Pharmacokinetics, Pharmacodynamics, Tolerability, and Food Effect of Cenerimod, a Selective S1P1 Receptor Modulator in Healthy Subjects

The pharmacokinetics, pharmacodynamics, tolerability, and food effect of cenerimod, a potent sphingosine-1-phosphate subtype 1 receptor modulator, were investigated in three sub-studies. Two double-blind, placebo-controlled, randomised studies in healthy male subjects were performed. Cenerimod was administered either as single dose (1, 3, 10 or 25 mg; Study 1) or once daily for 35 days (0.5, 1, 2 or 4 mg; Study 2). A two-period cross-over, open-label study was performed to assess the food effect (1 mg, Study 3). The pharmacokinetic profile of cenerimod was characterised by a tmax of 5.0–6.2 h. Terminal half-life after single and multiple doses ranged from 170 to 199 h and 283 to 539 h, respectively. Food had no relevant effect on the pharmacokinetics of cenerimod. A dose-dependent decrease in lymphocyte count was observed after initiation of cenerimod and reached a plateau (maximum change from baseline: −64%) after 20–23 days of treatment. Lymphocyte counts returned to baseline values at end-of-study examination. One serious adverse event of circulatory collapse (25 mg dose group, maximum tolerated dose: 10 mg) and adverse events of mild-to-moderate intensity were reported. Treatment initiation was associated with transient decreases in heart rate and blood pressure at doses >1 and ≥10 mg, respectively.


Introduction
Sphingosine-1-phosphate (S1P) is synthesised and secreted by many cell types. This phospholipid is a ligand of and binds to G-protein-coupled receptors (S1P 1 -S1P 5 ) to modulate a wide range of physiological systems [1][2][3]. Repeated administration of S1P 1 receptor (S1P 1 R) modulators triggers a sustained internalisation of this receptor and induces a long-lasting inhibition of the egress of lymphocytes from lymphoid organs [4]. This reduction in lymphocyte count in peripheral blood is considered a therapeutic approach to treat autoimmune diseases. In this respect, the non-selective S1P receptor modulator fingolimod (Gilenya ® ) is used to treat patients with relapsing multiple sclerosis [5] and selective S1P 1 R modulators have achieved proof-of-concept in multiple sclerosis [6] and psoriasis [7]. Whereas cardiodynamic (heart rate [HR] reduction, delay in atrio ventricular (AV) conduction) and pulmonary effects had initially been related to S1P 3 agonism [8][9][10], these effects were also observed with selective modulation of S1P 1 R [11][12][13][14][15].
Cenerimod (ACT-334441) is a potent S1P 1 R modulator. In vitro and in vivo preclinical studies revealed that cenerimod is highly selective for the human S1P 1 R. Indeed, compared to S1P, cenerimod is 16-fold more potent on the S1P 1 R (EC 50 : 1 vs. 16 nM for cenerimod vs. S1P) and 2000-fold less active on the S1P 3 receptor (EC 50 : 228 vs. 0.1 nM for cenerimod vs. S1P) [16]. In animal species and man, the binding of cenerimod to plasma proteins is >99.9%. In rats and dogs, single-and multiple-dose administration of cenerimod at doses >0.3 mg/kg induced a dose-dependent and reversible reduction of lymphocyte count. In a mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis), preventive and therapeutic treatment with cenerimod (6 mg/kg/day) led to clinical and histological efficacy. In rats, a decrease in HR was observed at doses ≥0.3 mg/kg, this reduction was similar at doses of 1 and 3 mg/kg. The pharmacokinetic (PK) profile of cenerimod (0.1 mg/kg) revealed a rapid absorption and a terminal half-life (t 1/2 ) of 12 and 7.5 h in male rats and dogs, respectively. Multiple-dose administration led to a greater than dose-proportional exposure in both rats and dogs between 3 and 10 mg/kg.
The aim of the studies reported here was to assess for the first time in humans the PK, pharmacodynamics (PD), food effect (only after single dose), safety, and tolerability of cenerimod as a single oral dose or once daily (o.d.) multiple oral doses for 35 days.
Lymphocyte count in each subject returned to at least the lower limit of the normal range (i.e., 80% of baseline) at end-of-study.
The data are expressed as mean (SD). AUEC: area under the effect-time curve, t max : time to nadir (minimum lymphocyte count), and E max : nadir value in lymphocyte count (×10 9 cells/L).

Pharmacokinetics/Pharmacodynamics
The PK/PD relationship has been investigated and is depicted in Figure 3. Based on Study 2 data, concentration and decrease in lymphocyte count vs. time follow similar profiles.

Pharmacokinetics/Pharmacodynamics
The PK/PD relationship has been investigated and is depicted in Figure 3. Based on Study 2 data, concentration and decrease in lymphocyte count vs. time follow similar profiles.

Safety and Tolerability
Apart from a severe and serious adverse event of circulatory collapse reported in Study 1 (6 h after administration of 25 mg cenerimod, lowest supine blood pressure (BP): 39/23 mmHg, pulse rate: 60 bpm, resolved without sequelae within 24 h following administration of atropine and ephedrine), all adverse events (AEs) were of mild-to-moderate intensity and resolved without sequelae. Most of the observed AEs were considered unrelated to study drug. The serious adverse event defined the maximum tolerated single dose of 10 mg. Pooling of the 3 studies revealed a similar number of subjects experiencing at least one AE following cenerimod (36 subjects, 64%) or placebo (11 subjects, 69%).
The most frequently reported AEs were headache, dizziness, chest pain (this combined chest pain, chest discomfort, musculoskeletal chest pain, and non-cardiac chest pain), and nasopharyngitis (Table 3). There were no apparent drug-related effects on body temperature, clinical chemistry variables, coagulation variables, or physical examination.

Safety and Tolerability
Apart from a severe and serious adverse event of circulatory collapse reported in Study 1 (6 h after administration of 25 mg cenerimod, lowest supine blood pressure (BP): 39/23 mmHg, pulse rate: 60 bpm, resolved without sequelae within 24 h following administration of atropine and ephedrine), all adverse events (AEs) were of mild-to-moderate intensity and resolved without sequelae. Most of the observed AEs were considered unrelated to study drug. The serious adverse event defined the maximum tolerated single dose of 10 mg. Pooling of the 3 studies revealed a similar number of subjects experiencing at least one AE following cenerimod (36 subjects, 64%) or placebo (11 subjects, 69%).

Discussion
This study provides the first PK, PD, safety, and tolerability data for cenerimod, a selective oral S1P 1 R modulator, in healthy male subjects. Single and multiple o.d. oral doses of up to 10 and 4 mg (up to 35 days), respectively, were well tolerated. The nature and severity of AEs reported in the present studies were similar to those observed following administration of other representatives of this drug class, e.g., ponesimod [7,11,12], fingolimod [17,18], or siponimod [19]. Furthermore, decreases in BP were triggered following single high doses of cenerimod as previously observed with ponesimod [11].
Pharmacokinetic evaluation revealed that exposure to cenerimod was slightly more than dose-proportional for single doses and dose-proportional across the entire dose range for multiple doses. The intersubject variability was low to moderate for C max (9.1-27.3%), AUC 0-24 (11.0-27.6%), and t 1/2 (5.3-28.6%). Multiple-dose PK of cenerimod are characterised by steady-state concentrations reached 20-32 days following o.d. administration and exposure is approximately 5-to 9-fold greater than after the initial dose. Compared to single-dose, multiple-dose PK revealed similar t max (4-6 h), but longer, dose-dependent t 1/2 (283-539 h), and higher intersubject variability (≤45.0%). These PK properties of cenerimod resemble those of fingolimod [20] or amiselimod [21] more than of ponesimod [11,12] or siponimod [19]. Based on the present data and its lipophilic properties, it is plausible that cenerimod accumulates in several tissues.
As expected, based on its pharmacological mode of action, cenerimod decreased the lymphocyte count in peripheral blood. Following single-dose administration, this reduction was different from placebo at doses ≥3 mg and was dose-dependent. Single-and multiple-dose administration of 25 and 2 mg led to a maximal decrease of circulating lymphocytes of 76% and 64%, respectively. This is in good agreement with previous data obtained from fingolimod and ponesimod administered at supra-therapeutic doses [11][12][13]17,18]. The effect was sustained and reversible within 7 and 40 days after single-and multiple-dose administration of cenerimod. In line with multiple-dose administration of ponesimod or fingolimod, lymphocyte counts decreased and reached a plateau [12,13,17]. Interestingly, the percent change from baseline and AUEC were slightly higher in the 2 mg compared to the 4 mg dose group. This may be explained by a lower mean baseline of lymphocyte count in the 4 mg group compared to the 2 mg group since the same threshold was reached (~0.6 × 10 9 cells/L) as shown in Table 2. Experience with other selective S1P 1 R modulators such as siponimod and ponesimod has shown that a lymphocyte count reduction of 60-70% from baseline is associated with a plateau of efficacy for treatment of multiple sclerosis, while a 20-30% reduction in lymphocyte count is associated with minimal efficacy [6,19].
The overall pattern of reported AEs is similar to the one observed with the non-selective S1P receptor modulator fingolimod or the selective S1P 1 R modulators ceralifimod, ozanimod and ponesimod [11,12,17,18,22,23]. Nevertheless, incidence of AEs such as bradycardia and dyspnoea was lower with cenerimod compared to fingolimod [17,18] or ponesimod [11,12]. Following single doses of 10 and 25 mg and multiple doses of 1 mg, a slight decrease in FEV 1 was observed. This effect might be related to agonism of S1P 1 R although mainly S1P 3 receptors have been shown to mediate S1P effects on the lung [24].
Although the role of S1P 3 receptors in S1P-induced HR effect has been described [8,10], another signalling might be involved based on the HR effect of highly selective S1P 1 R modulators [11,12,19]. Previous studies have revealed that both S1P 1 and S1P 3 agonism prior to receptor internalisation [8,10,25] lead to a reduction in HR via stimulation of the inward rectifier potassium current (I K.ACh ) [26]. Internalisation of S1P 1 R and their desensitisation explain that treatment initiation triggers a transient decrease in HR [27].
Cenerimod displays advantages over fingolimod and ponesimod in terms of selectivity on the S1P 1 R. In addition, the long t 1/2 of cenerimod, compared to ponesimod, will allow a built-in natural uptitration and a longer duration for drug holiday [15].
Single doses of cenerimod up to 10 mg were well tolerated and the tolerability profile of cenerimod is similar to that of ponesimod. In conclusion, the present promising results warrant further development of cenerimod in patients suffering from autoimmune disorders, e.g., systemic lupus erythematosus (NCT02472795).

Subjects
In these three sub-studies, healthy male subjects aged between 18 and 47 years were included. The health of the subjects was assessed at the screening visit, which included recording of the medical history, medications taken during the 3 months preceding the screening visit, a physical examination, measurement of body weight and height, clinical laboratory tests, recording of vital signs, and standard electrocardiogram (ECG). At screening, subjects had to have PR interval <200 ms, HR 55-90 bpm, systolic and diastolic blood pressure (BP) 100-150 and 50-90 mm Hg, respectively, (FEV 1 ) and (FVC) >80% of the predicted value, and a normal total lymphocyte count (>10 9 lymphocytes/L). Written informed consent was obtained from each individual participating in the study prior to any study procedure and after adequate explanation of the aims, methods, objectives and potential hazards of the studies. The single-ascending dose study (Study 1) was performed prior to and in a different centre than the multiple-ascending dose (Study 2) and the food effect study (Study 3). All protocols were approved by the Reading and Plymouth Independent Ethics Committees (UK) (date of approval of initial protocol from Institutional Review Board (IRB), 10 November 2010 for SAD, 20 April 2012 for MAD and food effect). These studies were performed according to good clinical practice and in accordance with the principles of the Declaration of Helsinki.

Study Design
This work was performed in two centres located in the UK. Study 1 and Study 2 were single-centre, randomised, double-blind, placebo-controlled, parallel-group single-and multiple-ascending oral dose studies, respectively. Study 3 was a two-way cross-over, open-label, food effect study. A total of 72 male subjects (32, 32 and 8 subjects in Study 1, Study 2 and Study 3, respectively) were enrolled, 8 subjects were assigned per dose group (ratio 3:1 for active:placebo in Study 1 and Study 2). Female subjects were excluded based on the potential teratogenic risk of cenerimod. Cenerimod (capsule formulation) was administered at a single dose of 1, 3, 10 or 25 mg in Study 1 and o.d. for 35 days at doses of 0.5, 1, 2 and 4 mg in Study 2. In Study 3, a single dose of 1 mg cenerimod was administered either in fed (i.e., a high-fat and high-calorie (as described in the Food and Drug Administration (FDA) guidance [28], breakfast 30 min before cenerimod administration) or in fasted conditions, with a washout period of at least 45 days between the study drug administrations. All treatments were administered in the morning (fasted condition in Study 1 and Study 2) with approximately 240 mL of water. Based on the ratio of the lowest-observed-adverse-effect-levels in the 4-week rat and dog toxicology studies and human equivalent doses at the planned starting dose of 1 mg, the safety margins were 33.8 and 116.8, respectively. PK and PD profiles after multiple oral o.d. doses were predicted from data obtained during Study 1 and supported the selection of doses in Study 2.
Subjects were admitted to the clinical centre on Day −1 and remained in the clinic until Day 7 (Study 1 and Study 3) or Day 41 (Study 2) when they were discharged if this was allowed on the basis of their medical condition (HR ≥45 bpm or ≥70% of baseline, no clinically relevant treatment-emergent ECG abnormalities, FEV 1 and FVC ≥80% of baseline, and absolute lymphocyte count ≥10 9 lymphocytes/L). Subjects returned to the clinic for weekly outpatient visits (until Day 21 in Study 3, and in the 10 and 25 mg dose groups in Study 1, Day 56 for the 0.5 mg dose group in Study 2, and Day 84 in the 1, 2 and 4 mg dose groups in Study 2). An end-of-study visit comprising the same examinations as at the screening visit was conducted at least 28 days after the last administration of cenerimod.

Pharmacokinetic Assessments
In Study 1 and Study 3, blood samples of about 3 mL were collected in ethylene di-amine tetra acetic acid (EDTA) tubes pre-dose and at 0.5, 1, 1. 5, 2.5, 4, 6, 8, 12, 16, 24, 36, 48, 72, 96, 120 and 144 h after study drug administration. In addition, blood PK samplings were performed on Day 14, Day 21 and Day 28 in Study 3, and in the 10 and 25 mg dose group in Study 1. In Study 2, blood sampling was performed pre-dose and 1, 2.5, 4, 6, 8, 12, 16 h on Day 1 and Day 35 for a full PK profile after the first and the last dose. Samples were collected pre-dose from Day 2 to Day 34 and also at each outpatient visit until the end-of-study examination. After centrifugation, plasma was transferred into a polypropylene tube and stored at −21 • C (±5 • C) pending analysis. Plasma concentrations of cenerimod were determined using a validated liquid chromatography coupled to tandem mass spectrometry assay with a lower limit of quantification of 0.1 ng/mL and using a pentadeuterated form of cenerimod as internal standard. The method was linear in the concentration range 0.1-100 ng/mL. Analysis of quality-control samples of all runs showed that inter-batch coefficients of variation (precision) were <8.8%, whereas the average intra-batch accuracy was in the range 96.0-102.7%. Noncompartmental PK analyses were performed using Professional WinNonlin 6.1 software (Pharsight Corp., Mountain View, CA, USA). The variables C max and t max were directly obtained from the plasma concentration-time profiles, AUC 0-t was calculated using the trapezoidal method [29], and t 1/2 was calculated as ln 2/λz, where λz is the terminal elimination rate constant estimated by log-linear regression analysis.

Pharmacodynamic Assessments
Analysis of the lymphocyte count in peripheral blood was performed at the same time points as PK samples in Study 1 and Study 3. In Study 2, lymphocyte count was assessed on Day 1, every 3 days from Day 2 to Day 35, and at each outpatient visit. Assessment of lymphocyte count was part of the clinical haematology evaluation. To assess lymphocyte count, blood samples of 2.7 mL were collected into a K 3 -EDTA polypropylene tube and analysis was performed using a cell counter. The measured individual whole blood lymphocyte counts were used to establish the effect-time curve, lymphocyte nadir (E max ), and time to nadir (t max ). The area under the effect-curve (AUEC) was calculated according to the linear trapezoidal rule using the measured lymphocyte count (effect)-time values.

Safety and Tolerability Assessments
Safety and tolerability were evaluated by monitoring AEs, vital signs measurements (supine BP), 12-lead ECG recordings, pulmonary function tests (PFTs), clinical laboratory, physical, and neurological examinations. ECG variables and BP were recorded pre-dose and 0. 5

Statistical Analysis
Pharmacokinetic variables were analysed descriptively providing the geometric mean and 95% CIs for C max , AUC 0-24 , AUC 0-∞ , and t 1/2 , and the median with the range for t max . Dose proportionality of log transformed C max , AUC 0-24 , and AUC 0-∞ values was explored by the power model [30]. Pharmacodynamic and cardiodynamic data are expressed as mean ± SD. The Student's t-test was used to analyse the effects of cenerimod on the lymphocyte count and HR. Comparisons of baseline versus nadir (i.e., lowest value measured) and placebo vs. treatment were performed. Safety and tolerability data were analysed descriptively by treatment group and data from placebo subjects were pooled. Differences were considered to be statistically significant at p < 0.05. SAS ® software, version 9.2 (SAS Institute, Cary, NC, USA) was used for the statistical analysis and descriptive statistics of clinical and PK data.

Acknowledgments:
The authors wish to express their thanks to Mark Enzler and Christoph Siethoff at Swiss BioQuant AG, Switzerland for the bio-analytical assay and the project managers Sandrine Gioria and Isabelle Naëije. This study was sponsored by Actelion Pharmaceuticals Ltd.