WO2024046469A1 - 环肽及其制备方法、包括其的复合物、及其用途 - Google Patents

环肽及其制备方法、包括其的复合物、及其用途 Download PDF

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WO2024046469A1
WO2024046469A1 PCT/CN2023/116515 CN2023116515W WO2024046469A1 WO 2024046469 A1 WO2024046469 A1 WO 2024046469A1 CN 2023116515 W CN2023116515 W CN 2023116515W WO 2024046469 A1 WO2024046469 A1 WO 2024046469A1
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cancer
acid
cyclic peptide
group
radionuclide
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PCT/CN2023/116515
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English (en)
French (fr)
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单长宇
曾德兴
陈银飞
李文杰
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核欣(苏州)医药科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present application relates to the field of medicine, and more specifically to a cyclic peptide and its preparation method, a complex including the same, and its use.
  • Theranostics have long been the main development direction in the field of nuclear medicine, and radiotherapy and diagnosis are also the most mature and widespread clinical applications in the field of theranostics.
  • a significant advantage of radiotherapy diagnostics is that the treatment site is the diagnostic image of the patient's lesion. Imaging and therapeutic intervention are closely related, so the development of specific radiotherapy with high affinity and specificity, low non-specific uptake, sufficient retention and effective penetration Sexual imaging tracers have become a key research direction in the field of nuclear medicine.
  • Aminopeptidase N is a Zn 2+ -dependent membrane-bound metalloprotease that can cleave neutral amino acids at the N-terminus of proteins or peptides.
  • CD13 was first purified in 1963 and later found to be overexpressed in tumors, tumor angiogenesis, and cardiac angiogenesis.
  • CD13 is expressed on a range of different human cells, such as macrophages, stromal cells, smooth muscle cells and fibroblasts. Due to involvement in peptide cleavage, viral infection, endocytosis and cell signaling. Abnormally high levels of CD13 expression occur in a variety of cancers, including breast, ovarian, thyroid, pancreatic, colorectal, and non-small cell lung cancer. In gastric cancer, CD13 and TGF- ⁇ 1 expression levels are related to tumor size, lymph node metastasis, and tumor differentiation. Similarly, in pancreatic cancer, serum CD13 levels correlate with tumor size, lymph node metastasis, and metastatic stage.
  • CD13 can be used as a cancer biomarker for clinical detection and evaluation of tumors.
  • CD13 is not expressed on the surface of normal blood vessels, but is usually highly expressed in blood vessels undergoing angiogenesis, such as tumor blood vessels, and certain peptide structures can bind to the active site of CD13 but are not cleaved and degraded by it.
  • the most famous peptide sequence is asparagine-glycine-arginine (NGR)-containing peptide, which can target tumor vascular tissue through interaction with CD13.
  • NGR peptides were obtained through phage display peptide library screening in 1998.
  • peptides containing NGR structures can bind to CD13 receptor-positive blood vessels in tumor tissues, but cannot bind to other CD13 receptor-rich tissues.
  • NGR fragment sequences As carriers to deliver chemotherapy drugs, nanoparticles and radioactive isotopes to tumors.
  • Preclinical experiments and clinical trials have shown that radiolabeled NGR peptides are useful in tumor vascular diagnostic imaging and targeted radioactivity.
  • Cyclic peptides are a type of cyclic compounds with special structure, wide range of biological activities and unique mechanism of action. They are a type of peptide molecules with stable and uniform conformation. They have high selective affinity for receptors and metabolic stability. powerful. As drug molecules, cyclic peptide compounds have a wide range of biological activities such as anticancer, antiviral, antibacterial, antifungal, and enzyme inhibitors. Therefore, research on cyclic peptide drugs is receiving more and more attention.
  • cyclic peptide compounds Due to the restriction of conformational changes caused by the cyclic structure, cyclic peptide compounds generally have a large surface area, which results in high affinity and recognition specificity for the target protein.
  • the limitation of the conformational flexibility of the macrocyclic structure also reduces the entropy value of the drug binding to the target and improves the stability of the binding.
  • the characteristics of amino acid composition determine that cyclic peptide compounds often have extremely low or even no cytotoxicity.
  • cyclic peptides are easy to produce through automated chemical synthesis processes, and are easy to carry out modification, processing and monitoring. These characteristics are very beneficial to the drug development process.
  • cyclo(CNGRC) cyclic peptide is cyclo(CNGRC) cyclic peptide.
  • This cyclic peptide has been used in previous studies to carry out the detection of 99m Tc, 68 Ga, 64 Cu and other radionuclides. Markers for molecular imaging of tumor neovasculature.
  • disulfide bond in the cyclo(CNGRC) cyclic peptide structure is easily affected by biodegradation or chemical modification, and its biological stability and retention time in the body still need to be further optimized, which limits its use to a certain extent. .
  • a cyclic peptide is provided with the sequence cyclo(X 1 X 2 X 3 X 4 X 5 X 6 ), wherein,
  • the X 1 is asparagine, especially L-asparagine or D-asparagine, especially L-asparagine;
  • the X 2 is glycine or sarcosine
  • the X 3 is arginine, especially L-arginine or D-arginine, especially L-arginine;
  • Said X5 is lysine, especially L-lysine or D-lysine, especially L-lysine;
  • a method for preparing a cyclic peptide including: coupling the C-terminal of X 5 to the N-terminal of X 6 ; and coupling the C-terminal of X 4 to the N-terminal of X 5 Coupling; the C terminus of X 3 is coupled with the N terminus of X 4 ; the C terminus of X 2 is coupled with the N terminus of X 3 ; the C terminus of X 1 is coupled with the N terminus of X 2 end coupling; and the C-terminus of X 6 is coupled to the N-terminus of X 1 .
  • the method includes N-terminally protecting the C-terminus of X 5 and performing a condensation reaction with the N-terminus of X 6 that is C-terminally protected or coupled to a solid phase material. In some embodiments, the method includes N-terminally protecting the C-terminus of X 4 and performing a condensation reaction with the N-terminus of X 5 that is C-terminally protected or coupled to a solid phase material. In some embodiments, the method includes N-terminally protecting the C-terminus of X 3 and performing a condensation reaction with the N-terminus of X 4 that is C-terminally protected or coupled to a solid phase material.
  • the method includes N-terminally protecting the C-terminus of X 2 and performing a condensation reaction with the N-terminus of X 3 that is C-terminally protected or coupled to a solid phase material. In some embodiments, the method includes N-terminally protecting the C-terminus of X 1 and performing a condensation reaction with the N-terminus of X 2 that is C-terminally protected or coupled to a solid phase material. In some embodiments, the method includes N-terminally protecting the C-terminus of X 6 and performing a condensation reaction with the N-terminus of X 1 that is C-terminally protected or coupled to a solid phase material.
  • a complex including the cyclic peptide, a linker, and a chelating agent.
  • a radionuclide formulation is provided that includes the complex and a radionuclide chelated with a chelating agent of the complex.
  • the use of the cyclic peptides or complexes of the present application in radionuclide labeling is provided. In some embodiments, the use of the cyclic peptide or complex of the present application in preparing radionuclide-labeled targeting molecules is provided. In some embodiments, the use of the cyclic peptides or complexes of the present application in the preparation of radionuclide labeling reagents is provided. In some embodiments, the use of the cyclic peptide or complex of the present application in the preparation of a pharmaceutical carrier is provided. In some embodiments, use of the cyclic peptides or complexes of the present application as pharmaceutical carriers is provided.
  • the use of the cyclic peptide, complex or radionuclide preparation of the present application in the preparation of a medicament for detecting cancer, diagnosing cancer, monitoring cancer progression, monitoring cancer treatment or treating cancer is provided. In some embodiments, the use of the cyclic peptide, complex or radionuclide preparation of the present application in detecting cancer, diagnosing cancer, monitoring cancer progression, monitoring cancer treatment or treating cancer is provided.
  • cyclic peptides or complexes of the present application are provided for radionuclide labeling.
  • radionuclide-labeled targeting molecules are provided that include or consist of a cyclic peptide or complex of the present application.
  • a radionuclide labeling agent is provided that includes or consists of a cyclic peptide or complex of the present application.
  • a pharmaceutical carrier is provided that includes or consists of a cyclic peptide or complex of the present application.
  • a formulation for detecting cancer, diagnosing cancer, monitoring cancer progression, monitoring cancer treatment, or treating cancer including or consisting of a cyclic peptide, complex, or radionuclide formulation of the present application.
  • cyclic peptides, complexes, or radionuclide formulations of the present application are provided for use in detecting cancer, diagnosing cancer, monitoring cancer progression, monitoring cancer treatment, or treating cancer.
  • a method of radionuclide labeling comprising contacting the complex that chelates the radionuclide, or the radionuclide formulation, with an object to be labeled with the radionuclide.
  • a method of detecting cancer, diagnosing cancer, monitoring cancer progression, and monitoring cancer treatment comprising: administering the complex of chelating a radionuclide, or the radionuclide preparation, to detecting Cancer, diagnosing cancer, monitoring cancer progression, or monitoring cancer treatment in a subject; detecting radionuclides and determining the levels and locations of radionuclides in said subjects; and comparing said levels and locations to those from untreated subjects.
  • the level and location of the radionuclide in an otherwise identical location in an affected subject or in an unaffected area of the subject is compared with the level and location of the radionuclide in an otherwise unaffected area of the subject.
  • Higher levels of said radionuclide in said subject compared to levels and locations in said sample from an affected subject or from an unaffected area of said subject or various locations indicating that the subject has cancer, thereby detecting cancer, diagnosing cancer, monitoring cancer progression, or monitoring cancer treatment.
  • Figure 1 is an electrospray mass spectrum of APN21-Bn-SCN-NOTA in some embodiments of the present application.
  • Figure 2 is a radiation HPLC chart of 68 Ga-APN21-Bn-SCN-NOTA in some embodiments of the present application.
  • Figure 3A shows the injection of only Comparative Example 68 Ga-CG6-Bn-SCN-NOTA (CG6 group), the injection of only Comparative Example 68 Ga-KE5-Bn-SCN-NOTA (KE5 group), and the injection of only Example 68 Ga-APN21- Bn-SCN-NOTA (APN21 group), and HT1080 tumor-bearing mice co-injected with Example 68 Ga-APN21-Bn-SCN-NOTA and unlabeled NGR peptide (APN21 blocking group), after injection of 0.5, 1 and Static PET/CT image 2 hours later.
  • Figure 3B shows the injection of only Comparative Example 68 Ga-CG6-Bn-SCN-NOTA (CG6 group), the injection of only Comparative Example 68 Ga-KE5-Bn-SCN-NOTA (KE5 group), and the injection of only Example 68 Ga-APN21- Bn-SCN-NOTA (APN21 group) and HT1080 tumor-bearing mice co-injected with Example 68 Ga-APN21-Bn-SCN-NOTA and unlabeled NGR peptide (APN21 blocking group), at injections of 0.5, 1 and 2 Hours later, tumor uptake calculated from 68 Ga-APN21-Bn-SCN-NOTA signal intensity.
  • the term “about” refers to an approximation, a range of about or in the vicinity. When the term “about” is used in connection with a numerical range, it modifies the range by extending the limit above or below the numerical value provided. In general, the term “about” is used herein to refer to a numerical value plus or minus 10% from the value provided. In one aspect, the term “about” means plus or minus 20% of the numerical value of the number it modifies. For example, “about 50%” means in the range of 45%–55%. Reference herein to numerical ranges by endpoints includes all integers and fractions subsumed within that range (eg, "1 to 5" includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all integers and fractions thereof are deemed to be modified by the term "about.”
  • compositions such as a device, a composition, a method, etc.
  • elements such as each unit of a device, each component of a composition, or substantive steps of a method, etc.
  • Consisting essentially of is meant to exclude other elements that are of any importance in combination for the stated purpose. Therefore, combinations consisting essentially of elements as defined herein do not exclude other elements that do not materially affect the basic and novel characteristics of the claimed invention.
  • Consisting of means a combination of other elements (unit components and substantial process steps) to the exclusion of other elements. Embodiments defined by each of these transitional terms are within the scope of the invention.
  • amino acid is used interchangeably with “amino acid residue” and may refer to both free amino acids and amino acid residues of peptides. It will be clear from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.
  • amino acid is intended to include natural and synthetic amino acids, and includes D- and L-form amino acids.
  • Standard amino acid refers to any of the twenty standard L-amino acids (including glycine) commonly found in naturally occurring peptides.
  • D-form and “L-form” amino acids are not intended to exclude amino acids that are not chiral, such as glycine, unless otherwise stated.
  • Non-standard amino acid residue refers to any amino acid other than a standard amino acid, whether or not synthetically prepared or derived from natural sources.
  • synthetic amino acid also includes chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (eg, amides), and substitutions.
  • Amino acids comprised in the peptides of the invention, and particularly located at the C-terminus, may be modified by methylation, amidation, acetylation or substitution with other chemical groups that alter the circulating half-life of the peptide without deleteriously affecting its activity. or N-terminal amino acid. Additionally, disulfide bonds may or may not be present in the peptides of the invention.
  • the term "pharmaceutical composition” refers to a composition containing at least one active ingredient that is acceptable for the study of a specific, effective outcome in a mammal, such as, but not limited to, a human. of. Based on the needs of the skilled artisan, those of ordinary skill in the art will understand and appreciate techniques suitable for determining whether an active ingredient has the desired effective effect.
  • the term "pharmaceutically acceptable carrier” refers to a chemical composition with which a suitable compound or derivative can be combined and which, in combination, can be used to administer the suitable compound to a subject.
  • physiologically acceptable ester or salt refers to an ester or salt form of an active ingredient that is compatible with any other ingredient of the pharmaceutical composition in such form as the subject to whom the composition is to be administered. harmless.
  • pharmaceutically acceptable means physiologically tolerable for human or veterinary use.
  • compositions include formulations for human and veterinary use.
  • plurality means at least two.
  • polynucleotide refers to single strands or parallel and antiparallel strands of nucleic acids.
  • a polynucleotide can be a single-stranded or double-stranded nucleic acid.
  • polypeptide refers to amino acid residues linked by peptide bonds, their related naturally occurring structural variants and their synthetic non-naturally occurring analogs, their related naturally occurring structural variants and Synthetic polymers composed of non-naturally occurring analogues.
  • synthetic peptide or polypeptide refers to a peptide or polypeptide that is not naturally occurring.
  • synthetic peptides or polypeptides can be synthesized using an automated peptide synthesizer.
  • N-terminal protection refers to the terminal amino group of a peptide coupled to any of the various N-terminal protecting groups conventionally employed in peptide synthesis.
  • C-terminal protection refers to the terminal amino group of a peptide coupled to any of the various C-terminal protecting groups conventionally employed in peptide synthesis.
  • the term "high expression” respectively means that the expression level of a subject is higher than that of a normal individual; in particular, the value or level of a specific substance, such as a specific biomarker or protein, in a biological sample of a subject , which is higher than the value or level of that particular substance detected in a biological sample obtained from a healthy or wild-type (normal) individual.
  • the term “low expression” respectively means that the expression level of the subject is lower than the expression level of a normal individual; in particular, the value or level of a specific substance, such as a specific biomarker or protein, in a biological sample of the subject , which is lower than the value or level of that particular substance detected in a biological sample obtained from a healthy or wild-type (normal) individual.
  • the terms “high expression” and “low expression” may indicate a "differential level” or “differential value” or “different expression” compared to a "normal” expression level or value of a biomarker, and may include quantification in expression levels Differences and qualitative differences.
  • the X1 is L-asparagine. In some embodiments, the X1 is D-asparagine. In some embodiments, X3 is L-arginine. In some embodiments, X3 is D-arginine. In some embodiments, the X5 is L-lysine. In some embodiments, the X5 is D-lysine. In some embodiments, the X 4 is L-threonine. In some embodiments, X is L-tyrosine. In some embodiments, the X 4 is L-threonine and the X 6 is L-tyrosine.
  • the cyclic peptide is a compound of formula (I) or a derivative thereof. In some embodiments, the cyclic peptide is a compound of Formula (I).
  • the preparation method of the cyclic peptide further includes: deprotecting the N-terminus of the N-terminally protected X 1 to obtain the X 1 ; deprotecting the N-terminus of the N-terminally protected X 2 Protect to obtain the X 2 ; deprotect the N-terminal of the N-terminal protected X 3 to obtain the X 3 ; deprotect the N-terminal of the N-terminal protected X 4 to obtain the X 4 ; Deprotect the N-terminal of the N-terminal protected X 5 to obtain the X 5 ; and/or deprotect the N-terminal of the N-terminal protected X 6 to obtain the X 6 .
  • the preparation method of the cyclic peptide further includes: deprotecting the N-terminus of X 1 protected at the N-terminus and protected at the C-terminus to obtain the X 1 with C-terminal protection; protecting the N-terminus and The N-terminal of X 2 protected by the C-terminal is deprotected to obtain the X 2 protected by the C-terminal; the N-terminal of the X 3 protected by the N-terminal and C-terminal is deprotected to obtain the C-terminal protected X 2 X 3 ; deprotect the N end of X 4 with N end protection and C end protection to obtain the X 4 with C end protection; deprotect the N end of X 5 with N end protection and C end protection. , obtain the C-terminal protected X 5 ; and/or deprotect the N-terminal of the N-terminal protected and C-terminal protected X 6 to obtain the C-terminal protected X 6 .
  • the preparation method of the cyclic peptide further includes: deprotecting the C-terminal of X 1 with N-terminal protection and C-terminus protection to obtain the N-terminus protected X 1 ; protecting the N-terminus and The C-terminal of X 2 protected at the C-terminal is deprotected to obtain the X 2 protected at the N-terminal; the C-terminal of the X 3 protected at the N-terminal and C-terminal is deprotected to obtain the X 2 protected at the N-terminal X 3 ; deprotect the C terminal of X 4 with N terminal protection and C terminal protection to obtain the N terminal protected X 4 ; deprotect the C terminal of X 5 with N terminal protection and C terminal protection. , obtaining the N-terminal protected X 5 ; and/or deprotecting the C-terminal of the N-terminal protected and C-terminal protected X 6 to obtain the N-terminal protected X 6 .
  • the preparation method of the cyclic peptide further includes: deprotecting the C-terminal of the C-terminal protected X 1 to obtain the X 1 ; deprotecting the C-terminal of the C-terminal protected X 2 Protection, the X 2 is obtained; the C-terminal of the X 3 protected by the C-terminal is deprotected, to obtain the X 3 ; the C-terminal of the X 4 protected by the C-terminal is deprotected, to obtain the X 4 ; Deprotect the C-terminal of X 5 with C-terminal protection to obtain X 5 ; and/or deprotect the C-terminus of X 6 with C-terminal protection to obtain X 6 .
  • the preparation method of the cyclic peptide further includes: separating the C-terminal of X 1 of the C-terminal coupled solid phase material from the solid phase material to obtain the X 1 ;
  • the C-terminal of the X 2 of the solid-phase material is separated from the solid-phase material to obtain the X 2 ;
  • the C-terminal of the X 3 coupled to the solid-phase material is separated from the solid-phase material to obtain the X 3 ;
  • the C-terminal of X 1 undergoes a condensation reaction with the N-terminal of X 2 , including contacting the N-terminal protected X 1 , X 2 , HBTU and DIEA.
  • the C-terminal of X 2 and the N-terminal of X 3 undergo a condensation reaction, including contacting the N-terminal protected X 2 , X 3 , HBTU and DIEA.
  • the C terminus of X 3 and the N terminus of X 4 undergo a condensation reaction, including contacting the N terminus protected X 3 , X 4 , HBTU and DIEA.
  • the C-terminus of X 4 and the N-terminus of X 5 undergo a condensation reaction, including contacting the N-terminally protected X 4 , X 5 , HBTU and DIEA.
  • the C-terminus of X 5 undergoes a condensation reaction with the N-terminus of X 6 , including contacting the N-terminally protected X 5 , X 6 , HBTU and DIEA.
  • the C-terminus of X 6 undergoes a condensation reaction with the N-terminus of X 1 , including contacting the N-terminally protected X 6 , X 1 , HBTU and DIEA.
  • N-terminal protection is Fmoc protection.
  • the solid phase material is resin.
  • the complex sequesters radionuclides include 44 Sc, 47 Sc, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr, 99m Tc , At least one of or consisting of the group consisting of 110m In, 111 In, 113m In, 114m In, 177 Lu, 203 Pb, 212 Pb, 212 Bi, 213 Bi, and 225 Ac.
  • the radionuclides include 44 Sc, 47 Sc, 64 Cu, 67 Cu , 67 Ga, 68 Ga, 90 Y, 99m Tc, 111 In, 177 Lu, 212 Pb, 213 Bi, At least one of or consisting of the group consisting of and 225 Ac.
  • the radionuclide includes or consists of at least one selected from the group consisting of 99m Tc, 68 Ga, and 64 Cu.
  • the linker includes or consists of polyethylene glycol (PEG).
  • the radionuclide includes 68 Ga.
  • the chelating agent includes a chelating agent selected from the group consisting of: 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (1,4,7 ,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid, DOTA), 1,4,7-triazacyclononane-N,N′,N′′-triacetic acid (1,4 ,7-triazacyclononane-N,N′,N′′-triacetic acid (NOTA), diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid (diethylenetriamine-N,N,N′ ,N′′,N′′-pentaacetic acid, DTPA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraazacyclotetradecane- 1,4,8,11 ⁇ te
  • the cyclic peptide is coupled to a linker.
  • X5 in the cyclic peptide is coupled to a linker.
  • the linker is coupled to a chelating agent.
  • the linker is coupled to the cyclic peptide and the chelating agent, particularly a first end of the linker is coupled to the cyclic peptide, and a second end of the linker that is different from the first end is coupled to the chelating agent.
  • the cancer is a tumor with high expression of CD13.
  • the cancer is selected from the group consisting of head and neck cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, endometrial cancer, cervical cancer, prostate cancer, adrenal cancer, lymphoma tumor, salivary gland cancer, bone cancer, brain cancer, cerebellar cancer, colon cancer, rectal cancer, colorectal cancer, oronasopharyngeal cancer, kidney cancer, bladder cancer, skin cancer, melanoma, basal cell carcinoma, hard palate cancer, tongue squamous cell carcinoma
  • the cancer is selected from the group consisting of breast cancer, ovarian cancer, thyroid cancer, pancreatic cancer, colorectal cancer, non-small cell lung cancer, and osteosarcoma. In some embodiments, the cancer is selected from the group consisting of esophageal cancer, pancreatic cancer, and gastric cancer.
  • NGR cyclic peptide which has higher in vivo stability, stronger affinity for the CD13 receptor, stronger targeting, and can accurately locate the CD13 receptor in the body after being labeled with radionuclides. body, to achieve the purpose of diagnosing tumor molecular effects through PET imaging,
  • an NGR cyclic peptide with a new structure uses the polypeptide for the preparation and application of radiopharmaceuticals.
  • the radiopharmaceutical and its labeling technology of the present application can be used for molecular imaging and treatment of malignant tumors targeting CD13 receptors in new blood vessels.
  • the receptor of the present application has good targeting properties, strong stability, simple labeling, and has high application value.
  • the filtrate is obtained, and then a large amount of diethyl ether is added to the filtrate to precipitate the crude product, which is centrifuged and washed to obtain the crude product of a linear polypeptide sequence with a protective group.
  • a protecting group cutting solution (composed of 95% trifluoroacetic acid (TFA), 2% ethanedithiol, 2% triisopropylsilane, and 1% water), add it to the cyclic peptide container in the previous step, and shake for 120 minutes.
  • TFA trifluoroacetic acid
  • 2% ethanedithiol 2% triisopropylsilane, and 1% water
  • the filtrate is obtained, and then a large amount of diethyl ether is added to the filtrate to precipitate the crude product, which is centrifuged and washed to obtain the crude product of the target cyclic peptide sequence.
  • the crude polypeptide sequence was purified using high-performance liquid chromatography (HPLC), lyophilized, and the molecular weight of the target product was confirmed using liquid chromatography-mass spectrometry (LC-MS).
  • HPLC high-performance liquid chromatography
  • LC-MS liquid chromatography-mass spectrometry
  • the human fibrosarcoma cell line HT ⁇ 1080 was cultured in Gibco's Dulbecco's modified Eagle medium (DMEM) mixed with 10% sterile filtered fetal bovine serum (FBS) and 1% penicillin–streptomycin antibiotics.
  • DMEM Gibco's Dulbecco's modified Eagle medium
  • FBS sterile filtered fetal bovine serum
  • penicillin–streptomycin antibiotics 1% penicillin–streptomycin antibiotics.
  • Cell culture was maintained in a controlled environment with a temperature of 37°C and 5% CO2 , and subcultures were performed every 3–4 days. 24 hours before the experiment, the cells were seeded in a 96-well plate (5000 cells per well) and cultured overnight until the cells adhered. Before the experiment, wash the cells twice with 1 mL of PBS balanced salt solution to remove the growth medium.
  • the cyclic peptides of Examples 1-28 (the sequences are respectively as shown in SEQ ID NO: 1-28, hereinafter respectively referred to as APN1-APN28, collectively referred to as APN) or the cyclic peptide of Comparative Example 1 (the sequences are as shown in SEQ ID NO: 29 (hereinafter referred to as KE5) was condensed with Cy3-NHS fluorescent dye in DMF solution, and prepared and purified by HPLC to obtain the APN-Cy3 fluorescent peptide of Examples 1-28 or the KE5-Cy3 fluorescent peptide of Comparative Example 1.
  • KE5-Cy3 fluorescent peptide treatment groups of Comparative Example 1 were established according to concentration gradients, respectively: 3.125 ⁇ mol/L, 6.25 ⁇ mol/L, 12.5 ⁇ mol/L, 25 ⁇ mol/L, 50 ⁇ mol/L, 100 ⁇ mol/L group, 6 wells in each group.
  • HT ⁇ 1080 cells were treated with comparative example ⁇ Cy3 fluorescent peptide.
  • HT-1080 cells were treated with the APN-Cy3 fluorescent peptide of each example, where the concentration of each APN-Cy3 fluorescent peptide was the optimal concentration of the KE5-Cy3 fluorescent peptide of the comparative example uptake by the HT-1080 cells.
  • RCU relative cell uptake
  • Orn represents ornithine
  • Dab represents 2,4-diaminobutyric acid
  • Sar represents sarcosine
  • Nme represents N-methyl-asparagine
  • each lowercase letter represents D-amino acid.
  • Example 2 Example 3, Example 4, Example 5, Example 6, Example 9, Example 15, Example 16, Example 18, Example 19, Example 21, and Example Example 22
  • the relative cellular uptake rate of the APN-Cy3 fluorescent peptide is at least 2 times the cellular uptake rate of the KE5-Cy3 fluorescent peptide of the comparative example, wherein Example 9, Example 15, Example 19, Example 21, and In Example 22, the relative cellular uptake rate of the APN-Cy3 fluorescent peptide is at least 4 times that of the KE5-Cy3 fluorescent peptide.
  • the relative cellular uptake rate of the APN21-Cy3 fluorescent peptide of Example 9 is about 5.03 times that of the KE5-Cy3 fluorescent peptide, while the relative cellular uptake rate of the APN21-Cy3 fluorescent peptide of Example 21 is KE5-Cy3 fluorescent peptide.
  • the cell uptake rate is approximately 5.68 times, indicating that these cyclic peptides can be particularly efficiently taken up by cells.
  • X 1 can be asparagine. Compared with X 1 being N-methyl-asparagine, its Cy3 fluorescent peptide can obtain a higher relative cellular uptake rate.
  • X 2 can be glycine or sarcosine to obtain a higher relative cellular uptake rate.
  • X2 can especially be sarcosine, whose Cy3 fluorescent peptide can obtain a higher relative cellular uptake rate.
  • X 4 can be selected from the group consisting of threonine, tyrosine, and phenylalanine to obtain a higher relative cellular uptake rate.
  • X 5 can be lysine. Compared with X 5 being ornithine, 2,4-diaminobutyric acid, or cysteine, the Cy3 fluorescent peptide can obtain a higher relative cellular uptake rate.
  • X 6 can be selected from the group consisting of tyrosine, valine, and glutamic acid to obtain a higher relative cellular uptake rate.
  • X 6 can be specifically tyrosine, more specifically L-tyrosine, and its Cy3 fluorescent peptide can obtain a higher relative cellular uptake rate.
  • the experiment used an in vitro constant temperature (37°C) incubation method to study the stability of the cyclic peptide APN21 in Example 21 and the cyclic peptide KE5 (1.0 ⁇ g/mL) in Comparative Example 1 in mouse plasma or PBS.
  • the above two cyclic peptides were dissolved in 1 mL of mouse serum and incubated at constant temperature in vitro for 0 h, 0.5 h, 1 h, 4 h, 24 h and 48 h, and then the remaining percentage of the drug was measured.
  • PBS was used as a negative control group.
  • the LC-MS/MS method was used to measure the peak area of the drug and the internal standard, and the ratio was used instead of the drug concentration for calculation.
  • the cyclic peptide of Example 21 has a significantly higher drug remaining percentage after in vitro constant temperature incubation in mouse plasma or PBS, which means that its stability is significantly higher.
  • the stationary phase is a semi-preparative C18 column.
  • the mobile phase uses a gradient elution method with a flow rate of 4mL/min and changes from 5% acetonitrile to 50% within 18 minutes. Acetonitrile.
  • Electrospray mass spectrometry (ESI-MS) measurement results: m/z[M+H] + 1184.87 (chemical formula: C 53 H 82 N 12 O 16 , calculated molecular weight 1183.54), the mass spectrum is as shown in Figure 1;
  • 68 Ga-APN21-Bn-SCN-NOTA conjugate of Comparative Example 1 and the 68 Ga of Comparative Example 2 were prepared according to the above method for preparing the 68 Ga-APN21-Bn-SCN-NOTA conjugate.
  • -CG6-Bn-SCN-NOTA conjugate (where "CG6” means a cyclic peptide with the sequence cyclo(CNGRC), as shown in SEQ ID NO: 30).
  • PET/CT 4.Positron emission computed tomography
  • PET/CT and image analysis were performed using a small animal NovelMedcal PET/CT scanner (Yongxin, Beijing).
  • the maximum tangential and radial half-width at the center of the field of view is 1.5mm, and the maximum tangential and radial half-width at the edge of the field of view is 1.8mm.
  • CG6 group approximately 3.7 MBq (100 ⁇ Ci) of the 68 Ga-CG6-Bn-SCN-NOTA compound of Comparative Example 2 was injected into HT1080 tumor-bearing mice through the tail vein under isoflurane anesthesia. Static PET/CT images were obtained for 15 minutes after 0.5, 1, and 2 hours of intravenous injection.
  • the signal intensity of 68 Ga-APN21-Bn-SCN-NOTA was significantly weaker than that of the unblocked APN21 group, and closer to the signal intensity of the KE5 group. Therefore, it can be confirmed that the target of 68 Ga-APN21-Bn-SCN-NOTA is blocked when NGR peptide blocks the CD13 receptor, which also proves that 68 Ga-APN21-Bn-SCN-NOTA targets the CD13 receptor. sex.
  • PET and CT images were acquired using NMSoft workstation software (Yongxin, Beijing), and data are given as percent of injected dose per gram of tissue or organ (ID/g) and corrected by decay for each sample (normalized to represent the injected dose). known weight).

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Abstract

提供了一种环肽，其序列为cyclo(X1X2X3X4X5X6)，其中，所述X1是天冬酰胺；所述X2是甘氨酸或肌氨酸；所述X3是精氨酸；所述X4选自由苏氨酸、酪氨酸、和苯丙氨酸所组成的组；所述X5是赖氨酸；且所述X6选自由酪氨酸、缬氨酸、和谷氨酸所组成的组。提供了一种环肽的制备方法。提供了一种复合物，包括所述环肽、连接子以及螯合剂。提供了一种复合物作为放射性核素标记的靶向分子的用途。提供了一种放射性核素标记的方法，包括使螯合放射性核素的复合物接触放射性核素所要标记的对象。

Description

环肽及其制备方法、包括其的复合物、及其用途 技术领域
本申请涉及药物领域，更具体涉及一种环肽及其制备方法、包括其的复合物、及其用途。
背景技术
长期以来，治疗诊断学一直是核医学领域的主要发展方向，同时放射治疗诊断也是治疗诊断学领域最成熟和广泛的临床应用。放射治疗诊断学的一个显著优势是治疗部位即为患者病灶的诊断图像，影像学和治疗干预密切相关，因此开发具有高亲和力和特异性、低非特异性摄取、足够的滞留和有效渗透性的特异性成像示踪剂成为了核医学领域的重点研究方向。
氨肽酶N
氨肽酶N(APN，也被称为CD13)是一种Zn2+依赖的膜结合金属蛋白水解酶，能够切割蛋白或多肽N末端的中性氨基酸。CD13于1963年首次纯化，后来发现在肿瘤、肿瘤血管新生和心脏血管新生中过表达。
CD13在一系列不同的人类细胞中表达，如巨噬细胞、基质细胞、平滑肌细胞和成纤维细胞。由于参与肽切割、病毒感染、内吞作用和细胞信号转导。异常高水平的CD13表达发生于各种癌症中，包括乳腺癌、卵巢癌、甲状腺癌、胰腺癌、结直肠癌和非小细胞肺癌。在胃癌中，CD13和TGF‐β1表达水平与肿瘤大小、淋巴结转移、肿瘤分化相关。同样，在胰腺癌中，血清CD13水平与肿瘤大小、淋巴结转移和转移分期相关。它是早期胰腺癌诊断和预后的生物标志物，能够预测胰腺癌患者的死亡率和总生存期。在结直肠癌中，患者血浆中CD13活性越高，总生存率越低。此外，有研究表明CD13的表达与骨肉瘤具有相关关系，其中免疫组化显示77％的骨肉瘤患者CD13表达阳性，且较高的CD13表达与较差的总生存率相关。综上所述，CD13能够作为癌症的生物标志物，用于肿瘤的临床检测和评估。
NGR配体
研究表明，CD13不表达于正常血管的表面，但通常在肿瘤血管等发生血管新生的血管中高表达，而且某些肽结构可以与CD13活性位点结合，但不被其切割降解。而其中最著名的肽序列为含天冬酰胺‐甘氨酸‐精氨酸(NGR)的多肽，其能够通过与CD13的相互作用靶向肿瘤血管组织。NGR多肽于1998年通过噬菌体呈现肽库筛选得到，实验表明，含NGR结构的多肽可以结合至肿瘤组织中CD13受体阳性的血管，但不能结合其他富含CD13受体的组织。这些结果进一步表明了利用含NGR的多肽作为一种潜在的肿瘤靶向诊断和治疗药物的可行性。
目前，已有很多研究将包含NGR片段序列的多肽作为向肿瘤传递化疗药物、纳米颗粒和放射性同位素的载体，临床前实验和临床试验均显示放射性标记的NGR肽在肿瘤血管诊断成像和靶向放射性核素或离子治疗方面具有巨大的潜力。
环肽
环肽类化合物是一类结构特殊、生物活性广泛、作用机理独特的环状化合物，是肽类分子中构象稳定和均一的一类，对受体具有高的选择性亲和能力、代谢稳定性强。环肽类化合物作为药物分子，具有抗癌、抗病毒、抗菌、抗真菌、酶抑制剂等广泛的生物活性。因此，针对环肽类药物的研究正在受到人们越来越多的关注。
由于环状结构导致的构象变化限制，环肽类化合物一般具有较大的表面积，从而使其与靶点蛋白间具有高度亲和力及识别特异性。大环结构构象灵活性的限制也降低了药物与靶点结合的熵值，提高了结合的稳定性。其次，氨基酸组成特点决定了环肽类化合物往往具有极低的、甚至没有细胞毒性。并且，环肽类化合物很容易通过自动化的化学合成流程实现生产，并且便于进行各项修饰、处理及监控，这些特点都非常有利于药物开发过程。
目前，在靶向NGR的肿瘤放射显影示踪剂研究当中，使用最多的环肽是cyclo(CNGRC)环肽，前期研究已利用该环肽进行99mTc、68Ga、64Cu等放射性核素的标记，用于肿瘤新生血管的分子显像。但值得注意的是，cyclo(CNGRC)环肽结构中的二硫键易受生物降解或化学修饰的影响，其生物稳定性和体内滞留时间仍需要进一步优化，这在一定程度上限制了其使用。
发明内容
在一些实施方式中，提供了一种环肽，其序列为cyclo(X1X2X3X4X5X6)，其中，
所述X1是天冬酰胺，特别是L‐天冬酰胺或D‐天冬酰胺，特别是L‐天冬酰胺；
所述X2是甘氨酸或肌氨酸；
所述X3是精氨酸，特别是L‐精氨酸或D‐精氨酸，特别是L‐精氨酸；
所述X4选自由苏氨酸、酪氨酸、和苯丙氨酸所組成的組，特别是选自由L‐苏氨酸、D‐苏氨酸、L‐酪氨酸、D‐酪氨酸、L‐苯丙氨酸、和D‐苯丙氨酸所組成的組，特別是苏氨酸，更特別是L‐苏氨酸或D‐苏氨酸，更特別是L‐苏氨酸；
所述X5是赖氨酸，特别是L‐赖氨酸或D‐赖氨酸，特别是L‐赖氨酸；且
所述X6选自由酪氨酸、缬氨酸、和谷氨酸所組成的組，特别是选自选自由L‐酪氨酸、D‐酪氨酸、L‐缬氨酸、D‐缬氨酸、L‐谷氨酸、和D‐谷氨酸所組成的組，特別是酪氨酸，更特別是L‐酪氨酸或D‐酪氨酸，更特別是L‐酪氨酸。
在一些实施方式中，提供了一种环肽的制备方法，包括：所述X5的C端与所述X6的N端偶合；所述X4的C端与所述X5的N端偶合；所述X3的C端与所述X4的N端偶合；所述X2的C端与所述X3的N端偶合；所述X1的C端与所述X2的N端偶合；以及所述X6的C端与所述X1的N端偶合。
在一些实施方式中，所述方法包括N端保护的所述X5的C端、与C端保护或偶联固相材质的所述X6的N端，进行缩合反应。在一些实施方式中，所述方法包括N端保护的所述X4的C端、与C端保护或偶联固相材质的所述X5的N端，进行缩合反应。在一些实施方式中，所述方法包括N端保护的所述X3的C端、与C端保护或偶联固相材质的所述X4的N端，进行缩合反应。在一些实施方式中，所述方法包括N端保护的所述X2的C端、与C端保护或偶联固相材质的所述X3的N端，进行缩合反应。在一些实施方式中，所述方法包括N端保护的所述X1的C端、与C端保护或偶联固相材质的所述X2的N端，进行缩合反应。在一些实施方式中，所述方法包括N端保护的所述X6的C端、与C端保护或偶联固相材质的所述X1的N端，进行缩合反应。
在一些实施方式中，提供了一种复合物，包括所述环肽、连接子、以及螯合剂。在一些实施方式中，提供了一种放射性核素制剂，包括所述复合物以及与所述复合物的螯合剂螯合的放射性核素。
在一些实施方式中，提供了本申请的环肽或复合物在放射性核素标记中的用途。在一些实施方式中，提供了本申请的环肽或复合物在制备放射性核素标记的靶向分子的用途。在一些实施方式中，提供了本申请的环肽或复合物在制备放射性核素标记试剂中的用途。在一些实施方式中，提供了本申请的环肽或复合物在制备药物载体中的用途。在一些实施方式中，提供了本申请的环肽或复合物作为药物载体的用途。在一些实施方式中，提供了本申请的环肽、复合物或放射性核素制剂在制备检测癌症、诊断癌症、监控癌症进展、监控癌症治疗或治疗癌症的药物中的用途。在一些实施方式中，提供了本申请的环肽、复合物或放射性核素制剂在检测癌症、诊断癌症、监控癌症进展、监控癌症治疗或治疗癌症的用途。
在一些实施方式中，提供了用于放射性核素标记的本申请的环肽或复合物。在一些实施方式中，提供了放射性核素标记的靶向分子，包括本申请的环肽或复合物，或由其组成。在一些实施方式中，提供了放射性核素标记试剂，包括本申请的环肽或复合物，或由其组成。在一些实施方式中，提供了药物载体，包括本申请的环肽或复合物，或由其组成。在一些实施方式中，提供了检测癌症、诊断癌症、监控癌症进展、监控癌症治疗或治疗癌症的制剂，包括本申请的环肽、复合物或放射性核素制剂，或由其组成。在一些实施方式中，提供了用于检测癌症、诊断癌症、监控癌症进展、监控癌症治疗或治疗癌症的，本申请的环肽、复合物或放射性核素制剂。
在一些实施方式中，提供了一种放射性核素标记的方法，包括使螯合放射性核素的所述复合物，或所述放射性核素制剂，接触放射性核素所要标记的对象。在一些实施方式中，提供了一种检测癌症、诊断癌症、监控癌症进展、监控癌症治疗的方法，包括：施用螯合放射性核素的所述复合物、或所述放射性核素制剂，到检测癌症、诊断癌症、监控癌症进展或监控癌症治疗的受试者；检测放射性核素和确定放射性核素在所述受试受治疗者体内的水平和位置；以及将所述水平和位置与来自未受影响的受治疗者的其他方面相同的位置或所述受试受治疗者的未受影响的区域的所述放射性核素的水平和位置进行对比，其中与所述放射性核素在来自未受影响的受治疗者或来自所述受试受治疗者的未受影响的区域的所述样品中的水平和位置相比，所述放射性核素在所述受试受治疗者体内的更高水平或不同位置表明所述受试受治疗者患有癌症，从而检测癌症、诊断癌症、监控癌症进展或监控癌症治疗。
附图说明
图1为本申请的一些实施例中APN21‐Bn‐SCN‐NOTA的电喷雾质谱图。
图2为本申请的一些实施例中68Ga‐APN21‐Bn‐SCN‐NOTA的放射HPLC图。
图3A为仅注射比较例68Ga‐CG6‐Bn‐SCN‐NOTA(CG6组)、仅注射比较例68Ga‐KE5‐Bn‐SCN‐NOTA(KE5组)、仅注射实施例68Ga‐APN21‐Bn‐SCN‐NOTA(APN21组)、以及共注射实施例68Ga‐APN21‐Bn‐SCN‐NOTA和未标记的NGR肽(APN21阻断组)的HT1080荷瘤小鼠，在注射0.5、1和2小时后的静态PET/CT图像。
图3B为仅注射比较例68Ga‐CG6‐Bn‐SCN‐NOTA(CG6组)、仅注射比较例68Ga‐KE5‐Bn‐SCN‐NOTA(KE5组)、仅注射实施例68Ga‐APN21‐Bn‐SCN‐NOTA(APN21组)以及共注射实施例68Ga‐APN21‐Bn‐SCN‐NOTA和未标记的NGR肽(APN21阻断组)的HT1080荷瘤小鼠，在注射0.5、1和2小时后的，根据68Ga‐APN21‐Bn‐SCN‐NOTA信号强度计算的肿瘤摄取量。
具体实施方式
本文使用单数术语指一个或多于一个。举例来说，“元件”或“一个元件”皆指一个元件或多于一个的元件。
如本文所述，术语“约”指近似，在大约或附近的范围。当结合数值范围使用术语“约”时，其通过扩大界限高于或低于所提供数值来修改该范围。一般来讲，本文使用术语“约”来使数值与所提供值上下变化10％。一方面，术语“约”指加上或减去其所修饰的数的数值的20％。举例来说，“约50％”指在45％–55％的范围内。本文通过端点提及的数值范围包括包含在该范围内的所有整数和分数(例如“1至5”包括1、1.5、2、2.75、3、3.90、4和5)。还应当理解其所有整数和分数视为被术语“约”修饰。
“包含”或“包括”旨在表示组合(例如装置、组合物、或方法等)包括所列举的要素(例如装置的各单元、组合物的各组分、或方法的实质性步骤等)，但不排除其他要素。当用于定义组合物和方法时，“基本上由……组成”意味着排除对于所述目的的组合具有任何重要意义的其他要素。因此，基本上由本文定义的要素组成的组合不排除不会实质上影响要求保护的本发明的基本和新颖特征的其他要素。“由……组成”是指排除其他要素的组合(单元组分和实质性的方法步骤。由这些过渡术语中的每一个定义的实施方案都在本发明的范围内。
术语“氨基酸”可与“氨基酸残基”互换地使用，且可指游离氨基酸和肽的氨基酸残基。从术语使用的上下文将清楚，其是指游离氨基酸还是肽的残基。如本文所述，“氨基酸”意在包括天然的和合成的氨基酸，并包括D‐型和L‐型氨基酸。“标准氨基酸”指通常发现于天然形成的肽中的二十个标准L‐氨基酸(包括甘氨酸)的任何一个。如本文所述的“D‐型”和“L‐型”氨基酸，除非另有说明，均非意图排除不具有手性的胺基酸，如甘氨酸。“非标准氨基酸残基”指除了标准氨基酸以外的任何氨基酸，无论其是否经合成制备或源自天然来源。如本文所用的，“合成氨基酸”还包括化学修饰的氨基酸，包括但不限于盐、氨基酸衍生物(例如酰胺)和取代物。可通过甲基化、酰胺化、乙酰化或用可改变肽的循环半衰期而不有害地影响其活性的其他化学基团取代来修饰包含在本发明的肽中的氨基酸，且特别是位于C端或N端的氨基酸。另外，本发明的肽中可存在或不存在二硫键。
如本文所述，术语“药物组合物”是指包含至少一种活性成分的组合物，其中对于在哺乳动物(例如但不限于人)中研究特定的、有效的结果，该组合物是可接受的。基于技术人员的需要，本领域普通技术人员将理解和了解适合确定活性成分是否具有期望的有效效果的技术。
如本文所述，术语“药学可接受的载体”指一种化学组合物，其可与合适的化合物或衍生物组合且组合后其可用于对受治疗者施用合适的化合物。
如本文所用的，术语“生理可接受的”酯或盐是指可与药物组合物的任何其他成分相容的活性成分的酯或盐形式，这种形式对待施用该组合物的受治疗者是无害的。
如本文所述，“药学可接受的”是指对于人或兽医应用是生理可耐受的。
如本文所述，“药物组合物”包括用于人和兽医用途的制剂。
如本文所述，“多个”是指至少两个。
如本文所述，“多核苷酸”是指核酸的单链或平行和反向平行链。因此，多核苷酸可以是单链或双链核酸。
如本文所述，“多肽”是指由通过肽键、其相关的天然形成的结构变体和其合成的非天然形成的类似物连接的氨基酸残基、其相关的天然形成的结构变体和合成的非天然形成的类似物组成的聚合物。
如本文所述，“合成肽或多肽”是指非天然形成的肽或多肽。例如，可以使用自动多肽合成仪合成合成肽或多肽。
如本文所述的“N端保护”指肽的末端氨基，与肽合成中常规采用的各种N端保护基中的任何一种偶联。如本文所述的“C端保护”指肽的末端氨基，与肽合成中常规采用的各种C端保护基中的任何一种偶联。
如本文所用，术语“高表达”分别表示受试者的表达水平高于正常个体的表达水平；特别是，受试者的生物样品中特定物质，例如特定的生物标记物或蛋白的值或水平，其高于从健康或野生型(正常)个体获得的生物样品中检测到的该特定物质的值或水平。如本文所用，术语“低表达”分别表示受试者的表达水平低于正常个体的表达水平；特别是，受试者的生物样品中特定物质，例如特定的生物标记物或蛋白的值或水平，其低于从健康或野生型(正常)个体获得的生物样品中检测到的该特定物质的值或水平。与生物标记物的“正常”表达水平或值相比，术语“高表达”和“低表达”可以指示“差异水平”或“差异值”或“不同表达”，并且可以包括表达水平中的定量差异和定性差异。
在一些实施方式中，所述X1是L‐天冬酰胺。在一些实施方式中，所述X1是D‐天冬酰胺。在一些实施方式中，所述X3是L‐精氨酸。在一些实施方式中，所述X3是D‐精氨酸。在一些实施方式中，所述X5是L‐赖氨酸。在一些实施方式中，所述X5是D‐赖氨酸。在一些实施方式中，所述X4是L‐苏氨酸。在一些实施方式中，所述X6是L‐酪氨酸。在一些实施方式中，所述X4是L‐苏氨酸，且所述X6是L‐酪氨酸。
在一些实施方式中，所述环肽为式(I)化合物或其衍生物。在一些实施方式中，所述环肽为式(I)化合物。
在一些实施方式中，所述环肽的制备方法还包括：将N端保护的所述X1的N端脱保护，得到所述X1；将N端保护的所述X2的N端脱保护，得到所述X2；将N端保护的所述X3的N端脱保护，得到所述X3；将N端保护的所述X4的N端脱保护，得到所述X4；将N端保护的所述X5的N端脱保护，得到所述X5；和/或将N端保护的所述X6的N端脱保护，得到所述X6。
在一些实施方式中，所述环肽的制备方法还包括：将N端保护且C端保护的所述X1的N端脱保护，得到C端保护的所述X1；将N端保护且C端保护的所述X2的N端脱保护，得到C端保护的所述X2；将N端保护且C端保护的所述X3的N端脱保护，得到C端保护的所述X3；将N端保护且C端保护的所述X4的N端脱保护，得到C端保护的所述X4；将N端保护且C端保护的所述X5的N端脱保护，得到C端保护的所述X5；和/或将N端保护且C端保护的所述X6的N端脱保护，得到C端保护的所述X6。
在一些实施方式中，所述环肽的制备方法还包括：将N端保护且C端保护的所述X1的C端脱保护，得到N端保护的所述X1；将N端保护且C端保护的所述X2的C端脱保护，得到N端保护的所述X2；将N端保护且C端保护的所述X3的C端脱保护，得到N端保护的所述X3；将N端保护且C端保护的所述X4的C端脱保护，得到N端保护的所述X4；将N端保护且C端保护的所述X5的C端脱保护，得到N端保护的所述X5；和/或将N端保护且C端保护的所述X6的C端脱保护，得到N端保护的所述X6。
在一些实施方式中，所述环肽的制备方法还包括：将C端保护的所述X1的C端脱保护，得到所述X1；将C端保护的所述X2的C端脱保护，得到所述X2；将C端保护的所述X3的C端脱保护，得到所述X3；将C端保护的所述X4的C端脱保护，得到所述X4；将C端保护的所述X5的C端脱保护，得到所述X5；和/或将C端保护的所述X6的C端脱保护，得到所述X6。
在一些实施方式中，所述环肽的制备方法还包括：将C端偶联固相材质的所述X1的C端与固相物质分离，得到所述X1；将C端偶联固相材质的所述X2的C端与固相物质分离，得到所述X2；将C端偶联固相材质的所述X3的C端与固相物质分离，得到所述X3；将C端偶联固相材质的所述X4的C端与固相物质分离，得到所述X4；将C端偶联固相材质的所述X5的C端与固相物质分离，得到所述X5；或将C端偶联固相材质的所述X6的C端与固相物质分离，得到所述X6。
在一些实施方式中，所述X1的C端与所述X2的N端进行缩合反应，包括使N端保护的所述X1、所述X2、HBTU和DIEA接触。在一些实施方式中，所述X2的C端与所述X3的N端进行缩合反应，包括使N端保护的所述X2、所述X3、HBTU和DIEA接触。在一些实施方式中，所述X3的C端与所述X4的N端进行缩合反应，包括使N端保护的所述X3、所述X4、HBTU和DIEA接触。在一些实施方式中，所述X4的C端与所述X5的N端进行缩合反应，包括使N端保护的所述X4、所述X5、HBTU和DIEA接触。在一些实施方式中，所述X5的C端与所述X6的N端进行缩合反应，包括使N端保护的所述X5、所述X6、HBTU和DIEA接触。在一些实施方式中，所述X6的C端与所述X1的N端进行缩合反应，包括使N端保护的所述X6、所述X1、HBTU和DIEA接触。
在一些实施方式中，N端保护是Fmoc保护。在一些实施方式中，固相材质为树脂。
在一些实施方式中，所述复合物螯合放射性核素。在一些实施方式中，所述放射性核素包括选自由44Sc、47Sc、62Cu、64Cu、67Cu、66Ga、67Ga、68Ga、86Y、90Y、89Zr、99mTc、110mIn、111In、113mIn、114mIn、177Lu、203Pb、212Pb、212Bi、213Bi、和225Ac所组成的组中的至少一个或由其组成。在一些实施方式中，所述放射性核素包括选自由44Sc、47Sc、64Cu、67Cu、67Ga、68Ga、90Y、99mTc、111In、177Lu、212Pb、213Bi、和225Ac所组成的组中的至少一个或由其组成。在一些实施方式中，所述放射性核素包括选自由99mTc、68Ga、和64Cu所组成的组中的至少一个或由其组成。在一些实施方式中，所述连接子包括聚乙二醇(PEG)或由其组成。在一些实施方式中，所述放射性核素包括68Ga。在一些实施方式中，所述螯合剂包括选自由：1,4,7,10‐四氮杂环十二烷‐N,N′,N″,N″′‐四乙酸(1,4,7,10‐tetraazacyclododecane‐N,N′,N″,N″′‐tetraacetic acid，DOTA)、1,4,7‐三氮杂环壬烷‐N,N′,N″‐三乙酸(1,4,7‐triazacyclononane‐Ν,Ν′,Ν″‐triacetic acid，NOTA)、二亚乙基三胺‐N,N,N′,N″,N″‐五乙酸(diethylenetriamine‐N,N,N′,N″,N″‐pentaacetic acid，DTPA)、1,4,8,11‐四氮杂环十四烷‐1,4,8,11‐四乙酸(1,4,8,11‐tetraazacyclotetradecane‐1,4,8,11‐tetraacetic acid，TETA)、2,2′‐((6‐氨基‐1‐(4,7‐双(羧甲基)‐1,4,7‐三氮烷‐1‐基)己烷‐2‐基)氮杂二酰基)二乙酸(2,2′‐((6‐amino‐1‐(4,7‐bis(carboxymethyl)‐1,4,7‐triazonan‐1‐yl)hexan‐2‐yl)azanediyl)diacetic acid，NETA)、1,4,7,10‐四氮杂环十二烷‐1,4,7‐三乙酸(1,4,7,10‐tetraazacyclododecane‐1,4,7‐triacetic acid，DO3A)、亚乙基双(邻羟基苯基)甘氨酸(ethylenebis(o‐hydroxyphenyl)glycine，EHPG)、N,N′‐双(2‐羟基苄基)乙二胺‐N,N′‐二乙酸(N,N′‐bis(2‐hydroxybenzyl)ethylenediamine‐N,N′‐diacetic acid，HBED)、1,4,7,10‐四氮杂环十二烷‐α,α′,α″,α″′‐四甲基‐N,N′,N″,N″′‐四乙酸(1,4,7,10‐tetraazacyclododecane‐α,α′,α″,α″′‐tetramethyl‐N,N′,N″,N″′‐tetraacetic acid，DOTMA)、1,4,8,11‐四氮杂环十四烷‐1,4,8,11‐(甲基四乙酸)(1,4,8,11‐tetraazacyclotetradecane‐1,4,8,11‐(methyl tetraacetic acid)，TETMA)、乙二胺四乙酸(ethylenediamine tetraacetic acid，EDTA)、1,3‐丙二胺四乙酸(1,3‐propylenediaminetetraacetic acid，PDTA)、三乙烯四胺六乙酸(triethylenetetraaminehexaacetic acid，TTHA)、1,5,10‐N,N′,N″‐三(2,3‐二羟基苯甲酰基)‐三邻苯二酚(1,5,10‐N,N′,N″‐tris(2,3‐dihydroxybenzoyl)‐tricatecholate，LICAM)、1,3,5‐N,N′,N″‐三(2,3‐二羟基苯甲酰基)氨基甲基苯(1,3,5‐N,N′,N″‐tris(2,3‐dihydroxybenzoyl)aminomethylbenzene，MECAM)、及6‐肼烟酸(6‐hydrazinonicotinic acid，HYNIC)，以及其减少一个或更多个氢离子所形成的离子所组成的组中的至少一个或由其组成。
在一些实施方式中，环肽与连接子偶联。在一些实施方式中，环肽中的X5与连接子偶联。在一些实施方式中，环肽中的赖氨酸，特别是X5的赖氨酸，特别是X5的赖氨酸的ε‐氨基，与连接子偶联。在一些实施方式中，连接子与螯合剂偶联。在一些实施方式中，连接子与环肽和螯合剂偶联，特别是连接子的第一端与环肽偶联，连接子的不同于第一端的第二端与螯合剂偶联。
在一些实施方式中，所述癌症是CD13高表达的肿瘤。在一些实施方式中，所述癌症选自由头颈癌、肝癌、胰腺癌、食道癌、胃癌、肺癌、乳腺癌、卵巢癌、子宫癌、子宫内膜癌、宫颈癌、前列腺癌、肾上腺癌、淋巴瘤、唾液腺癌、骨癌、脑癌、小脑癌、结肠癌、直肠癌、结肠直肠癌、口鼻咽癌、肾癌、膀胱癌、皮肤癌、黑色素瘤、基底细胞癌、硬腭癌、舌鳞状细胞癌、脑膜瘤、多形性腺瘤、星形细胞瘤、软组织肉瘤、软骨肉瘤、皮质腺瘤、间皮瘤、鳞状细胞癌和腺癌所组成的组。在一些实施方式中，所述癌症选自由乳腺癌、卵巢癌、甲状腺癌、胰腺癌、结直肠癌、非小细胞肺癌和骨肉瘤所组成的组。在一些实施方式中，所述癌症选自由食道癌、胰腺癌和胃癌所组成的组。
开发全新结构的NGR环肽，使其具备更高的体内稳定性、对CD13受体具有更强的亲和力，具有更强的靶向性，并且通过放射性核素标记后能够在体内准确定位CD13受体，通过PET显像实现肿瘤分子影响诊断目的，
通过标记治疗性放射性核素，实现肿瘤靶向治疗目标。
提供一种新型结构的NGR环肽，及利用该多肽进行放射性药物的制备与应用。在一些实施方式中，本申请的放射性药物及其标记技术，能够用于靶向新生血管中CD13受体的恶性肿瘤的分子显像和治疗。在一些实施方式中，本申请的受体靶向性好、稳定性强、标记简单，具有较高的应用价值。
为更进一步阐述本申请为了达成预定目的所采取的技术手段及功效，以下结合附图及较佳实施例，对依据本申请的具体实施方式、结构、特征及其功效，详细说明如下。
环肽的合成
使用固相连续流多肽全自动合成仪，从序列C端到N端合成多肽。
称取1n当量带9‐芴甲氧羰基(Fmoc)保护基的第一个氨基酸(C端氨基酸)CTC树脂放入反应器，加入二氯甲烷(DCM)溶胀半小时。抽掉DCM，加入2％哌啶N,N′‐二甲基甲酰胺(DMF)溶液去除树脂上第一个氨基酸的N端的Fmoc保护基。在一些实施方式中，序列为cyclo(X1X2X3X4X5X6)的环肽中，X1、X2、X3、X4、X5、和X6的任意一个可为所述的第一个氨基酸。
向反应器泵入3n当量的下一个氨基酸、3n当量HBTU(O‐苯并三氮唑‐N,N,N′,N′‐四甲基脲六氟磷酸盐)及10n当量DIEA(N,N‐二异丙基乙基胺)，连续回流进行缩合反应5min。然后用DMF洗涤反应器中的树脂4次洗净。加入2％哌啶DMF溶液去除下一个氨基酸的N端的Fmoc保护基，DMF洗涤4次洗净。如此依次完成多肽序列。
将树脂用氮气吹干后从反应柱中取下，倒入烧瓶中，然后往烧瓶中加一定量(每g树脂约10mL切割液)的切割液(由30％三氟乙醇和70％二氯甲烷组成)，震荡，滤掉树脂。
得到滤液，然后向滤液中加入大量乙醚析出粗产物，离心、清洗，得到带有保护基的线性多肽序列粗产物。
将保护肽段用DCM溶解，加入2n当量的PyBop，10n当量的DIEA，45摄氏度回流反应过夜，旋转蒸发仪旋去溶剂，得到带有保护基的环肽。
配制保护基切割液(由95％三氟乙酸(TFA)、2％乙二硫醇、2％三异丙基硅烷、1％水组成)，加入上步环肽容器中，震荡反应120min。
得到滤液，然后向滤液中加入大量乙醚析出粗产物，离心、清洗，得到目标环肽序列粗产物。
使用高效液相色谱(HPLC)纯化多肽序列粗产物，冻干，并使用液相色谱–质谱联用(LC‐MS)确认目标产物分子量。
环肽与CD13蛋白间的亲和力评价
通过细胞摄取研究，确定实施例以及比较例多肽在HT‐1080细胞系中的结合亲和力。
人纤维肉瘤细胞系HT‐1080的培养
人纤维肉瘤细胞系HT‐1080在混有10％无菌过滤胎牛血清(FBS)和1％青霉素–链霉素抗生素的Gibco的Dulbecco改良Eagle培养基(DMEM)中培养。细胞培养保持在温度37℃、5％CO2的控制环境中，每3–4天进行一次传代培养。在实验前24小时，将细胞接种在96孔板中(每孔5000个细胞)，培养过夜至细胞贴壁。实验前，用1mL的PBS平衡盐溶液清洗细胞两次，去除生长培养基。
Cy3荧光肽的制备
实施例1–28的环肽(序列分别如SEQ ID NO:1–28所示，以下分别称APN1–APN28，合称APN)或比较例1的环肽(序列如SEQ ID NO:29所示，以下称KE5)在DMF溶液中与Cy3‐NHS荧光染料缩合反应，通过HPLC制备纯化得到实施例1–28的APN‐Cy3荧光肽或比较例1的KE5‐Cy3荧光肽。
荧光肽处理HT‐1080细胞
除空白对照组外，按浓度梯度设立6个比较例1的KE5‐Cy3荧光肽处理组，分别为：3.125μmol/L、6.25μmol/L、12.5μmol/L、25μmol/L、50μmol/L、100μmol/L组，每个组6个孔。
用比较例‐Cy3荧光肽处理HT‐1080细胞。
37℃孵育30min后用PBS洗2次，荧光酶标仪(发射波长488nm、激发波长520nm)读数检测细胞内荧光强度变化来评估HT‐1080细胞摄取KE5‐Cy3荧光肽的情况。
每组细胞内加入CCK‐8 10μL孵育细胞4h，再用酶标仪读数(波长为490nm)，通过CCK‐8校正每组荧光强度因细胞数量差异而导致的误差。
各浓度组重复3次。测定得到HT‐1080细胞摄取KE5‐Cy3荧光肽的最佳浓度。
用每个实施例的APN‐Cy3荧光肽处理HT‐1080细胞，其中每个APN‐Cy3荧光肽的浓度是根据HT‐1080细胞摄取比较例的KE5‐Cy3荧光肽的最佳浓度。
37℃孵育30min后用PBS洗2次，荧光酶标仪(发射波长488nm、激发波长520nm)读数检测细胞内荧光强度变化来评估HT‐1080细胞摄取每个APN‐Cy3荧光肽的情况。
每组细胞内加入CCK‐8 10μL孵育细胞4h，再用酶标仪读数(波长为490nm)，通过CCK‐8校正每组荧光强度因细胞数量差异而导致的误差。
以如下公式计算每个APN‐Cy3荧光肽与KE5‐Cy3荧光肽之间的相对细胞摄取率(Relative cell uptake，RCU)。
请参照表1，为申请人合成的比较例1的环肽以及实施例1–28的环肽的具体序列，以及计算出的每个实施例的APN‐Cy3荧光肽相较于比较例1的KE5‐Cy3荧光肽的相对细胞摄取率(即，以KE5‐Cy3荧光肽的细胞摄取率为1.00计算出的倍数值)。
[表1]环肽具体序列以及荧光肽的相对细胞摄取率

在表1中：Orn表示鸟氨酸；Dab表示2,4‐二氨基丁酸；Sar表示肌氨酸；Nme表示N‐甲基‐天冬酰胺；各小写字母代表D‐氨基酸。
可以看到，实施例2、实施例3、实施例4、实施例5、实施例6、实施例9、实施例15、实施例16、实施例18、实施例19、实施例21、以及实施例22，其APN‐Cy3荧光肽的相对细胞摄取率至少为比较例的KE5‐Cy3荧光肽的细胞摄取率的2倍以上，其中实施例9、实施例15实施例19、实施例21、以及实施例22，其APN‐Cy3荧光肽的相对细胞摄取率至少为KE5‐Cy3荧光肽的细胞摄取率的4倍以上。实施例9的APN21‐Cy3荧光肽的相对细胞摄取率为KE5‐Cy3荧光肽的细胞摄取率的约5.03倍，而实施例21的APN21‐Cy3荧光肽的相对细胞摄取率为KE5‐Cy3荧光肽的细胞摄取率的约5.68倍，代表这些环肽能够特别有效地被细胞摄取。
从表1可见，对于序列为cyclo(X1X2X3X4X5X6)的环肽：
–X1可以是天冬酰胺，相较于X1是N‐甲基‐天冬酰胺，其Cy3荧光肽可以获得较高的相对细胞摄取率。
–X2可以是甘氨酸或肌氨酸，可以获得较高的相对细胞摄取率。其中X2尤其可以是肌氨酸，其Cy3荧光肽可以获得更高的相对细胞摄取率。
–X4可以选自由苏氨酸、酪氨酸、和苯丙氨酸所組成的組，可以获得较高的相对细胞摄取率。其中X4特别可以是苏氨酸，更特别可以是L‐苏氨酸，其Cy3荧光肽可以获得更高的相对细胞摄取率。
–X5可以是赖氨酸，相较于X5是鸟氨酸、2,4‐二氨基丁酸、或半胱氨酸，其Cy3荧光肽可以获得较高的相对细胞摄取率。
–X6可以选自由酪氨酸、缬氨酸、和谷氨酸所組成的組，可以获得较高的相对细胞摄取率。其中X6特別可以是酪氨酸，更特別可以是L‐酪氨酸，其Cy3荧光肽可以获得更高的相对细胞摄取率。
稳定性研究
实验采用体外恒温(37℃)孵育法研究实施例21的环肽APN21和比较例1的环肽KE5(1.0μg/mL)在小鼠血浆或PBS中的稳定性。上述两种环肽溶解在1mL小鼠血清中，体外恒温孵育0h、0.5h、1h、4h、24h和48h，然后测定药物剩余百分率。采用PBS作为阴性对照组。采用LC‐MS/MS法测定药物与内标的峰面积，用其比值代替药物浓度进行计算。
结果如表2。
[表2]实施例21和比较例的环肽在小鼠血浆或PBS中体外恒温孵育后的药物剩余百分率

可以看到，相较于比较例1的环肽，实施例21的环肽在小鼠血浆或PBS中体外恒温孵育后的药物剩余百分率明显较高，也就是说其稳定性明显较高。
动物实验
1.APN21‐Bn‐SCN‐NOTA的合成
1)称取实施例21的环肽(APN21)3.8mg(5.18μmol)，用50μL的DMF溶解，得到APN21的DMF溶液。将6.5mg的N,N‐二异丙基乙胺(DIEA)(52μmol)添加到以上APN21溶液中。
2)称取NOTA‐Bn‐SCN酯三盐酸盐2.9mg(5.18μmol)，用50μL DMF溶解。
3)在振荡条件下将NOTA‐Bn‐SCN溶液缓慢滴加至APN21溶液中，将反应混合物在室温下搅拌4小时。
4)使用高效液相色谱层析(HPLC)分离产物，固定相为半制备C18柱，流动相采用梯度洗脱法，流速为4mL/min，并在18分钟内从5％乙腈变为50％乙腈。
5)通过高效液相色谱和质谱分析鉴定产物。电喷雾质谱(ESI‐MS)测定结果：m/z[M+H]+＝1184.87(化学式：C53H82N12O16，计算分子量1183.54)，质谱图具体如图1；
6)将目标产物馏分冻干过夜处理。
2.APN21‐PEG4‐DOTA的68Ga标记程序
1)用1.5mL的0.25M醋酸钠溶液溶解0.75μmol的APN21‐Bn‐SCN‐NOTA。
2)采用4mL 0.05M盐酸溶液洗脱锗镓发生器，制备淋洗液68GaCl3。
3)取1mCi活度的淋洗液，加入到APN21‐Bn‐SCN‐NOTA溶液中，置60℃加热器反应10min，获得68Ga‐APN21‐Bn‐SCN‐NOTA缀合物。所得到的混合物，通过放射高效液相色谱进行监测和定量标记，纯度>97％，放射性色谱图具体如图2。
此外，另外以上述制备实施例的68Ga‐APN21‐Bn‐SCN‐NOTA缀合物的方法对应制备比较例1的68Ga‐KE5‐Bn‐SCN‐NOTA缀合物和比较例2的68Ga‐CG6‐Bn‐SCN‐NOTA缀合物(此处“CG6”意指序列为cyclo(CNGRC)的环肽，如SEQ ID NO:30所示)。
3.人纤维肉瘤异种移植瘤模型构建
正常NCr裸鼠(18–25g，4–6周龄，n＝3)取HT‐1080细胞，于200μL磷酸盐缓冲液和基质凝胶(v/v，1/1)混合液中，植入2×106个细胞于小鼠右肩皮下。在平均1.5周之后，肿瘤直径约10mm，已经足以进行生物分布和PET成像研究。
4.小型动物的正电子发射型计算机断层显像(PET/CT)
PET/CT和图像分析使用小型动物NovelMedcal PET/CT扫描仪(北京永新)进行。其在视野中心的切向和径向半宽最大值为1.5mm，在视野边缘的切向和径向半宽最大值为1.8mm。
对于CG6组，在异氟醚麻醉下，将约3.7MBq(100μCi)的比较例2的68Ga‐CG6‐Bn‐SCN‐NOTA化合物通过尾静脉注射至HT1080荷瘤小鼠体内。静脉注射0.5、1和2小时后，分别获得15分钟的静态PET/CT图像。
对于KE5组，在异氟醚麻醉下，将约3.7MBq(100μCi)的比较例1的68Ga‐KE5‐Bn‐SCN‐NOTA化合物通过尾静脉注射至HT1080荷瘤小鼠体内。静脉注射0.5、1和2小时后，分别获得15分钟的静态PET/CT图像。
对于APN21组，在异氟醚麻醉下，将约3.7MBq(100μCi)的实施例21的68Ga‐APN21‐Bn‐SCN‐NOTA化合物通过尾静脉注射至HT1080荷瘤小鼠体内。静脉注射0.5、1和2小时后，分别获得15分钟的静态PET/CT图像。
对于APN21阻断组，将约3.7MBq(100μCi)的实施例21的68Ga‐APN21‐Bn‐SCN‐NOTA和未标记的NGR肽(每个肽15mg/kg)共注射至HT1080荷瘤小鼠(每组n＝3)0.5、1和2小时后，分别获得15分钟的静态PET/CT图像。
请参照图3A和图3B，为仅注射68Ga‐CG6‐Bn‐SCN‐NOTA(CG6组)、仅注射68Ga‐KE5‐Bn‐SCN‐NOTA(KE5组)、仅注射68Ga‐APN21‐Bn‐SCN‐NOTA(APN21组)以及共注射68Ga‐APN21‐Bn‐SCN‐NOTA和未标记的NGR肽(APN21阻断组)的HT1080荷瘤小鼠，在注射0.5、1和2小时后的静态PET/CT图像，以及根据68Ga‐APN21‐Bn‐SCN‐NOTA信号强度计算的肿瘤摄取量。如图3A和3B所示，在APN21阻断组中，68Ga‐APN21‐Bn‐SCN‐NOTA的信号强度相较于未阻断的APN21组明显较弱，而更接近KE5组的信号强度，因此可以证实在NGR肽阻断CD13受体时68Ga‐APN21‐Bn‐SCN‐NOTA的作用靶点被阻断，亦即证明68Ga‐APN21‐Bn‐SCN‐NOTA对CD13受体的靶向性。
PET和CT图像利用NMSoft工作站软件(北京永新)进行采集，数据以每克组织或器官的注射剂量百分比(ID/g)给出，并通过每个样本的衰变校正(标准化到代表注射剂量的已知重量)来确定。
上述实施方式仅为本申请的优选实施方式，不能以此来限定本申请保护的范围，本领域的技术人员在本申请的基础上所做的任何非实质性的变化及替换均属于本申请所要求保护的范围。

Claims (10)

  1. 一种环肽，其特征在于，序列为cyclo(X1X2X3X4X5X6)，其中，
    所述X1为天冬酰胺；
    所述X2为甘氨酸或肌氨酸；
    所述X3为精氨酸；
    所述X4选自由苏氨酸、酪氨酸、和苯丙氨酸所組成的組；
    所述X5为赖氨酸；且
    所述X6选自由酪氨酸、缬氨酸、和谷氨酸所組成的組。
  2. 如权利要求1所述的环肽，其特征在于，
    所述X4为L‐苏氨酸或D‐苏氨酸；且/或
    所述X6为L‐酪氨酸或D‐酪氨酸。
  3. 如权利要求1或2所述的环肽，其特征在于，
    所述X1为L‐天冬酰胺；
    所述X3为L‐精氨酸；
    所述X4为L‐苏氨酸；
    所述X5为L‐赖氨酸；且/或
    所述X6为L‐酪氨酸；
    可选地，所述X1为L‐天冬酰胺，所述X2为肌氨酸，所述X3为L‐精氨酸，所述X4为L‐苏氨酸，所述X5为L‐赖氨酸，且所述X6为L‐酪氨酸；
    可选地，所述X1为L‐天冬酰胺，所述X2为甘氨酸，所述X3为L‐精氨酸，所述X4为L‐苏氨酸，所述X5为L‐赖氨酸，且所述X6为L‐酪氨酸；或
    可选地，所述X1为L‐天冬酰胺，所述X2为肌氨酸，所述X3为L‐精氨酸，所述X4为L‐苏氨酸，所述X5为L‐赖氨酸，且所述X6为L‐酪氨酸。
  4. 一种复合物，包括：
    权利要求1至3任一所述的环肽；
    连接子，偶联所述环肽；以及
    螯合剂，偶联所述连接子。
  5. 如权利要求4所述的复合物，其特征在于，
    所述连接子为聚乙二醇；且/或
    所述螯合剂包括选自由：1,4,7,10‐四氮杂环十二烷‐N,N′,N″,N″′‐四乙酸、1,4,7‐三氮杂环壬烷‐N,N′,N″‐三乙酸、二亚乙基三胺‐N,N,N′,N″,N″‐五乙酸、1,4,8,11‐四氮杂环十四烷‐1,4,8,11‐四乙酸、2,2′‐((6‐氨基‐1‐(4,7‐双(羧甲基)‐1,4,7‐三氮烷‐1‐基)己烷‐2‐基)氮杂二酰基)二乙酸、1,4,7,10‐四氮杂环十二烷‐1,4,7‐三乙酸、亚乙基双(邻羟基苯基)甘氨酸、N,N′‐双(2‐羟基苄基)乙二胺‐N,N′‐二乙酸、1,4,7,10‐四氮杂环十二烷‐α,α′,α″,α″′‐四甲基‐N,N′,N″,N″′‐四乙酸、1,4,8,11‐四氮杂环十四烷‐1,4,8,11‐(甲基四乙酸)、乙二胺四乙酸、1,3‐丙二胺四乙酸、三乙烯四胺六乙酸、1,5,10‐N,N′,N″‐三(2,3‐二羟基苯甲酰基)‐三邻苯二酚、1,3,5‐N,N′,N″‐三(2,3‐二羟基苯甲酰基)氨基甲基苯、及6‐肼烟酸，以及其失去一个或更多个氢离子所形成的离子，所组成的组中的至少一个或由其组成，
    可选地，所述螯合剂包括1,4,7,10‐四氮杂环十二烷‐N,N′,N″,N″′‐四乙酸。
  6. 如权利要求1至3任一所述的环肽、或权利要求4至5任一所述的复合物，在放射性核素标记、制备放射性核素标记试剂、或制备药物载体中的用途。
  7. 一种放射性核素制剂，其特征在于，包括：
    权利要求4至5任一所述的复合物；以及
    放射性核素，与所述复合物的螯合剂螯合。
  8. 如权利要求7所述的放射性核素制剂，其特征在于，所述放射性核素选自由44Sc、47Sc、64Cu、67Cu、67Ga、68Ga、99mTc、90Y、111In、177Lu、212Pb、213Bi、和225Ac所组成的组中的至少一个。
  9. 如权利要求1至4任一所述的环肽、或权利要求5至6任一所述的复合物、或如权利要求7至8任一所述的放射性核素制剂，在制备检测癌症、诊断癌症、监控癌症进展、监控癌症治疗或治疗癌症的药物中的用途；
    可选地，所述癌症选自由头颈癌、肝癌、胰腺癌、食道癌、胃癌、肺癌、乳腺癌、卵巢癌、子宫癌、子宫内膜癌、宫颈癌、前列腺癌、肾上腺癌、淋巴瘤、唾液腺癌、骨癌、脑癌、小脑癌、结肠癌、直肠癌、结肠直肠癌、口鼻咽癌、肾癌、膀胱癌、皮肤癌、黑色素瘤、基底细胞癌、硬腭癌、舌鳞状细胞癌、脑膜瘤、多形性腺瘤、星形细胞瘤、软组织肉瘤、软骨肉瘤、皮质腺瘤、间皮瘤、鳞状细胞癌和腺癌所组成的组。
  10. 如权利要求9所述的用途，其特征在于，
    所述癌症为CD13高表达；且/或
    所述癌症选自由乳腺癌、卵巢癌、甲状腺癌、胰腺癌、结直肠癌、非小细胞肺癌和骨肉瘤所组成的组。
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