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Flavopiridol (Alvocidib, HMR-1275) CDK inhibitor

Name: Flavopiridol (Alvocidib, HMR-1275)
Brand: Selleck Chemicals
SKU: S1230
Rating: 5 (146 reviews)

Cat.nr.: S1230

Flavopiridol (Alvocidib) competes with ATP to inhibit CDKs including CDK1, CDK2, CDK4, CDK6, and CDK9 with IC50 values in the 20-100 nM range. It is more selective for CDK1, 2, 4, 6, 9 versus CDK7. Flavopiridol is initially found to inhibit EGFR and PKA. Flavopiridol induces autophagy and ER stress. Flavopiridol blocks HIV-1 replication. Phase 1/2.

Flavopiridol (Alvocidib, HMR-1275) CDK inhibitor Chemical Structure

Chemische structuur

Molecuulgewicht: 401.84

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Kwaliteitscontrole (Quality Control)

Batch: Zuiverheid: 99.92%

99.92

Gerelateerde doelwitten	HSP PD-1/PD-L1 ROCK Wee1 DNA/RNA Synthesis Microtubule Associated Ras KRas Aurora Kinase Casein Kinase
Overig CDK Inhibitoren	AS2863619 Ro-3306 SEL120 (SEL120-34A) hydrochloride INX-315 Tagtociclib (PF-07104091) PF-07220060 (CDK4/6-IN-6) AZD4573 ON123300 Enitociclib (BAY 1251152) Samuraciclib (ICEC0942) hydrochloride

Celkweek, behandeling & werkzame concentratie
(Cell Culture, Treatment & Working Concentration)

Cellijnen	Assaytype	Concentratie	Incubatietijd	Activiteitsbeschrijving	PMID
MCF-7 tumor cell	Proliferation assay			Inhibition of MCF-7 tumor cell proliferation	10843211
PC3 cell	Function assay			Inhibition of PC3 cell clonogenic assay, IC50=10 μM	11063609
HCT116 cell	Function assay			Inhibition of HCT116 cell clonogenic assay, IC50=13 μM	11063609
A2780 cell	Function assay			Inhibition of A2780 cell clonogenic assay， IC50=15 μM	11063609
Mia PaCa-2 cell	Function assay			Inhibition of Mia PaCa-2 cell clonogenic assay, IC50=36 μM	11063609
LNCaP human prostate carcinoma cell	Proliferation assay			Inhibition of LNCaP human prostate carcinoma cell proliferation	12190313
HCT116/VP35 human colon carcinoma cell	Proliferation assay			Inhibition of HCT116/VP35 human colon carcinoma cell proliferation, IC50=17 nM	12190313
HCT116 human colon carcinoma cell	Proliferation assay			Inhibition of HCT116 human colon carcinoma cell proliferation, IC50=18 nM	12190313
HCT116/VM46 human colon carcinoma cell	Proliferation assay			Inhibition of HCT116/VM46 human colon carcinoma cell proliferation, IC50=21 nM	12190313
A2780/DDP-R human ovarian carcinoma cell	Proliferation assay			Inhibition of A2780/DDP-R human ovarian carcinoma cell proliferation, IC50=38 nM	12190313
ABAE human fibroblast cell	Proliferation assay			Inhibition of ABAE human fibroblast cell proliferation, IC50=45 nM	12190313
HL60 human leukemia cell	Proliferation assay			Inhibition of HL60 human leukemia cell proliferation, IC50=46 nM	12190313
Hs 27 human fibroblast cell	Proliferation assay			Inhibition of Hs 27 human fibroblast cell proliferation, IC50=51 nM	12190313
CCRF-CEM human leukemia cell	Proliferation assay			Inhibition of CCRF-CEM human leukemia cell proliferation, IC50=52 nM	12190313
OVCAR-3 human ovarian carcinoma cell	Proliferation assay			Inhibition of OVCAR-3 human ovarian carcinoma cell proliferation, IC50=54 nM	12190313
A2780/DDP-S human ovarian carcinoma cell	Proliferation assay			Inhibition of A2780/DDP-S human ovarian carcinoma cell proliferation, IC50=56 nM	12190313
A2780/TAX-S human ovarian carcinoma cell	Proliferation assay			Inhibition of A2780/TAX-S human ovarian carcinoma cell proliferation, IC50=65 nM	12190313
LS174T human colon carcinoma cell	Proliferation assay			Inhibition of LS174T human colon carcinoma cell proliferation, IC50=65 nM	12190313
MCF-7 human breast carcinoma cell	Proliferation assay			Inhibition of MCF-7 human breast carcinoma cell proliferation, IC50=66 nM	12190313
PC3 human prostate carcinoma cell	Proliferation assay			Inhibition of PC3 human prostate carcinoma cell proliferation, IC50=66 nM	12190313
MLF mouse lung fibroblast cell	Proliferation assay			Inhibition of MLF mouse lung fibroblast cell proliferation, IC50=72 nM	12190313
LX-1 human lung carcinoma	Proliferation assay			Inhibition of LX-1 human lung carcinoma proliferation, IC50=75 nM	12190313
A431 human squamous cell	Proliferation assay			Inhibition of A431 human squamous cell carcinoma cell proliferation, IC50=75 nM	12190313
SKBR-3 human breast carcinoma cell	Proliferation assay			Inhibition of SKBR-3 human breast carcinoma cell proliferation, IC50=77 nM	12190313
A2780/TAX-R human ovarian carcinoma cell	Proliferation assay			Inhibition of A2780/TAX-R human ovarian carcinoma cell proliferation, IC50=78 nM	12190313
M109 mouse lung carcinoma cell	Proliferation assay			Inhibition of M109 mouse lung carcinoma cell proliferation, IC50=80 nM	12190313
CACO-2 human colon carcinoma cell	Proliferation assay			Inhibition of CACO-2 human colon carcinoma cell proliferation, IC50=86 nM	12190313
A549 human lung carcinoma cell	Proliferation assay			Inhibition of A549 human lung carcinoma cell proliferation, IC50=96 nM	12190313
MIP human colon carcinoma cell	Function assay			Inhibition of MIP human colon carcinoma cell line, IC50=0.12 μM	12190313
K562 human leukemia cell	Proliferation assay			Inhibition of K562 human leukemia cell proliferation, IC50=0.13 μM	12190313
human A2780 cell line	Proliferation assay		72 h	Antiproliferative effect against human A2780 cell line was determined in a whole cell 72 hr cytotoxicity assay, IC50=71 nM	15027863
human ovarian (A2780) cancer cell	Cytotoxic assay			Cytotoxic effect on human ovarian (A2780) cancer cell line, IC50=71 nM	15125971
ID8 cells	Proliferation assay			Antiproliferative activity against ID8 cells, IC50=7 nM	17123821
MCF7 cells	Proliferation assay			Antiproliferative activity against MCF7 cells, IC50=26 nM	17123821
Sf9 cells	Function assay			Inhibition of recombinant cyclin A/CDK2 expressed in Sf9 cells, IC50=12 nM	17904366
human A2780 cells	Function assay			Inhibition of cdk-mediated NPM phosphorylation at thr199 in human A2780 cells	18469809
human A2780 cells	Function assay		24 h	Inhibition of cdk-mediated Rb phosphorylation at thr821 in human A2780 cells after 24 hrs	18469809
human NCI60 cells	Proliferation assay		72 h	Antiproliferative activity against human NCI60 cells after 72 hrs by sulforhodamine B assay, GI50=74.7 nM	21080703
human NCI60 cells	Proliferation assay		72 h	Antiproliferative activity against human NCI60 cells assessed as lethal effect after 72 hrs by sulforhodamine B assay, LC50=0.904 μM	21080703
human A2780 cells	Cytotoxic assay		24 h	Cytotoxicity against human A2780 cells after 24 hrs by MTT assay, GI50=23 nM	23301767
human MRC5 cells	Cytotoxic assay		72 h	Cytotoxicity against human MRC5 cells after 72 hrs by MTT assay, GI50=28 nM	23301767
human A2780 cells	Cytotoxic assay		72 h	Cytotoxicity against human A2780 cells after 72 hrs by MTT assay, GI50=29 nM	23301767
human A2780 cells	Cytotoxic assay		48 h	Cytotoxicity against human A2780 cells after 48 hrs by MTT assay, GI50=31 nM	23301767
human MRC5 cells	Cytotoxic assay		48 h	Cytotoxicity against human MRC5 cells after 48 hrs by MTT assay, GI50=39 nM	23301767
human MRC5 cells	Cytotoxic assay		24 h	Cytotoxicity against human MRC5 cells after 24 hrs by MTT assay, GI50=49 nM	23301767
human HMEC1 cells	Cytotoxic assay		24 h	Cytotoxicity against human HMEC1 cells after 24 hrs by MTT assay, GI50=61 nM	23301767
human HMEC1 cells	Cytotoxic assay		48 h	Cytotoxicity against human HMEC1 cells after 48 hrs by MTT assay, GI50=62 nM	23301767
human HMEC1 cells	Cytotoxic assay		72 h	Cytotoxicity against human HMEC1 cells after 72 hrs by MTT assay, GI50=66 nM	23301767
A2780	Function assay	330 nM	24 hr	Percentage A2780 cells in sb-G1 after 24 hr r at 330 nM (IC50), Cell cycle = 0.001 μM.	12190313
A2780	Apoptosis assay	330 nM	24 hr	Apoptotic A2780 cells after 24 hr r at 330 nM (IC50), Cell cycle = 0.001 μM.	12190313
A2780	Function assay	2980 nM	24 hr	Percentage A2780 cells in sub-G1 after 24 hr r at 2980 nM (IC90), Cell cycle = 0.002 μM.	12190313
A2780	Apoptosis assay	2980 nM	24 hr	Apoptotic A2780 cells after 24 hr r at 2980 nM (IC90), Cell cycle = 0.009 μM.	12190313
A2780	Function assay	2980 nM	24 hr	Percentage A2780 cells in S-phase after 24 hr r at 2980 nM (IC90), Cell cycle = 0.012 μM.	12190313
A2780	Function assay	330 nM	24 hr	Percentage A2780 cells in S-phase after 24 hr r at 330 nM (IC50), Cell cycle = 0.016 μM.	12190313
A2780	Function assay	330 nM	24 hr	Percentage A2780 cells in G2/M after 24 hr r at 330 nM (IC50), Cell cycle = 0.023 μM.	12190313
A2780	Function assay	2980 nM	24 hr	Percentage A2780 cells in G2/M after 24 hr r at 2980 nM (IC90), Cell cycle = 0.025 μM.	12190313
A2780	Function assay	330 nM	24 hr	Percentage A2780 cells in G1 after 24 hr r at 330 nM (IC50), Cell cycle = 0.06 μM.	12190313
A2780	Function assay	2980 nM	24 hr	Percentage A2780 cells in G1 after 24 hr r at 2980 nM (IC90), Cell cycle = 0.061 μM.	12190313
A2780	Function assay		24 hrs	Inhibition of cdk-mediated Rb phosphorylation at ser807/811 in human A2780 cells after 24 hrs	18469809
A2780	Function assay		24 hrs	Inhibition of cdk9-mediated RNA pol2 CTD phosphorylation at ser2 in human A2780 cells after 24 hrs	18469809
A2780	Function assay		24 hrs	Inhibition of cdk7-mediated RNA pol2 CTD phosphorylation at ser5 in human A2780 cells after 24 hrs	18469809
A2780	Apoptosis assay		24 hrs	Induction of apoptosis in human A2780 cells assessed as appearance of Mcl1 protein level after 24 hrs	18469809
DR-U2OS-GFP	Function assay	0.1 uM	56 hrs	Reduction of homologous recombination in human DR-U2OS-GFP cells expressing I-SceI nuclease assessed as reduction of RAD51 level at 0.1 uM after 56 hrs by immunoblotting	21417417
A2780	Function assay		24 hrs	Inhibition of CDK9 in human A2780 cells assessed as reduction of RNAPII CTD phosphorylation at Ser2 at GI50 concentration after 24 hrs by Western blotting analysis	23301767
A2780	Cell cycle assay		24 hrs	Cell cycle arrest in human A2780 cells assessed as accumulation at G2/M phase at less than GI50 after 24 hrs by flow cytometric analysis	23301767
A2780	Function assay		24 hrs	Inhibition of CDK9 in human A2780 cells assessed as downregulation of MCL1 at GI50 to 5XGI50 concentration after 24 hrs by Western blotting analysis	23301767
A2780	Apoptosis assay		24 hrs	Induction of apoptosis in human A2780 cells assessed as induction of PARP cleavage at GI50 to 5XGI50 concentration after 24 hrs by Western blotting analysis	23301767
A2780	Function assay		24 hrs	Inhibition of CDK9 in human A2780 cells assessed as downregulation of HDM2 at GI50 to 5XGI50 concentration after 24 hrs by Western blotting analysis	23301767
MT4	Antiproliferative assay			Antiviral activity against Human immunodeficiency virus 1 NL 4-3 infected in MT4 cells measured on day 4 post infection by p24 assay, EC50 = 0.015 μM.	25914804
MT4	Cytotoxicity assay			Cytotoxicity against human MT4 cells, IC50 = 0.067 μM.	25914804
Sf9	Function assay		15 mins	Inhibition of CDK2/cyclin E1 (unknown origin) expressed in Sf9 insect cells using UlightCFFKNIVTPRTPPPSQGK-amide substrate after 15 mins by autoradiography, IC50 = 0.13 μM.	25914804
A549	Antiproliferative assay		3 days	Antiproliferative activity against human A549 cells after 3 days by SRB method, GI50 = 0.14 μM.	25914804
DU145	Antiproliferative assay		3 days	Antiproliferative activity against human DU145 cells after 3 days by SRB method, GI50 = 0.15 μM.	25914804
KB	Antiproliferative assay		3 days	Antiproliferative activity against human KB cells after 3 days by SRB method, GI50 = 0.16 μM.	25914804
KBVIN	Antiproliferative assay		3 days	Antiproliferative activity against human KBVIN cells after 3 days by SRB method, GI50 = 0.18 μM.	25914804
HCT116	Antiproliferative assay		72 hrs	Antiproliferative activity against human HCT116 cells after 72 hrs by Celltiter-Glo reagent based assay in presence of 10% fetal bovine serum, EC50 = 0.034 μM.	26985305
HCT116	Antiproliferative assay		72 hrs	Antiproliferative activity against human HCT116 cells after 72 hrs by Celltiter-Glo reagent based assay in presence of 0.625% fetal bovine serum, EC50 = 0.059 μM.	26985305
Sf9	Function assay		10 mins	Inhibition of human His6-tagged CDK9/cyclin T1 expressed in baculovirus infected sf9 cells using GST-CTD as substrate after 10 mins in presence of [gamma-32P]ATP by SDS-PAGE analysis, IC50 = 0.0025 μM.	27171036
Sf21	Function assay			Inhibition of recombinant human full length C-terminal His6-tagged CDK9/cyclin T1 expressed in baculovirus infected Sf21 insect cells using PDKtide as substrate, IC50 = 0.011 μM.	27171036
HeLa	Cytotoxicity assay		72 hrs	Cytotoxicity against human HeLa cells assessed as decrease in cell viability after 72 hrs by MTT assay, CC50 = 0.12 μM.	27171036
sf21	Function assay			Inhibition of full length human N-terminal His6-tagged CDK6/N-terminal GST-tagged cyclin D3 expressed in sf21 cells using histone H1 substrate, IC50 = 0.395 μM.	27171036
Sf21	Function assay			Inhibition of recombinant human full length C-terminal His6-tagged CDK7/cyclin H/N-terminal GST-tagged MAT1 expressed in baculovirus infected Sf21 insect cells using cdk7 substrate peptide, IC50 = 0.514 μM.	27171036
HepG2	Antiproliferative assay		72 hrs	Antiproliferative activity against human HepG2 cells after 72 hrs by CelTiter-Glo assay, EC50 = 0.1464 μM.	29407975
KOPN8	Antiproliferative assay		72 hrs	Antiproliferative activity against human KOPN8 cells after 72 hrs by CelTiter-Glo assay, EC50 = 0.1926 μM.	29407975
SEM	Antiproliferative assay		72 hrs	Antiproliferative activity against human SEM cells after 72 hrs by CelTiter-Glo assay, EC50 = 0.2043 μM.	29407975
UOCB1	Antiproliferative assay		72 hrs	Antiproliferative activity against human UOCB1 cells after 72 hrs by CelTiter-Glo assay, EC50 = 0.2084 μM.	29407975
KOPN8	Apoptosis assay	0.5 uM	3 to 24 hrs	Induction of apoptosis in human KOPN8 cells assessed as upregulation of cleaved PARP level at 0.5 uM after 3 to 24 hrs by Western blot analysis	29407975
KOPN8	Apoptosis assay	0.5 uM	1 hr	Induction of apoptosis in human KOPN8 cells assessed as upregulation of cleaved PARP level at 0.5 uM pre-treated with NAC for 1 hr and measured after 3 to 24 hrs by Western blot analysis	29407975
Klik om meer experimentele gegevens over cellijnen te bekijken

Chemische informatie, opslag en stabiliteit (Chemical Information, Storage & Stability)

Molecuulgewicht	401.84	Formule	C₂₁H₂₀ClNO₅	Opslag (vanaf de datum van ontvangst)	3 jaar-20°Cpoeder
CAS-nr.	146426-40-6	SDF downloaden		Opslag van stamoplossingen	1 jaar-80°Cin oplosmiddel 1 maand-20°Cin oplosmiddel
Synoniemen	NSC 649890, HMR-1275, L86-8275			Smiles	CN1CCC(C(C1)O)C2=C(C=C(C3=C2OC(=CC3=O)C4=CC=CC=C4Cl)O)O

Oplosbaarheid (Solubility)

In vitro
Batch:

DMSO : 80 mg/mL (199.08 mM)
(Met vocht verontreinigd DMSO kan de oplosbaarheid verminderen. Gebruik verse, watervrije DMSO.)

Water : Insoluble

Ethanol : Insoluble

Molariteitscalculator

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Verdunningscalculator Molecuulgewichtcalculator

In vivo Batch:
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Clear solution	5%DMSO 1 30%PEG300 2 65%ddH2O Gevalideerd door Selleck labs. Mocht u aanpassingen aan deze formulering nodig hebben, neem dan contact op met ons verkoopteam voor aangepaste tests.	2.500mg/ml (6.22mM)	Taking the 1 mL working solution as an example, add 50 μL of 50 mg/ml clarified DMSO stock solution to 300 μL of PEG 300, mix evenly to clarify it; then continue to add 650 μL of ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.

In vivo formulatiecalculator (heldere oplossing)

Stap 1: Voer onderstaande informatie in (Aanbevolen: een extra dier om rekening te houden met verlies tijdens het experiment)

Dosering: mg/kg Gemiddeld gewicht van dieren: g Doseervolume per dier:μL Aantal dieren:

Stap 2: Voer de in vivo formulering in (Dit is alleen de calculator, geen formulering. Neem eerst contact met ons op als er geen in vivo formulering is in de sectie oplosbaarheid.)

% DMSO + % + % Tween 80 + % ddH₂O

%DMSO+ %

Berekeningsresultaten:

Werkconcentratie: mg/ml;

Methode voor het bereiden van DMSO-moedervloeistof: mg geneesmiddel vooropgelost in μL DMSO ( Concentratie moedervloeistof mg/mL, Neem eerst contact met ons op als de concentratie de DMSO-oplosbaarheid van de batch van het geneesmiddel overschrijdt. )

Methode voor het bereiden van in vivo formulering: Neem μL DMSO moedervloeistof, voeg daarna toeμL PEG300, mengen en verhelderen, daarna toevoegenμL Tween 80, mengen en verhelderen, daarna toevoegen μL ddH₂O, mengen en verhelderen.

Methode voor het bereiden van in vivo formulering: Neem μL DMSO moedervloeistof, voeg daarna toe μL Maïsolie, mengen en verhelderen.

Opmerking: 1. Zorg ervoor dat de vloeistof helder is voordat u het volgende oplosmiddel toevoegt.
2. Zorg ervoor dat u het/de oplosmiddel(en) in de juiste volgorde toevoegt. U moet ervoor zorgen dat de verkregen oplossing, bij de vorige toevoeging, een heldere oplossing is voordat u verdergaat met het toevoegen van het volgende oplosmiddel. Fysieke methoden zoals vortexen, ultrasoon of een warmwaterbad kunnen worden gebruikt om het oplossen te bevorderen.

Werkingsmechanisme (Mechanism of Action)

Kenmerken	First CDK inhibitor to be used in human clinical trials.
Targets/IC₅₀/Ki	CDK9 (Cell-free assay) 20 nM CDK1 (Cell-free assay) 30 nM CDK4 (Cell-free assay) 20-40 nM CDK2 (Cell-free assay) 40 nM CDK6 (Cell-free assay) 60 nM CDK7 (Cell-free assay) 875 nM
In vitro	Flavopiridol (Alvocidib) vertoont minder activiteit tegen niet-verwante kinasen zoals MAP, PAK, PKC en EGFR met een IC50 van >14 μM. Het remt significant de kolonie groei van HCT116, A2780, PC3 en Mia PaCa-2 cellen met IC50 van respectievelijk 13 nM, 15 nM, 10 nM en 36 nM. Deze verbinding remt ook potent de activiteit van Glycogeen synthase kinase-3 (GSK-3) met een IC50 van 280 nM. In vergelijking met andere CDK's remt het de activiteit van CDK7 minder potent met een IC50 van 875 nM. Bij 0.5 μM remt het zowel pSer807/811 Rb als pThr199 NPM, terwijl milde veranderingen worden waargenomen bij pThr821 Rb. Het verlaagt ook het algehele RNA polymerase II niveau, evenals de fosforylering van RNA polymerase II op de CTD-herhalingen bij Ser2 Ser5. Als een breed-spectrum CDK-remmer kan het de Cell Cycle progressie remmen in zowel G1 als G2. Bij 0.3 μM induceert het G1-arrest in MCF-7 of MDA-MB-468 cellen door remming van de CDK4 of CDK2 kinase-activiteit. Het vertoont potente cytotoxiciteit tegen een breed scala aan tumorcellijnen met IC50-waarden variërend van 16 nM voor LNCAP tot 130 nM voor K562.
Kinase Assay	CDK kinase-test
Kinase Assay	Voor de CDK1/cycline B1 kinase-test bestaan de kinase-reacties uit 100 ng van baculovirus-uitgedrukt GST-CDK1/cycline B1 (humaan) complex, 1 μg histone HI, 0,2 μCi [γ-³³P]ATP, 25 μM ATP in 50 μL kinase-buffer (50 mM Tris, pH 8.0, 10 mM MgCl₂, 1 mM EGTA, 0,5 mM DTT). Voor de CDK2/cycline E kinase-test bestaan de kinase-reacties uit 5 ng van baculovirus-uitgedrukt GST-CDK2/cycline E (humaan) complex, 0,5 μg GST-RB fusie-eiwit (aminozuren 776-928 van retinoblastoma-eiwit), 0,2 μCi [γ-³³P]ATP, 25 μM ATP in 50 μL kinase-buffer (50 mM Hepes, pH 8.0, 10 mM MgCl₂, 1 mM EGTA, 2 mM DTT). Voor de CDK4/cycline D1 kinase-test bestaan de kinase-reacties uit 150 ng van baculovirus-uitgedrukt GST-CDK4/cycline D1 (humaan), 280 ng Stag-cycline D1, 0,5 μg GST-RB fusie-eiwit (aminozuren 776-928 van retinoblastoma-eiwit), 0,2 μCi [γ-³³P]ATP, 25 μM ATP in 50 μL kinase-buffer (50 mM Hepes, pH 8.0, 10 mM MgCl₂, 1 mM EGTA, 2 mM DTT). Reacties worden 45 minuten voor CDK1 en CDK2, of 1 uur voor CDK4 bij 30 °C geïncubeerd en gestopt door toevoeging van koud trichloorazijnzuur (TCA) tot een eindconcentratie van 15%. TCA-precipitaten worden verzameld op GF/C unifilterplaten met behulp van een Filtermate universele harvester en de filters worden gekwantificeerd met behulp van een TopCount 96-well vloeistofscintillatieteller. Flavopiridol (Alvocidib) wordt opgelost in 10 mM dimethylformamide (DMF) en geëvalueerd bij zes concentraties, elk in drievoud. De uiteindelijke concentratie DMF in de test = 2%. IC50-waarden worden afgeleid door niet-lineaire regressieanalyse en hebben een variatiecoëfficiënt = 16%. Om de activiteit op CDK6 te testen, wordt een filterbindingsassay opgezet. De volgende componenten worden gecombineerd in het reactiemengsel: 2 μL CDK6 (0,7 mg/μL), 5 μL histone H1 (6 mg/mL), 14 μL kinasebuffer (60 mM β-glycerofosfaat, 30 mM p-nitrofenylfosfaat, 25 mM MOPS (pH 7.0), 5 mM EGTA, 15 mM MgCl₂, 1 mM DTT, 0,1 mM Na-vanadate), 3 μL toenemende concentraties van deze verbinding verdund in 50% DMSO, en 6 μL ³³P-ATP (1 mCi/mL) in niet-radioactief ATP bij 90 μM concentratie (eindconcentratie: 15 μM). De assay wordt gestart door toevoeging van ³³P-ATP. De reactie wordt 20 minuten bij 30°C geïncubeerd. Een 25 μL aliquot van het supernatant wordt vervolgens op Whatman P81 fosfocellulosepapier gespot. Filters worden 5 keer gewassen met 1% fosforzuuroplossing. Natte filters worden geteld in aanwezigheid van 1 mL scintillatievloeistof. Cdk9-activiteit wordt gemeten met 50 nM recombinant Cdk9/cyclin T in 50 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM DTT, 3 μM Na₃VO₄, 150 μM RNA polymerase CDT-peptide en 80 μM ATP. Cdk7-assay wordt uitgevoerd in dezelfde buffer met 37 nM gezuiverde kinase in aanwezigheid van 200 μM ATP en 10 μM myelinebindend eiwit als substraat. De potentie van het middel voor CDK9 en CDK7 wordt bepaald met behulp van een sterke anionenwisselaar (Dowex 1-X8 hars, formiaatvorm) -gebaseerde assay of een scintillatieproximiteitsassay. IC50-waarden worden berekend uit de dosis-respons-curven.
In vivo	Toediening van S1230 in een dosis van 7,5 mg/kg gedurende 7 dagen vertoont een lichte antitumoractiviteit tegen P388 murine leukemie, wat resulteert in een %T/C-waarde van 110, en is actief tegen het humane A2780 ovariumcarcinoom dat subcutaan is geïmplanteerd in naakte muizen, wat resulteert in 1,5 log celvernietiging (LCK). Behandeling met S1230 in een dosis van 1-2,5 mg/kg gedurende 10 dagen onderdrukt significant collageen-geïnduceerde artritis bij muizen op een dosisafhankelijke manier, door synoviale hyperplasie en gewrichtsdestructie te remmen, terwijl serumconcentraties van anti-collageen type II (CII) Abs en proliferatieve reacties op CII behouden blijven. In de p21-intacte Hct116 xenografts in naakte muizen remt toediening van CPT-11 (100 mg/kg) gevolgd door S1230 (3 mg/kg) 7 en 16 uur later de tumorregressie significant met respectievelijk 86% en 82%, wat een >2-voudige remming vertoont in vergelijking met CPT-11 alleen met 40%. De combinatie produceert een compleet responspercentage (CR) van ~30%, in tegenstelling tot CPT-11 alleen waarbij geen CR wordt gevonden.
Referenties	[1] https://pubmed.ncbi.nlm.nih.gov/11063609/ [2] https://pubmed.ncbi.nlm.nih.gov/15689157/ [3] https://pubmed.ncbi.nlm.nih.gov/18469809/ [4] https://pubmed.ncbi.nlm.nih.gov/8674031/ [5] https://pubmed.ncbi.nlm.nih.gov/12190313/ [6] https://pubmed.ncbi.nlm.nih.gov/18209094/ [7] https://pubmed.ncbi.nlm.nih.gov/11751522/ [8] https://pubmed.ncbi.nlm.nih.gov/18469809/ [9] https://pubmed.ncbi.nlm.nih.gov/11311660/

Toepassingen (Applications)

Methoden	Biomarkers	Afbeeldingen	PMID
Western blot	Cleaved caspase-8 / Cleaved caspase-9 / Cleaved caspase-3 p-RNAPII / p-eIF4E / Mnk1 p-ERK / ERK / p-p38 / p-4EBP1 / 4EBP1 / p-S6 CDK2 / CDK4 / Cyclin A / p21 / p27 / Rb		31193061
Growth inhibition assay	Cell viability		31193061

Informatie over klinische proeven (Clinical Trial Information)

(gegevens van https://clinicaltrials.gov, bijgewerkt op 2024-05-22)

NCT-nummer	Werving	Aandoeningen	Sponsor/medewerkers	Startdatum	Fasen
NCT03441555	Completed	Acute Myeloid Leukemia (AML)	AbbVie\|Sumitomo Pharma America Inc.	May 30 2018	Phase 1
NCT03298984	Completed	Acute Myeloid Leukemia	Sumitomo Pharma America Inc.	September 25 2017	Phase 1

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