Functionalized azirine based scaffolds as endothelin inhibitors for the selective anti-angiogenic activity.

Anti-angiogenic therapy has long been used as an adjunct therapy for the resolution of tumor burden. The current findings describe the synthesis of novel marine-based azirine-containing compounds that exhibit anti-angiogenic mediated anti-tumor activity. Azirine-2-carboxylate inhibited HUVEC-mediated tubulogenesis without causing cell death in a dose-dependent manner. Ex-vivo CAM, in-vivo Matrigel implantation, and ear angiogenesis experiments have all shown that azirine-2-carboxylate effectively inhibits angiogenesis. Furthermore, azirine-2-carboxylate inhibits the migration of ECs without disrupting the preformed tubule network. Azirine-2-carboxylate had adequate intramuscular systemic exposure and inhibited tumor growth in a xenograft mouse model. DARTS analysis, competitive binding assay, and gene expression investigations revealed that azirine-2-carboxylate inhibits endothelin-1-mediated angiogenesis. Overall, the discovery of azirine-2-carboxylate demonstrated a potent inhibition of angiogenesis targeting ET1 and a possible application in anti-angiogenic therapy.

2,3-Difunctionalized Benzo[b]thiophene Scaffolds Possessing Potent Antiangiogenic Properties.

A new class of 2-anilino-3-cyanobenzo[b]thiophenes (2,3-ACBTs) was studied for its antiangiogenic activity for the first time. One of the 2,3-ACBTs inhibited tubulogenesis in a dose-dependent manner without any toxicity. The 2,3-ACBTs significantly reduced neovascularization in both ex vivo and in vivo angiogenic assays without affecting the proliferation of endothelial cells. Neovascularization was limited through reduced phosphorylation of Akt/Src and depolymerization of f-actin and ß-tubulin filaments, resulting in reduced migration of cells. In addition, the 2,3-ACBT compound disrupted the preformed angiogenic tubules, and docking/competitive binding studies showed that it binds to VEGFR2. Compound 2,3-ACBT had good stability and intramuscular profile, translating in suppressing the tumor angiogenesis induced in a xenograft model. Overall, the present study suggests that 2,3-ACBT arrests angiogenesis by regulating the Akt/Src signaling pathway and deranging cytoskeletal filaments of endothelial cells.

Elucidation of plasma protein binding, blood partitioning, permeability, CYP phenotyping and CYP inhibition studies of Withanone using validated UPLC method: An active constituent of neuroprotective herb Ashwagandha.

ETHNOPHARMACOLOGICAL RELEVANCE: Withanone (WN), an active constituent of Withania somnifera commonly called Ashwagandha has remarkable pharmacological responses along with neurological activities. However, for a better understanding of the pharmacokinetic and pharmacodynamic behavior of WN, a comprehensive in-vitro ADME (absorption, distribution, metabolism, and excretion) studies are necessary. AIM OF THE STUDY: A precise, accurate, and sensitive reverse-phase ultra-performance liquid chromatographic method of WN was developed and validated in rat plasma for the first time. The developed method was successfully applied to the in-vitro ADME investigation of WN. MATERIAL AND METHODS: The passive permeability of WN was assayed using PAMPA plates and the plasma protein binding (PPB) was performed using the equilibrium dialysis method. Pooled liver microsomes of rat (RLM) and human (HLM) were used for the microsomal stability, CYP phenotyping, and inhibition studies. CYP phenotyping was evaluated using the specific inhibitors. CYP inhibition study was performed using specific probe substrates along with WN or specific inhibitors. RESULTS: WN was found to be stable in the simulated gastric and intestinal environment and has a high passive permeability at pH 4.0 and 7.0 in PAMPA assay. The PPB of WN at 5 and 20 µg/mL concentrations were found to be high i.e. 82.01 ± 1.44 and 88.02 ± 1.15%, respectively. The in vitro half-life of WN in RLM and HLM was found to be 59.63 ± 2.50 and 68.42 ± 2.19 min, respectively. CYP phenotyping results showed that WN was extensively metabolized by CYP 3A4 and1A2 enzymes in RLM and HLM. However, the results of CYP Inhibition studies showed that none of the CYP isoenzymes were potentially inhibited by WN in RLM and HLM. CONCLUSION: The in vitro results of pH-dependent stability, plasma stability, permeability, PPB, blood partitioning, microsomal stability, CYP phenotyping, and CYP inhibition studies demonstrated that WN could be a better phytochemical for neurological disorders.

Determination of permeability, plasma protein binding, blood partitioning, pharmacokinetics and tissue distribution of Withanolide A in rats: A neuroprotective steroidal lactone.

Preclinical Research & Development Withanolide A (WA), a steroidal lactone is a major bioactive constituent of Withania somnifera (L.) with remarkable neuropharmacological activity. In this study, we investigated the permeability, plasma protein binding (PPB), blood partitioning, intravenous (i.v.), and oral pharmacokinetics as well as i.v. tissue distribution (TD) of pure WA in a rat model. The PPB, RBCs partitioning, and permeability of WA were determined by Ultra Performance Liquid Chromatography (UPLC) method. However, the pharmacokinetics and TD of WA were evaluated by validated and sensitive liquid chromatography coupled mass spectrometry (LC-ESI-MS/MS) method. The PPB and permeability of WA were determined by equilibrium dialysis and parallel artificial membrane permeability assay method, respectively. The results demonstrated that WA has high PPB and passive permeability. Furthermore, WA was found to have fast equilibration between RBCs and plasma. Following i.v. (2 mg/kg) and per-oral (25 mg/kg) administration of WA, the max concentration (Cmax ) in plasma was found as 85.53 ± 6.54 and 48.04 ±5.78 ng/mL, respectively. The TD study results indicated that WA has a rapid and wide TD. The maximum concentration in various tissues was found in following order: Clung > Cliver > Ckidney ≈ Cspleen > Cheart > Cbrain . The preclinical in vitro, as well as pharmacokinetics and TD results, are anticipated to support the future preclinical and clinical application of WA.

Evaluation of oral pharmacokinetics, in vitro metabolism, blood partitioning and plasma protein binding of novel antidiabetic agent, S009-0629 in rats.

S009-0629 [methyl-8-(methylthio)-2-phenyl-6-p-tolyl-4,5-dihydro-2H-benzo[e]indazole-9-carboxylate] is a novel antidiabetic agent with PTP1B inhibitory activity. In this study, we have investigated the in vitro metabolic stability, plasma protein binding, blood partitioning, and oral pharmacokinetic study of S009-0629 in rats. The plasma protein binding, blood partitioning, and metabolic stability were determined by HPLC method. The oral pharmacokinetic study was analyzed by liquid chromatography coupled mass spectrometry (LC-MS/MS) method. The plasma protein binding of S009-0629 using modified charcoal adsorption method at 5 and 10 µg/mL was 80.58 ± 1.04% and 81.95 ± 1.15%, respectively. The KRBC/PL of S009-0629 was independent of concentration and time. The in-vitro half-life of S009-0629 at 5 and 10 µM using rat liver microsomes was determined as 273 ± 24.46 and 281.67 ± 26.53 min, respectively. After oral administration, S009-0629 exhibited Cmax 55.51 ± 1.18 ng/mL was observed at 18 hr (tmax ). S009-0629 was found to have the large apparent volume of distribution (1,894.93 ± 363.67 L/kg). Oral in-vivo t1/2 of S009-0629 was found to be 41.23 ± 5.96 hr. A rapid and highly sensitive LC-MS/MS method was validated for S009-0629 in rat plasma. S009-0629 has high plasma protein binding and low hepatic extraction. S009-0629 has no affinity with human P-gp and BCRP in ATPase assay. After oral dosing, S009-0629 has slow absorption and elimination in rats.

Validation of a Rapid and Sensitive UPLC-MS-MS Method Coupled with Protein Precipitation for the Simultaneous Determination of Seven Pyrethroids in 100 µL of Rat Plasma by Using Ammonium Adduct as Precursor Ion.

United States Environmental Protection Agency has recommended estimating pyrethroids' risk using cumulative exposure. For cumulative risk assessment, it would be useful to have a bioanalytical method for quantification of one or several pyrethroids simultaneously in a small sample volume to support toxicokinetic studies. Therefore, in the present study, a simple, sensitive and high-throughput ultraperformance liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous analysis of seven pyrethroids (fenvalerate, fenpropathrin, bifenthrin, lambda-cyhalothrin, cyfluthrin, cypermethrin and deltamethrin) in 100 µL of rat plasma. A simple single-step protein precipitation method was used for the extraction of target compounds. The total chromatographic run time of the method was 5 min. The chromatographic system used a Supelco C18 column and isocratic elution with a mobile phase consisting of methanol and 5 mM ammonium formate in the ratio of 90 : 10 (v/v). Mass spectrometer (API 4000) was operated in multiple reaction monitoring positive-ion mode using the electrospray ionization technique. The calibration curves were linear in the range of 7.8-2,000 ng/mL with correlation coefficients of ≥ 0.99. All validation parameters such as precision, accuracy, recovery, matrix effect and stability met the acceptance criteria according to the regulatory guidelines. The method was successfully applied to the toxicokinetic study of cypermethrin in rats. To the best of our knowledge, this is the first LC-MS-MS method for the simultaneous analysis of pyrethroids in rat plasma. This validated method with minimal modification can also be utilized for forensic and clinical toxicological applications due to its simplicity, sensitivity and rapidity.