The proliferation of KASUMI-1 cells was considerably suppressed, dose-wise, by kevetrin hydrochloride (85, 170, 340 μM; 6 h), but not that of MOLM-13 [1]. Kevetrin hydrochloride (340 μM; 6) induces metallothionein (MT) expression in acute myeloid leukemia (AML) cells and downregulates p53 activity. Kevetrin hydrochloride (340 μM; 24 hours) induces KASUMI-1 cell line cells without causing changes to the cell cycle. Kevetrin hydrochloride is the forkhead box K2 regulator of WNT/β-catenin signaling Regulator-related gene signal transducer and regulatory activator 5A (STAT5A). P53 is increased by kevetrin hydrochloride (100, 200, and 400 μM; 48 h)[1].

The proliferation of KASUMI-1 cells was considerably suppressed, dose-wise, by kevetrin hydrochloride (85, 170, 340 μM; 6 h), but not that of MOLM-13 [1]. Kevetrin hydrochloride (340 μM; 6) induces metallothionein (MT) expression in acute myeloid leukemia (AML) cells and downregulates p53 activity. Kevetrin hydrochloride (340 μM; 24 hours) induces KASUMI-1 cell line cells without causing changes to the cell cycle. Kevetrin hydrochloride is the forkhead box K2 regulator of WNT/β-catenin signaling Regulator-related gene signal transducer and regulatory activator 5A (STAT5A). P53 is increased by kevetrin hydrochloride (100, 200, and 400 μM; 48 h)[1].

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Kevetrin induced dose-dependent decreases in cell viability in acute myeloid leukemia (AML) cell lines (TP53 wild-type: OCI-AML3 and MOLM-13; TP53-mutant: KASUMI-1 and NOMO-1) after 48 hours of continuous treatment at concentrations of 85, 170, and 340 µM. The mutant cell lines (KASUMI-1 and NOMO-1) showed higher sensitivity.[1]
Kevetrin induced significant apoptosis (Annexin V+ cells) in all tested AML cell lines after 48-hour treatment, particularly at 340 µM. For example, in MOLM-13 cells, apoptosis increased to 54.95±5.63% (vs. 12.53±6.15% in control); in NOMO-1 cells, to 60.93±2.63% (vs. 22.90±4.63% in control); and in KASUMI-1 cells, to 79.70±4.57% (vs. 13.18±0.80% in control).[1]
Kevetrin treatment (340 µM, 48 hours) led to mitochondrial membrane depolarization, DNA fragmentation (TUNEL assay), and caspase-3 activation in MOLM-13 and KASUMI-1 cells, confirming apoptosis induction.[1]
Cell cycle analysis revealed that Kevetrin did not alter the cell cycle in MOLM-13 and KASUMI-1 cells, but induced G0/G1 accumulation and reduced S-phase cells in OCI-AML3 and NOMO-1 cells after 24- and 48-hour treatments.[1]
In primary AML blasts from patients (including one TP53-mutant sample), Kevetrin (85-340 µM, 48 hours) caused a dose-dependent decrease in viability and increase in apoptosis, with preferential cytotoxicity against blast cells compared to monocytes and lymphocytes.[1]
Gene expression profiling after 48-hour treatment with 340 µM Kevetrin showed upregulation of genes involved in apoptosis, autophagy, NF-κB pathway, and MAPK activity, and downregulation of genes related to cell cycle, DNA repair, glycolysis, translation, and splicing.[1]
Western blot and immunofluorescence analyses indicated that Kevetrin treatment increased nuclear p53 localization and upregulated p21 protein levels in a dose-dependent manner in TP53 wild-type models.[1]
Short-term treatment (6 hours) with Kevetrin upregulated metallothionein (MT1/2) genes in both MOLM-13 and KASUMI-1 cells, and downregulated STAT5A, E2F4, GRWD1, and ELANE in KASUMI-1 cells.[1]

In tumor xenograft models, kevetrin hydrochloride (150–200 mg/kg; i.p.; 20 days) suppresses tumor development and prolongs focus, and it promotes about 40% cell death in OV-90 or OVCAR-3 xenograft tumors.

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In xenograft models, Kevetrin inhibited tumor growth in the A2780 (p53 wild-type) model.[2]
In the SKOV-3 (p53 partially deleted) xenograft ascites model, Kevetrin treatment significantly increased animal survival.[2]
Immunohistochemistry (IHC) analysis indicated that Kevetrin induced approximately 40% cell death in OV-90 and OVCAR-3 xenograft tumors.[2]
Transcriptomic analysis of xenograft tumor tissues showed that Kevetrin modulated the p53 signaling pathway in SKOV-3 tumors, as demonstrated by mRNAseq and small RNAseq with KEGG pathway analysis. This modulation was not observed in OV-90 xenograft tumors after treatment.[2]

Cell Viability Assay[1]
Cell Types: MOLM-13 and KASUMI-1 cells
Tested Concentrations: 85, 170 and 340 µM
Incubation Duration: 6 h, 6 h + mRNA and protein levels, and induce A2780 cells to produce p21 protein[1]. 66 h Wash-out (wo,×1), 6 h + 66 h wo (×2), 6 h + 66 h wo (×3)
Experimental Results: Only inhibited the cell viability of KASUMI-1 cells, reducing the cell viability dose and time dependent manner.

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Apoptosis analysis[1]
Cell Types: MOLM-13, KASUMI-1, TP53-wt OCI-AML3 and TP53 mutant NOMO-1 Cell
Tested Concentrations: 85, 170 and 340 µM
Incubation Duration: 24, 48 and 72 hrs (hours)
Experimental Results: Induces apoptosis of KASUMI-1 cells at 340 μM concentration for 24 hrs (hours) and inhibits MOLM-13 at 340 μM concentration for 48 hrs (hours).

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Cell cycle analysis[1]
Cell Types: MOLM-13, KASUMI-1, TP53-wt OCI-AML3 and TP53 mutant NOMO-1 Cell
Tested Concentrations: 340 µM
Incubation Duration: 24 and 48 hrs (hours)
Experimental Results: Cell cycle arrest OCI-AML3 and NOMO-1 cells are in the G0/G1 phase, and does not change the cell cycle of MOLM-13 and KASUMI-1 cells.

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Cell viability was assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay. Cells were seeded in 96-well plates and treated with Kevetrin (85, 170, 340 µM) for 24, 48, and 72 hours. Absorbance was measured at 490 nm.[1]
Apoptosis was evaluated by Annexin V-FITC/PI staining. Cells were treated with Kevetrin, incubated with Annexin V-FITC, and analyzed by flow cytometry. For primary samples, Annexin V staining was combined with surface marker antibodies (CD45, CD33, CD14, CD3, CD19).[1]
Mitochondrial membrane potential (ΔΨm) was assessed using a JC-1 based assay. Treated cells were incubated with JC-1 working solution and analyzed by flow cytometry.[1]
DNA fragmentation was detected by TUNEL assay. Cells were fixed, permeabilized, incubated with TdT and FITC-dUTP, counterstained with PI/RNase, and analyzed by flow cytometry.[1]
Active caspase-3 was measured using a FITC-conjugated anti-active caspase-3 antibody. Cells were fixed, permeabilized, incubated with the antibody, and analyzed by flow cytometry.[1]
Cell cycle analysis was performed by fixing cells in ethanol, staining with PI/RNase/NP40, and analyzing DNA content by flow cytometry.[1]
Gene expression profiling was performed using Human Transcriptome Array 2.0. RNA was isolated from cells untreated or treated with 340 µM Kevetrin for 6 and 48 hours. Data were analyzed with Expression Console and Transcriptome Analysis Console software.[1]
Western blot analysis was conducted on protein extracts from cells treated for 48 hours. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-p53 (Ser15), p53, p21, and β-actin.[1]
Immunofluorescence for p53 was performed on cells fixed after 48-hour treatment, incubated with anti-p53 antibody and Alexa Fluor 594 secondary antibody, mounted with DAPI, and imaged by confocal microscopy.[1]

Animal/Disease Models: A2780 nude mouse xenograft tumor model [2]
Doses: 200 mg/kg
Route of Administration: intraperitoneal (ip) injection; time [2]. 3 times a week for 20 days
Experimental Results: Inhibition of tumor growth and suppression of tumor volume.

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Animal/Disease Models: Mouse SKOV-3 xenograft ascites model [2]
Doses: 150 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: Prolonged mouse survival time, maintaining 100% survival rate for more than 35 days.

[1]. Kevetrin induces apoptosis in TP53 wildtype and mutant acute myeloid leukemia cells. Oncol Rep. 2020 Oct;44(4):1561-1573.

[2]. Kevetrin induces p53-dependent and independent cell cycle arrest and apoptosis in ovarian cancer cell lines representing heterogeneous histologies.

See also: 4-Isothiourea-butyronitrile (note moved to).

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Kevetrin is a small molecule compound that has shown p53-dependent and p53-independent activity in solid tumor models (lung cancer, breast cancer, colon cancer, ovarian cancer). A phase I clinical trial (NCT01664000) in advanced solid tumors reported that the drug was well tolerated and that 48% of patients had an increase of ≥10% in peripheral blood p21 expression within 7–24 hours after treatment initiation. [1]
This study suggests that Kevetrin may be a promising treatment option for both wild-type and TP53-mutant AML patients, particularly for the latter subgroup with limited treatment options and poor prognosis. [1]
The drug modulates a core transcriptional program that affects glycolysis, DNA repair, UPR, and key leukemia pathways (MYC, MYB, BCL11A). [1]

C5H10CLN3S

179.67

179.028

C, 33.43; H, 5.61; Cl, 19.73; N, 23.39; S, 17.84

66592-89-0

Kevetrin hydrochloride-13C2,15N3;2300178-72-5

49778916

white solid powder

125-127 ºC

2.518

3

3

4

10

134

0

Cl[H].S(/C(=N\[H])/N([H])[H])C([H])([H])C([H])([H])C([H])([H])C#N

NCXJZJFDQMKRKM-UHFFFAOYSA-N

InChI=1S/C5H9N3S.ClH/c6-3-1-2-4-9-5(7)8;/h1-2,4H2,(H3,7,8);1H

3-cyanopropyl carbamimidothioate;hydrochloride

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, avoid exposure to moisture.

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 (13.91 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 (13.91 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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 (13.91 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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Solubility in Formulation 4: 100 mg/mL (556.58 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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