Proteasome (IC50 = 5 nM)

体外活性：Carfilzomib 抑制多种细胞系和患者来源的肿瘤细胞（包括多发性骨髓瘤）的增殖，并诱导内在和外在的细胞凋亡信号通路以及 c-Jun-N 末端激酶 (JNK) 的激活。与硼替佐米相比，卡非佐米具有增强的抗 MM 活性，克服了对硼替佐米和其他药物的耐药性，并与地塞米松 (Dex) 具有协同作用。卡非佐米对 β5 亚基中的 ChT-L 活性具有优先的体外抑制效力，在 10 nM 剂量下抑制率超过 80%。短时间暴露于低剂量卡非佐米会导致β5组成型20S蛋白酶体和β5i免疫蛋白酶体亚基的优先结合特异性。用卡非佐米脉冲的 ANBL-6 细胞中 caspase 活性的测量显示，8 小时后 caspase-8、caspase-9 和 caspase-3 活性显着增加，分别比对照增加 3.2、3.9 和 6.9 倍8小时后的细胞。在卡非佐米脉冲处理的细胞中，线粒体膜完整性降低至 41% (Q1 + Q2)，而在媒介物处理的对照细胞中为 75%。在另一项研究中，卡非佐米还显示出针对血液系统和实体恶性肿瘤的临床前有效性。卡非佐米直接抑制破骨细胞形成和骨吸收。激酶测定：将 ANBL-6 细胞（2 × 106/孔）接种于 96 孔板中，并用 0.001 至 10 μM 剂量的卡非佐米处理 1 小时。然后裂解细胞（20 mM Tris-HCl、0.5 mM EDTA），并将澄清的裂解物转移至聚合酶链式反应 (PCR) 板。使用未处理的 ANBL-6 细胞裂解物生成标准曲线，起始浓度为 6 μg 蛋白质/μL。活性位点探针[生物素-(CH2)4-Leu-Leu-Leu-环氧酮；添加20 μM]并在室温下孵育1小时。然后通过添加 1% 十二烷基硫酸钠 (SDS) 并加热至 100°C 使细胞裂解物变性，然后在 96 孔多屏 DV 板中与每孔 20 μL 链霉亲和素-琼脂糖高性能珠混合并孵育 1 小时。然后用酶联免疫吸附测定 (ELISA) 缓冲液（PBS、1% 牛血清白蛋白和 0.1% Tween-20）洗涤这些珠子，并在平板摇床上与蛋白酶体亚基抗体在 4°C 下孵育过夜。使用的抗体包括小鼠单克隆抗β1、抗β2、抗β1i和抗β5i、山羊多克隆抗β2i和兔多克隆抗β5（针对KLH-CWIRVSSDNVADLHDKYS肽的亲和纯化抗血清）。洗涤珠子并与辣根过氧化物酶缀合的二抗山羊抗兔、山羊抗小鼠或兔抗山羊抗体一起孵育 2 小时。清洗后，使用 Supersignal ELISA 皮化学发光底物对珠子进行显色。进行发光检测。通过与标准曲线比较，将原始发光转换为μg/mL，并表示为相对于载体对照的抑制百分比。使用以下非 S 型剂量反应方程生成曲线拟合：Y = Bottom + (Top-Bottom)/(1 + 10̂((LogEC50 − X) × HillSlope))，其中 X 是浓度的对数，Y 是 %抑制作用，EC50是显示50%效果的剂量。细胞测定：WST-1 用于测定蛋白酶体抑制剂卡非佐米对细胞增殖的影响。相对于仅接受媒介物的平行对照细胞计算增殖抑制。使用 XLfit 4 软件，使用线性样条函数插值中值抑制浓度 (IC50)。耐药程度（DOR）的计算公式为：DOR = IC50（耐药细胞）/IC50（敏感细胞）。将用 100 nM 卡非佐米脉冲的 ANBL-6 细胞洗涤并悬浮在含有 5 μg/mL JC-1 的 PBS 中，JC-1 在线粒体中表现出电位依赖性积累。在 FacScan 上对从 525 nm 到 590 nm 的线粒体膜电位依赖性色移进行分析，并使用 CellQuest 软件对数据进行分析。

---

随着硼替佐米（一种用于复发/难治性多发性骨髓瘤（MM）的第一类可逆蛋白酶体抑制剂）的批准，蛋白酶体已成为癌症治疗的重要靶点。然而，许多患者的疾病对硼替佐米没有反应，而另一些患者则产生了耐药性，这表明需要其他活性增强的抑制剂。因此，我们评估了一种新型的、不可逆的、与环氧霉素相关的蛋白酶体抑制剂Carfilzomib。在多发性骨髓瘤模型中，该药物能有效结合并特异性抑制胰凝乳蛋白酶样蛋白酶体和免疫蛋白酶体的活性，导致泛素化底物的积累。Carfilzomib诱导了剂量和时间依赖性的增殖抑制，最终导致细胞凋亡。程序性细胞死亡与c-Jun-N-末端激酶的激活、线粒体膜去极化、细胞色素c的释放以及内源性和外源性半胱氨酸天冬氨酸蛋白酶途径的激活有关。该药物还抑制了患者来源的MM细胞和其他血液系统恶性肿瘤患者的肿瘤细胞的增殖并激活了凋亡。重要的是，与硼替佐米相比，carfilzomib显示出更高的疗效，并且对硼替佐米布耐药的MM细胞系和临床硼替佐密布耐药患者的样本具有活性。Carfilzomib还克服了对其他常规药物的耐药性，并与地塞米松协同作用以增强细胞死亡。综上所述，这些数据为carfilzomib在多发性骨髓瘤中的临床评估提供了理论基础。[1]

---

连续或生理性短暂给予Carfilzomib或丙佐米在体外对人MM细胞具有细胞毒性。在48小时的连续药物孵育下，Carfilzomi和丙佐米对10个与硼替佐米相似的人MM细胞系产生了细胞毒性作用。与之前的报告一致，硼替佐米的IC50约为2 nM，卡氟佐米为3 nM，丙佐米为25 nM（图1a）。然而，药代动力学数据表明，口服丙佐米后体内暴露于药物约4小时，静脉注射卡氟佐米或硼替佐米后约1小时。为了在体外更准确地复制这种生理情况，细胞用丙佐米短暂处理4小时，用卡氟佐米或硼替佐米处理1小时，然后在无药物培养基中再培养48小时。在短期治疗条件下，骨髓瘤细胞系仍然容易受到蛋白酶体抑制（图1b），尽管需要增加剂量才能达到类似的疗效（8 nM硼替佐米、6 nM卡氟佐米和50 nM丙佐米）。有效瞬时剂量仍远低于患者达到的最大血清水平（Cmax）（硼替佐米：0.162μM（静脉注射1.3mg/m2）；卡氟佐米：0.95μM（静脉注射20mg/m2）；丙佐米：3.8μM（口服30mg））。carfilzomib和丙佐米降低MM存活率的原因是抑制增殖和诱导凋亡（数据未显示），这与之前检查这些PI的报告一致。
阿曲佐米和卡氟佐米在体外抑制OC分化和功能。 Carfilzomib和丙佐米在体外促进成骨分化和矿化[3]。

---

在套细胞淋巴瘤（MCL）细胞中，体外和体内检测了蛋白酶体抑制剂Carfilzomib与组蛋白脱乙酰酶（HDAC）抑制剂vorinostat和SNDX-275之间的相互作用。将极低、毒性轻微的卡氟佐米布浓度（如3-4nmol/L）与最低致死浓度的伏利诺司他或SNDX-275联合给药，可诱导多个MCL细胞系和原代MCL细胞的线粒体损伤和凋亡急剧增加。致死性增强与c-jun-NH，-激酶（JNK）1/2激活、DNA损伤增加（诱导λH2A.X）以及ERK1/2和AKT1/2失活有关。联合使用carfilzomib和组蛋白脱乙酰酶抑制剂（HDACI）可显著增加活性氧（ROS）的产生和G（2）-M阻滞。值得注意的是，自由基清除剂四（4-苯甲酸）卟啉（TBAP）阻断了carfilzomib/HDACI介导的ROS产生λH2A。X形成、JNK1/2激活和致死性。JNK1/2的遗传（短发夹RNA）敲除显著减轻了carfilzomib/HDACI诱导的细胞凋亡，但并不能阻止ROS的产生或DNA损伤。Carfilzomib/HDACI方案对硼替佐米耐药的MCL细胞也有活性[4]。

卡非佐米/Carfilzomib在体内异种移植模型中适度降低肿瘤生长。在连续或短暂的模拟治疗后，卡非佐米可有效降低多发性骨髓瘤细胞的活力。卡非佐米可增加非肿瘤小鼠的骨小梁体积，减少骨吸收并增强骨形成。

---

基于环氧酮的PI对非荷瘤小鼠具有骨合成代谢作用[3]
体外证据表明，PI对OC和OB都有细胞自主作用。为了检查它们对非骨髓瘤骨的影响，将PI给药于非荷瘤免疫活性C57Bl/6小鼠两周。与硼替佐米相似，Carfilzomib或丙佐米治疗可增加骨小梁参数（图5a和b）。通过骨吸收导致的胶原蛋白分解产物（羧基末端端肽-胶原蛋白交联）的血清水平降低来衡量，所有三种PI都相对抑制了OC功能（图5c）。此外，与对照组相比，所有药物都显著增加了OB活性，这是通过血清中I型前胶原N-末端前肽（骨形成的标志物）水平的升高来衡量的（图5d）。值得注意的是，carfilzomib对I型前胶原N端前肽的增加明显大于硼替佐米。一致的是，双钙黄绿素标记表明PI增加了骨形成率（图5e）。这些数据表明，基于环氧酮的PIs carfilzomib和丙佐米通过合成代谢和抗分解代谢特性增强健康小鼠的骨体积，这些特性与硼替佐米相当甚至优于硼替佐密。

---

Carfilzomib和丙佐米可减轻MM肿瘤负担，保护小鼠免受骨破坏[3]
为了研究carfilzomib和丙佐米联合治疗已建立的骨髓瘤的抗肿瘤和保骨作用，我们利用了两种体内小鼠模型。将5TGM1-GFP小鼠骨髓瘤细胞静脉注射到免疫功能正常的同基因C57Bl/KaLwRij小鼠体内，在28天内产生具有明显骨破坏的播散性肿瘤。51,52个5TGM1肿瘤建立了14天，之后按照与每种药物临床剂量相关的时间表给药硼替佐米、卡氟佐米或丙佐米（见材料和方法）。根据克隆型抗体IgG2b的血清水平（图6a）或由表达GFP的肿瘤细胞组成的BM或脾脏的百分比（图6b和c），所有PI均显著降低了肿瘤负担。所有PI治疗组的microCT对肿瘤诱导的骨丢失的保护作用都很明显（图6d和e），骨转换的血清标志物显示出显著的抗吸收（图6f）和骨合成代谢（图6g）作用。值得注意的是，尽管PI内的差异没有统计学意义，但与硼替佐米相比，卡非佐米和丙佐米观察到I型前胶原活性N端前肽增加的趋势。

---

Carfilzomib/vorinostat方案在体内Granta异种移植物模型中的体内活性[4]
为了评估carfilzomib/vorinostat方案的体内活性，采用了类似于我们描述的DLBCL模型的Granta萤光细胞异种移植物侧翼模型（24）。动物侧腹接种10×106个细胞，肿瘤出现后，用2.0mg/kg carfilzomib（IV，BIW-第1、2天）±70mg/kg vorinostat（IP，TIW-第2、3天）治疗动物，然后每周监测两次肿瘤大小。值表示独立进行的两个单独实验的结果，通过结合两个实验的肿瘤生长数据计算每组的平均肿瘤体积。如图6A所示，单独使用伏利诺司坦的效果很小，而carfilzomib在第20天适度降低了肿瘤生长。然而，伏利诺司他/卡氟佐米布联合用药几乎消除了肿瘤生长。在接种了萤光素酶表达细胞的动物中进行了平行研究，并通过IVIS生物成像仪监测了肿瘤进展。与用单一药物或对照治疗的动物相比，联合治疗导致生物发光明显减少（图6B）。联合治疗的毒性，如脱发、体重减轻（<10%）很小（图6C）。最后，从切除的肿瘤中提取的蛋白质中获得的蛋白质印迹分析显示，磷酸化JNK，γH2A明显增加。X、 与单一药物或对照组相比，从用两种药物治疗的动物中获得的肿瘤中切割的胱天蛋白酶-3（图6D）与体外结果一致。

ANBL-6 细胞（以 2 × 10⁶/孔铺板）接受卡非佐米（Carfilzomib）处理 1 小时，剂量范围为 0.001 至 10 μM。下一步涉及裂解细胞（20 mM Tris-HCl、0.5 mM EDTA），然后将澄清的裂解物置于 PCR 板上。使用未经处理的 ANBL-6 细胞裂解物创建标准曲线，浓度为 6 μg 蛋白质/μL。添加活性位点探针（生物素-(CH2)4-Leu-Leu-Leu-环氧酮；20 μM）后，将混合物在室温下孵育一小时。将细胞裂解物加热至 100°C 并添加 1% 十二烷基硫酸钠 (SDS) 后，将混合物与 96 孔多屏 DV 板中每孔 20 μL 的链霉亲和素-琼脂糖高性能珠混合，并孵育混合物一个小时。在含有 PBS、1% 牛血清白蛋白和 0.1% Tween-20 的溶液中洗涤珠子后，将珠子与抗蛋白酶体亚基的抗体在平板摇床上于 4°C 下孵育整晚。使用的抗体包括山羊多克隆抗-β2i、兔多克隆抗-β5（针对KLH-CWIRVSSDNVADLHDKYS肽的亲和纯化抗血清）和小鼠单克隆抗-β1、抗-β2、抗-β1i和抗-β5i。将山羊抗兔、山羊抗小鼠或与辣根过氧化物酶缀合的兔抗山羊二抗应用于珠子，然后孵育 2 小时。 Supersignal ELISA 皮化学发光底物用于在清洗后对珠子进行显色。一种进行发光检测。原始发光表示为与载体对照相比的抑制百分比，并通过与标准曲线比较转换为 μg/mL。以下非 S 形剂量反应方程用于创建曲线拟合：Y = Bottom + (Top-Bottom)/(1 + 10̂((LogEC50 − X) × HillSlope))，其中 EC50 是表现出 50% 效应的剂量，X为浓度的对数，Y为抑制百分比。

---

Carfilzomib亚基分析的酶联免疫吸附试验 将ANBL-6细胞（2×106/孔）铺在96孔板上，用0.001至10μM剂量的carfilzomib处理1小时。然后裂解细胞（20mM Tris-HCl，0.5mM EDTA），并将清除的裂解物转移到聚合酶链式反应（PCR）板上。使用未经处理的ANBL-6细胞裂解物以6μg蛋白质/μL的浓度开始生成标准曲线。加入活性位点探针[生物素-（CH2）4-Leu-Leu-Leu-Eu-环氧酮；20μM]，在室温下孵育1小时。然后通过添加1%十二烷基硫酸钠（SDS）并加热至100°C使细胞裂解物变性，然后在96孔多筛DV板中与每孔20μL链霉抗生物素蛋白琼脂糖高性能珠混合并孵育1小时。

---

Carfilzomib亚基分析的竞争性结合[1]
用于通过竞争性结合确定carfilzomib亚基特异性的方案改编自Berkers等人。简而言之，在37°C下用增加的carfilzomi剂量预孵育ANBL-6细胞，然后加入半抗原标记的细胞渗透性乙烯砜（VS）蛋白酶体抑制剂VS-L3-AHx3-danysl。然后按照下一节中的详细说明制备蛋白质印迹，并用多克隆抗反义抗体进行检测。

WST-1 用于评估蛋白酶体抑制剂卡非佐米如何影响细胞生长。增殖抑制的计算基于单独给予媒介物的平行对照细胞。 XLfit 4 软件用于使用线性样条函数插值中值抑制浓度 (IC50)。使用以下公式确定耐药程度（DOR）：DOR = IC50（耐药细胞）/IC50（敏感细胞）。用 100 nM 卡非佐米脉冲后，清洁 ANBL-6 细胞并将其悬浮在含有 5 μg/mL JC-1 的 PBS 中，JC-1 是一种以电位依赖性方式在线粒体中积累的酶。使用 FacScan，检查从 525 到 590 nm 的线粒体膜电位依赖性色移。 CellQuest 软件用于分析数据。

---

凋亡DNA断裂分析[1]
对于凋亡实验，将细胞接种到96孔板上，用300 nM（RPMI 8226，ANBL-6）或100 nMCarfilzomib（KAS-6/1，U266）的1小时脉冲处理，并允许其恢复24小时，然后根据制造商的规范使用细胞死亡检测ELISAPLUS试剂盒进行分析。DNA断裂的倍数增加表示为相对于载体处理的对照细胞的平均值。

---

线粒体膜电位（ΔΨm）[1]
用100 nMCarfilzomib脉冲的ANBL-6细胞被洗涤并悬浮在含有5μg/mL JC-1 的PBS中，JC-1在线粒体中表现出潜在的依赖性积累。在FacScan上分析了线粒体膜从525到590 nM的潜在依赖性色移，并用CellQuest软件分析了数据。

---

可行性分析[3]
共接种5×104个细胞/ml，进行标准MTT法。对于瞬时给药实验，用磷酸缓冲盐水洗涤细胞两次，并在1小时（硼替佐米，Carfilzomib）或4小时（丙佐米）后用无药物培养基替换。

Beige-nude-XID mice are used in animal research. After pelleting 10×10⁶ Granta514 cells and twice washing them in 1X PBS, the cells are subcutaneously injected into the right flank. Following the appearance of tumors, Carfilzomib-vorinostat is administered to five to six mice, and the growth or regression of the tumors is tracked throughout treatment. In DMSO and 10% sulfobutylether betacyclodextrin at a pH of 10 mM citrate buffer, stock vorinostat and carfilzomib are dissolved, respectively. Before injection, they are diluted and kept in small aliquots at -80°C for storage.

---

In vivo drug treatment [3]
PIs were administered to mice on the following weekly schedules: bortezomib (1 mg/kg intravenously days 1 and 4); Carfilzomib (5 mg/kg for C57Bl/6, 3 mg/kg for KaLwRij, intravenously days 1 and 2); oprozomib (30 mg/kg by oral gavage once daily for 5 consecutive days followed by 2 days of rest). Vehicle mice were administered both oral 1% carboxy-methylcellulose (oprozomib schedule) and intravenous 10% Captisol in 10 mM citrate buffer, pH 3.5 (Carfilzomib schedule). In Figure 5f, following 14 days of drug treatment, three doses of 1 mg/kg of RANKL were given intraperitoneally at 24 h intervals as described in Tomimori et al.34 Serum was collected 90 min after the final RANKL injection.

---

Animal Studies [4]
Animal studies were performed utilizing Beige-nude-XID mice. 10×106 Granta514 cells were pelleted, washed twice with 1X PBS, injected subcutaneously into the right flank. Once the tumors were visible, 5 to 6 mice were treated with Carfilzomib ± vorinostat and progress of tumor growth or regression was monitored as described earlier. Stock vorinostat and Carfilzomib was dissolved in DMSO and 10% sulfobutylether betacyclodextrin in 10mM citrate buffer pH respectively. They were stored in −80°C in small aliquots and diluted before injection as described earlier.

Absorption, Distribution and Excretion
Cmax, single IV dose of 27 mg/m^2 = 4232 ng/mL; AUC, single IV dose of 27 mg/m^2 = 379 ng•hr/mL; Carfilzomib does not accumulation in the systemic. At doses between 20 and 36 mg/m2, there was a dose-dependent increase in exposure.
Vd, steady state, 20 mg/m^2 = 28 L
Systemic clearance = 151 - 263 L/hour. As this value exceeds hepatic blood flow, it suggests that carfilozmib is cleared extrahepatically.

---

Metabolism / Metabolites
Carfilzomib was rapidly and extensively metabolized by the liver. The predominant metabolites were the peptide fragments and the diol of carfilzomib which suggests that the main metabolic pathways are peptidase cleavage and epoxide hydrolysis. The cytochrome P450 enzyme system is minimally involved in the metabolism of carfilzomib. All metabolites are inactive.

---

Biological Half-Life
Following intravenous administration of doses ≥ 15 mg/m^2, carfilzomib was rapidly cleared from the systemic circulation with a half-life of ≤ 1 hour on Day 1 of Cycle 1.

Hepatotoxicity
In large clinical trials of carfilzomib, elevations in serum aminotransferase levels were common, occurring in 8% to 13% of patients. However, values greater than 5 times the upper limit of normal (ULN) were uncommon, occurring in 1% to 2% of recipients. In several studies there were reports of clinically apparent liver injury including acute liver failure in patients receiving carfilzomib; however, in most instances multiple concomitant medications were being taken (such as lenalidomide) and the specific role of carfilzomib in causing the liver injury was not always clear. The onset of injury was typically during the first cycle of therapy. The clinical features and pattern of injury in clinically apparent cases of liver injury due to carfilzomib have not been described in the published literature. Hepatotoxicity is listed as a warning in the product label for carfilzomib and monitoring of serum enzymes during treatment is recommended.
Likelihood score: D (Possible cause of clinically apparent liver injury).

---

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of carfilzomib during breastfeeding. Because carfilzomib is 97% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during carfilzomib therapy and for 2 weeks after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

---

Protein Binding
Over the concentration range of 0.4 - 4 micromolar, carfilzomib was 97% protein bound.

[1]. Blood . 2007 Nov 1;110(9):3281-90.

[2]. Curr Cancer Drug Targets . 2011 Mar;11(3):285-95.

[3]. Leukemia . 2013 Feb;27(2):430-40.

[4]. Mol Cancer Ther . 2011 Sep;10(9):1686-97.

Carfilzomib is a synthetic tetrapeptide consisting of morpholin-4-acetyl, L-2-amino-4-phenylbutanoyl, L-leucyl and L-phenylalanyl residues joined in sequence with the C-terminus connected to the amino group of (2S)-2-amino-4-methyl-1-[(2R)-2-methyloxiran-2-yl]-1-oxopentan-1-one via an amide linkage. Used for the treatment of patients with multiple myeloma It has a role as an antineoplastic agent and a proteasome inhibitor. It is a tetrapeptide, a member of morpholines and an epoxide.
Carfilzomib is an injectable antineoplastic agent (IV only). Chemically, it is a modified tetrapeptidyl epoxide and an analog of epoxomicin. It is also a selective proteasome inhibitor. FDA approved carfilzomib in July 2012 for the treatment of adults with relapsed or refractory multiple myeloma as monotherapy or combination therapy.
Carfilzomib is a Proteasome Inhibitor. The mechanism of action of carfilzomib is as a Proteasome Inhibitor.
Carfilzomib is an irreversible proteasome inhibitor and antineoplastic agent that is used in treatment of refractory multiple myeloma. Carfilzomib is associated with a low rate of serum enzyme elevations during treatment and has been implicated to rare instances of clinically apparent, acute liver injury some of which have been fatal.
Carfilzomib is an epoxomicin derivate with potential antineoplastic activity. Carfilzomib irreversibly binds to and inhibits the chymotrypsin-like activity of the 20S catalytic core subunit of the proteasome, a protease complex responsible for degrading a large variety of cellular proteins. Inhibition of proteasome-mediated proteolysis results in an accumulation of polyubiquinated proteins, which may lead to cell cycle arrest, induction of apoptosis, and inhibition of tumor growth.

---

Drug Indication
Carfilzomib is indicated for the treatment of adult patients with relapsed or refractory multiple myeloma who have received one to three lines of therapy in combination with lenalidomide and dexamethasone; or dexamethasone; or daratumumab and dexamethasone; or daratumumab and hyaluronidase-fihj and dexamethasone; or isatuximab and dexamethasone. It is also indicated as a single agent for the treatment of patients with relapsed or refractory multiple myeloma who have received one or more lines of therapy.
FDA Label
Kyprolis in combination with daratumumab and dexamethasone, with lenalidomide and dexamethasone, or with dexamethasone alone is indicated for the treatment of adult patients with multiple myeloma who have received at least one prior therapy.
Treatment of acute lymphoblastic leukaemia
Treatment of Multiple Myeloma

---

Mechanism of Action
Carfilzomib is made up of four modified peptides and acts as a proteasome inhibitor. Carfilzomib irreversibly and selectively binds to N-terminal threonine-containing active sites of the 20S proteasome, the proteolytic core particle within the 26S proteasome. This 20S core has 3 catalytic active sites: the chymotrypsin, trypsin, and caspase-like sites. Inhibition of the chymotrypsin-like site by carfilzomib (β5 and β5i subunits) is the most effective target in decreasing cellular proliferation, ultimately resulting in cell cycle arrest and apoptosis of cancerous cells. At higher doses, carfilzomib will inhibit the trypsin-and capase-like sites.

---

Pharmacodynamics
Intravenous carfilzomib administration resulted in suppression of proteasome chymotrypsin-like activity when measured in blood 1 hour after the first dose. On Day 1 of Cycle 1, proteasome inhibition in peripheral blood mononuclear cells (PBMCs) ranged from 79% to 89% at 15 mg/m2, and from 82% to 83% at 20 mg/m2. In addition, carfilzomib administration resulted in inhibition of the LMP2 and MECL1 subunits of the immunoproteasome ranging from 26% to 32% and 41% to 49%, respectively, at 20 mg/m2. Proteasome inhibition was maintained for ≥ 48 hours following the first dose of carfilzomib for each week of dosing. Resistance against carfilzomib has been observed and although the mechanism has not been confirmed, it is thought that up-regulation of P-glycoprotein may be a contributing factor. Furthermore, studies suggest that carfilzomib is more potent than bortezomib.

---

The ubiquitin-proteasome pathway (UPP) is an attractive chemotherapeutic target due to its intrinsically stringent regulation of cell cycle, pro-survival, and anti-apoptotic regulators that disproportionately favor survival and proliferation in malignant cells. A reversible first-in-class proteasome inhibitor, bortezomib, is Food and Drug Administration approved for multiple myeloma and relapsed/refractory mantle cell lymphoma and has proven to be extremely effective, both as a single agent and in combination. An irreversible second generation proteasome inhibitor, Carfilzomib, has shown preclinical effectiveness against hematological and solid malignancies both in vitro and in vivo. Carfilzomib, a peptidyl-epoxyketone functions similarly to bortezomib through primary inhibition of chymotrypsin-like (ChT-L) activity at the b5 subunits of the core 20S proteasome. Carfilzomib is also currently achieving successful response rates within the clinical setting. In addition to conventional proteasome inhibitors, a novel approach may be to specifically target the hematological-specific immunoproteasome, thereby increasing overall effectiveness and reducing negative off-target effects. The immunoproteasome-specific inhibitor, IPSI-001, was shown to have inhibitory preference over the constitutive proteasome, and display enhanced efficiency of apoptotic induction of tumor cells from a hematologic origin. Herein, we discuss the preclinical and clinical development of carfilzomib and explore the potential of immunoproteasome-specific inhibitors, like IPSI-001, as a rational approach to exclusively target hematological malignancies. [2]

---

Proteasome inhibitors (PIs), namely bortezomib, have become a cornerstone therapy for multiple myeloma (MM), potently reducing tumor burden and inhibiting pathologic bone destruction. In clinical trials, Carfilzomib, a next generation epoxyketone-based irreversible PI, has exhibited potent anti-myeloma efficacy and decreased side effects compared with bortezomib. Carfilzomib and its orally bioavailable analog oprozomib, effectively decreased MM cell viability following continual or transient treatment mimicking in vivo pharmacokinetics. Interactions between myeloma cells and the bone marrow (BM) microenvironment augment the number and activity of bone-resorbing osteoclasts (OCs) while inhibiting bone-forming osteoblasts (OBs), resulting in increased tumor growth and osteolytic lesions. At clinically relevant concentrations, carfilzomib and oprozomib directly inhibited OC formation and bone resorption in vitro, while enhancing osteogenic differentiation and matrix mineralization. Accordingly, carfilzomib and oprozomib increased trabecular bone volume, decreased bone resorption and enhanced bone formation in non-tumor bearing mice. Finally, in mouse models of disseminated MM, the epoxyketone-based PIs decreased murine 5TGM1 and human RPMI-8226 tumor burden and prevented bone loss. These data demonstrate that, in addition to anti-myeloma properties, carfilzomib and oprozomib effectively shift the bone microenvironment from a catabolic to an anabolic state and, similar to bortezomib, may decrease skeletal complications of MM.[3]

C40H57N5O7

719.91

719.425

C, 66.73; H, 7.98; N, 9.73; O, 15.56

868540-17-4

Carfilzomib-d8;1537187-53-3

11556711

White solid powder

1.2±0.1 g/cm3

975.6±65.0 °C at 760 mmHg

204-208°C

543.8±34.3 °C

0.0±0.3 mmHg at 25°C

1.551

6.71

172.43

4

8

20

52

1180

5

C([C@@]1(OC1)C)(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)CN1CCOCC1)CCC1C=CC=CC=1)CC1C=CC=CC=1

BLMPQMFVWMYDKT-NZTKNTHTSA-N

InChI=1S/C40H57N5O7/c1-27(2)22-32(36(47)40(5)26-52-40)42-39(50)34(24-30-14-10-7-11-15-30)44-38(49)33(23-28(3)4)43-37(48)31(17-16-29-12-8-6-9-13-29)41-35(46)25-45-18-20-51-21-19-45/h6-15,27-28,31-34H,16-26H2,1-5H3,(H,41,46)(H,42,50)(H,43,48)(H,44,49)/t31-,32-,33-,34-,40+/m0/s1

(2S)-4-methyl-N-[(2S)-1-[[(2S)-4-methyl-1-[(2R)-2-methyloxiran-2-yl]-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]-2-[[(2S)-2-[(2-morpholin-4-ylacetyl)amino]-4-phenylbutanoyl]amino]pentanamide

PR-171; PR 171; PR171; Carflizomib; brand name: Kyprolis

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 该产品在溶液状态不稳定，请现配现用。

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 (3.47 mM) = 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), 悬浮液；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。

---

配方 2 中的溶解度: ≥ 2.5 mg/mL (3.47 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900 μL 玉米油中，混合均匀。=

---

View More

配方 3 中的溶解度: 2.5 mg/mL (3.47 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% 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。
*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。

---

配方 4 中的溶解度: 2% DMSO+castor oil: 10 mg/mL

---

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。