JAK2; STAT3/5
Butyzamide targets the thrombopoietin receptor Mpl (also known as TPO-R or CD110). Mpl is a cytokine receptor that plays a critical role in megakaryopoiesis and platelet production. By acting as a potent and specific non-peptidyl agonist, Butyzamide binds to human Mpl and activates downstream signaling pathways including JAK2, STAT3, STAT5, and MAPK. It shows species specificity for human Mpl and does not stimulate other cytokine receptors such as EPO, G-CSF, GM-CSF, or IL-3.

In Ba/F3-hMpl cells, butyzamide (3 μM; 15 min) phosphorylates JAK2, STAT3, STAT5, and MAPK [1]. Human CD34+ hematopoietic progenitors are stimulated by butyzamide (3 μM; 48 hours) to produce polyploid megakaryocytes and megakaryocytes that form colony-forming units [1].

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In vitro, Butyzamide promotes the proliferation of Ba/F3 cells expressing human Mpl (Ba/F3-hMpl) with an EC50 of 10 nM. It does not promote the proliferation of parental Ba/F3 cells that do not express human Mpl. At 3 μM for 15 minutes, Butyzamide induces the phosphorylation of JAK2, STAT3, STAT5, and MAPK in Ba/F3-hMpl cells. It stimulates human CD34+ hematopoietic progenitor cells to produce polyploid megakaryocytes and colony-forming unit-megakaryocytes at 3 μM for 48 hours.

Butanamide (10 mg/kg, 50 mg/kg; lateral; once daily for 20 days) elevates human prospective levels in NOG implanted with human fetal liver-derived CD34+ cells [1]. < br/> Butyzamide increased human platelets in FL-CD34+-transplanted NOG mice[1]
To evaluate the ability of butyzamide to produce human platelets in vivo, FL-CD34 cells were transplanted into immunodeficient NOG mice which do not have lymphocytes or natural killer cells, and have low complement activity.28 Several investigators have used NOG mice to analyze hematopoiesis of stem cells, mast cells and megakaryocytes, because the engraftment rate of human cells is higher in NOG mice than in NOD/SCID mice.26,29 When butyzamide was administered at a dose of 10 or 50 mg/kg once daily for 20 days to NOG mice transplanted with FL-CD34 cells, it increased the number of human platelets but did not affect the number of murine platelets (Figure 4A, B). Before the increase in human platelets, reticulated human CD41 platelets, which were stained by thiazole orange, were also increased by butyzamide (Figure 4C). After treatment with butyzamide for 21 days, the mice were sacrificed and bone marrow cells were collected by flushing the femora. The number of human CD41 megakaryocytes in the bone marrow was increased by the administration of butyzamide (Figure 4D). The femora on the other side were stained with anti-human CD42b antibody to visualize human megakaryocytes in paraffin sections (Figure 4E). Treatment with butyzamide increased human CD42b cells compared with the controls, which indicates that butyzamide stimulates the proliferation and maturation of human megakaryocytes in vivo. These data show that oral administration of butyzamide is effective at increasing the number of human platelets in vivo.[1]

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In vivo, oral administration of Butyzamide (10 or 50 mg/kg; once daily for 20 days) increases human platelet levels in NOG mice transplanted with human fetal liver-derived CD34+ cells. It increased the number of human CD41+ megakaryocytes in the bone marrow and stimulated the proliferation and maturation of human megakaryocytes in vivo. Butyzamide did not affect the number of murine platelets, demonstrating specificity for human thrombopoiesis.

For the establishment of Ba/F3-hMpl, Ba/F3-mMpl and Ba/F3-hMpl(H499L) cells, Ba/F3 cells were transfected with each constructed plasmid by electroporation. The cells were cloned by a limiting dilution method in the presence of 2 mg/mL geneticin. For the establishment of Ba/F3-mMpl(L490H) cells, 293GP2 packaging cells were transfected with pQCXIP-mMpl(L490H) and pVSV-G vector by Fugene 6, according to the manufacturer’s protocol. The culture supernatant was collected and used to infect Ba/F3 cells. These established stable cell lines proliferated in response to rmIL-3, as well as parental Ba/F3 cells, and appeared to maintain the character of parental Ba/F3 cells [1].

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The in vitro enzyme assay for Butyzamide typically involves measuring its binding affinity or activation of the Mpl receptor. For example, in a cell-based proliferation assay, Ba/F3-hMpl cells are cultured in the presence of varying concentrations of Butyzamide, and cell proliferation is measured using a colorimetric method such as MTT or CellTiter-Glo. EC50 values are calculated from dose-response curves. Phosphorylation of downstream signaling proteins is assessed by Western blotting using phospho-specific antibodies.

Cells were cultured at a density of 7.5×10 cells/200 μL in 96-well plates with various concentrations of butyzamide or recombinant cytokines. The plates were incubated for 48 h at 37°C in a humidified chamber with 5% CO2, and 10 μL WST-8 reagent were added to each well during the last 4 h of culture. The absorbance was measured at a wavelength of 450 nm using a 96-well microplate reader, Model 680 [1].

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Liquid culture of human bone marrow-derived CD34+ cells[1]
Human bone marrow-derived CD34 cells were cultured at a density of 5.0×10 cells/mL in 24-well plates. As a serum-free medium, we used Iscove’s modified Dulbecco’s medium supplemented with 20% BIT9500. Cells were treated with 1 nM rhTPO or 3 μM butyzamide in triplicate for 10 days at 37°C in a humidified chamber with 5% CO2.

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In vitro cellular activity is assessed in Ba/F3-hMpl cells and human CD34+ hematopoietic progenitor cells. Ba/F3-hMpl cells are treated with Butyzamide at concentrations ranging from 0.1 to 10 μM, and cell proliferation is measured after 48-72 hours using a cell viability assay. For signaling studies, cells are treated with 3 μM Butyzamide for 15 minutes, and protein phosphorylation is analyzed by Western blotting. Megakaryocyte differentiation is assessed by culturing human CD34+ cells with Butyzamide for 48 hours and counting colony-forming units.

Immunodeficient NOD/Shi-scid,IL-2Rγ (NOG) mice (8–10 weeks old) were irradiated with 2.4 Gy (125 kV, 10 mA, 0.45 Gy/min) by MBR-1520R-3 (Hitachi Medico, Tokyo, Japan), and on the next day, 6.7×10 human fetal liver (FL)-derived CD34 cells were transplanted intravenously. Two months after transplantation, the NOG mice were randomized into three groups, based on the number of human platelets and body weight. The vehicle or butyzamide at a dose of 10 or 50 mg/kg was administered orally for 21 days. The numbers of human platelets and megakaryocytes were calculated as described below [1].

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In vivo efficacy studies are conducted in immunodeficient NOD/Shi-scid,IL-2Rγnull (NOG) mice that have been transplanted with human fetal liver-derived CD34+ cells. Butyzamide is administered orally at doses of 10 or 50 mg/kg once daily for 20 days. Human platelet levels in peripheral blood are measured by flow cytometry using a human-specific anti-CD41 or anti-CD42b antibody. Bone marrow cells are harvested to quantify human CD41+ megakaryocytes.

Pharmacokinetic data for Butyzamide indicates it is orally bioactive. As a small molecule, it is expected to have favorable oral bioavailability. Detailed PK parameters such as half-life, Cmax, and AUC are not extensively reported in the available literature. The compound is soluble in DMSO at 10 mM and is typically administered orally for in vivo studies.

In vivo toxicity data for Butyzamide is not extensively reported. In mouse studies, Butyzamide was well-tolerated at doses of 10 and 50 mg/kg administered orally once daily for 20 days. No significant adverse effects were reported in these studies. As with all research compounds, Butyzamide is intended for research use only and is not for human therapeutic applications. Comprehensive toxicology studies would be required for clinical development.

[1]. The effect of a novel, small non-peptidyl molecule butyzamide on human thrombopoietin receptor and megakaryopoiesis. Haematologica. 2008 Oct;93(10):1495-504.

Background: Thrombocytopenia is a common complication of treatment for cancer and other hematopoietic cell disorders. Previous clinical trials have shown that polyethylene glycol (PEG) conjugated recombinant human megakaryocyte growth and development factor (rHMGF) can increase platelet counts in patients with idiopathic thrombocytopenic purpura and solid tumors undergoing chemotherapy. However, antibodies against PEG-conjugated rHMGF are generated in healthy volunteers and patients undergoing chemotherapy, and these antibodies cross-react with endogenous thrombopoietin. Therefore, clinical development of PEG-conjugated recombinant human megakaryocyte growth and development factor was terminated in 1998. This study aimed to find an Mpl activator with high oral bioavailability that does not generate autoantibodies against endogenous thrombopoietin. [1]
Design and Methods: We screened a library of compounds and synthesized a novel non-peptide thrombopoietin receptor Mpl activator, named butyzamide. We evaluated the effect of butyzamide on megakaryocyte generation in vitro using Mpl-expressing Ba/F3 cells and artificial hematopoietic stem cells. To assess its in vivo effects, we orally administered butyramide to immunodeficient NOD/Shi-scid,IL-2R γ (NOG) mice transplanted with human fetal liver-derived CD34⁽⁺⁾ cells and investigated human platelet production. [1] Results: Butyramide specifically reacted with human MPl and activated the same signal transduction pathway as thrombopoietin. However, unlike thrombopoietin, butyramide did not react with mouse MPl, and its agonistic activity required histidine residues in the transmembrane domain of MPl. Butyramide induced human CD34⁽⁺⁾ hematopoietic progenitor cells to differentiate into colony-forming units—megakaryocytes and polyploid megakaryocytes, with effects comparable to thrombopoietin. When butyramide was administered orally at doses of 10 and 50 mg/kg for 20 consecutive days to NOG mice transplanted with human fetal liver-derived CD34(+) cells, human platelet counts increased by 6.2-fold and 22.9-fold, respectively. [1] Conclusion: Butyramide is an MPl activator with high oral bioavailability and has clinical development potential as a treatment for thrombocytopenia. [1]

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Butyzamide is a novel non-peptidyl thrombopoietin receptor agonist with potential for treating thrombocytopenia. It was discovered as a small molecule that mimics the activity of thrombopoietin by binding to and activating the Mpl receptor. The compound shows species specificity for human Mpl, making it a valuable tool for studying human thrombopoiesis in mouse xenograft models. Butyzamide is for research use only and is not approved for therapeutic use.

C29H32CL2N2O5S

590.14

1110767-45-7

44602781

White to off-white solid

7.6

2

7

11

39

857

0

CCCOC(C1=CC=CC(=C1OC)C2=CSC(=N2)NC(=O)C3=CC(=C(C(=C3)Cl)/C=C(\C)/C(=O)O)Cl)C(C)(C)C

BBEZGQPYGPBSLA-FOWTUZBSSA-N

InChI=1S/C29H32Cl2N2O5S/c1-7-11-38-25(29(3,4)5)19-10-8-9-18(24(19)37-6)23-15-39-28(32-23)33-26(34)17-13-21(30)20(22(31)14-17)12-16(2)27(35)36/h8-10,12-15,25H,7,11H2,1-6H3,(H,35,36)(H,32,33,34)/b16-12+

(E)-3-[2,6-dichloro-4-[[4-[3-(2,2-dimethyl-1-propoxypropyl)-2-methoxyphenyl]-1,3-thiazol-2-yl]carbamoyl]phenyl]-2-methylprop-2-enoic acid

Butyzamide; 1110767-45-7; (E)-3-(2,6-Dichloro-4-((4-(3-(2,2-dimethyl-1-propoxypropyl)-2-methoxyphenyl)thiazol-2-yl)carbamoyl)phenyl)-2-methylacrylic acid; SCHEMBL3059522; SCHEMBL3059524;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~169.05 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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