Selenium nanoparticles coated with pH responsive silk fibroin complex for fingolimod release and enhanced targeting in thyroid cancer.

Cancer-targeted drug delivery systems based on nanoparticles (NPs) have been considered promising therapies. In this study, we developed a pH-responsive smart NPs drug delivery system using silk fibroin (SF), selenium nanoparticles (Se NPs), fingolimod (FTY720), and heptapeptide (T7). The prepared FTY720@T7-SF-Se NPs were spheres with an average diameter of 120 nm, which would contribute to the enhanced permeability and retention effects in tumour regions. The encapsulation efficiency (EE) of the FTY720@T7-SF-Se NPs was 71.95 ± 3.81%. The release of FTY720 from the nanocarriers was pH-dependent, and the release of FTY720 was accelerated in an acidic environment. Both in vitro and in vivo studies showed that FTY720@T7-SF-Se NPs had an enhanced cellular uptake selectivity and antitumor activity for thyroid cancer. The bio-distribution study in vivo further demonstrated that FTY720@T7-SF-Se NPs could effectively accumulate in the tumour region, thereby enhancing the ability to kill cancer cells in vivo. In addition, studies of histology and immunohistochemistry showed that FTY720@T7-SF-Se NPs had low toxicity to the major organs of tumour-bearing mice, indicating the prepared NPs has good biocompatibility in vivo. These results suggest that the tumour-targeted NPs delivery system (FTY720@T7-SF-Se NPs) has great potential as a new tool for thyroid cancer therapy.

Chitosan nanocarrier for FTY720 enhanced delivery retards Parkinson's disease via PP2A-EzH2 signaling in vitro and ex vivo.

Parkinson's disease (PD) develops due to oxidative stress, mitochondrial aberrations, posttranslational modification, and α-Synuclein (α-Syn) aggregation. The α-synucleinopathy is attributed to phosphorylation and aggregation of α-Syn. A strategy to degrade or reduce phosphorylated protein paves the way to develop PD therapy. Hence, the neuroprotective efficiency of PP2A (Protein phosphatase 2) activator FTY720, loaded chitosan nanoformulation has been evaluated in vitro and ex vivo experimental PD models. Bio-compatible chitosan-based nanocarriers have been utilized to enhance the bio-availability and neuroprotective effect of FTY720. The neuroprotective effect of characterized nanoformulation was determined by the downregulation of PD hallmark phospho-serine 129 (pSer129) α-Syn, with anti-oxidative and anti-inflammatory potentials. The neuroprotective mechanism uncovered novel physical interaction of PP2A and polycomb group of protein Enhancer of zeste homolog 2 to mediate ubiquitination and degradation of agglomerated pSer129 α-Syn. Indeed, this study establishes the neuroprotective potential of chitosan based FTY720 nanoformulations by PP2A mediated epigenetic regulation for PD prevention.

In vitro anticancer evaluation of micelles containing N-(4-(2-((4-methoxybenzyl)amino)ethyl)phenyl)heptanamide, an analogue of fingolimod.

Fingolimod has been evaluated for use as an anticancer agent. However, many steps are required to synthesize fingolimod because of its intricate structure. A fingolimod analogue, N-(4-(2-((4-methoxybenzyl)amino)ethyl)phenyl)heptanamide (MPH), also has anti-cancer effects and is easier to synthesize but is poorly soluble in water. To compensate for its poor water solubility, MPH-loaded polymeric micelles were prepared by thin film hydration method using various polymers and the physicochemical properties of the MPH-loaded micelles such as particle size, drug-loading (DL, %), and encapsulation efficiency (EE, %) were evaluated. A storage stability test was conducted to select the final formulation and the release profile of the MPH-loaded micelles was confirmed by in vitro release assay. MPH-loaded mPEG-b-PLA micelles were selected for further testing based on their stability and physicochemical properties; they were stable for stable for 14 days at 4 °C and 25 °C and for 7 days at 37 °C. They showed anti-cancer efficacy against both A549 and U87 cancer cells. Encapsulation of MPH in polymeric micelles did not decrease the in vitro cytotoxicity of MPH. The findings of this study lay the groundwork for future formulations that enable the effective and stable delivery of poorly water-soluble agents.

Pharmacokinetics and Tolerability of the Novel Non-immunosuppressive Fingolimod Derivative, OSU-2S, in Dogs and Comparisons with Data in Mice and Rats.

In this study, we characterized the pharmacokinetics of OSU-2S, a fingolimod-derived, non-immunosuppressive phosphatase activator, in mice, rats, and dogs, as well as tolerability and food effects in dogs. Across all species tested, plasma protein binding for OSU-2S was > 99.5%, and metabolic stability and hepatic intrinsic clearance were in the moderate range. OSU-2S did not significantly modulate CYP enzyme activity up until 50 µM, and Caco-2 data suggested low permeability with active efflux at 2 µM. Apparent oral bioavailability in mice was 16% and 69% at 10 and 50 mg/kg, respectively. In rats, bioavailability was 24%, 35%, and 28% at 10, 30, and 100 mg/kg, respectively, while brain/plasma ratio was 36 at 6-h post-dose at 30 mg/kg. In dogs, OSU-2S was well tolerated with oral capsule bioavailability of 27.5%. Plasma OSU-2S exposures increased proportionally over a 2.5-20 mg/kg dose range. After 4 weeks of 3 times weekly, oral administration (20 mg/kg), plasma AUClast (26.1 µM*h), and Cmax (0.899 µM) were nearly 2-fold greater than those after 1 week of dosing, and no food effects were observed. The elimination half-life (29.7 h), clearance (22.9 mL/min/kg), and plasma concentrations of repeated oral doses support a 3-times weekly dosing schedule in dogs. No significant CBC, serum biochemical, or histopathological changes were observed. OSU-2S has favorable oral PK properties similar to fingolimod in rodents and dogs and is well tolerated in healthy animals. This work supports establishing trials of OSU-2S efficacy in dogs with spontaneous tumors to guide its clinical development as a cancer therapeutic for human patients.

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.

Improving the in-vivo biological activity of fingolimod loaded PHBV nanoparticles by using hydrophobically modified alginate.

Uncontrolled distribution of nanoparticles (NPs) within the body can significantly decrease the efficiency of drug therapy and is considered among the main restrictions of NPs application. The aim of this study was to develop a depot combination delivery system (CDS) containing fingolimod loaded poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) NPs dispersed into a matrix of oleic acid-grafted-aminated alginate (OA-g-AAlg) to minimize the nonspecific biodistribution (BD) of PHBV NPs. OA-g-AAlg was synthesized in two step; First, Alg was aminated by using adipic dihydrazide (ADH). The degree of hyrazide group substitution of Alg was determined by trinitro-benzene-sulfonic acid (TNBS) assay. Second, OA was attached to AAlg through formation of an amide bond. Chemical structure of OA-g-AAlg was confirmed with FTIR and HNMR spectroscopy. Furthermore, rheological properties of OA-g-AAlg with different grafting ratios were evaluated. In-vitro release studies indicated that 47% of fingolimod was released from the CDS within 28 days. Blood and tissue samples were analyzed using liquid chromatography/tandem mass spectrometry following subcutaneous (SC) injection of fingolimod-CDS into Wistar rats. The elimination phase half-life of CDS-fingolimod was significantly higher than that of fingolimod (∼32 d vs. ∼20 h). To investigate the therapeutic efficacy, lymphocyte count was assessed over a 40 day period in Wistar rats. Peripheral blood lymphocyte count decreased from baseline by 27 ± 8% in 2 days after injection. Overall, the designed CDS represented promising results in improving the pharmacokinetic properties of fingolimod. Therefore, we believe that this sustained release formulation has a great potential to be applied to delivery of various therapeutics.

An update on sphingosine-1-phosphate receptor 1 modulators.

Sphingolipids represent an essential class of lipids found in all eukaryotes, and strongly influence cellular signal transduction. Autoimmune diseases like asthma and multiple sclerosis (MS) are mediated by the sphingosine-1-phosphate receptor 1 (S1P1) to express a variety of symptoms and disease patterns. Inspired by its natural substrate, an array of artificial sphingolipid derivatives has been developed to target this specific G protein-coupled receptor (GPCR) in an attempt to suppress autoimmune disorders. FTY720, also known as fingolimod, is the first oral disease-modifying therapy for MS on the market. In pursuit of improved stability, bioavailability, and efficiency, structural analogues of this initial prodrug have emerged over time. This review covers a brief introduction to the sphingolipid metabolism, the mechanism of action on S1P1, and an updated overview of synthetic sphingosine S1P1 agonists.

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.

Targeted co-delivery of Beclin 1 siRNA and FTY720 to hepatocellular carcinoma by calcium phosphate nanoparticles for enhanced anticancer efficacy.

PURPOSE: FTY720, known as fingolimod, is a new immunosuppressive agent with effective anticancer properties. Although it was recently confirmed that FTY720 inhibits cancer cell proliferation, FTY720 can also induce protective autophagy and reduce cytotoxicity. Blocking autophagy with Beclin 1 siRNA after treatment with FTY720 promotes apoptosis. The objective of this study was to enhance the anticancer effect of FTY720 in hepatocellular carcinoma (HCC) by targeted co-delivery of FTY720 and Beclin 1 siRNA using calcium phosphate (CaP) nanoparticles (NPs). MATERIALS AND METHODS: First, the siRNA was encapsulated within the CaP core. To form an asymmetric lipid bilayer structure, we then used an anionic lipid for the inner leaflet and a cationic lipid for the outer leaflet; after removing chloroform by rotary evaporation, these lipids were dispersed in a saline solution with FTY720. The NPs were analyzed by transmission electron microscopy, dynamic light scattering and ultraviolet-visible spectrophotometry. Cancer cell viability and cell death were analyzed by MTT assays, fluorescence-activated cell sorting analysis and Western blotting. In addition, the in vivo effects of the NPs were investigated using an athymic nude mouse subcutaneous transplantation tumor model. RESULTS: When the CaP NPs, called LCP-II NPs, were loaded with FTY720 and siRNA, they exhibited the expected size and were internalized by cells. These NPs were stable in systemic circulation. Furthermore, co-delivery of FTY720 and Beclin 1 siRNA significantly increased cytotoxicity in vitro and in vivo compared with that caused by treatment with the free drug alone. CONCLUSION: The CaP NP system can be further developed for co-delivery of FTY720 and Beclin 1 siRNA to treat HCC, enhancing the anticancer efficacy of FTY720. Our findings provide a new insight into HCC treatment with co-delivered small molecules and siRNA, and these results can be readily translated into cancer clinical trials.

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.

Characterization and Prediction of Cardiovascular Effects of Fingolimod and Siponimod Using a Systems Pharmacology Modeling Approach.

Sphingosine 1-phosphate (S1P) receptor agonists are associated with cardiovascular effects in humans. This study aims to develop a systems pharmacology model to identify the site of action (i.e., primary hemodynamic response variable) of S1P receptor agonists, and to predict, in a quantitative manner, the cardiovascular effects of novel S1P receptor agonists in vivo. The cardiovascular effects of once-daily fingolimod (0, 0.1, 0.3, 1, 3, and 10 mg/kg) and siponimod (3 and 15 mg/kg) were continuously recorded in spontaneously hypertensive rats and Wistar-Kyoto rats. The results were analyzed using a recently developed systems cardiovascular pharmacology model, i.e. the CVS model; total peripheral resistance and heart rate were identified as the site of action for fingolimod. Next, the CVS model was interfaced with an S1P agonist pharmacokinetic-pharmacodynamic (PKPD) model. This combined model adequately predicted, in a quantitative manner, the cardiovascular effects of siponimod using in vitro binding assays. In conclusion, the combined CVS and S1P agonist PKPD model adequately describes the hemodynamic effects of S1P receptor agonists in rats and constitutes a basis for the prediction, in a strictly quantitative manner, of the cardiovascular effects of novel S1P receptor agonists.

Preclinical Metabolism, Pharmacokinetics and In Vivo Analysis of New Blood-Brain-Barrier Penetrant Fingolimod Analogues: FTY720-C2 and FTY720-Mitoxy.

Parkinson's disease (PD) is a neurodegenerative aging disorder in which postmortem PD brain exhibits neuroinflammation, as well as synucleinopathy-associated protein phosphatase 2A (PP2A) enzymatic activity loss. Based on our translational research, we began evaluating the PD-repurposing-potential of an anti-inflammatory, neuroprotective, and PP2A stimulatory oral drug that is FDA-approved for multiple sclerosis, FTY720 (fingolimod, Gilenya®). We also designed two new FTY720 analogues, FTY720-C2 and FTY720-Mitoxy, with modifications that affect drug potency and mitochondrial localization, respectively. Herein, we describe the metabolic stability and metabolic profiling of FTY720-C2 and FTY720-Mitoxy in liver microsomes and hepatocytes. Using mouse, rat, dog, monkey, and human liver microsomes the intrinsic clearance of FTY720-C2 was 22.5, 79.5, 6.0, 20.2 and 18.3 µL/min/mg; and for FTY720-Mitoxy was 1.8, 7.8, 1.4, 135.0 and 17.5 µL/min/mg, respectively. In hepatocytes, both FTY720-C2 and FTY720-Mitoxy were metabolized from the octyl side chain, generating a series of carboxylic acids similar to the parent FTY720, but without phosphorylated metabolites. To assess absorption and distribution, we gave equivalent single intravenous (IV) or oral doses of FTY720-C2 or FTY720-Mitoxy to C57BL/6 mice, with two mice per time point evaluated. After IV delivery, both FTY720-C2 and FTY720-Mitoxy were rapidly detected in plasma and brain; and reached peak concentrations at the first sampling time points. After oral dosing, FTY720-C2 was present in plasma and brain, although FTY720-Mitoxy was not orally bioavailable. Brain-to-plasma ratio of both compounds increased time-dependently, suggesting a preferential partitioning to the brain. PP2A activity in mouse adrenal gland increased ~2-fold after FTY720-C2 or FTY720-Mitoxy, as compared to untreated controls. In summary, FTY720-C2 and FTY720-Mitoxy both (i) crossed the blood-brain-barrier; (ii) produced metabolites similar to FTY720, except without phosphorylated species that cause S1P1-mediated-immunosuppression; and (iii) stimulated in vivo PP2A activity, all of which encourage additional preclinical assessment.

Interstitial Fluid Sphingosine-1-Phosphate in Murine Mammary Gland and Cancer and Human Breast Tissue and Cancer Determined by Novel Methods.

The tumor microenvironment is a determining factor for cancer biology and progression. Sphingosine-1-phosphate (S1P), produced by sphingosine kinases (SphKs), is a bioactive lipid mediator that regulates processes important for cancer progression. Despite its critical roles, the levels of S1P in interstitial fluid (IF), an important component of the tumor microenvironment, have never previously been measured due to a lack of efficient methods for collecting and quantifying IF. The purpose of this study is to clarify the levels of S1P in the IF from murine mammary glands and its tumors utilizing our novel methods. We developed an improved centrifugation method to collect IF. Sphingolipids in IF, blood, and tissue samples were measured by mass spectrometry. In mice with a deletion of SphK1, but not SphK2, levels of S1P in IF from the mammary glands were greatly attenuated. Levels of S1P in IF from mammary tumors were reduced when tumor growth was suppressed by oral administration of FTY720/fingolimod. Importantly, sphingosine, dihydro-sphingosine, and S1P levels, but not dihydro-S1P, were significantly higher in human breast tumor tissue IF than in the normal breast tissue IF. To our knowledge, this is the first reported S1P IF measurement in murine normal mammary glands and mammary tumors, as well as in human patients with breast cancer. S1P tumor IF measurement illuminates new aspects of the role of S1P in the tumor microenvironment.

Pharmacokinetics of fingolimod and metabolites in subjects with severe renal impairment: An open-label, single-dose, parallel-group study.

OBJECTIVE: This study assessed the pharmacokinetics and tolerability of fingolimod and its metabolites in severe renal impairment and healthy subjects. METHODS: In this single-dose, open-label study, 9 severe renal impairment subjects and 9 demographically matched healthy subjects were included. Each subject received a single oral dose of fingolimod 1.25 mg, and their blood and urine samples were assessed. The pharmacokinetics of fingolimod and its metabolites, fingolimod-phosphate (active metabolite, fingolimod-P), M2, and M3, were compared in both groups. Safety and tolerability were also assessed. RESULTS: In severe renal impairment subjects, mean±standard deviation values of Cmax (ng/mL) of fingolimod and fingolimod-P were 0.878±0.256 and 1.13±0.293 vs. 0.653±0.138 and 0.904±0.229 in healthy subjects, respectively. The overall drug exposures (AUCinf (ngxh/mL)) for fingolimod and fingolimod-P were 131±90.7 and 75.5±33.6 in severe renal impairment subjects vs. 82.3±36.9 and 65.9±30.6 in healthy subjects, respectively. t1/2 (hours) for fingolimod and fingolimod-P was comparable in severe renal impairment subjects (94±53 and 95±50) and healthy subjects (85±25 and 101±46). All adverse events were as expected for fingolimod 1.25 mg. CONCLUSIONS: The exposure to fingolimod and fingolimod-P was moderately increased (90% CI, 0.94-2.18) in severe renal impairment subjects, while half-lives and protein binding were similar to those in healthy subjects. Given that these changes are not clinically meaningful, fingolimod dose adjustment is considered unnecessary in patients with mild, moderate, or severe renal impairment.

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.