DETERMINATION OF THE SPHINGOLIPID BIOSYNTHESIS INHIBITOR MYRIOCIN BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (LC-MS/MS). PILOT APPLICATIONS IN BIOLOGICAL MODELS

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 points (10, 30, 60, 120 min of observation).The lower intracellular concentration of Myr was reached after 120 min (103 pmol/mg prot). The reported Myr concentrations corresponded to about 7-34 pmol/well against the nanomoles added into the media (10 nmol/mL, i.e. 30 nmol/well). Validation of TVRM4 model of Retinitis pigmentsa. In Tvrm4 mice control (DMSO) the light exposure induced a 60% increase of the total Cer (1934 pmol/mg prot; SEM= ± 110) compared with the total Cer content in wild type mice (1209 pmol/mg prot; SEM= ± 154). Overall, injection of Myr-DMSO induced a 28% reduction in mean Cer content, from 1934 pmol/mg prot (SEM= ± 110) to 1394 pmol/mg prot (SEM= ± 107) (p<0,05). Conclusions. We have optimized original mass spectrometric and chromatographic conditions for Myr quantification, to obtain satisfactory resolution and sensibility for the intended use. The method here presented proved to be suitable for dose-efficacy studies in lung mice and demonstrated the activity of this molecule in decreasing CEr levels. These preliminary results open a novel scenario on the activity and metabolism of this molecule, in the view of its use as candidate drug in a number of different sphingolipid-related diseases. We validated the accuracy of Myr administration by intravitreal route. We developed the extraction procedure of Myr from minute amounts of tissue such as a single retina (1 mg) proving a good accuracy of the measurement. The “in vitro” study on Myr uptake highlights an unexpected behaviour of the molecule, with intracellular amounts ranging between 1000-4000 fold lower than the one incubated. As it appears, only pmoles of Myr were found into the cells against nanomols added into the media, in accordance also with the “in vivo” observations. It will be necessary additional pharmacokinetics and biochemical studies to clarify the mechanistic basis by which Myr crosses the cellular membrane and achieve its activity. We demonstrated the validity of a new mouse model of RP by by following the behavior of all the Cer fingerprint. The perspective is to optimize a procedure able to assess, starting from a unique mouse retina, both Myr and Cer by LC-MS/MS, and therefore to allow dose-effect studies.