载三氧化二砷脑胶质瘤靶向纳米递药系统i RGD/TGN-PEG-PAMAM/ATO的构建及体外研究

目的以树状大分子为载体材料并修饰血脑屏障(blood brain barrier,BBB)靶向短肽TGN和肿瘤靶向短肽i RGD构建脑胶质瘤靶向递药系统(i RGD/TGN-PEG-PAMAM/ATO),旨在解决三氧化二砷(arsenictrioxide,As2O3,ATO)在治疗脑胶质瘤过程中分布缺乏特异性、透BBB难等问题,使其具有更好抗脑胶质瘤作用。方法核磁共振图谱(1H-NMR)、透射电子显微镜(TEM)等考察载体的理化性质;电感耦合等离子发射光谱(ICP)、透析袋法分析其包封率及体外释放情况;通过激光共聚焦及流式细胞仪分析i RGD、TGN对细胞摄取的影响;MTT法考察纳米载体对脑微血管内皮细胞(HBMEC)和脑胶质瘤U87细胞的毒性及BBB模型中递药系统抑制U87细胞生长的情况。结果成功合成了i RGD/TGN-PEG-PAMAM载体,其形态规整,大小均匀,测得其粒径(24.87±0.84)nm,电位(17.26±1.64)m V;该载体对HBMEC和U87细胞均具有较小的毒性;递药系统i RGD/TGN-PEG-PAMAM/ATO的包封率为(71.92±1.17)%,体外释放表明ATO经载体包载后呈现一种缓慢释放趋势,且在酸性条件下更有利于ATO的释放;细胞摄取结果提示iRGD/TGN的修饰有利于U87细胞对递药系统的摄取;体外跨BBB抑制U87细胞生长实验结果表明,i RGD/TGN-PEG-PAMAM/ATO组具有更好的跨BBB抑制U87细胞生长效果。结论 i RGD/TGN-PEG-PAMAM/ATO脑胶质瘤靶向递药系统具有较好的体外跨BBB抑制U87细胞生长的效果,为脑胶质瘤治疗提供了新的策略。     

载As2O3 pH敏感钙砷复合物脂质体的制备及体外评价

目的 制备pH敏感释药的As₂O₃脂质体，并进行体外评价。方法 采用薄膜分散法制备含钙离子脂质体，然后用离子沉淀法孵育制备钙砷复合物脂质体（CaAs-LP）。测定CaAs-LP的粒径、Zeta电位及多分散系数（PDI）；透射电子显微镜观察脂质体的形态；电感耦合等离子体发射光谱仪测定纳米药物的载药量与包封率；透析袋法考察其体外释药特性。噻唑蓝（MTT）法考察未载药脂质体及CaAs-LP对人源性乳腺癌MCF-7细胞、人源性脑胶质瘤U87细胞和人源性肝癌HepG2细胞的毒性；共聚焦显微镜考察U87细胞对CaAs-LP的摄取效率。结果 制备的CaAs-LP呈规整类球型，粒径约为（117.16±1.94）nm，包封率和载药量分别为（74.31±2.11）%、（8.31±0.13）%。体外释放研究表明，CaAs-LP具有明显的缓释以及pH响应释药特征。未载药的脂质体在MCF-7、U87、HepG2和L02细胞中的生物相容性良好；CaAs-LP抑制肿瘤细胞生长的作用较原药有所上升，半数抑制浓度（IC₅₀）值分别为11.91、4.90、19.41、27.59 μmol/L。细胞摄取研究表明肝癌细胞对脂质体具有良好的摄取。结论 CaAs-LP具备显著的缓释以及pH响应释药的特性，在肿瘤治疗方面具有较好的应用前景。     

Time course of arsenic species in red blood cells of acute promyelocytic leukemia (APL) patients treated with single agent arsenic trioxide

Background:Arsenic trioxide (ATO) is widely applied to treat acute promyelocytic leukemia (APL). To elucidate metabolism and toxicity of arsenic, we analyzed time course of arsenic species in red blood cells (RBCs) of APL patients.

Methods:Nine APL patients received ATO (0.16 mg/kg/day) through 18-h infusion. Blood was collected before daily administration (days 2 to 9), and at different time points on day 8. Inorganic arsenic (iAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were detected by HPLC-ICP-MS.

Results:Arsenic species reached C_max at 18 h on day 8. Arsenicals gradually accumulated during days 2 to 9, whereas their percentages remained almost constant. The general trend in red blood cells (RBCs) was iAs > MMA > DMA. MMA was consistently the predominant methylated arsenic metabolite in RBCs. iAs, MMA, and tAs (tAs = iAs + DMA + MMA) concentrations (P < 0.0001), MMA/DMA ratios (P = 0.0016) and iAs% (P = 0.0013) were higher in RBCs than in plasma.

Conclusions:Time course of arsenic species reveal kinetic characteristic of ATO metabolites in RBCs. Arsenic species accumulated with administration frequency. Arsenic species in RBCs were remarkably different from those in plasma. Time course of arsenic species in RBCs is important in ATO clinical application.     

Arsenic trioxide replacing or reducing chemotherapy in consolidation therapy for acute promyelocytic leukemia (APL2012 trial)

As all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are widely accepted in treating acute promyelocytic leukemia (APL), deescalating toxicity becomes a research hotspot. Here, we evaluated whether chemotherapy could be replaced or reduced by ATO in APL patients at different risks. After achieving complete remission with ATRA-ATO–based induction therapy, patients were randomized (1:1) into ATO and non-ATO groups for consolidation: ATRA-ATO versus ATRA–anthracycline for low-/intermediate-risk patients, or ATRA-ATO–anthracycline versus ATRA–anthracycline–cytarabine for high-risk patients. The primary end point was to assess disease-free survival (DFS) at 3 y by a noninferiority margin of –5%; 855 patients were enrolled with a median follow-up of 54.9 mo, and 658 of 755 patients could be evaluated at 3 y. In the ATO group, 96.1% (319/332) achieved 3-y DFS, compared to 92.6% (302/326) in the non-ATO group. The difference was 3.45% (95% CI –0.07 to 6.97), confirming noninferiority (P < 0.001). Using the Kaplan–Meier method, the estimated 7-y DFS was 95.7% (95% CI 93.6 to 97.9) in ATO and 92.6% (95% CI 89.8 to 95.4) in non-ATO groups (P = 0.066). Concerning secondary end points, the 7-y cumulative incidence of relapse (CIR) was significantly lower in ATO (2.2% [95% CI 1.1 to 4.2]) than in non-ATO group (6.1% [95% CI 3.9 to 9.5], P = 0.011). In addition, grade 3 to 4 hematological toxicities were significantly reduced in the ATO group during consolidation. Hence, ATRA-ATO in both chemotherapy-replacing and -reducing settings in consolidation is not inferior to ATRA–chemotherapy (https://www.clinicaltrials.gov/, {"type":"clinical-trial","attrs":{"text":"NCT01987297","term_id":"NCT01987297"}}NCT01987297).

The treatment of all-trans retinoic acid (ATRA) combined with anthracycline-based chemotherapy has remarkably improved the prognosis of patients with acute promyelocytic leukemia (APL), achieving over 90% complete remission (CR) and 60 to 80% long-term survival (1–6). For patients relapsed from ATRA-chemotherapy, arsenic trioxide (ATO) was initially used as salvage therapy and showed a satisfactory outcome (7–9). Then, the treatment of newly diagnosed APL with an ATRA-ATO combination therapy was reported in 2004, which demonstrated curative effects in 90% of patients (10–13). The advantage of ATO as the front-line treatment of APL has been further validated by a number of international working groups (14–18). Meanwhile, an exploratory study on ATRA-ATO with or without gemtuzumab ozogamicin (GO, the cytotoxic agent calicheamicin linked an anti-CD33 monoclonal antibody) by the MD Anderson Cancer Center suggested that a deescalating cytotoxic regimen might be feasible for APL patients (14, 19).A large body of evidence has been obtained to show that both ATRA and ATO target the APL-specific PML-RARA oncoprotein and the two agents may exert a synergistic effect in achieving a curative clinical effect in most APL (2, 9). However, in our previous studies, though ATRA-ATO were used as main therapeutic agents for induction, the consolidation was based on chemotherapy rather than ATO, which could cause life-threatening myelosuppression and cardiotoxicity (10, 11). Besides, risk-stratified treatment had not been introduced, leading to probable overtreatment for low- risk (a white blood cell [WBC] count ≤ 10 × 10⁹/L and a platelet count > 40 × 10⁹/L) to intermediate-risk (a WBC count ≤ 10 × 10⁹/L and a platelet count ≤ 40 × 10⁹/L) patients (20). These issues warranted further clinical investigations to address the role of ATRA-ATO in consolidation and to adapt the treatment protocols to distinct clinical risks. In order to optimize the treatment protocols by reducing their relevant toxicities and costs, as well as further improving therapeutic efficacy and tolerance, we proposed a multicenter randomized trial, APL2012, deriving from our previous ATRA-ATO–based therapy taking into consideration of Sanz risk stratification (20). The objective of this study was to examine whether chemotherapy could be replaced or reduced in consolidation therapy by ATO in patients with APL at different risks.     

3,4‐Dihydroxybenzaldehyde lowers ROS generation and protects human red blood cells from arsenic(III) induced oxidative damage

Arsenic (As) is a potent environmental toxicant and chronic exposure to it results in various malignancies in humans. Oxidative stress has been implicated in the etiopathogenesis of As‐induced toxicity. This investigated the protective effect of plant antioxidant 3,4‐dihydroxybenzaldehyde (DHB) on sodium meta‐arsenite (SA), an As‐(III) compound, induced oxidative damage in human red blood cells (RBC). The RBC were first incubated with different concentrations of DHB and then treated with SA at 37°C. Hemolysates were prepared and assayed for various biochemical parameters. Treatment of RBC with SA alone enhanced the generation of reactive oxygen species and increased lipid and protein oxidation. Reduced glutathione levels, total sulfhydryl content and cellular antioxidant power were significantly decreased in SA alone treated RBC, compared to the untreated control cells. This was accompanied by membrane damage, alterations in activities of antioxidant enzymes and deranged glucose metabolism. Incubation of RBC with DHB, prior to treatment with SA, significantly and dose‐dependently attenuated the SA‐induced changes in all these parameters. Scanning electron microscopy of RBC confirmed these biochemical results. Treatment of RBC with SA alone converted the biconcave discoids to echinocytes but the presence of DHB inhibited this conversion and the RBC retained their normal shape. These results show that DHB protects human RBC from SA‐induced oxidative damage, most probably due to its antioxidant character.     

不同来源牛黄与雄黄配伍的牛黄解毒片对小鼠毒性的影响

目的:比较不同来源牛黄与雄黄配伍的牛黄解毒片对小鼠的毒性反应,探讨临床上牛黄解毒片引起慢性砷中毒的机制。方法:按2015年版《中国药典》的配方用天然牛黄、人工牛黄、体外培育牛黄分别制成牛黄解毒片,并用同法制成无牛黄成分、无雄黄成分的缺味组,另设同剂量单味雄黄作为对照,每种牛黄解毒片组再分为0.5,1.0,2.0 g·kg~(-1)剂量组,单味雄黄组分为0.06,0.12,0.24 g·kg~(-1)剂量组,对ICR小鼠进行灌胃给药28 d,观察毒性反应;给药结束后解剖取血,分离血清检测肝肾功能,取心、肝、脾、肺、肾做组织病理学检查和电镜下超微结构观察。结果:给药期间各给药组未观察到毒性反应,给药结束亦未观察到大体内脏及体表皮肤病变,血液生化指标检查未能明确雄黄对肝肾的毒性作用。组织病理学和电镜下超微结构检查各牛黄解毒片组未发现异常毒性病理改变,但苏木素-伊红(HE)染色显示单味雄黄高剂量组肝脏出现小叶中心性肝细胞肥大和糖原聚集,过碘酸希夫(PAS)染色表明肝细胞内糖原聚集,电镜下超微结构检查发现肝细胞内局灶性胞浆溶解,内有数量不等糖原颗粒,提示雄黄可致小鼠肝脏毒性。结论:用不同来源牛黄与雄黄配伍的牛黄解毒片对小鼠进行28 d灌胃给药,均未发现对小鼠有明显的毒性反应。而用单味雄黄却引起肝脏毒性,提示牛黄解毒片中的其他成分与雄黄配伍能显著降低可溶性砷和价态砷的含量,从而降低雄黄的毒性。     

冬虫夏草中砷及其安全性评价研究进展

传统名贵中药冬虫夏草Cordyceps sinensis中砷超标及其安全性问题近年来备受关注,针对冬虫夏草中砷的研究逐渐增加并不断深入。在文献查阅的基础上,从冬虫夏草砷及砷形态的分析方法,砷的含量、分布、存在形态,安全性评价,以及野生和人工仿生繁育冬虫夏草中砷含量等方面进行归纳总结,并对冬虫夏草中砷的认知及研究进行展望,以期为冬虫夏草安全性评价的深入研究提供参考。     

三氧化二砷对人舌鳞癌细胞CAL-27侵袭能力的影响

目的 检测三氧化二砷对人舌鳞癌细胞CAL-27体内、外侵袭能力的影响并探讨其相关作用机制。方法 体外采用黏附实验、划痕实验、Transwell侵袭实验、免疫荧光染色和蛋白质印迹法检测三氧化二砷对CAL-27细胞黏附、迁移及侵袭能力的影响;体内采用免疫组织化学和蛋白质印迹法检测三氧化二砷对裸鼠移植瘤中CD44和基质金属蛋白酶（MMP）-2和MMP-9表达的影响。结果 三氧化二砷作用后,实验组CAL-27细胞的黏附、迁移及侵袭能力明显低于对照组（P〈0.05）,细胞骨架微丝解聚,微管结构模糊、紊乱,MMP-2和MMP-9的表达下降;三氧化二砷降低了裸鼠移植瘤中CD44、MMP-2和MMP-9的表达。结论 低浓度的三氧化二砷可降低人舌鳞癌细胞CAL-27细胞黏附能力,改变细胞骨架排列,下调CD44、MMP-2和MMP-9的表达,进而抑制癌细胞的侵袭能力。     

三氧化二砷新剂型的研究进展

三氧化二砷是急性早幼粒细胞白血病的一线用药,对多种实体瘤也具有抑制作用。然而,三氧化二砷肾脏清除速率快,无组织特异性分布,靶向性较差,易产生心、肝和肾等毒性作用,限制了其在实体瘤的临床应用。近年来,科学家开发大量三氧化二砷新剂型以提高其疗效,降低其不良反应,包括脂质体、微球、纳米粒、乳剂和悬浮剂等。本文对三氧化二砷的新剂型研究进展进行梳理,以期为三氧化二砷新剂型的合理开发和应用提供新思路。