JAK2; STAT3/5

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].

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]

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].

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.

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].

[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 problem in the management of patients with cancer and other conditions that affect hematopoietic cells. In previous clinical trials, the polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor increased platelet counts in patients with idiopathic thrombocytopenic purpura and solid tumors undergoing chemotherapy. However, antibodies to polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor develop in healthy volunteers and patients undergoing chemotherapy and cross-react with endogenous thrombopoietin. As a result, clinical development of polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor was discontinued in 1998. The aim of this study was to identify an orally bioavailable human Mpl activator that does not develop autoantibodies against endogenous thrombopoietin.[1]
Design and methods: We screened our chemical library and created a novel non-peptidyl thrombopoietin receptor, Mpl activator named butyzamide. We evaluated the effect of butyzamide on megakaryopoiesis in vitro using Ba/F3 cells expressing Mpl and human hematopoietic stem cells. For the evaluation of its in vivo effect, we administered butyzamide orally to immunodeficient NOD/Shi-scid,IL-2R gamma(null) (NOG) mice transplanted with human fetal liver-derived CD34(+) cells and investigated the production of human platelets.[1]
Results: Butyzamide specifically reacted with human Mpl and activated the same signal transduction pathway as thrombopoietin. However, unlike thrombopoietin, butyzamide did not react with murine Mpl and was shown to require the histidine residue in the transmembrane domain of Mpl for its agonistic activity. Butyzamide induced colony-forming unit-megakaryocytes and polyploid megakaryocytes from human CD34(+) hematopoietic progenitor cells, and its effects were comparable to those of thrombopoietin. When butyzamide was administered orally at the doses of 10 and 50 mg/kg for 20 days to NOG mice transplanted with human fetal liver-derived CD34(+) cells, the human platelet count increased by 6.2- and 22.9-fold, respectively.[1]
Conclusions: Butyzamide is an orally bioavailable human Mpl activator, and appears to have potential for clinical development as a therapeutic agent for patients with thrombocytopenia.[1]

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.)