p70S6K (IC50 = 4 nM)

LY-2584702 (LY2584702), having an IC50 of 0.1-0.24 μM, suppresses the phosphorylation of S6 ribosomal protein (pS6) in HCT116 colon cancer cells [1]. The S6K1 enzyme test yields an IC50 value of 2 nM for LY-2584702 (LY2584702). In cells, IC50=100 nM for pS6 inhibition. At high concentrations (IC50=58-176 nM in the enzyme assay), LY-2584702 exhibits specific action against the S6K-related kinases MSK2 and RSK. The phosphorylation of LY-2584702's downstream effector S6 determines how dose-dependently it suppresses S6K activity in EOMA cells [2]. When administered at 0.1 μM for more than 24 hours, LY-2584702 (LY2584702) can considerably limit the proliferation of A549 (P<0.05); additional time under treatment and/or higher drug concentrations make the downward trend more pronounced (both P<0.05). In SK-MES-1, comparable outcomes were noted, but at 0.6 μM Significantly more potent than A549, LY-2584702 showed an inhibitory effect (P<0.05)[3].

Under 2.5 mg/kg twice daily (BID) and 12.5 mg/kg BID, LY-2584702 showed noteworthy single-agent activity in HCT116 colon cancer and U87MG glioblastoma xenograft models. At TMED50 (threshold minimum effective dose 50%) (2.3 mg/kg) and TMED90 (10 mg/kg), LY-2584702 demonstrated statistically significant tumor growth decrease in the HCT116 colon cancer xenograft model [1]. LY-2584702 or rapamycin were administered to shAkt3-expressing EOMA cells implanted into nu/nu mice for a period of 14 days in order to investigate the function of S6K in vivo. LY-2584702 suppressed S6 phosphorylation nearly as well as rapamycin, according to analysis of tumors removed after 14 days. In contrast to pLKO, tumor development was enhanced by Akt3 loss. The growth of pLKO tumors was not substantially impacted by LY-2584702 treatment alone. LY-2584702, however, dramatically slowed the growth of shAkt3 tumors [2].

LY-2584702 is completely dissolved in 20 mL of 10% DMSO and stored at -80°C. When conducting the experiments in vitro, LY-2584702 is further diluted in 0.5% Tween 80, 5% propylene glycol, and 30% PEG400 to achieve various DMSO concentrations of 0.1 μM, 0.2 μM, 0.6 μM, and 1.0 μM. In vitro cell proliferation is assessed using the Cell Counting Kit-8 (CCK-8). A549 and SK-MES-1 cell lines that have been exposed to LY-2584702 at various concentrations for 24 hours are seeded in 96-well plates at a density of 5 103 cells per well with six repetitions. The concentration of LY-2584702 at zero is used as a negative control, or DMSO treated. Every 24 hours after seeding, cells' absorbance at 450 nm is measured to gauge their proliferative activities.

Pharmacokinetics [1]
Pharmacokinetic analysis was performed on both Part A and Part B, and the data are summarized in Table 4. The initial regimen started with a dose of 300 mg twice daily, but two patients taking 300 mg BID experienced severe nausea and vomiting. Analysis of plasma LY2584702 exposure levels indicated that we had exceeded the predicted range of effective target inhibition. In addition, the metabolic clearance was 10 L/h, while the predicted value was 26 L/h. Therefore, we implemented a new once-daily dosing regimen with a dose range of 25 to 200 mg. After analyzing the exposure of the once-daily dosing regimen, we established a twice-daily dosing group to determine whether twice-daily dosing could increase the total daily exposure. In the twice-daily dosing group, the maximum tolerated dose (MTD) was determined to be 75 mg. The maximum tolerated dose in the once-daily dosing group was 100 mg. The half-life remained stable at 5.96 hours in all groups, but there were differences in exposure (AUC) and Cmax. Exposure to LY2584702 was not dose-dependent, but increased with increasing dose. With the once-daily dosing regimen, no cumulative exposure to LY2584702 was observed, with a median cumulative ratio [Day 8 AUC(0–24)/Day 1 AUC(0–24)] of 0.61 (range: 0.52–1.7). With the twice-daily dosing regimen, drug accumulation was observed, with a median accumulation ratio of 1.98 (range: 1.1–2.69), but there was no evidence of time-dependent exposure. The median time dependence (AUC(0–24)/AUC(0–∞)) for the once-daily dosing regimen was 0.45 (range: 0.41–1.03), and the median time dependence (AUC(0–24)/AUC(0–∞)) for the twice-daily dosing regimen was 1.12 (range: 0.61–1.34).

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Pharmacokinetics [2]
The pharmacokinetic parameters of LY2584702 were analyzed, and the results are summarized in Table 4. When LY2584702 was used in combination with erlotinib, the exposure increased in a dose-proportional manner. The dose-standardized AUCs for once-daily 50 mg and twice-daily 50 mg were 88.73 ng·h/mL/mg and 86.16 ng·h/mL/mg, respectively. The AUC increased slightly at twice-daily 75 mg (107.89 ng·h/mL/mg), but decreased at twice-daily 100 mg (86.11 ng·h/mL/mg). When LY2584702 was used in combination with everolimus, the exposure also increased in a dose-proportional manner. The dose-standardized AUC values for 50 mg twice daily, 50 mg once daily, and 100 mg once daily were 121.95, 114.00, and 114.10 ng·h/mL/mg, respectively. For erlotinib and everolimus dosing regimens, the median cumulative ratios for the once-daily dosing regimen were 1.09 and 1.07 (range 0.98–1.16), respectively, while the median cumulative ratios for the twice-daily dosing regimen were 2.16 and 1.98 (range 1.85–3.41), respectively. There was a significant difference in LY2584702 exposure between the once-daily and twice-daily dosing regimens (p = 0.0145, t-test), with cumulative ratios of 1.08 and 2.46, respectively. The V/F ratio showed high variability across all dose groups, at 34.52 L (coefficient of variation 47%).

Toxicity[1]
34 patients received at least one dose of LY2584702, of whom 13 (38%) experienced serious adverse events (SAEs) during treatment. Of the 13 patients who experienced SAEs, 3 were related to the study drug. One patient experienced grade 3 hypophosphatemia, one patient experienced grade 3 vomiting and grade 3 pancreatitis, and another patient experienced grade 3 pancreatitis. 3 patients (9%) discontinued treatment due to adverse events. One patient died during a 30-day follow-up period following discontinuation of the study drug (at the physician's discretion). This patient received one dose of 100 mg twice daily of the study drug but withdrew from the study due to disease progression prior to death. Five patients in Part A and two patients in Part B experienced dose-limiting toxicities (DLTs). All DLTs were grade 3 and included vomiting, elevated lipase, nausea, hypophosphatemia, fatigue, and pancreatitis (Table 2). Of the 34 patients, 31 reported at least one treatment-adverse event (TEAE), of which 21 (62%) reported TEAEs that were likely related to the study drug. The most common TEAEs that were likely related to the study drug were nausea (26%), fatigue (18%), and vomiting (15%) (Table 3). Of the 55 adverse events related to the study drug, 22 were grade ≥ 3.

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Toxicity[2]
29 patients were enrolled, of whom 4 experienced grade ≥ 3 dose-limiting toxicities (DLT): 1 case of hypophosphatemia, 1 case of vomiting, 1 case of thromboembolic event, and 1 case of decreased coagulation factor V level (Table 2). 8 patients (47%) in group A experienced serious adverse events (SAEs), and 6 patients (50%) in group B experienced SAEs. Possible treatment-related adverse events (TEAEs) associated with the study drug included: grade 3 nausea, grade 3 vomiting, grade 3 anorexia, grade 3 gastritis, grade 3 pulmonary embolism, grade 3 elevated international normalized ratio (INR), grade 2 interstitial lung disease, and grade 2 deep vein thrombosis. Three patients (17.6%, n = 17) in group A and two patients (16.7%, n = 12) in group B discontinued treatment due to adverse events. The most common treatment-related adverse events (TEAEs) associated with the drug in group A were fatigue (88%), anorexia (71%), diarrhea (65%), nausea (53%), acne-like rash (53%), and vomiting (41%); in group B (Table 3), the incidence of fatigue (83%), anorexia (67%), nausea (58%), diarrhea (50%), and stomatitis (50%) was higher. In group A (n=17), 11 patients and in group B (n=12), 8 patients experienced grade 3/4 treatment-induced adverse events (TEAEs). Three patients in group A and one patient in group B experienced coagulation abnormalities. One patient in group A experienced a thromboembolic event (pulmonary embolism), and one patient in group B experienced a potentially related serious adverse event (SAE)—deep vein thrombosis. The third patient experienced a decrease in coagulation factor V levels during the first treatment cycle, from 60% on day 1 to 24% on day 8, then recovered to 85% on day 15, before decreasing again to 35% on day 22. No clinically significant changes were observed in intrinsic coagulation pathway factors IX, XI, XII, and thromboplastin (TP). The fourth patient in group A experienced a dose-limiting toxicity (DLT) manifested as hypophosphatemia with elevated INR and decreased levels of coagulation factors II, V, VII, and X (Figure 1). These changes were treatment-induced and improved upon discontinuation of the drug. Several patients in both groups experienced weight loss during treatment, ranging from 3% to 10% in group A and 4% to 11% in group B. Three patients (18%) in group A and six patients (50%) in group B experienced weight loss ≥10% (Figure 2). Dose escalation was discontinued in group B following the occurrence of thromboembolic events and the observation of other toxicities (fatigue and weight loss) in both groups.

[1]. A phase I trial of LY2584702 tosylate, a p70 S6 kinase inhibitor, in patients with advanced solid tumors. Eur J Cancer. 2014 Mar;50(5):867-75.

[2]. Akt1 and akt3 exert opposing roles in the regulation of vascular tumor growth. Cancer Res. 2015 Jan 1;75(1):40-50.

[3]. Hyperphosphorylation of RPS6KB1, rather than overexpression, predicts worse prognosis in non-small cell lung cancer patients. PLoS One. 2017 Aug 9;12(8):e0182891.

Angiomas are endothelial cell tumors, and their tumorigenesis mechanisms are not fully understood. Furthermore, current treatments, particularly those targeting malignant lesions, have limited clinical efficacy. This study demonstrates that endothelial cell activation of Akt1 kinase is sufficient to drive tumorigenesis. Mechanistic studies revealed that different Akt isoforms play opposing roles in this regulation: Akt1 promotes angiomas growth, while Akt3 inhibits it. Akt3 negatively impacts the growth and migration of tumor endothelial cells by regulating Rictor expression and inhibiting the activation of the translational regulatory kinase S6 kinase (S6K). Conversely, S6K inhibits Akt3 expression through a negative feedback loop. In contrast, the S6K signaling pathway is enhanced in Akt3-silenced angiomas, and a novel S6K inhibitor was able to suppress the growth of these tumor cells. Overall, our findings provide preclinical proof-of-concept for the therapeutic application of S6K inhibitors in angiomas such as angiosarcomas. [2]

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RPS6KB1 is a kinase of the ribosomal protein S6, which has a molecular weight of 70 kDa and is essential for protein translation. Although aberrant activation of RPS6KB1 has been found in various diseases, its role and clinical significance in non-small cell lung cancer (NSCLC) have not been fully investigated. In this study, we found that RPS6KB1 hyperphosphorylation (p-RPS6KB1) was present in NSCLC, and that p-RPS6KB1 was an independent poor prognostic marker for NSCLC patients. Although immunohistochemical staining (IHC) showed expression of both total RPS6KB1 and p-RPS6KB1 in NSCLC specimens, only p-RPS6KB1 was associated with clinicopathological features of NSCLC patients. Kaplan-Meier survival analysis showed that elevated p-RPS6KB1 expression was associated with poorer 5-year overall survival (OS) in NSCLC patients, while there was no statistically significant difference between the RPS6KB1 positive and negative groups. Subsequent univariate and multivariate Cox regression analyses confirmed the independent prognostic value of p-RPS6KB1. To elucidate the potential mechanism of RPS6KB1 phosphorylation in NSCLC, we specifically inhibited RPS6KB1 phosphorylation in the lung adenocarcinoma cell line A549 and the squamous cell carcinoma cell line SK-MES-1 using LY2584702. As expected, CCK-8 assays showed that RPS6KB1 dephosphorylation significantly inhibited cell proliferation, and cell cycle analysis revealed that RPS6KB1 dephosphorylation promoted more cells to arrest in the G0/G1 phase. Furthermore, apoptosis was significantly increased in A549 cells with RPS6KB1 dephosphorylation, and this trend also increased in SK-MES-1 cells, suggesting that RPS6KB1 phosphorylation may be involved in inducing apoptosis. In conclusion, our data indicate that in non-small cell lung cancer (NSCLC), RPS6KB1 is overactivated in the form of phosphorylated RPS6KB1 (p-RPS6KB1), and not just in terms of total protein overexpression. The phosphorylation level of RPS6KB1 may serve as a novel prognostic biomarker for NSCLC patients. [3]

C21H20CLF4N7

481.877016067505

481.14

1082948-81-9

LY-2584702 tosylate salt;1082949-68-5;LY-2584702 free base;1082949-67-4

66650363

Typically exists as solid at room temperature

2

9

3

33

644

0

CN1C=C(C2=CC=C(F)C(C(F)(F)F)=C2)N=C1C3CCN(C4=C5C(NN=C5)=NC=N4)CC3.[H]Cl

GDGYRKDHQORLNT-UHFFFAOYSA-N

InChI=1S/C21H19F4N7.ClH/c1-31-10-17(13-2-3-16(22)15(8-13)21(23,24)25)29-19(31)12-4-6-32(7-5-12)20-14-9-28-30-18(14)26-11-27-20;/h2-3,8-12H,4-7H2,1H3,(H,26,27,28,30);1H

4-[4-[4-[4-fluoro-3-(trifluoromethyl)phenyl]-1-methylimidazol-2-yl]piperidin-1-yl]-1H-pyrazolo[3,4-d]pyrimidine;hydrochloride

LY-2584702 hydrochloride; 1082948-81-9; LY-2584702 (hydrochloride); 4-(4-(4-(4-Fluoro-3-(trifluoromethyl)phenyl)-1-methyl-1H-imidazol-2-yl)piperidin-1-yl)-1H-pyrazolo[3,4-d]pyrimidine hydrochloride; 4-[4-[4-[4-fluoro-3-(trifluoromethyl)phenyl]-1-methylimidazol-2-yl]piperidin-1-yl]-1H-pyrazolo[3,4-d]pyrimidine;hydrochloride; 4-{4-[4-fluoro-3-(trifluoromethyl)phenyl]-1-methyl-1H-imidazol-2-yl}-1-{1H-pyrazolo[3,4-d]pyrimidin-4-yl}piperidine hydrochloride; LY2584702 Hydrochloride; SCHEMBL312564;

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.)