Introduction

The first clinical episode of multiple sclerosis (MS), a chronic immune-mediated disorder of the central nervous system (CNS), typically occurs at 20 to 30 years of age.1. An initial relapsing-remitting disease course (RRMS, 85%) is predominant and followed by a phase of insidious and irreversible worsening of neurologic function independent from relapses.2 A minor proportion of individuals (15%) have a progression from the onset, called primary progressive MS (PPMS).1 The disease stage developing in the aftermath of relapsing-remitting MS is termed secondary progressive MS (SPMS) and develops after a median of 15 years in a subgroup of untreated individuals.3 Common neurological manifestations include unilateral optic neuritis, motor and sensory disturbances, and myelopathic, brainstem, and cerebellar syndromes.4 The symptoms of the relapse resolve within a couple of weeks or months, and there is a higher chance of complete resolution earlier in the disease course. An epidemiological study from South-East Norway evaluated 1032 individuals diagnosed with MS between 1998 and 2017.5 They found a significantly longer time lag from disease onset to an Expanded Disability Status Scale (EDSS) of 6, which reflects the need for a walking aid, in people with MS (pwMS) who were diagnosed after 2006. Still, 692 (33%) individuals in their cohort had reached EDSS 6, which was after a median time lag of 29.8 (95% confidence interval (CI) 28.5–31.1) years. The clues are continuously growing about the crucial role of environmental, lifestyle, and genetic factors in the condition’s occurrence, clinical course, and outcome.6–10 MS is also associated with numerous comorbidities, which hamper early diagnosis and negatively impact the disease course.11

Over the past decade, the number of approved disease-modifying drugs (DMD) for treating MS has substantially increased.12 The distinct modes of action and levels of efficacy serve as the foundation for selecting the most appropriate DMD. However, proof of prognostic reliability is awaited for combined treatment goals, including ‘no evidence of disease activity (NEDA).13,14 The decision for a certain DMD is aided by the recent individual clinical and radiological dynamics of the inflammatory and degenerative processes within the CNS.15 The distinction between relapse-dependent and independent progression of long-term disease remission might be an additional variable to consider. In addition, the possibility of a DMD selection according to personal preferences and life circumstances is critical in maintaining adherence.16 More recently, there have been efforts to develop second-generation therapeutics with an improved risk-benefit profile.17

In the pivotal, placebo-controlled Phase III DEFINE and CONFIRM trials in adults with RRMS, twice-daily 240 mg dimethyl fumarate (DMF, Tecfidera®, Biogen, USA) reduced clinical and magnetic resonance imaging (MRI) measures of inflammatory disease activity and improved some aspects of health-related quality of life (HR-QoL).18,19 The DEFINE study also disclosed reduced disability progression with DMF.18 Sustained DMF safety/efficacy was observed in patients followed up to 13 years in the long-term follow-up of the ENDORSE trial.20 In the pivotal trials of DMF, gastrointestinal (GI) adverse events (AE) were reported by 42% of the patients, and 4% discontinued the study medication owing to these events.18,19 Even higher discontinuation rates of up to 19% are observed in real-life despite the implementation of symptom management and mitigation strategies.21–23 Of note, more than 560,000 patients have been treated with DMF, representing more than 1.100.000 patient years of exposure.24 In this regard, diroximel fumarate (DRF, Vumerity®, Biogen USA), a second-generation DMD, was developed to achieve improved gastrointestinal (GI) tolerability and has undergone testing in clinical trials. The United States Food and Drug Administration (FDA) granted marketing authorization for DRF in October 2019 to treat adults with relapsing forms of MS, including clinically isolated syndrome (CIS), RRMS, and active SPMS.25 The approval of the European Medicines Agency (EMA) in November 2021 respected RRMS.26 In April 2020, the FDA approved MMF (Bafiertam®, Banner Life Sciences, USA), the active metabolite of DMF and DRF, for relapsing forms of MS.27 Currently, DMF is being studied in other neurological (amyotrophic lateral sclerosis, glioblastoma multiforme) and non-neurological conditions (cutaneous T cell lymphoma, obstructive sleep apnea, rheumatoid arthritis).28,29

In this article, we review the existing scientific evidence for fumaric acid esters (FAEs) and the emerging preclinical and trial data leading to the approval of DRF in MS.

Fumaric Acid Esters: Historical Developments

FAEs were initially extracted from fungi, lichen, and moss (eg Fumaria officinalis). In medieval times, remedies containing FAE were used for blood cleansing, eye disorders, digestive/hepatobiliary complaints, and rheumatoid arthritis in Europe, Asia, and the Middle East.30 From the late 1950s, FAE was studied for its use in psoriasis, a chronic immune-mediated and non-contagious disease characterized by raised areas of abnormal skin.31 A mixture of FAEs, primarily consisting of DMF, was approved in Germany to treat psoriasis (Fumaderm®, Fumapharm, Switzerland) in 1994. The understanding of psoriasis as a T-cell-mediated autoimmune disease and the identification of the immunomodulatory activity of FAE as a central mode of action fostered the repurposing of DMF in MS.

Historically, the biocidal activity of FAEs, with the capability to prevent mold growth, was used in the production of clothes, furniture, and shoes. Of note, allergic skin reactions, severe eye irritation, and respiratory irritation can occur on direct contact.32 Thus, its use for consumer products has been forbidden in the European Union since 1998 under the Biocide Directive 98/8/EC.33

Fumaric Acid Esters: Chemistry, Pharmacodynamics, and Kinetics

DMF and DRF are both prodrugs and have distinct chemical structures and molecular weights, as shown in Figure 1. After oral intake, both compounds undergo rapid esterase cleavage into MMF, the only active metabolite.34 In detail, DMF undergoes rapid hydrolysis by esterase cleavage within the small intestine. DMF is converted to MMF, and the byproduct methanol (MeOH). In contrast, DRF is hydrolyzed via a major (>90%) and a minor pathway (>10%). The main path leads to the generation of MMF and the inactive metabolite 2-hydroxyethyl succinimide (HES). There is no evidence so far for the pharmacological activity of HES. Around 58–63% of the total dose of DRF is excreted as HES via the renal route. In the minor pathway, DRF metabolizes into the inactive RDC-8349 and MeOH. The stoichiometric ratio of MMF to MeOH is 1:1 after esterase hydrolysis of DMF.35 In contrast, MMF and MeOH are generated in a balance of 9:1 in the case of DRF metabolism. MMF crosses the BBB and is likely to exert action within the CNS.36 Drug penetration across the BBB was evaluated in people with SPMS, revealing that CSF MMF concentration was 11% of plasma.37 Moreover, the Tmax of CSF MMF peaked only 2 hours later than that of plasma.

Figure 1 Chemical structure and metabolism of dimethyl fumarate and diroximel fumarate.

The terminal half-life of MMF is around one hour.38 MMF is metabolized by the tricarboxylic acid cycle to carbon dioxide, fumaric acid, citric acid, and glucose. The elimination of MMF is primarily maintained through the exhalation of carbon dioxide (60% of a dose).39,40 Only a minor proportion is eliminated via the renal (15.5%) or fecal (0.9%) route.

DMF was designed as an oral delayed-release (DR) preparation for optimized pharmacokinetics with predominant release in the small intestine.40 The compound requires reapplication every 12 hours, ie, twice daily intake (2x240 mg). Most importantly, for DRF, an oral intake at the dose of 2×462 mg results in systemic MMF concentrations that are bioequivalent to DR DMF 2×240 mg.41 Peak plasma concentrations after oral intake of DR DMF are reached after 2–2.5 hours, and for DRF after 2.5–3 hours.42 The administration of DR DMF with a high-fat, high-calorie meal does not affect the area under the curve (AUC) of MMF.25 However, there is a decrease in the peak plasma concentration by 40%, with a delay of 3.5 hours; yet, this does not modify the AUC.43 Of note, co-administration with food significantly decreases the incidence and severity of nausea, vomiting, and abdominal pain.44 Indeed, intake of DR DMF with high-fat, high-protein, and low-starch foods can mitigate GI AEs.45 Moreover, the use of acid secretion blockers, antacids, or antihistamines is among further GI AE management strategies.

A population pharmacokinetic (PK) model for MMF and HES was established with pooled concentration-time data derived from 11 clinical studies of DRF.46 DRF metabolites showed linear kinetics across dose levels and over time. Variables that impacted PK were meal fat content, evening dosing, body weight, and extent of kidney dysfunction. MMF shows dose-proportional PK, with exposure increasing proportionally over a daily dose range of 462–924 mg. Moreover, MMF does not accumulate in the system over multiple doses of DRF.

Fumaric Acid Esters: Current Understanding of the Mode of Action Molecular Pathways

The therapeutic mode of action of FAEs in MS is not entirely unraveled. FAEs are presumed to impact different steps of MS pathophysiology. Both, inflammatory and neurodegenerative properties of immune and brain resident cells are targeted by FAEs, as shown in Figure 2.47,48

Figure 2 Presumed effect of MMF/DMF/DRF on immune cells and within the central nervous system.

There is evidence that the cytoprotective effects on neurons and glial cells are, in part, mediated through the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcriptional pathway, which leads to the activation of antioxidant and detoxifying genes.49–51 These include heme oxygenase-1 (HO-1, NAD(P)H quinone dehydrogenase (NQO-1), and glutathione S-transferase-1 (GST-1).52 While HO-1 activates anti-inflammatory, anti-proliferative, and anti-apoptotic pathways, NQO-1 and GST-1 can prevent cellular oxidative stress.53,54

FAEs also activate several Nrf2-independent pathways. MMF is an exogenous ligand for the hydroxycarboxylic acid receptor 2 (HCAR2), also known as the niacin receptor-1. Activation of HCAR2 was shown to inhibit microglia activation and thus attenuate neuroinflammation.55 Activated HCA2R2 inhibits chemoattractant-mediated macrophage migration via Gβγ/PKC/ERK1/2 pathway and heterologous receptor desensitization.56 FAE derivatives can inhibit the Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway, which controls cytokine production and cell survival, in an Nrf2-independent manner. MMF can also exert anti-inflammatory action through the 5’ adenosine monophosphate-activated protein kinase (AMPK)/NAD-dependent deacetylase sirtuin-1 (Sirt1) pathway.57

Mode of Action in Multiple Sclerosis

The treatment with DMF is associated with changes of peripheral immune cell subset composition and activation. Not all subpopulations of immune cells in the peripheral blood are affected equally. There is evidence for anti-inflammatory action, with a relative shift of immune cell populations toward anti-inflammatory phenotypes.58 DMF also interferes with lymphocyte signaling, attenuating proinflammatory cytokine and chemokine production.59 In-vivo, a relative decrease of the following immune cell populations is observed: proinflammatory Th1/17 cells, central and effector memory CD4/8 T cells, plasmablasts, IgD± memory, and CD138 plasma cells, CD56dim NK cells. Conversely, a relative increase in the following immune cell populations is observed in-vivo. The subsets involved include anti-inflammatory Th2 cells, naïve CD4/CD8 cells, naïve regulatory T cells, transitional B cells, and CD56bright NK cells. In addition, DMF treatment in MS results in a reduction of terminally differentiated effector memory T cells and decreased numbers of interferon-gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) producing CD4+ and CD8+ T cells. Moreover, DMF targets the intracellular stress response in activated T cells, thereby restricting mitochondrial function and energetic capacity.60 The subsequently enhanced mitochondrial reactive oxygen species levels result in enhanced T-cell apoptosis. DMF attenuates the migratory potential of CD4+ T cells, whereas CD8+ T cells are less affected.61

There is a decline in the absolute B-cell number with DMF treatment, with preferential depletion of memory B cells and a concurrent increase in naïve B cells.62 In addition, there is a significant reduction in GM-CSF, tumor-necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) secreting B cells with DMF treatment. Treatment with DMF is also associated with decreased expression of costimulatory CD40, antigen presentation molecule major histocompatibility complex class 2 and B cell activating factor receptor on B cells.63 This anti-inflammatory shift of B cell responses was corroborated in another study.64

The understanding of the effects of DMF on the distribution of lymphocyte subsets within the CNS of pwMS is limited. The knowledge about DMF/MMF effects on CNS resident cells derives mostly from in-vitro studies and preclinical models. In people with primary progressive MS, there were reduced numbers of CD4+ T cells and increased concentrations of IL-7 in CSF.65 IL-7 is a hematopoietic growth factor that stimulates the expansion of CD4+ T cells. Thus, the increase of IL-7 could be related to the peripheral lowering of CD4+ T cell counts by DMF and subsequently reduced transmigration to the CNS or the direct impact of MMF on CD4+ T cells within the CNS. In the CSF, the absolute number of mononuclear cells was significantly lower in DMF-treated pwMS than with other DMDs, and there was a disproportionate decrease of plasmablasts.66

Mode of Action in in-vitro Studies and Preclinical Models

After oral DMF administration in rodents, MMF rapidly distributes throughout different organs. MMF is detected in the plasma, brain, kidney, jejunum, and spleen.67 A recent study in rodents disclosed that FAE derivatives undergo a different biodistribution and may elicit distinct biological responses.67 In detail, the Nrf2-mediated oxidative stress response pathway was exclusively regulated by DMF, whereas MMF activated apoptosis pathways.

Immune cell migration to the CNS is attenuated by inhibition of the adhesion of immune cells to endothelium.68 A combined ex-vivo analysis of lymphocytes derived from DMF-treated pwMS and in-vitro MMF treatment of endothelial cells revealed dual action.55 There was VCAM-1 downregulation on the endothelial side and reduction in T cells with a migratory phenotype on the lymphocyte side. The binding of MMF to brain endothelium and activation of G-protein-coupled receptor HCA2 with subsequent inhibition of NF-κB signaling is involved in this process. An in-vitro study also confirmed a reduced extent of transendothelial migration of monocytes upon MMF treatment and increased levels of endogenous antioxidant proteins in brain endothelial cells.69

Preclinical and in-vitro studies corroborate that FAE can protect neurons and oligodendrocytes via activation of the Nrf2 pathway. In-vitro studies found that DMF/MMF alters the function of CNS resident cells, suppresses inflammatory cytokine production by activated microglia and astrocytes, and increases the number of oligodendrocyte precursor cells.51,70,71 In experimental autoimmune encephalomyelitis (EAE), an experimental model of MS, treatment with DMF was associated with decreased microglial activation in chronic active brain lesions.72 Moreover, in EAE, the mode of action of DMF includes the suppression of inducible nitric oxide synthetase (iNOS)+ proinflammatory macrophages, complement C3 positive astrocytes, and deposition of C3 in the CNS.73 DMF acts directly on CD4+ T cells and suppresses GM-CSF-producing Th1 or single GM-CSF+ T cells in EAE.74 In-vitro, DMF is capable of directly inducing apoptosis of B-cells.63

DMF alters lipid metabolism, which may support the ability of cells to survive oxidative stress:51,75 Most recently, DMF was shown to change the metabolic profile of human CD4+ and CD8+ T cells by restricting their antioxidative properties.60 The enhanced mitochondrial reactive oxygen species levels result in enhanced T-cell apoptosis and potentially contribute to the immunomodulatory function of DMF.

Clinical Efficacy of Dimethyl Fumarate in Multiple Sclerosis

The efficacy of orally administered DR DMF over two years was demonstrated in the pivotal, placebo-controlled phase III DEFINE and CONFIRM trials in adults with RRMS.18,19 In the long-term extension trial (ENDORSE), 1736 of 2079 DEFINE/CONFIRM completers participated and followed a median of 8.5 years (range 2.1–10.8).76 The adjusted annualized relapse rate (ARR) remained low in the extension study (⩽0.20). Moreover, almost 75% remained free of 24-week confirmed disability progression. A posthoc analysis of DEFINE/CONFIRM revealed that a significantly higher percentage of patients treated with DMF (26%) achieved NEDA status over two years compared with placebo (12%).77 The relative risk reduction was 42.7% (hazard ratio, 0.57; 95% CI, 0.48–0.69; P < 0.0001). Results of APEX, a pivotal trial in a predominantly East Asian population, were reassuring of those seen in DEFINE and CONFIRM.78 Real-world evidence corroborates the efficacy of DR DMF in RRMS concerning lowering the relapse rate and slowing disability progression. A recent review article identified several observational studies.79 Amongst were studies with large cohorts (n > 500), which were either prospective (n = 7) or retrospective (n = 11).

Tolerability and Safety of Dimethyl Fumarate in Multiple Sclerosis

The tolerability profile of DR DMF 240 mg twice daily is well studied. Of note, AEs were frequent in the phase III trials regardless of assignment for DMF (86–96%) or placebo/glatiramer acetate (GA, 77–95%) but were mainly in the mild to moderate severity range. The most common AEs (≥10% incidence) of DMF with a ≥ 2% greater incidence vs placebo included flushing (40% vs 6%), nausea (10% vs 5%), abdominal pain (18% vs 10%) and diarrhea (14% vs 11%). MS relapse was the most frequently reported serious AE in the pivotal trials. Relapses occurred in the DMF and placebo/GA arm in 10–11% and 14–15%, respectively. All other serious AEs occurred at a frequency of ≤2%. The absolute lymphocyte count (ALC) declines four weeks after DMF initiation. The ALC declines by 30% on average but remains above the lower limit of normal (LLN; ie, ALC ⩽ 0.91×109/L) in about two-thirds of the patients. Severe prolonged lymphopenia developed in 2% of patients; prolonged lymphopenia with moderate severity developed in 10%. Infections were also common in DEFINE (64% for DMF and 65% for placebo) and CONFIRM (56% and 50% for GA, respectively). The most common infections included nasopharyngitis, urinary tract infection, and upper respiratory tract infection. Few patients (≤2%) had serious infections, with no opportunistic infections in the main period of the pivotal trials.

In the long-term extension study of the DEFINE and CONFIRM trials (EXPAND), the incidence of SAEs was 30% among those exposed to DMF after the elimination of the GA-treated patients.76 The most common SAEs in this follow-up study over nine years were MS relapse, nasopharyngitis, and flushing. There were nine deaths reported in the long-term study. A fatal case of progressive multifocal leukoencephalopathy (PML) developed after severe lymphopenia over 3.5 years. A recent study calculated a DMF-related PML risk of 0.02 per 1000 patients.80 The FDA label for DMF recommends interruption of DMF therapy in patients with ALCs <0.5×109/L, if persisting over more than six months. In real-life, DMF-associated PML occurred in patients with ALCs above the guideline threshold, suggesting that changes in specific subsets might be more important than the total ALC.80 The peripheral immune cell pattern with DMF is characterized by a relative shortage of T cells in lymphopenic patients.81 In this regard, CCR4-expressing T helper cells were identified as a prognostic biomarker for developing relevant lymphopenia in patients treated with DMF.81 Severe lymphopenia is more prevalent in older patients, and age increases the risk for DMF-associated PML.82 Hepatic complications occurred in 11% of the DMF/DMF-treated patients.76 Proteinuria, microalbuminuria, and hematuria were the front runners of renal AEs (incidence ⩾5% in any treatment group). The incidence of malignancies was 3% in both treatment groups in the overall safety population and did not exceed the expected background rate.

A recent analysis of the FDA Adverse Event Reporting System disclosed several opportunistic infections beyond PML in causal association with DMF.83 These included 11 cases of CNS and 23 systemic infections. The first group consisted of five encephalitis cases (Herpes simplex virus n = 1, Varicella-zoster virus (VZV) n = 3, Listeria monocytogenes n = 1), three meningitis cases (West Nile virus, Streptococcus pneumoniae, Cryptococcus neoformans, n = 1 each), two cases of myelitis due to VZV, and one cerebral abscess caused by Nocardia.

GI AE Aspects of Dimethyl Fumarate

DMF-related GI AEs typically emerge shortly after treatment initiation. The real-world incidence of GI AEs was 88% when self-assessed GI symptoms were analyzed.84,85 The consequences of GI AEs include dose reduction, interruption, or premature discontinuation. In real-world studies with DMF, GI AE-related dose interruption or reduction was carried out in 7–21% of the patients.22,84 Up to 19% discontinued the treatment due to GI AEs. Such a dosing change or termination is hazardous to sufficient drug levels required for disease control. From a patient’s perspective, the successful mitigation of GI AEs is critical for treatment satisfaction.86 The extent of GI AEs is mitigated by taking DMF with a meal, eg, high-fat food.87 For some patients, this strategy may be irresolvable or even an offense to their otherwise healthy lifestyle. Of note, treatment with acetylsalicylic acid alleviates flushing associated with DMF, with no adverse or beneficial effects on GI events.88

COVID-19 and DMF

Knowledge about the COVID-19 disease course and immune responses to SARS-CoV-2 vaccination in pwMS is critical to maintaining a favorable risk-benefit profile of individual DMDs. In this regard, older age, obesity, progressive MS, and a higher degree of neurological disability are associated with severe COVID-19 outcomes.89–91 A systematic review of the available literature did not disclose any higher risk of acquiring a SARS-CoV-2 infection for pwMS on DMDs.92 However, there was an increased risk for severe COVID-19 (ICU admission, mortality) with CD20-depleting therapies. No increased chance for unfavorable outcome was identified so far for DMF treatment in pwMS.91,93

DMDs approved for MS differentially affect the development of SARS-CoV-2 vaccine-induced humoral and T-cell immunity.92,94–96 In this regard, there is consistent study evidence that pwMS treated with DMF can generate robust humoral and T cell responses and are not at a higher chance of COVID-19 breakthrough disease.97–99

GI Tolerability of Fumaric Esters

MeOH release upon the esterase hydrolysis of DMF is blamed as the critical determinant for GI-tolerability. Therefore, the aforementioned lower rate of GI irritation with DRF may be related to significantly lower MeOH production in the gut. The lower molecular weight of DMF and subsequent off-target activity on proteins and receptors may be another issue for the occurrence of GI AEs. Moreover, the chemical structure of DRF is more complex and presumably interacts with fewer off-target proteins.35 Eventually, also the differences in electrophilicity and half-life may be further factors involved in the distinct extent of GI AE.35

MMF acts as an agonist of the HCAR2, which is associated with dual action. Activation of the HCAR2 can elicit signals that, on the one hand, dampen cell activation and, on the other hand, promote inflammation. Subsequent gastrointestinal inflammation and skin flushing reaction are the main off-target effects.57 Differences in DMF and DRF metabolism and systemic uptake may also play a role in the extent and duration of GI side effects.

Lower doses of MMF (95 mg twice a day) than a single dose of 240 mg DR DMF are needed to yield equivalent plasma MMF levels.100 Promising data concerning tolerability were provided by a randomized, double-blind, head-to-head, 5-week study evaluating the GI AEs of MMF 190 mg vs DMF 240 mg in healthy subjects.101

Study Evidence for Diroximel Fumarate: Trials and Real-World Evidence

The clinical efficacy of DRF in relapsing forms or relapsing-remitting MS has not been studied by phase III trials. With proven bioequivalence between DMF and DRF for the active metabolite MMF, the approvals were based on the pivotal trials of DMF.41,102 In this regard, early scientific evidence corroborates DRF’s improved GI tolerability, as shown in Table 1.

Table 1 Pivotal Trials of DRF in MS

EVOLVE-MS-1 is an ongoing, 2-year, open-label, 96-week, Phase 3 study assessing DRF safety, tolerability, and efficacy in adults with RRMS.35,102,103 For this trial, patients were either newly enrolled or continued from the EVOLVE-MS-2 study. The interim analysis as of March 2018 included 696 patients and referred to a median exposure time of 59.9 weeks (range 0.1–98.9). AEs occurred in 84.6%; the severity of the events mainly was mild (31.2%) or moderate (46.8%).102 Overall treatment discontinuation was 14.9%; in detail, 6.3% were due to AEs and <1% due to GI AEs. At week 48, the mean number of Gd+ lesions was significantly lower when compared to baseline (−77%; p < 0.0001), and the adjusted ARR was low (0.16; 95% confidence interval (CI) 0.13–0.20). Another EVOLVE-1 interim analysis concerned treatment-emergent adverse events (TEAE).35 GI TEAEs developed in 30.9% of patients; the severity was mild or moderate in the vast majority (96%). When GI AEs did occur, the time of occurrence was early, the duration was short with a median of 7.5 (range 1–87) days, and resolved (88.8%) in most patients. GI TEAEs led to 1% of patients discontinuing treatment. The third interim analysis of EVOLVE-1 comprised 1057 EVOLVE-MS-2 patients (as of September 2020) and evaluated efficacy and safety outcomes in patients continuing on DRF (DRF-rollover) or switching from DMF (DMF-rollover).103 The control group consisted of patients who previously were on either GA or IFN-β and switched to DRF. Patients continuing on FAE had significantly lower rates of new or recurrent GI AEs. After 2 years of DRF exposure, patients with prior GA, IFN, or fumarate treatment had safety outcomes consistent with previous fumarate studies. The data suggest that transition to DRF from GA, IFN, or DMF is a reasonable treatment strategy, with low rates of discontinuation due to GI AEs.

The EVOLVE-MS-2 trial compared the GI tolerability of twice-daily administered DRF 462 mg and DMF 240 mg over five weeks in patients with RRMS.104,105 This Phase III, randomized-controlled, double-blind, head-to-head comparison study used two self-administered gastrointestinal symptoms scales.104 These were the Individual Gastrointestinal Symptom and Impact Scale (IGISIS) and the Global Gastrointestinal Symptom and Impact Scale (GGISIS). The primary endpoint was the number of days with an IGISIS intensity score of two or more relative to exposure. Other endpoints included the degree of gastrointestinal symptom severity measured by IGISIS/GGISIS and the evaluation of safety/tolerability. DRF treatment led to a statistically significant reduction in the number of days with an IGISIS symptom intensity score of two or more compared to DMF (−46%). Furthermore, lower rates of GI AE events, including diarrhea, nausea, vomiting, and abdominal pain, were observed with DRF than with DMF (34.8% vs 49.0%). In addition, treatment discontinuation was less frequent with DRF compared to DMF because of AE (1.6% vs 5.6%) and GI AE (0.8% vs 4.8%). A post hoc analysis elucidated the impact of GI tolerability events on QoL in EVOLVE-MS-2.105 Patients experienced a lesser effect on daily life and work and required less frequent concomitant symptomatic medication use. Thus, the improved GI tolerability with DRF translated into clinically meaningful benefits in quality-of-life measures.

A retrospective analysis of 160 pwMS from the AcariaHealth Specialty Pharmacy Program evaluated the persistence and adherence in DRF-treated patients in real-world clinical practice.106 The median patient age was 51 (range 20–79) years, 80.6% were women, and 16.3% had been on DMF treatment. The median treatment duration was 7.6 (range 0.1–10.4) months. The estimated proportion of patients continuing on DRF treatment eight months later was 88.6% (95% CI 82.5–2.7). Overall, 3.8% discontinued DRF due to GI AEs. The frequency of days covered was 91.4% (95% CI 89.1–93.7). Among the patients that switched from DMF to DRF, 92.3% remained on DRF, and 3.8% discontinued DRF due to GI AEs.

Limitations

The tolerability of DMDs and subsequent adherence is crucial for long-term disease control. DRF, a second-generation FAE, showed an improved GI tolerability profile in the EVOLVE-MS trials and a high level of adherence in a real-world study. Therefore, the EVOLVE-MS trials provide a solid basis for corroborating the improved tolerability of DRF in comparison to DMF. However, as a limitation, the GI events are based on patient-reported outcomes and need to be amended by further observations in the clinical routine. Furthermore, the relapse rates were patient-reported at baseline in EVOLVE-MS-1 and the final analysis is eagerly awaited. Importantly, whether the efficacy and safety data are interchangeable with the experience gained for DMF in pivotal trials and in the real-world setting is unclear. While MMF is the presumed active metabolite of both substances and prior studies confirmed MMF bioequivalence of 2×240 DMF and 2×462 mg DRF, emerging preclinical data disclosed a different biodistribution and distinct biological responses of FAE derivatives.

Conclusion

Taken together, DRF is an emerging first- and second-line oral DMD option for relapsing (FDA) and relapsing-remitting MS (EMA) with an improved tolerability profile compared to the precursor DMF. To be considered as a meaningful treatment option, further studies need to clarify, on the one hand, whether patients with initial or ongoing tolerability issues with DMF will benefit from a switch to DRF. On the other hand, DRF’s long-term tolerability, efficacy, and safety profiles must be characterized as for any other newly approved DMD. Moreover, direct head-to-head comparison trials and cost-effectiveness analyses will be critical to defining the position of DRF in the emerging spectrum of DMDs for the treatment of relapsing/relapsing-remitting MS. Of note, the FDA approved MMF to treat relapsing forms of MS. Also, the introduction of DMF generics can weaken the unique selling point of DRF. In the meantime, further efforts should be directed toward preventing lymphopenia with FAE treatment and implementing risk stratification algorithms. Also, a better understanding of FAE-related opportunistic infections and the immunopathogenesis of PML is indispensable.

Abbreviations

AE, adverse event; ALC, absolute lymphocyte count; AMPK, 5’ adenosine monophosphate-activated protein kinase; AUC, area under curve; ARR, annualized relapse rate; BBB, blood–brain barrier; CNS, central nervous system; COVID-19, coronavirus infectious disease 2019; CSF, cerebrospinal fluid; CUALs, combined unique active lesions; DMF, dimethyl fumarate; DMD, disease-modifying drug; DR, delayed release; DRF, diroximel fumarate; EDSS, expanded disability status scale; FAE, fumaric acid ester; FDA, United States Food and Drug Administration; EMA, European Medicines Agency; EBV, Epstein-Barr virus; EDSS, Expanded Disability Status Scale; GA, glatiramer acetate; Gd+, gadolinium-enhancing; GGSIS, Global Gastrointestinal Symptom and Impact Scale; GM-CSF, granulocyte-macrophage colony-stimulating factor; GI, gastrointestinal; HCAR2, hydroxycarboxylic acid receptor 2; HES, 2-hydroxyethyl succinimide; IGISIS, Individual Gastrointestinal Symptom and Impact Scale; MRI, magnetic resonance imaging; IFN, interferon; IL, interleukin; LLN, lower limit of normal; MeOH, methanol; MMF, monomethyl fumarate; MS, multiple sclerosis; NEDA, no evidence of disease activity; PK, pharmacokinetics; PML, progressive multifocal leukoencephalopathy; PPMS, primary-progressive MS; pwMS people with MS; QoL, quality of life; RMS, relapsing forms of MS; RRMS, relapsing-remitting MS; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; Sirt1, NAD-dependent deacetylase sirtuin-1; SPMS, secondary-progressive MS; TEAE, treatment-emergent adverse events; TNF-α, tumor-necrosis factor-alpha.

Disclosure

LH has nothing to disclose. JS received honoraria for presentations and the assembly of educational material from Alexion, Angelini, BMS, Biogen, Boehringer, Immunic, Janssen, Merck, Novartis, Pfizer, Sandoz and Sanofi. He is a member of scientific advisory boards of Alexion, BMS, Biogen, Boehringer, Novartis, Sandoz and Sanofi.

References

1. Dobson R, Giovannoni G. Multiple sclerosis - A review. Eur J Neurol. 2019;26(1):27–40. doi:10.1111/ene.13819

2. Cree BAC, Arnold DL, Chataway J, et al. Secondary progressive multiple sclerosis: new insights. Neurology. 2021;97(8):378–388. doi:10.1212/WNL.0000000000012323

3. Scalfari A, Neuhaus A, Degenhardt A, et al. The natural history of multiple sclerosis: a geographically based study 10: relapses and long-term disability. Brain. 2010;133(Pt7):1914–1929. doi:10.1093/brain/awq118

4. McGinley MP, Goldschmidt CH, Rae-Grant AD. Diagnosis and treatment of multiple sclerosis: a review. JAMA. 2021;325(8):765–779. doi:10.1001/jama.2020.26858

5. Simonsen CS, Flemmen HO, Broch L, et al. The course of multiple sclerosis rewritten: a Norwegian population-based study on disease demographics and progression. J Neurol. 2021;268(4):1330–1341. doi:10.1007/s00415-020-10279-7

6. Lanz TV, Brewer RC, Ho PP, et al. Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM. Nature. 2022;603(7900):321–327. doi:10.1038/s41586-022-04432-7

7. Bjornevik K, Cortese M, Healy BC, et al. Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis. Science. 2022;375(6578):296–301. doi:10.1126/science.abj8222

8. Feige J, Moser T, Bieler L, Schwenker K, Hauer L, Sellner J. Vitamin D supplementation in multiple sclerosis: a critical analysis of potentials and threats. Nutrients. 2020;12(3):783. doi:10.3390/nu12030783

9. Amato MP, Derfuss T, Hemmer B, et al. Environmental modifiable risk factors for multiple sclerosis: report from the 2016 ECTRIMS focused workshop. Mult Scler. 2018;24(5):590–603. doi:10.1177/1352458516686847

10. Waubant E, Lucas R, Mowry E, et al. Environmental and genetic risk factors for MS: an integrated review. Ann Clin Transl Neurol. 2019;6(9):1905–1922. doi:10.1002/acn3.50862

11. Hauer L, Perneczky J, Sellner J. A global view of comorbidity in multiple sclerosis: a systematic review with a focus on regional differences, methodology, and clinical implications. J Neurol. 2021;268(11):4066–4077. doi:10.1007/s00415-020-10107-y

12. Rommer P, Zettl UK. Treatment options in multiple sclerosis and neuromyelitis optica spectrum disorders. Curr Pharm Des. 2022;28(6):428–436. doi:10.2174/1381612827666210920151231

13. Brown JWL, Coles A, Horakova D, et al. Association of initial disease-modifying therapy with later conversion to secondary progressive multiple sclerosis. JAMA. 2019;321(2):175–187. doi:10.1001/jama.2018.20588

14. Kaunzner UW, Al-Kawaz M, Gauthier SA. Defining disease activity and response to therapy in MS. Curr Treat Options Neurol. 2017;19(5):20. doi:10.1007/s11940-017-0454-5

15. Chitnis T, Prat A. A roadmap to precision medicine for multiple sclerosis. Mult Scler. 2020;26(5):522–532. doi:10.1177/1352458519881558

16. Inojosa H, Proschmann U, Akgun K, Ziemssen T. The need for a strategic therapeutic approach: multiple sclerosis in check. Ther Adv Chronic Dis. 2022;13:20406223211063032. doi:10.1177/20406223211063032

17. Findling O, Sellner J. Second-generation immunotherapeutics in multiple sclerosis: can we discard their precursors? Drug Discov Today. 2021;26(2):416–428. doi:10.1016/j.drudis.2020.11.022

18. Gold R, Kappos L, Arnold DL, et al. Placebo-controlled phase 3 study of oral BG-12 for relapsing multiple sclerosis. N Engl J Med. 2012;367(12):1098–1107. doi:10.1056/NEJMoa1114287

19. Fox RJ, Miller DH, Phillips JT, et al. Placebo-controlled phase 3 study of oral BG-12 or glatiramer in multiple sclerosis. N Engl J Med. 2012;367(12):1087–1097. doi:10.1056/NEJMoa1206328

20. Gold R, Arnold DL, Bar-Or A, et al. Long-term safety and efficacy of dimethyl fumarate for up to 13 years in patients with relapsing-remitting multiple sclerosis: final ENDORSE study results. Mult Scler. 2022;28(5):801–816. doi:10.1177/13524585211037909

21. Sejbaek T, Nybo M, Petersen T, Illes Z. Real-life persistence and tolerability with dimethyl fumarate. Mult Scler Relat Disord. 2018;24:42–46. doi:10.1016/j.msard.2018.05.007

22. Vollmer B, Nair KV, Sillau SH, Corboy J, Vollmer T, Alvarez E. Comparison of fingolimod and dimethyl fumarate in the treatment of multiple sclerosis: two-year experience. Mult Scler J Exp Transl Clin. 2017;3(3):2055217317725102. doi:10.1177/2055217317725102

23. Min J, Cohan S, Alvarez E, et al. Real-world characterization of dimethyl fumarate-related gastrointestinal events in multiple sclerosis: management strategies to improve persistence on treatment and patient outcomes. Neurol Ther. 2019;8(1):109–119. doi:10.1007/s40120-019-0127-2

24. Biogen. New Data at AAN 2022 Highlight Real-World Evidence from Biogen’s MS Portfolio and Emerging Research on Disease Progression; 2022. Available from: https://investors.biogen.com/news-releases/news-release-details/new-data-aan-2022-highlight-real-world-evidence-biogens-ms. Accessed November3, 2022.

25. NDA 204063 Tecfidera (dimethyl fumerate) Approved Labeling Text. Available from: https://www.accessdata.fda.gov/drugsatfda_docs/label/2016/204063s014lbl.pdf. Accessed November3, 2022.

26. Tecfidera (dimethyl fumarate) An overview of Tecfidera and why it is authorised in the EU. Available from: https://www.ema.europa.eu/en/documents/overview/tecfidera-epar-medicine-overview_en.pdf. Accessed November3, 2022.

27. Drug Approval Package: BAFIERTAM. Available from: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2020/210296Orig1s000TOC.cfm. Accessed November3, 2022.

28. Rommer PS, Sellner J. Repurposing multiple sclerosis drugs: a review of studies in neurological and psychiatric conditions. Drug Discov Today. 2019;24(7):1398–1404. doi:10.1016/j.drudis.2019.05.009

29. Kourakis S, Timpani CA, de Haan JB, Gueven N, Fischer D, Rybalka E. Dimethyl fumarate and its esters: a drug with broad clinical utility? Pharmaceuticals. 2020;13(10):306. doi:10.3390/ph13100306

30. Hoogendoorn A, Avery TD, Li J, Bursill C, Abell A, Grace PM. Emerging therapeutic applications for fumarates. Trends Pharmacol Sci. 2021;42(4):239–254. doi:10.1016/j.tips.2021.01.004

31. Mrowietz U, Barker J, Boehncke WH, et al. Clinical use of dimethyl fumarate in moderate-to-severe plaque-type psoriasis: a European expert consensus. J Eur Acad Dermatol Venereol. 2018;32(Suppl 3):3–14. doi:10.1111/jdv.15218

32. Susitaival P, Winhoven SM, Williams J, et al. An outbreak of furniture related dermatitis (‘sofa dermatitis’) in Finland and the UK: history and clinical cases. J Eur Acad Dermatol Venereol. 2010;24(4):486–489. doi:10.1111/j.1468-3083.2009.03429.x

33. Substance Infocard - Dimethyl fumarate. Available from: https://echa.europa.eu/substance-information/-/substanceinfo/100.009.863. Accessed November3, 2022.

34. Werdenberg D, Joshi R, Wolffram S, Merkle HP, Langguth P. Presystemic metabolism and intestinal absorption of antipsoriatic fumaric acid esters. Biopharm Drug Dispos. 2003;24(6):259–273. doi:10.1002/bdd.364

35. Palte MJ, Wehr A, Tawa M, et al. Improving the gastrointestinal tolerability of fumaric acid esters: early findings on gastrointestinal events with diroximel fumarate in patients with relapsing-remitting multiple sclerosis from the phase 3, open-label EVOLVE-MS-1 study. Adv Ther. 2019;36(11):3154–3165. doi:10.1007/s12325-019-01085-3

36. Correale J, Halfon MJ, Jack D, Rubstein A, Villa A. Acting centrally or peripherally: a renewed interest in the central nervous system penetration of disease-modifying drugs in multiple sclerosis. Mult Scler Relat Disord. 2021;56:103264. doi:10.1016/j.msard.2021.103264

37. Edwards KR, Kamath A, Button J, et al. A pharmacokinetic and biomarker study of delayed-release dimethyl fumarate in subjects with secondary progressive multiple sclerosis: evaluation of cerebrospinal fluid penetration and the effects on exploratory biomarkers. Mult Scler Relat Disord. 2021;51:102861. doi:10.1016/j.msard.2021.102861

38. Cada DJ, Levien TL, Baker DE. Dimethyl fumarate. Hosp Pharm. 2013;48(8):668–679. doi:10.1310/hpj4808-668

39. Deeks ED. Dimethyl fumarate: a review in relapsing-remitting MS. Drugs. 2016;76(2):243–254. doi:10.1007/s40265-015-0528-1

40. Linker RA, Haghikia A. Dimethyl fumarate in multiple sclerosis: latest developments, evidence and place in therapy. Ther Adv Chronic Dis. 2016;7(4):198–207. doi:10.1177/2040622316653307

41. Center for drug evaluation and research - Dimethyl fumarate. Available from: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2019/211855Orig1s000SumR.pdf. Accessed November3, 2022.

42. Jonasson E, Sejbaek T. Diroximel fumarate in the treatment of multiple sclerosis. Neurodegener Dis Manag. 2020;10(5):267–276. doi:10.2217/nmt-2020-0025

43. Narapureddy B, Dubey D. Clinical evaluation of dimethyl fumarate for the treatment of relapsing-remitting multiple sclerosis: efficacy, safety, patient experience and adherence. Patient Prefer Adherence. 2019;13:1655–1666. doi:10.2147/PPA.S187529

44. Phillips JT, Agrella S, Fox RJ. Dimethyl fumarate: a review of efficacy and practical management strategies for common adverse events in patients with multiple sclerosis. Int J MS Care. 2017;19(2):74–83. doi:10.7224/1537-2073.2015-086

45. Mayer L, Fink MK, Sammarco C, Laing L. Management strategies to facilitate optimal outcomes for patients treated with delayed-release dimethyl fumarate. Drug Saf. 2018;41(4):347–356. doi:10.1007/s40264-017-0621-3

46. Kuchimanchi M, Bockbrader H, Dolphin N, et al. Development of a population pharmacokinetic model for the diroximel fumarate metabolites monomethyl fumarate and 2-hydroxyethyl succinimide following oral administration of diroximel fumarate in healthy participants and patients with multiple sclerosis. Neurol Ther. 2022;11(1):353–371. doi:10.1007/s40120-021-00316-6

47. Mills EA, Ogrodnik MA, Plave A, Mao-Draayer Y. Emerging understanding of the mechanism of action for dimethyl fumarate in the treatment of multiple sclerosis. Front Neurol. 2018;9:5. doi:10.3389/fneur.2018.00005

48. Rommer PS, Milo R, Han MH, et al. Immunological aspects of approved MS therapeutics. Front Immunol. 2019;10:1564. doi:10.3389/fimmu.2019.01564

49. Linker RA, Lee DH, Ryan S, et al. Fumaric acid esters exert neuroprotective effects in neuroinflammation via activation of the Nrf2 antioxidant pathway. Brain. 2011;134(Pt3):678–692. doi:10.1093/brain/awq386

50. Gopal S, Mikulskis A, Gold R, Fox RJ, Dawson KT, Amaravadi L. Evidence of activation of the Nrf2 pathway in multiple sclerosis patients treated with delayed-release dimethyl fumarate in the Phase 3 DEFINE and CONFIRM studies. Mult Scler. 2017;23(14):1875–1883. doi:10.1177/1352458517690617

51. Brennan MS, Matos MF, Richter KE, Li B, Scannevin RH. The NRF2 transcriptional target, OSGIN1, contributes to monomethyl fumarate-mediated cytoprotection in human astrocytes. Sci Rep. 2017;7:42054. doi:10.1038/srep42054

52. Ma Q. Role of nrf2 in oxidative stress and toxicity. Annu Rev Pharmacol Toxicol. 2013;53:401–426. doi:10.1146/annurev-pharmtox-011112-140320

53. Siegel D, Gustafson DL, Dehn DL, et al. NAD(P)H:quinone oxidoreductase 1: role as a superoxide scavenger. Mol Pharmacol. 2004;65(5):1238–1247. doi:10.1124/mol.65.5.1238

54. da Fonseca RR, Johnson WE, O’Brien SJ, Vasconcelos V, Antunes A. Molecular evolution and the role of oxidative stress in the expansion and functional diversification of cytosolic glutathione transferases. BMC Evol Biol. 2010;10:281. doi:10.1186/1471-2148-10-281

55. Offermanns S, Schwaninger M. Nutritional or pharmacological activation of HCA(2) ameliorates neuroinflammation. Trends Mol Med. 2015;21(4):245–255. doi:10.1016/j.molmed.2015.02.002

56. Shi Y, Lai X, Ye L, et al. Activated niacin receptor HCA2 inhibits chemoattractant-mediated macrophage migration via Gbetagamma/PKC/ERK1/2 pathway and heterologous receptor desensitization. Sci Rep. 2017;7:42279. doi:10.1038/srep42279

57. Parodi B, Sanna A, Cedola A, Uccelli A, Kerlero de Rosbo N. Hydroxycarboxylic acid receptor 2, a pleiotropically linked receptor for the multiple sclerosis drug, monomethyl fumarate. possible implications for the inflammatory response. Front Immunol. 2021;12:655212. doi:10.3389/fimmu.2021.655212

58. Longbrake EE, Mao-Draayer Y, Cascione M, et al. Dimethyl fumarate treatment shifts the immune environment toward an anti-inflammatory cell profile while maintaining protective humoral immunity. Mult Scler. 2021;27(6):883–894. doi:10.1177/1352458520937282

59. Longbrake EE, Cantoni C, Chahin S, Cignarella F, Cross AH, Piccio L. Dimethyl fumarate induces changes in B- and T-lymphocyte function independent of the effects on absolute lymphocyte count. Mult Scler. 2018;24(6):728–738. doi:10.1177/1352458517707069

60. Liebmann M, Korn L, Janoschka C, et al. Dimethyl fumarate treatment restrains the antioxidative capacity of T cells to control autoimmunity. Brain. 2021;144(10):3126–3141. doi:10.1093/brain/awab307

61. Holm Hansen R, Hojsgaard Chow H, Christensen JR, Sellebjerg F, von Essen MR. Dimethyl fumarate therapy reduces memory T cells and the CNS migration potential in patients with multiple sclerosis. Mult Scler Relat Disord. 2020;37:101451. doi:10.1016/j.msard.2019.101451

62. Smith MD, Martin KA, Calabresi PA, Bhargava P. Dimethyl fumarate alters B-cell memory and cytokine production in MS patients. Ann Clin Transl Neurol. 2017;4(5):351–355. doi:10.1002/acn3.411

63. Montes Diaz G, Fraussen J, Van Wijmeersch B, Hupperts R, Somers V. Dimethyl fumarate induces a persistent change in the composition of the innate and adaptive immune system in multiple sclerosis patients. Sci Rep. 2018;8(1):8194. doi:10.1038/s41598-018-26519-w

64. Li R, Rezk A, Ghadiri M, et al. Dimethyl fumarate treatment mediates an anti-inflammatory shift in B cell subsets of patients with multiple sclerosis. J Immunol. 2017;198(2):691–698. doi:10.4049/jimmunol.1601649

65. Talbot J, Hojsgaard Chow H, Holm Hansen R, von Essen MR, Sellebjerg F. Immunological effects of dimethyl fumarate treatment in blood and CSF of patients with primary progressive MS. J Neuroimmunol. 2021;361:577756. doi:10.1016/j.jneuroim.2021.577756

66. Hoglund RA, Polak J, Vartdal F, Holmoy T, Lossius A. B-cell composition in the blood and cerebrospinal fluid of multiple sclerosis patients treated with dimethyl fumarate. Mult Scler Relat Disord. 2018;26:90–95. doi:10.1016/j.msard.2018.08.032

67. Wipke BT, Hoepner R, Strassburger-Krogias K, et al. Different fumaric acid esters elicit distinct pharmacologic responses. Neurol Neuroimmunol Neuroinflamm. 2021;8(2):e950. doi:10.1212/NXI.0000000000000950

68. Breuer J, Herich S, Schneider-Hohendorf T, et al. Dual action by fumaric acid esters synergistically reduces adhesion to human endothelium. Mult Scler. 2018;24(14):1871–1882. doi:10.1177/1352458517735189

69. Lim JL, van der Pol SM, Di Dio F, et al. Protective effects of monomethyl fumarate at the inflamed blood-brain barrier. Microvasc Res. 2016;105:61–69. doi:10.1016/j.mvr.2015.12.003

70. Galloway DA, Williams JB, Moore CS. Effects of fumarates on inflammatory human astrocyte responses and oligodendrocyte differentiation. Ann Clin Transl Neurol. 2017;4(6):381–391. doi:10.1002/acn3.414

71. Paraiso HC, Kuo PC, Curfman ET, et al. Dimethyl fumarate attenuates reactive microglia and long-term memory deficits following systemic immune challenge. J Neuroinflammation. 2018;15(1):100. doi:10.1186/s12974-018-1125-5

72. Zinger N, Ponath G, Sweeney E, et al. Dimethyl fumarate reduces inflammation in chronic active multiple sclerosis lesions. Neurol Neuroimmunol Neuroinflamm. 2022;9(2):e1138. doi:10.1212/NXI.0000000000001138

73. Yadav SK, Ito N, Soin D, Ito K, Dhib-Jalbut S. Dimethyl fumarate suppresses demyelination and axonal loss through reduction in pro-inflammatory macrophage-induced reactive astrocytes and complement C3 deposition. J Clin Med. 2021;10(4):857. doi:10