TPO/thrombopoietin receptor

By specifically acting on human TPO, lutetrombopag effectively receives and initiates signal transmission, thereby driving bone marrow cell growth and necrosis into megakaryocytes and elevating the warning level [1].

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To investigate the proliferative activity and efficacy of megakaryocytic colony formation via human TPOR, lusutrombopag was applied to cultured human c-Mpl-expressing Ba/F3 (Ba/F3-hMpl) cells and human bone marrow-derived CD34-positive cells, respectively. Lusutrombopag caused a robust increase in Ba/F3-hMpl cells by activating pathways in a manner similar to that of thrombopoietin and induced colony-forming units-megakaryocyte and polyploid megakaryocytes in human CD34-positive cells. [2]

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Lusutrombopag exhibits agonist activity for human TPO receptor c-Mpl [2]
Lusutrombopag promoted the proliferation of Ba/F3-hMpl cells. The 50% EC50 values of lusutrombopag and rhTPO in Ba/F3-hMpl cells were 84.0 and 0.08 nmol/L, respectively (Fig. 2A), whereas lusutrombopag exhibited no proliferative activity in Ba/F3-mMpl cells (Fig. 2B). These results indicate that lusutrombopag promotes the proliferation of Ba/F3-hMpl cells via human c-Mpl. To investigate the signal transduction pathway of lusutrombopag, we evaluated the phosphorylation of JAK2, STAT3, STAT5 and p44/42 MAPK in Ba/F3-hMpl cells. Lusutrombopag phosphorylated these molecules similarly to rhTPO (Fig. 2C). These results suggest that lusutrombopag activates the same signal transduction pathways activated by rhTPO.

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Differentiation of CD34-positive hematopoietic cells promoted by lusutrombopag [2]
CFU-Mk activity of lusutrombopag was investigated using HuBM-CD34-positive cells. After incubation with lusutrombopag or rhTPO for 12 days, the CFU-Mk colonies were detected by anti-human CD41 antibody, and the CFU-Mk colonies were counted. The mean number of CFU-Mk colonies was 147.3 in the rhTPO (1.846 nmol/L)-treated group. The activity of rhTPO was defined as 100% activity, and the EC50 value of lusutrombopag was 0.31 µmol/L (Fig. 3A). CFU-Mk colonies of rhTPO and lusutrombopag were immunohistochemically detected using anti-human CD41 antibody. CFU-Mk colonies did not morphologically differ between rhTPO and lusutrombopag (Fig. 3B). For further investigation of megakaryocyte maturation, we examined megakaryocytic ploidization induced by lusutrombopag using a flow cytometer. HuBM-CD34-positive cells were cultured under serum-free conditions with lusutrombopag or rhTPO for 10 days. DNA ploidy of megakaryocytes in lusutrombopag had a distribution similar to that in rhTPO (Fig. 3C). These findings indicate that lusutrombopag is able to differentiate mature megakaryocytes from human hematopoietic stem cells.

Thrombopoietic activity of lusutrombopag in TPOR-Ki/Shi mice [2]
Lusutrombopag induced proliferative activity in human TPO receptor-expressing cells, but induced no proliferative activity in murine TPO receptor-expressing cells in vitro. Platelet production activity of lusutrombopag was evaluated in TPOR-Ki/Shi mice or wild-type mice. Lusutrombopag or vehicle (0.5% MC) was orally administered to TPOR-Ki/Shi mice or wild-type mice once a day for 14 days and the platelets were counted on days 7, 14, 21, and 28. Fig. 6 illustrates the development of platelet production after lusutrombopag administration. Platelet production was used to determine the platelet increase ratio (dividing platelet count on treatment by basal platelet count). Platelet increase ratios of TPOR-Ki/Shi mice treated with lusutrombopag were significantly higher than those of the vehicle-treated TPOR-Ki/Shi. The platelet number was increased 2.5 times by the treatment with lusutrombopa. After discontinuation of lusutrombopag, the platelets decreased by day 21 and returned to close to the basal level on day 28. In contrast, the platelets of wild-type mice treated with lusutrombopag did not increase during the experimental period. To clarify its minimum thrombocytopoietic effect, lusutrombopag at doses of 0.3, 1, 3, and 10 mg/kg/day or vehicle (0.5% MC) was orally administered to TPOR-Ki/Shi mice once daily for 21 consecutive days. Blood samples were collected from veins at group assignment (day 0), prior to dosing on days 8 and 15, and 24 hours after dosing on day 21 (day 22), and then the platelets in each sample were counted. Lusutrombopag significantly increased platelet count on day 8, in a dose-dependent manner, as compared with the vehicle control group. The platelet numbers continued to increase on days 15 and 22 (Fig. 7). The minimum effective dose of lusutrombopag was 0.3 mg/kg/day in the 21-day repeated oral administration study.

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Because lusutrombopag has high species specificity for human TPOR, there was no suitable experimental animal model for drug evaluation, except for immunodeficient mouse-based xenograft models. Therefore, a novel genetically modified knock-in mouse, TPOR-Ki/Shi, was developed by replacing mouse Mpl with human-mouse chimera Mpl. In TPOR-Ki/Shi mice, lusutrombopag significantly increased circulating platelets in a dose-dependent manner during 21-day repeated oral administration. Histopathological study of the TPOR-Ki/Shi mice on day 22 also revealed a significant increase in megakaryocytes in the bone marrow. These results indicate that lusutrombopag acts on human TPOR to upregulate differentiation and proliferation of megakaryocytic cells, leading to platelet production.[2]

Proliferation assay [2]
Ba/F3-hMpl cells and Ba/F3-mMpl cells were engineered from the murine interleukin (IL)-3-dependent pro-B-cell line Ba/F3, as previously described. Cells were cultured at a density of 7.5 × 103 cells/200 µL in 96-well plates with lusutrombopag (4.88–5000 nmol/L) or recombinant human TPO (rhTPO: 4.88–5000 pmol/L). The plates were incubated for 3 days at 37°C in a humidified chamber with 5% CO2; 10 µL WST-8 reagent was added to each well during the last 2–8 hours of culture. The absorbance was measured at a wavelength of 450 nm using a 96-well microplate reader.

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Megakaryocytic colony formation and ploidy assay [2]
Human bone marrow-derived CD34-positive (HuBM-CD34-positive) cells were obtained from human tissue with informed consent. This study was approved by the relevant institutional ethics committee. A colony-forming unit-megakaryocyte (CFU-Mk) assay was performed using the MegaCult-C kit. HuBM-CD34-positive cells were cultured on chamber slides with lusutrombopag or rhTPO for 12 days, the slides were stained with anti-human CD41 antibody, and the CFU-Mk colonies were counted. The 50% effective concentration (EC50) of lusutrombopag was calculated using the sigmoid maximum pharmacological effect (Emax) model. To investigate megakaryocyte maturation, HuBM-CD34 positive cells were cultured with lusutrombopag or hTPO for 10 days, and megakaryocytic ploidization was detected using a flow cytometer. More details of methods can be found in the Supplementary Methods (online only, available at www.exphem.org).

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Western blotting [2]
Ba/F3-hMpl cells were cultured in RPMI containing 0.5% bovine serum albumin (BSA, Wako, Osaka, Japan) for 5 hours and stimulated with 3 µM lusutrombopag or 1 nM rhTPO at 37°C for 15 minutes. After stimulation with lusutrombopag or rhTPO, the cells were washed with ice-cold PBS and lysed at 4°C for 30 minutes in lysis buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail. The protein samples were added to equal volume of 2 × Laemmli's sample buffer / 10% 2-mercaptoethanol, and boiled at 95°C for 5 min. The protein samples were separated by SDS-PAGE and transferred onto Immun-Blot PVDF Membrane. The blots were blocked by 5% BSA in TBS-T (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.05% Tween20) for 2 hours, and then incubated overnight at 4°C with anti-phospho JAK2 (Tyr1007/1008), anti-phospho STAT3 (Tyr705), anti-phospho STAT5 and anti-phospho p44/42 MAP kinase (Thr202/204) antibodies. The blots were stripped with Re-blot plus mild solution and re-probed with anti-JAK2, anti-STAT3, anti-STAT5 and anti-p44/42 MAP kinase antibodies. The blots were incubated with ECL Western blotting detection reagent and analysis system and exposed to Amersham Hyperfilm ECL.

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CFU-MK assay [2]
HuBM-CD34 positive cells were cultured with lusutrombopag (0.0923–9.23 µM) or rhTPO (1.8 nM) and after 12 days of incubation, the slides were stained with anti-human CD41 antibody and the CFU-Mk colonies were counted. The maximum CFU-Mk activity of rhTPO was defined as 100%, and 50% effective concentration (EC50) of lusutrombopag was calculated using the sigmoid maximum pharmacological effect (Emax) model.

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Megakaryocytes in liquid culture and ploidy analysis [2]
HuBM-CD34 positive cells were cultured at a density of 7.5 × 104 cells/mL in 24-well plates. As a serum-free medium, we used Iscove's modified Dulbecco's medium supplemented with 20% BIT9500 and 40 µg/mL human low density lipoprotein (LDL). Cells were treated with 3 µM lusutrombopag or 1 nM rhTPO in triplicate for 10 days at 37°C in a humidified chamber with 5% CO2. The cells were resuspended in Hank's Balanced Salt Solutions (HBSS) containing 13.6 mM sodium citrate, 2 µM prostaglandin E-1, 1 mM theophylline, 3% BSA, 11 mM glucose (Mk medium). Cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-human CD41 antibody and propidium iodide as described previously. The cells were analyzed using an EPICS-XL flow cytometer. Data was obtained by electronically gating PI-stained CD41 positive cells, and at least 10,000 cells were analyzed for each sample. The ploidy distribution was determined by setting markers at the nadirs between the peaks.

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
Following oral administration, lusutrobopal is rapidly absorbed. Its pharmacokinetic profile is dose-proportional across single doses ranging from 1 mg to 50 mg, with similar pharmacokinetic characteristics in healthy subjects and patients with chronic liver disease. In healthy subjects, after administration of 3 mg lusutrobopal, the geometric mean (%CV) of its peak concentration (Cmax) and area under the curve (AUC) was 111 (20.4) ng/mL and 2931 (23.4) ng·hr/mL, respectively. With repeated once-daily dosing, the cumulative ratio of Cmax to AUC was approximately 2, and steady-state plasma lusutrobopal concentrations were reached after day 5. In patients with chronic liver disease, the time to peak plasma concentration (Tmax) after oral administration was approximately 6 to 8 hours. Food intake has been reported not to affect the absorption and bioavailability of lusutrobopal. Approximately 1% of the administered dose is excreted in the urine. 83% of the total dose is excreted in the feces, of which 16% is excreted unchanged. The mean (%CV) apparent volume of distribution of rusetrobopap in healthy adult subjects was 39.5 (23.5) L. The mean (%CV) clearance of rusetrobopap in patients with chronic liver disease was estimated to be approximately 1.1 (36.1) L/hr. Metabolism/Metabolites: CYP4 enzymes, particularly CYP4A11, are primarily involved in the metabolism of rusetrobopap. Rusetrobopap has been reported to undergo primarily ω- and β-oxidation, as well as glucuronidation. Biological Half-Life: In healthy adult subjects, the terminal elimination half-life (t1/2) was approximately 27 hours.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
There is currently no information regarding the use of lusutrombopag during lactation. The manufacturer recommends avoiding breastfeeding during lusutrombopag use and for at least 28 days after the last dose.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Protein Binding
The plasma protein binding rate of lusutrombopag exceeds 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
Studies have found that the AUC of russutrobappa is associated with an increase in platelet count. In patients with chronic liver disease and thrombocytopenia, after daily administration of 3 mg russutrobappa, the mean (standard deviation) maximum platelet count in patients (N=74) who did not receive platelet transfusions was 86.9 (27.2) × 10^9/L, and the median time to reach maximum platelet count was 12.0 (5 to 35) days. Rusutrobappa at 8 times the recommended dose did not show any clinically significant QTc interval prolongation. Rusutrobappa belongs to the cinnamic acid class of drugs. Rusutrobappa is an orally bioavailable thrombopoietin receptor (TPOR) agonist developed by Shionogi & Co., Ltd. (Osaka), Japan. TPOR is a regulatory target of endogenous thrombopoietin, a major cytokine that promotes megakaryocyte proliferation and differentiation and affects other hematopoietic lineages, including erythroid, granulocytic, and lymphoid. Thrombocytopenia (abnormally low platelet count) is a common complication of chronic liver disease. This hematological abnormality, especially in severe thrombocytopenia (platelet count <50,000/μL), poses a challenge to patients requiring invasive medical procedures due to the high risk of spontaneous bleeding. Rusutrobappa binds to the transmembrane domain of TPOR expressed on the surface of megakaryocytes, inducing hematopoietic stem cell proliferation and differentiation into megakaryocyte progenitors. In September 2015, Rusutrobappa received its first global marketing authorization in Japan for reducing the need for platelet transfusions before invasive medical procedures in adult patients with chronic liver disease and thrombocytopenia. On July 31, 2018, russutrobappa, under the brand name Mulpleta, received approval from the U.S. Food and Drug Administration (FDA) for the same therapeutic indication. In two randomized, double-blind, placebo-controlled trials, russutrobappa was administered orally to patients with chronic liver disease and severe thrombocytopenia who underwent invasive medical procedures and had platelet counts below 50 × 10⁹/L. Compared to the placebo group, a higher proportion (65-78%) of patients in the russutrobappa group did not require platelet transfusions prior to their first invasive medical procedure. Rusutrobappa is currently in Phase III clinical development in several European countries, including Austria, Belgium, Germany, and the United Kingdom. Rusutrobappa is an orally administered thrombopoietin (TPO) receptor (TPOR; MPL) agonist with potential megakaryocyte-stimulating activity. After administration, russutrobappa binds to and interacts with the transmembrane domain of the human TPO receptor expressed on the surface of megakaryocytes, thereby promoting the differentiation of hematopoietic stem cells into megakaryocyte progenitor cells. This can increase platelet production and may prevent or treat thrombocytopenia in patients with chronic liver disease. TPOR is a cytokine receptor belonging to the hematopoietic receptor superfamily. Lusutrombopag is a small molecule drug that has completed Phase IV clinical trials (covering all indications) and was first approved in 2018 for the treatment of thrombocytopenia and bleeding, with one investigational indication. Lusutrombopag (Mulpleta®) is an orally bioavailable small molecule thrombopoietin (TPO) receptor agonist developed by Shionogi & Co., Ltd. for the treatment of patients with chronic liver disease (CLD) and thrombocytopenia prior to elective interventional procedures. Lusutrombopag selectively acts on human TPO receptors, activating signal transduction pathways and promoting the proliferation and differentiation of bone marrow cells into megakaryocytes, thereby increasing platelet levels. In September 2015, lusutrombopag received the first global marketing authorization in Japan for improving chronic liver disease-related thrombocytopenia in patients undergoing elective interventional procedures. This article summarizes the key milestones in the development of lusutrombopag, which ultimately led to its approval. [1]

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TPO-Ki/Shi mice are a suitable animal model for evaluating the systemic effects of synthetic TPOR agonists, the activity of which depends on histidine residues in the c-Mpl transmembrane region. The results showed that lusutrombopag promotes platelet production by acting on TPOR, upregulating megakaryocyte differentiation and proliferation. [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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.23 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (4.23 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.23 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)