WO2018102974A1 - 一种天然活性成分强心甾类固醇与奥曲肽的偶联物及其制法和用途 - Google Patents

一种天然活性成分强心甾类固醇与奥曲肽的偶联物及其制法和用途 Download PDF

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WO2018102974A1
WO2018102974A1 PCT/CN2016/108630 CN2016108630W WO2018102974A1 WO 2018102974 A1 WO2018102974 A1 WO 2018102974A1 CN 2016108630 W CN2016108630 W CN 2016108630W WO 2018102974 A1 WO2018102974 A1 WO 2018102974A1
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steroid
octreotide
conjugate
cardiotonic
tumor
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徐希明
张辉云
余江南
徐文倩
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Jiangsu University
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Jiangsu University
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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
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin

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  • the invention relates to a conjugate of a natural active ingredient cardiotonic steroid and octreotide, a preparation method thereof and use as an antitumor drug.
  • Steroidal steroids have a variety of anti-tumor mechanisms, which are important for the prevention and treatment of malignant tumors. They are an emerging lead compound for screening tumor therapeutic drugs. China's traditional Chinese medicine plant resources are abundant. The screening and mechanism research on the activity of traditional Chinese medicine for cardiac steroids, and the investment and development of new anti-tumor drug development are of great significance for the prevention and clinical treatment of tumors.
  • Different cardiotonic steroid components have different physical and chemical properties. Some components have strong hydrophobicity and low water solubility. Some components are hydrophilic and water soluble, but they also have common characteristics, such as narrow therapeutic window and system. Sexual toxicity, instability, irritation, etc. These characteristics often result in significant limitations in clinical administration and treatment.
  • Octreotide a widely studied somatostatin analogue that selectively binds to the somatostatin receptors SSTR-2, SSTR-3, and SSTR-5, has a weak affinity for SSTR-1 and SSTR-4 (see: Huo M, Zou A, Yao C, et al. Biomaterials. 2012, 33:6393-6407.). Due to its good stability in humans (half-life of 100 min), octreotide is clinically used for the treatment and diagnosis of malignant tumors with high expression of SSTRs (see: Sclafani F, Carnaghi C, Di T L, et al. Tumori. 2011, 97: 620-628.).
  • octreotide As it is swollen Successful use in angiography, octreotide is also used in drug delivery systems, and octreotide improves tumor targeting by coupling with anti-tumor drugs to reduce its toxicity (see: De J M, Breeman W A, Kwekkeboom D J) , et al. Accounts of Chemical Research. 2009, 42. Huang C M, Wu Y T, and Chen S T. Chemistry & biology. 2000, 7: 453-461.
  • One of the objects of the present invention is to provide a conjugate of a natural active ingredient, cardiotonic steroid and octreotide, and a process for the preparation thereof.
  • a second object of the present invention is to provide an in vitro anti-tumor evaluation of a conjugate of a cardiac steroid and octreotide.
  • a third object of the present invention is to provide the use of the conjugate nanoparticles described in the treatment of liver cancer.
  • a conjugate of a cardiac steroid and octreotide which is a cardiotonic steroid linked to a cardiac steroid by a carboxyl group of a dicarboxylic acid, and then a dicarboxylic acid is then linked to octreotide to obtain a cardiac steroid and octreotide. Conjugate.
  • the cardiotonic steroid may be a leucoside, a leucovorin or a lycopene.
  • a method for preparing the above-mentioned conjugate of cardiotonic steroid and octreotide comprising the steps of:
  • Step 1 Weigh the amount of cardiotonic steroids and anhydrides, the molar ratio of cardiotonic steroids and anhydrides is 1/2 to 1/10, add dichloromethane to dissolve, add a certain amount of triethylamine and 4-di
  • the molar ratio of methylaminopyridine (Dmap), triethylamine and 4-dimethylaminopyridine to cardiotonic steroid is 1/1/10 ⁇ 1/1/5. After stirring for 24 hours, it is rotated by a rotary evaporator.
  • carboxylated cardiotonic steroid derivative which may be succinic anhydride, maleic anhydride, etc.
  • the dibasic acid anhydride, the obtained carboxylated cardiotonic steroid derivative is characterized by infrared and ESI-MS;
  • Step 2 Weigh the product of step 1, dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) in an appropriate amount to 2:1 by volume of dichloromethane (DCM) and tetrahydrofuran (THF).
  • DCC dicyclohexylcarbodiimide
  • NHS N-hydroxysuccinimide
  • N-hydroxysuccinimide is 1/2/2 ⁇ 1/2/4, sealed at room temperature, magnetically stirred for 2-24 hours, then blown dry with nitrogen, added with 50% acetonitrile-water sonicated Purified by C8 column, the eluent was in different proportions of acetonitrile-0.05% trifluoroacetic acid (TFA) aqueous solution, detected by TLC, spots were collected, and directly lyophilized to obtain NHS-PPG samples.
  • TFA trifluoroacetic acid
  • Step 3 Weigh the appropriate amount of the product NHS-PPG in step 2, dissolve it with 50% acetonitrile-containing 0.1 M 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), add octreotide, molar ratio of octreotide to NHS-PPG. It is 1.5/1 ⁇ 5/1, N-methylmorpholine is adjusted to pH 8.0-9.0, sealed, room temperature, magnetic stirring reaction for 12-24 hours, and analyzed by liquid chromatography-mass spectrometry, the purification of the reaction product is highly efficient.
  • HEPES 4-hydroxyethylpiperazineethanesulfonic acid
  • cardiotonic steroid-octreotide conjugates were prepared by liquid chromatography, which were octreotide (alanine)-cardiotonic steroid conjugate and octreotide (lysine)-cardiotonic steroid conjugate. And cardiotonic steroid-octreotide-cardiotonic steroid conjugate.
  • the cardiac steroids described in the step 1 include cardiotonic substances such as perilla aglycone, periplocin, and leucovorin.
  • the purification method in the step 1 or the step 2 is not limited to C8, C18 packed column chromatography and liquid phase preparative purification, and may also be purified by silica gel column chromatography.
  • the molar ratio of the cardiotonic steroid and the acid anhydride in the step 1 is preferably 1/2 to 1/10.
  • the organic solvent in step 1 may be selected from one or more mixed solvents of DMSO, ethyl acetate and DMF in addition to dichloromethane.
  • the inventors of the present invention evaluated the toxicity of the natural active ingredient cardiac steroid and octreotide on the tumor cells and normal cells, and found that the cardiac steroid-octreotide (alanine) conjugate was significantly toxic to tumor cells. It is better than the strong heart steroid prototype drug, and the toxicity to normal cells is significantly weaker than the cardiotonic steroid prototype drug.
  • cardiac steroid-octreotide (alanine) conjugate in the treatment of liver cancer.
  • cardiac steroid-octreotide (alanine) conjugate In vivo anti-tumor activity experiments were performed using H 22 liver cancer solid tumors as a model. The experimental results show that the cardiotonic steroid-octreotide (alanine) conjugate inhibits the growth of H 22 liver tumor solid tumors stronger than the cardiotonic steroid prototype drug, which can be used to treat liver cancer.
  • the synthetic cardiac steroid-octreotide conjugate of the present invention is a conjugate of perilla aglycone-octreotide and a leucovorin-octreotide, respectively, wherein the cardiac steroid-octreotide (alanine) conjugate is the most active. Strong.
  • the preparation of the cardiac steroid-octreotide (alanine) conjugate prepared by the invention has obvious tumor cell selectivity and tumor tissue targeting, improves the antitumor activity of the cardiac steroid and inhibits the drug in vivo. In vivo toxicity.
  • FIG. 1 Characterization of periplocin-octreotide conjugate: A: LC-MS total ion pattern of the reaction solution; B: 1 H-NMR chart, where a is PPG; b is OCT(Phe)-S-PPG; It is OCT(Lys)-S-PPG; d is OCT-2S-2PPG; e is OCT.
  • Figure 4 Characterization of the scutellarin-octreotide conjugate: A, HPLC; B, PPM (a), OCT (b), SPPM (c), OCT (Phe) - S-PPM (d), OCT Infrared spectra of (Lys)-S-PPM(e) and OCT-2S-2PPM(f); C, 1 H-NMR chart.
  • FIG. 1 In vitro cytotoxicity of OCT, PPG, OCT(Phe)-S-PPG, OCT(Lys)-S-PPG and OCT-2S-2PPG: A is a test drug applied to HepG2 cells for 72 h; B is a test The drug acts on normal human liver cells L-02 for 72 h.
  • FIG. 9 Efficacy diagram of OCT(Phe)-S-PPG and PPG on H 22 solid tumor of liver cancer: (A) graph of volume growth of solid tumor after administration; (B) graph of body weight change after administration (C) Solid tumor after 11 days of administration and its weight (D).
  • FIG. Pharmacological map of OCT(Phe)-S-PPM and PPM against H 22 solid tumor of liver cancer: (A) graph of volume growth of solid tumor after administration; (B) graph of body weight change after administration (C) Solid tumor after 11 days of administration and its weight (D).
  • FIG. 1 Pathological section of heart and liver tissue of the OCT (Phe)-S-PPG and PPG groups (H&E staining, x 100or x 400).
  • FIG 12 PPM and OCT (Phe) -S-PPM group after treatment of cardiac solid tumor H 22, and Liver Histology FIG tumors (H & E stained, ⁇ 20).
  • Dichloromethane, 50% methanol-0.1% formic acid water was dissolved in the sample, and purified by C8 reversed-phase silica gel column, 50% methanol-0.1% formic acid water and 55% methanol-0.1% formic acid water 100 mL, respectively, detected by TLC, LC,
  • the TLC conditions were dichloromethane with 10:1, 5% phosphomolybdic acid, LC conditions, column, waters ODS C18 (150 mm * 4.6 mm), mobile phase 58% methanol - 0.1% formic acid,
  • the flow rate was 1 mL/min, the wavelength was 220 nm, and Rt was 12.3 min.
  • the color point was collected and concentrated to obtain 12 mg, which was characterized by carboxylation and ESI-MS as carboxylated perilla aglycone (PPS).
  • HOOC-PPM (12.8 mg, 0.02 mmol), DCC (7.79 mg, 0.0408 mmol, 2 ⁇ excess), NHS (5.0 mg, 0.0408 mmol, 2 ⁇ excess) was weighed and dissolved in 3 mL of DCM+THF (2:1). Sealed, room temperature, magnetic stirring reaction for 2h, nitrogen drying, adding 20% acetonitrile-water sonicated, purified by C8 column, the eluent is 20% acetonitrile-0.05% TFA water and 50% acetonitrile-0.05% TFA water Each 100 mL, TLC detected NHS-PPM, collected spots, and directly lyophilized to obtain 10.5 mg of NHS-PPM sample, and the yield was 71.27%.
  • the NHS-PPM (7.4 mg, 0.01 mmol) was weighed, dissolved in 50% acetonitrile-water (0.1 M HEPES), 20.3 mg (0.021 mmol, 2 ⁇ excess) of octreotide was added, and the pH was adjusted to 8.0 by N-methylmorpholine.
  • the cardiac steroid-octreotide conjugate was characterized by LC-MS, 1 H-NMR and infrared. The results are shown in Figures 3 and 4.
  • the human hepatoma cell line HepG2 and human normal liver cell L-02 were cultured to cover 80%-90% of the bottom of the flask, and the cells were digested with 0.25% trypsin for 5 min, then added to the medium containing calf serum; centrifuged at 1000 rpm for 5 min. Thereafter, a cell suspension was prepared, and 4.5 mL of a cell suspension (1 ⁇ 10 5 /mL) was added to the flask to continue the culture.
  • the HepG2 and L-02 cell lines were inoculated into 96-well plates with 2 cell lines of 10 cells/well, respectively, and cultured for 24 hours. 100 ⁇ L of the medium containing the test drug was sequentially added to each well, and the concentrations of the cardiac steroid-octreotide conjugate were 0.005, 0.05, 0.5, 1, 2.5, 5, and 50.0 ⁇ M, respectively. Three replicate holes are provided for each concentration. Cell morphology was observed after 72 h.
  • IR inhibition rate
  • the cardiac steroid-octreotide (alanine) conjugate was significantly more toxic to tumor cells than the cardiac steroid steroid, and the toxicity to normal cells was significantly weaker than that of cardiac sputum.
  • Steroid prototype drug As shown in Figures 5 and 6, the cardiac steroid-octreotide (alanine) conjugate was significantly more toxic to tumor cells than the cardiac steroid steroid, and the toxicity to normal cells was significantly weaker than that of cardiac sputum. Steroid prototype drug.
  • mice 60 Kunming mice, weighing 18-22g, were male (purchased from Jiangsu University Animal Center). After acclimatization the mice 2d, H 22 containing from mouse ascites tumor ascites was clarified by centrifugation and treated erythrocyte lysis buffer, PBS buffer configuration with the amount of liquid to 1 ⁇ 107 / mL single cell suspension.
  • mice The right axillary skin of the mice was disinfected with iodophor cotton balls.
  • mice One week after the inoculation, the right subcutaneous subcutaneous tumor volume of the mouse grew to a size of about 1 cm3, and the tumor formation rate was 100%, and the body weight of the mice was weighed one by one.
  • Sixty male H 22 solid tumor Kunming mice were randomly divided into groups. Five mice in each group were subjected to tissue distribution experiments. During the environmental adaptation period, they were fed normally with water. They were fasted for 12 hours before the experiment and were given free access to water.
  • the dose administered was 20.5 ⁇ mol/kg.
  • the injection volume of mice weighing 20 g was 0.2 mL, and the injection volume was adjusted according to the actual body weight of the mice.
  • mice Each group of mice was injected intravenously with PPG and OCT (Phe)-S-PPG at a dose of 20.5 ⁇ mol/kg.
  • the rats were sacrificed at 0.16, 0.50, 1, 2, 3 and 4 hours after administration.
  • Blood, heart, liver, spleen, lung, kidney and tumor were taken.
  • the blood of the mice was placed in a 2 mL plastic centrifuge tube with appropriate amount of heparin sodium. After half an hour, the plasma was centrifuged at 4000 rpm for 10 min, and stored at -20 °C until the sample was determined.
  • mice Each group of mice was injected intravenously with PPM and OCT (Phe)-S-PPM at a dose of 4 mg/kg and 12.23 mg/kg, respectively.
  • the other steps are as described above.
  • the cardiac steroid-octreotide (alanine) conjugate significantly increased the distribution of cardiotonic steroids in tumor tissues, while reducing the distribution in heart and liver tissues. It indicates that the cardiac steroid-octreotide (alanine) conjugate has tumor targeting properties.
  • mice One week after inoculation, the diameter of the right subcutaneous subcutaneous tumor of the mouse was about 0.5 cm in diameter, and the tumor formation rate was 100%.
  • the body weight of the mice was weighed one by one, and they were divided into 4 groups by random number table, each group 6 only.
  • Blank model group 0.1 mL of blank injection was injected into the tail vein of mice, and administered once every 2 days.
  • OCT group The mice were injected with 20.5 ⁇ mol/kg in the tail vein and administered once every 2 days.
  • mice were injected with 20.5 ⁇ mol/kg in the tail vein and administered once every 2 days.
  • mice were injected with 20.5 ⁇ mol/kg in the tail vein and administered once every 2 days.
  • Blank model group The mouse tail vein was injected with a blank injection of 5 mL/kg once a day for 2 days.
  • OCT group The mice were injected with 7.62 mg/kg in the tail vein and administered once every 2 days.
  • PPM group 4 mg/kg was injected into the tail vein of mice, and administered once every 2 days.
  • mice were injected with 12.23 mg/kg in the tail vein and administered once every 2 days.
  • mice The body weight of the mice was weighed every 2 days and recorded.
  • Tumor inhibition rate (control unit average tumor weight - mean tumor weight of the medication group) / control group mean tumor weight x 100%.
  • H&E hematoxylin-eosin
  • the cardiotonic steroid-octreotide (alanine) conjugate inhibited the growth of H 22 liver tumor solid tumors more strongly than the cardiotonic steroid prototype drug, and was used for treating liver cancer.
  • the cardiac steroid-octreotide (alanine) conjugate reduced liver and cardiotoxicity of the cardiotonic steroid prototype.

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Abstract

一种天然活性成分强心甾类固醇与奥曲肽的偶联物及其制备方法，它是以丁二酸酐为连接剂，合成得到强心甾类固醇-奥曲肽偶联物。该偶联物在体外细胞毒实验中，明显提高了强心甾类固醇对肿瘤细胞的毒性(包括人肝癌细胞HepG2、人乳腺癌细胞MCf-7)，同时降低了对正常细胞的毒性，同时肿瘤靶向性提高、心脏和肝脏毒性降低、肿瘤治疗效果增强。

Description

一种天然活性成分强心甾类固醇与奥曲肽的偶联物及其制法和用途 技术领域
本发明涉及天然活性成分强心甾类固醇与奥曲肽的偶联物及其制备方法和作为抗肿瘤药物的用途。
背景技术
近年来，基于临床上化疗药物的毒副作用、耐药性和经济性的多重考虑，人们纷纷把目光转向从植物中获取安全、高效、低毒的活性先导化合物。强心甾类固醇在过去几百年来一直作为强心剂而倍受关注，进入20世纪以来，随着植物化学、生物化学、药物化学和生物医学的发展及中药分离技术的进步，大量的强心甾类固醇物质被证实具有良好体内外抗肿瘤活性，且与其他临床上常用化疗药物的抗肿瘤机理不同，是一种多效或者多机制的抗癌药物。
强心甾类固醇成分具有多种抗肿瘤机制，其对恶性肿瘤的预防及治疗具有重要意义，是一种新兴的筛选肿瘤治疗药物的先导化合物。我国中药植物资源丰富，关于强心甾类固醇的中药活性筛选、机理研究、抗肿瘤新药开发的投入和发展对肿瘤的预防和临床治疗具有重大的意义。不同的强心甾类固醇成分，其理化性质各异，部分成分的疏水性强、水溶解性低，有些成分亲水强、水溶解性好，但也存在共有的特性，如治疗窗窄、系统性毒性强、不稳定性、刺激性等。这些特性常导致其在临床给药和治疗受到极大的限制。选择合适的前体药物设计，将先进的药物合成技术、前体药物设计技术运用于高效活性成分强心甾类固醇的产品开发中，合成出靶向性好、选择性高和毒性低的前体药物，对此类物质的新药开发和临床应用十分关键，而此类研究势必为中药活性成分强心甾类固醇在临床的应用提供新思路和新方法。
奥曲肽，一种被广泛研究的生长激素抑制素类似物，选择性结合生长抑素受体SSTR-2、SSTR-3和SSTR-5而对SSTR-1和SSTR-4的亲和力较弱(参见：Huo M,Zou A,Yao C,et al.Biomaterials.2012,33:6393-6407.)。由于奥曲肽在人体内的稳定性好(半衰期为100min)而被临床应用于治疗和诊断SSTRs高表达的恶性肿瘤(参见：Sclafani F,Carnaghi C,Di T L,et al.Tumori.2011,97:620-628.)。随着其在肿 瘤造影中的成功利用，奥曲肽亦被用于药物肿瘤传递系统中，奥曲肽通过与抗肿瘤药物偶联来降低其毒性而提高肿瘤靶向性(参见：De J M,Breeman W A,Kwekkeboom D J,et al.Accounts of Chemical Research.2009,42.Huang C M,Wu Y T,and Chen S T.Chemistry&biology.2000,7:453-461.)
中国专利申请CN201010583339.0《一种杠柳次苷的制备方法》，是关于杠柳次苷的制备方法，未涉及到该药在抗肿瘤活性方面的应用。
发明内容
本发明的目的之一是，提供天然活性成分强心甾类固醇与奥曲肽的偶联物及其制备方法。
本发明的目的之二是，提供强心甾类固醇与奥曲肽的偶联物的体外抑瘤评价。
本发明的目的之三是，提供所述的偶联物纳米粒在治疗肝癌中的应用。
本发明的技术方案如下：
一种强心甾类固醇与奥曲肽的偶联物，它是强心甾类固醇通过二元羧酸的羧基与强心甾固醇连接，随后二元羧酸再与奥曲肽连接得到强心甾类固醇与奥曲肽的偶联物。
上述的偶联物，所述的强心甾类固醇可以是杠柳苷元、杠柳次苷或杠柳毒苷等。
一种制备上述的强心甾类固醇与奥曲肽的偶联物的方法，它包括如下步骤：
步骤1.称取强心甾类固醇和酸酐适量，强心甾类固醇和酸酐的摩尔比为1/2～1/10，加二氯甲烷混匀至溶解，加入一定量三乙胺和4-二甲氨基吡啶(Dmap)，三乙胺和4-二甲氨基吡啶与强心甾类固醇的摩尔比为1/1/10～1/1/5，搅拌反应24h后，采用减压旋转蒸发仪旋干二氯甲烷，采用C8柱纯化，洗脱剂为不同比例甲醇-0.1％甲酸水，得到羧基化强心甾类固醇衍生物(SPPG)，所述的酸酐可以是丁二酸酐、马来酸酐等二元酸酐，所得到的羧基化强心甾类固醇衍生物经红外及ESI-MS表征；
步骤2.称取步骤1产物、二环己基碳二亚胺(DCC)和N-羟基琥珀酰亚胺(NHS)适量加入体积比为2:1的二氯甲烷(DCM)与四氢呋喃(THF)的混合溶剂溶解羧基化强心甾类固醇衍生物(SPPG)、二环己基碳二亚胺(DCC)和 N-羟基琥珀酰亚胺(NHS)的摩尔比为1/2/2～1/2/4，密封室温，磁力搅拌反应2～24h后，氮气吹干，加入50％乙腈-水超声混溶，采用C8柱纯化，洗脱剂为不同比例的乙腈-0.05％三氟乙酸(TFA)水溶液，TLC检测，收集斑点，直接冻干，得NHS-PPG样品。
步骤3.称取步骤2产物NHS-PPG适量，用加入50％乙腈-含0.1M 4-羟乙基哌嗪乙磺酸(HEPES)的水溶液溶解，加入奥曲肽，奥曲肽与NHS-PPG的摩尔比为1.5/1～5/1，N-甲基吗啉调节pH值为8.0～9.0，密封，室温，磁力搅拌反应12～24h后，用液相色谱-质谱进行分析，反应产物的纯化通过高效液相色谱半制备得到三种强心甾类固醇-奥曲肽偶联物，它们分别是奥曲肽(丙氨酸)-强心甾类固醇偶联物、奥曲肽(赖氨酸)-强心甾类固醇偶联物和强心甾类固醇-奥曲肽-强心甾类固醇偶联物。
上述的方法，步骤1所述的强心甾类固醇包括杠柳苷元、杠柳次苷、杠柳毒苷等强心甾类固醇类物质。
上述的方法，步骤1或步骤2中的纯化方法不限于C8、C18填料柱层析及液相制备纯化，也可以采用硅胶柱层析纯化。
上述的方法，步骤1中强心甾类固醇和酸酐的摩尔比优选为1/2～1/10。
上述的方法，步骤1中有机溶剂除二氯甲烷外，也可以选自DMSO、乙酸乙酯、DMF中的一种或者多种混合溶剂
本发明人对天然活性成分强心甾类固醇与奥曲肽的偶联物体外作用于肿瘤细胞和正常细胞的毒性评价，发现强心甾类固醇-奥曲肽(丙氨酸)偶联物对肿瘤细胞的毒性明显强于强心甾类固醇原型药，而对正常细胞的毒性明显弱于强心甾类固醇原型药。
进一步发现强心甾类固醇-奥曲肽(丙氨酸)偶联物的组织分布特性。以H22肝癌实体瘤为模型进行组织分布实验，结果表明，强心甾类固醇-奥曲肽(丙氨酸)偶联物具有肿瘤靶向性。
强心甾类固醇-奥曲肽(丙氨酸)偶联物在治疗肝癌中的应用。以H22肝癌实体瘤为模型进行体内抗肿瘤活性实验。实验结果表明，强心甾类固醇-奥曲肽(丙氨酸)偶联物抑制H22肝癌实体瘤的生长的作用强于强心甾类固醇原型药，可用于治疗肝癌。
有益效果
(1)本发明合成强心甾类固醇-奥曲肽偶联物，分别是杠柳苷元-奥曲肽和杠柳次苷-奥曲肽偶联物，其中强心甾类固醇-奥曲肽(丙氨酸)偶联物活性最强。
(2)本发明制备得到强心甾类固醇-奥曲肽(丙氨酸)偶联物具有明显肿瘤细胞选择性和肿瘤组织靶向性，提高了强心甾类固醇的体内抑瘤活性，降低了药物的体内毒性。
附图说明：
图1、OCT(Phe)-S-PPG合成路线图。
图2、OCT(Phe)-S-PPM合成路线图。
图3、杠柳苷元-奥曲肽偶联物的表征:A:反应液的LC-MS总离子图；B:1H-NMR图，其中a为PPG；b为OCT(Phe)-S-PPG；c为OCT(Lys)-S-PPG；d为OCT-2S-2PPG；e为OCT。
图4、杠柳次苷-奥曲肽偶联物的表征：A，HPLC图；B，PPM(a)、OCT(b)、SPPM(c)、OCT(Phe)–S-PPM(d)、OCT(Lys)-S-PPM(e)和OCT-2S-2PPM(f)的红外图谱；C，1H-NMR图。
图5、OCT，PPG，OCT(Phe)-S-PPG，OCT(Lys)-S-PPG和OCT-2S-2PPG的体外细胞毒性：A为受试药物作用于HepG2细胞72h；B为受试药物作用于正常人肝细胞L-02 72h。
图6、杠柳次苷-奥曲肽偶联物作用于HepG-2、MCF-7和L-02细胞72h后细胞毒性。
图7、PPG和OCT(Phe)-S-PPG尾静脉注射给药后各个时间点后血浆及各个脏器中药物含量(μg/g)(n＝5)。
图8、PPM和OCT(Phe)-S-PPM尾静脉注射给药后各个时间点后血浆及各个脏器中药物含量(μg/g)(n＝5)。
图9、OCT(Phe)-S-PPG和PPG对H22肝癌实体瘤的药效图：(A)给药后实体瘤体积生长变化曲线图；(B)给药后小鼠的体重变化图；(C)给药11天后的实体瘤及其重量(D)。
图10、OCT(Phe)-S-PPM和PPM对H22肝癌实体瘤的药效图：(A)给药后实体瘤体积生长变化曲线图；(B)给药后小鼠的体重变化图；(C)给药11天后的 实体瘤及其重量(D)。
图11、OCT(Phe)-S-PPG和PPG组的心脏和肝脏组织的病理切片图(H&E staining,×100or×400)。
图12、PPM和OCT(Phe)-S-PPM组治疗H22实体瘤小鼠后的心脏、肝脏和肿瘤的病理切片图(H&E染色，×20)。
具体实施方式
以下所列实施例有助于本领域技术人员更好地理解本发明，但不以任何方式限制本发明。
实施例1.奥曲肽-杠柳苷元偶联物的制备
称取杠柳苷元20mg和丁二酸酐30mg，加3mL二氯甲烷混匀至溶解，加入一定量三乙胺和4-二甲氨基吡啶(Dmap)，搅拌24h后，采用减压旋转蒸发仪旋干二氯甲烷，加50％甲醇-0.1％甲酸水溶解样品，上C8反相硅胶柱纯化，50％甲醇-0.1％甲酸水和55％甲醇-0.1％甲酸水各100mL，通过TLC、LC检测，TLC条件为二氯甲烷比甲醇为10:1，5％磷钼酸显色，LC条件为，色谱柱，waters ODS C18(150mm*4.6mm)，流动相为58％甲醇-0.1％甲酸水，流速为1mL/min，波长为220nm，Rt＝12.3min，收集显色点，合并浓缩得到12mg，经红外及ESI-MS表征为羧基化杠柳苷元(PPS)。
称取PPS(10mg，0.0204mmol)，DCC(7.79mg，0.0408mmol，2×excess)，NHS(5.0mg，0.0408mmol，2×excess)加入3ml DCM+THF(2:1，v/v)溶解，密封，室温，磁力搅拌反应2h后，氮气吹干，加入50％乙腈-水超声混溶，加入C8柱纯化，洗脱剂为20％乙腈-0.05％TFA水和50％乙腈-0.05％TFA水各100mL，TLC检测，收集斑点，直接冻干，得NHS-PPG样品8mg，得率为74.20％。
取NHS-PPG(3.5mg，0.0059mmol)，用加入50％乙腈-水(0.1M HEPES)溶解，加入OCT 12.1mg(0.0119mmol，2excess)，N-甲基吗啉调节PH值为8.0左右，密封室温，磁力搅拌反应24h后，用液相-质谱进行分析。反应产物的纯化通过高效液相色谱半制备完成。流速4mL/min，流动相溶剂从0.01min的30％A(0.05％TFA水溶液)和70％溶剂B(0.0.5％TFA-乙腈溶液)到90min的80％溶剂A和20％溶剂B。制备流程如图1所示。
实施例2.奥曲肽-杠柳次苷偶联物的制备
称取杠柳次苷(27.1mg，0.05mmol)和丁二酸酐(30mg，0.3mmol)，加入3mL二氯甲烷混匀至溶解，加入一定量三乙胺和Dmap，搅拌24h后，采用减压旋转蒸发仪旋干二氯甲烷，加20％乙腈-0.05％TFA水溶解样品，上C8反相硅胶柱纯化，洗脱液为20％乙腈-0.05％TFA水和40％乙腈-0.05％TFA水各150mL，通过TLC、LC检测，TLC条件为二氯甲烷比甲醇为10︰1，5％磷钼酸显色，LC条件为，色谱柱，waters ODS C18(150mm*4.6mm)，流动相为40％乙腈-0.05％TFA水，流速为1mL/min，Rt＝5.1min，收集显色点，合并浓缩得到HOOC-PPM(26mg)。
称取HOOC-PPM(12.8mg，0.02mmol)，DCC(7.79mg，0.0408mmol，2×excess)，NHS(5.0mg，0.0408mmol，2×excess)加入3mL DCM+THF(2:1)溶解，密封，室温，磁力搅拌反应2h后，氮气吹干，加入20％乙腈-水超声混溶，采用C8柱纯化，洗脱剂为20％乙腈-0.05％TFA水和50％乙腈-0.05％TFA水各100mL，TLC检测NHS-PPM，收集斑点，直接冻干，得NHS-PPM样品10.5mg，得率为71.27％。
称取NHS-PPM(7.4mg，0.01mmol)，加入50％乙腈-水(0.1M HEPES)溶解，加入奥曲肽20.3mg(0.021mmol，2×excess)，N-甲基吗啉调节PH值为8.0左右，密封，室温，磁力搅拌反应2h，反应结束后，过C8柱纯化分离，洗脱程序为10％乙腈-0.05％TFA水、20％乙腈-0.05％TFA水、40％乙腈-0.05％TFA水、50％乙腈-0.05％TFA、90％乙腈-0.05％TFA水依次洗脱。LC检测收集液，合并相同出峰时间收集液，分别得OCT(Phe)-S-PPM、OCT(Lys)-S-PPM和OCT-2S-2PPM。制备流程图如图2所示。
实施例3强心甾类固醇-奥曲肽偶联物的表征
采用LC-MS、1H-NMR和红外对强心甾类固醇-奥曲肽偶联物进行表征，结果如图3和图4所示。
实施例4.细胞毒性评价
(1)细胞培养
将人肝癌细胞株HepG2、人正常肝细胞L-02培养至覆盖培养瓶瓶底80％～90％，用0.25％的胰蛋白酶消化细胞5min后加入含小牛血清的培养基终止；1000rpm离心5min后制成细胞悬液，在培养瓶中加入4.5mL细胞悬液(1×105个/mL)继续培养。
(2)强心甾类固醇-奥曲肽偶联物对人肝癌细胞株HepG2、人正常肝细胞L-02的毒性评价
分别将HepG2、L-02细胞株分别以2胞株l04个细胞/孔接种于96孔板，培养24h。各孔依次加入含有受试药物的培养基100μL，强心甾类固醇-奥曲肽偶联物的浓度分别为0.005、0.05、0.5、1、2.5、5、50.0μM。每个浓度设3个复孔。72h后观察细胞形态。
(3)MTT法测定细胞生存率
各孔加入浓度为5mg/mL的MTT 20μL培养4h后弃上清，再加入150μL DMSO反应10min，于570nm波长测定吸光度值(OD值)。按如下公式计算抑制率(inhibition rate IR)：IR(％)＝(1-受试药物组吸光度/阴性组吸光度)×100％。采用Curve-Expert软件计算样品半数抑制浓度(IC50)。
结果如图5、6所示，强心甾类固醇-奥曲肽(丙氨酸)偶联物对肿瘤细胞的毒性明显强于强心甾类固醇原型药，而对正常细胞的毒性明显弱于强心甾类固醇原型药。
实施例5强心甾类固醇-奥曲肽(丙氨酸)偶联物在H22实体瘤小鼠中体内组织分布研究
1、H22实体瘤模型建立
(1)昆明小鼠60只，标重18-22g，均为雄性(购自江苏大学动物中心)。小鼠适应环境2d后，从含H22腹水瘤小鼠抽取腹水，经离心及红细胞裂解液处理过，用适量PBS缓存液配置成1×107/mL单细胞悬液。
(2)小鼠右侧腋下皮肤用碘伏棉球消毒。
(3)在小鼠右侧腋下皮下用1mL注射器接种H22单细胞悬液0.1mL/只。
2、动物及药物准备
接种1周后，小鼠右侧腋下皮下瘤体积长到约1cm3左右大小，成瘤率为 100％，逐个称取小鼠体重。60只雄性H22实体瘤昆明种小鼠随机分组，每组5只小鼠，进行组织分布实验，环境适应期间正常进水进食，于实验前12小时禁食，自由饮水。分别制备浓度为2.05μM的PPG和OCT(Phe)-S-PPG注射液(乙醇/聚氧乙烯蓖麻油，v/v＝1︰1，加水20倍稀释而成)，尾静脉注射给药，给药剂量为20.5μmol/kg。体重为20g的小鼠注射体积为0.2mL，按小鼠实际体重调整注射体积。
3、组织分布实验方法
各组小鼠以20.5μmol/kg的剂量尾静脉注射PPG和OCT(Phe)-S-PPG注射液。于给药后0.16、0.50、1、2、3和4小时处死，取血、心、肝、脾、肺、肾、瘤，小鼠血放入加适量肝素钠的2mL塑料离心管中静置半小时以后4000rpm离心10min取血浆，-20℃保存直至样品测定；精密称取一定量小鼠各脏器，按0.1g·mL-1加入生理盐水，高速剪切机捣碎组织样品后取适量至2mL塑料离心管，-20℃保存直至样品测定。
4、OCT(Phe)-S-PPM在H22实体瘤小鼠中体内组织分布研究
各组小鼠分别以4mg/kg和12.23mg/kg的剂量尾静脉注射PPM和OCT(Phe)-S-PPM注射液。其他步骤如上所述。
结果如图7、8所示，强心甾类固醇-奥曲肽(丙氨酸)偶联物显著提高了强心甾类固醇在肿瘤组织中的分布，而降低了在心脏和肝脏组织中的分布。表明强心甾类固醇-奥曲肽(丙氨酸)偶联物具有肿瘤靶向性
实施例6.强心甾类固醇-奥曲肽(丙氨酸)偶联物在H22实体瘤小鼠中药效学研究
1、OCT(Phe)-S-PPG的分组及药物处理
接种1周后，小鼠右侧腋下皮下瘤直径长到约0.5cm左右大小，成瘤率为100％，逐个称取小鼠体重，用随机数字表法将其分成4组，每组6只。
(1)空白模型组：小鼠尾静脉注射空白注射液0.1mL，每2天给药1次。
(2)OCT组：小鼠尾静脉注射20.5μmol/kg，每2天给药1次。
(3)PPG组：小鼠尾静脉注射20.5μmol/kg，每2天给药1次。
(4)OCT(Phe)-S-PPG组：小鼠尾静脉注射20.5μmol/kg，每2天给药1次。
2、OCT(Phe)-S-PPM的分组及药物处理
(1)空白模型组:小鼠尾静脉注射空白注射液5mL/kg，每2天给药1次。
(2)OCT组:小鼠尾静脉注射7.62mg/kg，每2天给药1次。
(3)PPM组:小鼠尾静脉注射4mg/kg，每2天给药1次。
(4)OCT(Phe)-S-PPM组:小鼠尾静脉注射12.23mg/kg，每2天给药1次。
3、观察指标与取材
(1)肿瘤生长的观察
每隔2天观察小鼠H22移植瘤的生长情况。用毫米游标卡尺测量小鼠H22移植瘤的长径(a)和短径(b)的长度，并记录。肿瘤体积公式：v＝0.52×a×b2。
(2)每2天电子天平称量小鼠体重，并记录。
(3)计算抑瘤率：各组药物分别给药11d后，腹腔注射戊巴比妥钠麻醉，用心电图测定仪测定心跳频率，颈椎脱臼法处死小鼠。摘取小鼠腋下皮下H22移植瘤及其心脏和肝脏组织，电子天秤称量肿瘤重量。
抑瘤率＝(对照组平均瘤重量-用药组平均瘤重量)/对照组平均瘤重量×100％。
(4)采用苏木精-伊红(H&E)染色进行组织病理学观察心脏和肝脏组织病变情况。
结果如图9、10所示，强心甾类固醇-奥曲肽(丙氨酸)偶联物抑制H22肝癌实体瘤的生长的作用强于强心甾类固醇原型药，可用于治疗肝癌。如图11、12所示，强心甾类固醇-奥曲肽(丙氨酸)偶联物能降低强心甾类固醇原型药的肝脏和心脏毒性。

Claims (9)

  1. 一种强心甾类固醇与奥曲肽的偶联物，其特征是：它是强心甾类固醇通过二元羧酸的羧基与强心甾固醇连接，随后二元羧酸再与奥曲肽连接得到强心甾类固醇与奥曲肽的偶联物。
  2. 根据权利要求1所述的偶联物，其特征是：所述的强心甾类固醇是杠柳苷元、杠柳次苷或杠柳毒苷。
  3. 一种制备上述的强心甾类固醇与奥曲肽的偶联物的方法，其特征是它包括如下步骤：
    步骤1.称取强心甾类固醇和酸酐适量，强心甾类固醇和酸酐的摩尔比为1/2～1/10，加二氯甲烷混匀至溶解，加入一定量三乙胺和4-二甲氨基吡啶(Dmap)，三乙胺和4-二甲氨基吡啶与强心甾类固醇的摩尔比为1/1/10～1/1/5，搅拌反应24h后，采用减压旋转蒸发仪旋干二氯甲烷，采用C8柱纯化，洗脱剂为不同比例甲醇-0.1％甲酸水，得到羧基化强心甾类固醇衍生物(SPPG)，所述的酸酐是丁二酸酐、马来酸酐等二元酸酐，所得到的羧基化强心甾类固醇衍生物经红外及ESI-MS表征；
    步骤2.称取步骤1产物、二环己基碳二亚胺(DCC)和N-羟基琥珀酰亚胺(NHS)适量加入体积比为2:1的二氯甲烷(DCM)与四氢呋喃(THF)的混合溶剂溶解羧基化强心甾类固醇衍生物(SPPG)、二环己基碳二亚胺(DCC)和N-羟基琥珀酰亚胺(NHS)的摩尔比为1/2/2～1/2/4，密封，室温，磁力搅拌反应2～24h后，氮气吹干，加入50％乙腈-水超声混溶，采用C8柱纯化，洗脱剂为不同比例的乙腈-0.05％三氟乙酸(TFA)水溶液，TLC检测，收集斑点，直接冻干，得NHS-PPG样品；
    步骤3.称取步骤2产物NHS-PPG适量，用加入50％乙腈-含0.1M 4-羟乙基哌嗪乙磺酸(HEPES)的水溶液溶解，加入奥曲肽，奥曲肽与NHS-PPG的摩尔比为1.5/1～5/1，N-甲基吗啉调节pH值为8.0～9.0，密封，室温，磁力搅拌反应12～24h后，用液相色谱-质谱进行分析，反应产物的纯化通过高效液相色谱半制备得到三种强心甾类固醇-奥曲肽偶联物，它们分别是奥曲肽(丙氨酸)-强心甾类固醇偶联物、奥曲肽(赖氨酸)-强心甾类固醇偶联物和强心甾类固醇-奥 曲肽-强心甾类固醇偶联物。
  4. 根据权利要求3所述的方法，其特征是：步骤1所述的强心甾类固醇包括杠柳苷元、杠柳次苷或杠柳毒苷强心甾类固醇类物质。
  5. 根据权利要求3所述的方法，其特征是：步骤1或步骤2中的纯化方法采用硅胶柱层析纯化。
  6. 根据权利要求3所述的方法，其特征是：步骤1中强心甾类固醇和酸酐的摩尔比为1/2～1/10。
  7. 根据权利要求3所述的方法，其特征是：步骤1中有机溶剂选自二氯甲烷、DMSO、乙酸乙酯或DMF中的一种或者它们多种的混合溶剂。
  8. 权利要求1所述的强心甾类固醇-奥曲肽(丙氨酸)偶联物在治疗肿瘤的应用。
  9. 根据权利要求6所述的应用，其特征是：所述肿瘤为H22肝癌实体瘤。
PCT/CN2016/108630 2016-12-06 2016-12-06 一种天然活性成分强心甾类固醇与奥曲肽的偶联物及其制法和用途 Ceased WO2018102974A1 (zh)

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