FLT3 in culture medium (IC50 = 132 nM); FLT3 in plasma (IC50 = 9.8 μM)

In the 100–500 nM dose range, CGP52421 (200, 400, 600, 800, and 1000 nM) exhibits greater cytotoxicity than PKC412 [1].

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CGP52421 may be less protein bound in plasma compared with PKC412[1]
We first adjusted the curve shown in Figure 3B to account for the presence of the active metabolite CGP62221 (by simply adding its concentration to that of PKC412), but the PIA data points still fell to the left of this adjusted curve (not shown). Because of this continuing discrepancy between the PIA assay results and the pharmacokinetic data, and because of the remarkably high levels of CGP52421 in the plasma of trial patients, we further investigated the FLT3-inhibitory properties of CGP52421. In culture medium, CGP52421 inhibits FLT3 autophosphorylation 22-fold less potently than the parent compound, with an IC50 of approximately 132 nM (Figure 5A). In plasma, the IC50 of CGP52421 is 9.8 μM, compared with 1.8 μM for PKC412 (compare Figure 5B with Figure 1D). Therefore, while CGP52421 is 22-fold less potent than PKC412 in culture medium containing 10% serum, it is roughly 5-fold less potent than the parent compound in plasma, possibly because it is less protein bound. CGP52421 levels at steady state ranged from 20 to 30 μM, 2- to 3-fold over the IC50 and likely sufficient to contribute significantly to FLT3 inhibition. Using a formula of the concentration of PKC412 plus the concentration of CGP6221 plus the concentration of CGP52421/5.4, the levels of PKC412 and its metabolites can be expressed as an adjusted value in terms of FLT3 inhibition. When the PIA results are plotted against the adjusted values, the points now fall along the standard curve for PKC412-mediated FLT3 inhibition in plasma (Figure 6A). When the results of all of the PIA assays are averaged and plotted along with the adjusted plasma concentrations, the remarkably effective, sustained FLT3 inhibition by PKC412 and its metabolites is illustrated (Figure 6B-C).[1]

FLT3 phosphorylation[1]
Leukemia cells were washed in PBS and then lysed by resuspending them in lysis buffer (20 mM Tris [pH 7.4], 100 mM NaCl, 1% Igepal [Sigma, St Louis, MO], 1 mM EDTA, 2 mM NaVO4, plus Complete protease inhibitor for 30 minutes while rocking. The extract was clarified by centrifugation at 14 000 rpm, and the supernatant was assayed for protein. Anti-FLT3 antibody was added to the extract for overnight incubation, and then protein A-Sepharose was added for 2 additional hours. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to Immobilon membranes, immunoblotting was performed with antiphosphotyrosine antibody (4G10) to detect phosphorylated FLT3 and then the blots were stripped and reprobed with anti-FLT3 antibody to measure total FLT3. Proteins were visualized using chemiluminescence and scanned using a Bio-Rad GS800 densitometer.

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Plasma inhibitory activity[1]
Frozen plasma samples were thawed and clarified by centrifugation at 16 000g for 2 minutes. There was no difference in results when fresh plasma was used in comparison with frozen/thawed, nor was there any difference in results if samples were allowed to incubate at room temperature (protected from light) for as much as 1 week (data not shown). We have, however, found that there is some loss of plasma inhibitory activity in CEP-701/plasma samples stored at -80°C for more than 12 months (data not shown). All assays described herein were performed within 12 months of collection. For each time point, 4 × 106 TF/ITD cells were incubated with 1 mL plasma at 37°C for 2 hours. We have found that a 2-hour incubation period yields the most consistent results with the indolocarbazole FLT3 inhibitors. For other, more soluble, inhibitors, a 1-hour incubation period is sufficient. Pretreatment plasma is used as a baseline. The TF/ITD cells were washed twice with ice-cold PBS and then lysed and analyzed by immunoprecipitation for FLT3 and antiphosphotyrosine immunoblotting as described above, under “FLT3 phosphorylation.” After immunoblotting, densitometric analysis was performed on the bands, and the PIA for a given plasma sample was calculated by expressing the density of its corresponding band as a percent decrease from the density of the baseline band, which was arbitrarily set at 100%. For example, if the densitometric analysis of a sample band reveals it to be 10% of the pretreatment/baseline sample, the FLT3 PIA for that sample is 90%. While we have performed this assay successfully and with equivalent results using other human cell lines (Molm-14, EOL-1, SEMK2), the results shown here are for TF/ITD cells only.

CGP52421 is less selective but more cytotoxic than PKC412 Because CGP52421 contributes significantly to FLT3 inhibition in patients treated with PKC412, we wished to better characterize its cytotoxic properties against leukemia cells. We first tested it against BaF3-ITD cells, a model cell line derived by transfecting murine Ba/F3 cells, which are IL-3 dependent, with a FLT3/ITD construct.16 While the resultant BaF3-ITD cells are IL-3 independent, IL-3 can be used to “rescue” the cells from the cytotoxic effects of FLT3 inhibitors. Using an MTT assay, we determined that PKC412 induces a cytotoxic effect in BaF3-ITD cells with an IC50 of 29 nM in the absence of IL-3 and 155 nM in the presence of IL-3. The cytotoxic effects seen in the presence of IL-3 are presumably due to inhibition of other, unidentified targets of PKC412. Using the same assay, CGP52421 induces cytotoxicity in BaF3-ITD cells with an IC50 of 613 nM in the absence of IL-3 and 1053 nM in the presence of IL-3. The difference between the IC50 values in the absence and presence of IL-3—4.3-fold versus 1.7-fold higher for PKC412 and CGP52421, respectively—suggests that CGP52421 is less selective than PKC412 at concentrations that are inhibitory for FLT3. For CEP-701 the IC50 is 8 nM in the absence of IL-3 and 24 nM in the presence of IL-3, which places it between PKC412 and CGP52421 in this assessment of selectivity. The results of the IL-3 rescue assays are summarized in Table 1.[1]

[1]. Plasma inhibitory activity (PIA): a pharmacodynamic assay reveals insights into the basis for cytotoxic response to FLT3 inhibitors. Blood. 2006 Nov 15;108(10):3477-83.

We have developed a useful surrogate assay for monitoring the efficacy of FLT3 inhibition in patients treated with oral FLT3 inhibitors. The plasma inhibitory activity (PIA) for FLT3 correlates with clinical activity in patients treated with CEP-701 and PKC412. Using the PIA assay, along with in vitro phosphorylation and cytotoxicity assays in leukemia cells, we compared PKC412 and its metabolite, CGP52421, with CEP-701. While both drugs could effectively inhibit FLT3 in vitro, CEP-701 was more cytotoxic to primary samples at comparable levels of FLT3 inhibition. PKC412 appears to be more selective than CEP-701 and therefore less effective at inducing cytotoxicity in primary acute myeloid leukemia (AML) samples in vitro. However, the PKC412 metabolite CGP52421 is less selective than its parent compound, PKC412, and is more cytotoxic against primary blast samples at comparable levels of FLT3 inhibition. The plasma inhibitory activity assay represents a useful correlative tool in the development of small-molecule inhibitors. Our application of this assay has revealed that the metabolite CGP52421 may contribute a significant portion of the antileukemia activity observed in patients receiving oral PKC412. Additionally, our results suggest that nonselectivity may constitute an important component of the cytotoxic effect of FLT3 inhibitors in FLT3-mutant AML[1].

C36H32N4O5

600.66

586.222

C, 71.98; H, 5.37; N, 9.33; O, 13.32

179237-49-1

3-Hydroxy Midostaurin-d5

137552093

Light yellow to yellow solid powder

5.726

2

5

3

44

1180

4

WMQSFYBLPAXEDG-VPROBEFOSA-N

InChI=1S/C36H32N4O5/c1-36-18-25(44-3)24(38(2)35(43)19-11-5-4-6-12-19)17-26(45-36)39-22-15-9-7-13-20(22)27-29-30(34(42)37-33(29)41)28-21-14-8-10-16-23(21)40(36)32(28)31(27)39/h4-16,24-26,34,42H,17-18H2,1-3H3,(H,37,41)/t24-,25-,26-,34?,36+/m0/s1

N-((5S,7S,8S,10R)-15-hydroxy-8-methoxy-10-methyl-17-oxo-6,7,8,9,10,15,16,17-octahydro-5H-18-oxa-4b,10a,16-triaza-5,10-methanodibenzo[b,h]cyclodeca[jkl]cyclopenta[e]-as-indacen-7-yl)-N-methylbenzamide

CGP 52421; CGP-52421; 3-Hydroxy Midostaurin; CGP52421

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)