With GI50 values less than 100 nM, AZD1208 hydrochloride has strong antiproliferative action in the megakaryoblastic leukemia cell line MOLM-16[1]. The proliferation of Ramos cells is inhibited by AZD1208 hydrochloride (10 μM), and at 1 μM, it substantially inhibits PIM kinases in all cells. Apoptosis is induced by AZD1208 hydrochloride, and PIM2 knockdown mostly results in a change in the cell cycle[2]. When combined, AZD1208 hydrochloride and AZD2014 significantly block AKT and 4EBP1 activation, reduce polysome formation, and quickly activate AMPKα, a negative regulator of translation machinery through mTORC1/2 signaling in AML cells[3].

With GI50 values less than 100 nM, AZD1208 hydrochloride has strong antiproliferative action in the megakaryoblastic leukemia cell line MOLM-16[1]. The proliferation of Ramos cells is inhibited by AZD1208 hydrochloride (10 μM), and at 1 μM, it substantially inhibits PIM kinases in all cells. Apoptosis is induced by AZD1208 hydrochloride, and PIM2 knockdown mostly results in a change in the cell cycle[2]. When combined, AZD1208 hydrochloride and AZD2014 significantly block AKT and 4EBP1 activation, reduce polysome formation, and quickly activate AMPKα, a negative regulator of translation machinery through mTORC1/2 signaling in AML cells[3].

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AZD1208 inhibited proliferation and induced cell death in a dose-dependent manner in several AML cell lines (MOLM-16, OCI-AML3, MV4;11, MOLM-13), though MOLM-14 cells were relatively resistant. Sensitivity varied among cell lines; MOLM-16 was the most sensitive. [3]
In primary AML samples (with or without FLT3-ITD mutation) co-cultured with bone marrow-derived mesenchymal stem cells (MSCs) to mimic the stromal microenvironment, AZD1208 induced moderate growth inhibition in 2 out of 6 samples. Sensitivity to AZD1208 appeared independent of FLT3 mutation status. [3]
Combination of AZD1208 with the dual mTORC1/2 inhibitor AZD2014 synergistically reduced cell viability and increased apoptosis in AML cell lines and primary samples, as calculated by the Chou-Talalay method (Combination Index, CI). Synergy was strong in MOLM-16 (CI <0.01 at ED50, ED75, ED90) and MV4;11 (CI average 0.25), moderate in MOLM-14 (CI average 0.36) and OCI-AML3 (CI average 0.31), and slightly antagonistic in MOLM-13 (CI average 1.2). [3]
AZD1208 alone or in combination reduced phosphorylation of 4EBP1 (Thr37/46) and S6 (Ser240/244), key markers of mTOR pathway activity, in AML cell lines (OCI-AML3, MOLM-16, MV4;11) and primary AML cells. [3]
In the FLT3-ITD cell line MV4;11, AZD1208 reduced c-Myc protein levels. In the FLT3-WT cell line MOLM-16, AZD1208 also reduced c-Myc. [3]
Proteomic (iTRAQ) analysis in MOLM-16 and OCI-AML3 cells showed that AZD1208 treatment altered protein expression profiles, including downregulation of translation elongation factor EF1A1. Pathway analysis indicated that AZD1208 altered the ribosome biogenesis pathway in sensitive MOLM-16 cells. [3]
Polysome profile analysis in MOLM-16 cells showed that AZD1208 altered the polysomal profile, indicating reduced CAP-dependent translation. [3]
Clonogenic assays using primary AML mononuclear cells showed that AZD1208 alone had moderate or no effect in some samples, but in combination with AZD2014, it showed complementary or additive inhibitory effects on colony formation regardless of FLT3 mutation status. [3]

AZD1208 suppresses the growth of MOLM-16 and KG-1a xenograft tumors in vivo in a dose proportional manner.

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In a human cholangiocarcinoma (CC) cell xenograft model in CD1 nude mice, treatment with AZD7507 (or GW2580, another CSF1R inhibitor) resulted in a significant decrease in the number of CD68-positive macrophages within the xenografts. [2]
For CC cell lines CC-LP-1 and SNU-1079 (but not WITT-1), treatment with AZD7507 (or GW2580) led to reduced tumor volume and mass. [2]
In the CC-LP-1 group, only 3 out of 8 initially palpable tumors were detectable after 6 weeks of treatment with CSF1R inhibitors, suggesting reduced tumor burden. [2]
Treatment with AZD7507 (or GW2580) in xenograft models resulted in reduced expression of murine Wnt7b mRNA and human pro-proliferation genes (BIRC5, CCND2, CCNE), and an increase in expression of the pro-apoptotic gene BAX1 in CC-LP-1 and SNU-1079 xenografts, indicating reduced proliferation and induced apoptosis. [2]

Cell Viability and Apoptosis Assay: AML cell lines or primary mononuclear cells were treated with AZD1208, AZD2014, or their combination for 72 hours. Viable cell numbers were determined by trypan blue exclusion. Apoptosis was assessed by Annexin V/7AAD staining followed by flow cytometry analysis. [3]
Western Blot Analysis: Cells were lysed in buffer containing protease and phosphatase inhibitors. Protein concentration was determined, and 40 µg of total protein was separated by SDS-PAGE, transferred to PVDF membranes, and probed with specific primary and secondary antibodies. Signals were detected using chemiluminescence. Antibodies included those against PIM1, PIM2, PIM3, phospho-4EBP1, total 4EBP1, phospho-S6, c-Myc, AKT, phospho-AKT, AMPKα, phospho-AMPKα, and loading controls (α-tubulin, β-actin, GAPDH). [3]
Flow Cytometry for Intracellular Phospho-proteins: Cells were treated, fixed with paraformaldehyde, permeabilized with ice-cold methanol, and stained with fluorochrome-conjugated antibodies against phospho-ERK, phospho-S6, phospho-AKT, and CXCR4. Analysis was performed using a flow cytometer. [3]
Clonogenic Assay: Primary AML mononuclear cells were seeded in methylcellulose-based medium. AZD1208 (1 or 3 µM), AZD2014 (0.25 or 0.5 µM), or the combination was added to the medium before plating. Cells were plated in triplicate and incubated for 14 days. Colonies were counted using a stereoscope. [3]
Proteomic Analysis (iTRAQ): Cell lysates were digested with trypsin, and the resulting peptides were labeled with iTRAQ reagents. Labeled peptides were pooled, fractionated by strong cation exchange chromatography, and analyzed by nano liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein identification and relative quantification were performed using specialized software, searching against a protein database. Significantly altered proteins were subjected to pathway enrichment analysis. [3]
Polysomal Assay: Cells were treated with AZD1208, AZD2014, or combination in the presence of cycloheximide. Cells were lysed in hypotonic buffer containing cycloheximide, dithiothreitol, protease inhibitor, and RNase inhibitor. After centrifugation, the supernatant was layered onto a linear sucrose density gradient and ultracentrifuged. Gradients were fractionated, and RNA from polysomal fractions was extracted for quantitative RT-PCR analysis of specific transcripts (e.g., CCND1). [3]

Dissolved in 0.5% hydroxypropyl methylcellulose; 30 mg/kg twice per week; oral gavageFemale CB17 SCID mice implanted with MOLM-16 cells (5 × 106) or KG-1a cells (6 × 106)

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Human CC Xenograft Model: CD1 nude mice were subcutaneously engrafted with human CC cells (e.g., SNU-1079, CC-LP-1). Once palpable tumors formed (approximately 3 weeks), mice were treated. AZD7507 was formulated in sterile water containing 0.5% methylcellulose and 0.1% Tween-80. It was administered orally (by gavage) at a dose of 100 mg/kg, twice daily. Control animals received the vehicle (water with 0.5% methylcellulose and 0.1% Tween-80). Treatment duration and frequency beyond the stated dosing regimen are not specified in the provided text. [2]

[1]. Discovery of novel benzylidene-1,3-thiazolidine-2,4-diones as potent and selective inhibitors of the PIM-1, PIM-2, and PIM-3 protein kinases. Bioorg Med Chem Lett. 2012 Jul 15;22(14):4599-604.

[2]. Loss of PIM2 enhances the anti-proliferative effect of the pan-PIM kinase inhibitor AZD1208 in non-Hodgkin lymphomas. Mol Cancer. 2015 Dec 8;14:205.

[3]. The novel combination of dual mTOR inhibitor AZD2014 and pan-PIM inhibitor AZD1208 inhibits growth in acute myeloid leukemia via HSF pathway suppression. Oncotarget. 2015 Nov 10;6(35):37930-47.

AZD7507 is a small molecule CSF1R inhibitor used in preclinical studies to investigate the role of macrophages in the progression of cholangiocarcinoma. [2]
AZD7507, in combination with GW2580 and liposomal clodronate, demonstrated that macrophage clearance (and the resulting attenuation of the macrophage-derived WNT signaling pathway) reduced tumor burden in a specific cholangiocarcinoma model, supporting the hypothesis that inflammatory macrophages promote cholangiocarcinoma growth through the WNT signaling pathway. [2]
This study indicates that systemic macrophage clearance (e.g., by clodronate) is not a viable treatment approach, emphasizing that in this study, such inhibitors should be used as research tools to understand disease mechanisms rather than as direct clinical candidates. [2]

C21H22CLN3O2S

415.936282634735

415.112

1621866-96-3

AZD1208;1204144-28-4

76962885

Typically exists as solid at room temperature

3

5

3

28

602

1

C1C[C@H](CN(C1)C2=C(C=CC=C2C3=CC=CC=C3)/C=C\4/C(=O)NC(=O)S4)N.Cl

KPQHIFXAXSIKOA-SLWUYDEESA-N

InChI=1S/C21H21N3O2S.ClH/c22-16-9-5-11-24(13-16)19-15(12-18-20(25)23-21(26)27-18)8-4-10-17(19)14-6-2-1-3-7-14;/h1-4,6-8,10,12,16H,5,9,11,13,22H2,(H,23,25,26);1H/b18-12-;/t16-;/m1./s1

(5Z)-5-[[2-[(3R)-3-aminopiperidin-1-yl]-3-phenylphenyl]methylidene]-1,3-thiazolidine-2,4-dione;hydrochloride

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)

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.)