Purpose: Etrasimod is a next-generation, oral S1P receptor 1, 4, and 5 selective modulator in clinical development for the treatment of immune-mediated inflammatory disorders. In vitro studies were conducted to: 1) determine the CYP450 (CYP) enzymes involved in the metabolism of etrasimod in both recombinant human CYP450 (rCYP) enzymes and human liver microsomes (HLMs); 2) evaluate the ability of etrasimod to inhibit the major CYP enzymes in HLMs in a direct and metabolism-dependent manner and to also directly inhibit the major uridine diphosphate glucuronosyltransferase (UGT) enzymes in HLMs; and 3) investigate the inductive effects of treating primary cultures of cryopreserved human hepatocytes with etrasimod on the expression of inducible CYPs.
Methods: 1) Reaction Phenotyping: Two approaches to CYP reaction phenotyping (rCYP enzymes and chemical inhibition experiments with selective CYP inhibitors in HLMs) were assessed. To evaluate potential of rCYP enzymes to metabolize etrasimod, the compound (1 and 10 µM) was incubated with a panel of rCYP enzymes (rCYP1A2, rCYP2B6, rCYP2C8, rCYP2C9, rCYP2C19, rCYP2D6 and rCYP3A4; 50 pmol CYP/mL). Etrasimod (10 µM) was also incubated for 30 min with HLMs (0.5 mg protein/mL) in the presence and absence of selective CYP inhibitors. 2) CYP and UGT Inhibition: HLMs were incubated with marker substrates of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7 and UGT2B17 in the presence or absence of etrasimod (1 or 10 µM). For metabolism dependent inhibition of CYP enzymes, etrasimod was preincubated with HLMs for 30 min with an NADPH regenerating system, prior to the incubation with marker substrates. 3) CYP Induction: Three preparations of cultured human hepatocytes from three separate livers were treated once daily for three consecutive days with vehicle control, flumazenil (25 µM, negative control), etrasimod (1 or 10 µM), or with known human CYP enzyme inducers. After treatment, cells were harvested to isolate mRNA, and analyzed by qRT-PCR to assess effect of etrasimod on CYP1A2, CYP2B6, and CYP3A4 mRNA levels.
Results: 1) Reaction Phenotyping: After 120 minutes of incubation with rCYP enzymes, etrasimod loss was observed to the greatest extent with rCYP2C8 (up to 75%) and ranged from only 12 to 36% loss with rCYP2C9, rCYP2C19 and rCYP3A4. In HLMs without chemical inhibitors, overall loss of etrasimod was negligible (0 to 13.0%). A selective CYP2C8 inhibitor (gemfibrozil) inhibited, by 85%, 47%, and 60%, the observed limited (very low) conversion of etrasimod to two different oxidative metabolites and a ketone metabolite, respectively. The observed limited conversion of etrasimod to one of the two oxidative metabolites was also inhibited (72%) by a CYP2C9 inhibitor (tienilic acid). 2) CYP and UGT Inhibition: Direct inhibition of CYP2C8 activity was 22% at 1 µM and ~100% at 10 µM in the presence of etrasimod. Little or no direct inhibition of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 or CYP3A4/5 activities was observed in the presence of etrasimod concentrations up to 10 µM. In addition, little or no evidence of metabolism dependent inhibition of CYPs was seen. Etrasimod directly inhibited UGT1A1 and UGT1A6 activities by up to 28% and 48%, respectively. Little or no evidence of direct inhibition of UGT1A3, UGT1A4, UGT1A9, UGT2B7 or UGT2B17 was observed with etrasimod. 3) CYP Induction: Treatment of cultured human hepatocytes with etrasimod was found to have no induction potential on CYP1A2 mRNA. Additionally, no induction potential for CYP2B6 and CYP3A4 was seen, based on induction criteria being a > 2-fold increase in mRNA and also ≥ 20% of the mRNA increase seen with positive control.
Conclusion: Based on the results from two approaches to CYP reaction phenotyping, CYP2C8 and CYP2C9 were established to play major roles in conversion of etrasimod to oxidative metabolites, and CYP2C8 also plays a major role in formation of a ketone metabolite. However, the overall conversion of etrasimod to metabolites was negligible in HLMs and there was little difference in the loss of etrasimod seen in the presence and absence of direct-acting and metabolism-dependent selective CYP inhibitors. Direct inhibition of CYP2C8 activity was 22% at 1 μM and ~100% at 10 μM in the presence of etrasimod. However, there was little or no direct inhibition observed for any other CYPs with etrasimod. Etrasimod directly inhibited UGT1A1 and UGT1A6 activities by up to 28% and 48%, respectively, at the highest concentration tested (10 μM), but there was no evidence of direct inhibition of UGT1A3, UGT1A4, UGT1A9, UGT2B7 or UGT2B17. Lastly, etrasimod (up to 10 µM) was not a potential inducer of CYP1A2, CYP2B6, and CYP3A4/5. The results of these and other in vitro experiments, along with clinical pharmacokinetic evaluations with etrasimod, will provide the mechanistic information needed to help inform the need for, and design of, potential future clinical drug-drug interaction studies involving etrasimod.
D. Alexander Oh– Arena Pharmaceuticals, Inc.
Ronald Christopher– Parallel 33 Consulting
Yong-Hee Lee– Consultant
Yong Tang– Arena Pharmaceuticals, Inc.
John S. Grundy– Arena Pharmaceuticals, Inc.
John S. Grundy– Arena Pharmaceuticals, Inc.
Dooman Oh– Senior Director, Arena Pharmaceuticals, Inc., San Diego