WO2017054640A1 - 延胡索乙素在制备抗顺铂毒性药物中的应用 - Google Patents

延胡索乙素在制备抗顺铂毒性药物中的应用 Download PDF

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WO2017054640A1
WO2017054640A1 PCT/CN2016/098657 CN2016098657W WO2017054640A1 WO 2017054640 A1 WO2017054640 A1 WO 2017054640A1 CN 2016098657 W CN2016098657 W CN 2016098657W WO 2017054640 A1 WO2017054640 A1 WO 2017054640A1
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cisplatin
thp
cells
hoct2
mdck
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李丽萍
蒋惠娣
周慧
曾苏
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Zhejiang University ZJU
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine

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  • the invention relates to a pharmaceutical use and relates to the use of tetrahydropalmatine in reducing cisplatin-induced toxicity, including nephrotoxicity, ototoxicity and neurotoxicity.
  • Cisplatin is one of the most effective broad-spectrum anti-tumor drugs in clinical practice. It is widely used in the treatment of various solid tumors such as ovarian cancer, prostate cancer, lung cancer and breast cancer (Ciarimboli G. Membrane transporters as mediators of cisplatin effects and side effects). .Scientifica (Cairo), 2012, 2012: 473829), but its severe side effects, including nephrotoxicity, ototoxicity, neurotoxicity, etc., limit its clinical application, especially with the most serious nephrotoxicity.
  • cisplatin nephrotoxicity
  • its high concentration in renal tubular epithelial cells is critical for its toxicity.
  • cisplatin is distributed along the blood circulation to various tissues and organs of the whole body.
  • the highest concentration is renal tissue, especially renal tubular epithelial cells.
  • uptake and efflux drug transporters are expressed on the renal tubular epithelial membrane, including the organic cation transporter (OCT2), which is highly expressed on the basement membrane side, and the apical membrane multidrug and toxin efflux protein.
  • OCT2 organic cation transporter
  • MATEs multidrug and toxin extrusion proteins, MATEs
  • OCT2 organic cation transporter 2
  • cimetidine Under low dose conditions, it can significantly inhibit the renal efflux of MATEs on cisplatin, so that cisplatin accumulates in renal tubular cells, but may make it smooth. Platinum nephrotoxicity is enhanced; in addition, cimetidine also inhibits the metabolism of some cisplatin chemotherapeutic drugs, such as: paclitaxel (Ito, S., et al., Competitive inhibition of the luminal efflux by multidrug and toxin extrusions, but Not basolateral uptake by organic cation transporter 2, is the likely mechanism underlying the pharmacokinetic drug-drug interactions caused by cimetidine in the kidney.
  • paclitaxel Ito, S., et al.
  • Tetrahydropalmatine including L-tancoidin ((-)-THP), dextro-homolysin ((+)-THP), and racemic tetrahydropalmatine (( ⁇ )-THP),
  • Corydalis Yuanhu
  • Yuanhu analgesic and anti-oxidation
  • Han, Y., et al., l-Tetrahydropalmatine an active component of Corydalis yanhusuo WTWang, protects against myocardial ischaemia -reperfusion injury in rats.
  • (-)-THP is often used to relieve headache, chest pain and other pains, and has the advantages of non-addictive and toxic side effects (Cao, FL, et al., Antinociceptive effects of intragastric DL-tetrahydropalmatine on visceral and somatic persistent Nociception and pain hypersensitivity in rats.
  • THP The inhibitory effect of THP on OCT2 and MATE1 can reduce the accumulation of intracellular cisplatin, thereby antagonizing the cytotoxicity induced by cisplatin.
  • (-)-THP is used to study the accumulation and toxicity of cisplatin in mouse kidney.
  • Antagonistic effect to elucidate the significance of tetrahydropalmatine as a cisplatin-induced nephrotoxicity attenuating drug, and provide tumor adjuvant therapy for cancer patients with both attenuated, analgesic and non-addictive.
  • the toxicity includes nephrotoxicity, ototoxicity and neurotoxicity.
  • tetrahydropalmatine including (-)-THP, (+)-THP and ( ⁇ )-THP, inhibits OCT2 and MATE1 and antagonizes cisplatin-induced cytotoxicity in primary cultured renal tubular epithelium.
  • the cell model is well validated.
  • (-)-THP as the representative, it was found that tetrahydropalmatine has a good protective effect on cisplatin-induced kidney injury in mice. Therefore, the combination of tetrahydropalmatine and cisplatin may reduce the accumulation of cisplatin in the kidneys, thereby achieving the expected result of preventing or reducing the toxicity of cisplatin.
  • Tetrahydropalmatine is the main active ingredient of Yuanhu, one of the "Zhebawei", and is also the main active ingredient of Yuanhu Zhitong Recipe
  • (-)-THP is a commonly used analgesic in clinical practice. It has been used to treat pain caused by tumors and is not recessive.
  • the present invention will expand the new use of Yuanhu, Yuanhuzhitong and (-)-THP; (2) Although researchers have hoped to inhibit OCT2 by cisplatin in combination with drugs to reduce cisplatin nephrotoxicity, clinical Attempts of OCT2 inhibitors (such as cimetidine) are often more potent due to MATE1-mediated cisplatin efflux, which may result in more accumulation of cisplatin in the kidney and greater toxicity.
  • OCT2 inhibitors such as cimetidine
  • FIG. 7 (-)-THP, (+)-THP and ( ⁇ )-THP concentration-dependently reduce cisplatin-induced leakage of lactate dehydrogenase (LDH) in MDCK-hOCT2/hMATE1 cells.
  • Figure 8 Effect of (-)-THP on the accumulation and transport of cisplatin on MDCK-hOCT2/pcDNA3.1 monolayer cells.
  • Figure 9 Effect of (-)-THP on the accumulation and transport of cisplatin on MDCK-hOCT2/hMATE1 monolayer cells.
  • Figure 14 Effect of (-)-THP (20 mg/kg, tail vein injection, iv) on body weight of mice administered cisplatin (10 mg/kg, iv).
  • Figure 15 Effect of (-)-THP (20 mg/kg, tail vein injection, iv) on renal function in cisplatin (10 mg/kg, iv) mice.
  • the median biochemical index of the control group was 100%.
  • Figure 16 Representative map of pathological examination of mouse kidney tissue.
  • A solvent control control mouse kidney sections;
  • B cisplatin group (10 mg/kg, iv) mouse kidney sections;
  • C combined administration of cisplatin (10 mg/kg, iv) and
  • Pathological changes in the kidney tissue the black arrow indicates the protein tube type in the renal tubule, and the green arrow indicates the necrosis of the renal tubular epithelial cells.
  • Leica DM2500 microscope 10*20 times photo.
  • Example 1 Concentration-dependent toxicity of cisplatin on MDCK-hOCT2, MDCK-hOCT2/hMATE1 and mock cells
  • MDCK-hOCT2, MDCK-hOCT2/hMATE1 and mock cells in logarithmic growth phase were selected (constructed by Zhejiang University School of Pharmacy. For specific construction methods, see a: Lei Hongmei, etc., stably expressing hMATE1 and co-expressing hMATE1 with hOCT1 or hOCT2 cells. Construction of the model. Journal of Pharmaceutical Sciences, 2015, 50(7): 842-847. b: Wang K., et al., Involvement of organic cation transporter 2inhibition in potential mechanisms of antidepressant action. Prog Neuropsychopharmacol Biol Psychiatry, 2014, 53: 90-98), digest and collect cells. The cells were resuspended in fresh medium, adjusted to a cell density of 4.0 ⁇ 10 4 /mL, and seeded in a 96-well cell plate at 0.2 mL / well.
  • MTT assay was carried out for 48 hours after administration, and each well was added with 5 mg/mL MTT (thiazole blue tetrazolium bromide) reagent 20 ⁇ L, gently shaken, and after incubating for 4 h, the incubation solution was discarded, 150 ⁇ L of DMSO was added to each well, and the mixture was shaken slightly. The instrument was shaken for 10 min.
  • MTT thiazole blue tetrazolium bromide
  • Example 2 Inhibition of Tetrahydropalmatine, including (-)-THP, (+)-THP and ( ⁇ )-THP on the accumulation of MPP + on MDCK-hOCT2 and MDCK-hMATE1 cells
  • MDCK-hOCT2, MDCK-hOCT2/hMATE1 and MDCK-hMATE1 cells in logarithmic growth phase were selected (constructed by Zhejiang University School of Pharmacy. For specific construction methods, see a: Lei Hongmei, etc., stable expression of hMATE1 and co-expression of hMATE1 and hOCT1 or Construction of the hOCT2 cell model. Journal of Pharmaceutical Sciences, 2015, 50(7): 842-847. b: Wang K., et al., Involvement of organic cation transporter 2inhibition in potential mechanisms of antidepressant action. Prog Neuropsychopharmacol Biol Psychiatry, 2014, 53:90-98), digest and collect cells. The cells were resuspended in fresh medium, adjusted to a cell density of 2.0 ⁇ 10 5 cells/mL, and seeded in a 24-well cell plate at 0.5 mL/well. The cell growth was 90% confluency and used for accumulation experiments.
  • MDCK-hOCT2 and MDCK-hMATE1 cells are MDCK cells that highly express human OCT2 and MATE1 transporters.
  • the study investigated 0.001-100 ⁇ mol/L (-)-THP, (+)-THP and ( ⁇ )-THP to accumulate in MPP + (classical substrate of OCT2 and MATE1 transporter, 1 ⁇ mol/L, 3 min) in the above cells. Impact.
  • the results showed that (-)-THP, (+)-THP and ( ⁇ )-THP significantly inhibited the accumulation of MPP + on MDCK-hOCT2 and MDCK-hMATE1 cells, and the IC 50 for OCT2 inhibition constant was 2.6, 9.5, respectively.
  • Example 3 (-)-THP, (+)-THP and ( ⁇ )-THP on the toxicity of cisplatin on MDCK-hOCT2 and MDCK-hOCT2/hMATE1 cells
  • MDCK-hOCT2 and MDCK-hOCT2/hMATE1 in logarithmic growth phase were selected (constructed by Zhejiang University School of Pharmacy. For specific construction methods, see a: Lei Hongmei, etc., stable expression of hMATE1 and co-expression of hMATE1 and hOCT1 or hOCT2 cell model construction Journal of Pharmaceutical Sciences, 2015, 50(7): 842-847.
  • b Wang K., et al., Involvement of organic cation transporter 2 inhibition in potential mechanisms of antidepressant action. Prog Neuropsychopharmacol Biol Psychiatry, 2014, 53:90- 98), digest and collect cells.
  • the cells were resuspended in fresh medium, adjusted to a cell density of 4.0 ⁇ 10 4 mL -1 , and seeded in a 96-well cell plate at 0.2 mL/well. After the cells were cultured for 24 hours, the medium was discarded, and a cisplatin medium solution containing 1 to 100 ⁇ mol/L of (-)-THP, (+)-THP and ( ⁇ )-THP was added to contain the corresponding concentration of solvent (DMSO). The medium was a blank control group.
  • the MTT assay was carried out for 48 hours, and each well was added with 5 mg/mL MTT (thiazole blue tetrazolium bromide) reagent 20 ⁇ L, gently shaken, and after incubating for 4 h, the incubation solution was discarded, and 150 ⁇ L of DMSO was added to each well. The oscillator was shaken for 10 min. After the formazan crystals were completely dissolved, the absorbance of each sample at 570 nm (referenced to 630 nm) was measured by an enzyme-linked immunosorbent assay, and the viability of the sample cells was expressed by the absorbance value/absence of the absorbance of the blank control group. .
  • MTT thiazole blue tetrazolium bromide
  • the cell culture medium sample collected after 48 hours of culture administration was subjected to LDH activity measurement, and the LDH activity in the cell culture liquid was measured according to the requirements of the lactate dehydrogenase (LDH) assay kit. Relative quantification was carried out with a solvent control enzyme activity of 100% administered at each concentration.
  • LDH lactate dehydrogenase
  • MDCK-hOCT2/pcDNA3.1 and MDCK-hOCT2/hMATE1 cells in logarithmic growth phase were selected (constructed by Zhejiang University School of Pharmacy. For specific construction methods, see: Lei Hongmei, etc., stable expression of hMATE1 and co-expression of hMATE1 and hOCT1 or hOCT2 Construction of a cell model. Journal of Pharmaceutical Sciences, 2015, 50(7): 842-847.), digesting and collecting cells. The cells were resuspended in fresh medium, adjusted to a cell density of 3.0 ⁇ 10 5 /mL, and seeded in a 12-well Tanswell cell transport plate at 0.5 mL/well for 3-5 days, when the cells grew tightly into a monolayer.
  • the cell culture medium was pre-incubated with incubation buffer at 37 ° C for 20 min, and the resistance value of each well was measured with a Millicell-ERS cell resistance meter. When the resistance value was ⁇ 250 ⁇ cm 2 , the pore cell link was tightly used for transcellular cells.
  • Transport experiment In the cisplatin transport experiment, 0.5 mL buffer (pH 6.5) was added to the top side of the transfer plate insert, and 1.5 mL buffer (pH 7.4) was added to the bottom side, and the buffer contained 50 ⁇ mol/L cisplatin. The corresponding blank buffer was added to the relative measurement.
  • Healthy ICR mice male, ⁇ 25g were immersed in 75% alcohol for 1 minute after ether anesthesia and fixed on the operating table. Cut open the abdominal cavity, to a solution in HBSS without Ca 2+ from the hepatic portal vein perfusion kidney now to off-white, rapidly remove the kidneys placed in sterile double-antibody added (1% + 1% penicillin-streptomycin) in PBS. Wash it in a sterile clean bench with PBS solution supplemented with double-antibody several times, remove the kidney capsule, and cut it; after washing for 3 to 5 times, chop it with a sterile surgical blade and place it in 0.1% type IV collagenase. The mixture was vortexed for about 40 min in a mixed solution with 0.1% trypsin.
  • the upper layer was filtered on an 80 mesh screen and centrifuged twice with DMEM/F12 medium (containing DMEM/F12 medium, 10% fetal bovine serum, 1% double antibody, 1% insulin-transferrin-selenium triplet). Resuspend in the medium, pass through a 200 mesh screen and plant in a 96-well plate, and change the solution every other day. After about 5 to 6 days of growth, it can be used for cytotoxicity experiments.
  • the absorbance of each sample at 570 nm was measured by an enzyme-linked immunosorbent assay, and the viability of the sample cells was expressed by the absorbance value/absence of the absorbance of the blank control group. .
  • Healthy ICR mice male, ⁇ 25g were immersed in 75% alcohol for 1 minute after ether anesthesia and fixed on the operating table. Cut open the abdominal cavity, to a solution in HBSS without Ca 2+ from the hepatic portal vein perfusion kidney now to off-white, rapidly remove the kidneys placed in sterile double-antibody added (1% + 1% penicillin-streptomycin) in PBS. Wash it in a sterile clean bench with PBS solution supplemented with double-antibody several times, remove the kidney capsule, and cut it; after washing for 3 to 5 times, chop it with a sterile surgical blade and place it in 0.1% type IV collagenase. The mixture was vortexed for about 40 min in a mixed solution with 0.1% trypsin.
  • the upper layer was filtered on an 80 mesh screen and centrifuged twice with DMEM/F12 medium (containing DMEM/F12 medium, 10% fetal bovine serum, 1% double antibody, 1% insulin-transferrin-selenium triplet). Resuspend in the medium, pass through a 200 mesh screen and plant in a 96-well plate, and change the solution every other day. After about 5 to 6 days of growth, it can be used for cell accumulation experiments.
  • DMEM/F12 medium containing fetal bovine serum, 1% double antibody, 1% insulin-transferrin-selenium triplet. Resuspend in the medium, pass through a 200 mesh screen and plant in a 96-well plate, and change the solution every other day. After about 5 to 6 days of growth, it can be used for cell accumulation experiments.
  • Mouse primary renal tubular cells express Oct2 and Mate1 transporters and can be used in Oct2/Mate1 mediated cisplatin cell accumulation assays.
  • the effect of 100 ⁇ mol/L(-)-THP on the accumulation of cisplatin in mouse primary renal tubular cells 50 ⁇ mol/L, 2 h was investigated.
  • ICR mice 25-30 g
  • male type provided by the Animal Center of Zhejiang Academy of Medical Sciences (clean secondary, animal license number: SCXK (Zhejiang) 20140001). All animals were kept according to the requirements of the experimental animal operation instructions of Zhejiang University. The environment was controlled at a suitable temperature (20 ⁇ 23 ° C) and humidity (50 ⁇ 60%). Fasting 12 hours before the experiment, free drinking water.
  • ICR mice Male, weighing 25-30 g were randomly divided into 10 mg/kg cisplatin group and 10 mg/kg cisplatin + 20 mg/kg(-)-THP group. After a single injection of the (-)-THP solution in the tail vein, a 10 mg/kg cisplatin solution was intravenously administered. After 72 hours of administration, the kidney tissue was dissected and the blood was washed away with physiological saline, and the filter paper was blotted and stored at -80 ° C for testing.
  • mice Male, body weight 25-30 g
  • the (-)-THP solution was administered first, then 10 mg/kg cisplatin solution was intravenously administered, and the control group was given the same volume of physiological saline.
  • the body weight of the mice was recorded daily, and the changes in the skin and hair of the mice and the general activities were closely observed.
  • (-)-THP After intravenous injection of 20mg/kg(-)-THP solution in mice, (-)-THP is rapidly distributed in kidney tissue, which can reach 22.6 ⁇ M at 10min, which is about 5 times of plasma concentration (4.5 ⁇ M). 12. Tips for (-)-THP concentration in kidney tissue Higher, may interact with the transporter OCT2/MATE1 in the kidney to affect the kidney concentration of cisplatin, which may affect its renal toxicity.
  • mice given 20 mg/kg (-)-THP the weight loss caused by cisplatin was significantly relieved, suggesting that (-)-THP can inhibit cisplatin-induced renal toxicity in mice, see Figure 14.

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Abstract

本发明提供了延胡索乙素在制备抗顺铂毒性药物中的应用，所述毒性包括肾脏毒性、耳毒性及神经毒性。

Description

延胡索乙素在制备抗顺铂毒性药物中的应用 技术领域
本发明属药物用途，涉及延胡索乙素在降低顺铂所致毒性药物中的应用，包括肾脏毒性、耳毒性和神经毒性。
技术背景
顺铂是目前临床上最有效的广谱抗肿瘤药物之一，广泛用于卵巢癌、前列腺癌、肺癌及乳腺癌等多种实体肿瘤治疗(Ciarimboli G.Membrane transporters as mediators of cisplatin effects and side effects.Scientifica(Cairo),2012,2012:473829)，但其严重的毒副反应，包括肾毒性、耳毒性、神经毒性等限制了其临床应用，尤其是以肾毒性最为严重。临床尽管采用水化疗法或强迫利尿等防护措施，顺铂的肾毒性发生率仍高达25～35％(Miller,R.P.,et al.,Mechanisms of cisplatin nephrotoxicity.Toxins(Basel),2010,2(11):2490-2518)，且在治疗初期随即发生。通过氧化应激、DNA损伤及炎症反应等机制，顺铂可导致肾小管细胞死亡，肾组织损伤，从而使肾小球滤过率下降，肾脏排泄显著减少，导致其在肾脏中大量蓄积，引起严重的肾脏衰竭。顺铂所致肾脏毒性是临床化疗过程中亟待解决的问题，其防护措施的研究也是近年来的研究热点。临床常用抗炎、抗氧化等措施，以保护顺铂引起的肾脏损伤，但效果并不理想。
顺铂肾毒性的产生是一个复杂的多因子参与过程，但其在肾小管上皮细胞中的高度浓集是其毒性的关键。顺铂经静脉给药后，随血液循环分布至全身各组织器官，其中浓度最高的是肾组织，尤其是肾小管上皮细胞。肾小管上皮细胞膜上表达多种摄取型及外排型药物转运体，包括基底膜侧特异性高表达的有机阳离子转运体2(organic cation transporter,OCT2)及顶膜侧多药及毒素外排蛋白(multidrug and toxin extrusion proteins,MATEs)，在阳离子药物的肾脏跨膜转运及肾脏处置中发挥着重要作用。顺铂经OCT2由血液摄取入肾小管细胞，在肾小管蓄积(Filipski,K.K.,et al.,Contribution of organic cation transporter 2(OCT2)to cisplatin-induced nephrotoxicity.Clin Pharmacol Ther,2009,86(4):396-402)；外排型转运体MATEs(MATE1与MATE2-K)将顺铂泵出肾小管细胞，降低其在肾细胞的浓度(Ciarimboli,G.,Membrane transporters as mediators of cisplatin side-effects.Anticancer Res,2014,34(1):547-550)。由于OCT2对顺铂的大量摄取，而MATEs对其较弱的外排，使顺铂在肾脏严重蓄积，从而导致肾脏毒性(Yokoo,S.,et al.,Differential contribution of organic cation transporters,OCT2 and MATE1,in platinum agent-induced nephrotoxicity.Biochem Pharmacol, 2007,74(3):477-487)。临床尝试以OCT2抑制剂西咪替丁等，通过抑制OCT2对顺铂肾细胞摄取，以降低顺铂所致的肾脏毒性。然而，西咪替丁竞争性抑制MATEs作用强于对OCT2的抑制，在低剂量条件下即能显著抑制MATEs对顺铂的肾小管外排，使顺铂在肾小管细胞蓄积，反而可能使顺铂肾毒性增强；另外，西咪替丁也会抑制一些顺铂合用化疗药物，如：紫杉醇等的代谢(Ito,S.,et al.,Competitive inhibition of the luminal efflux by multidrug and toxin extrusions,but not basolateral uptake by organic cation transporter 2,is the likely mechanism underlying the pharmacokinetic drug-drug interactions caused by cimetidine in the kidney.J Pharmacol Exp Ther,2012,340(2):393-403；Sprowl,J.A.,et al.,Conjunctive therapy of cisplatin with the OCT2 inhibitor cimetidine:influence on antitumor efficacy and systemic clearance.Clin Pharmacol Ther,2013,94(5):585-592),致使合用药物血药浓度升高，由此可能引起一些毒副反应。因此，寻找对OCT2强抑制，对MATEs无抑制或弱抑制的药物，以减少顺铂在肾脏积聚，从而降低其毒性是拓展顺铂临床应用的很好途径。
延胡索乙素(tetrahydropalmatine,THP)，包括左旋延胡索乙素((-)-THP)，右旋延胡索乙素((+)-THP)，及消旋延胡索乙素((±)-THP)，是延胡索(元胡)的主要效应成分之一，具有镇痛、抗氧化等多种生理活性(Han,Y.,et al.,l-Tetrahydropalmatine,an active component of Corydalis yanhusuo W.T.Wang,protects against myocardial ischaemia-reperfusion injury in rats.PLoS One,2012,7(6):e38627；Chu,H.,et al.,Recent development in studies of tetrahydroprotoberberines:mechanism in antinociception and drug addiction.Cell Mol Neurobiol,2008.28(4):491-499)。其中(-)-THP常用于缓解头痛、胸痛等多种疼痛，且具有非成瘾性、毒副作用小的优点(Cao,F.L.,et al.,Antinociceptive effects of intragastric DL-tetrahydropalmatine on visceral and somatic persistent nociception and pain hypersensitivity in rats.Pharmacol Biochem Behav,2011,100(1):199-204；Liao,Z.G.,et al.,Correlation between synergistic action of Radix Angelica dahurica extracts on analgesic effects of Corydalis alkaloid and plasma concentration of dl-THP.J Ethnopharmacol,2010.129(1):115-120)。本研究首先在稳定表达人OCT2，MATE1，共表达人OCT2和MATE1的转基因细胞以及原代培养的小鼠肾上皮细胞考察延胡索乙素，包括(-)-THP，(+)-THP及(±)-THP对OCT2与MATE1的抑制作用，能减少细胞内顺铂积聚，从而拮抗顺铂所致细胞毒性；进一步以(-)-THP为代表研究其对顺铂在小鼠肾脏蓄积及毒性的拮抗作用，以阐明延胡索乙素作为顺铂诱发的肾毒性减毒药物的意义，为肿瘤患者提供兼有减毒、镇痛、无成瘾性的肿瘤辅助治疗药物。延胡索乙素结构式：
Figure PCTCN2016098657-appb-000001
发明内容
本发明的目的是提供延胡索乙素在制备抗顺铂毒性药物中的应用。所述毒性包括肾脏毒性、耳毒性及神经毒性。
研究表明，延胡索乙素，包括(-)-THP，(+)-THP及(±)-THP对OCT2与MATE1具有抑制作用，能拮抗顺铂引起的细胞毒性，并在原代培养的肾小管上皮细胞模型上得到较好的验证。且以(-)-THP为代表，研究发现延胡索乙素对顺铂所致小鼠肾损伤具有很好的保护作用。因此，延胡索乙素与顺铂合用，可能减少顺铂在肾脏蓄积，从而达到预防或降低顺铂毒性的预期结果。
本发明的有益之处是：(1)延胡索乙素是“浙八味”之一元胡的主要有效成分，也是元胡止痛方主要有效成分，(-)-THP更是临床常用镇痛药，已用于治疗肿瘤引起的疼痛，且无成隐性。因此本发明将拓展元胡、元胡止痛方及(-)-THP的新用途；(2)虽然已有研究者希望通过顺铂联用药物对OCT2的抑制以降低顺铂肾毒性，但是临床尝试的OCT2抑制剂(如西咪替丁)往往由于对MATE1介导的顺铂外排具有更强的抑制，可能使顺铂在肾脏蓄积更多，毒性更强。本研究发现，延胡索乙素对OCT2的抑制显著大于对MATE1的抑制，能显著降低顺铂的肾毒性；(3)由于肾脏组织高表达OCT2，而肿瘤细胞上往往缺少OCT2的表达，由于顺铂具有一定的被动透过性，因此，延胡索乙素与顺铂合用，减弱顺铂所致肾脏毒性的同时，并不会降低顺铂的抗肿瘤效果。
附图说明
图1.顺铂在MDCK-hOCT2，MDCK-hOCT2/hMATE1和mock细胞上浓度依赖性毒性。以溶剂对照组细胞活性均值为100％，图中数据以mean±SD表示,n＝3；与mock细胞比较，*P<0.05，**P<0.01和***P<0.001。
图2.(-)-THP,(+)-THP及(±)-THP浓度依赖性抑制MPP+在MDCK-hOCT2细胞上的积聚。以溶剂对照组MPP+摄取值为100％，图中数据以mean±SD表示，n＝3。
图3.(-)-THP,(+)-THP及(±)-THP浓度依赖性抑制MPP+在MDCK-hMATE1细胞上的积聚。以溶剂对照组数值为100％，图中数据以mean±SD表示，n＝3。
图4.(-)-THP,(+)-THP及(±)-THP对顺铂在MDCK-hOCT2细胞上毒性的浓度依赖性抑制作用。以溶剂对照组数值为100％，图中数据以mean±SD表示，n＝3；与对照组比较，###P<0.001；与顺铂组比较，***P<0.001。
图5.(-)-THP,(+)-THP及(±)-THP对顺铂在MDCK-hOCT2/hMATE1细胞上毒性的浓度依赖性抑制作用。以溶剂对照组数值为100％，图中数据以mean±SD表示，n＝3；与对照组比较，###P<0.001；与顺铂组比较，*P<0.05，**P<0.01和***P<0.001。
图6.(-)-THP,(+)-THP及(±)-THP浓度依赖性减少顺铂引起的MDCK-hOCT2细胞乳酸脱氢酶(LDH)的渗漏。以溶剂对照组LDH活力为100％，图中数据以mean±SD表示，n＝3；与对照组比较,###P<0.001；与顺铂组比较，**P<0.01和***P<0.001。
图7.(-)-THP,(+)-THP及(±)-THP浓度依赖性减少顺铂引起MDCK-hOCT2/hMATE1细胞乳酸脱氢酶(LDH)的渗漏。以溶剂对照组LDH活力为100％，图中数据以mean±SD表示，n＝3；与对照组比较,###P<0.001；与顺铂组比较，*P<0.05，**P<0.01和***P<0.001。
图8.(-)-THP对顺铂在MDCK-hOCT2/pcDNA3.1单层细胞上积聚及转运的影响。以溶剂对照组MDCK-hOCT2/pcDNA3.1(AP→BL)数值为100％(对照组)，图中数据以mean±SD表示，n＝3；与对照组比较，**P<0.01和***P<0.001。
图9.(-)-THP对顺铂在MDCK-hOCT2/hMATE1单层细胞上积聚及转运的影响。以溶剂对照组MDCK-hOCT2/pcDNA3.1(AP→BL)数值为100％(对照组)，图中数据以mean±SD表示，n＝3；与对照组比较，**P<0.01。
图10.(-)-THP,(+)-THP及(±)-THP对顺铂在小鼠原代肾小管上皮细胞上毒性的浓度依赖性抑制作用。以溶剂对照组数值为100％，图中数据以mean±SD表示，n＝3；与对照组比较，###P<0.001；与顺铂组比较，**P<0.01和***P<0.001。
图11.(-)-THP对顺铂在小鼠原代肾小管上皮细胞上积聚的抑制作用。以溶剂对照组数值为100％，图中数据以mean±SD表示，n＝3；与对照组比较，***P<0.001。
图12.(-)-THP在小鼠血浆及肾脏组织的分布。图中数据以mean±SD表示，n＝5。
图13.(-)-THP(20mg/kg，尾静脉注射，iv)对顺铂(10mg/kg，iv)在小鼠肾脏浓度的影响。图中数据以mean±SD表示,n＝6-10；与顺铂组比较，***P<0.001。
图14.(-)-THP(20mg/kg，尾静脉注射，iv)对顺铂(10mg/kg，iv)给药小鼠体重的影响。图中数据以mean±SD表示，n＝6-10；与对照组比较，###P<0.001；与顺铂组比较，*P<0.05 和**P<0.01。
图15.(-)-THP(20mg/kg，尾静脉注射，iv)对顺铂(10mg/kg，iv)小鼠肾功能的影响。以溶剂对照组生化指标均值为100％，图中数据以mean±SD表示,n＝6-10；与对照组比较,###P<0.001；与顺铂组比较，***P<0.001。
图16.小鼠肾组织病理学检查的代表性图谱。(A)溶剂对照组(control)小鼠肾脏切片；(B)顺铂组(10mg/kg，iv)小鼠肾脏切片；(C)合并给予顺铂(10mg/kg，iv)与(-)-THP(20mg/kg，iv)组小鼠肾脏切片。肾脏组织病理改变：黑色箭头所指肾小管内蛋白管型，绿色箭头所指肾小管上皮细胞坏死。leica DM2500显微镜，10*20倍拍照。
具体实施方式
本发明结合附图和实施例作进一步的说明。
实施例1顺铂在MDCK-hOCT2，MDCK-hOCT2/hMATE1和mock细胞上浓度依赖性毒性
1.1在96孔板上接种细胞
选取处于对数生长期的MDCK-hOCT2，MDCK-hOCT2/hMATE1和mock细胞(由浙江大学药学院构建，具体构建方法见a:雷红梅等，稳定表达hMATE1及共表达hMATE1与hOCT1或hOCT2细胞模型的构建。药学学报，2015,50(7)：842-847。b:Wang K.,et al.,Involvement of organic cation transporter 2inhibition in potential mechanisms of antidepressant action.Prog Neuropsychopharmacol Biol Psychiatry,2014,53:90-98)，消化、收集细胞。用新鲜培养基重新悬浮细胞，调整细胞密度为4.0×104个/mL，以0.2mL/孔接种于96孔细胞板培养。
1.2MTT实验
细胞培养24h后，弃除培养基，加入含不同浓度药物(顺铂)的培养基，以含相应浓度溶剂(DMSO)的培养基为空白对照组。给药后培养48h进行MTT实验，每孔加5mg/mLMTT(溴化噻唑蓝四氮唑)试剂20μL，轻轻摇匀，孵育4h后，弃去孵育液，于每孔加入150μL DMSO，微量振荡仪振荡10min，待甲臜结晶完全溶解后，酶联免疫检测仪测定570nm波长(以630nm为参照)处各样本的吸光度，用吸光度值/空白对照组吸光度的均值表示该样本细胞的存活率。数据以mean±SD(n＝3)表示，以不加顺铂的对照组(control)细胞存活率为100％；与对照组比较，*P<0.05，**P<0.01和***P<0.001。
1.3结果
MTT实验结果表明，MDCK-hOCT2，MDCK-hOCT2/hMATE1及mock细胞与顺铂(0.01-1000μmol/L)共孵育48h后，MDCK-hOCT2与MDCK-hOCT2/hMATE1细胞对顺铂 的毒性敏感性远远高于mock细胞，其LD50值分别为8.71±0.21μmol/L，7.31±0.18μmol/L和15.2±3.5μmol/L，参见图1(以溶剂对照组细胞活性均值为100％，图中数据以mean±SD表示,n＝3；与mock细胞比较，*P<0.05，**P<0.01和***P<0.001)。上述结果提示hOCT2介导顺铂细胞摄取使细胞积聚增加，导致其细胞毒性增强。
实施例2延胡索乙素，包括(-)-THP，(+)-THP及(±)-THP对MPP+在MDCK-hOCT2和MDCK-hMATE1细胞上积聚的抑制作用
选取处于对数生长期的MDCK-hOCT2，MDCK-hOCT2/hMATE1和MDCK-hMATE1细胞(由浙江大学药学院构建，具体构建方法见a:雷红梅等，稳定表达hMATE1及共表达hMATE1与hOCT1或hOCT2细胞模型的构建。药学学报，2015,50(7)：842-847。b:Wang K.,et al.,Involvement of organic cation transporter 2inhibition in potential mechanisms of antidepressant action.Prog Neuropsychopharmacol Biol Psychiatry,2014,53:90-98)，消化、收集细胞。用新鲜培养基重新悬浮细胞，调整细胞密度为2.0×105个/mL，以0.5mL/孔接种于24孔细胞板培养，当细胞生长达90％融合度即可用于积聚实验。
MDCK-hOCT2和MDCK-hMATE1细胞为高表达人OCT2与MATE1转运体的MDCK细胞。研究考察了0.001～100μmol/L(-)-THP，(+)-THP及(±)-THP对MPP+(OCT2与MATE1转运体的经典底物，1μmol/L，3min)在上述细胞中积聚的影响。结果显示：(-)-THP，(+)-THP及(±)-THP均能显著抑制MPP+在MDCK-hOCT2和MDCK-hMATE1细胞上的积聚，对OCT2抑制常数IC50分别为2.6，9.5，0.24μmol/L，对MATE1抑制常数IC50分别为21.0，8.6，2.6μmol/L，参见图2、图3，以溶剂对照组MPP+摄取值为100％，图中数据以mean±SD(n＝3)表示。上述结果提示：(-)-THP，(+)-THP及(±)-THP对OCT2和MATE1均具有较强的抑制作用，且(-)-THP和(±)-THP对MATE1抑制能力弱于对OCT2的抑制。
实施例3(-)-THP,(+)-THP及(±)-THP对顺铂在MDCK-hOCT2及MDCK-hOCT2/hMATE1细胞上毒性的影响
4.1MTT实验
选取处于对数生长期的MDCK-hOCT2和MDCK-hOCT2/hMATE1(由浙江大学药学院构建，具体构建方法见a:雷红梅等，稳定表达hMATE1及共表达hMATE1与hOCT1或hOCT2细胞模型的构建。药学学报，2015,50(7)：842-847。b:Wang K.,et al.,Involvement of organic cation transporter 2 inhibition in potential mechanisms of antidepressant action.Prog Neuropsychopharmacol Biol Psychiatry,2014,53:90-98)，消化、收集细胞。用新鲜培养基重新悬浮细胞，调整细胞密度为4.0×104mL-1，以0.2mL/孔接种于96孔细胞板培养。细胞培 养24h后，弃除培养基，加入含1～100μmol/L(-)-THP，(+)-THP及(±)-THP的顺铂培养基溶液，以含相应浓度溶剂(DMSO)的培养基为空白对照组。给药后培养48h进行MTT实验，每孔加5mg/mL MTT(溴化噻唑蓝四氮唑)试剂20μL，轻轻摇匀，孵育4h后，弃去孵育液，于每孔加入150μL DMSO，微量振荡仪振荡10min，待甲臜结晶完全溶解后，酶联免疫检测仪测定570nm波长(以630nm为参照)处各样本的吸光度，用吸光度值/空白对照组吸光度的均值表示该样本细胞的存活率。
4.2细胞培养液中LDH活力的测定
给药培养48h后收集的细胞培养基样品进行LDH活力测定，按照乳酸脱氢酶(LDH)测定试剂盒说明书要求测定测定细胞培养液中LDH活力。以每个浓度给药的溶剂对照组酶活力为100％进行相对定量。
4.3结果
以MTT实验和细胞培养液中的LDH活力考察(-)-THP，(+)-THP及(±)-THP对顺铂在MDCK-hOCT2及MDCK-hOCT2/hMATE1细胞上毒性的影响。结果显示，顺铂显著降低细胞存活率，而(-)-THP，(+)-THP及(±)-THP浓度依赖性(1～100μmol/L)对抗顺铂(cisplatin，15μmol/L)诱导的细胞存活率降低，参见图4、图5(图中以溶剂对照组数值为100％，图中数据以mean±SD表示，n＝3；与对照组比较，###P<0.001；与顺铂组比较，*P<0.05，**P<0.01和***P<0.001)。同时，(-)-THP，(+)-THP及(±)-THP(1～100μmol/L)可明显降低顺铂引起的细胞LDH活力的升高，参见图6、图7，图中以溶剂对照组LDH活力为100％，图中数据以mean±SD表示，n＝3；与对照组比较,###P<0.001；与顺铂组比较，*P<0.05，**P<0.01和***P<0.001。
实施例4(-)-THP对顺铂在MDCK-hOCT2/pcDNA3.1及MDCK-hOCT2/hMATE1单层细胞上积聚及转运的影响
选取处于对数生长期的MDCK-hOCT2/pcDNA3.1及MDCK-hOCT2/hMATE1细胞(由浙江大学药学院构建，具体构建方法见:雷红梅等，稳定表达hMATE1及共表达hMATE1与hOCT1或hOCT2细胞模型的构建。药学学报，2015,50(7)：842-847。)，消化、收集细胞。用新鲜培养基重新悬浮细胞，调整细胞密度为3.0×105个/mL，以0.5mL/孔接种于12孔Tanswell细胞转运板培养3-5天，当细胞生长紧密成单层膜。将细胞培养基换成孵育缓冲液37℃预孵育20min，用Millicell-ERS细胞电阻仪测定每孔的电阻值，当电阻值为≥250Ω·cm2时，说明该孔细胞链接紧密可用于跨细胞转运实验。顺铂转运实验中，在转运板插件顶侧加入0.5mL缓冲液(pH6.5)，底侧加入1.5mL缓冲液(pH7.4)，缓冲液中含50μmol/L顺铂， 并于他们的相对测加入相应的空白缓冲液。抑制试验中，分别于顶侧和底侧均加入顺铂溶液含或不含抑制剂100μmol/L(-)-THP。37℃预孵育2.0h后，弃除孵育溶液，以冰冷的PBS溶液清洗细胞3遍。用0.5％的胰酶将插件上的细胞消化并收集，于细胞中加入浓硝酸：过氧化氢＝1:2在80℃硝解2.0h，加入适量的氢氧化铵溶液中和酸性，以0.1％的硝酸溶液定容至2.0mL，过滤，ICP-MS分析铂的含量。
结果显示：顺铂在MDCK-hOCT2/pcDNA3.1细胞单层膜上底侧到顶侧方向的积聚明显高于顶侧到底侧方向，同时显著高于MDCK-hOCT2/hMATE1细胞上底侧到顶侧的积聚；加入抑制剂100μmol/L(-)-THP后，能显著抑制顺铂在两个细胞上底侧到顶侧方向的积聚，参见图8、图9，图中数据以mean±SD(n＝3)表示，以溶剂对照组hOCT2/pcDNA3.1(AP→BL)数值为100％；与hOCT2/pcDNA3.1(AP→BL)对照组比较，**P<0.01和***P<0.001。上结果提示：(-)-THP对OCT2的抑制作用明显强于对MATE1抑制，显著抑制细胞中顺铂的积聚。
实施例5(-)-THP,(+)-THP及(±)-THP对顺铂在小鼠原代肾小管上皮细胞上毒性的影响
5.1小鼠原代肾小管上皮细胞的分离与培养
健康ICR小鼠(雄性，～25g)，乙醚麻醉后75％酒精浸泡1分钟，固定于手术台。剖开腹腔，以无Ca2+的HBSS溶液从肝门静脉灌流至肾脏现灰白色，迅速取下双肾置于无菌加有双抗(1％青霉素+1％链霉素)的PBS溶液中。于无菌超净台中用加有双抗的PBS溶液清洗数遍，去肾脏包膜，剪碎；在清洗3～5遍后，用无菌手术刀片切碎，置于0.1％Ⅳ型胶原酶与0.1％胰酶的混合溶液中震荡消化约40min。上层液体于80目筛网过滤，用DMEM/F12培养基(含DMEM/F12培养基，10％胎牛血清，1％双抗，1％胰岛素-转铁蛋白-硒三联剂)离心清洗2遍，重悬于培养基中，过200目筛网后种于96孔板，并隔天换液，生长约5～6天后即可用于细胞毒性实验。
5.2MTT实验
原代肾细胞培养约5天后，弃除培养基，加入含1～100μmol/L(-)-THP，(+)-THP及(±)-THP的顺铂培养基溶液，以含相应浓度溶剂(DMSO)的培养基为空白对照组。给药后培养48h进行MTT实验，每孔加5mg/mL MTT(溴化噻唑蓝四氮唑)试剂20μL，轻轻摇匀，孵育4h后，弃去孵育液，于每孔加入150μL DMSO，微量振荡仪振荡10min，待甲臜结晶完全溶解后，酶联免疫检测仪测定570nm波长(以630nm为参照)处各样本的吸光度，用吸光度值/空白对照组吸光度的均值表示该样本细胞的存活率。
5.3结果
MTT实验结果显示，(-)-THP，(+)-THP及(±)-THP均能浓度依赖性地(1～100μmol/L)降低顺铂(cisplatin，15μmol/L)在小鼠原代肾细胞上的毒性，增加细胞存活率，参见图10，图中以溶剂对照组数值为100％，图中数据以mean±SD表示，n＝3；与对照组比较，###P<0.001；与顺铂组比较，***P<0.001。
实施例6(-)-THP对顺铂在小鼠原代肾小管上皮细胞上积聚的影响
6.1小鼠原代肾小管上皮细胞的分离与培养
健康ICR小鼠(雄性，～25g)，乙醚麻醉后75％酒精浸泡1分钟，固定于手术台。剖开腹腔，以无Ca2+的HBSS溶液从肝门静脉灌流至肾脏现灰白色，迅速取下双肾置于无菌加有双抗(1％青霉素+1％链霉素)的PBS溶液中。于无菌超净台中用加有双抗的PBS溶液清洗数遍，去肾脏包膜，剪碎；在清洗3～5遍后，用无菌手术刀片切碎，置于0.1％Ⅳ型胶原酶与0.1％胰酶的混合溶液中震荡消化约40min。上层液体于80目筛网过滤，用DMEM/F12培养基(含DMEM/F12培养基，10％胎牛血清，1％双抗，1％胰岛素-转铁蛋白-硒三联剂)离心清洗2遍，重悬于培养基中，过200目筛网后种于96孔板，并隔天换液，生长约5～6天后即可用于细胞积聚实验。
6.2原代肾细胞积聚实验
小鼠原代肾小管细胞表达Oct2与Mate1转运体，可用于Oct2/Mate1介导的顺铂细胞积聚试验。考察了100μmol/L(-)-THP对顺铂在小鼠原代肾小管细胞中积聚(50μmol/L，2h)的影响。结果显示：(-)-THP显著抑制顺铂在小鼠原代肾小管细胞中的积聚，100μmol/L(-)-THP降低顺铂细胞积聚至溶剂对照组的21.1％，参见图11，以溶剂对照组数值为100％，图中数据以mean±SD(n＝3)表示。上述结果提示：(-)-THP能显著抑制Oct2/Mate1介导的顺铂转运入原代肾小管细胞内。
实施例7.(-)-THP对顺铂致小鼠肾毒性的影响
7.1实验动物
ICR小鼠(25～30g)，雄型，由浙江省医学科学院动物中心提供(清洁二级，动物许可证号：SCXK(浙)20140001)。所有动物均按照浙江大学实验动物操作指导的要求饲养，饲养环境控制合适温度(20～23℃)和湿度(50～60％)，实验前12h禁食，自由饮水。
7.2(-)-THP肾脏组织分布
7.2.1动物给药及样品釆集
ICR小鼠25只，随机分成5组，每组5只。尾静脉注射给予20mg/kg(-)-THP溶液，于给药后10min，20min，30min，60min及120min时间点分别取血浆和肾脏。将 组织以生理盐水洗去血液，滤纸吸干并精确称重，使用组织匀浆分散机制成1g/15mL的组织匀浆液(乙腈：水＝1:1)，-80℃保存，待测。
7.2.2生物样品预处理
-80℃保存的血浆或组织样品，室温解冻，取血浆和组织匀浆液各50μL置1.5mL离心管中，分别加200μL及450μL乙腈(含内标氯雷他定50ng/mL)，血浆或组织样品均旋振荡2min沉淀蛋白，于13000rpm离心15min，取上清液，进行LC-MS/MS分析。用待测物与内标峰面积比，代入随行标准曲线，进行定量分析。
7.3(-)-THP对顺铂在肾脏中浓度的影响
7.3.1动物给药及样品釆集
ICR小鼠16只(雄性，体重25～30g)，随机分为10mg/kg顺铂组和10mg/kg顺铂+20mg/kg(-)-THP组。尾静脉单次注射给予(-)-THP溶液后，再静注10mg/kg顺铂溶液。给药72h后，剖杀取肾脏组织，以生理盐水洗去血液，滤纸吸干，-80℃保存，待测。
7.3.2肾脏样品预处理
-80℃保存的肾脏样品，室温解冻，精密称定肾脏组织50mg置2.0mL离心管中，加200μL硝酸及400μL H2O2，在80℃水浴振荡消解1.5h，取出置于冰浴中冷却至室温。再加入200μL氨水进行中和，0.22μM水性滤膜过滤，并用0.1％硝酸定容至3.0mL，样品经超声后进行ICP-MS分析铂的浓度。
7.4(-)-THP对顺铂所致小鼠肾毒性的影响
7.4.1动物给药及样品釆集
22只ICR小鼠(雄性，体重25～30g)，随机分为control组，10mg/kg顺铂组和10mg/kg顺铂+20mg/kg(-)-THP组(n＝6-10)。尾静脉单次注射先给予(-)-THP溶液后，再静注10mg/kg顺铂溶液，control组给予同体积的生理盐水。每天记录小鼠的体重，密切观察记录小鼠皮、毛的改变及一般活动情况。于给药前及给药后72h，眼眶内眦静脉丛采血，取血清检测肾功能生化指标(包括尿素氮BUN，肌酐CRE，尿酸URA)，并剖杀小鼠，取出肾脏，精密称重，部分组织用40％福尔马林缓冲液固定后，石蜡包埋，做病理切片，进行HE染色，显微镜下观察肾组织结构的病理性变化。
7.5实验结果
7.5.1(-)-THP肾脏组织分布
小鼠静脉注射20mg/kg(-)-THP溶液后，(-)-THP迅速在肾脏组织中分布，10min时即可达22.6μM，约为血浆中浓度(4.5μM)的5倍，参见图12。提示肾组织中(-)-THP浓度 较高，可能会与肾脏中转运体OCT2/MATE1发生相互作用而影响顺铂的肾脏浓度，由此可能影响其肾脏毒性。
7.5.2(-)-THP降低顺铂的肾脏浓度
为了阐明(-)-THP是否会通过抑制OCT2/MATE1介导的顺铂肾脏转运而影响顺铂在肾脏的浓度，我们进一步比较了单次给予10mg/kg顺铂，以及合并给予顺铂及20mg/kg(-)-THP72h后，顺铂在肾脏中浓度的差异。结果表明，单独给予顺铂组小鼠肾脏中顺铂的浓度高达5.65ppm/g，而合并给予(-)-THP组小鼠肾脏浓度为2.82ppm/g，参见图13，说明(-)-THP能显著抑制顺铂的肾脏摄取而降低其肾脏浓度。
7.5.3顺铂毒性试验中动物一般状况
以10mg/kg剂量的顺铂给小鼠尾静脉单次注射后24h后，小鼠鼠皮毛开始竖起，并出现萎靡、对外界刺激反应迟钝，活动减少，食欲减退，体重开始明显下降，而对照组大鼠的行为及活动反应无异常，且体重稳步增加，两者存在显著的差异，说明顺铂对小鼠鼠整体水平具有一定的毒性。而合并给予20mg/kg(-)-THP的小鼠，顺铂引起的体重降低明显缓解，提示(-)-THP能抑制顺铂所致的小鼠肾脏毒性，参见图14。
7.5.4血清生化指标
检测给药前及给药后72h小鼠血清的尿素氮(BUN)、肌酐(CRE)及尿酸(URA)值。由图15可见，单独给予顺铂组小鼠血清肾功能指标BUN值显著高于溶剂对照组(control)，顺铂合用20mg/kg的(-)-THP后，BUN值明显降低且接近于control组。上述结果提示，静脉注射10mg/kg顺铂在小鼠体内能引起严重的的肾毒性；20mg/kg剂量的(-)-THP对顺铂引起的肾毒性具有较强的缓解作用。
7.5.5肾组织病理检查
HE染色，切片病理学分析，顺铂给药组小鼠肾组织病理检查发现有受损情况：主要的病理学改变为肾小管内管型，部分肾小管上皮细胞坏死，而合用20mg/kg(-)-THP组肾组织损伤显著好转，未见明显病理性改变。溶剂对照组(control)组肾组织病理检查未见任何异常，参见图16。

Claims (3)

  1. 一种延胡索乙素在制备抗顺铂毒性药物中的应用，所述延胡索乙素，包括左旋延胡索乙素，右旋延胡索乙素及消旋延胡索乙素，结构式为：
    Figure PCTCN2016098657-appb-100001
    其特征在于，所述毒性包括肾脏毒性、耳毒性及神经毒性。
  2. 根据权利要求1所述的应用，其特征在于，所述药物由延胡索乙素与制剂允许的辅料制成。
  3. 根据权利要求2所述的应用，其特征在于，所述药物的给药途径为静注或口服。
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