Effects of Ponesimod, a Selective S1P1 Receptor Modulator, on the Pharmacokinetics Of

Effects of ponesimod, a selective S1P1 receptor modulator, on the pharmacokinetics of a hormonal combination contraceptive

Supplementary material

Bioanalytical assay

Ponesimod and the internal standard, a deuterated analogue of ponesimod, were extracted by protein precipitation using a mixture of acetonitrile and ethanol (1:1, vol/vol). After protein precipitation, plasma samples were filtered through a protein precipitation plate and the filtrate was diluted with an equal volume of water containing 0.1% formic acid prior to injection onto the HPLC column. The analysis was done by separation using reverse phase chromatography followed by detection with a triple-stage quadrupole MS/MS in thepositive ion mode. The calibration range for the validated method was 1.00 ng/mL to 1000 ng/mL for ponesimod.

Plasma samples (500μL) were fortified with 25 μL of internal standard working solution. Ethinyloestradiol and NET and their deuteratedanalogues (17α-ethinyloestradiol-2,4,16,16-d4 and norethindrone-2,2,4,6,6-d5) were extractedby liquid-liquid extraction using a mixture of ethyl acetate and hexane (10:90 v/v). Thesolvent was evaporated and the remaining residue derivatised with dansyl chloride.The derivatisedanalytes were extracted into hexane, the solvent was evaporated, and the remaining residue was reconstituted with 100 μL of acetonitrile and 200 μL of water. The final extract was analysed via HPLC with MS/MS detection. The chromatographic system consisted of two Agilent 1100 LC pumps (Agilent Technologies, Santa Clara, CA, USA), aPursuit PFP, 2 mm x 50 mm, 5 μmtrapping column (Varian Medical Systems, Inc., Palo Alto, CA, USA) and a ODS-AQ, 2 mm x 100 mm, 3 μm, 120Å analytical column (YMC America, Inc., Allentown, PA, USA). Mobile phases to elute the columns consisted of20:80 acetonitrile / 0.1% formic acid and 98:2 acetonitrile / 0.1% formic acid. Mass spectrometric detection was performed with a triple quadrupole mass spectrometer (Sciex API 4000, AB Sciex, Framingham, MA, USA) operating in electron spray-ionization positive-ion mode. Samples were quantified using peak area ratios. The calibration range was 2.00-500 pg/mL for EE and 50.0-25,000 pg/mL for NET.The LLOQ was 2 pg/mL and 50 pg/mL for EE and NET, respectively.

Results

Safety and tolerability

Clinically relevant decreases in pulmonary function (defined as decreases of more than 12% and 0.20 L in FEV1from baseline, and a more than 5% decrease in FEV1/FVC ratio) were observed during treatment with ponesimod. Overall, 22 of 23 subjects experienced at least one clinically relevant decrease in FEV1 during treatment with ponesimod. The incidence increased as the dose was up-titrated, with clinically relevant FEV1decreases in five subjects on Days 1–3 (10 mg dose period), 14 subjects on Days 4–6 (20 mg dose period), and 20 subjects on Days 7–14 (40 mg dose period). Episodes of dyspnoea were associated with decreases in FEV1 at 40 mg ponesimod. Seven of the 23 subjects experienced non-clinically relevant transaminase elevations during treatment with ponesimod (in all cases < 2 upper limit of normal [ULN]). No other relevant changes in laboratory variables were observed.

Two subjects experienced asymptomatic 1st degree atrioventricular (AV) block: one subject on Day 1 (10 mg) 2.5 h post-dose (with increased PQ interval for the remainder of the study) and another subject on Day 11 (40 mg) 4 h post-dose. No other relevant ECG abnormalities were reported in subjects who received ponesimod or ponesimod in combination with the oral contraceptive.

Discussion

The PD effect of ponesimod on lymphocyte count was demonstrated in all exposed subjects. Sphingosine-1-phosphate signalling has been implicated in various functions, including human lung vascular barrier function and cardiac function [1-6]. The observed effects on HR and AV conduction in this study were also reported in the single ascending dose study with ponesimod[7]. Other S1P receptor agonists such as fingolimod and BAF312 reported similar effects on HR [3, 8]. In a previous ponesimod study with an up-titration (10 mg for 4 days, 20 mg for 4 days, and 40 mg for 7 days), the first-dose effects led to similar reductions in HR [9]. As observed in this study, the effect on HR is more pronounced after the first dose and diminishes upon repeated administration of ponesimod, suggesting desensitization with repeated exposure.

Decreases in FEV1 and FEV1/FVC ratio were associated with symptoms of intermittent dyspnoea at the 40mg dose level. Sphingosine 1-phosphate and its receptors are also reported to be associated with vascular epithelial barrier integrity, and in vivo experiments in S1P3-deficient mice have shown pulmonary oedema upon S1P administration through the airways[2, 4, 10]. Fingolimod has also shown reductions in FEV1, suggesting a drug class effect[8, 11, 12].A previous study with multiple doses of ponesimod showed a decline in FEV1 compared to placebo, but rapid reversibility was shown with a return to baseline FEV1 within 6-10 days after drug discontinuation [9, 13]. The mechanism by which pulmonary function may be altered by drugs that target S1P receptors remains to be determined.

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