Posaconazole (POS) is a sterol C14ɑ demethylase inhibitor (IC50: 0.25 nM).

C14ɑ demethylase:0.25 nM

Posaconazole的生物利用度可被食物,尤其是高脂肪膳食显著增加.与高脂和脱脂食物一起消耗时,全身接触Posaconazole分别增加其4倍和2.6倍的消耗.Posaconazole（≥15 mg/kg,b.i.d）可使小鼠寿命延长,并减少组织负担.在感染的动物中,胺碘酮单独使用可减少寄生虫血症,增加60天生存期(未处理的对照组为0%,胺碘酮处理的动物为40%),与Posaconazole联用可延缓寄生虫血症的进程.Posaconazole 和胺碘酮可能产生有效的抗T. cruzi治疗,且副作用低.在禁食状态,Posaconazole与Boost Plus同时服用增加药物暴露.

Posaconazole对临床相关的细胞内无鞭毛体寄生虫形式的效能更好。Posaconazole的最低抑菌浓度和IC50值分别为3 nM和0.25 nM。Posaconazole对耐氟康唑、伏立康唑和两性霉素B的念珠菌和曲霉菌株具有活性,且效果比其它三唑类抗接合菌药更好。胺碘酮与Posaconazole可产生协同效果。Posaconazole还会影响并打乱T. cruzi 中Ca2+内稳态。Posaconazole对前鞭体(细胞外)阶段的增殖具有明显的剂量依赖性作用,最低抑菌浓度为20 nM,IC50为14 nM。

In vitro Aβ reduction assays : Human embryonic kidney cells (American Type Culture Collection CRL-1573), transfected with the gene for APP751 (HEK 293) are used for routine Aβ reduction assays. Cells are plated in 96-well plates and allowed to adhere overnight in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum. DAPT are diluted from stock solutions in dimethylsulfoxide (DMSO) to yield a final concentration equal to 0.1% DMSO in media. Cells are pre-treated for 2 hours at 37 °C with DAPT, media are aspirated off and fresh compound solutions applied. After an additional 2-hour treatment period, conditioned media is drawn off and analyzed by a sandwich ELISA (266–3D6) specific for total Aβ. Reduction of Aβ production is measured relative to control cells treated with 0.1% DMSO and expressed as a percentage inhibition. Data from at least six doses in duplicate are fitted to a four-parameter logistical model using XLfit software in order to determine potency. Human and PDAPP mouse neuronal cultures are grown in serum-free media to enhance their neuronal characteristics, and appeared to be greater than 90% neurons after maturation prior to use. Conditioned media to establish baseline Aβ values are collected by adding fresh media to each well and incubated for 24 hours at 37 °C in the absence of DAPT. Cultures are then treated with fresh media containing DAPT at the desired range of concentrations for an additional 24 hours at 37 °C, and conditioned media collected. For the measurement of total Aβ, samples are analyzed with the same ELISA (266–3D6) as used for the HEK 293 cell assays. Analyses of samples for Aβ42 production are performed by a separate ELISA (21F12–3D6) that utilizes a capture antibody specific for the Aβ42 C-terminus. Inhibition of production for both total Aβ and Aβ42 are determined by the difference between the values for the compound treatment and baseline periods.After plotting percentage inhibition versus DAPT concentration, data are analyzed with XLfit software, as above, to determine potency.

The epimastigote form of the parasite is cultivated in liver infusion tryptose medium,supplemented with 10% new born calf serum at 28 °C with strong (120 rpm) agitation.Cultures are initiated at a cell density of 2 × 106 epimastigotes/mL,and Posaconazole is added at a cell density of 0.5−1.0 × 107 epimastigotes/mL.Cell densities are measured by using an electronic particle counter as well as by direct counting with a hemocytometer.Cell viability is followed by Trypan blue exclusion,using light microscopy.Amastigotes are cultured in Vero cells maintained in minimal essential medium supplemented with 1% fetal calf serum in a humidified atmosphere (95% air−5% CO2) at 37 °C.Cells are infected with 10 tissue culture-derived trypomastigotes per cell for 2 hours and then washed three times with phosphate-buffered saline (PBS) to remove nonadherent parasites.Fresh medium with and without Posaconazole is added,and the cells are incubated for 96 hours with a medium change at 48 hours.The percent of infected cells and the numbers of parasites per cell are determined directly using light microscopy,and a statistical analysis of the results is carried out.IC50 values are calculated by nonlinear regression,using the program GraFit.Fractional inhibitory concentrations (FIC) are calculated.Cytoplasmic free Ca2+ concentrations in control and drug-treated extracellular epimastigotes are determined by fluorimetric methods using Fura-2.Subcellular Ca2+ levels and mitochondrial membrane potentials are monitored on individual Vero cells infected with T.cruzi amastigotes by using time-scan confocal microscopy.Briefly,Vero cells heavily infected (72 hours) with T.cruzi amastigotes are plated onto 22 × 40 mm glass coverslips (0.15 mm thickness) and incubated simultaneously with 10 μM cell-permeant Rhod-2 and 10 μg/mL Rhodamine-123 for 50 minutes at 37 °