CN112055586A - 苯磺酰胺衍生物及调节脂膜筏的方法 - Google Patents
苯磺酰胺衍生物及调节脂膜筏的方法 Download PDFInfo
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Abstract
本公开涉及可用于调节癌细胞的脂膜筏完整性的苯磺酰胺衍生物和苯磺酰胺衍生物在预防或治疗可通过改变受试者的脂膜筏完整性而有所改善的疾病或病症中的用途。
Description
技术领域
本公开涉及一种调节脂膜筏完整性的方法，尤其涉及一种通过苯磺酰胺衍生物调节脂膜筏完整性的方法。本公开也涉及一种通过将苯磺酰胺衍生物给予有需要的受试者，以预防或治疗癌症的方法。
背景技术
前列腺癌已被公认为男性最重要的医疗难题之一。当晚期前列腺癌进展且尽管用雄激素剥夺疗法进行医学治疗时出现转移时，其最终会发展为去势抵抗性前列腺癌(castration-resistant prostate cancer，CRPC)。由于患者目前几乎没有治疗选择，因此在CRPC治疗中需要新的治疗剂。鉴于前列腺癌细胞能够适应许多细胞应激(cellularstress)，因此极度需要针对不同于对抗多种适应性机制的前列腺癌的有前景的治疗，以提供替代治疗的选择[1]。
雄激素剥夺疗法是晚期转移性前列腺癌的主要治疗方法；然而，其最终将会进展到CRPC。已经确定数种导致CRPC发生的机制，包括雄激素受体基因的扩增或点突变、雄激素受体和生长因子之间的相互作用，以及补偿性存活信号途径的活化[2-4]。在调节细胞存活和肿瘤转化中扮演关键角色的磷酸肌醇3-激酶(phosphoinositide 3-kinase，PI3K)/Akt信号传导途径，在大多数CRPC中被组成性地活化。在CRPC中，PI3K/Akt的活化最常见于补偿性存活信号途径的类别中[5-7]。肿瘤抑制因子PTEN(染色体10上所缺失的磷酸酶和张力蛋白同源物)是PI3K/Akt活性的负调节因子，其在50％至80％的前列腺腺癌(prostateadenocarcinoma)患者中发生突变或缺失[2]。此外，PTEN能力的下降和PI3K/Akt活性的增加与高Gleason评分和晚期病理阶段疾病密切相关[8]。此外，PTEN表达的缺失与前列腺癌治疗的较差存活率和较短时间相关，例如阿比特龙(abitaterone)治疗[87]。此外，Akt可通过转译后修饰来负调节转录因子FOXO3A(肿瘤抑制因子)，并造成细胞质积累量增加，同时导致DNA结合降低，最终诱导细胞存活。已注意到在前列腺肿瘤标本中，检测到FOXO3A的深层细胞质积累并伴随Gleason评分增加[9]。总之，这些研究显示PI3K/Akt在CRPC进展中的关键角色。
胆固醇是维持细胞膜完整性和流动性的重要结构成分，对于激素、维生素D和胆汁酸的合成以及多细胞信号传导的调节至关重要[10、11]。脂膜筏是膜微域，优先与胆固醇、饱和脂质和激酶结合，以调节许多细胞信号途径[12、13]。大量研究证实，脂膜筏中胆固醇的减少或消耗能够破坏PI3K/Akt信号传导[14-16]，这显示膜胆固醇含量和脂膜筏完整性的重要性。例如，据报告乳腺癌和前列腺癌细胞的脂膜筏数量相当丰富，导致其对于通过耗竭胆固醇所引起的促凋亡刺激具更高的敏感性[17]。此外，小窝蛋白(caveolin)、阀蛋白(flotillin)、神经节苷脂(GM1)和糖基磷脂肌醇(glycosylphosphatidylinositol，GPI)-锚定物的胎盘碱性磷酸酶(placental alkaline phosphatase，PLAP)被视为脂膜筏标记物[57-60]，先前研究显示在一些诸如前列腺癌[57、61-63]、肺癌[57、64-69]、胰腺癌[70、71]、黑色素瘤[57、69、72-74]、肾癌[57、63、65、69、75]、膀胱癌[76]、精原细胞瘤[60]、卵巢癌[60]、子宫颈癌[60]、乳癌[63、69]、结肠癌[63、77]、肝癌[32、58]、食道癌[62、63、78、79]、口腔癌[80]、舌癌[62]、甲状腺癌[62]、脑膜瘤[86]、胆管癌[70]、下咽癌[81]、鼻咽癌[64、82]、胃癌[83、84]、或外阴癌[85]的癌症中，会增加这些脂膜筏标记物的表达。
在体外和体内研究中，对甲苯磺酰胺(para-toluenesulfonamide，p-TSA)是一种可对抗数种癌症的小分子，包括肝细胞癌、非小细胞肺癌和舌鳞状细胞癌[18-21]。此外，通过局部注射治疗，其在临床试验中展现对抗晚期肝细胞癌和非小细胞肺癌的有效抗肿瘤活性[20、21]。
本文提供一种意料之外的脂膜筏调节剂，p-TSA及其衍生物，以及这些化合物在治疗易于通过干扰或破坏脂膜筏完整性，尤其是通过调节脂膜筏中胆固醇含量而改善的疾病的用途。
发明内容
本公开至少部分地基于用于调节脂膜筏完整性的医药组合物的发现，包括苯磺酰胺衍生物及其药学上可接受的赋形剂。
本公开也提供一种用于预防或治疗癌症的方法，包括将医药组合物给药至有需要的受试者。
本公开证实在CRPC细胞中，脂膜筏和胆固醇的含量在数种存活激酶的经p-TSA介导的再分布和活性中的角色。首次显示胆固醇含量的扰动以及与脂膜筏相关的Akt/mTOR/p70S6K途径的改变导致经p-TSA诱导的抗CRPC作用。
附图说明
通过参考附图并阅读以下实施方案的详细描述，可以更全面地理解本公开，其中：
图1A至1D显示p-TSA对人类去势抵抗性前列腺癌细胞和初代前列腺细胞的抗增殖作用的影响。将细胞在缺乏或存在有p-TSA的情况下培养指定的时间。处理后，将细胞固定并染色，以用于SRB分析(图1A)和群落形成分析(图1B)。将添加或不添加有p-TSA的PC-3细胞和DU-145细胞进行培养。处理后，收集细胞以用于CFSE染色的流式细胞术分析(图1C和1D)。通过Modfit LT 3.2版本和WinList 5.0版本的软体计算母代或不同世代的增殖指数和细胞群。定量数据以三至四次独立实验的平均值±SEM表示。相较于对照组，***P<0.001。
图2A至2D显示p-TSA对细胞周期停滞和粒线体的功能障碍的影响。将PC-3和DU-145细胞在缺乏或存在有p-TSA的情况下培育24小时。收集细胞以用于碘化丙啶染色以分析在细胞周期阶段中细胞群的分布(图2A)，或用于JC-1染色以使用FACScan流式细胞术分析检测粒线体膜电位(图2B)。绿色荧光显示用于粒线体膜电位的定量(图2B)。将细胞收集并裂解，并通过西方墨点法分析检测数种Bcl-2家族成员的蛋白质表达(图2C和2D)。数据以三至四次所测定的平均值±SEM表示。相较于对照组，*P<0.05、**P<0.01和***P<0.001。
图2E和2F显示Z-VAD-FMK对经PTS诱导的PC-3细胞凋亡的影响。在缺乏或存在有指定试剂的情况下，将细胞培养24小时。收集细胞以用于核小体DNA片段分析的PI染色(图2F)的流式细胞术分析(图2E)。定量数据以三次独立实验的平均值±SD表示。相较于对照组，**P<0.01和***P<0.001。
图3A至3C显示p-TSA对细胞周期调节因子和激酶表达的影响。将细胞在缺乏或存在有6mM p-TSA的情况下培养指定的时间。处理后，将细胞收集并裂解，并通过西方墨点法分析检测细胞周期调节因子的蛋白质表达(图3A)和Akt/mTOR/p70S6K途径信号(图3B)。利用Image Lab Software 6.0(BIO-RAD)对表达进行定量(图3C)。数据以三次测定的平均值±SEM表示。相较于对照组，*P<0.05、**P<0.01和***P<0.001。
图4A至4D显示Akt的存在下，对经PTS诱导后数种蛋白质的改变及影响。利用Myr-Akt质粒转染PC-3细胞。然后，将细胞在缺乏或存在有p-TSA的情况下培养3小时(图4A)或24小时(图4B)。将细胞收集并裂解，并通过西方墨点法分析检测所指定的蛋白质。蛋白质表达利用Image Lab软体6.0(BIO-RAD)进行定量(图4C和图4D)。数据以三次独立实验的平均值±SEM表示。
图4E和4F显示PTS在PC-3细胞中的抗增殖作用。在指定条件的存在下进行细胞培养。将细胞用PTS处理48小时或10天或不处理，并分别进行SRB(图4E)和群落形成分析(图4F)。处理后，将细胞固定并进行染色，以用于分析。定量数据以三次独立实验的平均值±SD表示。相较于个别的对照组，*P<0.05和**P<0.01。
图5A至5C显示p-TSA对于数种激酶的脂膜筏相关表达的影响。在图5A中，在缺乏或存在有6mM p-TSA的情况下，培养PC-3和DU-145细胞2小时。处理后，将细胞于1％Triton X-100中进行裂解，并如实施例4所述，通过离心分级。通过西方墨点法检测蛋白质的表达。在图5B和5C中，蛋白质表达利用Image Lab软体6.0(BIO-RAD)进行定量。
图6A至6E显示胆固醇的补充对经p-TSA介导的作用的影响。将PC-3和DU-145细胞在缺乏或存在有指定药剂下，培养1小时。处理后，将细胞收集并裂解，并通过西方墨点法分析检测蛋白质的表达(图6A和6C)。蛋白质表达利用Image Lab软件6.0(BIO-RAD)进行定量(图6B和6D)。将PC-3细胞在指定的试剂(PTS，1.5mM)的存在下培养10天。处理后，将细胞固定并染色，以进行群落形成测定(图6E)。数据以三次测定的平均值±SEM表示。相较于单独的p-TSA，*P<0.05、**P<0.01和***P<0.001。
图7A至7D显示p-TSA在体内抗肿瘤异种移植模型中的作用。将PC-3细胞以皮下注射的方式注射至裸鼠皮下(107个细胞/小鼠)。每天测量肿瘤。当肿瘤达到体积为100mm3时，将小鼠分成两组且开始进行腹腔内注射p-TSA。测量肿瘤的长度(l)和宽度(w)，并以lw2/2计算肿瘤的体积(图7A和7B)，也测量体重(图7C)。体内研究的方案由台湾大学的实验动物照护及使用委员会批准。所有的动物程序和方案皆经国际实验动物管理评鉴及认证协会(AAALAC)认可的机构批准。已检测在对照组和PTS组中随机筛选的六个肿瘤的p-Akt表达(图7D)。数据以平均值±SD表示。
具体实施方式
以下实施例用于举例说明本公开，所属技术领域技术人员能够基于本公开的说明书而思及本公开的其他优点。本公开亦可以所述的不同实施例来实现或应用。对于不同的方面和应用，可能在不违背本公开的精神和范围下，修改和/或改变用于实施本公开的上述实施例。
应进一步注意，如本说明书中所使用的单数形式“一(a)”、“一(an)”和“该(the)”除非特意地且明确地局限于一个指示物，包括复数指示物。除非上下文另有明确说明，否则术语“或”可与术语“和/或”互换使用。
本公开提供包含苯磺酰胺衍生物和其药学上可接受的赋形剂的医药组合物。
在本公开的一个实施方案中，苯磺酰胺衍生物由式(I)表示：
或其药学上可接受的盐，
其中，R1至R7独立地选自由下列所组成的组：H、C1至C6直链或支链烷基、C1至C6直链或支链烷氧基、C3至C6环烷基、C3至C6环杂烷基、胺基和卤素基团，或R6和R7相互连接以形成一个环。
在本公开的一个实施方案中，R1至R7中的烷基、烷氧基、环烷基、环杂烷基和环独立地未经取代或经一个或多个取代基取代。在本公开的一个实施方案中，取代基选自苯基、卤素、氧代、醚、羟基、羧基、胺基、磺基和磺酰胺基。
在本公开的一个实施方案中，苯磺酰胺衍生物包括，但不限于，对-甲苯磺酰胺、邻-甲苯磺酰胺、间-甲苯磺酰胺、N-乙基-对-甲苯磺酰胺、N-乙基-邻-甲苯磺酰胺、N-环己基-对-甲苯磺酰胺、
在本公开的一个实施方案中，苯磺酰胺衍生物是对-甲苯磺酰胺(p-TSA)。
在本公开的一个实施方案中，药学上可接受的赋形剂可为填充剂、粘合剂、防腐剂、崩解剂、润滑剂、悬浮剂、润湿剂、溶剂、表面活性剂、酸、调味剂、聚乙二醇(PEG)、烷基乙二醇、癸二酸、二甲基亚砜、醇或其组合。
在本公开的一个实施方案中，以重量计，苯磺酰胺衍生物的含量范围为约5％至约60％。
在本公开的一个实施方案中，医药组合物为适合用于肠胃外给药、注射、连续输注、舌下给药、皮下给药或口服给药的形式。
在本公开的一个实施方案中，医药组合物的形式包括，但不限于，注射制剂、干粉、片剂、口服液、薄片、薄膜、锭剂、胶囊、颗粒或丸剂。
本公开亦提供一种用于调节细胞的脂膜筏完整性的方法，包括将医药组合物给药至需要治疗的受试者，以调节脂膜筏完整性。
在本公开的一个实施方案中，医药组合物会干扰或破坏脂膜筏的完整性。在本公开的一个实施方案中，医药组合物会耗竭细胞膜中的胆固醇。
在本公开的一个实施方案中，该方法可用于通过降低脂膜筏完整性的水平(level)来治疗易于改善的疾病。在本公开的另一个实施方案中，所述疾病是癌症。
本公开亦提供一种用于预防或治疗癌症的方法，包括将医药组合物给药至有需要的受试者。
在本公开的一个实施方案中，医药组合物中的苯磺酰胺衍生物以每天约20mg至约4000mg的治疗有效量给药至受试者。
在本公开的一个实施方案中，医药组合物通过肿瘤内、静脉内、皮下、皮内、口服、鞘内、腹腔内、鼻内、肌肉内、胸膜内或通过雾化给药至受试者。
在本公开的一个实施方案中，苯磺酰胺衍生物作为用于抑制肿瘤生长的脂膜筏调节剂。在本公开的另一个实施方案中，在肿瘤细胞中的脂膜筏完整性的水平被降低。在本公开的另一个实施方案中，所有形式或磷酸化形式的脂膜筏相关激酶的水平在肿瘤细胞中被降低。在本公开的另一个实施方案中，癌症选自下列所组成组的至少一种：肺癌(包括，但不限于，非小细胞肺癌、肺鳞状细胞癌、小细胞肺癌、肺腺癌)、乳腺癌、肝癌(包括但不限于，肝细胞癌)、前列腺癌、皮肤癌、胰腺癌(包括但不限于，胰脏腺癌(pancreaticadenocarcinoma))、黑色素瘤、肾癌(包括，但不限于，肾细胞癌)、膀胱癌(包括但不限于，尿路上皮癌)、精原细胞瘤、卵巢癌、子宫颈癌、大肠癌、食道癌(包括但不限于，食道鳞状细胞癌)、口腔癌(包括但不限于，口腔鳞状细胞癌)、舌癌(包括但不限于，舌鳞状细胞癌)、甲状腺癌、脑膜瘤、胆管癌、下咽癌(包括但不限于，下咽鳞状细胞癌)、鼻咽癌、胃癌和外阴癌(包括但不限于，外阴的鳞状细胞癌)。
在本公开的一个实施方案中，苯磺酰胺衍生物作为抑制肿瘤生长的胆固醇消耗剂。在本公开的另一个实施方案中，肿瘤细胞脂膜筏中胆固醇的水平被降低。在本公开的另一个实施方案中，癌症选自下列所组成组的至少一种：肺癌、乳腺癌、肝癌、皮肤癌、前列腺癌、骨髓性白血病和口腔癌。
在本公开的一个实施方案中，苯磺酰胺衍生物破坏脂膜筏/胞膜窖(caveolae)平台结构的完整性，以抑制特定的信号传导途径，包括，但不限于Akt的去活化。
在本公开的一个实施方案中，该方法还包括向受试者给药至少一种额外的抗癌剂。
在本公开的一个实施方案中，该方法还包括向受试者施用至少一种额外的抗癌疗法，其中所述的抗癌疗法包括，但不限于，全身化学疗法、放射疗法或热疗法。
以下是进一步证明本公开的功效的具体实施方式，但不限制本公开的范围。
[实施例]
去势抵抗性前列腺癌(CRPC)细胞可抵抗许多细胞应激以确保存活。存在未满足的医疗需求来对抗细胞中的多种适应性机制，以实现患者的最佳治疗。根据以下实施方案，本公开证明对-甲苯磺酰胺(p-TSA)是通过细胞周期的p21-和p27-非依赖性G1停滞来抑制PC-3和DU-145(两种CRPC细胞株)的细胞增殖的小分子，该细胞周期中的周期蛋白D1是下降的，且Rb磷酸化是受到抑制的。p-TSA亦诱导粒线体膜电位的显著丧失，其归因于两种促凋亡的Bcl-2家族成员Bak和PUMA的上升，导致细胞凋亡。p-TSA会抑制两种细胞株中m-TOR、4E-BP1和p70S6K的磷酸化。持续活化型Akt的过度表达挽救了PC-3细胞中mTOR/p70S6K信号传导的抑制，显示Akt依赖性途径。相反地，在DU-145细胞中观察到Akt非依赖性效应。脂膜筏作为多细胞信号传导和运输过程的功能平台，两种细胞株均会表达脂膜筏相关的Akt、mTOR和p70S6K，p-TSA会诱导降低这些与脂膜筏相关激酶的全部形式和磷酸化形式的表达。经p-TSA诱导的抑制作用可通过补充胆固醇(脂膜筏中的必要组成)来反转，表明胆固醇含量的关键角色。此外，肿瘤异种移植模型显示p-TSA会以T/C(治疗/对照)为0.44及抑制56％的生长速率来抑制肿瘤生长，并表明体内功效。总之，数据显示p-TSA是一种通过抑制Akt依赖性和非依赖性mTOR/p70S6K途径而在体外和体内具有功效的有效抗肿瘤剂。此外，脂膜筏和胆固醇含量的扰动可解释经p-TSA介导的抗肿瘤过程。
在本公开的实施方案中所使用的材料来源如下所示。人类前列腺腺癌细胞株PC-3和DU-145从美国典型培养物保藏中心(Rockville，MD，美国)取得。RPMI 1640培养基、胎牛血清(FBS)、青霉素和链霉素从GIBCO/BRL Life Technologies(Grand Island，NY，美国)购得。PARP-1、Bcl-2、Bak、Mcl-1、细胞凋亡的p53上调调节子(PUMA)、α-微管蛋白、细胞周期蛋白E、细胞周期蛋白A、细胞周期蛋白B、细胞周期蛋白依赖性激酶(Cdk)4、Cdk2、Cdk1、GAPDH、p27和小窝蛋白-1的抗体从Santa Cruz Biotechnology，Inc.(Santa Cruz，CA，美国)取得。Rb、p-RbSer807/811、p21、Akt、p-AktThr308、p-AktSer473、Bid、cyclin D1、mTOR、p-mTORSer2448、4E-BP1、p-4E-BP1Thr37/46、p-p70S6KThr389和p-IκB-αSer32的抗体从Cell SignalingTechnologies(Boston，MA，美国)获得。P70S6K来自于Abcam(Cambridge，英国)。凋亡蛋白酶来自Imgenex公司(San Diego，CA，美国)。羧基荧光素琥珀酰亚胺酯(CFSE)来自MolecularProbes Inc.(Eugene，OR，美国)。抗小鼠和抗兔IgG来自Jackson ImmunoResearchLaboratories，Inc.(West Grove，PA，美国)。对甲苯磺酰胺(p-TSA)、磺酰罗丹明B(SRB)、亮抑酶肽、NaF、NaVO4、二硫苏糖醇、苯甲基磺酰氟(PMSF)、三氯乙酸(TCA)、米托蒽醌(mitoxantrone)、水溶性胆固醇、碘化丙啶(PI)和所有其他的化合物从Sigma-Aldrich(St.Louis，MO，美国)购得。
实施例1：检测在去势抵抗性前列腺癌(CRPC)细胞内，对甲苯磺酰胺(p-TSA)抑制细胞增殖的影响
PC-3和DU-145的两种去势抵抗性前列腺癌(CRPC)细胞株在含有5％FBS(v/v)、青霉素(100单位/ml)和链霉素(100μg/ml)的RPMI1640培养基中培养。将培养物保持在含有5％CO2的37℃培养箱中，且当其等达到约80％群聚时，利用0.05％胰蛋白酶-EDTA继代培养附着的培养物。来自人类体组织样本的前列腺细胞是在捐赠者知情同意下以及在台湾大学医院的伦理审查委员会进行全面审查后获得。前列腺标本来自男性经尿道前列腺切除术。所有具有前列腺肥大性疾病病史的患者都通过直肠指检、前列腺的经直肠超声波检查和尿路动力学研究诊断为良性的前列腺增生。在先前的研究[22]中描述自前列腺组织外植体中分离人类前列腺细胞。相较于前列腺癌细胞，前列腺细胞具有较少量的脂膜筏。
SRB测定、群落形成分析和羧基荧光素琥珀酰亚胺酯(CFSE)染色用于抗增殖检测。SRB分析是基于细胞蛋白的定量SRB染色的准确且可重复的分析，且SRB分析的过程如下进行。将单个的PC-3和DU-145细胞接种在含有10％FBS培养基的96孔盘中。24小时后，利用10％TCA固定PC-3和DU-145细胞，以代表添加p-TSA时的细胞群。在DMSO或p-TSA的额外培养48或72小时后，用10％TCA固定细胞，然后加入0.4％(w/v)的SRB(于1％乙酸中)，以将PC-3和DU-145细胞染色。用1％乙酸洗去未结合的SRB，且用10mM Tris碱溶解经SRB结合的细胞。在加入p-TSA时(下文称为零时段，TZ)，以及在额外的DMSO(下文称为对照生长，CTL)或p-TSA培养之后(以下简称Tx)，于515nm的波长下检测SRB的吸光度。通过[1-(Tx-TZ)/(CTL-TZ)]×100％的公式计算抑制细胞生长的百分比。在p-TSA浓度下检测细胞生长的半抑制浓度(IC50)，其会造成在p-TSA培养期间内，对照细胞中减少50％的总蛋白质增量。
为了分析锚定依赖性群落形成作用，将个别的PC-3和DU-145细胞(150个细胞/孔)接种在6孔板中。用p-TSA处理10天后，用磷酸盐缓冲盐水(PBS)冲洗细胞群落，用溶解在20％甲醇中的0.4％(w/v)结晶紫进行染色，并利用溶于50％乙醇中的50mM柠檬酸钠进行裂解。在595nm的波长下读取吸光度。
结果如图1A所示，p-TSA显示PC-3和DU-145细胞株的浓度依赖性抑制的IC50值约为3mM。此外，数据亦显示p-TSA在正常的前列腺细胞中展现出较低的抗增殖活性。此外，如图1B所示的结果，群落形成分析证明p-TSA在PC-3和DU-145细胞中均表现出长期抗增殖的作用(10天)。
通过CFSE染色进一步检测抗增殖的作用。CFSE是一种用于细胞追踪的荧光细胞染剂，其会与胞内的蛋白质结合，且在细胞增殖后均匀地被分裂的细胞遗传。因此，随着继代时间的推移，荧光染色会被分配到后代细胞中。CFSE染色的过程如下进行。将CFSE溶解在DMSO中，以形成10mM的储存溶液，且直至使用前保持在-20℃。将PC-3和DU-145细胞调整为106个细胞/ml的密度，并在试管中以最终浓度为10μM的CFSE处理。在37℃下培养10分钟后，通过加入含10％FBS的RPMI培养基来阻断标记。将试管置于冰上5分钟，然后洗涤。将试管进行离心后，将细胞接种于含有10％FCS且缺乏或存在p-TSA的RPMI培养基中，并于37℃、5％CO2/95％空气条件下培养48小时。通过流式细胞术分析确定荧光强度。通过监测在子细胞中所降低的标记强度来评估细胞增殖。通过使用Modfit LT 3.2版本和WinList 5.0版本软件来计算亲本或不同世代的增殖指数和细胞群。
流式细胞术分析如下进行。通过胰蛋白酶水解来收集细胞，在4℃下利用70％(v/v)乙醇固定30分钟，并用PBS洗涤。离心后，在室温下，将细胞培养于磷酸盐-柠檬酸缓冲液(0.2mol/l NaHPO4、0.1mol/l柠檬酸，pH 7.8)中30分钟。然后将细胞离心并用含有TritonX-100(0.1％v/v)、RNase(100μg/ml)和PI(80μg/ml)的0.5ml碘化丙啶(PI)溶液再次悬浮。利用FACScan和CellQuest软件(Becton Dickinson，Mountain View，CA，美国)分析DNA含量。
如图1C和图1D所示，p-TSA显著抑制细胞增殖，诱导前几代的细胞群的增生。基于CFSE染色分析的PC-3和DU-145细胞中的增殖指数显示对p-TSA作用的浓度依赖性抑制。由于p-TSA是一种分子量为171道尔顿(Dalton)的简单小分子，所以p-TSA在毫摩尔浓度范围内有效是合理的。已报告还有数种其他化合物和药物是以毫摩尔浓度来显示活性，例如清除活性氧物质(ROS)的N-乙酰半胱氨酸和水溶性维生素E(trolox)，以及诱导细胞周期停滞和促凋亡细胞死亡的阿司匹林和表没食子儿茶素-3-没食子酸酯(epigallocatechin-3-gallate)[23、24]。
由上可知，p-TSA会抑制CRPC细胞的增殖。
实施例2：检测p-TSA对细胞周期进展和粒线体应激的影响
如图2A所示，p-TSA会诱导PC-3细胞和DU-145细胞在细胞周期的G1期积累并加速细胞凋亡。细胞周期检查点是确保正确细胞分裂的关键机制。G1检查点是细胞致力于进入细胞周期的主要检查点。当发生DNA受损或某些细胞压力时，会进行G1停滞，直到修复受损为止。如果没有适当地修复，将通过抑制促存活成分或活化凋亡途径来启动细胞凋亡，其中粒线体是协调这些信号最敏感的胞器。
粒线体产生大部分ATP以合成诸如蛋白质、脂质和核苷酸的生物活性成分，以用于细胞生长和增殖。粒线体的应激和对ATP产生的干扰已经显示会诱导细胞周期停滞[25]。粒线体膜电位(ΔΨm)的检测如下进行。JC-1是一种粒线体染色剂，以膜电位依赖性方式对活细胞中的粒线体进行染色，以用于确定粒线体膜电位(ΔΨm)。PC-3和DU145细胞用p-TSA处理或不处理。在终止培养之前30分钟，将细胞与JC-1(最终浓度5μM)在37℃培养30分钟。最后收集细胞，并利用流式细胞术分析确定JC-1的累积。如图2B所示，p-TSA会导致粒线体膜电位的浓度依赖性降低，表明粒线体应激会导致凋亡蛋白酶依赖性细胞凋亡。由于使用PI染色的流式细胞术分析和核小体DNA片断分析两者下，泛凋亡蛋白酶抑制剂Z-VAD-FMK极度地抑制经PTS诱导的细胞凋亡(图2E和图2F)，导致凋亡因子释放的粒线体外膜通透性的增加是细胞凋亡的关键。粒线体膜通透性由Bcl-2蛋白家族直接控制[26]。
图2C和图2D所示的数据证实p-TSA会诱导PC-3和DU-145细胞中两种促凋亡Bcl-2家族成员PUMA和Bak表达的增加。此外，通过西方墨点法与如下所述的方法进行各Mcl-1、Bcl-2、Bid、PUMA和Bak的蛋白质表达分析。将PC-3和DU-145细胞处理p-TSA后，用胰蛋白酶处理、离心，并于0.1ml含有10mM Tris-HCl(pH 7.4)、150mM NaCl、1mM EGTA、1％Triton X-100、1mM PMSF、10μg/ml亮抑酶肽、10μg/ml抑肽酶、50mM NaF和100μM正钒酸钠的裂解缓冲液中裂解并收集细胞。对总蛋白质进行定量，与样品缓冲液混合，并在90℃下煮沸5分钟。在8％或12％的SDS-PAGE中通过电泳分离等量的蛋白质(30μg)，转移至PVDF膜上，并各用Mcl-1、Bcl-2、Bid、PUMA和Bak蛋白质的特异性抗体(1：1000稀释)检测。经适当标记的二级抗体(1：3000稀释)培养后的免疫反应蛋白，以增强的化学发光检测试剂盒(Amersham，Buckinghamshire，英国)进行检测。此外，从图2D中观察到，p-TSA显著降低DU-145细胞中Mcl-1(抗凋亡的Bcl-2家族成员)的蛋白质表达。这些影响与粒线体应激有关。
在细胞周期的G1期，细胞周期蛋白D1/Cdk4复合物导致通过Rb蛋白的磷酸化进展至S期。细胞周期蛋白D1/Cdk4复合物能够降低诸如p21和p27的Cdk抑制剂(CDKIs)的活性，从而触发细胞周期蛋白E/Cdk2复合物的后续活化，以活化许多与DNA合成相关的蛋白质[27]。
通过使用细胞死亡检测ELISAplus试剂盒(Roche，Mannheim，德国)检测DNA片段化以确定细胞死亡。该分析是基于在诱导细胞死亡后，细胞质组蛋白相关的DNA片段(单核小体和寡核小体)的定量体外测定。经化合物处理后，将细胞裂解并离心，并根据制造商的步骤准则，将上清液用于检测核小体DNA。
如图3A所示，其证明p-TSA诱导降低PC-3细胞中细胞周期蛋白D1的蛋白表达和Rb磷酸化，但仅降低Rb-突变的DU-145细胞中的细胞周期蛋白D1的表达。此外，在PC-3和DU-145两者细胞中，p-TSA均未修饰p21和p27的表达。经p-TSA诱导的细胞周期蛋白D1的下调是与细胞周期的G1停滞相关。然而，p21和p27在经p-TSA介导的G1期停滞中的独立性可能由其他因素解释，并将在下面讨论。
以上可知，p-TSA提供诱导细胞周期的G1停滞和粒线体应激的作用。
实施例3：检测p-TSA对于调节Akt/mTOR/p70S6K途径的影响
在G1期，细胞尺寸增长，并合成DNA合成时所必需的mRNA和蛋白质。通过影响数种下游转译因子的磷酸化或活性来调控蛋白质合成(mRNA转译)的丝氨酸/苏氨酸激酶mTOR和p70S6K是G1期的关键调控因子[28-30]。通过西方墨点法进行各Akt、mTOR 4E-BP1和p70S6K的蛋白质表达分析。如图3B和图3C所示，它证实在PC-3和DU-145两者细胞中，p-TSA会诱导mTOR、4E-BP1(mRNA转译的抑制子)和p70S6K蛋白的磷酸化的抑制作用，其解释在这些细胞上的蛋白质合成的抑制作用和抗癌作用。此外，p-TSA会抑制Akt的磷酸化，在PC-3细胞中，Akt是mTOR、4E-BP1和p70S6K信号途径的关键参与者，表明Akt/mTOR/p70S6K轴活化的抑制可能导致经p-TSA介导的作用。相反地，在DU-145细胞中，p-TSA未修饰Akt磷酸化，显示Akt非依赖性mTOR/p70S6K信号传导。
为了进一步证实Akt的作用，用以下所示的方法制备经Myr-Akt过度表达的PC-3细胞。编码Myr-Akt的质粒(N末端肉豆蔻酰化信号附着Akt)是受惠自Mien-Chie Hung教授(德州大学，M.D.安德森癌症中心)。为了转染，将PC-3细胞接种到具有30％群聚的60-mm组织培养皿中并生长24小时至50至60％群聚。用不含血清的Opti-MEM(Life Technologies)洗涤各培养皿，然后加入2ml不含血清的Opti-MEM。按照制造商的说明，利用Lipofectamine2000(Invitrogen)将含有Myr-Akt表达载体的等分试样或在不含血清的Opti-MEM中的对照质粒转染到细胞中。在37℃培养6小时后，用不含血清的Opti-MEM洗涤细胞，并在含有10％FBS的RPMI-1640培养基中培养48小时。细胞用p-TSA处理或不处理，然后进行相关的分析。如图4A至4D所示，PC-3细胞中的持续活化型Myr-Akt的过度表达显著地挽救对mTOR和4E-BP1两者磷酸化的抑制作用，并抑制凋亡蛋白酶-3的活化和下游底物PARP-1的裂解。此外，确定Myr-Akt的功能性拯救。数据显示，利用SRB分析和群落形成分析，在PC-3细胞中组成性活化型Myr-Akt的过度表达适度地但显著地减弱经PTS介导的生长抑制(图4E和图4F)；IC50值分别从1.97±0.01mM显著偏移至2.18±0.10mM(P<0.05)和从0.58±0.02mM显著偏移至0.68±0.02mM(P<0.01)。然而，Myr-Akt的过度表达对经p-TSA诱导的细胞周期蛋白D1的下调的影响很小。数据显示Akt主要影响下游mTOR途径，而非通过调节细胞周期蛋白D1。
由上可知，p-TSA会抑制Akt/mTOR/p70S6K途径的活化。
实施例4：检测脂膜筏和胆固醇对p-TSA作用的影响
脂膜筏是含有鞘糖脂和许多受体的细胞质膜，而受体排列在脂膜筏特定的糖脂蛋白微域中，聚集各种信号分子、膜液和蛋白质运输[31]。有人提出包括Akt、mTOR和p70S6K的数种与脂膜筏相关的信号途径成分会参与调节细胞存活[32]。为了检测脂膜筏对p-TSA作用的影响，通过蔗糖梯度分离法以自完整的细胞中分离出脂膜筏，随后通过免疫印迹法分析数种丝氨酸/苏氨酸激酶在脂膜筏区分部分中的定位。脂膜筏分离的详细方法如下进行。
利用裂解条件分离出脂膜筏，且以不连续的蔗糖梯度进行离心。简而言之，细胞经p-TSA处理或不处理后，用冰冷的PBS洗涤细胞，且在冰上以含有1％Triton X-100的TNEV缓冲液(10mM Tris-HCl，pH 7.5、150mM NaCl、5mM EDTA、1mM Na3VO4和1mM PMSF)裂解30分钟。利用Biovision组织匀浆机并以15冲程，将细胞进行匀浆。以200g离心8分钟后，将细胞核和细胞碎片沉淀，并将400μl的上清液与400μl含有85％(w/v)蔗糖的TNEV缓冲液混合，然后转移至Beckman 13×51mm离心管底部。将稀释的溶胞产物以2.4ml含有35％(w/v)蔗糖的TNEV缓冲液覆盖，并在最后以1.4ml含有5％(w/v)蔗糖的TNEV缓冲液覆盖。在Beckman OptimaL100K超速离心机(Beckman Instruments，Palo Alto，CA，美国)中，在4℃下，将样品在SW55旋转器中以200,000g转速离心18小时。然后，从顶部梯度收集各体积为350μl的区分部分。将15μl的各区分部分进行西方墨点法分析，以确定在不连续的蔗糖梯度中靶蛋白的位置。
结果显示于图5A、5B和5C中。数据显示，PC-3和DU-145两种细胞株均表达不同水平的三种与脂膜筏相关的激酶，其中以p70S6K含量最多。此外，p-TSA会导致与脂膜筏相关的全部形式和磷酸化形式激酶两者的表达降低，其中磷酸化的p70S6K表达在PC-3和DU-145两种细胞株中完全消除。
此外，胆固醇是由所有的动物细胞经生物合成的独特脂质分子，是细胞膜中必要的结构成分，以维持其结构的完整性和流动性。在外部外质小叶中，脂膜筏是由神经鞘脂质和胆固醇所组成，与脂质双层的内部细胞质小叶中的磷脂和胆固醇相连[33、34]。近来的研究显示，脂膜筏的胆固醇耗竭与数种癌症的细胞凋亡有关。为了检测胆固醇对p-TSA作用的影响，将胆固醇补充于经p-TSA处理的PC-3和DU-145细胞。结果显示于图6A至6D中，数据显示适当浓度的胆固醇补充显著地挽救经p-TSA诱导所降低的Akt和p70S6K磷酸化，在PC-3和DU-15两者细胞中的细胞周期蛋白D1则无此现象。胆固醇本身会诱导增加Akt磷酸化，同时降低细胞周期蛋白D1蛋白的表达，尤其是在DU-145细胞中。亦确定在细胞生长中胆固醇的功能性拯救，且结果如图6E所示。数据显示，通过使用群落形成分析，胆固醇显著地挽救经PTS诱导而抑制的细胞生长。
由上可知，脂膜筏和胆固醇对于数种激酶对p-TSA作用的活性是至关重要的。
实施例5：检测p-TSA作用在小鼠异种移植模型中的作用
体内抗肿瘤研究如下进行。裸鼠PC-3异种移植作为体内模型。向裸鼠皮下注射PC-3细胞(107个细胞/小鼠)。每天测量肿瘤。当肿瘤达到100mm3的体积时，将小鼠分成两组(n＝8至10)，并开始进行化合物治疗。将p-TSA溶于15％的1-甲基-2-吡咯烷酮(NMP)中。每隔一天通过腹腔内给药媒介物(15％NMP)或p-TSA。检测肿瘤的长度(l)和宽度(w)，以lw2/2计算肿瘤体积。体内研究的方案是由台湾大学的动物护理和使用委员会批准。所有的动物程序和方案皆经AAALAC认可的机构批准。数据分析如下所示。数据代表单独实验的指示数值的平均值±平均值的标准误差(SEM)。利用方差分析(ANOVA)对多个样品组进行统计分析。利用Student’s t检定进行适当组别的单一比较。P值小于0.05在统计学上被认为是显著的。
在皮下接种PC-3细胞的裸鼠模型肿瘤异种移植，用于进行p-TSA的体内功效评估。操作员对实验处理分组完全不知情。当肿瘤达到100mm3(p-TSA组121±40mm3 vs.对照组101±48mm3)的尺寸时，给予初始腹腔内注射75mg/kg p-TSA。首先，利用肿瘤生长抑制治疗/对照(T/C)比率来定量p-TSA在小鼠异种移植模型中的治疗效果。如图7A所示，数据证实在p-TSA处理结束时，肿瘤生长抑制T/C比为0.44。此外，停止p-TSA治疗会引起肿瘤的反弹生长，显示p-TSA是抑制肿瘤生长的完全原因。另外，如图7B所示，对照组的生长速度vs.p-TSA组的生长速度为37.8mm3/天vs.16.8mm3/天，显示p-TSA会抑制56％。相较于对照组的中位肿瘤尺寸为765mm3，p-TSA组的中位肿瘤尺寸为403mm3，显示p-TSA的抑制率为47％。此外，如图7C所示，尽管没有显示组间差异的显著，体内研究显示对照组和p-TSA组的体重会逐渐减轻，其亦显示相较于p-TSA组在第22天达到体重减轻20％，对照组在第12天达到体重减轻20％。此外，如图7D所示，肿瘤中p-Akt表达的检测亦显示出PTS的显著抑制作用。
由上可知，p-TSA在小鼠异种移植模型中显示出抗CRPC的体内功效。
自主的细胞增殖是癌细胞的标志之一，是由活化的存活(survival)和促生长(growth-promoting)癌基因所驱动。磷脂肌醇-3-激酶(PI3K)/Akt/mTOR/p70S6K信号传导途径通常是前列腺癌细胞中的活化途径。PTEN肿瘤抑制因子的丧失经常被报为该途径异常活化的因素，不仅涉及前列腺癌细胞的存活和生长，亦涉及肿瘤转移[5、36、37]。因此，这些途径的去活化作用可能有机会治疗前列腺癌。一些研究报告PI3K抑制剂具有前景的临床前结果，而临床试验的数据较不具有说服力。因此，已经提出阻断PI3K/Akt和mTOR两者的双PI3K/mTOR抑制剂以获得更好的抗癌结果[36、38]。然而，表现出多种生物学和临床行为的前列腺癌代表极端的遗传异质性的附带现象[37]。基于多种因素而检测多种特定的分子改变和开发最合适的治疗能提供更好的治疗机会。
本公开通过使用PC-3和DU-145两种CRPC细胞株，阐述经p-TSA介导的多种机制，其在体外和体内皆能有效阻断细胞的生长和存活。p-TSA通过诱导G1期展示出有效且长期稳定的抗增殖活性，而最终诱导细胞凋亡。通常，DNA受损或某些细胞压力倾向于导致G1检查点停滞，以允许修复来自程序性细胞死亡的拯救细胞的损伤。然而，经p-TSA诱导的粒线体损伤，显示在p-TSA的处理期间，并未显著修复细胞的损伤，最终导致细胞凋亡。粒线体膜的完整性和通透性受到Bcl-2蛋白家族的严格调控[26]。PUMA是Bcl-2家族的唯一BH3亚群的p53依赖性和p53非依赖性促凋亡成员，且已被鉴定为通过其BH3结构域直接结合抗凋亡Bcl-2成员，以诱导活化促凋亡Bcl-2成员和增加粒线体外膜通透性，导致粒线体的功能障碍和凋亡蛋白酶活化[39]。近来，数个证据显示类似于Bim、Noxa和tBid的PUMA是直接的Bak活化剂，可启动Bak的低聚合作用和活化[40]。p-TSA显著地诱导明显增加PUMA和Bak的表达，显示其等对粒线体功能障碍的贡献。p-TSA亦显著地抑制DU-145细胞中的Mcl-1表达。已报告，在与PUMA共同表达的期间，Mcl-1和PUMA共同定位于粒线体且能增加Mcl-1的水平，显示PUMA能够稳定Mcl-1。相反地，数个研究显示，PUMA与Mcl-1的结合并不足以防止Mcl-1的快速降解[41]。我们的数据显示类似的结果，PUMA并未防止Mcl-1降解；此外，粒线体的功能障碍部分地归因于DU-145细胞内的Mcl-1降解。
在G1期，细胞尺寸会伴随着为了合成DNA而强烈合成mRNA和蛋白质而增加，其中mTOR通过诱导磷酸化和数种下游转译因子的活化而扮演关键角色[28-30]。p-TSA显著抑制C-末端调节区内Ser2448中的mTOR磷酸化，其为mTOR活化的关键标志[28、42]。此种磷酸化可通过Akt依赖性或非依赖性途径而活化[42、43]。Myr-Akt(一种Akt的持续活化型形式)的过度表达几乎完全消除对PC-3细胞内mTORSer2448和4E-BP1Thr37/46(mTOR的直接底物)两者经p-TSA介导的磷酸化抑制，显示Akt依赖性mTOR活性对p-TSA的抑制作用。相反地，DU-145细胞内的Akt活性并不明显的原因可能是由于PTEN(PI3K/Akt活性的负调节因子)的存在，因为除了PC-3之外，DU-145会在mRNA和蛋白质水平上表达PTEN[44]。下面讨论在DU-145细胞中通过Akt非依赖性途径调节mTOR的活性。
细胞周期蛋白D1是细胞周期进展的G1期的关键调节因子，且异常的细胞周期蛋白D1表达与许多人类肿瘤的发生、转移和进展有关[45、46]。细胞周期蛋白D1的过度表达与前列腺癌的发生和加重的骨转移有关[45]。p-TSA会诱导细胞周期G1停滞，且有效地阻断在源自骨转移的PC-3和源自脑转移的DU-145细胞内细胞周期蛋白D1的表达，显示p-TSA抑制前列腺癌转移的潜力。然而，过度表达Myr-Akt并未挽救细胞周期蛋白D1的下调，显示存在Akt非依赖性调节途径。已提出数种途径会参与细胞周期蛋白D1的下调，包括降低细胞的ATP水平、活化蛋白激酶C和磷酸酶PP2A、消耗腺嘌呤核苷酸转位酶2和下调c-Myc[47-49]。明确的途径需要进一步阐明。经p-TSA诱导的细胞周期G1的停滞与p21和p27无关。在许多研究中已报导类似的作用。Vaziri等人报导经丁酸盐诱导的G1的停滞发生在来自于转基因的p21“敲除(knockout)”小鼠(p21-/-)的成纤维细胞的原代培养物中，显示p21诱导的独立性[50]。Berns等人报导在缺乏p27和p21两者的胚胎成纤维细胞中，c-Myc的显性负突变可诱导细胞周期小鼠的G1停滞[51]。这些研究支持p21和p27两者对于经p-TSA介导的G1期阻滞并非为速率决定的细胞周期调节因子。
近来，由于包括Akt、mTOR和p70S6K等与脂膜筏相关的信号途径成分参与了细胞存活的调控[32]，因此，脂膜筏在抗癌研究中的功能受到很多关注。由于前列腺癌细胞含有更多的脂膜筏，因此，其对治疗前列腺癌是很重要的[34,52]。胆固醇对于保持膜的完整性和流动性是必要的，且对于脂膜筏/胞膜窖的形成是至关重要的。细胞的胆固醇含量的变化可能改变脂膜筏的特性[53]，且来自细胞膜上的胆固醇耗损会诱导细胞凋亡，尤其，在具有更高的细胞膜胆固醇含量的前列腺癌细胞中[52]。因此，针对脂膜筏/胆固醇的靶标治疗将会是一种潜在的策略。数据证实PC-3和DU-145两者细胞株均会表达与脂膜筏相关的Akt、mTOR和p70S6K。p-TSA会诱导降低这几种蛋白质的全部和磷酸化两者形式的表达。数个研究显示脂膜筏的完整性对于这些激酶的活性是必需的。脂膜筏的干扰或破坏能够损害这些激酶的磷酸化[54-56]。因此，p-TSA可能引起脂膜筏的干扰或破坏，导致这些激酶离开脂膜筏和去活化作用。胆固醇的补充显著地挽救这些激酶经p-TSA诱导的去活化作用，并进一步关联胆固醇对经p-TSA介导的脂膜筏破坏的作用。然而，胆固醇补充剂并不能阻止细胞周期蛋白D1的下调，因为其并非是与脂膜筏相关的成分。数据亦支持细胞周期蛋白D1的下降并非是Akt/mTOR/p70S6K途径的下游事件。最后，裸鼠异种移植模型是用于检测p-TSA的体内抗肿瘤功效。通过测量T/C和生长速率，p-TSA的腹腔内治疗会诱导抑制56％的肿瘤生长，以及通过检测中位肿瘤尺寸，p-TSA的腹腔内治疗会诱导抑制47％。PTS亦会显著地抑制肿瘤中的Akt磷酸化。数据表明p-TSA的体内功效。
总之，数据显示p-TSA是一种具有体外和体内两者功效的有效抗肿瘤剂。p-TSA会通过诱导细胞周期停滞在G1期而达到抗增殖作用，以及在PC-3和DU-145细胞中，透过Bak和PUMA诱发的粒线体功能障碍，而诱导细胞凋亡。此外，Akt对于调控细胞凋亡的mTOR/p70S6K途径，在PTEN缺陷/Akt正常的PC-3的细胞中是必须的，与在PTEN正常的DU-145细胞中不同。脂膜筏和胆固醇含量的干扰能至少部分地解释在两种细胞株中，Akt、mTOR和p70S6K从脂膜筏的离开和去活化作用。
本公开已经使用例示性实施例描述。然而，应理解，本公开的范围不限于所公开的实施方案。相反地，其意图在涵盖各种修饰和类似的重组。因此，所请权利要求范围应当与最广泛的解释相一致，以涵盖所有这些修饰和类似的重组。
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Claims (15)
1.一种调节细胞的脂膜筏完整性的方法，包括将医药组合物给药至需要治疗的受试者，以调节脂膜筏完整性，其中所述医药组合物包含苯磺酰胺衍生物及其药学上可接受的赋形剂。
2.根据权利要求1所述的方法，其中，所述苯磺酰胺衍生物由式(I)表示：
或其药学上可接受的盐，
其中，R1至R7独立地选自由下列所组成的组：H、C1至C6直链或支链烷基、C1至C6直链或支链烷氧基、C3至C6环烷基、C3至C6环杂烷基、胺基及卤素基团，或R6和R7相互连接形成一个环，
其中，所述烷基、所述烷氧基、所述环烷基、所述环杂烷基和所述环未经取代或经一个或多个取代基取代，以及
其中，所述取代基选自由苯基、卤素、氧代、醚基、羟基、羧基、胺基、磺基和磺酰胺基所组成的组。
3.根据权利要求2所述的方法，其中，所述苯磺酰胺衍生物选自由下列所组成的组的至少一种：对-甲苯磺酰胺、邻-甲苯磺酰胺、间-甲苯磺酰胺、N-乙基-对-甲苯磺酰胺、N-乙基-邻-甲苯磺酰胺、N-环己基-对-甲苯磺酰胺、
4.根据权利要求1所述的方法，所述方法消耗所述细胞中细胞膜的胆固醇。
5.根据权利要求1所述的方法，所述方法通过降低所述脂膜筏完整性的水平来治疗易于改善的疾病。
6.根据权利要求5所述的方法，其中，所述疾病是癌症。
7.根据权利要求6所述的方法，其中，所述癌症富含脂膜筏，且是选自由下列所组成的组的至少一种：乳腺癌、前列腺癌、皮肤癌、肺癌、胰腺癌、黑色素瘤、肾癌、膀胱癌、精原细胞瘤、卵巢癌、子宫颈癌、大肠癌、肝癌、食道癌、口腔癌、舌癌、甲状腺癌、脑膜瘤、胆管癌、下咽癌、鼻咽癌、胃癌和外阴癌。
8.一种预防或治疗癌症的方法，包括将医药组合物给药至有其需要的受试者，其中，所述医药组合物包含苯磺酰胺衍生物和其药学上可接受的赋形剂。
9.根据权利要求8所述的方法，其中，所述癌症富含脂膜筏，且是选自由下列所组成的组的至少一种：乳腺癌、前列腺癌、皮肤癌、肺癌、胰腺癌、黑色素瘤、肾癌、膀胱癌、精原细胞瘤、卵巢癌、子宫颈癌、大肠癌、肝癌、食道癌、口腔癌、舌癌、甲状腺癌、脑膜瘤、胆管癌、下咽癌、鼻咽癌、胃癌和外阴癌。
10.根据权利要求8所述的方法，还包括向受试者施用至少一种额外的抗癌疗法。
11.根据权利要求10所述的方法，其中，所述额外的抗癌疗法是全身化疗、放疗或热疗。
12.根据权利要求8所述的方法，其中，所述医药组合物中的所述苯磺酰胺衍生物以每天约20mg至约4000mg的治疗有效量给药至所述受试者。
13.根据权利要求8所述的方法，其中，所述医药组合物调节癌细胞的脂膜筏完整性。
14.根据权利要求13所述的方法，其中，所述医药组合物消耗所述癌细胞中细胞膜的胆固醇。
15.根据权利要求13所述的方法，其中，所述医药组合物干扰或破坏所述癌细胞的所述脂膜筏完整性。
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| EP (1) | EP3731839A4 (zh) |
| JP (1) | JP7164784B2 (zh) |
| KR (2) | KR20200094215A (zh) |
| CN (1) | CN112055586A (zh) |
| AU (1) | AU2018395281B2 (zh) |
| TW (2) | TW202402267A (zh) |
| WO (1) | WO2019133797A1 (zh) |
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| US11752160B2 (en) * | 2020-09-24 | 2023-09-12 | Gongwin Biopharm Co., Ltd | Method for reducing fat by administering benzenesulfonamide compositions |
| US11752118B2 (en) | 2020-12-02 | 2023-09-12 | Gongwin Biopharm Co., Ltd | Method for treating melanoma |
| US20230098310A1 (en) * | 2021-09-29 | 2023-03-30 | Gongwin Biopharm Co., Ltd | Methods for treating mast cell tumors |
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- 2018-12-28 EP EP18897388.7A patent/EP3731839A4/en active Pending
- 2018-12-28 CN CN201880084805.5A patent/CN112055586A/zh active Pending
- 2018-12-28 WO PCT/US2018/067838 patent/WO2019133797A1/en unknown
- 2018-12-28 JP JP2020536515A patent/JP7164784B2/ja active Active
- 2018-12-28 KR KR1020207020747A patent/KR20200094215A/ko not_active IP Right Cessation
- 2018-12-28 TW TW112135547A patent/TW202402267A/zh unknown
- 2018-12-28 AU AU2018395281A patent/AU2018395281B2/en active Active
- 2018-12-28 KR KR1020237027960A patent/KR20230127355A/ko not_active Application Discontinuation
- 2018-12-28 TW TW107147889A patent/TWI822719B/zh active
- 2018-12-28 US US16/959,054 patent/US20200338028A1/en active Pending
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Also Published As
| Publication number | Publication date |
|---|---|
| TW201929845A (zh) | 2019-08-01 |
| AU2018395281B2 (en) | 2022-03-10 |
| WO2019133797A1 (en) | 2019-07-04 |
| JP7164784B2 (ja) | 2022-11-02 |
| US20200338028A1 (en) | 2020-10-29 |
| EP3731839A1 (en) | 2020-11-04 |
| KR20200094215A (ko) | 2020-08-06 |
| TWI822719B (zh) | 2023-11-21 |
| TW202402267A (zh) | 2024-01-16 |
| EP3731839A4 (en) | 2021-11-10 |
| AU2018395281A1 (en) | 2020-07-16 |
| KR20230127355A (ko) | 2023-08-31 |
| JP2021509404A (ja) | 2021-03-25 |
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