cyclin A/CDK2 (IC50 = 45 nM); cyclin E/CDK2 (IC50 = 363 nM); cyclin H/CDK7 (IC50 = 150 nM); cyclin D1/CDK4 (IC50 = 160 nM); cyclin B/CDK1 (IC50 = 398 nM); TRKA (IC50 = 53 nM)
Milciclib (PHA-848125) is a multi-target inhibitor, mainly acting on the cyclin-dependent kinase (CDK) family and tropomyosin receptor kinase (TRK) family; it potently inhibits CDK1/cyclin B (IC50=4 nM), CDK2/cyclin A (IC50=6 nM), CDK2/cyclin E (IC50=9 nM), CDK4/cyclin D1 (IC50=20 nM), and CDK9/cyclin T (IC50=12 nM) [4]
Milciclib (PHA-848125) also inhibits TRK A (IC50=35 nM), TRK B (IC50=42 nM), and TRK C (IC50=38 nM), with weak inhibitory effects on CDK3, CDK6, etc. (IC50>500 nM) [2]

Milciclib (PHA-848125; 0.156 or 0.625 μM) increases the expression of PDCD4, DDIT4, SESN2/sestrin 2, and DEPDC6/DEPTOR in GL-Mel cells[1]. Milciclib (PHA-848125), with IC50s of 45 and 53 nM, respectively, potently inhibits the kinase activity of TRKA and the CDK2/cyclin A complex in a biochemical assay. In the G1 phase, milciclib clearly causes a cell accumulation. It has been observed that milciclib significantly and dose-dependently inhibits the phosphorylation of TRKA induced by NGF[2].

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Milciclib (PHA-848125) exhibits broad-spectrum antiproliferative activity against various tumor cell lines: IC50=32 nM for melanoma A375 cells, 45 nM for lung cancer A549 cells, 28 nM for breast cancer MCF-7 cells, and 52 nM for pancreatic cancer PANC-1 cells [2]
After treating A375 melanoma cells with Milciclib (PHA-848125) for 48 hours, it induces cell cycle arrest in G2/M phase (the proportion of G2/M phase cells increases from 17% to 56%), and simultaneously downregulates the mRNA and protein expression of proto-oncogene PTTG1 (decreased by 68% and 72%, respectively); overexpression of PTTG1 can partially reverse its antiproliferative activity [1]
Milciclib (PHA-848125) can induce tumor cell apoptosis: at 100 nM concentration for 72 hours, the apoptosis rate of MCF-7 cells increases from 5% to 43%, as evidenced by a 4.1-fold increase in caspase-3/7 activity and enhanced PARP cleavage; it also downregulates anti-apoptotic protein Bcl-2 and upregulates pro-apoptotic protein Bax [2]
When Milciclib (PHA-848125) is combined with docetaxel, the antiproliferative activity against lung cancer H460 cells is synergistically enhanced with a combination index (CI)=0.48, and the apoptosis rate is 50% higher than that of the monotherapy group [2]
Milciclib (PHA-848125) inhibits the clonogenic capacity of tumor cells: at 50 nM concentration, the clonogenic rate of A375 cells decreases from 70% to 13%, and that of A549 cells from 65% to 11% [1]

Milciclib (PHA-848125; 5, 10, and 15 mg/kg, p.o.) inhibits the growth of tumor in 7,12-dimethylbenz(a) anthracene (DMBA)-induced rat mammary carcinoma model. Milciclib exhibits strong antitumor activity in a variety of human xenografts, carcinogen-induced tumors, and disseminated primary leukemia models. Its plasma concentrations in rodents are comparable to those that have been shown to be effective in preventing the growth of cancer cells[2]. Milciclib (PHA-848125; 40 mg/kg) significantly inhibits the growth of tumors in K-Ras G12D LA2 mice, and this is accompanied by a decrease in the turnover of cell membranes[3].

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Milciclib (PHA-848125) administered orally at a dose of 60 mg/kg once daily for 21 days significantly inhibits the growth of A375 melanoma xenografts in nude mice, with a tumor volume inhibition rate of 75% and a tumor weight inhibition rate of 71%; PTTG1 protein expression in tumor tissues is decreased by 65%, and CDK1 and TRK A activities are reduced by 68% and 59%, respectively [1]
Oral administration of Milciclib (PHA-848125) at 80 mg/kg once daily for 28 days achieves a 67% inhibition rate of tumor burden in K-Ras(G12D)LA2 transgenic lung cancer mouse models, and multimodal imaging shows a significant reduction in tumor metabolic activity (decreased by 62%) [3]
Oral administration of Milciclib (PHA-848125) (50 mg/kg once daily) combined with docetaxel (10 mg/kg intraperitoneally once weekly) for 3 weeks results in an 83% inhibition rate of H460 lung cancer xenografts in nude mice, which is significantly higher than that of the monotherapy groups (Milciclib 62%, docetaxel 45%) [2]

The assessment of PHA-848125's inhibition of kinase activity is conducted through a robotized format assay utilizing a strong anion exchanger (Dowex 1X8 resin) on 384-well plates. This assay involves the use of optimal buffers and cofactors to facilitate the transphosphorylation of particular peptides or protein substrates by their respective kinase in the presence of ATP traced with [γ-33P]ATP. PHA-848125's potency against CDKs and 38 other kinases from an internal Kinase Selectivity Screening panel is assessed, and the corresponding IC50s are found. After calculating the absolute KM values for ATP and the particular substrate for each enzyme, the assays are conducted at optimized concentrations of ATP (2KM) and substrate (5KM). This configuration facilitates the direct comparison of PHA-848125's IC50 values across the panel in order to assess its biochemical profile.

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Recombinant CDK1/cyclin B, CDK2/cyclin A/E, CDK4/cyclin D1, CDK9/cyclin T, and TRK A/B/C kinase complexes were prepared. Gradient concentrations of Milciclib (PHA-848125) were mixed with kinase complexes, ATP substrate, and specific fluorescent peptides, and incubated at 37°C for 60 minutes; the amount of phosphorylated peptides was detected by fluorescence resonance energy transfer (FRET) to calculate the kinase activity inhibition rate and IC50 value [4]
Radioactive phosphorylation assay was used to verify CDK activity: Milciclib (PHA-848125) was pre-incubated with CDK2/cyclin A complex for 15 minutes, then [γ-³²P]ATP and substrate peptides were added. After reacting at 30°C for 45 minutes, the substrate was separated by gel electrophoresis and autoradiographed, and the phosphorylation level was quantitatively analyzed to determine the IC50 [2]

Melanoma cells are cultured at a density of 2 × 10 4 cells/mL in media, then 50 μL aliquots are added to flat-bottom 96-well plates and left to adhere for an entire night at 37 °C. The wells (4 wells per point) in 50 μL of CM are then filled with graded amounts of PHA-848125 or TMZ, and the plates are incubated for 5 days at 37 °C in a humidified atmosphere with 5% CO₂. Together with the MGMT inhibitor BG, the cytotoxic effects of TMZ are also assessed. To achieve this, 2 hours prior to TMZ, 10 μM BG is added to the plates, and they are left in culture for the duration of the cells' exposure to the medication. Cells treated with BG or DMSO alone, as well as untreated cells, represent the ContS1017rol groups. There is no discernible difference in the growth of cells treated with BG or DMSO alone compared to untreated cells. The inhibitor PHA-848125 is added to BG-treated cells two hours later, and the MGMT activity of these cells is virtually undetectable until the assay is finished. Normal melanocytes are plated (50 μL/well) and exposed to either PHA-848125 or TMZ + BG, as described for melanoma cells, after being suspended in MGM at a concentration of 1.6 × 10 5 cells/mL. Using the MTT assay, cell growth is assessed at the conclusion of the incubation period. In a nutshell, each well receives 0.1 mg of MTT (in 20 μL of PBS), and the cells are incubated for 4 hours at 37 °C. After that, cells are lysed using a buffer (0.1 mL/well) that has a pH 4.7 mixture of 50% N,N-dimethylformamide and 20% SDS. Using a 3550-UV microplate reader, the absorbance is measured at 595 nm following an overnight incubation. The drug concentration that produces a 50% inhibition of cell growth, or IC50, is a measure of how sensitive cells are to treatment. It is derived from plotting absorbance values at 595 nm against the logarithm of drug concentration to create a regression line.

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Tumor cells were seeded in 96-well plates (5×10³ cells/well) and cultured for 24 hours, then gradient concentrations of Milciclib (PHA-848125) (0.01-10 μM) were added and cultured for another 72 hours; the CellTiter-Glo luminescent method was used to detect cell viability, and the IC50 value was calculated by curve fitting [2]
After treating A375 cells with Milciclib (PHA-848125) (50 nM) for 48 hours, the cells were collected and fixed, stained with PI, and the cell cycle distribution was analyzed by flow cytometry; total RNA and protein were extracted, and PTTG1 mRNA expression was detected by qPCR, while PTTG1 and cell cycle-related proteins (phosphorylated Rb, cyclin B1) were detected by Western blot [1]
After treating MCF-7 cells with the drug for 72 hours, the apoptosis rate was detected by Annexin V-FITC/PI double staining; caspase-3/7 activity was determined by a caspase-3/7 activity assay kit, and the expression of Bcl-2, Bax, and PARP proteins was detected by Western blot [2]
Tumor cells were seeded in 6-well plates (1×10³ cells/well) and cultured for 24 hours, then Milciclib (PHA-848125) (0.01-1 μM) was added and cultured for another 14 days; after fixation with methanol and staining with crystal violet, clones with ≥50 cells were counted to calculate the clonogenic rate [1]

Once a mammary tumor reaches a diameter of 0.5 cm, rats are randomly assigned to the study. Ten animal groups receive oral treatment twice daily for a duration of ten days, either with glucose as a vehicle or with Milciclib at doses of 5, 10, or 15 mg/kg. Another group of animals receives two cycles of Milciclib at a dose of 20 mg/kg twice daily for five days, separated by a one-week rest period. Throughout the experiment, the tumor volume is periodically measured with a caliper.

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Female nude mice (6-8 weeks old) were subcutaneously inoculated with A375 cell suspension (2×10⁶ cells/mouse) on the right back. Drug administration started when the tumor volume reached 100-150 mm³; Milciclib (PHA-848125) was dissolved in normal saline containing 0.5% hydroxypropyl methylcellulose and 0.1% Tween 80, and administered orally at 60 mg/kg once daily for 21 days; tumor volume and mouse weight were measured every 3 days, and tumors were excised and weighed at the end of the experiment to detect PTTG1 expression and CDK/TRK activity in tumor tissues [1]
K-Ras(G12D)LA2 transgenic lung cancer mice (8 weeks old, male) were randomly divided into groups; the administration group was given oral Milciclib (PHA-848125) 80 mg/kg once daily for 28 days; tumor volume and metabolic activity were monitored by magnetic resonance imaging (MRI) and positron emission tomography (PET), and tumor infiltration in lung tissue was detected at the end of the experiment [3]
Nude mice with H460 lung cancer xenograft models (tumor volume reached 150 mm³) were divided into monotherapy and combination groups: Milciclib (PHA-848125) 50 mg/kg orally once daily; docetaxel 10 mg/kg intraperitoneally once weekly; the combination group was administered synchronously for 3 weeks, and the tumor inhibition rate was calculated at the end of the experiment [2]

After oral administration of 60 mg/kg Milcilib (PHA-848125) to rats, the time to peak concentration (Tmax) was 2.2 hours, the peak plasma concentration (Cmax) was 890 ng/mL, and the oral bioavailability was 52% [4]. The elimination half-life (t1/2) of Milcilib (PHA-848125) in mice was 6.9 hours, and the elimination half-life in rats was 8.3 hours. It is mainly metabolized in the liver, with 70% excreted in feces and 19% in urine [4]. Milcilib (PHA-848125) is widely distributed in mice. The drug concentration in tumor tissue is 1.6 times that in plasma, and the drug concentration in liver and kidney tissue is 4.1 times and 2.7 times that in plasma, respectively [4].

The oral median lethal dose (LD50) of mircidyl (PHA-848125) in mice and rats was 590 mg/kg and 550 mg/kg, respectively, indicating that its acute toxicity was low[4]. When rats were given mircidyl (PHA-848125) orally at a dose of 100 mg/kg (once daily for 28 days), no obvious hepatotoxicity or nephrotoxicity was observed, and there were no statistically significant differences in serum ALT, AST, BUN and Cr levels compared with the control group; the peripheral blood leukocyte count also did not decrease significantly (change ≤±12%)[4]. The human plasma protein binding rate of mircidyl (PHA-848125) was 95%±2%[4].

[1]. Down-regulation of the PTTG1 proto-oncogene contributes to the melanoma suppressive effects of the cyclin-dependent kinase inhibitor PHA-848125. Biochem Pharmacol. 2012 Sep 1;84(5):598-611.

[2]. Dual targeting of CDK and tropomyosin receptor kinase families by the oral inhibitor PHA-848125, an agent with broad-spectrum antitumor efficacy. Mol Cancer Ther. 2010 Aug;9(8):2243-54.

[3]. Efficacy of PHA-848125, a cyclin-dependent kinase inhibitor, on the K-Ras(G12D)LA2 lung adenocarcinoma transgenic mouse model: evaluation by multimodality imaging. Mol Cancer Ther. 2010 Mar;9(3):673-81.

[4]. Identification of N,1,4,4-tetramethyl-8-{[4-(4-methylpiperazin-1-yl)phenyl]amino}-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (PHA-848125), a potent, orally available cyclin dependent kinase inhibitor. J. Med. Chem. 52(16), 5152-5163 (2009).

Milciclib is an orally bioavailable inhibitor of cyclin-dependent kinase (CDK) and tropomyosin receptor kinase A (TRKA) with potential antitumor activity. The CDK2/TRKA inhibitor PHA-848125 AC potently inhibits cyclin-dependent kinase 2 (CDK2) and is active against other CDKs, including CDK1 and CDK4, as well as TRKA. Inhibition of these kinases may lead to cell cycle arrest and apoptosis in tumor cells expressing these kinases. CDKs are serine/threonine kinases involved in cell cycle regulation and may be overexpressed in some cancer cell types. The neurotrophic factor receptor TRKA is mutated in a variety of cancer cell types. See also: Mircilib maleate (note moved to). Mircilib (PHA-848125) is an orally potent dual-target inhibitor that exerts a synergistic antitumor effect by simultaneously inhibiting the CDK family (which regulates cell cycle and transcription) and the TRK family (which regulates tumor proliferation signaling) [2].
Mircea (PHA-848125) has significant inhibitory activity against K-Ras mutant lung cancer, and its mechanism is related to the downregulation of the Ras-MAPK signaling pathway [3].
Preclinical studies have shown that Mircea (PHA-848125) has broad-spectrum anti-tumor efficacy, especially suitable for tumors with high CDK/TRK expression or K-Ras mutation, and is currently in the preclinical development stage. [4]
The inhibitory effect of mircea (PHA-848125) on melanoma depends in part on the downregulation of PTTG1 proto-oncogene expression, and PTTG1 can be used as a predictive marker of its efficacy [1]

C25H32N8O

460.57

460.269

C, 65.19; H, 7.00; N, 24.33; O, 3.47

802539-81-7

16718576

Light yellow to yellow solid powder

1.3±0.1 g/cm3

1.692

1.75

2

7

4

34

718

0

O=C(C1C2=C(C3C(CC2(C)C)=CN=C(NC2C=CC(N4CCN(C)CC4)=CC=2)N=3)N(C)N=1)NC

RXZMYLDMFYNEIM-UHFFFAOYSA-N

InChI=1S/C25H32N8O/c1-25(2)14-16-15-27-24(29-20(16)22-19(25)21(23(34)26-3)30-32(22)5)28-17-6-8-18(9-7-17)33-12-10-31(4)11-13-33/h6-9,15H,10-14H2,1-5H3,(H,26,34)(H,27,28,29)

N,1,4,4-tetramethyl-8-[4-(4-methylpiperazin-1-yl)anilino]-5H-pyrazolo[4,3-h]quinazoline-3-carboxamide

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)

Solubility in Formulation 1: ≥ 2 mg/mL (4.34 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 20.0 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 mg/mL (4.34 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 20.0 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: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30mg/mL

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