Selective detection of phospholipids using molecularly imprinted fluorescent sensory core-shell particles.

Sphingosine-1-phosphate (S1P) is a bioactive sphingo-lipid with a broad range of activities coupled to its role in G-protein coupled receptor signalling. Monitoring of both intra and extra cellular levels of this lipid is challenging due to its low abundance and lack of robust affinity assays or sensors. We here report on fluorescent sensory core-shell molecularly imprinted polymer (MIP) particles responsive to near physiologically relevant levels of S1P and the S1P receptor modulator fingolimod phosphate (FP) in spiked human serum samples. Imprinting was achieved using the tetrabutylammonium (TBA) salt of FP or phosphatidic acid (DPPA·Na) as templates in combination with a polymerizable nitrobenzoxadiazole (NBD)-urea monomer with the dual role of capturing the phospho-anion and signalling its presence. The monomers were grafted from ca 300 nm RAFT-modified silica core particles using ethyleneglycol dimethacrylate (EGDMA) as crosslinker resulting in 10-20 nm thick shells displaying selective fluorescence response to the targeted lipids S1P and DPPA in aqueous buffered media. Potential use of the sensory particles for monitoring S1P in serum was demonstrated on spiked serum samples, proving a linear range of 18-60 µM and a detection limit of 5.6 µM, a value in the same range as the plasma concentration of the biomarker.

Bioequivalence Study of 2 Capsule Formulations of Fingolimod 0.5 mg Assessing Both Parent Drug and Active Metabolite in New Zealand Healthy Subjects (Truncated Design).

Fingolimod is indicated for the treatment of patients with the relapsing-remitting form of multiple sclerosis. The primary study objective was to evaluate the bioequivalence of a test formulation, 0.5 mg fingolimod HCl capsule (Lebrina, Asofarma Sociedad Anónima Industrial y Comercial, Argentina) relative to a reference formulation, 0.5 mg fingolimod capsule (Gilenya, Novartis Pharmaceutical, Australia). In a single-center, randomized, single-dose, single-blinded, 2-way crossover study, 33 New Zealand healthy subjects of both sexes were enrolled to receive a 0.5-mg dose of 3 capsules of each fingolimod formulation under fasting conditions, with a 42-day washout period between administrations. Additional pharmacokinetic information regarding its main active metabolite, fingolimod phosphate, was also provided. The point estimate and 90% confidence intervals of the ratios of maximum concentration and area under the plasma concentration-time curve from time 0 to 72 hours were 99.07 (95.83-102.41) and 97.64 (95.33-100.00) for fingolimod, and 95.60 (90.95-100.49) and 98.54 (96.19-100.96), for fingolimod phosphate. Primary parameters, maximum concentration and area under the plasma concentration-time curve from time 0 to 72 hours for fingolimod and fingolimod phosphate were found to have no significant difference when test and reference formulations were compared. Fingolimod and fingolimod phosphate of both formulations were within the accepted 90% confidence interval limits of 80.00% and 125.00%. No significant differences between the test and reference drug products were detected in any of the pharmacokinetic parameters estimated. Notwithstanding the primary conclusion of bioequivalence is focused on the measurement of the parent compound, compliance with the same criteria by the active metabolite reinforces the comparability between the pharmacokinetic profiles of both formulations (ClinicalTrials.gov Identifier: NCT03757338).

A sensitive liquid chromatography-tandem mass spectrometry method for quantitative bioanalysis of fingolimod in human blood: Application to pharmacokinetic study.

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of fingolimod in human blood. The analyte and internal standard fingolimod-d4 were extracted from 300 µl of human blood using protein precipitation coupled with solid-phase extraction method. The chromatographic separation was achieved with a Kinetex biphenyl column (100 × 4.6 mm, 2.6 µm) under isocratic conditions at the flow rate of 0.8 ml/min and column temperature was maintained at 45°C. The detection of analyte and internal standard was carried out by tandem mass spectrometry, operated in positive ion and multiple reaction monitoring acquisition mode. The method was fully validated for its selectivity, precision, accuracy, linearity, stability, detection and quantification limit. The extraction recovery of fingolimod in human blood ranged from 98.39 to 99.54%. The developed method was linear over the concentration range of 5-2500 pg/ml with a detection limit of 1 pg/ml. The developed method was validated and successfully applied for pharmacokinetic study after oral administration of fingolimod capsules.

The involvement of blood cells in the phosphorylation of the selective sphingosine-1-phosphate receptor 1 agonist SYL-927.

SYL-927 is a selective sphingosine-1-phosphate receptor 1 (S1P1) agonist for autoimmune diseases. It undergoes phosphorylation to the active SYL-927-P in vivo, which activates S1P1 on lymphocytes, causing lymphopenia by retention of lymphocytes in the lymph nodes. The aim of this study was to identify the involvement of blood cells in the phosphorylation of SYL-927. In addition, pharmacokinetics of SYL-927 and SYL-927-P in blood and plasma were compared in rats. The results demonstrated that SYL-927 can be converted to SYL-927-P in rat blood, but not in rat plasma. However, both rat blood and plasma are capable of dephosphorylating SYL-927-P to SYL-927. SYL-927-P generation and release were observed after incubating SYL-927 with rat and human erythrocytes and platelets. The addition of sphingosine kinases (SPHKs) inhibitors N,N-dimethylsphingosine (DMS) and FTY720 significantly inhibited SYL-927-P generation, indicating the involvement of SPHKs. In addition, SYL-927 and SYL-927-P levels in blood were significantly higher than those in plasma after oral administration of SYL-927 in rats, suggesting the blood cells for the production of SYL-927-P. In summary, the blood cells such as erythrocytes and platelets contribute to the generation and release of SYL-927-P, which is important for maintaining plasma active phosphate levels for prolonged effects.

Pharmacokinetic Interaction Between Fingolimod and Carbamazepine in Healthy Subjects.

This open-label, single-sequence study in healthy subjects investigated the effects of steady-state carbamazepine on the pharmacokinetic (PK) profile of a single 2-mg dose of fingolimod. In period 1, a single oral dose of fingolimod 2 mg (day 1) was followed by PK and safety assessments up to 36 days. In period 2, carbamazepine was administered in flexible, up-titrated doses (600 mg twice daily maximum) for 49 days. Fingolimod was administered on day 35, followed by a study completion evaluation (day 71). The PK analysis included 23 of 26 of the enrolled subjects (88.5%). Coadministration of fingolimod at steady-state carbamazepine concentrations resulted in increased fingolimod CL/F by 67% through the induction of CYP3A4, a cytochrome with negligible involvement in fingolimod clearance in an uninduced state. Fingolimod Cmax was reduced by 18% and AUCinf by 40%, as was T1/2 (106 vs 163 hours). A similar trend was observed for fingolimod-P. Models linking fingolimod-P blood concentrations to lymphocyte count or annual relapse rate suggest that such a decrease would have a low impact on the treatment effect. However, in the absence of efficacy data of fingolimod at doses lower than the therapeutic dose, their coadministration should be used with caution.

Determination of Seminal Concentration of Fingolimod and Fingolimod-Phosphate in Multiple Sclerosis Patients Receiving Chronic Treatment With Fingolimod.

The safety profile of fingolimod 0.5 mg, approved therapy for relapsing multiple sclerosis, is well established in clinical and real-world studies. As fingolimod is teratogenic in rats, it was considered important to assess the concentrations of fingolimod and its active metabolite, fingolimod-phosphate, in the semen of male patients on treatment and the risk of harming a fetus in a pregnant partner. In this multicenter open-label study, 13 male patients receiving fingolimod for at least 6 months provided 1 semen and 1 blood sample for analyte concentration measurements. The steady-state seminal concentrations of fingolimod and fingolimod-phosphate were close to those simultaneously observed in blood. The amount of fingolimod-related material in 10 mL of ejaculate was estimated to be 47.5 ng. The estimated fingolimod and fingolimod-phosphate blood Cmax values in a woman having regular sexual intercourse with a male patient treated with fingolimod 0.5 mg were approximately 400 and 2400 times smaller than the estimated values in the embryo-fetal development study in rats at the no-observed-adverse-event level. Consequently, the risk of harming a fetus in a pregnant woman is considered extremely unlikely.

Incurred sample reanalysis of fingolimod and fingolimod phosphate in blood: stability evaluation and application to a rat pharmacokinetic study.

BACKGROUND: Incurred sample reanalysis (ISR) is an in-study validation parameter, which reinforces that the validated bioanalytical methods are reproducible. ISR of whole blood samples is complex when the test compounds can interconvert, ex vivo. Fingolimod and fingolimod phosphate are highly distributed in the blood cellular components and undergo rapid interconversion, both in vivo and ex vivo. An LC-MS/MS method capable of simultaneous quantification of fingolimod and fingolimod phosphate with the controlled sample preparation procedure is essential. RESULTS: The ex vivo analyte interconversion in blood was controlled by lysing the blood cells. CONCLUSION: Lysis of blood samples not only controlled the interconversion but also rendered homogeneity to the sample, which led to acceptable ISR results from the study.

Mixed hemimicelles solid-phase extraction based on sodium dodecyl sulfate (SDS)-coated nano-magnets for the spectrophotometric determination of Fingolomid in biological fluids.

In this study, mixed hemimicelles solid-phase extraction (SPE) based on sodium dodecyl sulfate (SDS)-coated nano-magnets Fe3O4 was investigated as a novel method for the separation and determination of Fingolimod (FLM) in water, urine and plasma samples prior to spectrophotometeric determination. Due to the high surface area of these new sorbents and the excellent adsorption capacity after surface modification by SDS, satisfactory extraction recoveries can be produced. The main factors affecting the adsolubilization of analysts, such as pH, surfactant and adsorbent amounts, ionic strength, extraction time and desorption conditions were studied and optimized. Under the selected conditions, FLM has been quantitatively extracted. The accuracy of the method was evaluated by recovery measurements on spiked samples, and good recoveries of 96%, 95% and 88% were observed for water, urine and plasma respectively. Proper linear behaviors over the investigated concentration ranges of 2-26, 2-17 and 2-13 mg/L with good coefficients of determination, 0.998, 0.997 and 0.995 were achieved for water, urine and plasma samples, respectively. To the best of our knowledge, this is the first time that a mixed hemimicelles SPE method based on magnetic separation and nanoparticles has been used as a simple and sensitive method for monitoring of FLM in water and biological samples.

A high throughput flow gradient LC-MS/MS method for simultaneous determination of fingolimod, fampridine and prednisone in rat plasma, application to in vivo perfusion study.

In this study a selective and high throughput liquid chromatography-mass spectrometry method was developed and validated for the simultaneous quantification of fingolimod (FLD), fampridine (FMP) and prednisone (PDN) in rat plasma using imipramine (IMP) as internal standard (ISTD). In this LC-MS method, following protein precipitation extraction (PPE), the analytes and ISTD were run on XBridge C18 column (150×4.6mm, 5µm) using gradient mobile phase consisting of 5mM ammonium formate in water (pH 9.0) and acetonitrile in a flow gradience program. The drug precursor and product ions were monitored on a triple quadrupole instrument that was operated in positive ionization mode. The method was validated over a concentration range of 0.1-100ng/mL for all the three analytes with relative recoveries ranging from 69 to 82%. The intra and inter batch precision (% CV) across four validation runs were less than 13.4%. The accuracy determined at four QC levels (LLOQ, LQC, MQC and HQC) were within ±6.5% of CV values. The method proved to be highly reproducible and sensitive that was successfully applied in a pharmacokinetic study after single dose oral administration to the rats and also in perfusion study sample analysis.

Effect of ceralifimod (ONO-4641) on lymphocytes and cardiac function: Randomized, double-blind, placebo-controlled trial with an open-label fingolimod arm.

This randomized, double-blind, placebo-controlled, 6-arm, parallel-design study investigated cardiac and hematological pharmacodynamic effects of ceralifimod (ONO-4641), a selective sphingosine-1-phosphate (S1P) receptor modulator, over a broad dose range in direct comparison with the nonselective S1P modulator fingolimod. Healthy subjects were assigned to ceralifimod (0.01, 0.025, 0.05, or 0.10 mg), fingolimod (0.5 mg), or placebo once daily for 14 days (n = 24 per group). After 14 days of treatment, mean absolute lymphocyte count percentage change from baseline was greatest in the fingolimod (-62%) and ceralifimod 0.10 mg (-56%) groups. On treatment cessation, lymphocyte recovery was faster in the ceralifimod versus the fingolimod group. Ceralifimod showed dose- and concentration-dependent chronotropic effect. Cardiac effects in the fingolimod group were dependent on fingolimod-P concentrations. Maximum mean heart rate (HR) effect on day 1 was larger with fingolimod (placebo-adjusted change from time-matched baseline HR [ΔΔHR], -14.9 beats per minute [bpm]) versus ceralifimod (ΔΔHR, -6.2 and -12.0 bpm for the 0.05- and 0.10-mg doses, respectively). Ceralifimod's effect on the PR interval was minor. Safety biomarker results suggest that potential therapeutic doses of ceralifimod, in particular the 0.05-mg dose, might result in reduced occurrence of bradycardia, atrioventricular block absolute lymphocyte count and grade 3/4 lymphopenia compared with fingolimod 0.5 mg.