1-2, A1-3, A1-4 and A1-5 of examples 1-5 in PET/CT imaging of HT29 tumor mice bearing renal carcinoma:

The solutions containing the products A1-1, A1-2, A1-3, A1-4 and A1-5 prepared in examples 1-5 were filtered through sterile filters, respectively, and diluted with physiological saline to obtain dilutions.

200. Mu.L of the dilutions (total radioactivity 100. Mu. Ci) were injected into mice via tail vein, respectively, and tumor mice were anesthetized with isoflurane. The mice were anesthetized, positioned for CT scan, and then PET scan. In the whole scanning process, 2L/min oxygen flow is ensured, and 1.5% isoflurane is mixed. Inveon Acquisition Workplace (IAW) control the whole scanning process. The image was reconstructed using the OSEM3D (Three-Dimensional Ordered Subsets Expectation Maximum) algorithm.

PET-CT imaging shows that PET probes A1-1, A1-2, A1-3, A1-4 and A1-5 are mainly metabolized in tumor mice through kidneys, livers and intestinal tracts, and the in vivo clearance speed is high. HT29 tumors have specific uptake of PET probes A1-1, A1-2, A1-3, A1-4 and A1-5, and tumor uptake is clearly visible. The marked products A1-1, A1-2, A1-3, A1-4 and A1-5 have good stability in mice, and no phenomenon of removing [ ⁶⁸ Ga ] gallium is found. Therefore, the marked products A1-1, A1-2, A1-3, A1-4 and A1-5 are PET probes targeting CAIX with great clinical application prospect, and can be used for early diagnosis of CA IX high-expression tumor.

The Standard Uptake Value (SUV) of the tumor after 2 hours of injection of PET probe A1-1 is 0.6, the uptake ratio of tumor to muscle is 10.9 at 2 hours, the uptake ratio of tumor to brain is 20.3 at 1 hour, the uptake ratio of tumor to heart is 10.5 at 2 hours, and the uptake ratio of tumor to lung is 7.9 at 2 hours, which are higher than the ¹²⁵ I and ¹⁸ F labeled structurally similar compounds as the products of the invention reported in the literature.

Claims (15)

1. A polypeptide compound as shown in Formula I, or a pharmaceutically acceptable salt thereof: ; Where R is: ; L is a _C1 - _C10 straight-chain alkylene group, a fragment as shown in Formula 2, a fragment as shown in Formula 3, a fragment as shown in Formula 4, or a fragment as shown in Formula 5; Formula 2; Formula 3; Equation 4; Formula 5; _n2 is an integer from 1 to 10; _n3 is an integer from 2 to 10; the A end of the segments shown in Equation 2, Equation 3, Equation 4, and Equation 5 is connected to R. NHVPLSPy is a polypeptide Asn-His-Val-Pro-Leu-Ser-Pro-(D-Tyr); It is linked to the α-amino group in Asn. 2. The polypeptide compound of Formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein L is a _C1 - _C10 straight-chain alkylene group. 3. The polypeptide compound of Formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that the _C1 - _C10 straight-chain alkylene group is a fragment of Formula I, wherein _n1 is 0-9; ; Formula 1. 4. The polypeptide compound of Formula I as described in claim 3, or a pharmaceutically acceptable salt thereof, wherein _n1 is 0 to 3. 5. The polypeptide compound of Formula I as claimed in claim 3, or a pharmaceutically acceptable salt thereof, characterized in that _n1 = 0. 6. The polypeptide compound of Formula I as claimed in claim 3, or a pharmaceutically acceptable salt thereof, characterized in that the _C1 - _C10 linear alkylene group is methylene. 7. The polypeptide compound of Formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that the polypeptide compound of Formula I is... , , , or . 8. The polypeptide compound of Formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that the polypeptide compound of Formula I is... . 9. A method for preparing a polypeptide compound of Formula I as described in any one of claims 1-8, comprising the following steps: reacting ^{68Ga3 +} with a compound of Formula II in a solvent to obtain the polypeptide compound of Formula I. ; Where R' is . 10. A pharmaceutical composition comprising a polypeptide compound of Formula I as described in any one of claims 1-8 or a pharmaceutically acceptable salt thereof, and a pharmaceutical excipient. 11. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition is a pharmaceutical composition for diagnosing tumors. 12. The pharmaceutical composition of claim 11, wherein the tumor satisfies one or two of the following conditions: (1) The tumors mentioned are colon cancer, rectal cancer, kidney cancer, cervical cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer; (2) The tumor mentioned is a CA IX-expressing tumor. 13. The pharmaceutical composition of claim 12, wherein the tumor is colon cancer or kidney cancer. 14. The use of a polypeptide compound of Formula I as described in any one of claims 1-8, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for diagnosing tumors; The tumor meets one or two of the following conditions: (1) The tumors mentioned are colon cancer, rectal cancer, kidney cancer, cervical cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer; (2) The tumor mentioned is a CA IX-expressing tumor. 15. The application as described in claim 14, wherein the tumor is colon cancer or kidney cancer.