DETERMINATION OF THE SPHINGOLIPID BIOSYNTHESIS INHIBITOR MYRIOCIN BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (LC-MS/MS). PILOT APPLICATIONS IN BIOLOGICAL MODELS
Tesi di Dottorato
Data di Pubblicazione:
2017
Citazione:
DETERMINATION OF THE SPHINGOLIPID BIOSYNTHESIS INHIBITOR MYRIOCIN BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (LC-MS/MS). PILOT APPLICATIONS IN BIOLOGICAL MODELS / G.m. Campisi ; tutore: R. Ghidoni ; direttore del dottorato: M. Clerici. DIPARTIMENTO DI FISIOPATOLOGIA MEDICO-CHIRURGICA E DEI TRAPIANTI, 2017 Feb 16. 29. ciclo, Anno Accademico 2016. [10.13130/campisi-giuseppe-matteo_phd2017-02-16].
Abstract:
Introduction. Myriocin (Myr) is an antifungal compound isolated in the 1972s from Myriococcum albomyces. Myr specifically inhibits serine palmitoyl transferase, the first enzyme involved in the de novo synthesis of Ceramide (Cer), which Intracellular accumulation yields inflammation and apoptosis. Targeting Cer biosynthesis, thus, may be a promising therapeutic treatment for all sphingolipid-related diseases. To test the efficacy of Myr as pharmacological tool able to drive the clinical outcome and rationalize the dose-effect relationship, Myr levels need to be accurately measured in minute amount of biological samples. Since the few available methods are inadequate, this study developed and validated the first method for the measurement of Myr by LC-MS/MS.
Methodology. Internal standard 14-OH-Myr (custom synthetized) and SPE were used for sample preparation and purification. A Dionex Ultimate 3000 UPLC coupled to an ABSciex 3200QTrap working in the MRM mode to monitor the analytical transitions (-400.4 →-104.0 for Myr; -402.4 → -104.0 for the IS) were used for analysis. Separation on Inertsil ODS-3 column (3μm, 3.0 x 150mm) lasted 9 min using a 50-100% 0.1% formic acid-acetonitrile gradient.
As preliminary study, we measured, in C57 Black6 mice, Myr administrated by intra-tracheal and intravitreal routes, two easy and common administration routes employed for lung and retina related diseases. In addition, for the intra-tracheal administration was used also Myr loaded in Solid Lipid Nanoparticles (Myr-SLN). The dose administrated were 4.69 nmoles (N. mice=6) of Myr-DMSO and 3.15 nmoles (N. mice=4) of Myr-SLN for lung study and 1.87 nmoles (N. mice=3) of Myr-DMSO for retina study. After sacrifice, we measured Myr levels (LC-MS/MS) in lung and retina at 24hrs and 4 hrs from administration, respectively.
In the same lung samples, we carried out dose-effect study quantifying Cer content, as a direct “readout”.
For “in vitro” uptake study of Myr we used the human C38 cell line as a model system. Cells were seeded into P100 or 6 multi-wells plate and after 24 h Myr was added into LHC-8 media at 10 nmol/mL. The time points checked were 5,10,30,60,120 min, cells were collected, washed and analysed.
As a mouse model of Retinitis Pigmentosa (RP) we chose Tvrm4 strain in which it is known that the exposure to high light originate the disease. As a preliminary validation of this model we tested the Cer increase (by LC-MS/MS) and their eventual inhibition by Myr-DMSO treatment (1.87 nmoles; N. mice=7).
Results
Method validation. As low as 20 fmoles can be detected on-column and quantified with ±15% precision and within ±20% of target level; the method was linear up to 25 pmoles in a typical 150 microL extracted. Examined samples (n>50; median level 0.25 pmoles) include organs of animal models, cultured human cells, Myr loaded into Solid Lipid Nanoparticles as an innovative pharmaceutical formulation (Myr-SLN).
Dose-effect in mice lung. The levels of Myr in lung homogenates were 11.0 ± 1.96 pmoles (mean ± SEM) and 4.36 ± 1.16 pmoles (mean ± SEM) for Myr-DMSO and Myr-SLN respectively. Both DMSO-Myr, and SLN-Myr produced a significant decrease in Cer levels vs the respective control.
IV
Set-up of Myr measurement in mouse retina. The levels of Myr in retina homogenates of wild type mice after intravitreal injection were 0.24 ± 0.08 mean ± SEM for Myr-DMSO.
In vitro uptake in C38 cells. The “in vitro” study of Myr-DMSO uptake in C38 cells already showed at the first observation point (5 min) the peak of Myr intracellular concentration (527 pmols/mg prot) followed by two different kinetics order of Myr disappearance. A fast kinetic was featured between 5-10 min, followed by slower trend for the other checked time
Tipologia IRIS:
Tesi di dottorato
Elenco autori:
G.M. Campisi
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