WO2019100973A1 - 一种谷氨酸多肽-雌激素/抗雌激素缀合物、合成方法、组合物及试剂盒 - Google Patents

一种谷氨酸多肽-雌激素/抗雌激素缀合物、合成方法、组合物及试剂盒 Download PDF

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WO2019100973A1
WO2019100973A1 PCT/CN2018/115290 CN2018115290W WO2019100973A1 WO 2019100973 A1 WO2019100973 A1 WO 2019100973A1 CN 2018115290 W CN2018115290 W CN 2018115290W WO 2019100973 A1 WO2019100973 A1 WO 2019100973A1
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estrogen
polypeptide
glutamate
conjugate
antiestrogens
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PCT/CN2018/115290
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English (en)
French (fr)
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金志明
杨敬文
井上登美夫
西蒙⋅塞尔吉奥·丹尼尔
石明启
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金志明
杨敬文
井上登美夫
西蒙⋅塞尔吉奥·丹尼尔
石明启
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Publication of WO2019100973A1 publication Critical patent/WO2019100973A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • 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

Definitions

  • the present invention relates to a novel method for molecular imaging and therapeutic conjugates and their synthesis, as well as compositions formed using the conjugates and kits therefor.
  • the conjugate is based on individual genetic composition, biochemical and molecular design related to the disease of each patient, and can be used for personalized treatment, and belongs to the fields of chemical synthesis, labeling, imaging, drug treatment, treatment of ER-positive diseases, and cancer treatment.
  • ROS reactive oxygen species
  • GSH glutathione
  • TXN thioredoxin
  • catalase catalase
  • All three pathways contain key enzymes that inhibit the antioxidant response in cancer cells.
  • GSH is affected by elevated glutamate and cysteine transport subsystems as well as NADPH.
  • GSH is derived from the metabolites glutamate and cysteine.
  • Glutamicase 1 (GLS1) and GLS2 produce glutamate, while the cystine/glutamate transporter XCT passes the glutamate-cysteine ligase modifier (glutamate–cysteine ligase modifier)
  • the subunit (GCLM) and the GCL catalytic subunit (GCLC) provide a cysteine that produces GSH.
  • GSH acts directly on the elimination of ROS through the action of glutathione peroxidase and glutathione s-transferase [see: Chen H, Tang X, Zhou B, Zhou Z, Xu N, Wang YA ROS-mediated mitochondrial Pathway and Nrf2 pathway activation are involved in BDE-47 induced apoptosis in Neuro-2a cells.
  • Poly-L-glutamic acid (PG) has been used as a tumor-targeting drug carrier for a variety of hydrophobic cancer chemotherapeutic agents [see: Batz HG, Ringsdorf H, Ritter H. Pharmacologically active polymers, 7. Cyclophosphamide-and steroid hormone containing polymers As potential anticancer compounds.Die Makromolekulare Chemie.1974;175(8):2229–2239.;Hurwitz E,Wilchek M,Pitha J.Soluble macromolecules as carriers for daunorubicin.J Appl Biochem.1980;2(1):25– 35.; Kato Y, Saito M, Fukushima H, Takeda Y, Hara T.
  • PG has additional advantages such as excellent water solubility, biocompatibility, non-immunogenicity, biodegradability to glutamate, and high drug loading capacity due to its multiple carboxyl groups [see: McCormick-Thomson LA ,Sgouras D,Duncan R.Poly(amino acid)copolymers as a potential soluble drug delivery system.2.Body distribution and preliminary biocompatibility testing in vitro and in vivo.J Bioact Compat Polym.1989;4(3):252–268 Sumi H, Kawabe K, Nakajima N.
  • reagents need to achieve high specific activity (Ci/umol). If the labeled reagent is contaminated by a different molecular weight, the image quality assurance will be problematic. In addition, it is not known at which site the drug is bound to the PG. This detailed GMC information is usually required for compliance.
  • the present invention discloses that a molecule is first reacted with a glutamate followed by a glutamic acid peptide. The final structure is to position the bound molecule to the first glutamate of the glutamate peptide.
  • the excitatory amino acid glutamate exerts its action via a variety of glutamate receptors. Metabolic glutamate receptors have been reported to interact with the membrane estrogen receptor (ER), and more specifically with the mGlu5 receptor subtype. 17 ⁇ -estradiol and mGlu5 antagonists in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methyl-4-phenyl-1,2,3) with Parkinson's disease , 6-tetrahydropyridine, MPTP) has a neuroprotective effect in a mouse model [see: Al-Sweidi S, Morissette M, Di Paolo T.
  • estradiol an estrogen receptor ligand
  • Glutamate peptides are known to stimulate bone resorption in vitro and specifically act on glutamate receptors [see: Chenu C, Serre CM, Raynal C, Burt-Pichat B, Delmas PD.
  • Glutamate receptors are expressed by bone cells and are Involved in bone resorption.Bone.1998;22(4):295-9.]. Glutamate peptides have demonstrated their ability to target kidney tissue [see: Chai HJ, Kiew LV, Chin Y, et al. Renal targeting potential of a polymeric drug carrier, poly-l-glutamic acid, in normal and diabetic rats. J Nanomedicine.2017;12:577-591.doi:10.2147/IJN.S111284.]. It is suitable to bind estradiol (EDL) to GAP, and GAP-EDL can enhance the binding efficacy of ER in cells.
  • EDL estradiol
  • GAP can chelate radiometallic isotopes for imaging and radiation therapy applications.
  • 99mTc-GAP-EDL and 68Ga-GAP-EDL have previously been reported to be useful compounds in tomography [see: Takahashi N, Yang DJ, Kohanim S, Oh CS, Yu DF, Azhdarinia A, Zhang XC, Chang JY, Kim EE. Targeted functional imaging of estrogen receptors with 99mTc-GAP-EDL.Eur J Nucl Med Mol Imaging.2007;34:354-362.;Takahashi N,Yang DJ,Kurihara H,Borne A,Kohanim S,Oh CS, Mawlawi O, Kim EE. Functional imaging of estrogen receptors with radiolabeled GAP-EDL in rabbits endometriosis model. Academic Radiology 2007; 14(9): 1050-7.].
  • Estrogen causes a genomic effect via alpha and beta estrogen receptors located primarily in the nucleus.
  • the role of ER ⁇ in mediating gene transcription is well documented in the literature, and studies using mouse models and human breast (cancer) cell lines have demonstrated the role of ER ⁇ in cell proliferation. In contrast, the role of ER ⁇ as a transcriptional regulator is not clear. Studies have revealed that ER ⁇ may reduce the activity of ER ⁇ by heterodimerization [see: Speirs V, Carder PJ, Lane S, Dodwell D, Lansdown MR, Hanby AM: Oestrogen receptor beta: what it means for patients with breast cancer.
  • ER mediates inhibition of NF- ⁇ B activity at several levels.
  • the interaction between these important regulators between these hormones and the immune system can be used to treat cancer, inflammatory and autoimmune diseases [see: Sas L, Lardon F, Vermeulen PB, Hauspy J, et al. The interaction between ER and NF ⁇ B in resistance to endocrine therapy. Breast Cancer Research 2012, 14:212].
  • estradiol is bound to PG by aqueous purification (Yang DJ, Oh CS, Kohanim S, Yu DF, Azhdarinia A, Kim EE. Poly (peptide) as a chelator: methods of manufacture and Uses. US Patent Application 20060246005, WO 2006107794 A2.), but with low cellular uptake, which may be due to its purity issues.
  • GAP-EDL glutamate peptide-estradiol
  • the discovery of estrogen receptors in binding and response to individual hormonal pathways benefits patients in the treatment of ROS, inflammation and cancer caused by oxidative stress. During morbid conditions, glutamate receptors are overexpressed due to the high demand for GSH.
  • Glutamate peptide is known to bind to a glutamate receptor/transporter (Yang DJ et al., U.S. Patent Application No. US20060246005A1; WO 2006107794 A2).
  • the overexpressed glutamate system enhances cellular uptake of labeled GAP-EDL. Once GAP-EDL enters the cell, GAP-EDL can target the ER gene.
  • the present invention describes the use of glutamate peptide-estrogen conjugates for precise ER positive (ER+) imaging and treatment.
  • ER+ ER positive
  • Molecular imaging of the ER+ pathway activation system serves as the basis for the management of hormonal-dependent diseases such as cancer and endometriosis.
  • ER+ patients respond better to endocrine therapy and have twice as long survival as ER-patients.
  • the tumor resistance results of clinically used aromatase inhibitors and antiestrogens are unpredictable.
  • Radiolabeled ER+ ligands are capable of quantifying ER+ tissue uptake, thereby facilitating staging and re-segmentation of cancer and endometriosis.
  • the differential diagnosis of ER+ lesions using labeled ligands helps to select patients who have the best response to endocrine therapy and to discontinue treatment when drug resistance occurs.
  • radiolabeled ER+ ligands serve as the basis for image-oriented response follow-up.
  • Existing literature reports have shown that strategies using polyglutamate as a drug carrier can alter drug solubility and potentiate estrogen receptor-ligand binding pockets. This technique helps to target intracellular genes through glutamate receptor-mediated processes.
  • the polyglutamate described is a blend of mixed polymers having a wide range of molecular weights. Thus, the purity of the product is not optimized.
  • the purification of the preparation may sometimes be problematic.
  • Purification under aqueous conditions can be achieved, for example, by size exclusion chromatography or membrane dialysis with a specific molecular weight sieve.
  • dialysis is usually most effective when separating substances having a molecular weight of 1000 g/mol or more.
  • this purification method usually separates not only the desired reagent but also any other substance that can pass through the membrane.
  • Receptor-based imaging agents require high specific activities (>0.1 Ci/umol) to overcome non-specific protein binding. Therefore, the introduction of impurities in receptor-based imaging agents can be problematic in their clinical applications.
  • an imaging agent containing a radionuclide is considered to be pure and actually contains an impurity that also contains a radionuclide
  • the measurement detection of the imaging agent may be erroneous due to the presence of impurities.
  • a polypeptide such as polyglutamate is used as the drug carrier.
  • polyglutamates have a wide range of molecular weights and comprise a mixture of various polyglutamates. This makes the quality assurance of the drug site in the molecule difficult.
  • the binding reaction is frequently carried out under aqueous conditions so that the purity is not optimized.
  • the method of synthesizing an organic compound in an organic solvent and the use of a protecting group are generally more advantageous for obtaining a relatively pure compound than aqueous purification.
  • the protecting group is required to facilitate the purification of the intermediate.
  • High purity compounds are more readily obtained by various purification methods using organic solvents.
  • methods of organic synthesis and purification of the developer will likely yield higher purity reagents than those obtained via aqueous purification. Therefore, it is necessary to develop organic synthesis techniques to obtain higher purity site-specific conjugates in a more efficient manner.
  • This developer, integrated with molecular imaging can more accurately understand the dynamic changes in pathway-activated cellular receptors that lead to tissue deterioration, inflammation, and proliferative disorders, and improve patient diagnosis, treatment, and prognosis.
  • the invention also provides methods of imaging and treating diseases in a subject comprising a composition employing a conjugate prepared in the present invention.
  • a glutamate polypeptide-estrogen/antiestrogenic conjugate comprising a polypeptide having 3 to 20 repeating amino acids and an estrogen or an antiestrogens, the estrogen or antiestrogens binding to the polypeptide A glutamate.
  • the polypeptide is a glutamic acid peptide, an aspartic acid peptide, or a polypeptide in which a glutamic acid peptide or an aspartic acid peptide is mixed.
  • the polypeptide is a polypeptide of 3 to 6 repeating amino acids, and further preferably, the polypeptide is a polypeptide of 6 repeating amino acids.
  • the polypeptide has a molecular weight of from 500 to 9000.
  • the estrogen is estradiol, estrone, estriol or clomiphene, and further preferably, the estrogen is estradiol.
  • the antiestrogens are non-steroidal tamoxifen, toremifene
  • the ammoniated estrogen binds to the 5-glutamic acid end with both an amino group and a 1-carboxylic acid-protected glutamic acid to form a glutaminyl-estrogen, the glutaminyl group.
  • - Estrogen binds to the amino terminus of all acid protected glutamate peptides.
  • the above conjugate structure is:
  • the amino estrogen is an estrogen whose amino group is located at the 3', 5', 17' position of estrogen.
  • the amino estrogen has an amino group at the 3' position of the steroid ring.
  • the amino estrogen is amino estradiol, amino estriol or aminoestrone.
  • the antiestrogens are non-steroidal tamoxifen, toremifene, tamoxifen, raloxifene or aminoglutethimide.
  • GAP-estradiol conjugate a protected monoglutamate is used as a precursor to prepare a 5-glutamyl-estradiol conjugate. 5-Glucoyl-estradiol then binds to the glutamate peptide (GAP) at the N-terminus to form GAP-estradiol (GAP-EDL), which is site specific at the first glutamate of GAP sexually combined.
  • GAP glutamate peptide
  • GAP-EDL GAP-estradiol
  • the above mixing method may be carried out in an organic solvent such as dimethylformamide, dimethyl sulfoxide, dioxane, methanol, ethanol, hexane, dichloromethane, chloride, acetonitrile, tetrahydrofuran, or a mixture thereof. achieve.
  • the mixing process can be carried out in an aqueous solvent.
  • One, two, three, four, five or all of the carboxylic acid groups or polypeptides of the amino acid may, for example, be protected by a t-butyl or benzyl group or may not be protected.
  • the amino acid is a glutamic acid protected by N-tert-butoxycarbonyl and 1-benzylcarboxy, such as 1-benzyl-N-tert-butoxycarbonyl-L-glutamate.
  • Protecting groups are commonly used for organic synthesis and not for aqueous synthesis.
  • the method of the invention may further comprise at least one purification step.
  • Any of the compounds of the invention can be purified by any method known to those skilled in the art. Those methods are familiar to those skilled in the art when those methods are available.
  • the purification step can be carried out after each synthesis step, after every few steps, at different points in the synthesis period, and/or at the end of the synthesis.
  • the one or more purification steps comprise a technique selected from the group consisting of silica gel column chromatography, high performance liquid chromatography (HPLC), and liquid chromatography (LC).
  • the purification method specifically does not include size exclusion chromatography and/or dialysis. The purification method will be described in more detail below.
  • the method can include purifying the glutamate-estradiol conjugate prior to coupling with a glutamate peptide.
  • the protected carboxylic acid of the glutamate-estradiol conjugate is selectively deprotected and a coupling chemistry (such as 1-hydroxybenzo) is employed.
  • a coupling chemistry such as 1-hydroxybenzo
  • composition for personal diagnosis or precise medical treatment comprising the above glutamic acid polypeptide-estrogen/antiestrogens conjugate and metal ions for labeling.
  • the metal ion is a radionuclide.
  • the metal ions are Tc-99m, Ga-68, Cu-60, Cu-64, In-111, Ho-166, Re-186, Re-188, Y-90, Lu-177, Ra-223. , Ac-225, or a metal used in the treatment of diseases.
  • composition is preferably 99m Tc-glutamic acid peptide-estradiol or 68 Ga-glutamic acid peptide-estradiol.
  • the present invention accurately provides a method of imaging a disease site in a given subject for pre- and post-treatment assessment and is capable of monitoring the subject as long as the subject is being treated or under treatment with estrogen.
  • the method comprises detecting a signal produced by a radionuclide-labeled chelator conjugate at a disease site in an individual subject, wherein the disease site, if present, is produced more intensely around the tissue signal of.
  • the metal ion is selected from the group consisting of cerium ions, tin ions, copper ions, indium ions, cerium ions, gallium ions, arsenic ions, cerium ions, cerium ions. , strontium ions, strontium ions, selenium ions, strontium ions, strontium ions, strontium ions, iron ions, manganese ions, strontium ions, cobalt ions, platinum ions, calcium ions, strontium ions.
  • the metal ion is a radionuclide, as well as any radionuclide known to those skilled in the art.
  • radionuclides include: 99m Tc, 117m Sn, 177 Lu, 188 Re, 186 Re, 153 Sm, 166 Ho, 90 Y, 89 Sr, 67 Ga, 68 Ga, 111 In, 183 Gd, 59 Fe, 225 Ac, 212 Bi, 211 At, 45 Ti, 60 Cu, 61 Cu, 67 Cu, 64 Cu, and 62 Cu.
  • the metal ion is a non-radioactive metal, such as 187 Re.
  • the site to be imaged may be a tumor or an ER-enriched tissue such as ovary and uterine tissue.
  • the method can also be defined as a method of treating a subject having cancer or endometriosis.
  • the cancer is breast cancer, lung cancer, prostate cancer, ovarian cancer, uterine cancer, cervical cancer or endometrial cancer.
  • the method can be further defined as a method of imaging a site within a subject, comprising detecting a signal from a metal ion labeled glutamate peptide-estrogen conjugate located at the site.
  • the signal can be detected using a technique selected from the group consisting of PET, PET/CT, SPECT, SPECT/CT, PET/MRI, SPECT/MRI, optical imaging in conjunction with a nuclear imaging device.
  • the present invention provides a kit comprising the above-described composition for personal diagnosis or precise medical treatment.
  • the invention also provides a conjugate composition or kit comprising a conjugate of the invention, for example comprising about 1 mg of a glutamate peptide-estradiol conjugate.
  • the conjugate composition or kit may also contain an antioxidant, a stabilizer, a preservative or a salt.
  • the conjugate composition or kit may additionally comprise ascorbic acid, mannitol, and/or tin (II) chloride.
  • the composition is an aqueous solution or a frozen and/or lyophilized solution.
  • a method of imaging, diagnosing, or treating a disease in a subject comprising administering to the patient a metal ion labeled glutamate peptide-estradiol conjugate.
  • the method can include a) obtaining a composition comprising a 68Ga-labeled glutamate peptide-estradiol conjugate; b) administering to the subject a pharmaceutically or diagnostically effective amount of the composition, wherein The site is imaged, the disease is diagnosed, or the disease is treated.
  • the site to be imaged may be tumor, heart, or endometrial tissue.
  • the method can also be defined as a method of imaging, diagnosing or treating a subject having a cardiovascular disease.
  • the cardiovascular disease may be myocardial infarction, congestive heart failure, cardiomyopathy, valvular heart disease, arrhythmia, congenital heart disease, angina pectoris, non-cardiac circulatory congestion, systolic heart failure, heart failure with normal function. Or right heart failure.
  • a glutamic acid polypeptide-estrogen/antiestrogens conjugate refers to a glutamic acid polypeptide-estrogen conjugate or a glutamic acid polypeptide-antiestrogens conjugate
  • estrogen or antibiotic Estrogen is a receptor ligand.
  • the conjugate is a glutamate polypeptide-estrogen conjugate
  • the receptor ligand selects an anti-estrogen
  • the conjugate is It is a glutamic acid polypeptide-antiestrogens conjugate.
  • the improved synthetic method of the present invention produces pure GAP-EDL and has greatly increased sensitivity and specificity in cell uptake experiments.
  • the conjugate of the present invention binds (labels) a metal ion to form a composition, such as 68 Ga-GAP-EDL, which has an average cell uptake of 5 times higher than previously reported synthesis and is capable of imaging efficiently And treatment-related diseases, such as methods of imaging ER-enriched tissues in a subject and methods of diagnosing endometriosis.
  • the technology platform of the present invention employs binding antagonists and agonists and sees its effects in a variety of disease forms.
  • the techniques and methods of manufacture disclosed herein can also be prepared as pharmaceutical dosage forms and kits using chemical processes known to those skilled in the art.
  • the synthetic method of the present invention avoids the addition of a protective agent to glutamate and glutamate pentapeptide (glutamate peptide) as compared to other methods described in U.S. Patent Application Nos. US20060246005A1 and WO 2006107794 A2, incorporated herein by reference. Improve process efficiency and purity of the final product.
  • Figure 1 is a GAP-EDL synthetic route map.
  • Figure 2 is a spectrum of the product obtained by the synthesis of GAP-EDL-1.
  • Figure 3 is a spectrum of the product obtained by the synthesis of GAP-EDL-2.
  • Figure 4 is a spectrum of the product obtained by the synthesis of GAP-EDL-3.
  • Figure 5 is a spectrum of the product obtained by the synthesis of GAP-EDL-4.
  • Figure 6 is a spectrum of the product obtained by the synthesis of GAP-EDL-5.
  • Figure 7 is a 1H-NMR of 1,5-di-tert-butyl GAP ester.
  • Figure 8 is the 1H-NMR of GAP-EDL-5.
  • Figure 9 shows the synthesis of GAP-EDL.
  • Figure 10 is a mass spectrum of GAP-EDL.
  • Figure 11 is a 1H-NMR of GAP-EDL.
  • Figure 12 is a 1H-NMR of GAP-EDL-4.
  • Figure 13 is the HPLC of GAP-EDL-4.
  • Figure 14 is the HPLC of GAP-EDL.
  • Figure 15 is an ITLC analysis of 68 GaCL3 (polyamide, eluent: saline).
  • Figure 16 is an ITLC analysis of 68 Ga-GAP (polyamide, eluent: physiological saline).
  • Figure 17 is an ITLC analysis of 68 Ga-GAP-EDL (polyamide, eluent: physiological saline).
  • Fig. 1 The synthesis scheme of the GAP-EDL of the present invention is shown in Fig. 1.
  • GAP-EDL-1 (2.10 g, 6.79 mmol) was dissolved in anhydrous THF (90 mL) under nitrogen atmosphere. The mixture was cooled to 0-5 ° C and lithium aluminum hydride (1.70 g, 44.80 mmol) was added portionwise. The reaction mixture was stirred at 0-5 ° C for 5-10 minutes and then at room temperature for 2 hours. After the reaction was over, the mixture was again cooled to 0-5 ° C and quenched with water. The suspension was filtered and washed with THF. The filtrate was evaporated and concentrated under reduced pressure.
  • reaction mixture was stirred at 0-5 ° C then GAP-EDL-2 (0.50 g, 1.585 mmol). The reaction mixture was stirred at 0-5 ° C for 10 minutes and at room temperature for 1 hour. After the reaction was completed, DMF was removed under reduced pressure and ethyl acetate (250 mL) was added. The organic phase was washed with 4% aqueous sodium bicarbonate (200 mL), dried over magnesium sulfate, filtered and concentrated under reduced pressure.
  • the crude compound was purified by column chromatography eluting elut elut elut elut elut elut elut elut elut elut elut elut elut elut elut elut elut elut elut elut elut , yield 42.5%).
  • GAP-EDL-4 (0.580 g, 1.06 mmol), 1-hydroxybenzotriazole (0.146 g, 1.08 mmol) and BOP (0.470 g, 1.06 mmol) were dissolved in anhydrous DMF (10 mL). The mixture was stirred under a nitrogen atmosphere and the temperature was cooled to 0 °C. Then N,N-diisopropylethylamine (DIPEA, 0.140 g, 1.08 mmol) and 1,5-di-tert-butyl-L-glutamate pentapeptide (GAP ester, 1.260 g, 1.06 mmmol) were added. The reaction mixture was stirred at 50 ° C for 16-18 hours and monitored by TLC.
  • DIPEA 1,3-diisopropylethylamine
  • GAP ester 1,5-di-tert-butyl-L-glutamate pentapeptide
  • 68 GaCl 3 was obtained from a 68 Ge/ 68 Ga generating device eluting with free HCL (ranging from 0.01 N to 1 N).
  • free HCL ranging from 0.01 N to 1 N
  • 68 GaCl 3 was eluted from a 68 Ge/ 68 Ga generating apparatus using 0.3 N and 0.6 N HCL (10 mL).
  • the elution volume 0.3 N or 0.6 N HCL, 6 mL
  • Each tube calculates its radioactivity. The highest activity in the portion between 4 and 6 is combined.
  • the generating device was eluted again with 6 mL of HCL and collected in these specific portions according to the previous elution.
  • breast tumor cells (50,000 cells/well) are treated with 0, 15, 150, 300 ⁇ mol/L cold estrone. (in DMSO) was treated for 30 minutes, then 68 Ga-GAP-EDL (4 ⁇ g/well, 4 ⁇ Ci/well) was added and incubated for 90 minutes. After the incubation, the cell supernatant was collected. The cells were washed with ice-cold PBS (1 mL) as a washing solution, and the washing solution and the cell supernatant were collected into a tube, and trypsin-EDTA (0.1 mL/well) was added to the cell culture well to digest the cells.
  • trypsin-EDTA 0.1 mL/well
  • the uptake of 68 Ga-GAP-EDL was significantly increased compared to the uptake of 68 GaCl 3 and 68 Ga-GAP (see Table 1).
  • the improved synthetic method produces pure GAP-EDL and has greatly enhanced sensitivity in cell uptake studies.
  • the average cellular uptake of 68 Ga-GAP-EDL of the improved synthetic method (as in Table 1) was at least 5 times higher than the previously reported synthetic method (Takahashi N et al, Academic Radiology, 2007). Higher ER(+) density MCF-7 has more uptake than lower ER(+)SKBR-3.
  • cellular uptake may be competitively inhibited by estrone, especially at 15, 150, 300 ⁇ mol/L. Furthermore, especially at 15 ⁇ mol/L of estrone, MCF-7 had a much lower uptake compared to the lower ER(+)SKBR-3 (see Table 2). Reduced uptake demonstrates cellular uptake of 68 Ga-GAP-EDL via an ER-mediated process.

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Abstract

一种谷氨酸多肽-雌激素/抗雌激素缀合物及其合成方法、组合物、试剂盒。该缀合物包括多肽和雌激素或抗雌激素，雌激素或抗雌激素结合至多肽的第一谷氨酸盐处，获得纯的GAP-EDL并提高了细胞摄取率。

Description

一种谷氨酸多肽-雌激素/抗雌激素缀合物、合成方法、组合物及试剂盒 技术领域
本发明涉及一种用于分子成像和治疗的缀合物(conjugate)及其合成的新方法，以及利用该缀合物形成的组合物及其试剂盒。该缀合物基于与各个病人的疾病相关的个体基因组成、生化和分子设计，可用于个性化治疗，属于化学合成、标记、成像、药物治疗、ER-阳性疾病的治疗以及癌症的治疗领域。
背景技术
活性氧(reactive oxygen species,ROS)的过量存在导致氧化应激。ROS引起DNA损伤，导致聚腺苷二磷酸-核糖聚合酶(poly-ADP ribose polymerase-1,PARP-1)的过度激活，从而引起细胞内NAD+的耗尽。ROS还激活促炎转录因子NF-kB，从而引起神经炎症。NAD+的耗尽引起糖酵解抑制，丙酮酸酯产出不足以及线粒体ATP产出降低。还有，NAD+的耗尽使乳酸酯转化成丙酮酸酯效率低下，而乳酸酯不再能够用作能量基质，从而引起细胞凋亡。主要有三条路径用于消除活性氧，而这些路径涉及降低的谷胱甘肽(GSH)、硫氧还蛋白(TXN)和过氧化氢酶。所有这三条路径都包含在癌细胞内抑制抗氧化响应的关键酶。在这些路径中，GSH受到升高的谷氨酸盐和半胱氨酸转运子系统以及NADPH的影响。GSH衍生自代谢物谷氨酸盐和半胱氨酸。谷氨酰胺酶1(GLS1)和GLS2产生谷氨酸盐，而胱氨酸/谷氨酸盐转运子XCT通过谷氨酸盐-半胱氨酸连接酶修饰物亚基(glutamate–cysteine ligase modifier subunit，GCLM)和GCL催化亚基(GCL catalytic subunit，GCLC)提供生成GSH的半胱氨酸。GSH通过谷胱甘肽过氧化物酶和谷胱甘肽s-转移酶的作用直接作用于消除ROS[参见：Chen H,Tang X,Zhou B,Zhou Z,Xu N,Wang Y.A ROS-mediated mitochondrial pathway and Nrf2 pathway activation are involved in BDE-47 induced apoptosis in Neuro-2a cells.Chemosphere.2017；184:679-686.doi:10.1016/j.chemosphere.2017.06.006.；Akanda MR,Kim MJ,Kim IS,Ahn D, Tae HJ,Rahman MM,Park YG,Seol JW,Nam HH,Choo BK,Park BY.Neuroprotective Effects of Sigesbeckia pubescens Extract on Glutamate-Induced Oxidative Stress in HT22 Cells via Downregulation of MAPK/caspase-3 Pathways.Cell Mol Neurobiol.2017.doi:10.1007/s10571-017-0496-y.；Habib E,Linher-Melville K,Lin HX,Singh G.Expression of xCT and activity of system xc(-)are regulated by NRF2 in human breast cancer cells in response to oxidative stress.Redox Biol.2015；5:33-42.doi:10.1016/j.redox.2015.03.003.]。谷氨酸盐在ROS期间引起Ca2+过载，从而导致GSH的生成。因此，谷氨酸盐受体/转运子系统与细胞内GSH生成有关。
聚左旋谷氨酸(PG)已用作多种疏水性癌症化学治疗剂的肿瘤靶向药物载体[参见：Batz HG,Ringsdorf H,Ritter H.Pharmacologically active polymers,7.Cyclophosphamide-and steroid hormone containing polymers as potential anticancer compounds.Die Makromolekulare Chemie.1974；175(8):2229–2239.；Hurwitz E,Wilchek M,Pitha J.Soluble macromolecules as carriers for daunorubicin.J Appl Biochem.1980；2(1):25–35.；Kato Y,Saito M,Fukushima H,Takeda Y,Hara T.Antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugated with polyglutamic acid and its derivative.Cancer Res.1984；44(1):25–30.；Morimoto Y,Sugibayashi K,Sugihara S.Antitumor agent poly(amino acid)conjugates as a drug carrier in cancer chemotherapy.J Pharmacobiodyn.1984；7(9):688–698.；Hoes CJT,Potman W,van Heeswijk WAR,et al.Optimization of macromolecular prodrugs of the antitumor antibiotic adriamycin.J Control Release.1985；2:205–213.；Mochizuki E,Inaki Y,Takemoto K.Synthesis of poly-L-glutamates containing 5-substituted uracil moieties.Nucleic acids Symp Ser.1985；16(16):121–124.；Zunino F,Pratesi G,Micheloni A.Poly(carboxylic acid)polymers as carriers  for anthracyclines.J Control Release.1989；10(1):65–73.；Hoes CJT,Grootoonk J,Duncan R,et al.Biological properties of adriamycin bound to biodegradable polymeric carriers.J Control Release.1993；23(1):37–53.；Kiew LV,Cheong SK,Ramli E,Sidik K,Lim TM,Chung LY.Efficacy of a poly-L-glutamic acid-gemcitabine conjugate in tumor-bearing mice.Drug Dev Res.2012；73(3):120–129.]。PG具有额外的优点，诸如优异的水溶性、生物相容性、非免疫原性、可生物降解至谷氨酸盐以及由于它的多个羧基导致的高药物装载能力[参见：McCormick-Thomson LA,Sgouras D,Duncan R.Poly(amino acid)copolymers as a potential soluble drug delivery system.2.Body distribution and preliminary biocompatibility testing in vitro and in vivo.J Bioact Compat Polym.1989；4(3):252–268.；Sumi H,Kawabe K,Nakajima N.Effect of various polyamino acids and D-and L-amino acids on the blood fibrinolytic system.Comp Biochem Physiol B.1992；102(1):159–162.；Li C.Poly(L-glutamic acid)–anticancer drug conjugates.Adv Drug Deliv Rev.2002；54(5):695–713.；Kenny AD.Evaluation of sodium poly-alpha,L-glutamate as a plasma expander.Proc Soc Exp Biol Med.1959；100(4):778–780.]。具有这些特点，PG-药物缀合物能够靶向细胞内基因。PG是具有相同重复单元的聚合物基材料。因此，在，PG-药物结合期间，一致性和纯度并不优化。在基于受体的成像中，试剂需要达成高比活度(Ci/umol)。如果被标记的试剂由不同的分子量污染，则成像质量保证会有问题。此外，不知道在哪个位点药物被结合至PG。合规性通常需要该详细的GMC信息。本发明公开了分子首先与谷氨酸盐反应，随后是谷氨酸肽。最终的结构是使该结合的分子设置至谷氨酸肽的第一谷氨酸盐。
兴奋性氨基酸谷氨酸盐经由多种谷氨酸盐受体发挥其作用。代谢性谷氨酸盐受体据报道与膜雌激素受体(ER)，更具体地与mGlu5受体亚型相互作用。17β-雌二醇和mGlu5拮抗剂在患有帕金森病的1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine，MPTP)小鼠模 型中具有神经保护效果[参见：Al-Sweidi S,Morissette M,Di Paolo T.Estrogen receptors modulate striatal metabotropic receptor type 5 in intact and MPTP male mice model of Parkinson's disease.J Steroid Biochem Mol Biol.2016；161:84-91.doi:10.1016/j.jsbmb.2016.02.004]。因此，雌二醇(一种雌激素受体配体)被选作结合至谷氨酸肽的分子。已知谷氨酸肽在体外刺激骨吸收且特异性作用于谷氨酸受体[参见：Chenu C,Serre CM,Raynal C,Burt-Pichat B,Delmas PD.Glutamate receptors are expressed by bone cells and are involved in bone resorption.Bone.1998；22(4):295-9.]。谷氨酸肽已证明其靶向肾组织的能力[参见：Chai HJ,Kiew LV,Chin Y,et al.Renal targeting potential of a polymeric drug carrier,poly-l-glutamic acid,in normal and diabetic rats.J Nanomedicine.2017；12:577-591.doi:10.2147/IJN.S111284.]。将雌二醇(EDL)结合至GAP是合适的，且GAP-EDL可增强细胞内ER的结合功效。采用来自GAP的酸残基，GAP可螯合放射性金属同位素(radiometallic isotopes)，以用于成像和放射治疗应用。先前已经报道过99mTc-GAP-EDL和68Ga-GAP-EDL都是断层扫描中有用的化合物[参见：Takahashi N,Yang DJ,Kohanim S,Oh C-S,Yu D-F,Azhdarinia A,Zhang X-C,Chang JY,Kim EE.Targeted functional imaging of estrogen receptors with 99mTc-GAP-EDL.Eur J Nucl Med Mol Imaging.2007；34:354-362.；Takahashi N,Yang DJ,Kurihara H,Borne A,Kohanim S,Oh C-S,Mawlawi O,Kim EE.Functional imaging of estrogen receptors with radiolabeled GAP-EDL in rabbits endometriosis model.Academic Radiology 2007；14(9):1050-7.]。
雌激素经由主要位于细胞核内的α和β雌激素受体引起基因组效应。ERα在介导基因转录中的作用在文献中有据可查，并且采用小鼠模型和人乳腺(癌)细胞系的研究已证明ERα在细胞增生方面所起的作用。相比之下，ERβ作为转录调控子的作用并不清晰。研究揭示ERβ可能通过异源二聚化可降低ERα的活性[参见：Speirs V,Carder PJ,Lane S,Dodwell D,Lansdown MR,Hanby  AM:Oestrogen receptor beta:what it means for patients with breast cancer.Lancet Oncol.2004,5:174-181.10.1016/S1470-2045(04)01413-5.；Hayashi SI,Eguchi H,Tanimoto K,Yoshida T,Omoto Y,Inoue A,Yoshida N,Yamaguchi Y:The expression and function of estrogen receptor alpha and beta in human breast cancer and its clinical application.Endocr Relat Cancer.2003,10:193-202.10.1677/erc.0.0100193.]。最近，处于膜上的雌激素受体已被认为是通过雌激素对若干细胞过程引起快速“非基因”作用。ER调节子，例如三苯氧胺是研究雌激素的作用机制以及临床实践中的重要工具。ER在若干水平介导NF-κB活性的抑制。在这些激素和免疫系统之间的这些重要调节子之间的交互作用可被用来治疗癌症、炎性和自身免疫性疾病[参见：Sas L,Lardon F,Vermeulen PB,Hauspy J,et al.The interaction between ER and NFκB in resistance to endocrine therapy.Breast Cancer Research 2012,14:212]。
在现有技术中，雌二醇采用水纯化(aqueous purification)结合至PG(Yang DJ,Oh CS,Kohanim S,Yu DF,Azhdarinia A,Kim EE.Poly(peptide)as a chelator:methods of manufacture and uses.U.S.Patent application 20060246005,WO 2006107794 A2.)，但是细胞摄取低，这可能是由于其纯度问题。因而，谷氨酸肽-雌二醇(GAP-EDL)采用改进的有机合成方法进行结合。雌激素受体在结合以及对各个荷尔蒙路径的响应方面的发现在治疗氧化应激引起的ROS、炎症和癌症方面使患者受益。在病态期间，由于GSH的高需求，谷氨酸盐受体被过表达。已知谷氨酸肽(GAP)结合至谷氨酸盐受体/转运体(Yang DJ等人，U.S.专利申请US20060246005A1；WO 2006107794 A2)。过表达的谷氨酸盐系统可增强标记的GAP-EDL的细胞摄取。一旦GAP-EDL进入细胞，GAP-EDL可靶向ER基因。
为了增进对雌激素受体(ER)相关疾病的理解，本发明描述了采用谷氨酸肽-雌激素缀合物进行精密ER阳性(ER+)成像和治疗。ER+路径激活系统的分子成像用作荷尔蒙依赖型疾病(如癌症和子宫内膜异位)管理的基础。ER+ 病人对内分泌治疗反应更好，且生存期是ER-病人的两倍长。然而，临床采用的芳香化酶抑制剂和抗雌激素的肿瘤抵抗性结果是不可预知的。放射性标记的ER+配体能够定量化ER+组织摄取，从而有助于对癌症和子宫内膜异位进行分期和再分期。通过采用标记配体的ER+损伤的鉴别诊断有助于选择对内分泌治疗有最佳反应的病人，以及当耐药性发生时中断治疗。此外，放射性标记的ER+配体用作成像导向的响应跟进的基础。现有文献报告已显示采用聚谷氨酸盐作为药物载体的策略可改变药物溶解性并强化雌激素受体-配体结合袋。该技术有助于通过谷氨酸盐受体介导的过程靶向细胞内基因。但是，所描述的聚谷氨酸盐为混合的聚合物的共混物，具有宽广范围的分子量。因而，产品的纯度是未被优化的。
相关技术描述:
关于用于金属标记的分子制剂的合成制备，当此类制剂在水性(湿)条件下制备时，制剂的纯化有时可能存在问题。在水性条件下，纯化可采用例如尺寸排阻层析(size exclusion chromatography)或用特定分子量筛截的膜透析来实现。例如，当分离分子量为1000g/mol或更高的物质时，透析通常是最有效的。然而，该纯化方法通常不仅仅分离所需的试剂，也分离可通过膜的任何其它物质。基于受体的成像剂需要高的比活度(>0.1Ci/umol)以克服非特异性蛋白质结合。因此，在基于受体的成像剂中引入杂质在它们的临床应用中可能是有问题的。例如，如果含有放射性核素的成像剂被认为是纯的，而实际上含有也包含放射性核素的杂质，则成像剂的测量检测可能会由于杂质的存在而出现错误。当采用多肽如聚谷氨酸盐作为药物载体时，情况尤其如此。商购的聚谷氨酸盐具有宽范围的分子量，其包含多种聚谷氨酸盐的混合物。这使得在分子中的药物位置的质量保证困难。此外，结合反应在水性条件下频繁进行，从而纯度得不到优化。
在有机溶剂中合成有机化合物的方法以及采用保护基通常相比水性纯化更有利于获得较纯的化合物。保护基需利于中间体的纯化。采用有机溶剂的各种纯化方法更加容易获得高纯度化合物。考虑到有机合成和纯化提供的相比水性纯化的益处，有机合成和纯化显影剂的方法将可能产出相比经由水性纯 化获得的那些试剂纯度更高的试剂。因此，有必要开发有机合成技术能够以更有效的方式获得更高纯度的位点特异性缀合物。这种显影剂与分子成像整合起来，可以更准确地理解导致组织恶化、发炎和增生病症的路径激活细胞受体的动态变化，并改进患者诊断、治疗和预后。
发明内容
本发明的目的是提供一种用于治疗和诊断应用的谷氨酸肽-雌激素/抗雌激素缀合物以及其有效合成方法，该改进的合成方法制备纯的GAP-EDL，并在细胞摄取实验中具有大大增加的敏感性和特异性。本发明还提供了包括采用本发明中制备的缀合物的组合物在受试者中的成像和治疗疾病的方法。
为了达到上述技术目的，本发明的技术方案是：
一种谷氨酸多肽-雌激素/抗雌激素缀合物，其包括具有3～20个重复氨基酸的多肽和雌激素或抗雌激素，所述雌激素或抗雌激素结合至上述多肽的第一谷氨酸盐处。
作为优选，所述多肽为谷氨酸肽、天冬氨酸肽，或为谷氨酸肽、天冬氨酸肽混合的多肽。所述多肽为3～6个重复氨基酸的多肽，作为进一步的优选，所述多肽为6个重复氨基酸的多肽。
作为优选，所述多肽的分子量为500到9000。
作为优选，所述雌激素为雌二醇、雌素酮、雌三醇或克罗米酚，作为进一步优选，所述雌激素为雌二醇。
作为优选，所述抗雌激素为非类固醇三苯氧胺、托瑞米芬
(toremiphene)、他莫昔芬、雷洛昔芬或氨鲁米特。
上述缀合物结构：氨化的雌激素采用氨基和1-羧酸保护的谷氨酸两者结合至5-谷氨酸末端，制成谷氨二酰基-雌激素，所述谷氨二酰基-雌激素结合至全部酸保护的谷氨酸肽的氨基末端。
作为一个实施例，上述缀合物结构为：
Figure PCTCN2018115290-appb-000001
一种谷氨酸多肽-雌激素/抗雌激素缀合物的合成方法：
将氨基雌激素或氨基抗雌激素与羧酸保护的谷氨酸盐(单谷氨酸盐)在有机溶剂中混合，制备谷氨酸盐-雌激素缀合物或谷氨酸盐-抗雌激素缀合物；将谷氨酸盐-雌激素缀合物或谷氨酸盐-抗雌激素缀合物与包含偶联剂的全部羧酸保护的多肽进行反应，相应制得谷氨酸多肽-雌激素缀合物或谷氨酸多肽-抗雌激素缀合物。根据选择的雌激素或抗雌激素的不同，分别获得谷氨酸多肽-雌激素或谷氨酸多肽-抗雌激素，此时所述雌激素位于谷氨酸多肽-雌激素的第一谷氨酸盐处，所述抗雌激素位于谷氨酸多肽-抗雌激素的第一谷氨酸盐处。
作为进一步限定，所述氨基雌激素为氨基位于雌激素的3’、5’、17’位的雌激素。所述氨基雌激素具有位于甾体环的3’位处的氨基。具体地，所述氨基雌激素为氨基雌二醇、氨基雌三醇或氨基雌素酮。
作为进一步限定，所述抗雌激素为非类固醇三苯氧胺、托瑞米芬(toremiphene)、他莫昔芬、雷洛昔芬或氨鲁米特。
具体地，上述缀合物优选的合成方法：
将氨基雌二醇和1-羧酸保护的谷氨酸盐在有机溶剂中混合，制备γ谷氨二酰基-雌二醇缀合物；将所述γ谷氨二酰基-雌二醇缀合物与包含偶联剂的全部羧酸保护的谷氨酸五肽(谷氨酸肽)进行反应制得谷氨酸多肽-雌二醇，雌二醇位于谷氨酸多肽-雌二醇的第一谷氨酸盐处。
或如谷氨酸肽(GAP)-雌二醇缀合物的有效合成方法：受保护的单谷氨酸盐用作前体来制备5-谷氨二酰基-雌二醇缀合物。5-谷氨二酰基-雌二醇然后在N端结合至谷氨酸肽(GAP)以形成GAP-雌二醇(GAP-EDL)，EDL在GAP的第一谷氨酸盐处位点特异性地结合。
上述混合方法可在有机溶剂如二甲基甲酰胺、二甲亚砜、二氧六环(dioxane)、甲醇、乙醇、己烷、二氯甲烷、氯化物、乙腈、四氢呋喃，或它们的混合物中实现。在其它方面，该混合方法可在水性溶剂中实现。氨基酸的一个、两个、三个、四个、五个或所有的羧酸基团或多肽可例如被叔丁基或苄基保护，或未被保护。在一个具体的例子中，该氨基酸为由N-叔丁氧羰基和1-苄基羧基保护的谷氨酸，例如1-苄基-N-叔丁氧羰基-L-谷氨酸酯。保护基通常用于有机合成而不用于水性合成。
本发明的方法可进一步包括至少一个纯化步骤。本发明的任何化合物可通过本领域的技术人员已知的任何方法纯化。当那些方法可采用时，本领域的技术人员熟悉该方法。例如，在一个目的在于获得一特定化合物的多步骤合成中，可在每一个合成步骤后、每几个步骤后、合成期间的不同点处、和/或在合成的末尾处进行纯化步骤。在一些方法中，一个或多个纯化步骤包括选自以下的技术：硅胶柱色谱法、高效液相层析(HPLC)和液相层析(LC)。在某些实施方式中，纯化方法明确不包括尺寸排阻色谱法和/或透析。纯化方法将在下面更详细描述。在一个特定方面，该方法可包括在与谷氨酸肽偶合反应之前纯化所述谷氨盐酸-雌二醇缀合物。
为了制备谷氨酸肽-雌二醇缀合物，谷氨酸酸-雌二醇缀合物的受保护的羧酸被选择性地解保护并采用偶联化学品(如1-羟基苯并三唑和苯并三唑-1-基氧基-三(二甲氨基)-磷六氟磷酸盐(Benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate，BOP))与全部酸保护的谷氨酸五肽(glutamate pentapeptide，谷氨酸肽)酯反应。
一种用于个人诊断或精确医疗的组合物，其包括上述谷氨酸多肽-雌 激素/抗雌激素缀合物和用于标记的金属离子。
作为优选，所述金属离子是放射性核素。例如，所述金属离子是Tc-99m、Ga-68、Cu-60、Cu-64、In-111、Ho-166、Re-186、Re-188、Y-90、Lu-177、Ra-223、Ac-225，或在疾病治疗中用于诊疗的金属。
所述组合物优选为 99mTc-谷氨酸肽-雌二醇或 68Ga-谷氨酸肽-雌二醇。
本发明精确地提供了在给定受试对象中的疾病部位成像的方法以进行预治疗/后治疗评估并能够监控该受试对象，只要受试对象正采用雌激素进行治疗或在治疗中。在某些方面，该方法包括检测由在个体受试对象中的疾病部位处的放射性核素标记的螯合剂缀合物产生的信号，其中疾病部位，如果存在的话，产生比该组织周围更强烈的信号。为了生成金属离子标记的谷氨酸肽-雌二醇缀合物，所述金属离子选自锝离子、锡离子、铜离子、铟离子、铊离子、镓离子、砷离子、铼离子、钬离子、钇离子、钐离子、硒离子、锶离子、钆离子、铋离子、铁离子、锰离子、镥离子、钴离子、铂离子、钙离子、铹离子。在一些方面，金属离子为一种放射性核素，以及任何本领域的技术人员已知的放射性核素。非限制性的放射性核素的例子包括： 99mTc、 117mSn、 177Lu、 188Re、 186Re、 153Sm、 166Ho、 90Y、 89Sr、 67Ga、 68Ga、 111In、 183Gd、 59Fe、 225Ac、 212Bi、 211At、 45Ti、 60Cu、 61Cu、 67Cu、 64Cu以及 62Cu。在其它方面，金属离子为非放射性金属，例如 187Re。
将要成像的位点可为肿瘤或富集ER的组织，诸如卵巢和子宫组织。该方法还可限定为治疗患有癌症或子宫内膜异位的受试个体的方法。在具体的方面，所述癌症为乳腺癌、肺癌、前列腺癌、卵巢癌、子宫癌、宫颈癌或子宫内膜癌。在其它方面，该方法可被进一步限定为成像受试个体内的位点的方法，包括检测来自于位于所述位点处的金属离子标记的谷氨酸肽-雌激素缀合物的信号。该信号可采用选自以下的技术检测：PET、PET/CT、SPECT、SPECT/CT、PET/MRI、SPECT/MRI、结合核成像装置的光学成像。
本发明提供一种试剂盒，其包括上述的一种用于个人诊断或精确医疗的组合物。
本发明还提供一种缀合物组合物或试剂盒，缀合物组合物或试剂盒包 括本发明的缀合物，例如包含约1mg的谷氨酸肽-雌二醇缀合物。缀合物组合物或试剂盒还可包含抗氧化剂、稳定剂、防腐剂或盐。例如，缀合物组合物或试剂盒可额外地包含抗坏血酸、甘露醇和/或氯化锡(II)。例如包含：a)每1mg谷氨酸肽-雌二醇缀合物约0.5至2.0mg的抗坏血酸；b)每1mg谷氨酸肽-雌二醇缀合物约10至20mg的甘露醇；和/或c)每1mg谷氨酸肽-雌二醇缀合物约0.05至0.1mg的氯化锡(II)。在一些方面中，所述组合物为水性溶液或已冰冻和/或冻干的溶液。
在再一个实施例中，提供了一种对一部位进行成像、诊断或治疗在受试个体内的疾病的方法，包括向患者施用金属离子标记的谷氨酸肽-雌二醇缀合物。例如，该方法可包括a)获得包含68Ga标记的谷氨酸肽-雌二醇缀合物的组合物；b)向受试个体施用药学上或诊断上有效量的所述组合物，其中所述部位被成像，所述疾病被诊断，或所述疾病被治疗。
要成像的位点可为肿瘤、心脏、子宫内膜组织。该方法还可限定为对患有心血管疾病的受试对象的成像、诊断或治疗的方法。所述心血管疾病可为心肌梗死、充血性心力衰竭、心肌病、心脏瓣膜病、心律不齐、先天性心脏病、心绞痛、非心脏性循环充血、收缩性心力衰竭、具有正常功能的心力衰竭或右侧心力衰竭。
本发明专利文件中，谷氨酸多肽-雌激素/抗雌激素缀合物指的是谷氨酸多肽-雌激素缀合物或谷氨酸多肽-抗雌激素缀合物，雌激素或抗雌激素为受体配体，当受体配体选择雌激素时，缀合物即为谷氨酸多肽-雌激素缀合物，而当受体配体选择抗雌激素时，缀合物即为谷氨酸多肽-抗雌激素缀合物。
本发明改进的合成方法制备纯的GAP-EDL并在细胞摄取实验中具有大大增加的敏感性和特异性。本发明的缀合物与金属离子结合(标记)形成组合物，如 68Ga-GAP-EDL，该改进的合成的组合物的平均细胞摄取为以前报道的合成的5倍高，能够有效地成像和治疗相关疾病，例如对受试者内的ER富集组织进行成像的方法以及对子宫内膜异位进行诊断的方法。本发明技术平台采用结合拮抗剂和激动剂且在多种疾病形式中看到其效果。本发明公开的技术和制造方 法还可采用本领域的技术人员已知的化学工艺制备为药剂剂型和试剂盒。本发明合成方法可避免向谷氨酸盐和谷氨酸五肽(谷氨酸肽)添加保护剂相比于美国专利申请US20060246005A1和WO 2006107794 A2(在此以引用方式并入)中描述的其它方法提高工艺效率和最终产物的纯度。
附图说明
图1为GAP-EDL合成路线图。
图2为GAP-EDL-1的合成获得产物光谱图。
图3为GAP-EDL-2的合成获得产物光谱图。
图4为GAP-EDL-3的合成获得产物光谱图。
图5为GAP-EDL-4的合成获得产物光谱图。
图6为GAP-EDL-5的合成获得产物光谱图。
图7为1,5-二-叔丁基GAP酯的1H-NMR。
图8为GAP-EDL-5的1H-NMR。
图9为GAP-EDL的合成。
图10为GAP-EDL的质谱。
图11为GAP-EDL的1H-NMR。
图12为GAP-EDL-4的1H-NMR。
图13为GAP-EDL-4的HPLC。
图14为GAP-EDL的HPLC。
图15为 68GaCL3的ITLC分析(聚酰胺，洗脱剂：生理盐水)。
图16为 68Ga-GAP的ITLC分析(聚酰胺，洗脱剂：生理盐水)。
图17为 68Ga-GAP-EDL的ITLC分析(聚酰胺，洗脱剂：生理盐水)。
具体实施方式
下面结合附图和具体实施方式对本发明作进一步详细的说明。
本发明GAP-EDL的合成方案示于图1中。
(1)(8R,9S,13S,14S)-3-氰基甲氧基-13-甲基-7,8,9,11,12,14,15,16-八氢-6H-环戊醇[a]菲-17-酮 ((8R,9S,13S,14S)-3-Cyanomethoxy-13-methyl-7,8,9,11,12,14,15,16-octah ydro-6H-cyclopenta[a]phenanthren-17-one)(GAP-EDL-1))的合成
(8R,9S,13S,14S)-3-羟基-13-甲基-7,8,9,11,12,14,15,16-八氢-6H-环-五[a]菲-17-酮(雌素酮，2.00g，7.40mmol)在氮气氛下溶解在无水四氢呋喃(THF，25mL)中。然后加入甲醇钠(0.80g，14.82mmol)。将溶解在12mL THF中的溴乙腈(1.78g，14.84mmol)逐滴加入。在室温下搅拌混合物1小时。加入另外的甲醇钠(0.60g，11.11mmol)和在4mL THF中的溴乙腈(1.21g，10.89mmol)。将反应混合物在室温下继续搅拌0.5小时。将饱和的氯化铵溶液(100mL)和乙酸乙酯(100mL)加入到反应混合物中。收集有机层。用另外的乙酸乙酯(2×50mL)提取水相。合并有机层并用饱和氯化钠溶液清洗。将有机提取物在硫酸镁上干燥并过滤。在降低的压力下将溶剂蒸发。将粗固体产物用二乙醚洗涤并获得2.11g(6.82mmol)固体产物，产率为92.2％。 1H-NMR(300MHz，DMSO):7.27(d，J＝8.6Hz，1H)，6.83(dd，J＝8.8Hz，2.6Hz，1H)，6.78(d，J＝2.5Hz，1H)，5.11(s，2H)，2.80-2.86(m，2H)，2.36-2.50(m，2H)，2.16-2.26(m，1H)，1.84-2.15(m，3H)，1.74-1.80(m，1H)，1.33-1.46(m，3H),1.46-1.60(m，3H)，0.84(s，3H)。LC-MS:针对C 20H 23NO 2计算，309.2；发现[M+H]为310.1。光谱示于图2中。其中光谱图右侧的参数的含义罗列如下：
Current Data Parameters…………………当前数据参数
Name…………………………………………名称
Team……………………………………………队
EXPNO…………………………………………实验号
PROCNO………………………………………处理号
F2-Acquisition Parameters……………获得参数
Date_..............................日期
Time…………………………………………时间
INSTRUM spect…………………………….仪器spect
PROBHD 5mm PABBO BB-………………BB-5毫米宽带探头
PULPROG zg……………………………..预饱和水峰压制脉冲序列
TD……………………………………………原始数据点数
SOLVENT……………………………………溶剂
NS…………………………………………….采样次数
DS…………………………………………….采样前扫描次数
SWH………………………………………….谱宽
FIDRES……………………………………….FID分辨率
AQ…………………………………………….取得时间(s)
RG……………………………………………..接收增益
DW……………………………………两次连续数据点之间的间隔时间
DE……………………………………………..扫描前延迟
TE………………………………………………温度
CHANNEL f1………………………………..通道f1
NUC1…………………………………………频率通道f1-f8用核
P1……………………………………………..90度高功率脉冲
PL1…………………………………………...脉冲功率水平(默认)
SF01…………………………………………..共振频率01
F2-Processing Parameters……………………..F2-处理参数
SI……………………………………………….数据点个数
SF……………………………………………….使用的共振频率
WDW EM…………………………………....FID窗口倍压模式
SSB…………………………………………….正弦贝尔移位
LB………………………………………………洛伦兹扩展系数
GB………………………………………………高斯扩展系数
PC……………………………………………….峰值检出敏感度
对于其它实例中光谱图右侧的参数的含义与如上所列相同。
(2)(8R,9S,13S,14S,17S)-3-氨基乙氧基-13-甲基 -6,7,8,9,11,12,14,15,16,17-十氢环戊二烯[a]菲-17-开环((8R,9S,13S,14S,17S)-3-Aminoethoxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthrene-17-ol(GAP-EDL-2))的合成
在氮气氛下将GAP-EDL-1(2.10g，6.79mmol)溶解在无水THF(90mL)中。将混合物冷却至0-5℃并将氢化铝锂(1.70g，44.80mmol)逐份加入。将反应混合物在0-5℃搅拌5-10分钟，然后在室温下搅拌2小时。在反应结束后，将混合物再冷却至0-5℃并用水淬火。将悬浮液过滤并用THF进行清洗。蒸发滤液并在降低的压力下浓缩。将粗化合物采用由甲醇/二氯化物(MeOH/DCM；1/30)洗脱的硅凝胶填充的柱层析进行纯化以提供为灰白(off-white)固体(1.22g，3.87mmol，产率57.0％)的3-氨基乙氧基雌二醇(GAP-EDL-2)。 1H-NMR(300MHz，CDCl3):7.20(d，J＝8.6Hz，1H)，6.71(dd，J＝8.6Hz，2.7Hz,1H)，6.64(d，J＝2.7Hz，1H)，3.98(t.J＝5.2Hz，2H)，3.73(t.J＝8.4Hz1H)，3.06(t.J＝5.2Hz，2H)，2.80-2.85(m，2H)，2.26-2.36(m，1H)，2.06-2.23(m，2H)，1.83-1.98(m，2H)，1.65-1.76(m，2H)，1.65-1.76(m，1H)，1.14-1.48(m，7H)，0.78(s，3H)。LC-MS:针对C 20H 29NO 2计算，315.2；发现[M+H]为316.1。光谱示于图3中。
(3)5-N-[氨基-3-乙氧基雌二醇]-1-苄基-N-叔-丁氧基羰基-L-谷氨酸盐5-酰胺(5-N-[Amino-3-ethoxyestradiol]-1-Benzyl-N-tert-butoxycarbonyl-L-Glutamate 5-Amide(GAP-EDL-3))的合成
将1-苄基-N-叔-丁氧基羰基-L-谷氨酸酯(0.50g，1.48mmol)、1,2,3-苯并三氮唑-1-醇(0.215g，1.59mmoml)和苯并三氮唑-1-氧基-三(二甲氨基)磷六氟磷酸盐(BOP，0.700g，1.58mmol)在氮气氛下溶解在无水二甲基甲酰胺(DMF，15mL)中。然后加入N,N-二异丙基乙胺(DIPEA，0.205g，1.59mmol)。将反应混合物在0-5℃下搅拌，然后加入GAP-EDL-2(0.50g，1.585mmol)。将反应混合物在0-5℃下搅拌10分钟并在室温下搅拌1小时。待反应完成后，在降低的压力下移除DMF并加入乙酸乙酯(250mL)。用4％的碳酸氢钠溶液(200mL)清洗有机相，在硫酸镁上干燥，过滤并在降低的压力下进行浓缩。通过柱层析 在用乙酸乙酯/二氯甲烷(EtOAc/DCM，5/1)洗脱的硅凝胶上纯化粗化合物，以提供呈白色固体的GAP-EDL-3(0.40g，0.63mmol，产率42.5％)。 1H-NMR(300MHz，CDCl3)：7.31-7.38(m，5H)，7.19(d，J＝8.7Hz，1H)，6.69(dd，J＝8.6Hz，2.7Hz，1H)，6.61(d，J＝2.6Hz，1H)，6.27(br，1H)，5.27-5.34(m，1H)，5.19(d，J＝12.2Hz，1H)，5.11(d，J＝12.2Hz，1H)，4.28-4.47(m，1H)，3.99(t，J＝5.1Hz，2H)，3.73(t，J＝8.5Hz，1H)，3.62(q，J＝5.7Hz，2H)，2.78-2.88(m，2H)，2.06-2.34(m，6H)，1.84-1.98(m，3H)，1.65-1.76(m，1H)，1.14-1.54(m，16H)，0.77(s，3H)。LC-MS：针对C37H50N2O7计算，634.4；发现[M+H]为635.2。光谱示于图4中。
(4)5-N-[氨基-3-乙氧基雌二醇]-N-叔-丁基羰基-L-谷氨酸5-酰胺(5-N-[Amino-3-ethoxyestradiol]-N-tert-butoxycarbonyl-L-Glutamic acid 5-Amide(GAP-EDL-4))的合成
在氮气氛下向GAP-EDL-3(0.39g，0.61mmmol)的THF(40mL)和甲醇(8mL)溶液中加入0.080g 5％的Pd/C。在氮气氛下将反应混合物在室温下搅拌1小时。然后将悬浮液在硅藻土上过滤并在降低的压力下浓缩。获得呈白色固体的脱酯(de-ester)产物(0.30g，0.55mmol，产率89.7％)。 1H-NMR(300MHz，CDCl3)：7.19(d，J＝8.7Hz，1H)，6.69(dd，J＝8.6Hz，2.7Hz，1H)，6.61(d，J＝2.6Hz，1H)，6.49(br，1H)，5.64-5.66(m，1H)，4.26(q，J＝6.6Hz1H)，4.02(t，J＝5.1Hz，2H)，3.73(t，J＝8.4Hz，1H)，3.62-3.70(m，2H)，2.81-2.86(m，2H)，2.38-2.60(m，2H)，2.26-2.36(m，1H)，2.06-2.22(m，3H)，1.82-2.04(m，3H)，1.64-1.76(m，1H)，1.17-1.55(m，16H)，0.77(s，3H)。LC-MS：针对C 30H 44N 2O 7计算，544.3；发现[M+H]为545.2。光谱示于图5中。
(5)5-N-[氨基-3-乙氧基雌二醇]-N-叔-丁基羰基-谷氨二酰基-1,5-二-叔丁基-L-谷氨酸五肽酯(5-N-[Amino-3-ethoxyestradiol]-N-tert-butoxycarbonyl-Glutamoyl-1,5-di-t-butyl-L-Glutamate pentapeptide ester(GAP-EDL-5))的合成
将GAP-EDL-4(0.580g，1.06mmol)、1-羟基苯并三唑(0.146g，1.08moml)和BOP(0.470g，1.06mmol)溶解在无水DMF(10mL)中。在氮气氛下将混合物搅拌，使温度冷却至0℃。然后加入N,N-二异丙基乙胺(DIPEA，0.140g,1.08mmol)和1,5-二-叔丁基-L-谷氨酸五肽酯(GAP酯，1.260g，1.06mmmol)(中国浙江省杭州市的浙江鸿拓生物技术有限公司)将反应混合物在50℃继续搅拌16-18小时，并用TLC进行监控。当TLC分析显示反应完成时，在降低的压力下去除DMF并添加DCM(250ml)。将有机相用饱和氯化钠溶液(200mL)进行清洗，在硫酸镁上干燥、过滤并在降低的压力下进行浓缩。通过采用梯度DCM和DCM/MeOH(100:0-100:1)洗脱的硅凝胶填充柱纯化粗化合物，以提供呈泡沫状白色固体的GAP-EDL-5(0.96g，0.56mmol，产率52.8％)。 1H-NMR(300MHz，DMSO+D2O)：7.80-8.20(m，7H)，7.12(d，J＝8.6Hz，1H)，7.03(brs，1H)，6.64(d，J＝8.6Hz，1H)，6.58(s，1H)，4.04-4.28(m，6H)，3.77-3.93(m，3H)，3.45-3.54(m，1H)，3.30-3.44(m，2H)，2.66-2.78(m，2H)，2.00-2.32(m，16H)，1.50-2.00(m18H)，1.35(s，72H)，1.05-1.28(m，7H)，0.63(s，3H)。光谱示于图6中。为了确定GAP-EDL-5的结构，对1,5-二-叔丁基-L-谷氨酸五肽酯(GAP酯)进行了质子NMR(核磁共振波谱法)(示于图7中)。将GAP-EDL-5的质子NMR与GAP酯进行了比较(分别详见图8、图7)。发现在GAP-EDL-5中7ppm处有化学位移，但在GAP酯中未发现。该化学位移为来自GAP-EDL-5的芳族质子。
(6)5-N-[氨基-3-乙氧基雌二醇]-谷氨二酰基-L-谷氨酸五肽(GAP-EDL)
在氮气氛下于0-5℃将GAP-EDL-5(0.560g，0.327mmol)溶解于无水DCM(11mL)中。加入三氟乙酸(2.8mL，37.702mmol)。将混合物在室温下搅拌16-18小时。在降低的压力下去除溶剂并用乙醚清洗残留物以得到粗产物。采用梯度洗脱VA:VB＝95:5-70:30(A相：0.1％的TFA水溶液；B相：0.1％的在乙腈中的TFA)通过制备型高效液相色谱(prep-HPLC)进行纯化。在冻干后，得到呈白色固体的GAP-EDL(0.214g，0.176mmol，产率53.7％)。 1H-NMR(300MHz， DMSO+D2O)：7.12(d，J＝8.6Hz，1H)，6.64(d，J＝8.6Hz，1H)，6.57(s，1H)，4.10-4.26(m，6H)，3.85-3.94(m，2H)，3.76-3.80(m，1H)，3.47-3.53(m，1H)，3.32-3.41(m，2H)，2.66-2.76(m，2H)，2.00-2.34(m，16H)，1.48-1.98(m，18H)，1.00-1.40(m，7H)，0.61(s，3H)；LC-MS：针对C 55H 78N 8O 23计算，1218.5；发现[M+H]为1219.7。质子NMR和质谱示于图9、图10(C 55H 78N 8O 23,1218.5；发现[M+H]为1219.7)中。为了确定GAP-EDL的结构，将GAP-EDL-4(单体)的质子NMR与GAP-DEL进行了比较(图11、图12)。发现在GAP-EDL-4和GAP-EDL中7ppm处都有来自芳族质子的化学位移。高效液相色谱(HPLC)分析表明GAP-EDL和GAP-EDL-4具有不同的吸收(210nm对239nm)和保留时间(图13、图14)。
(7) 68Ga-GAP-EDL的合成
68GaCl 3得自由HCL(范围为0.01N-1N)洗脱的 68Ge/ 68Ga发生装置。例如， 68GaCl 3从 68Ge/ 68Ga发生装置采用0.3N和0.6N的HCL(10mL)洗脱。在第二天，洗脱体积(0.3N或0.6N的HCL，6mL)被分配在12管中(0.5mL/管)。每个管计算其放射性。将在4和6之间部分中的最高活性进行组合。在连续的循环中，采用6mL HCL再次洗脱发生装置并根据前面的洗脱情况在这些特定的部分收集。将一小份 68GaCl 3(在0.6NHCL中，0.5ml，6.70mCi)加入至GAP-EDL(0.1mg)于0.8mL NaOAc(2.5M)的溶液中，且pH值为4-5。将溶液在70℃加热10分钟。冷却后，通过用盐水洗脱的ITLC(聚酰胺-6-层片材，cat.30149864，中国上海国药化学试剂公司)，美国密苏里州圣路易斯的奥德里奇-西格玛公司)测定放射化学纯度。放射化学纯度为100％，其中Rf值为0.01。在相同的ITLC条件下， 68Ga和 68Ga-GAP的Rf值都大于 68Ga-GAP-EDL的(分别示于图15、图16、图17)。
(8)体外细胞摄取实验
在乳腺肿瘤细胞中进行了 68Ga-GAP-EDL的体外细胞摄取实验。采用了具有高(MCF7)和低(SK-BR-3)雌激素受体浓度的乳腺癌细胞系来确定对细胞摄取分析的敏感性和特异性。简要地，对于敏感性分析，乳腺肿瘤细胞(50000 个细胞/孔，12孔)添加有 68GaCl 3、 68Ga-GAP和 68Ga-GAP-EDL(4μCi/80μL/孔,4μg/孔)。将所述孔温育0.5-2小时。为了证明 68Ga-GAP-EDL的细胞摄取是经由ER-介导的过程(特异性分析)，乳腺肿瘤细胞(50000个细胞/孔)用0、15、150、300μmol/L的冷雌素酮(在DMSO中)处理30分钟，随后添加 68Ga-GAP-EDL(4μg/孔,4μCi/孔)并温育达90分钟。温育后，收集细胞上清液。用冰冷PBS(1mL)作为清洗液清洗细胞后收集清洗液与细胞上清液合并为A管，细胞培养孔中加入胰蛋白酶-EDTA(0.1mL/孔)消化细胞。两分钟后，收集细胞。每孔用冷PBS(0.5mL)清洗两次和收集的细胞合并为B管。分别测定A管和B管的放射性计数。每个数据代表三次测量的平均值。计算摄取百分比(％)＝B管放射性计数/(A管放射性计数+B管放射性计数)×100％。
在敏感性分析中，在乳腺癌细胞摄取方面，相比 68GaCl 3和 68Ga–GAP的摄取， 68Ga-GAP-EDL的摄取有显著提高(如表1)。所述改进的合成方法制备纯的GAP-EDL并在细胞摄取研究中具有大大增强的敏感性。所述改进的合成方法的 68Ga-GAP-EDL的平均细胞摄取(如表1)至少为先前报道的合成方法(Takahashi N等人，Academic Radiology，2007)的5倍高。较高的ER(+)密度MCF-7相比较低的ER(+)SKBR-3具有更多的摄取。在特异性分析中，细胞摄取可能被雌素酮竞争性抑制，特别是在15、150、300μmol/L处。再有，尤其在15μmol/L的雌素酮处，相比于较低的ER(+)SKBR-3，MCF-7具有更加降低的摄取(如表2)。降低的摄取证明了 68Ga-GAP-EDL的细胞摄取经由ER-介导的过程。
表1.体外细胞摄取实验(三个测量结果的平均值)
Figure PCTCN2018115290-appb-000002
表2.体外细胞摄取特异性实验(三个测量结果的平均值)
雌素酮(umol/L) MCF-7 SK-BR-3
0 14.78±3.10 9.38±1.95
15 7.96±2.09 8.55±3.55
150 2.95±0.56 2.68±1.00
300 2.95±0.43 2.84±1.00
上述实施例不以任何方式限制本发明，凡是采用等同替换或等效变换的方式获得的技术方案均落在本发明的保护范围内。

Claims (21)

  1. 一种谷氨酸多肽-雌激素/抗雌激素缀合物，其特征在于包括具有3～20个重复氨基酸的多肽和雌激素或抗雌激素，所述雌激素或抗雌激素结合至上述多肽的第一谷氨酸盐处。
  2. 根据权利要求1所述的一种谷氨酸多肽-雌激素/抗雌激素缀合物，其特征在于：所述多肽为谷氨酸肽、天冬氨酸肽，或为谷氨酸肽、天冬氨酸肽混合的多肽。
  3. 根据权利要求1所述的一种谷氨酸多肽-雌激素/抗雌激素缀合物，其特征在于：所述多肽为3～6个重复氨基酸的多肽。
  4. 根据权利要求1所述的一种谷氨酸多肽-雌激素/抗雌激素缀合物，其特征在于：所述多肽为6个重复氨基酸的多肽。
  5. 根据权利要求1所述的一种谷氨酸多肽-雌激素/抗雌激素缀合物，其特征在于：所述多肽的分子量为500到9000。
  6. 根据权利要求1所述的一种谷氨酸多肽-雌激素/抗雌激素缀合物，其特征在于：所述雌激素为雌二醇、雌素酮、雌三醇或克罗米酚。
  7. 根据权利要求6所述的一种谷氨酸多肽-雌激素/抗雌激素缀合物，其特征在于：所述雌激素为雌二醇。
  8. 根据权利要求1所述的一种谷氨酸多肽-雌激素/抗雌激素缀合物，其特征在于：所述抗雌激素为非类固醇三苯氧胺、托瑞米芬、他莫昔芬、雷洛昔芬或氨鲁米特。
  9. 根据权利要求1所述的一种谷氨酸多肽-雌激素/抗雌激素缀合物，其特征在于：氨化的雌激素采用氨基和1-羧酸保护的谷氨酸两者结合至5-谷氨酸末端，制成谷氨二酰基-雌激素，所述谷氨二酰基-雌激素结合至全部酸保护的谷氨酸肽的氨基末端。
  10. 根据权利要求1所述的一种谷氨酸多肽-雌激素/抗雌激素缀合物，其特征在于其结构为：
    Figure PCTCN2018115290-appb-100001
  11. 一种谷氨酸多肽-雌激素/抗雌激素缀合物的合成方法，其特征在于：将氨基雌激素或氨基抗雌激素与羧酸保护的谷氨酸盐在有机溶剂中混合，制备谷氨酸盐-雌激素缀合物或谷氨酸盐-抗雌激素缀合物；将谷氨酸盐-雌激素缀合物或谷氨酸盐-抗雌激素缀合物与包含偶联剂的全部羧酸保护的多肽进行反应，相应制得谷氨酸多肽-雌激素缀合物或谷氨酸多肽-抗雌激素缀合物，所述雌激素位于谷氨酸多肽-雌激素的第一谷氨酸盐处，所述抗雌激素位于谷氨酸多肽-抗雌激素的第一谷氨酸盐处。
  12. 根据权利要求11一种谷氨酸多肽-雌激素/抗雌激素缀合物的合成方法，其特征在于：所述氨基雌激素为氨基位于雌激素的3’、5’、17’位的雌激素。
  13. 根据权利要求11一种谷氨酸多肽-雌激素/抗雌激素缀合物的合成方法，其特征在于：所述氨基雌激素具有位于甾体环的3’位处的氨基。
  14. 根据权利要求11一种谷氨酸多肽-雌激素/抗雌激素缀合物的合成方法，其特征在于：所述氨基雌激素为氨基雌二醇、氨基雌三醇或氨基雌素酮。
  15. 根据权利要求11一种谷氨酸多肽-雌激素/抗雌激素缀合物的合成方法，其特征在于：所述抗雌激素为非类固醇三苯氧胺、托瑞米芬、他莫昔芬、雷洛 昔芬或氨鲁米特。
  16. 根据权利要求11所述的谷氨酸多肽-雌激素/抗雌激素缀合物的合成方法，其特征在于：将氨基雌二醇和1-羧酸保护的谷氨酸盐在有机溶剂中混合，制备γ谷氨二酰基-雌二醇缀合物；将所述γ谷氨二酰基-雌二醇缀合物与包含偶联剂的全部羧酸保护的谷氨酸肽进行反应制得谷氨酸多肽-雌二醇，雌二醇位于谷氨酸多肽-雌二醇的第一谷氨酸盐处。
  17. 一种用于个人诊断或精确医疗的组合物，其特征在于包括权利要求1～10任一项所述的谷氨酸多肽-雌激素/抗雌激素缀合物或权利要求11～16任一项所述的谷氨酸多肽-雌激素/抗雌激素缀合物的合成方法获得的谷氨酸多肽-雌激素/抗雌激素缀合物和用于标记的金属离子。
  18. 根据权利要求17所述的一种用于个人诊断或精确医疗的组合物，其特征在于：所述金属离子是放射性核素。
  19. 根据权利要求19所述的一种用于个人诊断或精确医疗的组合物，其特征在于：所述金属离子是Tc-99m、Ga-68、Cu-60、Cu-64、In-111、Ho-166、Re-186、Re-188、Y-90、Lu-177、Ra-223、Ac-225，或在疾病治疗中用于诊疗的金属。、
  20. 根据权利要求17所述的一种用于个人诊断或精确医疗的组合物，其特征在于：所述组合物为 99mTc-谷氨酸肽-雌二醇或 68Ga-谷氨酸肽-雌二醇。
  21. 一种试剂盒，其特征在于包括权利要求17-20任一项所述的一种用于个人诊断或精确医疗的组合物。
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