Verteporfin (DB-00460, CL-318952, BPD-MA, BpdMA)   中文名称: 维替泊芬

YAP-TEAD interaction

PDX细胞筛选用于特异性选择维替泊芬。 PhLO、PhLH 和 PhLK 的 GI50 值分别为 228 nM、395 nM 和 538 nM，导致 50% 的生长抑制。相比之下，ALL-1、TCC-Y/sr 和 NPhA1 的 GI50 值分别为 3.93 μM、2.11 μM 和 5.61 μM。 GSH 显着降低了三个 PDX 细胞中的两个对维替泊芬的敏感性。在 PDX 细胞中，维替泊芬降低线粒体膜的电位 [1]。通过抑制 YAP 表达，维替泊芬降低 HCT-8/T 细胞的 PTX 耐药性。当 Verteporfin 和 NSC 125973 一起治疗时，它们具有抑制 YAP 并降低 HCT-8/T 细胞毒性的协同作用 [2]。

BMS-354825和维替泊芬（10 mg/kg，csc）均显着降低了白血病细胞的百分比，并且它们的联合治疗还降低了脾脏中白血病细胞的总量[1]。

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通过基于图像和分子分析，我们发现维替泊芬/verteporfin 抑制了吉西他滨刺激的自噬，吉西他滨是目前治疗PDAC的标准药物。BxPC-3异种移植物小鼠模型的药代动力学和疗效研究表明，维替泊芬/verteporfin 在肿瘤中以自噬抑制水平积聚，并在体内抑制自噬，但作为单一药物不会减少肿瘤体积或增加存活率。与单独使用吉西他滨相比，与吉西他滨联合使用，维替泊芬适度降低了肿瘤生长并提高了生存率。虽然我们的研究结果并不支持自噬抑制作为单一治疗方式可能对PDAC广泛有效的前提，但它们确实支持自噬抑制是使PDAC对吉西他滨敏感的一种方法。[3]

为了研究YAP与PTX耐药性之间的关系，通过转染YAP质粒或siYAP RNA产生稳定的YAP过表达或YAP沉默细胞系。进行WST-1测定以检测PTX对HCT-8和HCT-8/T细胞的细胞毒性。分别进行克隆形成试验和Transwell试验，以确定细胞增殖和侵袭能力。进行免疫荧光和蛋白质印迹分析以检测蛋白质。[2]
结果：YAP在HCT-8/T中的表达强于HCT-8，PTX耐药性与YAP表达水平呈正相关。VP是一种强YAP抑制剂，可以通过抑制YAP来降低HCT-8/T细胞对PTX的耐药性，而无需光激活。此外，VP和PTX联合治疗对YAP的抑制和对HCT-8/T的细胞毒性显示出协同作用。此外，维替泊芬和PTX组合治疗影响HCT-8/T细胞的侵袭和集落形成能力，并诱导其凋亡。[2]
结论：VP可以通过抑制YAP的表达来逆转YAP过表达在HCT-8/T细胞中诱导的PTX抗性，而无需光活化。[2]

维替泊芬对PDAC细胞系的体外作用[3]
将8个人PDAC细胞系暴露于0-10µM的维替泊芬中长达7天，并使用自动荧光显微镜计数细胞核或通过MTT法测量吸光度来定量活细胞。四条线（Capan 1、Capan 2、HS766T、CFPAC-1）在所有浓度下对维替泊芬均不敏感。Panc-1和MIA PaCa-2细胞在≤5µM维替泊芬中正常生长，但在10µM维他泊芬中细胞增殖受到显著抑制。值得注意的是，BxPC-3和SU86.86细胞的增殖在10µM维替泊芬中完全受到抑制，在5µM维他泊芬中减少了50%以上。[3]

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药物相互作用的估计[1]
与S17细胞共培养的PDX细胞用16种组合的维替泊芬（60 nM、120 nM、180 nM和240 nM）和达沙替尼（12 nM、24 nM、36 nM和48 nM）处理。48小时后，使用FACS Aria流式细胞仪测量用每种组合处理的细胞的存活率。为了评估维替泊芬和达沙替尼之间的药物相互作用，使用CompuSyn软件制作了归一化的等渗图和分数影响组合指数（CI）图。CI值大于1.0表示拮抗作用，等于1.0的加性作用，低于1.0的协同作用。

Estimation of in vivo drug effects[1]
PhLO cells (1.0 × 10~7 /mouse) were injected intravenously into 6-week-old male NOG mice, which were then treated with vehicle, verteporfin (140 milligram (mg)/kilogram (kg)/day), dasatinib (20 mg/kg/day), and a combination of these drugs from days 22 to 28. Verteporfin was administered by continuous subcutaneous infusion (c.s.c.) using Alzet osmotic pumps. An intraperitoneal injection (i.p.) was performed for dasatinib. All mice were sacrificed on day 28 and the chimerism of leukemia cells was investigated by flow cytometer using an anti-human CD19 antibody and anti-mouse CD45 antibody. Blood concentrations of verteporfin were calculated by LCMS-2020.[1]

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Verteporfin efficacy studies[3]
Female Rag2M mice (20-25g) were inoculated subcutaneously in the centre of the lower back with 5 x 106 BxPC-3 or SU86.86 cells (1:1 RPMI:matrigel; 100µL volume; expressed as day 0). Tumors appeared within three weeks of implantation. Once the tumors were palpable, tumor growth was monitored by measuring tumor dimensions with digital calipers. When tumors reached 100-150mg (calculated according to the equation (length X width2)/2 converted to tumor weight in mg for each 1mm3), mice were randomized in groups of eight animals and treatment was initiated.

Verteporfin was administered i.p. Monday, Wednesday, Friday for 4 weeks at a dose of 45mg/kg (injection volume 400µl/20g mouse). Gemcitabine was administered i.p. once weekly (Monday) for 4 weeks at 120mg/kg or 240mg/kg (injection volume 200µl/20g mouse). Groups treated with both verteporfin and gemcitabine received gemcitabine 6h after verteporfin administration. This time was selected because maximum verteporfin tumor levels were achieved 8h post-administration and maximum gemcitabine tumor levels were observed 2h post-administration. Animals in the control group were treated with the delivery vehicle DSPE-mPEG2000 at the same concentration and schedule as verteporfin. Care was taken to house animals treated with verteporfin in dark conditions until the morning after treatment because verteporfin is a photosensitizer and exposure to bright light could be harmful. A One-Way ANOVA with Tukey's multiple comparison test was used to compare differences in tumor growth.

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Pharmacokinetic studies of verteporfin DSPE-mPEG2000 micelles in BxPC-3 tumor-bearing mice[3]
Rag2M mice (20-25g; n=3) were inoculated subcutaneously with 5 x 106 BxPC-3 cells. Mice were injected i.p. with verteporfin at 45mg/kg when tumors were approximately 200-250mg. Mice were euthanized by CO2 inhalation, and blood and tumors were collected at 2, 8, 16 and 24h post administration of verteporfin. Plasma was prepared by centrifuging samples at 1000 x g for 15min at 4°C. Tumors were excised, rinsed in PBS, and snap-frozen in cryovials in liquid nitrogen and stored at -80°C. Tumors were sectioned while frozen and one half was used for determining verteporfin concentration by UPLC-MS/MS and the other for immunoblot analysis of p62 and LC3.

Plasma samples were thawed, homogenized and extracted with acetonitrile containing 0.1% formic acid. Protein precipitation and filtration was carried out using ISOLUTE® PPT+ protein precipitation plates (Biotage). Samples were analyzed using a Waters® ACQUITY® UPLC system with mass spectrometry detection. Separations were performed using an isocratic method where mobile phase A was 0.1% formic acid in water and B was 0.1% formic acid in acetonitrile (70% B for 2.5min followed by 95% B for the wash). Verteporfin regioisomer A (monoacid A form) was eluted at 1.77min and regioisomer B (monoacid B form) was eluted at 2.16min with a total run time of 4.5min per sample. The MS/MS system was operated with an ESI interface in a positive ionization mode. Quantification was performed using multiple reactions monitoring (MRM) mode with a precursor mass m/z of 719.27 and product mass m/z of 645.36. The levels of verteporfin were measured against external calibration standards prepared using the same process.

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Formulation of verteporfin for animal studies[3]
Verteporfin was formulated in DSPE-mPEG2000 micelles. Briefly, 200mg verteporfin dissolved in 2ml DMSO was slowly added, with stirring, to 1500mg DSPE-mPEG2000 dissolved in 50ml PBS at pH 7.4. Stirring was continued for 1h at 23°C followed by dialysis overnight against PBS using Spectra/Por dialysis membranes (15,000 MWCO). The concentration of verteporfin was measured in triplicate and quantified against an external standard curve using a Waters® ACQUITY® UPLC equipped with a PDA detector. Separations were done using a C18 column (Waters® BEH; column size 50 x 2.1mm, particle size 1.7µm) and a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile:methanol (1:1; B) [10-80% B over 2min at 0.5ml/min flow rate]. The concentration of verteporfin was adjusted to 2.25mg/ml with PBS followed by filter-sterilization and sterile vialing of the formulation. The concentration was reconfirmed before proceeding with the animal studies. The formulation was chemically and physically stable for an observation period of 4 weeks, which covered the duration of the animals studies as verified by UPLC and polarized light microscopy.

140 mg/kg; i.v. injection

Mice: PhLO cells (1.0×107/mouse) are injected intravenously into 6-week-old male NOG mice, which are then treated with vehicle, verteporfin (140 mg/kg/day), dasatinib (20 mg/kg/day), and a combination of these drugs from days 22 to 28. Verteporfin is administered by continuous subcutaneous infusion (c.s.c.) using Alzet osmotic pumps. An intraperitoneal injection (i.p.) is performed for dasatinib. All mice are sacrificed on day 28 and the chimerism of leukemia cells is investigated by flow cytometer using an anti-human CD19 antibody and antimouse CD45 antibody. Blood concentrations of verteporfin are calculated by LCMS-2020.

[1]. Morishita T, et al. The photosensitizer verteporfin has light-independent anti-leukemic activity for Ph-positive acute lymphoblastic leukemia and synergistically works with BMS-354825. Oncotarget. 2016 Aug 2.
[2]. Pan W, et al. Verteporfin can Reverse the NSC 125973 Resistance Induced by YAP Over-Expression in HCT-8/T Cells without Photoactivation through Inhibiting YAP Expression. Cell Physiol Biochem. 2016;39(2):481-90.
[3]. Donohue E, et al. The autophagy inhibitor verteporfin moderately enhances the antitumor activity of gemcitabine in a pancreatic ductal adenocarcinoma model.J Cancer. 2013 Aug 28;4(7):585-96

A benzoporphyrin derivative that is used in PHOTOCHEMOTHERAPY to treat wet type MACULAR DEGENERATION. A synthetic light-activated agent with photodynamic activity. Upon systemic administration, verteporfin accumulates in neovessels in the eye and, once stimulated by nonthermal red light in the presence of oxygen, produces highly reactive short-lived singlet oxygen and other reactive oxygen radicals, resulting in local damage to neovascular endothelium and blood vessel occlusion.

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An equimolar mixture of the 9-methyl ester and 13-methyl ester of trans-(+-)-18-ethenyl-4,4a-dihydro-3,4-bis(methoxycarbonyl)-4a,8,14,19-tetramethyl-23H,25H-benzo[b]porphine-9,13-dipropanoic acid. It is used as a photosensitizer in photodynamic therapy to eliminate the abnormal blood vessels in the eye associated with neovascular (wet) age-related macular degeneration. Verteporfin accumulates in these abnormal blood vessels and, when activated by red (693 nm) laser light in the presence of oxygen, produces highly reactive short-lived singlet oxygen and other reactive oxygen radicals, resulting in local damage to the endothelium and blockage of the vessels.

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Cell lines have been used for drug discovery as useful models of cancers; however, they do not recapitulate cancers faithfully, particularly from the viewpoints of microenvironmental independence. Patient-derived xenografts (PDX) are established by the transfer of primary tumor cells directly from patients into immunodeficient mice and can provide primary-like tumor cells of the amount needed at the desired time. We developed a high-throughput drug screening system using PDX cells and performed drug screening using the PDX cells of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). We established four Ph+ ALL PDX mice and performed high-throughput screening of 3440 compounds using leukemia cells from the PDX mice (PDX-cell screening). The profiles of drugs selected by PDX-cell screening were markedly different from those by screening using the Ph+ ALL cell line. We found that verteporfin, an FDA-approved drug, exhibited strong PDX cell-specific cytotoxicity. In the validation assay, its GI50 was 228 nM, 395 nM, and 538 nM in three PDX cells and 3.93 µM, 2.11 µM, and 5.61 µM in three cell lines. Although verteporfin is a photosensitizer activated by photoirradiation, its cytotoxic effects were mediated by the light-independent production of reactive oxygen species; therefore, its anti-leukemic effects were also exerted in vivo without photoirradiation. Furthermore, it exhibited synergistic effects with dasatinib, an ABL kinase inhibitor. These results indicated the potential of verteporfin as a new anti-leukemic reagent.[1]

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aclitaxel (PTX) is one of the most effective anti-cancer drugs. However, multiple drug resistance is still the main factor that hinders the effective treatment of cancer with PTX. Several factors including YAP over-expression can cause PTX resistance. In this study, we aimed to verify the role YAP plays in PTX resistance, explore the reversal of PTX resistance by verteporfin (VP) and investigate the effect of combination therapy of PTX and VP on the PTX resistant colon cancer cells (HCT-8/T).[2]

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Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to chemotherapy. It has been described as requiring elevated autophagy for growth and inhibiting autophagy has been proposed as a treatment strategy. To date, all preclinical reports and clinical trials investigating pharmacological inhibition of autophagy have used chloroquine or hydroxychloroquine, which interfere with lysosomal function and block autophagy at a late stage. Verteporfin is a newly discovered autophagy inhibitor that blocks autophagy at an early stage by inhibiting autophagosome formation. Here we report that PDAC cell lines show variable sensitivity to verteporfin in vitro, suggesting cell-line specific autophagy dependence. Using image-based and molecular analyses, we show that verteporfin inhibits autophagy stimulated by gemcitabine, the current standard treatment for PDAC. Pharmacokinetic and efficacy studies in a BxPC-3 xenograft mouse model demonstrated that verteporfin accumulated in tumors at autophagy-inhibiting levels and inhibited autophagy in vivo, but did not reduce tumor volume or increase survival as a single agent. In combination with gemcitabine verteporfin moderately reduced tumor growth and enhanced survival compared to gemcitabine alone. While our results do not uphold the premise that autophagy inhibition might be widely effective against PDAC as a single-modality treatment, they do support autophagy inhibition as an approach to sensitize PDAC to gemcitabine.[3]

C41H42N4O8

718.79

718.30

C, 68.51; H, 5.89; N, 7.79; O, 17.81

129497-78-5

Typically exists as Brown to black solid at room temperature

10.246

337.48

[C@]12(C)[C@@H](C(=O)OC)C(C(=O)OC)=CC=C1C1=CC3=NC(C(C=C)=C3C)=CC3NC(=C(C=3C)CCC(=O)O)C=C3C(CCC(=O)OC)=C(C)C(=N3)C=C2N1.[C@]12(C)[C@@H](C(=O)OC)C(=CC=C1C1NC2=CC2C(C)=C(CCC(=O)O)C(=CC3NC(=C(C)C=3CCC(=O)OC)C=C3C(C=C)=C(C)C(=N3)C=1)N=2)C(=O)OC |c:26,t:16,40,54,73,84,99,109,&1:0,2,53,55|

YTZALCGQUPRCGW-MXVXOLGGSA-N

InChI=1S/C41H42N4O8/c1-9-23-20(2)29-17-34-27-13-10-26(39(49)52-7)38(40(50)53-8)41(27,5)35(45-34)19-30-22(4)25(12-15-37(48)51-6)33(44-30)18-32-24(11-14-36(46)47)21(3)28(43-32)16-31(23)42-29/h9-10,13,16-19,38,42,44H,1,11-12,14-15H2,2-8H3,(H,46,47)/b28-16-,29-17-,30-19-,31-16-,32-18-,33-18-,34-17-,35-19-/t38-,41+/m0/s1

(1): 3-[(23S,24R)-14-ethenyl-5-(3-methoxy-3-oxopropyl)-22,23-bis(methoxycarbonyl)-4,10,15,24-tetramethyl-25,26,27,28-tetraazahexacyclo[16.6.1.13,6.18,11.113,16.019,24]octacosa-1,3,5,7,9,11(27),12,14,16,18(25),19,21-dodecaen-9-yl]propanoic acid.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 5 mg/mL (6.96 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 50.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 5 mg/mL (6.96 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 50.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。