Introduction

Siponimod (brand name Mayzent) is a sphingosine-1-phosphate (S1P) receptor modulator used in the treatment and management of relapsing forms of multiple sclerosis (MS) in adults. It works by targeting lymphocytes to decrease the number of circulating cells that are associated with MS symptomatic attacks and disease progression and may also have a direct neuroprotective impact. Siponimod strongly binds to the S1P type 1 and type 5 receptors that are abundantly expressed on lymphocytes and multiple other cell types in the central nervous system (CNS). Off-target interactions and effects on cardiac cells may occur, also. The use of a dose titration schedule is recommended to decrease the risk of bradycardia (see Table 1, Table 2) (1, 2). This medication is approved for multiple forms of relapsing MS (RMS) in the United States (1) and for active, secondary progressive disease in Europe and Canada (2, 3).

Siponimod is metabolized by members of the cytochrome P450 family, specifically CYP2C9 and, to a lesser extent CYP3A4. The CYP2C9 gene is polymorphic and activity scores are used to categorize diplotype into phenotype. Decreased CYP2C9 metabolic activity is associated with increased exposure to siponimod and increased risk of adverse effects. Therefore, individuals with the CYP2C9*3/*3 diplotype (activity score = 0) are contraindicated from taking siponimod (1, 2). Individuals with one copy of the no-function *3 allele (diplotype with activity scores of 0.5 or 1.0) are advised to take half the standard maintenance dose (1, 2). Consideration of genotype and activity score is essential for CYP2C9-based siponimod dosing because labeled dose recommendations are not categorized by phenotype. In the US, there is a modified titration schedule for individuals with a CYP2C9*3 allele (Table 1)(1); however, the European prescribing guidelines do not modify the titration schedule for individuals with a single copy of the CYP2C9*3 allele (heterozygous for CYP2C9*3) (Table 2) (2). The Dutch Pharmacogenetics Working Group (DPWG) of the Royal Dutch Association for the Advancement of Pharmacy similarly recommends a 50% reduced maintenance dosage for intermediate metabolizers (IM) (Table 3) (4). It should be noted that dose recommendations in the Siponimod package label are limited to diplotypes consisting of only CYP2C9 *1,*2, and *3 alleles due to lack of clinical data on the impact of other decreased or no-function alleles(1), while other medication and testing guidelines also consider*5, *6, *8, and *11 (5, 6).

Drug: Siponimod

Siponimod is a modulator of S1P receptors, specifically type 1 and type 5, used in the treatment of some forms of RMS. It is approved for use in the United States by the FDA, in the European Union by the European Medicines Agency (EMA), and in Canada by Health Canada. In the US, siponimod is authorized for all forms of RMS, including isolated syndrome, relapsing-remitting disease, and active secondary progressive MS (SPMS)(1). In contrast, the EMA and Health Canada have authorized it only for use in active SPMS (progressive MS with evidence of active disease based on relapses or imaging features of inflammatory activity) (2, 3). In all cases, the medication is approved for adults and is contraindicated in individuals with the CYP2C9*3/*3 genotype. (1, 2, 7) Due to its nonselective binding and adverse effects in cardiac cells, siponimod is also contraindicated in individuals with a recent history (previous 6 months) of myocardial infarction, unstable angina, stroke, transient ischemic attack, heart failure requiring hospitalization, or class III/IV heart failure (1). Cardiac conduction defects, such as Mobitz type II second- or third-degree atrioventricular (AV) block, or sick sinus syndrome without a functioning pacemaker, are also contraindications for siponimod therapy (1).

Siponimod is extensively absorbed (≥70%) with high oral bioavailability (84%), reaching maximum plasma concentration approximately 4 hours following dose administration (1). Following the prescribed dose titration period, siponimod usually reaches steady-state levels in the circulation after 6 days of the maintenance dosage. Although siponimod can cross the blood brain barrier, it is estimated that 68% of the medication remains in plasma (1). Siponimod is primarily metabolized via oxidation by the cytochrome P450 family members 2C9 (CYP2C9, accounting for 79% of the metabolism) and 3A4 (CYP3A4, accounting for 18.5%) (1, 8). The elimination half-life is 30 hours, and no unchanged siponimod is detected in urine, indicating it is fully metabolized before excretion. The main metabolites, M3 and M17, are not thought to contribute to the efficacy or safety of siponimod. Following discontinuation of therapy, complete elimination from the body may take up to 10 days, and the residual effect of reduced peripheral lymphocyte count may persist for 3 to 4 weeks. (1, 8, 9)

Differences in CYP2C9 activity, either due to genetic variation or inhibition from the dual CYP2C9/3A4 inhibitor fluconazole, are associated with reduced metabolism of siponimod (9) and increased overall exposure to the medication (1, 10). The most common CYP2C9 loss-of-function alleles are the CYP2C9*3 no-function allele and the *2 reduced reduced-function allele. Individuals with 2 copies of the CYP2C9*3 no-function allele are contraindicated for siponimod treatment, and those with reduced CYP2C9 enzyme activity due to a single *3 allele, specifically, the CYP2C9*1/*3 or *2/*3 diplotype, are advised to take a lower daily maintenance dose (1 mg) compared to individuals without these genotypes (2 mg daily dose) (1). Although the FDA-approved label mentions other CYP2C9 alleles with reduced or no functional activity that might have a similar effect on metabolism, there is inadequate evidence to justify a recommendation for altered dosing based on the presence of those alleles. In contrast, the DPWG guideline for siponimod recommends a similar dose adjustment for those with a genotype comparable to CYP2C9*3 heterozygous individuals, and avoidance of siponimod for those with a genotype comparable to homozygous CYP2C9*3. (4)

Multiple sclerosis is an immune-mediated disorder of the CNS, characterized by neuroinflammation, focal demyelination, and axonal damage (11). It affects more than 2 million people worldwide, with approximately 85% of these individuals having the relapsing-remitting form of MS, which is characterized by periodic attacks followed by partial or complete remission, at disease onset (12). The relapsing-remitting phenotype is usually followed by a secondary progressive phase (SPMS) consisting of gradual disability accrual, with or without overlapping relapses (13). Typically, MS affects individuals between the ages of 20 to 40 and can lead to significant disability (14). The specific symptoms and severity of the disease vary depending on the site and severity of lesions in the CNS. Common symptoms include fatigue, spasticity, weakness, tremor, visual impairment, pain, motor paralysis, cognitive impairment, and bladder and bowel problems (12). The clinical presentation of MS forms a spectrum, ranging from less severe forms of radiologically or clinically isolated syndrome to more aggressive primary progressive MS in which there is little to no relapse from symptoms, with persistent disease progression (15). Both genetics and environmental exposures are believed to play a role in determining the risk of developing MS, similar to other autoimmune disorders (11).

Receptors for S1P are expressed by a variety of cell types including astrocytes, oligodendrocytes, erythrocytes, myocytes, lymphocytes, and cells of the eyes and spleen (16). Prolonged activation of S1P1 receptors on lymphocytes leads to their internalization and prevents cellular exit from lymph nodes. This reduces migration of these immune cells from the peripheral bloodstream into the CNS, preventing inflammatory relapses (17) . Siponimod can also readily cross the blood brain barrier. Preclinical and clinical data suggest that binding of siponimod to S1P1 and S1P5 receptors in the CNS may also provide a direct protective and regenerative effect on neuronal cells, and this may be achieved independent of effects on peripheral lymphocyte counts (18, 19, 20).

Unlike a previous generation nonselective S1P modulator, fingolimod, siponimod targets only the type 1 and 5 receptors and does not require phosphorylation. This increased specificity reduces, but does not eliminate, the off-target adverse effects of siponimod on cardiac tissue (21). Specifically, siponimod can cause a transient and dose-dependent decrease in heart rate, which can be largely improved by following a specific dose titration schedule at the initiation of therapy (or following daily treatment interruption for more than 4 days) (22). Additionally, for individuals with preexisting cardiac conditions such as sinus bradycardia and first- or second-degree AV block, the drug labeling recommends a first dose monitoring period of 6 hours to address potential symptomatic bradycardia. (1, 2)

Compared to placebo, siponimod improves imaging-based metrics for disease progression (for example, lesion volume by MRI) and reduces the risk of confirmed disability progression, relapses, and the annual relapse rate (23). The benefits of siponimod are more substantial in individuals with active MS, as observed in the EXPAND extension study (24). Secondary analysis of the EXPAND trial reported a clinically meaningful improvement in cognitive function for individuals in the siponimod arm versus placebo (25). A systemic review of the BOLD (phase II) and core EXPAND (phase III) trials found low-certainty evidence supporting the benefit of 2 mg daily siponimod for the primary and secondary measures reported in those studies. However, the review called for additional trials to examine longer treatment duration for potential adverse effects and inclusion of other active controls(12). In contrast, a review of multiple disease-modifying therapies for RMS and active SPMS concluded that the EXPAND trial showed a significant reduction in disability progression and relapse rates (26). The neuroprotective effect of siponimod has led some groups to begin preclinical studies to assess its efficacy in autoimmune neuritis and optic nerve damage due to glaucoma (27, 28). Additional, ongoing clinical studies of siponimod can be found in the online database ClinicalTrials.gov, including a study for pediatric individuals (ages 10-17) with MS (NCT04926818) (29).

As siponimod specifically targets the immune system, it is essential to assess individuals before treatment for signs of active infection and to test for immunity to varicella zoster virus (either history of chicken pox or vaccination). Effective therapy with siponimod can lead to a 20–30% decrease in peripheral lymphocyte counts, increasing the individual’s risk for infection. A recent (within prior 6 months) complete blood count should be taken before initiating treatment (1). Similarly, individuals without immunity to varicella zoster infection should complete a vaccination course 4 weeks before starting siponimod (1). Studies have found that individuals on a S1P receptor modulator therapy (including siponimod and fingolimod) also have impaired responses to vaccination to SARS-CoV-2 and may be at an increased risk of break-through infection following vaccination (30, 31, 32). Siponimod may have a limited effect on the immune response following other vaccinations such as influenza or pneumococcal polysaccharide (33).

Other side effects including macular edema, elevated liver enzymes, and hypertension were reported in a higher proportion of individuals on siponimod compared to placebo (23). Thus, the FDA recommends an ophthalmic evaluation, as well as measurement of transaminase and bilirubin levels before starting treatment (1). Treatment with S1P receptor modulating therapies is also associated with a higher risk of cutaneous malignancies, such as basal cell carcinoma. Therefore, individuals should be monitored for suspicious skin lesions before starting treatment and periodically during treatment with siponimod (1). The extension of the EXPAND trial observed an increase in basal cell carcinoma incidence with increased time on siponimod, specifically for 3–5 years, with an incidence rate of 0.9 per “100 patient-years” compared to 0.7 for the placebo arm (24).

The use of siponimod in pediatric individuals, nursing mothers, pregnant women, or individuals aged 65 and over has not been sufficiently studied to decide if this medication is safe or if modified administration would be needed for these specific groups. Safety and efficacy in a pediatric population have not been established. Animal studies have suggested that siponimod may be able to cause fetal harm when taken by a pregnant woman. There is a pregnancy exposure registry available for enrollment: MotherToBaby (www.mothertobaby.org/join-study). Rat studies suggest siponimod, its metabolites, or both are detectable in milk (1). However, there are no data on the presence of siponimod in human breastmilk, its effects on breastfed infants, or the impact on milk production in lactating females. While it is unlikely that siponimod would be present in breast milk in significant amounts, it is potentially toxic to a breastfed infant. Based on the related drug fingolimod, experts recommend avoiding its use during breastfeeding (34). Individuals aged 65 or older should be managed with care due to the greater frequency of renal, cardiac, or hepatic dysfunction in this population. However, there is no specific recommendation in the drug labeling to suggest altered dosing for these individuals. (1)

Gene: CYP2C9

The cytochrome P450 superfamily (CYP450) is a large and diverse group of hepatic enzymes that form the major system for metabolizing lipids, hormones, toxins, and drugs. The CYP450 genes are highly polymorphic, and genetic variants can lead to reduced, absent, or increased enzyme activity.

The CYP2C9 enzyme metabolizes approximately 15–20% of clinically used drugs, and atypical metabolic activity caused by genetic variants in the CYP2C9 gene can play a significant role in adverse drug reactions (35, 36). Among CYP2C isoforms expressed in the liver, CYP2C9 is the most abundant (37, 38).

The CYP2C9 gene is polymorphic, with more than 80 known alleles (39). The wild-type allele is designated CYP2C9*1. In a test of multiple CYP2C9 variants, when none of the tested variants are detected, the genotype is assigned as CYP2C9*1, and the gene is predicted to produce a fully functional enzyme with normal enzyme activity (40). Individuals who have 2 normal-function alleles, for example, CYP2C9*1/*1, are classified as “normal metabolizers” (Table 4). Each allele is assigned an activity score of 0 (no-function), 0.5 (reduced-function), or 1 (normal-function), and the combined score for both alleles is used to determine the phenotype. This approach was initially described for standardization of phenotype for the gene CYP2D6 and is used for multiple CYP loci by the Clinical Pharmacogenetics Implementation Consortium (CPIC) (37, 41). A combination of alleles resulting in an activity score of 1.5 or 1 is classified as an IM phenotype, while a combined activity score of 0 or 0.5 is classified as a poor metabolizer (PM) phenotype (Table 4) (40). However, the effect of the reduced-function allele yielding a higher activity score than the no-function allele may result in different recommendations for IMs with activity scores of 1.5 and 1. For example, the CPIC guideline for the CYP2C9-phenytoin interaction recommends no adjustments are needed from typical dosing strategies for IMs with an activity score of 1.5, but recommends subsequent doses are reduced by 25% for IMs with an activity score of 1 (40).

Allelic variants associated with reduced, or no enzyme activity include CYP2C9*2 (p.Arg144Cys), CYP2C9*3 (p.Ile359Leu), CYP2C9*5 (p.Asp360Glu), CYP2C9*8 (p.Arg150His) and CYP2C9*11 (p.Arg335Trp). The *2 reduced-function allele is more common in European (13%), Near Eastern (13%), and Central/South Asian (11%) populations than in Latino (8%), Oceanian (3%), East Asian (0.2%), or African (1–2%) descent populations. The *3 no-function allele is less common (<10% in most populations, except for Central/South Asian, at approximately 11%), and it is extremely rare in African populations. In African descent populations, the CYP2C9*5 no-function (~1%), and *8 (6–8%), and *11 (1–3%) reduced-function alleles are also common. (42, 43, 44, 45)

Phenoconversion

Medications can alter CYP2C9 enzymatic activity, resulting in conversion to a different metabolizer phenotype than would be predicted from an individuals’ genotype. This phenomenon is referred to as phenoconversion. Any medication that is a substrate for CYP2C9 may compete with other concomitant CYP2C9 substrate medications, leading to a reduced effective enzyme activity level for all CYP2C9-metabolized medications. Other non-substrate medications that inhibit the CYP2C9 enzyme can alter the pharmacokinetic parameters of any drug that is a substrate of CYP2C9 through mechanisms other than competition for metabolism. An individual who is predicted from their genotype to be an IM and is taking more than one CYP2C9 substrate or inhibitor, such as amiodarone, fluconazole, metronidazole, sulfaphenazole, or voriconazole (37, 38), may present phenotypically like a PM. Conversely, CYP2C9 can be induced by other medications, leading to increased enzyme activity compared to the activity predicted by the genotype. Administration of rifampin and carbamazepine can induce CYP2C9 and the related enzyme CYP3A4 (1). Additional CYP2C9 inducers include nifedipine, hyperforin (found in St. John’s wort herbal supplement), phenobarbital, phenytoin, dicloxacillin, flucloxacillin, and tamoxifen (37). If an individual who is predicted to be an IM is taking a CYP2C9 substrate and an inducer, the individual may present phenotypically more like a normal metabolizer because the increased activity produced by the inducer may counteract some of the decreased activity produced by the reduced-function allele.

Linking CYP2C9 Genetic Variation with Treatment Response

The manufacturer provided data for EMA and FDA authorization indicates that individuals with CYP2C9*3/*3 genotype (activity score 0) should not take siponimod due to substantially elevated plasma levels of the medication, increased by 285% compared to CYP2C9*1/*1 exposure (1, 2). The DPWG also recommends avoiding siponimod in individuals with CYP2C9*3/*3 genotype (4). However, this contraindication is not extended to other genotypes with a PM phenotype, as the total exposure of CYP2C9*2/*3 (activity score 0.5) was only 91% higher (1). Citing a lack of experimental or clinical data on the impact of other no-function alleles (including alleles like CYP2C9*6), the FDA-approved label states it is likely that other no-function alleles “will have similar effects on siponimod pharmacokinetics” though clinical guidance is not provided(1).

Individuals with the reduced-function genotype of CYP2C9*1/*3 (activity score of 1) or CYP2C9*2/*3 (activity score of 0.5) are recommended to take half of the standard maintenance dose, as stated by the FDA, EMA drug labels, and the DPWG (1, 2, 4). Again, other alleles that confer an IM phenotype are not specifically addressed by the FDA drug labeling, and the reduced dosage recommendation is specific to these genotypes. It should be noted that the versions of the drug label approved in the US (1) and Canada specifically recommend that individuals with CYP2C9*2/*2 genotype be given the standard dose (2 mg daily maintenance dose).The European approved version of the drug label does not explicitly address dosing for individuals with CYP2C9*2/*2 genotype (2).

Currently, the dose recommended for CYP2C9*3 heterozygotes cannot be extrapolated to all IM-associated genotypes. Much of the available literature for siponimod pharmacokinetics was published before the use of activity scoring and phenotype assignments were standardized (5). In vitro metabolism studies with human liver microsomes reported a 2.7-fold decrease in siponimod metabolism in samples with CYP2C9*2/*2 genotype and simulations of plasma concentration of siponimod were elevated in both the *2/*2 and *3/*3 genotype (9). However, in vivo studies described in the drug label show a 25% increase in plasma concentration (measured as area under the curve) with the CYP2C9*2/*2 genotype compared to *1/*1 (1).The clearance of siponimod reported for CYP2C9*2/*2 was higher than CYP2C9*1/*3, and the CYP2C9*2/*3 clearance was well above CYP2C9*3/*3(47). The difference in in vitro and in vivo metabolism of siponimod raises a question as to whether the CYP2C9*2 variant may not confer as significant of a decrease in enzyme activity for siponimod as compared to other CYP2C9 substrates. Contrary to other medications, siponimod pharmacogenetic dosing recommendations are not made based on metabolizer phenotype but rather specific allele identification based on known effects of those alleles.

This model for pharmacogenetic recommendations highlights the need for more studies to assess the impact of CYP2C9 alleles found in populations with non-European genetic ancestry. Some authors have observed that the incidence of MS is higher in blacks (racial assignment extracted from health and military records, most likely reflecting self-reported identification (48, 49); incidence rates averaged to 10.2 per 100,000 as compared to 6.9 in whites, 2.9 in Hispanics and 1.4 in Asians) and the EXPAND and BOLD clinical trials for siponimod did not report race or ethnicity in their studies (50). The FDA review documentation for siponimod approval indicated that approximately 95% of the participants in EXPAND (also referred to as CBAF312A2304) were white (51). Additional decreased or no-function CYP2C9 alleles (for example, *5, *6, *8 and *11) are highly relevant across a broad range of racial and ethnic backgrounds, indicating the need for additional analysis and consideration for dosing adjustment (50). Based on altered pharmacokinetics of phenytoin and warfarin in individuals with a CYP2C9*11 allele, analysis of this allele is highly recommended before prescribing medications like siponimod, especially due to their narrow therapeutic window and CYP2C9 substrate status (52).

Due to the more rapid metabolism of siponimod, co-medication with drugs that strongly induce CYP2C9 and CYP3A4 is not recommended in all individuals. Specifically, co-medication with moderate or strong CYP3A4 inducers such as modafinil or efavirenz, is not recommended in individuals with CYP2C9*1/*3 or *2/*3 IM genotype. Conversely, co-medication with drugs that inhibit CYP2C9 and CYP3A4, such as fluconazole is also not recommended for all individuals due to the risk of increased exposure and potential adverse effects. (1)

Genetic Testing

The NIH’s Genetic Testing Registry provides examples of the genetic tests available for siponimod drug response and the CYP2C9 gene.

For CYP2C9, the Association for Molecular Pathology has recommended alleles that should be included in clinical genotyping assays (6). Testing results are typically reported as a diplotype, such as CYP2C9*1/*2, and may include an interpretation of the individual’s predicted metabolizer phenotype (normal, intermediate, or poor) and an activity score (Table 5). Testing laboratories will report an allele assignment of CYP2C9*1/*1 if no tested variant is detected in the sample. However, if an allele is not covered by the test, the *1/*1 diplotype does not mean that the individual does not have that variant; it only indicates the person does not have any of the variants included in the test. Therefore, reviewing the testing methods and alleles covered by the specific test used is crucial to understanding what a *1/*1 report truly means for the individual tested.

The CYP2C9 Gene Interactions with Medications Used for Additional Indications

The CYP2C9 enzyme is involved in the metabolism of a wide range of medications, making genotyping results informative for many drugs. Medications that may be affected by CYP2C9 genetic variation include:

• NSAIDs like celecoxib, flurbiprofen, piroxicam, all of which are metabolized by CYP2C9; thus individuals with reduced enzyme activity will experience increased exposure and have a higher risk of side effects.
• Cannabinoids like dronabinol, a synthetic form of delta-9-tetrahydrocannabinol, are metabolized by CYP2C9 and decreased enzyme activity increases an individual’s exposure to the active compound
• Anti-convulsant medications like phenytoin, which are metabolized by CYP2C9, and a loss of function in this enzyme increases exposure to the medication and the risk of adverse effects.
• Uric acid lowering medications like lesinurad; individuals who lack CYP2C9 activity have an increased exposure to lesinurad, and an increased risk of side effects.
• Anticoagulants like warfarin, individuals with reduced or no function CYP2C9 alleles require lower doses of this medication.

Additional information on gene-drug interactions for CYP2C9 are available from PharmGKB, CPIC and the FDA (search for “CYP2C9”).

Therapeutic Recommendations based on Genotype

This section contains excerpted¹ information on gene-based dosing recommendations. Neither this section nor other parts of this review contain the complete recommendations from the sources.

2023 Statement from the US Food and Drug Administration (FDA):

CYP2C9 Genotype Determination

Test patients for CYP2C9 variants to determine CYP2C9 genotype.

[…]

Recommended Dosage in Patients With CYP2C9 Genotypes *1/*1, *1/*2, or *2/*2

Maintenance Dosage

After treatment titration (see Treatment Initiation), the recommended maintenance dosage of [siponimod] is 2 mg taken orally once daily starting on Day 6.

[…]

Recommended Dosage in Patients With CYP2C9 Genotypes *1/*3 or *2/*3

Maintenance Dosage

In patients with a CYP2C9*1/*3 or *2/*3 genotype, after treatment titration (see Treatment Initiation), the recommended maintenance dosage of [siponimod] is 1 mg taken

orally once daily starting on Day 5.

Administer tablets whole; do not split, crush, or chew [siponimod] tablets.

Treatment Initiation

Initiate [siponimod] with a 4-day titration, as shown in Table 2... A 7-tablet starter pack should be used for patients who will be titrated to the 1-mg maintenance dosage.

[…]

[Siponimod] is contraindicated in patients who have:

A CYP2C9*3/*3 genotype

Please review the complete therapeutic recommendations that are located here: (1).

Nomenclature for Selected CYP2C9 Alleles

Acknowledgments

The author would like to acknowledge J. Shawn Jones, PhD, MS, Associate Professor, Texas Tech University Health Sciences Center, Dallas, TX, USA, Christine Kroger, PharmD, BCACP, Ambulatory Care Pharmacist & Pharmacogenomics Specialist, Allina Health Care Management, Minneapolis, MN, USA, and Radu Tanasescu, MD, Consultant Neurologist, Honorary Clinical Associate Professor of Neurology, MRC CARP, University of Nottingham School of Medicine, Queen’s Medical Centre, Nottingham, United Kingdom for their expert review of this summary.

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