A polypeptide compound targeting HSP90, a radioprobe-labeled precursor, a radioprobe and a preparation method and application thereof

Abstract

The present application relates to the technical field of radiopharmaceuticals, and discloses a polypeptide compound targeting HSP90, a radioactive probe labeling precursor, a radioactive probe and a preparation method and application thereof, wherein a polypeptide compound targeting HSP90 is obtained by screening a polypeptide specifically combined with HSP90 from a T7 phage display polypeptide library; the polypeptide compound targeting HSP90 is synthesized, and is subjected to a coupling reaction by being mixed with a linking arm and a chelating agent to obtain a crude product of a labeling precursor, and after purification, a radioactive probe labeling precursor targeting HSP90 is obtained; a nuclide eluent is eluted, and then is mixed with the radioactive probe labeling precursor targeting HSP90, and after incubation, a radioactive probe targeting HSP90 is obtained. The radioactive molecular probe prepared by the present application can be specifically combined with HSP90 protein, has high affinity and functional activity for HSP90, and can evaluate the expression level of HSP90 in real time in vivo.

Description

Technical Field

[0001] This invention belongs to the field of radiopharmaceutical technology, specifically relating to a polypeptide compound targeting HSP90, a radiolabeled precursor, a radioactive probe, its preparation method, and its application. Background Technology

[0002] Pancreatic cancer is a highly malignant gastrointestinal tumor that presents significant challenges in both diagnosis and treatment. Currently, conventional imaging diagnostic methods have low specificity for pancreatic cancer. Effectively differentiating pancreatic cancer from chronic pancreatitis (especially chronic mass pancreatitis) remains a long-standing and pressing problem in both imaging and clinical medicine.

[0003] Heat shock protein 90 (HSP90) is a class of highly conserved proteins widely distributed in the biological world, possessing unique molecular chaperone functions, with a molecular weight of approximately 83-90 kDa. HSP90 contains three highly conserved domains: an N-terminal adenosine triphosphate (ATP)-binding domain, a middle domain, and a C-terminal domain, existing as a homodimer and primarily involved in cell cycle and apoptosis regulation. In various malignant tumor cells, including pancreatic cancer, HSP90 exhibits abnormally high expression, and its expression level is closely related to tumor occurrence, development, grade, stage, and prognosis. Unlike normal cells where it is located only in the cytoplasm, HSP90 in tumor cells can be activated and translocated to the cell surface. Therefore, HSP90 has become a highly promising target for tumor therapeutic research, laying a solid foundation for its molecular imaging studies as a target.

[0004] Therefore, developing a radiopharmaceutical that can accurately locate HSP90-highly-expressing tumor cells and tissues to achieve HSP90-targeted tumor therapy has become an urgent problem to be solved. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a polypeptide compound targeting HSP90, a radiolabeled precursor, a radioactive probe, a method for preparing the same, and its application. The prepared radioactive molecular probe can specifically bind to the HSP90 protein, has high affinity and functional activity for HSP90, and can evaluate the expression level of HSP90 in real time in vivo. It can also be used as an imaging agent for the grouping of primary and metastatic tumor patients, monitoring of the treatment process, and evaluation of efficacy.

[0006] This invention provides the following technical solution:

[0007] In a first aspect, a polypeptide compound targeting HSP90 is provided, said polypeptide compound having one of the following structural formulas:

[0008] ;

[0009] ;

[0010] ;

[0011] .

[0012] In a second aspect, a radioactive probe labeling precursor targeting HSP90 is provided, the radioactive probe labeling precursor having the structure XRG; wherein X is a chelating agent, R is a linker arm, and G is a polypeptide compound as described in the first aspect; the amino side of the linker arm is connected to the chelating agent, and the carboxyl side of the linker arm is connected to the amino group of the first amino acid at the N-terminus of the polypeptide compound.

[0013] Furthermore, the connecting arm is selected from one of the following compounds:

[0014] ;

[0015] Where n is an integer in the range of 1 to 20.

[0016] Furthermore, the chelating agent is selected from one of the following compounds:

[0017] .

[0018] Thirdly, a radioactive probe targeting HSP90 is provided, wherein the radioactive probe is obtained by radiolabeling a radioactive probe-labeled precursor as described in any of the second aspects with a radionuclide.

[0019] Fourthly, a method for preparing the radioactive probe targeting HSP90 as described in the third aspect is provided, comprising the following steps:

[0020] T7 phage display peptide library was used to screen for peptides that specifically bind to HSP90, thus obtaining peptide compounds targeting HSP90.

[0021] The peptide compound targeting HSP90 was synthesized and coupled with a linker and a chelating agent to obtain a crude labeled precursor. After purification, a radioactive probe labeled precursor targeting HSP90 was obtained.

[0022] The elution buffer of the radionuclide was rinsed and then mixed with the radioactive probe labeled precursor targeting HSP90. After incubation, the radioactive probe targeting HSP90 was obtained.

[0023] Furthermore, the specific preparation method of the labeled precursor crude product includes:

[0024] A peptide compound targeting HSP90 was synthesized using a solid-phase synthesis method, retaining its nitrogen-terminal Fmoc group.

[0025] The synthesized solid resin containing the polypeptide compound was added to DMF for swelling, then the linker and chelating agent were added, and the reaction was carried out overnight. After the chelating agent was connected, the polymer was treated with a DMF solution of piperidine to remove the Fmoc group at the nitrogen end of the polypeptide compound.

[0026] The polypeptide compound was cleaved from the resin using a cleavage fluid, while removing the side chain protecting groups of the amino acids. Then, ether was added to precipitate the compound, followed by centrifugation and washing to obtain the crude labeled precursor.

[0027] Furthermore, the molar ratio of the polypeptide compound, the linker arm, and the chelating agent is 1:(1-5):(1-5).

[0028] Furthermore, the incubation conditions are as follows: adjust the pH to 3.5-10 with buffer solution and incubate at 20-110°C for 10 minutes.

[0029] Furthermore, the buffer solution is one of sodium acetate, water, ethanol, phosphate, salt buffer solution or dimethyl sulfoxide.

[0030] Furthermore, the nuclide is selected from... 18 F, 32 P, 33 P, 45 Ti、 47 Sc、 52 Fe、 59 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 75 Se、 75 Sc、 77 As、 86 Y, 89 Sr、 89 Zr、 90Y、 94 Tc, 99m Tc, 99 Mo、 105 Pd, 105 Rh、 109 Pd, 111 In、 123 I, 124 I, 125 I, 131 I, 142 Pr、 143 Pr、 149 Pm, 153 Sm、 161 Tb, 166 Dy、 166 Ho、 169 Er、 175 Lu、 177 Lu、 186 Re、 188 Re、 189 Re、 194 Ir、 198 Au、 199 Au、 211 At、 211 Pb, 212 Bi、 212 Pb, 213 Bi、 223 Ra or 225 One of Ac.

[0031] Fifthly, a pharmaceutical composition is provided, comprising the radioactive probe targeting HSP90 described in the third aspect, a diluent, a carrier, and / or an excipient.

[0032] Sixthly, the use of the HSP90-targeting polypeptide compound of the first aspect, or the radiolabeled precursor of any of the second aspects, or the HSP90-targeting radioprobe of the third aspect, or the pharmaceutical composition of the fifth aspect in the preparation of a medicament for the prevention, diagnosis and / or treatment of HSP90-mediated diseases.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] (1) The HSP90-targeting polypeptide compound provided by the present invention has a high affinity for HSP90. The chelating agent is linked to the HSP90-targeting polypeptide compound through a linker arm and labeled with a radionuclide. The resulting radioactive molecular probe can specifically bind to the HSP90 protein and has a high affinity and functional activity for HSP90. It can evaluate the expression level of HSP90 in vivo in real time and serve as an imaging agent for the grouping of primary and metastatic tumor patients, monitoring of treatment process and evaluation of efficacy.

[0035] (2) The radioactive probe or drug composition targeting HSP90 provided by the present invention can accurately locate HSP90-highly-expressing tumor cells and tissues through nuclear medicine methods, so as to achieve the purpose of HSP90-targeted tumor therapy. After further clinical trial evaluation, it is expected to play an important role in the clinical application of integrated diagnosis and treatment of HSP90-highly-expressing tumors. Attached Figure Description

[0036] Figure 1 The results of titer determination of the elution products of the T7 phage display 15-peptide library in rounds 1-3 of Example 1 of this invention;

[0037] Figure 2 This is a sequencing result comparison diagram of the Chinese library screening products in Example 1 of the present invention;

[0038] Figure 3 This is the HPLC chromatogram of DOTA-T3-1 in Example 2 of the present invention;

[0039] Figure 4 The LC-MS identification spectrum of DOTA-T3-1 in Example 2 of this invention;

[0040] Figure 5 This is the HPLC chromatogram of DOTA-T3-2 in Example 2 of the present invention;

[0041] Figure 6 The LC-MS identification spectrum of DOTA-T3-2 in Embodiment 2 of the present invention;

[0042] Figure 7 This is the HPLC chromatogram of DOTA-T3-3 in Example 2 of the present invention;

[0043] Figure 8 The LC-MS identification spectrum of DOTA-T3-3 in Example 2 of this invention;

[0044] Figure 9 This is the HPLC chromatogram of DOTA-T3-4 in Example 2 of the present invention;

[0045] Figure 10 The LC-MS identification spectrum of DOTA-T3-4 in Example 2 of this invention;

[0046] Figure 11 In Embodiment 3 of the present invention 68 Radiolabeled purity analysis diagram of Ga-DOTA-T3-1;

[0047] Figure 12 In Embodiment 3 of the present invention 68 Radiolabeled purity analysis diagram of Ga-DOTA-T3-2;

[0048] Figure 13 In Embodiment 3 of the present invention 68 Radiolabeled purity analysis diagram of Ga-DOTA-T3-3;

[0049] Figure 14 In Embodiment 3 of the present invention 68 Radiolabeled purity analysis diagram of Ga-DOTA-T3-4;

[0050] Figure 15 In Embodiment 4 of the present invention 68 Ga-DOTA-T3-1~ 68 PET / CT imaging results of Ga-DOTA-T3-4 in BxPC3 tumor-bearing mice. Detailed Implementation

[0051] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and should not be used to limit the scope of protection of the present invention.

[0052] In the description of this invention, it should be understood that, unless otherwise stated, "a plurality of" means two or more. The terms "comprising," "including," "having," "containing," etc., as used herein are open-ended terms, meaning that they include but are not limited to.

[0053] In the following embodiments, 68 Ga was obtained by eluting a germanium-gallium generator, and the radioactive high-performance liquid chromatography analysis of all radiotherapeutic drugs was performed using an HPLC system (Shimadzu, Japan).

[0054] Human pancreatic cancer BxPC3 cell line was used as a cell model. The culture medium was 1640 medium + 20% fetal bovine serum + 1% penicillin / streptomycin. The incubator was set at 37℃ and 5% CO2. Subculturing was performed every three days. The medium was changed every two days.

[0055] All animal experiments used in this embodiment were approved by the Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences. All animal experiments adhered to the 3R principle and were conducted under the guidance of the Laboratory Animal Ethics Committee. A tumor-bearing mouse model was established by subcutaneous injection of tumor cells; each mouse received a unilateral injection of 1×10⁻⁶ tumor cells into one axilla. 6 One tumor was constructed per mouse using individual cells. After 1-2 weeks, the tumor tissue grew to approximately 100-300 mm. 3 At that time, mice were given the drug and PET / CT imaging studies were performed.

[0056] The Chinese meanings of the English words and abbreviations used in the embodiments are as follows:

[0057] Fmoc: 9-Shuthylmethoxycarbonyl;

[0058] Rink Amide-MBHA Resin: 4-Methyldiphenylmethylamine resin hydrochloride;

[0059] DMF: N,N,-Dimethylformamide;

[0060] DCM: Dichloromethane;

[0061] TFA: Trifluoroacetic acid;

[0062] TIS: Triisopropylsilane;

[0063] Trt: Triphenylmethyl;

[0064] DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid;

[0065] DIEA: N,N-diisopropylethylamine;

[0066] Example 1

[0067] This embodiment provides a polypeptide compound targeting HSP90 and its preparation method.

[0068] Take a fresh BL21 single colony and inoculate it into LB medium containing antibiotics. Shake and incubate overnight. Transfer the overnight culture to LB or LB / carbenicillin medium for culture. Infect with phage library, shake and incubate until lysis is terminated. Centrifuge and collect the supernatant, which is the amplified library.

[0069] Using HSP90 as bait, a random 15-peptide library was screened in three rounds. The target phages were amplified and their titers were determined. The results are as follows: Figure 1 See Table 1.

[0070] Table 1. Relevant data from the three rounds of screening.

[0071]

[0072] In Table 1, T3 represents HSP90. (From...) Figure 1 As shown in Table 1, the sequence enrichment gradually increased after three rounds of screening.

[0073] Gene sequencing analysis of the amplified target phage yielded 12 polypeptides that specifically bind to HSP90 after translation, such as... Figure 2 As shown, T3-1, T3-2, T3-3, and T3-4 were enriched the most, and are the four preferred highly enriched polypeptide sequences, i.e., polypeptide compounds targeting HSP90. Their structural formulas are as follows:

[0074] T3-1:

[0075] ;

[0076] T3-2:

[0077] ;

[0078] T3-3:

[0079] ;

[0080] T3-4:

[0081] .

[0082] Example 2

[0083] Based on Example 1, this example provides four radioactive probe labeling precursors targeting HSP90 and their preparation methods.

[0084] The synthesis of steps S1, T3-1, T3-2, T3-3, and T3-4.

[0085] According to the polypeptide sequences T3-1, T3-2, T3-3, and T3-4 in Example 1, 0.025 mol of polypeptides were synthesized in solid phase using a fully automated microwave polypeptide synthesizer, retaining the Fmoc group at the nitrogen terminus of the polypeptides.

[0086] The synthesis of steps S2, DOTA-T3-1, DOTA-T3-2, DOTA-T3-3, and DOTA-T3-4.

[0087] The 0.025 mol solid resin containing the polypeptide compound synthesized in step S1 was added to 1.0 mL of DMF and swollen for 15 min. 2.0 eq DOTA-NHS and 3.0 eq DIEA were added to adjust the pH to about 8.0, and the reaction was carried out overnight at 37 °C. After DOTA linkage was completed, the mixture was treated with 20% piperidine DMF solution for 15 min to remove the Fmoc group at the nitrogen end of the polypeptide.

[0088] Step S3: Peptide cleavage and purification.

[0089] The polypeptide synthesized in step S2 was transferred to a centrifuge tube, and 5.0 mL of cleavage buffer (TFA:TIS:H2O=95:2.5:2.5, v:v:v) was added for cleavage to remove the polypeptide from the resin and remove the side chain protecting groups of the amino acids. After cleavage at room temperature for 2 h, ether was added to precipitate the polypeptide, and the mixture was centrifuged and washed three times to obtain crude polypeptide. Then, preparative chromatography was used to purify the polypeptide to obtain purified DOTA-T3-1, DOTA-T3-2, DOTA-T3-3, and DOTA-T3-4, respectively.

[0090] The prepared DOTA-T3-1, DOTA-T3-2, DOTA-T3-3, and DOTA-T3-4 were identified by LC-MS, and the results are as follows: Figures 3-10 As shown, the purity of all compounds is above 90%.

[0091] Example 3

[0092] This embodiment provides four radioactive probes targeting HSP90 and their preparation methods.

[0093] Dissolve 20 μg of DOTA-T3-1, DOTA-T3-2, DOTA-T3-3, and DOTA-T3-4 respectively in sodium acetate solution (0.25 mol / L, 1 mL), and then add... 68 GaCl2 (10 mCi) solution, adjusted to pH 3-5, incubated at 90℃ for 10 minutes to obtain... 68 Ga-DOTA-T3-1 68 Ga-DOTA-T3-2 68 Ga-DOTA-T3-3 68 Ga-DOTA-T3-4.

[0094] Diluted with physiological saline and filtered through a 0.22 μm filter for sterilization. Analyzed using Radio-HPLC with a C18 filter column. 68 Ga-DOTA-T3-1~ 68 The radiochemical purity of Ga-DOTA-T3-4 was determined under the following analytical conditions: solvent gradient: solvent A, deionized water; solvent B, acetonitrile (0.1% TFA); flow time: 15 min, acetonitrile from 1% to 100%; flow rate: 1.0 mL / min.

[0095] like Figures 11-14 As shown, 68 Ga-DOTA-T3-1~ 68The radiochemical purity of Ga-DOTA-T3-4 is greater than 95%.

[0096] Example 4

[0097] This embodiment provides 68 Ga-DOTA-T3-1~ 68 PET / CT imaging study of Ga-DOTA-T3-4 in BxPC3 tumor-bearing mice.

[0098] Micro-PET / CT imaging was performed on BxPC3 cell-bearing mice, and 100 μCi / mouse was injected via the tail vein. 68 Ga-DOTA-T3-1~ 68 Ga-DOTA-T3-4 was subjected to Micro-PET / CT imaging at 30 min, 60 min, 120 min, and 240 min post-injection, with an acquisition time of 10 minutes. Imaging results are shown below. Figure 15 As shown, 68 Ga-DOTA-T3-1~ 68 Ga-DOTA-T3-4 uptake was clearly observed in BxPC3 tumor tissue.

[0099] The HSP90-targeting polypeptide compound provided by this invention has a high affinity for HSP90. A chelating agent is linked to the HSP90-targeting polypeptide compound via a linker arm and labeled with a radionuclide. The resulting radioactive molecular probe can specifically bind to the HSP90 protein, exhibiting high affinity and functional activity for HSP90. It can evaluate HSP90 expression levels in real-time in vivo and can be applied to the preparation of drugs for the prevention, diagnosis, and / or treatment of HSP90-mediated diseases, such as as an imaging agent for patient grouping, treatment monitoring, and efficacy evaluation of primary and metastatic tumors. The HSP90-targeting radioactive probe or drug composition provided by this invention can accurately locate HSP90-highly-expressing tumor cells and tissues using nuclear medicine methods, achieving the goal of HSP90-targeted tumor therapy. After further clinical trial evaluation, it is expected to play an important role in the integrated diagnosis and treatment of HSP90-highly-expressing tumors.

[0100] Examples of HSP90-mediated diseases mentioned above are as follows: (1) Various solid tumors, including liver cancer, lung cancer, cervical cancer, skin cancer, prostate cancer, bladder cancer, gastric cancer, ovarian cancer, leukemia, breast cancer, kidney cancer, thyroid cancer, mesothelioma, endometrial cancer, intestinal cancer, head and neck cancer, head and neck squamous cell carcinoma, multiple glioma, thymic carcinoma, low-grade glioma of the brain, pheochromocytoma and paraganglioma, esophageal cancer, nasopharyngeal carcinoma, urothelial carcinoma of the bladder, pancreatic cancer, sarcoma, etc., as well as one or more of hematologic malignancies such as leukemia and lymphoma; neurodegenerative diseases, cardiovascular and cerebrovascular diseases, metabolic / endocrine dysfunction and autoimmune diseases, wherein the diseases are selected from Parkinson's disease, Alzheimer's disease, obesity, non-alcoholic fatty liver disease, insulin resistance, type 2 diabetes, atherosclerosis and polycystic ovary syndrome, etc. When the aforementioned radioactive molecular probes are used as imaging agents, PET imaging or SPECT imaging can be used.

[0101] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide compound targeting HSP90, characterized in that, The structural formula of the polypeptide compound is as follows: 。 2. A radioactive probe labeling precursor targeting HSP90, characterized in that, The structure of the radioactive probe-labeled precursor is XRG; wherein X is a chelating agent, R is a linker arm, and G is the polypeptide compound as described in claim 1; the amino side of the linker arm is connected to the chelating agent, and the carboxyl side of the linker arm is connected to the amino group of the first amino acid at the N-terminus of the polypeptide compound.

3. The radiolabeled precursor for HSP90 targeting according to claim 2, characterized in that, The connecting arm is: ； Where n is an integer in the range of 1 to 20.

4. The radiolabeled precursor for HSP90 targeting according to claim 2, characterized in that, The chelating agent is selected from one of the following compounds: 。 5. A radioactive probe targeting HSP90, characterized in that, The radioactive probe is obtained by radiolabeling the radioactive probe precursor according to any one of claims 2 to 4 with a radionuclide.

6. A method for preparing a radioactive probe targeting HSP90 as described in claim 5, characterized in that, Includes the following steps: T7 phage display peptide library was used to screen for peptides that specifically bind to HSP90, thus obtaining peptide compounds targeting HSP90. The peptide compound targeting HSP90 was synthesized and coupled with a linker and a chelating agent to obtain a crude labeled precursor. After purification, a radioactive probe labeled precursor targeting HSP90 was obtained. The elution buffer of the radionuclide was rinsed and then mixed with the radioactive probe labeled precursor targeting HSP90. After incubation, the radioactive probe targeting HSP90 was obtained.

7. The method for preparing a radioactive probe targeting HSP90 according to claim 6, characterized in that, The specific preparation method of the crude labeled precursor includes: A peptide compound targeting HSP90 was synthesized using a solid-phase synthesis method, retaining its nitrogen-terminal Fmoc group. The synthesized solid resin containing the polypeptide compound was added to DMF for swelling, then the linker and chelating agent were added, and the reaction was carried out overnight. After the chelating agent was connected, the polymer was treated with a DMF solution of piperidine to remove the Fmoc group at the nitrogen end of the polypeptide compound. The polypeptide compound was cleaved from the resin using a cleavage fluid, while removing the side chain protecting groups of the amino acids. Then, ether was added to precipitate the compound, followed by centrifugation and washing to obtain the crude labeled precursor.

8. The method for preparing a radioactive probe targeting HSP90 according to claim 6, characterized in that, The molar ratio of the polypeptide compound, the linker arm, and the chelating agent is 1:(1-5):(1-5).

9. The method for preparing a radioactive probe targeting HSP90 according to claim 6, characterized in that, The incubation conditions are as follows: adjust the pH to 3.5-10 with buffer solution and incubate at 20-110°C for 10 minutes.

10. The method for preparing a radioactive probe targeting HSP90 according to claim 6, characterized in that, The nuclide is selected from 18 F, 32 P, 33 P, 45 Ti、 47 Sc、 52 Fe、 59 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 75 Se、 75 Sc、 77 As、 86 Y, 89 Sr、 89 Zr、 90 Y、 94 Tc, 99m Tc, 99 Mo、 105 Pd, 105 Rh、 109 Pd, 111 In、 123 I, 124 I, 125 I, 131 I, 142 Pr、 143 Pr、 149 Pm, 153 Sm、 161 Tb, 166 Dy、 166 Ho、 169 Er、 175 Lu、 177 Lu、 186 Re、 188 Re、 189 Re、 194 Ir、 198 Au、 199 Au、 211 At、 211 Pb, 212 Bi、 212 Pb, 213 Bi、 223 Ra or 225 One of Ac.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition is used to prepare a medicament for the prevention, diagnosis and / or treatment of HSP90-mediated diseases, the pharmaceutical composition comprising the HSP90-targeting radioactive probe, diluent, carrier and / or excipient as described in claim 5.

12. The use of a polypeptide compound targeting HSP90 according to claim 1, or a radiolabeled precursor of any one of claims 2 to 4, or a radiolabeled probe targeting HSP90 according to claim 5, or the use of a pharmaceutical composition according to claim 11 in the preparation of a medicament for the prevention, diagnosis and / or treatment of HSP90-mediated diseases.

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