LC-MS/MS method for the quantitation of a dual PI3K/BRD4 inhibitor SF2523 in mouse plasma: application to plasma protein binding and metabolism studies.
作者: Veenu Bala ; Yashpal S Chhonker ; Guillermo A Morales ; Krishnaiah Maddeboina ; Dhananjaya Pal ; Donald L Durden ; Daryl J Murry
A sensitive and selective liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of dual PI3K/BRD4 inhibitor SF2523 in mouse plasma. The analysis was performed on a UPLC system connected to a Shimadzu 8060 mass spectrometer by electrospray in positive multiple reaction monitoring mode. Chromatographic separation was carried out on an ACE Excel C18 column with a gradient elution containing 0.1% formic acid and methanol as the mobile phase. The linearity was conducted in the concentration range of 0.1-500 ng/mL for SF2523 in 100μL plasma. The inter- and intra-batch precision (% RSD) were both lower than 13.5 %, with the accuracy (%Bias) ranged from varied from -10.03% to 11.56%. The validated method was successfully applied to plasma protein binding and in vitro metabolism studies. SF2523 was highly bound to mouse plasma proteins (>95% bound). Utilizing mouse S9 fractions, a total of seven phase I and II metabolites were identified with hydroxylation found to be the major metabolic pathway. Metabolites identification included analysis of retention behaviors, molecular weight changes and MS/MS fragment patterns of SF2523 and the metabolites. This newly developed and validated method allows the rapid and easy determination of the SF2523 concentration with high sensitivity in low sample volume and can be applied to future pre-clinical studies.
2023-03-27·Journal of orthopaedic research : official publication of the Orthopaedic Research Society
The Effectiveness and Mechanical Properties of Chemotherapy-Impregnated Cement in Ewing Sarcoma.
作者: Amanda N Goldin ; Robert M Healey
Bone cement is often used in the surgical treatment of Ewing sarcoma (ES). Chemotherapy-impregnated cement (CIC) has never been tested in slowing ES growth. The purpose of the study is to determine if CIC can decrease cell proliferation, and to assess changes in the mechanical qualities of the cement. Chemotherapeutic agents including doxorubicin, cisplatin, etoposide, and SF2523 were mixed with bone cement. ES cells were plated and exposed to cell growth media that had contained CIC or regular bone cement (RBC) as a control, and cell proliferation assays were performed daily for three days. Mechanical testing on RBC and CIC was also performed. There was a significant decrease (p<0.001) in cell proliferation among all cells treated with CIC compared to cells treated with RBC by 48 hours post-exposure. Additionally, there was a synergistic effectiveness of the CIC noted when multiple antineoplastic agents were combined. Three-point-bending tests did not reveal substantial reductions in tolerated maximum bending load and maximal displacement at maximal bending load between CIC and RBC. This article is protected by copyright. All rights reserved.
2023-01-05·Journal of controlled release : official journal of the Controlled Release Society
Targeting BRD4 and PI3K signaling pathways for the treatment of medulloblastoma.
作者: Bharti Sethi ; Virender Kumar ; Thilina D Jayasinghe ; Yuxiang Dong ; Donald R Ronning ; Haizhen A Zhong ; Donald W Coulter ; Ram I Mahato
Medulloblastoma (MB) is a malignant pediatric brain tumor which shows upregulation of MYC and sonic hedgehog (SHH) signaling. SHH inhibitor face acquired resistance, which is a major cause of relapse. Further, direct MYC oncogene inhibition is a challenging therapeutic target, inhibition of MYC upstream insulin-like growth factor/ phosphatidylinositol-4,5-bisphosphate 3-kinase (IGF/PI3K) is a promising alternative. While PI3K inhibition activates resistance mechanisms, simultaneous inhibition of bromodomain-containing protein 4 (BRD4) and PI3K can overcome resistance. We synthesized a new molecule 8-(2,3-dihydrobenzo[b] [1, 4] dioxin-6-yl)-2-morpholino-4H-chromen-4-one (MDP5) that targets both BRD4 and PI3K pathways. We used X-ray crystal structures and a molecular modeling approach to confirm the interactions between MDP5 with bromo domains (BDs) from both BRD2 and BRD4, and molecular modeling for PI3K binding. MDP5 was shown to inhibit target pathways and MB cell growth in vitro and in vivo. MDP5 showed higher potency in DAOY cells (IC50 5.5 μM) compared to SF2523 (IC50 12.6 μM), and its IC50 values in HD-MB03 cells were like SF2523. Treatment of MB cells with MDP5 significantly decreased colony formation, increased apoptosis, and halted cell cycle progression. Further, MDP5 was well tolerated in NSG mice bearing either xenograft or orthotopic MB tumors at the dose of 20 mg/kg, and significantly reduced tumor growth and prolonged animal survival.