TPO/thrombopoietin receptor

鲁曲波帕通过特异性作用于人TPO，有效接收并启动信号传递，从而驱动骨髓细胞生长并坏死为巨核细胞，提高警戒水平[1]。

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为了研究通过人TPOR形成巨核细胞集落的增殖活性和效果，我们将lusutrombopag分别应用于培养的表达Ba/F3 （Ba/F3- hmpl）的人c- mpl细胞和人骨髓来源的cd34阳性细胞。Lusutrombopag以类似于血小板生成素的方式激活途径，并在人cd34阳性细胞中诱导集落形成单位-巨核细胞和多倍体巨核细胞，从而导致Ba/F3-hMpl细胞的显著增加。[2]

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Lusutrombopag对人TPO受体c-Mpl 具有激动作用[2]
Lusutrombopag促进Ba/F3-hMpl细胞的增殖。lusutrombopag和rhTPO在Ba/F3-mMpl细胞中的50% EC50值分别为84.0和0.08 nmol/L（图2A），而lusutrombopag在Ba/F3-mMpl细胞中没有增殖活性（图2B）。上述结果表明lusutrombopag通过人c-Mpl促进Ba/F3-hMpl细胞的增殖。为了研究lusutrombopag的信号转导途径，我们评估了Ba/F3-hMpl细胞中JAK2、STAT3、STAT5和p44/42 MAPK的磷酸化。Lusutrombopag磷酸化这些分子类似于rhTPO（图2C）。这些结果表明lusutrombopag激活了与rhTPO激活的相同的信号转导途径。

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lusutrombopag 促进cd34阳性造血细胞的分化[2]
采用hubm - cd34阳性细胞检测lusutrombopag的CFU-Mk活性。与lusutrombopag或rhTPO孵育12 d后，用抗人CD41抗体检测CFU-Mk菌落，计数CFU-Mk菌落。rhTPO （1.846 nmol/L）处理组CFU-Mk菌落数平均为147.3个。rhTPO活性定义为100%活性，lusutrombopag EC50值为0.31µmol/L（图3A）。采用抗人CD41抗体对CFU-Mk中rhTPO和lusutrombopag菌落进行免疫组化检测。CFU-Mk菌落在rhTPO和lusutrombopag之间没有形态学差异（图3B）。为了进一步研究巨核细胞的成熟，我们使用流式细胞仪检测了lusutrombopag诱导的巨核细胞倍化。hubm - cd34阳性细胞在无血清条件下用lusutrombopag或rhTPO培养10天。lusutrombopag中巨核细胞的DNA倍性分布与rhTPO相似（图3C）。这些发现表明lusutrombopag能够将成熟的巨核细胞与人造血干细胞区分开来。

TPOR-Ki/Shi小鼠中lusutrombopag的血小板生成活性[2]
Lusutrombopag对人TPO受体表达细胞有增殖活性，但对小鼠TPO受体表达细胞无增殖活性。在TPOR-Ki/Shi小鼠和野生型小鼠中评价lusutrombopag的血小板生成活性。给TPOR-Ki/Shi小鼠或野生型小鼠口服Lusutrombopag或载药（0.5% MC），每天1次，连续14天，于第7、14、21和28天计数血小板。图6显示了lusutrombopag给药后血小板生成的发展。血小板生成用于测定血小板增加比（用基础血小板计数除以治疗后的血小板计数）。lusutrombopag处理的TPOR-Ki/Shi小鼠血小板增加率显著高于载药处理的TPOR-Ki/Shi小鼠。血小板数量比原波多巴组增加2.5倍。停药后，血小板在第21天下降，并在第28天恢复到接近基础水平。与此相反，lusutrombopag治疗野生型小鼠的血小板在实验期间没有增加。为了明确其最小的血小板生成作用，将0.3、1、3和10 mg/kg/天剂量的lusutrombopag或载药（0.5% MC）口服给TPOR-Ki/Shi小鼠，每天一次，连续21天。各组分配时（第0天）、给药前（第8天和第15天）和给药后第21天（第22天）24小时静脉采血，然后对每个样本的血小板进行计数。与载体对照组相比，Lusutrombopag在第8天以剂量依赖的方式显著增加血小板计数。血小板数量在第15天和第22天继续增加（图7）。在21天的重复口服研究中，lusutrombopag的最小有效剂量为0.3 mg/kg/天。 <人力资源> 由于lusutrombopag对人类TPOR具有很高的物种特异性，除了基于免疫缺陷小鼠的异种移植模型外，没有合适的实验动物模型用于药物评估。因此，我们用人鼠嵌合体Mpl代替小鼠Mpl，开发了一种新的基因修饰敲入小鼠TPOR-Ki/Shi。在TPOR-Ki/Shi小鼠中，lusutrombopag在21天的重复口服中以剂量依赖的方式显著增加循环血小板。TPOR-Ki/Shi小鼠在第22天的组织病理学研究也显示骨髓中巨核细胞显著增加。这些结果表明，lusutrombopag作用于人TPOR，上调巨核细胞的分化和增殖，导致血小板的产生。［2］

增殖试验[2]
如前所述，Ba/F3- hmpl细胞和Ba/F3- mmpl细胞由小鼠白细胞介素(IL)-3依赖性前b细胞系Ba/F3工程化。细胞在lusutrombopag (4.88-5000 nmol/L)或重组人TPO （rhTPO: 4.88-5000 pmol/L）的96孔板中以7.5 × 103个/200µL的密度培养。在加5% CO2的加湿室中，37°C孵育3天；在培养的最后2-8小时，每孔加入10µL WST-8试剂。采用96孔微孔板读卡器在450 nm波长处测量吸光度。

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巨核细胞集落形成及倍性测定[2]
在知情同意的情况下，从人体组织中获得人骨髓来源的cd34阳性（hubm - cd34阳性）细胞。本研究已获得相关机构伦理委员会的批准。使用MegaCult-C试剂盒进行集落形成单位-巨核细胞（CFU-Mk）测定。hubm - cd34阳性细胞在lusutrombopag或rhTPO的载玻片上培养12 d，对载玻片进行抗人CD41抗体染色，计数CFU-Mk菌落。采用乙状结肠最大药理效应（Emax）模型计算lusutrombopag的50%有效浓度（EC50）。为了研究巨核细胞的成熟情况，将HuBM-CD34阳性细胞与lusutrombopag或hTPO培养10天，用流式细胞仪检测巨核细胞的倍化。方法的更多细节可以在补充方法中找到（仅在线，可在www.exphem.org上找到）。

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Western blotting [2]
Ba/F3-hMpl细胞在含0.5%牛血清白蛋白（BSA, Wako, Osaka, Japan）的RPMI中培养5小时，用3µM lusutrombopag或1 nM rhTPO在37℃下刺激15分钟。在lusutrombopag或rhTPO刺激后，用冰冷的PBS洗涤细胞，在含有蛋白酶抑制剂鸡尾酒和磷酸酶抑制剂鸡尾酒的裂解缓冲液中4°C裂解30分钟。将蛋白样品加入等体积的2 × Laemmli样品缓冲液/ 10% 2-巯基乙醇中，95℃煮沸5 min。蛋白样品经SDS-PAGE分离后，转移到immune - blot PVDF膜上。印迹用5% BSA在TBS-T （20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.05% Tween20）中阻断2小时，然后与抗磷酸化JAK2 （Tyr1007/1008）、抗磷酸化STAT3 （Tyr705）、抗磷酸化STAT5和抗磷酸化p44/42 MAP激酶（Thr202/204）抗体在4℃下孵育过夜。用Re-blot +温和溶液剥离印迹，用抗jak2、抗stat3、抗stat5和抗p44/42 MAP激酶抗体重新探针。用ECL Western blotting检测试剂和分析系统孵育，并暴露于Amersham Hyperfilm ECL。

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CFU-MK检测[2]
HuBM-CD34阳性细胞用lusutrombopag（0.0923-9.23µM）或rhTPO （1.8 nM）培养，孵育12 d后，用抗人CD41抗体染色，计数CFU-Mk菌落。rhTPO的最大CFU-Mk活性定义为100%，采用s型最大药理效应（Emax）模型计算lusutrombopag的50%有效浓度（EC50）。

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巨核细胞液体培养及倍性分析[2]
HuBM-CD34阳性细胞在24孔板中以7.5 × 104个/mL的密度培养。作为无血清培养基，我们使用Iscove改良Dulbecco培养基，添加20% BIT9500和40µg/mL人低密度脂蛋白（LDL）。细胞用3µM lusutrombopag或1 nM rhTPO处理，一式三份，37°C， 5% CO2加湿室处理10天。细胞重悬于含有13.6 mM柠檬酸钠、2µM前列腺素E-1、1 mM茶碱、3%牛血清白蛋白、11 mM葡萄糖（Mk培养基）的汉克平衡盐溶液（HBSS）中。如前所述，用异硫氰酸荧光素（FITC）标记的抗人CD41抗体和碘化丙啶染色细胞。使用EPICS-XL流式细胞仪分析细胞。通过电子门控pi染色的CD41阳性细胞获得数据，每个样本至少分析10,000个细胞。倍性分布是通过在峰值之间的最低点设置标记来确定的。

Effect on platelet production in human TPOR-expressing mice [2]
To evaluate the thrombocytopoietic effect of lusutrombopag in vivo, the genetically modified mouse TPOR-Ki/Shi was developed using knocked-in technology that enabled replacement of the mouse TM domain of TPOR with a human–mouse chimera TM domain. TPOR-Ki/Shi mice were used in this study. Development of the genetically modified mouse is discussed in the Supplementary Methods. To clarify the effectiveness of TPOR-Ki/Shi mice, we investigated the effect on platelet production of oral administration of lusutrombopag to TPOR-Ki/Shi mice or C57BL/6 mice, the wild type of TPOR-Ki/Shi mice. Eight female TPOR-Ki/Shi mice and C57BL/6 mice in each group were administered 10 mg/kg/day lusutrombopag or vehicle (0.5% methylcellulose [MC] aqueous solution) daily for 14 days. Blood was collected from veins under anesthesia 1 day before the start of administration (day 0) and on days 7, 14, 21 and 28, and the platelets were counted using a K-4500 multi-automatic hemocytometer. Dose escalation of platelet production of lusutrombopag and megakaryocytopoiesis in bone marrow was investigated using TPOR-Ki/Shi mice.

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Thrombopoietic effect and morphometric analysis of megakaryocytes in TPOR-Ki/Shi mice [2]
The thrombocytopoietic effect of lusutrombopag (0.3, 1, 3 and 10 mg/kg) and an increase of megakaryocytopoiesis in the bone marrow and hematological changes of lusutrombopag (0.3 and 10 mg/kg/day) were examined in TPOR-Ki/Shi mice (female, eight per group, 11–12 weeks old). Lusutrombopag or vehicle (0.5% MC) was orally administered once daily for 21 consecutive days. Small amounts of blood samples were collected from the vein under anesthesia and the platelet number was then counted using a K-4500 multi automatic hemocytometer. For histological and hematological study, on the day following the final administration (Day 22), mice were anesthetized with pentobarbital and blood was collected from the posterior vena cava and whole blood treated with EDTA-2K was subjected to analysis using the ADVIA 120 Hematology System. The following tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin (H&E), and then five fields of bone marrow were randomly selected and photographed by computer-digitizing imaging system consisting of a light microscope with a camera. The megakaryocyte number was counted in the fields and average data were expressed as mean ± standard deviation (SD).

Absorption, Distribution and Excretion
Lusutrombopag is rapidly absorbed following oral administration. It exhibited a dose‐proportional pharmacokinetic profile over the single dose range of 1 mg to 50 mg, which was similar in both healthy subjects and those with chronic liver disease. A geometric mean (%CV) maximal concentration (Cmax) and area under the curve (AUC) in healthy subjects receiving 3 mg of lusutrombopag were 111 (20.4) ng/mL and 2931 (23.4) ng.hr/mL. The accumulation ratios of Cmax and AUC were approximately 2 with once‐daily multiple‐dose administration, and steady‐state plasma lusutrombopag concentrations were achieved after Day 5. The time to reach peak plasma concentrations (Tmax) were approximately 6 to 8 hours after oral administration in patients with chronic liver disease. Food consumption is not reported to affect the absorption and bioavailability of lusutrombopag.
About 1% of the administered dose of lusutrombopag undergoes urinary excretion. Fecal excretion accounted for 83% of the total dose, where 16% of the dose was excreted as unchanged parent compound.
The mean (%CV) lusutrombopag apparent volume of distribution in healthy adult subjects was 39.5 (23.5) L.
The approximate mean (%CV) clearance of lusutrombopag in patients with chronic liver disease is estimated to be 1.1 (36.1) L/hr.

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Metabolism / Metabolites
CYP4 enzymes predominantly contribute to the metabolism of lusutrombopag, especially CYP4A11. Lusutrombopag is reported to mainly undergo ω- and β-oxidation, as well as glucuronidation.

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Biological Half-Life
In healthy adult subjects, the terminal elimination half‐life (t1/2) was approximately 27 hours.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of lusutrombopag during breastfeeding. The manufacturer recommends avoiding breastfeeding during the use of lusutrombopag and for at least 28 days 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.

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Protein Binding
The plasma protein binding of lusutrombopag is more than 99.9%.

[1]. Lusutrombopag: First Global Approval. Drugs. 2016 Jan;76(1):155-8.

[2]. Development of a new knock-in mouse model and evaluation of pharmacological activities of lusutrombopag, a novel, nonpeptidyl small-molecule agonist of the human thrombopoietin receptor c-Mpl. Exp Hematol. 2018 Mar:59:30-39.e2.

Pharmacodynamics
The AUC of lusutrombopag was found to correlate the increased platelet counts. Following administration of 3 mg daily dose in patients with chronic liver disease and thrombocytopenia, the mean (standard deviation) maximum platelet count in patients (N=74) without platelet transfusion was 86.9 (27.2) × 10^9/L, and the median time to reach the maximum platelet count was 12.0 (5 to 35) days. Lusutrombopag was not shown to induce any clinically significant QTc prolongation at a dose 8 times the recommended dosage.

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Lusutrombopag is a member of cinnamic acids.

Lusutrombopag is an orally bioavailable thrombopoietin receptor (TPOR) agonist developed by Shionogi & Company (Osaka, Japan). TPOR is a regulatory target site for endogenous thrombopoietin, which acts as a primary cytokine to promote megakaryocyte proliferation and differentiation, and affect other hematopoietic lineages as well, including erythroid, granulocytic and lymphoid lineages. Thrombocytopenia, which indicates abnormally low levels of platelets, is a common complication related to chronic liver disease. This hematological abnormality, especially in cases of severe thrombocytopenia (platelet count <50,000/μL), creates challenges to patients requiring invasive medical procedures where there is a significant risk for spontaneous bleeding. Lusutrombopag binds to the transmembrane domain of TPOR expressed on megakaryocytes, and causes the proliferation and differentiation of megakaryocytic progenitor cells from hematopoietic stem cells. In September 2015, lusutrombopag received its first global approval in Japan to reduce the need for platelet transfusion in adults with chronic liver disease and thrombocytopenia who are schedule to undergo an invasive medical procedure. Lusutrombopag was approved by the FDA on July 31st, 2018 for the same therapeutic indication under the market name Mulpleta. In two randomized, double-blind, placebo-controlled trials, patients with chronic liver disease and severe thrombocytopenia who were undergoing an invasive procedure with a platelet count less than 50 x 10^9/L were administered lusutrombopag orally. Higher percentages (65-78%) of the patients receiving lusutrombopag required no platelet transfusion prior to the primary invasive procedure compared to those receiving placebo. Lusutrombopag is currently in phase III development in various European countries including Austria, Belgium, Germany, and the UK.

Lusutrombopag is an orally available thrombopoietin (TPO) receptor (TPOR; MPL) agonist, with potential megakaryopoiesis stimulating activity. Upon administration, lusutrombopag binds to and interacts with the transmembrane domain of human TPO receptor expressed on megakaryocytes, which leads to the proliferation and differentiation of megakaryocytic progenitor cells from hematopoietic stem cells. This increases the production of platelets and may prevent or treat thrombocytopenia in patients with chronic liver disease. TPOR is a cytokine receptor and member of the hematopoietic receptor superfamily.

LUSUTROMBOPAG is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2018 and is indicated for thrombocytopenia and hemorrhage and has 1 investigational indication.

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Lusutrombopag (Mulpleta®) is an orally bioavailable, small molecule thrombopoietin (TPO) receptor agonist being developed by Shionogi for chronic liver disease (CLD) patients with thrombocytopenia prior to elective invasive surgery. Lusutrombopag acts selectively on the human TPO receptor and activates signal transduction pathways that promote the proliferation and differentiation of bone marrow cells into megakaryocytes, thereby increasing platelet levels. In September 2015, lusutrombopag received its first global approval in Japan for the improvement of CLD-associated thrombocytopenia in patients scheduled to undergo elective invasive procedures. This article summarizes the milestones in the development of lusutrombopag leading to this first approval.[1]

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The TPO-Ki/Shi mouse could be a suitable animal model for evaluation of the systemic effects of synthetic TPOR agonists that require the histidine residue in the TM region of c-Mpl for their activity. The results indicate that lusutrombopag acts on TPOR to upregulate differentiation and proliferation of megakaryocytic cells leading to platelet production. [2]

C29H32CL2N2O5S

591.54

590.14

C, 58.88; H, 5.45; Cl, 11.99; N, 4.74; O, 13.52; S, 5.42

1110766-97-6

Lusutrombopag-d13

49843517

White to off-white solid powder

1.3±0.1 g/cm3

1.618

8.64

126

2

7

13

39

822

1

CCCCCCO[C@@H](C)C1=CC=CC(=C1OC)C2=CSC(=N2)NC(=O)C3=CC(=C(C(=C3)Cl)/C=C(\C)/C(=O)O)Cl

NOZIJMHMKORZBA-KJCUYJGMSA-N

InChI=1S/C29H32Cl2N2O5S/c1-5-6-7-8-12-38-18(3)20-10-9-11-21(26(20)37-4)25-16-39-29(32-25)33-27(34)19-14-23(30)22(24(31)15-19)13-17(2)28(35)36/h9-11,13-16,18H,5-8,12H2,1-4H3,(H,35,36)(H,32,33,34)/b17-13+/t18-/m0/s1

(2E)-3-(2,6-Dichloro-4-((4-(3-((1S)-1-(hexyloxy)ethyl)-2-methoxyphenyl)-1,3-thiazol-2-yl)carbamoyl)phenyl)-2-methylprop-2-enoic acid

S-888711; S888711; LUSUTROMBOPAG; 1110766-97-6; mulpleta; (S,E)-3-(2,6-Dichloro-4-((4-(3-(1-(hexyloxy)ethyl)-2-methoxyphenyl)thiazol-2-yl)carbamoyl)phenyl)-2-methylacrylic acid; UNII-6LL5JFU42F; 6LL5JFU42F; RSC888711; S 888711; Trade name: Mulpleta

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 (4.23 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 (4.23 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 (4.23 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));
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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；
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