Thrombopoietin Receptor; thrombopoietin receptor (TpoR)

When luciferase reporter gene is transfected into mouse BAF3 cells, eltrombopag (0.002-50 μM; 4 h) exhibits activity [1]. Eltrombopag (30 μM; 120 min) has an impact on the way that p-N2C-Tpo STAT5 is activated in cells [1]. In megakaryocytes, eltrombopag (30 μM; 120 minutes) stimulates p-STAT5[1]. BAF3/hTpoR cell proliferation is stimulated by eltrombopag (0.1 nM-10 μM; 30 min) [1]. Eltrombopag (0.03-3 μM; 10 days) promotes CD34+ cells in the bone marrow to differentiate into CD41+ megakaryocytes [1]. N2C-Tpo cell apoptosis is impacted by eltrombopag (0-3 μM; 72 h)[1]. With a MIC50 of 0.3 mg/L, eltrombopag efficiently prevents the growth of pneumococci, but it is ineffective against Gram-negative bacteria [3]. Eltrombopag (0 -200 mg/L; 24 hours; HepG2 and Caco-2 cells) has a MIC50 of 1.5 mg/L, which is less effective than that of vancomycin (MIC50 of 1.2 mg/L), when used in combination. L[3]. In Huh7 cells, eltrombopag (0 or 10 μg/mL; 72 h) strongly promotes G0/G1 phase arrest [5]. On HCC cell lines, eltrombopag (0.1-100 μg/mL; 72 h) exhibits anti-proliferative action [5].

Chimpanzees can tolerate Eltrombopag Olamine (10 mg/kg) when given orally once day for five days [1]. Mean S is greatly decreased by elotrombopag Olamine (17.6 mg/kg; i.p.; once daily for two days). Mice's nasal infections with aureus numbers [3].

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The in vivo activity of eltrombopag was demonstrated by an increase of up to 100% in platelet numbers when administered orally (10 mg/kg per day for 5 days) to chimpanzees. In conclusion, eltrombopag interacts selectively with the TpoR without competing with Tpo, leading to the increased proliferation and differentiation of human bone marrow progenitor cells into megakaryocytes and increased platelet production. These results suggest that eltrombopag and Tpo may be able to act additively to increase platelet production.[1]

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Repeat Dose Study in Chimpanzees [1]
Given the distinct species specificity for STAT activation exhibited by eltrombopag in human and chimpanzee platelets, a 5-day, repeat-dose safety and pharmacology study was conducted in five female chimpanzees. Chimpanzees were administered either vehicle alone (n = 2) or eltrombopag in vehicle (n = 3; 10 mg/kg per day) by oral gavage. Administration of eltrombopag was well tolerated following repeat oral doses. There were no adverse effects on hematology, coagulation, or clinical chemistry parameters. In chimpanzees treated with eltrombopag, platelet counts were increased over twofold approximately 1 week after the last dose for one chimpanzee and approximately 1.5-fold for the other two chimpanzees. Platelet counts returned to near baseline values approximately 2 weeks after peak values were reached (Fig. 6).

Luciferase Reporter Gene Assay [1]
BAF3/hTpoR or 32D-mpl cells were washed and starved of rmIL-3 or rhTpo overnight prior to treatment. Starved parental BAF3 cells (1 × 105 cells/ml) in Iscove's modified Dulbecco's medium (IMDM)/0.5% fetal bovine serum (FBS) and 30 μM ZnCl2 were treated with Eltrombopag (0.002–50 μM) or rhTpo (100 ng/ml) at 37°C, 5% CO2, for 4 hours. Cells were lysed in 100 μl lysis buffer (25 mM tris, 15% glycerol, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 1% lecithin, 1% bovine serum albumin, 4 mM EGTA, 8 mM MgCl2, 10 mM dithiothreitol, and 0.4 mM phenylmethylsulfonyl fluoride, pH 7.8) for 15 minutes and added to a 96-well plate (30 μl per well). Promega Steady Glow (100 μl) was added to each well immediately before the plates were read using a chemiluminometer. Luciferase assay data are presented as the mean and standard error of quadruplicate wells.

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Caspase-3 and Caspase-7 Assays [1]
The Caspase-Glo 3/7 assay (Promega) is a luminescent assay that measures caspase-3 and caspase-7 activity. The addition of the Caspase-Glo reagent results in cell lysis, followed by caspase cleavage of the substrate and generation of a luminescent signal; the amount of luminescence is proportional to the amount of caspase present. Cytokine-starved N2C-Tpo cells (1.4 × 105 cells/ml final concentration) were grown in a white view plate and exposed to Eltrombopag (0.003–3 μM) and/or rhTpo (1–100 ng/ml) for 72 hours at 37°C. Caspase-Glo (100 μl) was added, and cells were incubated for 90 minutes at room temperature.

Cell Viability Assay[1]
Cell Types: Murine BAF3 cells
Tested Concentrations: 0.002-50 μM
Incubation Duration: 4 h
Experimental Results: Effectively inhibited murine BAF3 cells with human TpoR with an EC50 value of 0.27 μM. [1][1]
Cell Types: N2C-Tpo cells and CD34+
Tested Concentrations: 30 μM for N2C-Tpo cells; 0, 1, 3 and 10 μM for CD34+
Incubation Duration: 120 min for N2C-Tpo cells; 30 min for CD34+
Experimental Results: Activated phospho-STAT5 and maximum signal intensity demonstrated at 60 minutes after treatment in N2C-Tpo cells. Dose-dependently activated STAT5 phosphorylation at 30 minutes after treatment in CD34+.

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Cell Proliferation Assay[1]
Cell Types: BAF3/hTpoR cells
Tested Concentrations: 0.1 nM-10 μM
Incubation Duration: 2 days
Experimental Results: Promoted BAF3/hTpoR cells proliferation after incubated for 2 days with an EC50 of 0.03 μM.

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Cell Differentiation Assay[1]
Cell Types: CD34+
Tested Concentrations: 0.003, 0.01, 0.03, 0.1, 0.3, 1 and 3 μM
Incubation Duration: 10 days
Experimental Results: Dose-dependently stimulated the differentiation from bone marrow CD34+ cells to CD

Animal/Disease Models: Female chimpanzees[1]
Doses: 10 mg/kg
Route of Administration: po (oral gavage); 10 mg/kg one time/day; for 5 days
Experimental Results: Appeared a goes up and then goes back tendency of platelet counts after treatment, and demonstrated no bad effects of hematology, coagulation, or clinical chemistry parameters on animal.

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Animal/Disease Models: C57BL/6 male mice (7 weeks, 20-22 g; injected S. aureus (5 × 108 CFU suspended in 40 µL PBS) into the nasal cavities )[3]
Doses: 17.6 mg/kg
Route of Administration: IP; one time/day for 2 days
Experimental Results: Dramatically decreased mean bacterial counts (5.0 × 106 CFU/lung) in the nasal infection model compared with control PBS (5.2 × 107 CFU/lung) lung) mice.

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Female chimpanzees (approximately 7–8 years of age) were given either Eltrombopag (10 mg/kg) in aqueous 2% hydroxypropyl methylcellulose with 0.2% sodium lauryl sulfate vehicle or vehicle alone by oral gavage at a dose volume of 1 ml/kg. Chimpanzees were given five daily doses of vehicle alone (n = 2) or Eltrombopag (n = 3). Platelet counts and reticulated platelet counts were performed prior to, during, and following the treatment regimen. At the end of the study, all chimpanzees were returned to the stock colony.[1]

Although only limited pharmacokinetic sampling in three chimpanzees was performed, the data suggest that the pharmacodynamic signal of a change in platelet count from baseline for eltrombopag in the chimpanzees was detected at minimum concentration (Cmin), Cmax, and area under the curve (AUC) values of approximately 0.107 μg/ml, 0.525 μg/ml, and 12.1 μg h/ml, respectively. [1]

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Minimal information is available on the use of eltrombopag during breastfeeding. One breastfed infant with thrombocytosis at birth that was possibly prolonged by eltrombopag in milk. Until more data become available, romiplostim should be used with careful infant monitoring of infant blood parameters during breastfeeding, especially while nursing a newborn or preterm infant. The manufacturer recommends avoiding breastfeeding during the use of eltrombopag. Based on the drug’s half-life, the drug should be eliminated by the mother 8 days after the last dose.
◉ Effects in Breastfed Infants
An infant was born to a mother taking eltrombopag in a maximum dose of 100 mg during pregnancy. At birth, the infant had thrombocytosis, which persisted for a few weeks while the mother was breastfeeding. The extent of breastfeeding and the maternal dose were not stated. The authors felt that the persistence of thrombocytosis in the infant was possibly caused by eltrombopag in milk.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

[1]. Preclinical activity of eltrombopag (SB-497115), an oral, nonpeptide thrombopoietin receptor agonist. Stem Cells. 2009 Feb;27(2):424-30.

[2]. Discovery and characterization of a selective, nonpeptidyl thrombopoietin receptor agonist. Exp Hematol. 2005 Jan;33(1):85-93.

[3]. Repurposing Eltrombopag for Multidrug Resistant Staphylococcus aureus Infections. Antibiotics (Basel). 2021 Nov 9;10(11):1372.

[4]. Identification of Eltrombopag as a Repurposing Drug Against Staphylococcus epidermidis and its Biofilms. Curr Microbiol. 2021 Feb 21.

[5]. The Eltrombopag antitumor effect on hepatocellular carcinoma. Int J Oncol. 2015 Nov;47(5):1696-702.

Eltrombopag is a hydrazine in which each nitrogen atom is substituted, one by a 3'-carboxy-2-hydroxy[1,1'-biphenyl]-3-yl group and the other by a 1-(3,4-dimethylphenyl)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazol-4-ylidene group. A small molecule agonist of the c-mpl (TpoR) receptor (the physiological target of the hormone thrombopoietin), it has been developed as a medication for conditions that lead to thrombocytopenia (abnormally low platelet counts). It has a role as a thrombopoietin receptor agonist and a xenobiotic. It is a member of hydrazines, a member of pyrazoles and a member of benzoic acids.
Eltrombopag is a Thrombopoietin Receptor Agonist. The mechanism of action of eltrombopag is as a Thrombopoietin Receptor Agonist, and Organic Anion Transporting Polypeptide 1B1 Inhibitor, and Breast Cancer Resistance Protein Inhibitor, and UGT1A1 Inhibitor, and UGT1A3 Inhibitor, and UGT1A4 Inhibitor, and UGT1A6 Inhibitor, and UGT1A9 Inhibitor, and UGT2B7 Inhibitor, and UGT2B15 Inhibitor. The physiologic effect of eltrombopag is by means of Increased Megakaryocyte Maturation, and Increased Platelet Production.
See also: Romiplostim (annotation moved to); Eltrombopag (annotation moved to).

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Drug Indication
Revolade is indicated for the treatment of adult patients with primary immune thrombocytopenia (ITP) who are refractory to other treatments (e. g. corticosteroids, immunoglobulins) (see sections 4. 2 and 5. 1). Revolade is indicated for the treatment of paediatric patients aged 1 year and above with primary immune thrombocytopenia (ITP) lasting 6 months or longer from diagnosis and who are refractory to other treatments (e. g. corticosteroids, immunoglobulins) (see sections 4. 2 and 5. 1). Revolade is indicated in adult patients with chronic hepatitis C virus (HCV) infection for the treatment of thrombocytopenia, where the degree of thrombocytopenia is the main factor preventing the initiation or limiting the ability to maintain optimal interferon-based therapy (see sections 4. 4 and 5. 1). Revolade is indicated in adult patients with acquired severe aplastic anaemia (SAA) who were either refractory to prior immunosuppressive therapy or heavily pretreated and are unsuitable for haematopoietic stem cell transplantation (see section 5. 1).

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Eltrombopag is a first-in-class, orally bioavailable, small-molecule, nonpeptide agonist of the thrombopoietin receptor (TpoR), which is being developed as a treatment for thrombocytopenia of various etiologies. In vitro studies have demonstrated that the activity of eltrombopag is dependent on expression of TpoR, which activates the signaling transducers and activators of transcription (STAT) and mitogen-activated protein kinase signal transduction pathways. The objective of this preclinical study is to determine if eltrombopag interacts selectively with the TpoR to facilitate megakaryocyte differentiation in platelets. Functional thrombopoietic activity was demonstrated by the proliferation and differentiation of primary human CD34(+) bone marrow cells into CD41(+) megakaryocytes. Measurements in platelets in several species indicated that eltrombopag specifically activates only the human and chimpanzee STAT pathways. [1]

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The continuous rise of antimicrobial resistance urgently demands new therapeutic agents for human health. Drug repurposing is an attractive strategy that could significantly save time delivering new antibiotics to clinics. We screened 182 US Food and Drug Administration (FDA)-approved drugs to identify potential antibiotic candidates against Staphylococcus aureus, a major pathogenic bacterium. This screening revealed the significant antibacterial activity of three small molecule drugs against S. aureus: (1) LDK378 (Ceritinib), an anaplastic lymphoma kinase (ALK) inhibitor for the treatment of lung cancer, (2) dronedarone HCl, an antiarrhythmic drug for the treatment of atrial fibrillation, and (3) eltrombopag, a thrombopoietin receptor agonist for the treatment of thrombocytopenia. Among these, eltrombopag showed the highest potency against not only a drug-sensitive S. aureus strain but also 55 clinical isolates including 35 methicillin-resistant S. aureus (Minimum inhibitory concentration, MIC, to inhibit 50% growth [MIC50] = 1.4-3.2 mg/L). Furthermore, we showed that eltrombopag inhibited bacterial growth in a cell infection model and reduced bacterial loads in infected mice, demonstrating its potential as a new antibiotic agent against S. aureus that can overcome current antibiotic resistance.[3]

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Staphylococcus epidermidis is a common cause of nosocomial infections, and readily adheres to medical apparatus to form biofilms consisting of highly resistant persister cells. Owing to the refractory infections caused by S. epidermidis biofilms and persisters in immunosuppressed patients, it is crucial to develop new antimicrobials. In the present study, we analyzed the antimicrobial effects of the thrombopoietin receptor agonist eltrombopag (EP) against S. epidermidis planktonic cells, biofilms, and persister cells. EP was significantly toxic to S. epidermidis with the minimal inhibitory concentration of 8 μg/ml, and effectively inhibited the biofilms and persisters in a strain-dependent manner. In addition, EP was only mildly toxic to mammalian cells after 12 to 24 h treatment. It also partially synergized with vancomycin against S. epidermidis, which enhanced its antimicrobial effects and reduced its toxicity to mammalian cells. Taken together, EP is a potential antibiotic for treating refractory infections caused by S. epidermidis.[4]

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Currently, sorafenib is the only available chemotherapeutic agent for advanced hepatocellular carcinoma (HCC), but it cannot be used in patients with liver cirrhosis (LC) or thrombocytopenia. In these cases, sorafenib is likely effective if given in combination with treatments that increase the number of platelets, such as thrombopoietin (TPO) receptor agonists. Increasing the platelet count via TPO treatment resulted in reduction of LC. Eltrombopag (EP), a TPO receptor agonist, has been reported to have antitumor effects against certain cancers, despite their lack of TPO receptor expression. We hypothesized that EP may possess antitumor activity against HCC in addition to its ability to suppress hepatic fibrosis by increasing the platelet count. In the present study, the antitumor activity of EP was examined by assessing the inhibition of cell proliferation and then ascertaining the ability of iron supplementation to reverse these effects in HepG2, Hep3B and Huh7 cells. In addition, a cell cycle assay was performed using flow cytometry, and signal transduction was evaluated by analyzing cell cycle-related protein expression. The results of EP were compared with those of the most common iron chelator, deferoxamine (DFO). The combined effect of EP and sorafenib was also assessed. The results revealed that EP exerts antitumor activity in HCC that is mediated by the modulation of intracellular iron content. EP suppressed the expression of the cell cycle-related protein cyclin D1 and elicited cell cycle arrest in the G0/G1 phase. The activity of EP was comparable to that of DFO in HCC, and EP did not compete with sorafenib at low concentrations. In conclusion, our findings suggest that EP is a good candidate chemotherapeutic agent for the treatment of HCC in patients with LC and thrombocytopenia.[5]

C25H22N4O4

442.47

442.164

C, 67.86; H, 5.01; N, 12.66; O, 14.46

496775-61-2

Eltrombopag Olamine;496775-62-3;(E/Z)-Eltrombopag-13C4;1217230-31-3

135449332

Typically exists as Yellow to orange or red solid at room temperature

1.3±0.1 g/cm3

656.8±65.0 °C at 760 mmHg

242-244ºC

351.0±34.3 °C

0.0±2.1 mmHg at 25°C

1.667

3.7

3

7

5

33

812

0

O=C1C(=C(C([H])([H])[H])N([H])N1C1C([H])=C([H])C(C([H])([H])[H])=C(C([H])([H])[H])C=1[H])/N=N/C1=C([H])C([H])=C([H])C(=C1O[H])C1C([H])=C([H])C([H])=C(C(=O)O[H])C=1[H]

SVOQIEJWJCQGDQ-UHFFFAOYSA-N

InChI=1S/C25H22N4O4/c1-14-10-11-19(12-15(14)2)29-24(31)22(16(3)28-29)27-26-21-9-5-8-20(23(21)30)17-6-4-7-18(13-17)25(32)33/h4-13,28,30H,1-3H3,(H,32,33)

3-[2-[(2Z)-1-(3,4-Dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazinyl]-2-hydroxy-[1,1-biphenyl]-3-carboxylic acid

Eltrombopag; SB497115; 496775-61-2; (Z)-3'-(2-(1-(3,4-Dimethylphenyl)-3-methyl-5-oxo-1H-pyrazol-4(5H)-ylidene)hydrazinyl)-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid; SB497,115; Eltrombopag [INN];SB-497115; SB 497115; SB497115GR; trade name: PROMACTA

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: ≥ 1 mg/mL (2.26 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 + to the above solution and mix evenly; then add 450 μL of 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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 (Please use freshly prepared in vivo formulations for optimal results.)