Salirasib (Farnesyl Thiosalicylic Acid)   中文名称: 沙利雷塞

RAS; PPMTase (Ki = 2.6 μM)

体外活性：Salirasib抑制人 Ha-ras 转化 Rat1 细胞的生长，这与其对 PPMTase 的抑制作用密切相关。 Salirasib抑制 Rat-1 成纤维细胞、Ras 转化的 Rat-1 和 B16 黑色素瘤细胞中的 Ras 甲基化。 Salirasib 还可以降低细胞膜中 Ras 的水平，并抑制 Ras 依赖性细胞生长，与甲基化无关，但通过调节 Ras-Raf 通讯来实现。在 Ras 转化的 EJ 细胞中，Salirasib 干扰 Raf-1 和 MAPK 的激活并抑制 DNA 合成。激酶测定：大鼠脑小脑的突触体膜或培养细胞系的总膜（100,000 × g 颗粒）用于无细胞系统中的甲基转移酶测定。甲基转移酶测定在 50 mM Tris-HCl 缓冲液（pH 7.4）中于 37°C 下进行，使用 100 μg 蛋白质、25 μM [methyl-3H]AdoMet (300,000 cpm/nmol) 和 50 μM AFC（作为储备溶液制备） DMSO 中），总体积为 50 μL。所有测定中的 DMSO 浓度均为 8%。如文中所示，多个实验中使用了不同的 AFC 浓度。 10 分钟后，通过添加 500 μL 氯仿:甲醇 (1:1) 终止反应，随后添加 250 μL H2O，混合并相分离。将 125 µL 氯仿相在 40°C 下干燥，并添加 200 µl 1 N NaOH、1% SDS 溶液。通过气相平衡法对由此形成的甲醇进行计数。典型的背景计数（未添加 AFC）为 50-100 cpm，而带有 AFC 的典型反应产量为 500-6,000 cpm。测定一式三份进行，并减去背景计数。进行内源底物的甲基化和凝胶电泳。使用 100 μCi/mL [甲基-3H]蛋氨酸测定完整细胞中的蛋白质羧甲基化。在含有 5 mL 标记培养基的 10 厘米平板中生长的近汇合培养物中对细胞进行分析。细胞测定：细胞在24孔板中生长。电镀后 2 小时，细胞接受从储备溶液中新鲜制备的溶剂或 FTS，以在 0.1% DMSO 中产生最终指定浓度。每 4 天更换一次含有溶剂或药物的新鲜培养基。单独的实验表明溶剂本身对细胞生长没有影响。在指定的日子里，通过胰蛋白酶/EDTA将细胞从平板上分离并在光学显微镜下计数。所有测定均一式四份进行。在平行实验中，细胞用台盼蓝或MTT染色，并在光学显微镜下检查染色的细胞。在一些 MTT 染色培养物中，将细胞溶解在 0.2 mL 100% DMSO 中，并使用酶联免疫吸附测定读数仪通过分光光度法测定染色程度。

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我们的研究结果表明，ELT3细胞中，Rheb。GTP水平在结构上很高（图2），比表达TSC2的ELT3细胞（图1）增殖更快，其中TSC2促进Rheb。GTP水解。我们发现FTS/Salirasib在体外抑制了TSC2缺失的ELT3细胞的增殖，并且这种抑制作用在很大程度上被TSC2表达所抵消（图1a-1c）。因此，FTS优先抑制快速增殖的细胞。此外，我们的研究结果表明，增殖的抑制主要归因于Rheb蛋白的抑制，而不是Ras的抑制。根据这一发现，DN Rheb（而非DN Ras）模拟了FTS对细胞增殖的生长抑制作用。FTS还降低了TSC2-null ELT3细胞中Rheb蛋白及其下游靶点S6K的量（图2a和2b）。

ELT3细胞中的大多数Rheb蛋白似乎都是GTP结合的形式。5,20我们发现ELT3细胞含有相对大量的Rheb。[32P]-GTP（约占Rheb总量的50%），其中唾液酸/FTS治疗减少了88%（图2e）。因此，这些新的结果表明，FTS/Salirasib通过抑制活性Rheb来下调Rheb蛋白（图2a）。GTP（图2e）和降低Rheb稳定性（图2d）。研究表明，Rheb定位对于Rheb介导的mTOR激活是必不可少的。28还表明，FTS不会破坏Rheb的亚细胞定位。28与这些发现一致，我们没有观察到FTS对Rheb的错误定位（数据未显示）。我们假设Rheb与细胞膜紧密结合，并附着在一个假定的锚定蛋白上；FTS干扰了推测的锚定蛋白和Rheb的相互作用。GTP，从而阻断Rheb GTP的负载和活性，而不影响其总体细胞定位。因此，我们提出了一个方案，描述了FTS在ELT3细胞中作用的可能机制（图6）。该方案结合了前面所述的Ras和Rheb信号29，以及本文所述的FTS对Ras和Rheb的影响。有趣的是，Ras的量。我们TSC2缺失的ELT3细胞中的GTP相对较小，并且正如预期的那样，FTS对Ras的影响（如果有的话）仅为轻微22（图2a）。这些结果表明：（i）活性Ras对ELT3细胞生长的重要性不如活性Rheb，以及（ii）FTS诱导的对ELT3肿瘤生长的抑制是其对Rheb而非Ras作用的结果。高Rheb之间可能存在关联。GTP和低Ras。GTP因观察到表达TSC2的ELT3细胞中Rheb的量如预期的那样显著低于TSC2缺失的ELT3电池中的Rheb量而得到加强，20而Ras的量。GTP高于亲本ELT3细胞（图2a）。这种相互关系的原因尚不清楚，但如果这是一种普遍现象，则可能与Ras和Rheb生物学以及与这些蛋白质相关的疾病（如LAM、2结节硬化症30和癌症）有关。这种现象的原因尚不清楚，需要进一步研究，但它并不干扰我们的主要结论。[1]

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在这项研究中，我们首次报道了新型异戊二烯基半胱氨酸类似物Salirasib通过干扰ras和mTOR抑制三种人类HCC细胞系细胞生长的作用。更重要的是，salirasib能够抑制EGF和IGF诱导的人HCC细胞系增殖，这可能会降低与一种生长因子途径激活相关的逃逸机制的可能性，以应对另一种途径的抑制。尽管三种受试细胞系在治疗三天后的IC50相似，但时间过程实验表明，在三种受测细胞系中，Hep3B细胞对唾液酸盐最敏感，而Huh7细胞则更具耐药性。重要的是，我们的研究结果还表明，在远低于估计的IC50的剂量下，长期使用salirasib治疗是有效的。

另一方面，凋亡也有助于Salirasib的生长抑制作用，与其他两种细胞系相比，Huh7的相对耐药性可能是由于这些细胞在处理后没有凋亡诱导。然而，至少在我们的实验条件下，凋亡的贡献似乎不如salirasib的抗增殖作用显著。事实上，半胱天冬酶在HepG2细胞中的激活比在更敏感的Hep3B细胞中更为明显。此外，在后一种细胞中，在50μM或100μM的salirasib下没有观察到凋亡诱导，尽管这些剂量已经随着时间的推移导致细胞计数急剧下降。

然而，高剂量Salirasib在两种细胞系中诱导了caspase-3/7的激活，这可能至少部分是由线粒体凋亡途径介导的。我们的细胞凋亡可能是由survivin的下调引起的，因为salirasib已被证明可以减少胶质母细胞瘤细胞中survivin的表达，这足以引发细胞凋亡。此外，反义寡核苷酸下调存活素已被证明可以抑制细胞生长并诱导包括HepG2在内的几种细胞系凋亡。然而，在抗凋亡的Huh7细胞中，它也受到抑制，这表明需要额外的事件来触发细胞死亡。我们的研究结果还表明，柳氮可能通过上调HepG2和Hep3B细胞中TRAIL受体DR4和DR5，以及HepG2细胞中Fas表达的增加和Hep3B电池中TNFα的诱导，使细胞对死亡受体诱导的凋亡敏感。然而，单独上调Fas和TRAIL受体可能不足以在体外诱导重大影响，因为它们的配体FasL和TRAIL主要在免疫细胞上表达，而免疫细胞在单一培养物中不存在。然而，通过Salirasib等治疗上调肿瘤细胞上的死亡受体及其与免疫细胞上各自配体的相互作用在体内可能具有重要意义，进一步增强了Salirasib的抗肿瘤作用[3]。

在 Panc-1 异种移植裸鼠中，Salirasib (5 mg/kg ip) 显着抑制肿瘤生长，且无全身毒性。在雄性 Wistar 大鼠中，Salirasib (5 mg/kg ip) 显着抑制硫代乙酰胺诱导的肝硬化。在先天性肌营养不良症 dy(2J)/dy(2J) 小鼠模型中，Salirasib（5 mg/kg ip）可减轻纤维化并提高肌肉力量。

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FTS/Salirasib抑制体内ELT3肿瘤的生长[1]
接下来，我们研究了FTS对裸鼠ELT3肿瘤生长的影响，即对体内模型中细胞转化的影响，如材料和方法所述。在三个不同FTS剂量的独立实验中，小鼠在右侧腹部皮下注射4×106个细胞，如上所述。21,27治疗开始于5天后，三个实验组（每组n=10）的小鼠每天口服FTS（40、60或80 mg/kg），而对照组（n=10）只接受赋形剂（羧甲基纤维素）。在治疗开始54天后杀死小鼠。如图4a所示，从第33天开始，80 mg/kg的FTS显著抑制了肿瘤生长，到第54天，与对照组相比，肿瘤生长抑制了约85%。这种生长抑制是剂量依赖性的（图4b）。在实验结束时（第54天），当肿瘤被切除并称重时，FTS（80mg/kg）使肿瘤重量减轻了65.8±12.5%（n=10，p<0.01；图4a）。值得注意的是，无论是对照组还是接受FTS治疗的小鼠，即使在2个月后也没有死亡。可能需要更详细的校准来检查FTS对小鼠存活的可能影响。

部分肿瘤也被切片并用苏木精和伊红染色（图5A），分别对核酸和细胞质进行染色。我们还对凋亡细胞进行了TUNEL染色（图5B）。病理检查显示，Salirasib治疗组的肿瘤细胞增殖速度比对照组慢得多。图5A显示了两只FTS治疗小鼠肿瘤的典型切片（放大4倍、10倍和40倍）；一个肿瘤相对较大（图5A-d–5A-f），另一个是几乎完全被FTS根除的小肿瘤（图5A-g–5A-i）。还显示了对照小鼠的肿瘤切片（图5A-d–5A-f）。每组另外四个部分得出了类似的结果（未显示）。如图5A所示，FTS治疗（60 mg/kg，40天）导致切片中肿瘤细胞数量显著减少（白色箭头），而与此同时，这些肿瘤中纤维化结缔组织的数量增加（黑色箭头）。每组5只小鼠的染色切片的统计分析表明，FTS治疗组的染色面积百分比明显小于对照组（14.2%±3.6%比25.7%±3.1%；n=5，p<0.05）。值得注意的是，TUNEL染色显示没有明显的细胞死亡，对照组和FTS治疗的小鼠之间也没有差异（n=5；图5B）。这些结果与体外实验的结果一致，表明FTS抑制了ELT3细胞的生长，但没有诱导细胞死亡（图1）。

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Ras表达和Ras-GTP[2]
WT和dy2J/dy2J小鼠用S/SalirasibFTS治疗12周。研究结束时，使用抗Ras抗体对骨骼肌进行免疫印迹。与野生型组相比，未经治疗的dy2J/dy2J小鼠显示出显著更高的Ras表达（555.08±66.32比100±22.45密度，对照百分比；P<0.01；图1A）。FTS治疗与dy2J/dy2J小鼠Ras表达显著降低有关（205.76±19.37；P<0.01）。用FTS治疗WT小鼠导致Ras表达增加，远低于dy2J/dy2J小鼠中观察到的增加（206.76±29.37）。除了Ras表达外，在研究结束时通过Ras结合结构域下拉分析测量Ras活性。在该测定中，GTP结合的Ras通过其优先结合Raf1的RBD结构域来检测，Raf1与琼脂糖珠结合[26]。我们发现dy2J/dy2J小鼠的Ras-GTP水平高于WT小鼠（157.40±14.53 vs.100±10.23；P<0.05。图1B）。FTS治疗的dy2J/dy2J组Ras-GTP水平显著降低（97.16±10.54；P<0.01）。此外，治疗组dy2J/dy2J中的Ras-GTP恢复正常，与WT组相当。用FTS治疗WT小鼠诱导Ras-GTP增加（141.72±12.4），与这些小鼠Ras表达的增加相当。在WT小鼠中观察到的这些增加的性质尚不清楚。
此外，我们测量了Ras下游蛋白ERK的磷酸化（图1C）。与WT组相比，dy2J/dy2J小鼠的ERK磷酸化非常高（424.97±63.85对100±21.56；P<0.02），而Salirasib/FTS治疗显著降低了这种磷酸化（175.62±21.53；P<0.002）。WT FTS中pERK略有增加（169.29±31.1），而总ERK根本没有变化。

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肌肉力量[2]
使用电子握力计每周测定一次总峰值力。在整个研究过程中，未经治疗的dy2J/dy2J和WT小鼠的后肢肌力存在显著差异（P<0.01；图2）。与未治疗的dy2J/dy2J小鼠相比，经Salirasib/FTS治疗的小鼠后肢肌肉力量显著增加（P<0.05）。在研究期间，接受治疗的dy2J/dy2J小鼠的肌肉力量从3.01±0.27增加到4.79±0.22（克力/克体重），而未接受治疗的dy2J/dy2U组的肌肉力量则从2.67±0.22保持不变，为2.72±0.19。此外，在试验结束时，dy2J/dy2J治疗的小鼠后肢肌肉力量完全恢复到两个WT组的水平（4.79±0.22 vs.WT:4.64±0.39；WT+FTS:4.77±0.25）。在dy2J/dy2J小鼠的前肢（更强壮）（图S1）以及治疗和未治疗WT组的前肢和后肢中没有检测到这种差异。

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肌肉组织学[2]
与正常WT相比（图3A），18周龄未经治疗的dy2J/dy2J小鼠股四头肌的苏木精和伊红染色显示严重的晚期营养不良变化，纤维大小、内核异常变化，严重纤维化过度（图3B）。Salirasib/FTS治疗的dy2J/dy2J肌肉显示出相当大的纤维化衰减（见下一段），但仍然存在异常的肌病性变化，纤维大小不同，中心核数量增加（图3C）。

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Salirasib在皮下异种移植物模型中抑制肿瘤生长[3]
最后，我们在裸鼠HepG2细胞的皮下异种移植物模型中评估了salirasib的体内抗肿瘤活性。从治疗的第5天开始，salirasib诱导肿瘤体积在统计学上显著减少（图8A）。治疗12天后，平均肿瘤重量为131.7±18.9 mg，而对照组（赋形剂）为297.5±48.2 mg，表明salirasib使肿瘤生长减少了56%（图8B）。此外，在对照组和治疗组之间没有观察到肿瘤重量的重叠，这意味着即使是对照组中最小的肿瘤也比治疗组中最大的肿瘤大（图8C）。在整个实验过程中，动物保持良好状态，治疗后没有观察到体重减轻，这表明该剂量方案对salirasib具有良好的耐受性（数据未显示）。

对于无细胞系统中的甲基转移酶测定，使用培养细胞系的总膜（100,000 × g 颗粒）或大鼠脑小脑的突触体膜。 50 μL 50 mM Tris-HCl 缓冲液（pH 7.4）、100 μg 蛋白质、25 μM [methyl-3H]AdoMet (300,000 cpm/nmol) 和 50 μM AFC（在 DMSO 中制备为储备溶液）用于甲基转移酶化验。测定在 37°C 下进行。所有测定均使用 8% DMSO。如文中所述，许多实验中使用了不同的 AFC 浓度。 10 分钟后，加入 500 μL 1:1 氯仿:甲醇混合物，然后混合并相分离，然后加入 250 μL H2O，终止反应。将 200 μl 1 N NaOH 和 1% SDS 溶液添加到已在 40°C 干燥的 125 μL 氯仿相中。使用气相平衡法来计算由此形成的甲醇。添加 AFC 后，典型的背景计数（无 AFC）范围为 50 至 100 cpm，而典型的反应产量为 500 至 6,000 cpm。每个测定进行三份重复，并扣除背景计数。进行凝胶电泳和内源底物的甲基化。在完整细胞中，使用 100 μCi/mL [甲基-3H]蛋氨酸测量蛋白质羧基甲基化。分析前，在 10 厘米平板中用 5 mL 标记培养基培养接近汇合的细胞培养物。

在时间依赖性反应研究中，每天使用 0.05% 胰蛋白酶-EDTA 收获细胞，持续 1 至 7 天。然后在显微镜下使用台盼蓝排除法对细胞进行计数。将细胞在补充有 Salirasib或 DMSO 的培养基中培养三天，以进行剂量反应研究。按照制造商的指示，使用比色 WST-1 测定来评估细胞活力。使用 GraphPad Prism 软件，使用非线性回归分析来确定 IC50 值，或与 DMSO 对照相比，50% 的细胞生长被抑制的点。

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荧光激活细胞分选仪分析[1]
对于细胞死亡评估，以每10 cm板2×105个细胞的密度接种细胞，用Salirasib/FTS处理72小时，然后根据制造商的说明收集细胞并用膜联蛋白-V/碘化丙啶（PI）双重染色进行检测。用CBA软件分析FACSCalibur流式细胞术获得的结果。

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克隆形成试验[1]
表达ETL3和TSC2的ELT3细胞以每24孔板2.5×104个细胞的密度铺板，生长24小时，然后用Salirasib/FTS或0.1%Me2SO4处理（对照）。3天后，将细胞分离并铺在10cm板上（1:50稀释）。四天后，用结晶紫对细胞进行染色，并对菌落进行计数。

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免疫印迹分析[1]
ETL3细胞以每10 cm板4×105个细胞的密度铺板，生长24小时，然后用Salirasib/FTS或0.1%Me2SO4（对照）处理。如上所述裂解细胞，14并用小鼠抗泛Ras抗体、兔抗Rheb抗体、兔抗pS6K抗体、抗Flag抗体、家兔抗S6K抗体、小鼠抗pERK抗体、兔抗ERK抗体和兔抗β-微管蛋白抗体对裂解物（100μg蛋白质）进行免疫印迹。免疫印迹暴露于适当的次级过氧化物酶偶联IgG并进行增强化学发光。使用图像EZQuant凝胶统计分析软件通过密度测定法对蛋白质条带进行定量。

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总RNA纯化和实时PCR分析[1]
使用RNeasy Plus迷你试剂盒从未处理或75μM Salirasib处理的ELT3细胞中分离总RNA。如前所述，纯化的RNA用于实时PCR。16使用以下引物靶向Rheb基因：正向5′-GCTTATCGGTGTGGGAA-3′，反向5′-CTGCCTGGTGTCTACA-3′，以及看家基因GAPDH：正向5’-CCAGAACATCATC CCTGC-3′，逆向5′-GGAAGGGCCAATGCCAGTAGGAGC-3′。靶基因的mRNA表达被标准化为GAPDH参考基因的表达。

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Rheb的测量。GTP[1]
实验按照其他描述进行。17简而言之，ETL3细胞以每10 cm板4×105个细胞的速度铺板，生长24小时，然后用Salirasib/FTS或0.1%Me2SO4（对照）处理。2天后，将细胞饥饿过夜，然后换成无磷酸盐培养基30分钟，然后用0.25 mCi/ml[32Pi]正磷酸盐标记4小时。收获细胞的裂解物用蛋白-G琼脂糖珠和sc-6341抗Rheb抗体免疫沉淀1小时。核苷酸在65°C下洗脱，并进行薄层色谱（TLC）。GTP和GDP被用作标记，并用碘进行检测。通过在-80°C下用增感屏暴露于X射线胶片6天来检测信号。GDP和GTP通过Image EZ Quant Gel统计分析软件进行密度定量。Rheb结合的GTP百分比计算为[GTP/（GDP×1.5）+GTP]×100%。

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生长抑制研究[3]
对于时间依赖性反应研究，每天用0.05%胰蛋白酶-EDTA收获细胞1至7天，并在显微镜下使用台盼蓝排除法进行计数。 对于剂量反应研究，细胞在添加了Salirasib或DMSO的培养基中孵育3天。根据制造商的说明，使用比色WST-1测定法测定细胞存活率。

Female athymic NMRI nu/nu mice, aged six weeks, are kept in cages with filters on top and are given unlimited access to food and drink. Twelve mice receive a subcutaneous injection of 5x10⁶ HepG2 cells suspended in 100 μL PBS to create tumors in their right lower flank. When palpable tumors have developed two weeks after cell inoculation, mice are divided into two groups: one for Salirasib treatment (n = 6) and the other for control (n = 4). Two of the animals had to be removed from the study because they do not develop tumors at that point. For 12 days, they are given daily intraperitoneal injections (10 mg/kg salirasib) or an equivalent volume of vehicle solution (PBS with 2.5% v/v ethanol, pH 8.0). A digital calliper is used to record tumor dimensions three times a week beginning on the first day of treatment. To estimate tumor volumes, use the formula V (mm³)=(length×width²)/2. To assess the effectiveness of treatment, tumour weights are noted at the moment of sacrifice.

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Athymic nude mice (6 weeks old) were housed in barrier facilities on a 12-hr light/dark cycle with unlimited food and water. Mice in the experimental group received orally administered Salirasib/FTS (0.1 ml) daily. Subcutaneous tumors were measured with a caliper, and animal weights were recorded every 4 days. Tumor volumes were calculated using the formula: [length × width] × [(length + width)/2]. [1]

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Following previous protocols in the rat model of liver cirrhosis, WT and dy2J/dy2J mice were injected intra-peritoneally 3 times a week with Salirasib/FTS 5 mg/kg or control solution (see below), for 12 weeks from the age of 6 weeks (n = 7/group, each group consisted of 4 males and 3 female mice). At the end of the study both hind limb muscles were dissected. Part of the muscle sample was frozen in liquid nitrogen and stored at −80°C for biochemical analysis. Quadriceps femoris muscle was rapidly frozen in isopentane pre-chilled by liquid nitrogen for cryostat sections and histology. [2]

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Six week old female athymic NMRI nu/nu mice were housed in filter-topped cages and received food and water ad libitum. Tumors were generated by subcutaneous injection into the right lower flank with 5 × 106 HepG2 cells suspended in 100 μl PBS in 12 mice. Two weeks after cell inoculation, when palpable tumours were established, mice were separated into Salirasib-treated (n = 6) and control group (n = 4). Two animals did not develop tumours at that time point and had to be excluded from the study. They received daily i.p. injections of 10 mg/kg Salirasib or a similar volume of vehicle solution (PBS containing 2.5% v/v ethanol, pH 8.0) for 12 days. Tumor dimensions were recorded three times per week with a digital calliper starting with the first day of treatment. Tumor volumes were estimated as follows: V (mm³) = (length × width²)/2. Tumour weights were recorded at the time of sacrifice in order to evaluate treatment response. [3]

Salirasib plasma pharmacokinetics [4]
Pharmacokinetic data were obtained and evaluable from 24 patients, involving 41 pharmacokinetic study periods (Table 4). The salirasib pharmacokinetic profile was characterized by slow absorption and a rapid elimination phase following oral administration (Fig. 1). Accumulation did not occur, as salirasib exposure (C max; day 1 AUCinf vs. day 15 AUC0–12 h) was similar between days 1 and 15 (P > 0.05). The T 1/2 (mean ± SD) was 3.6 ± 2.2 h on day 1. Salirasib had a ClS/F (mean ± SD) of 103.0 ± 112.5 and 73.3 ± 37.3 L/h on days 1 and 15, respectively, and showed extensive distribution in excess of blood volume with a V/F of 458.0 ± 466.1 and 255.7 ± 187.8 L on days 1 and 15, respectively (Table 4).
Despite the variability in the majority of the pharmacokinetic parameters, salirasib drug exposure (C max and AUC) and ClS/F were similar on days 1 and 15 (P > 0.05) according to matched pairs analysis. The lack of accumulation in C max or AUC is consistent with a dosing interval approximately four times longer (i.e., 12 h) than the T 1/2 (i.e., 3.6 h). C min concentrations appeared to reach a plateau at 8 days, whereas C 1 h values were consistent on days 1, 8, and 15. The systemic drug exposure (C max and AUC) increased in a manner proportionate with the increase in salirasib dose from 100 to 800 mg, as demonstrated by there being no significant difference between dose-normalized parameters by dose group (P > 0.05).

Toxicity [4]
To date, a total of 103 cycles of salirasib therapy have been administered. The median number of cycles per patient was 2 (range 1–13 cycles). All patients were treated at their initial dose level, except for one patient whose dose was escalated from 100 to 200 mg in the second cycle (total cycles, 2). The drug-related adverse events by dose are listed in Table 2. Only Grade 1–2 drug-related toxicities occurred. The most common toxic effect was diarrhea, which occurred in 79% of the patients (Grade 1 in 75% and Grade 2 in 4%). The duration of diarrhea increased with the salirasib dose. The frequency of diarrhea also was higher in patients treated with higher salirasib doses: 67% (2 of 3 patients), 67% (4 of 6), 100% (6 of 6), 67% (4 of 6), and 100% (3 of 3) in patients treated with 100, 200, 400, 600, and 800 mg of salirasib twice daily, respectively. Diarrhea did not result in abnormalities in clinical chemistry parameters, and it was usually reversible with oral antidiarrheal agents such as loperamide or diphenoxylate hydrochloride. Patients were instructed to use these medications if diarrhea occurred. Nineteen (79%) patients had no toxicities greater that Grade 1. Other toxicities included abdominal pain in 21% of patients and nausea, vomiting, and fatigue in 17% each. No classic DLT was reached at 800 mg twice daily, the highest dose tested. However, all three patients treated at this dose developed Grade 1–2 prolonged diarrhea, and it was decided that further dose escalation would not be tolerable. At 600 mg p.o. twice daily, no patients experienced a DLT or prolonged diarrhea, and therefore, it was felt that this dose would be optimal for the phase II study.

[1]. Farnesylthiosalicylic acid (salirasib) inhibits Rheb in TSC2-null ELT3 cells: a potential treatment for lymphangioleiomyomatosis. Int J Cancer. 2012 Mar 15;130(6):1420-9.

[2]. The Ras antagonist, farnesylthiosalicylic acid (FTS), decreases fibrosis and improves muscle strength in dy/dy mouse model of muscular dystrophy. PLoS One. 2011 Mar 22;6(3):e18049.

[3]. Salirasib inhibits the growth of hepatocarcinoma cell lines in vitro and tumor growth in vivo through ras and mTOR inhibition. Mol Cancer. 2010 Sep 22;9:256.

[4]. Phase 1 first-in-human clinical study of S-trans,trans-farnesylthiosalicylic acid (salirasib) in patients with solid tumors. Chemother Pharmacol. 2010 Jan;65(2):235-41.

Salirasib is a sesquiterpenoid.
Salirasib has been used in trials studying the diagnostic of Carcinoma, Non-Small-Cell Lung.
Salirasib is a salicylic acid derivative with potential antineoplastic activity. Salirasib dislodges all Ras isoforms from their membrane-anchoring sites, thereby preventing activation of RAS signaling cascades that mediated cell proliferation, differentiation, and senescence. RAS signaling is believed to be abnormally activated in one-third of human cancers, including cancers of the pancreas, colon, lung and breast.

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The small GTPase proteins, Ras and Rheb, serve as molecular switches regulating cell proliferation, differentiation and apoptosis. Ras also regulates Rheb by inactivating the tuberous sclerosis complex (TSC), which includes products of the TSC1 and TSC2 genes encoding hamartin (TSC1) and tuberin (TSC2), respectively, and acts as a Rheb-specific GTPase-activating protein. Loss of function of TSC1 or TSC2 results in an increase in active Rheb.GTP with the consequent translational abnormalities and excessive cell proliferation characteristic of the genetic disorders, tuberous sclerosis and lymphangioleiomyomatosis (LAM). To determine whether inactivation of Rheb, Ras or both might be a potential treatment for LAM, we used TSC2-null ELT3 cells as a LAM model. The cells were treated with the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS; salirasib), which mimics the C-terminal S-farnesyl cysteine common to Ras and Rheb. This C-terminus is critical for their attachment to cellular membranes and for their biological activities. Untreated, the ELT3 cells expressed significant amounts of Rheb but little Ras.GTP, and this phenotype was reversed by TSC2 reexpression. Treatment with FTS decreased Ras.GTP only slightly in the TSC2-null cells, but reduced their overactive Rheb as well as their proliferation, migration and tumor growth. Notably, TSC2 reexpression in these ELT3 cells rescued them from the inhibitory effect of FTS. Evidently, therefore, FTS blocks active Rheb in TSC2-null ELT3 cells and may have therapeutic potential for LAM.[1]

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The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy(2J)/dy(2J) mouse model of merosin deficient congenital muscular dystrophy. The dy(2J)/dy(2J) mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy(2J)/dy(2J) mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy(2J)/dy(2J) mouse model of congenital muscular dystrophy. [2]

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Background: Dysregulation of epidermal growth factor and insulin-like growth factor signaling play important roles in human hepatocellular carcinoma (HCC), leading to frequent activation of their downstream targets, the ras/raf/extracellular signal-regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) pathways. Salirasib is an S-prenyl-cysteine analog that has been shown to block ras and/or mTOR activation in several non hepatic tumor cell lines. We investigated in vitro the effect of salirasib on cell growth as well as its mechanism of action in human hepatoma cell lines (HepG2, Huh7, and Hep3B) and its in vivo effect in a subcutaneous xenograft model with HepG2 cells.
Results: Salirasib induced a time and dose dependent growth inhibition in hepatocarcinoma cells through inhibition of proliferation and partially through induction of apoptosis. A 50 percent reduction in cell growth was obtained in all three cell lines at a dose of 150 μM when they were cultured with serum. By contrast, salirasib was more potent at reducing cell growth after stimulation with EGF or IGF2 under serum-free conditions, with an IC50 ranging from 60 μM to 85 μM. The drug-induced anti-proliferative effect was associated with downregulation of cyclin A and to a lesser extent of cyclin D1, and upregulation of p21 and p27. Apoptosis induction was related to a global pro-apoptotic balance with caspase 3 activation, cytochrome c release, death receptor upregulation, and a reduced mRNA expression of the apoptosis inhibitors cFLIP and survivin. These effects were associated with ras downregulation and mTOR inhibition, without reduction of ERK and Akt activation. In vivo, salirasib reduced tumour growth from day 5 onwards. After 12 days of treatment, mean tumor weight was diminished by 56 percent in the treated animals.
Conclusions: Our results show for the first time that salirasib inhibits the growth of human hepatoma cell lines through inhibition of proliferation and induction of apoptosis, which is associated with ras and mTOR inhibition. The therapeutic potential of salirasib in human HCC was further confirmed in a subcutaneous xenograft model. [3]

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Purpose: This phase I first-in-human trial evaluated salirasib, an S-prenyl derivative of thiosalicylic acid that competitively blocks RAS signaling.
Methods: Patients with advanced cancers received salirasib twice daily for 21 days every 4 weeks. Doses were escalated from 100 to 200, 400, 600, and 800 mg.
Results: The most common toxicity was dose-related diarrhea (Grade 1-2, 79% of 24 patients). Other toxicities included abdominal pain, nausea, and vomiting. No Grade 3-4 toxicity was noted. Nineteen (79%) patients had no drug-related toxicity >Grade 1. Dose-limiting toxicity (DLT) was not reached, but all three patients treated with 800 mg experienced Grade 1-2 diarrhea, brogating dose escalation. Six patients were treated at a dose of 600 mg with no DLTs. Seven (29%) patients had stable disease on salirasib for ≥4 months (range 4-23+). The salirasib pharmacokinetic profile was characterized by slow absorption and a rapid elimination phase following oral administration. Salirasib exposure (C(max); day 1 AUC(inf) vs. day 15 AUC(0-12 h)) was similar between days 1 and 15 (P > 0.05). The T(1/2) (mean ± SD) was 3.6 ± 2.2 h on day 1.
Conclusions: Salirasib therapy was well tolerated. The recommended dose for phase II studies is 600 mg twice daily.[4]

C22H30O2S

358.54

358.196

C, 73.70; H, 8.43; O, 8.92; S, 8.94

162520-00-5

5469318

White to light yellow solid powder

1.1±0.1 g/cm3

486.0±45.0 °C at 760 mmHg

64-66°C

247.7±28.7 °C

0.0±1.3 mmHg at 25°C

1.559

8.53

62.6

1

3

10

25

498

0

S(C1=C([H])C([H])=C([H])C([H])=C1C(=O)O[H])C([H])([H])/C(/[H])=C(\C([H])([H])[H])/C([H])([H])C([H])([H])/C(/[H])=C(\C([H])([H])[H])/C([H])([H])C([H])([H])/C(/[H])=C(\C([H])([H])[H])/C([H])([H])[H]

WUILNKCFCLNXOK-CFBAGHHKSA-N

InChI=1S/C22H30O2S/c1-17(2)9-7-10-18(3)11-8-12-19(4)15-16-25-21-14-6-5-13-20(21)22(23)24/h5-6,9,11,13-15H,7-8,10,12,16H2,1-4H3,(H,23,24)/b18-11+,19-15+

2-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl]sulfanylbenzoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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: ≥ 2.5 mg/mL (6.97 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 25.0 mg/mL澄清DMSO储备液加入到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: 2.5 mg/mL (6.97 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 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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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.97 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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。