Targeting Demyelination and Virtual Hypoxia with High-Dose Biotin As a Treatment for Progressive Multiple Sclerosis

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Neuropharmacology 110 (2016) 644e653

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Neuropharmacology

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Invited review Targeting demyelination and virtual hypoxia with high-dose biotin as a treatment for progressive multiple sclerosis

* Fred eric Sedel a, , Delphine Bernard a, Donald M. Mock b, c, Ayman Tourbah d

a MedDay Pharmaceuticals, ICM-Brain and Spine Institute-IPEPs, Groupe Hospitalier Pitie Salp^etriere, 47 Boulevard de l'Hopital, 75013 Paris, France b Department of Biochemistry & Molecular Biology, University of Arkansas for Medical Sciences, 4301 W Markham Street, Little Rock, AR 72205, USA c Department of Pediatrics, University of Arkansas for Medical Sciences, 4301 W Markham Street, Little Rock, AR 72205, USA d Department of Neurology and FacultedeMedecine de Reims, CHU de Reims, URCA, 45 Rue Cognacq Jay, 51092 Reims Cedex, France

article info abstract

Article history: Progressive multiple sclerosis (MS) is a severely disabling neurological condition, and an effective Received 7 April 2015 treatment is urgently needed. Recently, high-dose biotin has emerged as a promising therapy for affected Received in revised form individuals. Initial clinical data have shown that daily doses of biotin of up to 300 mg can improve 24 July 2015 objective measures of MS-related disability. In this article, we review the biology of biotin and explore Accepted 18 August 2015 the properties of this ubiquitous coenzyme that may explain the encouraging responses seen in patients Available online 5 September 2015 with progressive MS. The gradual worsening of neurological disability in patients with progressive MS is caused by progressive axonal loss or damage. The triggers for axonal loss in MS likely include both in- Keywords: ﬂ Multiple sclerosis ammatory demyelination of the myelin sheath and primary neurodegeneration caused by a state of Biotin virtual hypoxia within the neuron. Accordingly, targeting both these pathological processes could be Promyelinogenic agent effective in the treatment of progressive MS. Biotin is an essential co-factor for ﬁve carboxylases involved Virtual hypoxia in fatty acid synthesis and energy production. We hypothesize that high-dose biotin is exerting a therapeutic effect in patients with progressive MS through two different and complementary mechanisms: by promoting axonal remyelination by enhancing myelin production and by reducing axonal hypoxia through enhanced energy production. This article is part of the Special Issue entitled ‘Oligodendrocytes in Health and Disease’. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction ...... 645 2. Etiology and pathogenesis of MS ...... 645 3. High-dose biotin as a treatment for MS ...... 646 4. The role of biotin in brain chemistry ...... 646 5. Hypothesized modes of action of high-dose biotin in the treatment of progressive MS ...... 647 5.1. High-dose biotin as a promoter of myelination through enhanced myelin formation in oligodendrocytes ...... 647 5.2. High-dose biotin in the reversal of virtual hypoxia ...... 648 6. Feasibility of high-dose biotin as a therapy for progressive MS ...... 649 7. Overview of ongoing studies with MD1003 and future directions ...... 650 Author contributions ...... 650

Abbreviations: ACC, acetyl-CoA carboxylase; AMN, adrenomyeloneuropathy; BBB, bloodebrain barrier; BTBGD, biotin-thiamine responsive basal ganglia disease; EDSS, Expanded Disability Status Scale; HLCS, holocarboxylase synthetase; LINGO1, leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 1; MCC, 3-methylcrotonyl-CoA carboxylase; MS, multiple sclerosis; OPC, oligodendrocyte precursor cell; PC, pyruvate carboxylase; PCC, propionyl-CoA carboxylase; PPMS, primary progressive multiple sclerosis; RRMS, relapsing-remitting multiple sclerosis; SPMS, secondary progressive multiple sclerosis; TCA, tricarboxylic acid; THTR2, thiamine transporter 2; TW25, time to walk 25 feet. * Corresponding author. E-mail addresses: [email protected] (F. Sedel), [email protected] (D. Bernard), [email protected] (D.M. Mock), [email protected] (A. Tourbah).

http://dx.doi.org/10.1016/j.neuropharm.2015.08.028 0028-3908/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). F. Sedel et al. / Neuropharmacology 110 (2016) 644e653 645

Conflict of interest ...... 650 Acknowledgments ...... 651 References...... 651

1. Introduction animal models of autoimmune encephalomyelitis results in the formation of new myelin sheaths (Mi et al., 2007). Phase I data from Multiple sclerosis (MS) affects an estimated 2.3 million people healthy volunteers and patients with MS indicate that inhibition of worldwide (Browne et al., 2014). MS is the most common disabling LINGO-1 with the monoclonal antibody BIIB033 (Biogen Idec) ap- neurological disease of young adults, with first symptoms typically pears to be well tolerated and a phase II trial in patients with RRMS manifesting between 20 and 40 years of age (Browne et al., 2014). is ongoing (EUDRACT #: 2011-006262-40). First results have been The condition is more common in women than men, and preva- presented at the American Academy of Neurology. In the per- lence generally increases with latitude (Simpson et al., 2011). In protocol population, the anti-LINGO-1 group showed significantly Europe, the highest prevalence of MS is seen in Nordic countries improved optic nerve conduction latency vs placebo at Week 32. No and the British Isles (Kingwell et al., 2013). A diagnosis of MS places statistical difference was observed in the ITT population or for the a high burden on the affected individual, both economically and secondary endpoints including visual acuity at low contrast with respect to MS-associated disability (Pike et al., 2012). (Cadavid et al., 2015). In addition, a recombinant form of a human In the majority (85%) of cases, patients experience an initial IgM (rHIgM22; Acorda Therapeutics) that binds to myelin and the phase of relapsing-remitting neurological dysfunction (RRMS), surface of oligodendrocytes has been shown to promote remyeli- which typically evolves into a secondary progressive disease at a nation in murine models of MS and other demyelinating diseases later point in the clinical course (SPMS) (Confavreux et al., 2000). (Mitsunaga et al., 2002; Warrington et al., 2007). The safety and Once MS is in the progressive phase, individuals experience a preliminary efficacy of rHIgM22 are currently being investigated in gradual worsening of neurological disability leading to problems patients with all forms of MS in a phase I trial (NCT01803867). with vision, walking, balance, incontinence, cognitive changes, fa- In this paper, we review recently published data that suggest tigue, and pain (Gibson and Frank, 2002). Primary progressive MS that high-dose biotin is an effective treatment for patients with (PPMS), characterized by disease progression from onset, is less progressive MS and propose two distinct (and complementary) common, affecting 10e15% of patients (Confavreux et al., 2000; mechanisms of action to explain this efficacy. Koch et al., 2009). Despite these different initial clinical pheno- types, the time to reach certain disability milestones and the ages at 2. Etiology and pathogenesis of MS which the milestones are reached are similar for all patients with progressive MS (Confavreux and Vukusic, 2006). Despite decades of research into the biology of MS, the etiology Treatment options for MS remain inadequate. Most currently of this progressive neurological disease remains incompletely un- approved therapies for MS target inflammatory processes and aim derstood. Multiple sclerosis is classically considered to be an to reduce the frequency of exacerbations in patients with RRMS. autoimmune demyelination disorder in which activated T-cells These include b-interferons, dimethyl fumarate, glatiramer acetate, migrate across the blood brain barrier and attack the insulating fingolimod, teriflunomide, and natalizumab (Wingerchuk and myelin sheath that surrounds the axons (Compston and Coles, Carter, 2014). b-interferon (Trojano et al., 2007), teriflunomide 2008). This leads to a progressive demyelination of the axons that (Confavreux et al., 2014), fingolimod (Kappos et al., 2010), alem- ultimately culminates in degeneration of the denuded neurons. tuzumab (Coles et al., 2012), natalizumab (Polman et al., 2006), and Indeed, there is abundant evidence supporting the inflammatory mitoxantrone (Martinelli et al., 2013) may also delay or reduce the component of MS. Acute inflammatory lesions are characterized by risk of disability progression. However, currently available treat- the presence of infiltrating activated T-cells (both CD4þ and CD8þ) ments have little to no efficacy in patients with progressive MS, (Denic et al., 2013; Traugott et al., 1983), and the frequency of particularly if superimposed relapses are absent, and there is no axonal damage is related to the degree of inflammation within le- pharmacological therapy capable of arresting or reversing MS- sions (Trapp et al., 1998). Inflammation is initially transient and related disability (Comi, 2013). Encouraging results have recently remyelination of axons occurs (Olsen and Akirav, 2015), which may been reported with the use of nonmyeloablative hematopoietic explain the relapsing-remitting nature of the signs and symptoms stem cell transplantation in patients with RRMS (Burt et al., 2015). of RRMS. If demyelination persists, denuded axons are vulnerable to This technique achieved a striking improvement in disability (as damage and these immune-mediated attacks lead to axonal injury measured by the Expanded Disability Status Scale; EDSS) in and neuronal death (Brugarolas and Popko, 2014; Podbielska et al., approximately half of the patients at 2-year follow-up; moreover, 2013). 80% of patients remained relapse free at 4-year follow-up. However, However, autoimmunity alone fails to fully explain the patho- no benefit was seen in patients who had progressive MS at the time physiology of MS. In particular, axonal degradation can occur in of transplant. For these individuals, an effective treatment remains normal-appearing white matter that has no evidence of myelin loss a significant unmet medical need. (Bjartmar et al., 2001; Lovas et al., 2000)orinflammation (Bitsch Two promyelinogenic agents are currently in development et al., 2000; Dutta and Trapp, 2011), and axonal degradation can as treatment for MS. LINGO-1 (leucine-rich repeat and occur at all stages of MS disease progression (De Stefano et al., immunoglobulin-like domain-containing nogo receptor-interacting 2001; Levin et al., 2014; Trapp et al., 1998). An alternative theory protein 1) inhibits the differentiation of oligodendrocyte precursor proposes that MS is primarily a neurodegenerative disorder in cells (OPCs) and consequently inhibits myelination (Jepson et al., which the inflammatory responses observed in patients with MS 2012). Upregulation of this receptor is seen in many neurological arise in response to highly antigenic components that are released disorders, including in OPCs found within areas of demyelination during degradation of the neurons (Bruck, 2005; Stys et al., 2012; from patients with MS (Mi et al., 2013). Inhibition of LINGO-1 in Trapp and Nave, 2008). Regardless of whether neurodegeneration 646 F. Sedel et al. / Neuropharmacology 110 (2016) 644e653 occurs secondary to demyelination or whether primary neuro- of MS because no therapeutic options have yet been validated to degeneration is the trigger for inflammatory demyelination, axonal treat the progressive (primary or secondary) forms of the disease loss or damage is undeniably the cause for the progressive neuro- and its sequelae-related symptoms. In each patient treated with logical disability seen in MS (Bjartmar et al., 2000, 2003; De Stefano MD1003, there was a delay between the onset of treatment and the et al., 1998; Dutta and Trapp, 2007). The exact mechanisms un- onset of objective clinical improvement; delays ranged from 2 to 8 derlying neurodegeneration remain poorly understood but are months. This tantalizing observation suggests that MD1003 is believed to include a combination of energy imbalance, CD8þ cells, triggering some form of slowly progressive repair and furthermore glutamate, nitric oxide, and loss of trophic interaction with oligo- may act as a disease-modifying therapy in patients with progres- dendrocytes (Bitsch et al., 2000; Bjartmar et al., 2003; Bruck, 2005). sive disease. Both the compassionate use open-label study and the placebo-controlled trial after 12 months' follow-up are however of 3. High-dose biotin as a treatment for MS insufficient duration to answer this question. Data from the 12- month extension phase of this trial (during which all participants MD1003 (MedDay Pharmaceuticals, Paris, France) is an oral receive high-dose biotin) are expected in January 2016 and will be formulation of high-dose pharmaceutical-grade biotin currently in important in assessing the impact of the drug on long-term clinical development as a treatment for progressive MS and adre- disability progression. nomyeloneuropathy (AMN). The daily dose of MD1003 currently being investigated in phase III trials (300 mg biotin) is 10,000-fold 4. The role of biotin in brain chemistry higher than the Adequate Intake (AI) e the daily dietary intake level of biotin that is considered sufficient for the maintenance of health Biotin (vitamin H) is a ubiquitous B-complex vitamin that acts as by the Food and Nutrition Board of the Institute of Medicine is an essential coenzyme for five essential carboxylases: pyruvate 30 mg/day for adults (IOM Standing Committee on the Scientific carboxylase (PC) 3-methylcrotonyl-CoA carboxylase (MCC), Evaluation of Dietary Reference Intervals, 1998). At 300 mg per propionyl-CoA carboxylase (PCC), and the two isoforms of acetyl- day, biotin is considered by regulators to be an active pharmaceu- CoA carboxylase (ACC1 and ACC2) (Tong, 2013; Zempleni et al., tical agent. Therefore, the efficacy and safety of high-dose biotin as 2009). Biotin is covalently bound to specific lysine residues on a therapeutic option in MS require careful investigation prior to apocarboxylases; the biotinylation reaction is catalyzed by hol- being made available for clinical use. ocarboxylase synthetase (HLCS), an ATP-dependent enzyme enco- Clinical data from the first patients treated with MD1003 have ded by the HLCS gene (Chapman-Smith and Cronan, 1999). Once recently been published (Sedel et al., 2015). This open-label pilot bound, biotin acts as a transporter of an activated carboxyl moiety trial investigated daily administration of high-dose biotin in a variety of carboxylation reactions involved in energy meta- (100e300 mg/day) in 23 patients with primary or secondary pro- bolism, fatty acid synthesis, and amino acid catabolism (Tong, gressive MS who received MD1003 for a mean duration of 9.2 2013). The central role of biotin in intermediary metabolism is months (range 2e36 months). Over 90% of the patients (21/23) likely responsible for the observation that biotin is required by all exhibited some degree of qualitative or quantitative clinical tissues of the body; however, biotin appears to play a particularly improvement following MD1003 therapy including reduced MS- important role in brain chemistry. In contrast to liver, kidney, and related disability. A beneficial effect was seen in all forms of pro- brain, biotin is present in relatively small amounts in many tissues gressive MS. Improvements in walking distance, EDSS values, and (a fact on which many (strept-)avidin-biotin histochemical tech- time to walk 25 feet (TW25) were observed in patients with niques rely); however, studies in the rat have demonstrated that prominent spinal cord involvement, and improvements in visual significant levels of covalently bound biotin are found in the brain, acuity and the progressive reappearance of visual evoked potentials particularly in the cerebellar motor system and the brainstem with normal latencies were recorded in patients with visual auditory system (McKay et al., 2004). Biotin is actively transported impairment related to optic nerve injury (Sedel et al., 2015). The across the human bloodebrain barrier (BBB) predominantly via the þ promising efficacy observed in this pilot study suggests that high- Na -dependent multivitamin transporter (SLC5A6/SMVT; Uchida dose biotin is effective in both reversing disease progression and et al. (2015)). This transporter has an estimated half-saturation reducing chronic disability. The study has important methodolog- concentration (Km) of roughly 100 mM in rats (Spector and Mock, ical limitations including the small number of patients, the small 1987), 35 mM in mice (Park and Sinko, 2005) and 20 mM in rab- number of centers, the lack of a placebo or control group, the bits (Spector and Mock, 1988a). By extrapolation of data obtained heterogeneity of patients enrolled, the heterogeneity of evaluation with smaller doses (Mock and Mock, 1997; Zempleni et al., 2001), criteria, and the fact that treating and assessing physicians were the assuming dose proportionality, high doses of biotin in the 300 mg/ same. Results of a double-blind, placebo-controlled, multisite study day range would result in plasma concentrations of biotin of about were presented in April at the American Academy of Neurology 5 mM, which theoretically would not be saturating the biotin (Tourbah et al., 2015a). 154 patients were randomized, (103 transporter (Spector and Johanson, 2007). Consistent with these received 300 mg of biotin per day and 51 received a placebo). Pa- data, biotin concentration was shown to increase from about 0.1 to tients had secondary or primary progressive MS with EDSS be- 6 nmol/g of brain tissue in juvenile rats given increasing oral doses tween 4.5 and 7 and evidence of EDSS progression within the past of biotin corresponding to about 0.3 mge3 mg/kg body weight in two years. Treatment duration was 48 weeks. The primary endpoint humans, the highest dose being closest to the human therapeutic was the proportion of patients who improved at month 9 and dose given in progressive MS (Sawamura et al., 2007). Biotin con- confirmed at month 12, defined as decreased EDSS (by at least 1 centration also increased in other tissues evaluated, namely skel- point for EDSS 5.5 and 0.5 point for EDSS 6) or improved TW25 etal muscle, heart, kidney, lung, spleen and testis. Within animal of at least 20%. A significant proportion of biotin-treated patients brain, limited data indicate that biotin is localized to the oligo- achieved the primary endpoint versus none of 51 placebo-treated dendrocytes (LeVine and Macklin, 1988; Wang and Pevsner, 1999) patients (p ¼ 0.0051). The primary endpoint was confirmed by a and in some neurons (McKay et al., 2004). Interestingly, levels of significant decrease in the mean change in EDSS and the Clinical covalently bound biotin and the expression of HCLS, biotin- Global Impression Scale assessed by the investigator and subject in dependent carboxylases, and genes involved in biotin transport the biotin group (Tourbah et al., 2015b). We are hopeful that these and biotin recycling are maintained in the brain during periods of results ultimately represent a major breakthrough in the treatment biotin deficiency at the expense of other tissues such as liver F. Sedel et al. / Neuropharmacology 110 (2016) 644e653 647

(Chiang and Mistry, 1974; Pacheco-Alvarez et al., 2004; Rodriguez- Melendez et al., 2001; Sander et al., 1982). Further evidence for the vital role of biotin in neuron function comes from the observation that neurological dysfunction is prominent in patients with biotin deﬁciency and inborn errors of biotin metabolism (Wolf, 2011; Wolf and Feldman, 1982). Biotin has a vital role in the treatment of the orphan neurological disease biotin-thiamine responsive basal ganglia disease (BTBGD). This autosomal recessive disorder is caused by inherited mutations in the gene encoding a transmembrane thiamine transporter that was originally termed solute carrier family 19 member 3 [SLC19A3] but now more commonly known as thiamine transporter 2 (THTR2) (Said et al., 2004; Zeng et al., 2005). The symptoms of BTBGD are severe and are characterized by recurrent subacute onset of en- cephalopathy that manifest as confusion, seizures, dysarthria, ataxia, dystonia, supranuclear facial palsy and even coma (Ozand et al., 1998; Tabarki et al., 2013). Despite the fact that THTR2 is not a biotin transporter in studies in hepatic, intestinal, or neuronal cell lines (Subramanian et al., 2006), the condition is effectively and rapidly treated with high-dose biotin (5e10 mg/kg/day) (Bindu et al., 2009; Ozand et al., 1998). Fig. 1. Biotin activates myelin formation in oligodendrocytes through its role as a cofactor for ACC. ACC is the rate limiting and committing step for the synthesis of 5. Hypothesized modes of action of high-dose biotin in the malonyl-CoA, the building block for fatty acids synthesis. ACC activity is detected at a high level in oligodendrocytes in the brain. It is hypothesized that high doses of biotin treatment of progressive MS may result in increased fatty acid synthesis required for myelin repair.

We hypothesize that high-dose biotin exerts its therapeutic effect in progressive MS through two primary mechanisms: 1) pro- catalyzed by ACC2 increase, carnitine palmitoyltransferase 1 (CPT1) motion of remyelination through enhanced myelin formation in is inhibited and the rate of fatty acid transport into mitochondria oligodendrocytes, and 2) enhancement of brain energy production, decreases leading to reduced fatty acid beta oxidation (Foster, 2012; thereby protecting demyelinated axons from degradation. McGarry et al., 1978). Therefore, high-dose biotin may be acting as a promyelinogenic agent through its role as a cofactor for ACC1 and 5.1. High-dose biotin as a promoter of myelination through ACC2. enhanced myelin formation in oligodendrocytes The normalization of visual evoked potential latency observed in one MS patient with chronic visual loss after 9 months' treat- Through its role as an essential cofactor for ACC1 and ACC2, ment with MD1003 is consistent with myelin repair (Sedel et al., high-dose biotin may be promoting remyelination by increasing 2015). A progressive decrease in the choline/creatine ratio was the availability of the fundamental building blocks required for observed with magnetic resonance spectroscopy in a second pa- myelin membrane synthesis. ACC1 and ACC2 are encoded by two tient with prominent optic nerve involvement; the ratio normal- distinct genes (ACACA and ACACB) and have distinct subcellular ized completely after 9 months of treatment with MD1003. locations and distinct metabolic roles. ACC1 catalyzes the rate Elevations of choline, the core component of the hydrophilic head limiting, committed step in fatty acid biosynthesis: the cytosolic of membrane phospholipids, are commonly observed in MS pla- synthesis of malonyl-CoA from acetyl-CoA (Fig. 1)(Foster, 2012; ques and are thought to result from the breakdown of the phos- Tong, 2013). A role for ACC in promoting myelination is supported pholipid membrane that occurs during inflammation, gliosis, and by several observations. Within the CNS, ACC appears to be pri- demyelination (Arnold et al., 1992; Narayana, 2005). We speculate marily expressed in oligodendrocytes, the cells specialized in that normalization of the choline/creatine ratio overtime after myelin synthesis (Chakraborty and Ledeen, 2003; Tansey and treatment with MD1003 reflects progressive myelin repair Cammer, 1988), not surprisingly, a significant proportion of cyto- accompanied by a decrease in free choline release from mem- solic ACC is detectable in purified myelin (Chakraborty and Ledeen, branes. In addition, the delay in onset of clinical benefit(2e8 2003) and the activity of ACC in oligodendrocytes in rats is highest months) seen in MS patients treated with MD1003 is consistent during the neonatal myelinating period (DeWille and Horrocks, with remodeling of the myelin sheath. Remodeling of the myelin 1992; Tansey et al., 1988). The relative contribution of ACC1 and sheath is believed to occur at a relatively slow rate. MRI studies in ACC2 to ACC activity in the brain is not known. However, in human patients with acute gadolinium (Gd)-enhancing lesions suggest adult brain (RNA pooled from multiple donors), ACC1 RNA was that the process of remyelination takes an average of 7 months found to be approximately twice as abundant as ACC2 RNA (Castle (Chen et al., 2008). et al., 2009). Furthermore, levels of murine ACC are significantly Remyelination of denuded axons is a logical and attractive reduced in the sciatic nerve of the trembler mouse model of pe- strategy for the treatment of progressive MS (Franklin and Ffrench- ripheral nervous system dysmyelination compared with normal Constant, 2008; Zawadzka and Franklin, 2007). Extensive regen- mice (Salles et al., 2003). Finally, malonyl-CoA, the product of the erative remyelination occurs in some patients with early MS but is reaction catalyzed by ACC, is used as the two-carbon building block typically reduced as the disease progresses (Goldschmidt et al., for fatty acid synthesis by the brain microsomal fatty acid elon- 2009; Miron et al., 2011; Prineas et al., 1993). Remyelination has gating system for deposition in myelin (DeWille and Horrocks, been documented in both active and inactive MS lesions (Miron 1992). Malonyl-CoA also coordinates the balance between fatty et al., 2011), and remyelination strategies stimulate functional re- acid synthesis and fatty acid oxidation. As cytosolic levels of covery in animal models of MS (Bai et al., 2012; Deshmukh et al., malonyl-CoA increase, the rate of fatty acid synthesis increases; as 2013; Duncan et al., 2009). The process underlying remyelination peri-mitochondrial levels of malonyl-CoA from the reaction is complex and is thought to involve the maturation and migration 648 F. Sedel et al. / Neuropharmacology 110 (2016) 644e653 of OPCs and numerous promoting and inhibitory factors (Munzel membrane potential at the nodes of Ranvier. Restoration of the and Williams, 2013). However, by increasing the supply of the resting membrane potential is mainly achieved by the ATP- þ þ þ þ two carbon units required for fatty acid elongation (the funda- dependent Na /K pump (Na /K ATPase) (Krishnan et al., 2009). mental building block of myelin), high-dose biotin acting through Loss of the insulating myelin sheath causes the loss of saltatory ACC is an attractive candidate for a novel promyelinogenic agent. conduction and thereby increases the energy required for nerve þ propagation. In denuded axons, voltage-gated Na channels are redistributed along the length of the axon (Fig. 2). This compen- 5.2. High-dose biotin in the reversal of virtual hypoxia satory mechanism reflects a switch from saltatory to continuous conduction (Craner et al., 2004; Levin et al., 2014). This switch is As well as promoting remyelination through its role as a cofactor accompanied by an increase in the number of mitochondria in the for ACC, high-dose biotin may also be targeting cellular energy demyelinated axons as a result of the neuron's attempt to supply levels. Several lines of evidence suggest that cellular energy the increased energy demand required to maintain the intracel- deprivation secondary to demyelination is responsible for the lular/extracellular ion gradients (Witte et al., 2009). However, evi- progressive irreversible neuronal degeneration observed in pro- dence suggests that, in MS patients, axonal ATP production is gressive MS (Luessi et al., 2012; Stys et al., 2012). In the normal, simultaneously compromised due to defects in their neuronal myelinated neuron, the nerve impulse is conducted along the mitochondria (Dutta et al., 2006; Mahad et al., 2009; Su et al., length of the axon in discreet jumps spreading along the chain of þ 2009). The resulting imbalance between energy supply and en- nodes of Ranvier, which contain high densities of voltage-gated Na ‘ ’ þ ergy demand causes a state of virtual hypoxia which may be the and K channels (Fig. 2)(Rasband and Trimmer, 2001). This pro- trigger for neuron degeneration (Luessi et al., 2012; Trapp and Stys, þ þ cess, termed saltatory conduction, not only increases the speed at 2009). If insufficient ATP is available for Na /K ATPase to restore which the nerve impulse is propagated compared with nerve im- the membrane potential, the neuron will enter a state of depolar- þ pulses in unmyelinated axons, but also reduces neuronal energy ization characterized by a further influx of Na . This state can þ þ expenditure because ATP is only needed to restore the resting activate the Na /Ca2 exchanger in reverse mode, leading to an þ þ increase in intra-axonal Ca2 as Na ions are removed from the þ neuron (Trapp and Stys, 2009). Excessive levels of Ca2 in the þ neuron can activate a number of Ca2 -mediated degenerative pathways (Trapp and Stys, 2009). In chronic MS lesions, axonal þ þ levels of Na /K ATPase are decreased, which may exacerbate the state of membrane depolarization (Young et al., 2008). We hypothesize that treatment with high-dose biotin reverses this state of virtual hypoxia through its role as a cofactor for PC, MCC, and PCC. These three enzymes are central to aerobic energy production and generate intermediates for the tricarboxylic acid (TCA) cycle (Fig. 3)(Tong, 2013). All three of these enzymes are known to be expressed in astrocytes and neurons (Ballhausen et al., 2009; Hassel, 2000). PC catalyzes the conversion of pyruvate to

Fig. 2. Increased axonal energy demand as a result from axon demyelination. Nerve impulses in myelinated axons propagate between the nodes of Ranvier. Depolarization Fig. 3. Biotin increases ATP production in axonal mitochondria. Biotin is a co-enzyme þ of the membrane is triggered by the transient opening of voltage-gated Na ion for PC, PCC, and MCC, three carboxylases involved in the metabolism of pyruvate and þ channels. Repolarization is achieved through voltage-gated K ion channels, which amino acids. These three enzymes are expressed in neurons and their actions ulti- restore electrical conditions, followed by restoration of the ionic distribution by the mately result in the production of key intermediates of the TCA cycle including þ þ ATP-dependent Na /K ATPase pump. ATP consumption is greatly increased in oxaloacetate, acetyl-CoA and succinyl-CoA. The TCA cycle produces one ATP molecule demyelinated axons due to redistribution of ion channels along the length of the for every acetyl-CoA used. We hypothesize that high doses of biotin may result in denuded axon. increased ATP production in demyelinating neurons. F. Sedel et al. / Neuropharmacology 110 (2016) 644e653 649 oxaloacetate, thus serving an essential anaplerotic role by replen- through control of gene expression by modification of chromatin ishing the 4-carbon “backbone” of the TCA cycle. PCC generates structure remains speculative. methylmalonyl-CoA from propionyl-CoA, which is then converted to succinyl-CoA by methylmalonyl-CoA mutase. MCC plays a role in the metabolism of leucine and ultimately leads to production of acetyl-CoA. Thus, these three biotin-dependent carboxylases feed the TCA cycle at three different entry points (oxaloacetate, succi- 6. Feasibility of high-dose biotin as a therapy for progressive nate, and acetyl-CoA) and could be expected to increase the levels MS of cellular ATP at the rate of one molecule of acetyl-CoA used by the TCA cycle producing one molecule of ATP. Indeed, nutritional biotin Several characteristics of high-dose biotin make it an attractive deficiency and biotin deficiency induced by knocking out the therapeutic option in progressive MS. Biotin demonstrates high recycling enzyme biotinidase cause severe ATP depletion (~100%) bioavailability (Zempleni and Mock, 1999) and is able to (Hernandez-Vazquez et al., 2012, 2013; Velazquez-Arellano et al., cross the blood brain barrier (Spector and Mock, 1988b). The ability 2011). By increasing the available intraneuronal pool of ATP, high- to dose orally will greatly facilitate treatment for individuals with dose biotin may reduce demyelinated neural dysfunction and the MS and consequently a high level of treatment compliance can be adverse effects of hypoxia (Lazzarino et al., 2010; Trapp and Stys, expected. While the body of preclinical toxicology data for high- 2009). dose biotin is limited, the available data indicates that only mar- In progressive MS, the proportion of mutant mitochondria in- ginal adverse effects are observed in animals treated with biotin creases through clonal expansion of mitochondrial DNA deletions doses in the hundreds of milligrams per kilogram of body weight (Campbell et al., 2011). As a consequence, some neurons still range (Hathcock, 2015). Adverse events in patients with progres- possess functional mitochondria, while others have damaged sive MS treated with MD1003 (100e300 mg/day) were infrequent; mitochondria or a mixture of functional and damaged mitochon- occurring in three of 23 (13%) patients treated in the compassionate dria (heteroplasmy; Mahad et al. (2015)). In this latter case, the use open-label trial (Sedel et al., 2015). Two patients experienced remaining pool of mitochondria with functional respiratory chain transient diarrhea and one patient died from cardiac failure 36 enzymes may not be sufficient to compensate for the increase in months after treatment onset. This patient exhibited mild aortic energy demand occurring in chronically demyelinated axons. It valvulopathy with dilatation of the ascending aorta together with a could be anticipated that only axons with some remaining healthy first-degree atrio-ventricular block which were found 18 months mitochondria would benefit from the treatment with high-dose after treatment onset; no relation could be established between biotin. treatment onset, mild cardiac abnormalities and death. Whether A similar hypothesis has been the basis for testing idebenone in these abnormalities existed before treatment is unknown. progressive MS (Spindler et al., 2009) (NCT00950248 and A significant teratogenic effect on rabbit fetuses during the NCT01854359). Increasing cellular energy production is also period of organogenesis was observed in pregnant rabbits treated ® thought to be the mechanism by which idebenone (Catena , San- with high-dose biotin (30 mg/kg/day; approximately twice the thera Pharmaceuticals), a synthetic analog of coenzyme Q10 exerts equivalent human dose currently under investigation based on the its therapeutic activity. This compound is a substrate for reduction surface area conversion table; unpublished data, MedDay Phar- by two NAD(P)H:quinone oxidoreductases (NQO1 and NQO2) maceuticals). These effects included a higher incidence of malro- (Haefeli et al., 2011). The reduced form of idebenone is believed to tated paw (11.8%), domed head (4.1%), hydrocephaly (4.1%) and pass electrons into the mitochondrial respiratory chain triggering liquid content in the cranial cavity (4.7%) compared with control an increase in cellular ATP levels. Of note, experiments in the rabbits and those treated with 15 mg/kg/day biotin. Consequently, chronic experimental autoimmune encephalomyelitis (EAE) mouse high-dose biotin may not be suitable for administration to women model failed to demonstrate any effect of idebenone in preventing with MS who are pregnant or considering pregnancy. Fingolimod, or attenuating disability (Fiebiger et al., 2013). teriflunomide and interferon beta-1a are also contraindicated in Several other mechanisms by which biotin may be exerting a pregnant women (although pregnancy may start while the patient therapeutic effect exist. Biotin is a key transcriptional regulator of is receiving interferon beta-1a) (Biogen Idec Ltd, 2014; Novartis several genes involved in glucose and lipid metabolism. In vitro and Europharm Ltd, 2011; Sanofi-Aventis Group, 2013). Control of in vivo studies have shown that elevated biotin levels can upregu- pregnancy is recommended prior to each infusion of natalizumab late the expression of hepatic and pancreatic glucokinase, insulin, and discontinuation of treatment should be considered if a women and the insulin receptor, and downregulate hepatic phosphoenol- becomes pregnant while taking this drug (Biogen Idec Ltd., 2011). pyruvate carboxykinase (Chauhan and Dakshinamurti, 1991; Overall, high-dose biotin appears to be a practical and well- Dokusova and Krivoruchenko, 1972; Leon-Del-Rio, 2005; Marshall tolerated therapeutic option in patients with progressive MS, et al., 1980; Pacheco-Alvarez et al., 2002; Romero-Navarro et al., particularly when compared with currently approved treatments 1999). Interestingly, biotin also regulates the expression of two of for MS that have some significant safety and tolerability concerns. the carboxylases for which it acts as a cofactor (PC and ACC1), and Patients receiving fingolimod require close monitoring due to the of HCLS (Pacheco-Alvarez et al., 2004). The mechanism by which risk of serious cardiovascular complications, infections, and mac- biotin regulates gene expression remains elusive. Biotin was ular edema (Wingerchuk and Carter, 2014). Monoclonal antibodies initially thought not to be a natural modifier of histones (Healy such as natalizumab and alemtuzumab require parenteral admin- et al., 2009), However, biotinylation sites are known to be present istration which can be inconvenient for patients, and these agents on histones H2A, H3, and H4 (Zempleni et al., 2012b) and several have potentially serious side effects, such as the risk of developing studies have demonstrated that biotinylation of histones by HCLS progressive multifocal leukoencephalopathy with natalizumab induces chromatin remodeling that can regulate gene expression (Bloomgren et al., 2012) and idiopathic thrombocytopenic purpura (Filenko et al., 2011; Gralla et al., 2008; Kuroishi et al., 2011; and antibody-mediated immune diseases for alemtuzumab (Cuker Wijeratne et al., 2010). However, more recent data suggests that et al., 2011). While nonmyeloablative hematopoietic stem cell HCLS itself may interact directly with chromatin as part of a multi- transplantation appears to be a promising treatment option for protein gene regulating complex (Liu and Zempleni, 2014; patients with RRMS (Burt et al., 2015), this procedure will not be Zempleni et al., 2012a). Currently, an effect of biotin on MS suitable for all patients and is associated with significant risks. 650 F. Sedel et al. / Neuropharmacology 110 (2016) 644e653

The efficacy and safety of MD1003 is currently under investigation in three multicenter phase III placebo-controlled trials in patients with progressive MS involving either the spinal cord or chronic visual loss after optic neuritis, and in patients with AMN (Table 1). The study in patients with spinal progressive MS repre- sents a particularly ambitious evaluation of high-dose biotin as a therapy for MS, because the patients recruited to this trial have relatively severe disease: PPMS or SPMS fulfilling revised McDonald criteria (Polman et al., 2011) and Lublin criteria (Lublin and Reingold, 1996) with clinical evidence of spastic paraparesis and significant disability (EDSS scores 4.5e7). Furthermore, this is the first trial of an investigational MS therapy that has chosen a reduction in MS-related disability as the primary efficacy endpoint of the trial, as measured by one of two validated clinical assessment scales (the EDSS and the TW25). Results from all three trials are expected in 2015e2016. Several in vivo studies that will further investigate the pharmacodynamic effects of high-dose biotin are also currently underway. The potential mechanism of action of high-dose biotin in progressive MS is still partly speculative and there is a striking lack of pre-clinical data on the exact mechanism of action. Some important gaps in knowledge remain to be elucidated experimentally:

Fig. 4. Overview of putative mechanism of action of high-dose biotin in multiple (1) Are biotin or malonyl-CoA limiting factors for remyelination sclerosis. Targets of biotin are: (a) ACC: acetyl-CoA carboxylase (b) PC: pyruvate in chronic MS lesions? This might be addressed using in vitro carboxylase, (c) PCC: propionyl-CoA carboxylase, (d) MCC: methylcrotonyl-CoA models such as OPCs. carboxylase. Activation of ACC may increase myelin synthesis in oligodendrocytes and remyelination whereas activation of PC, PCC, and MCC may lead to increase ATP (2) Does biotin have an impact on myelination or remyelination production in neurons (and astrocytes). in vivo? This could be assessed in relevant animal models with and without inﬂammation such as experimental autoimmune encephalomyelitis, cuprizone or toxin-induced 7. Overview of ongoing studies with MD1003 and future demyelination. directions (3) Does high-dose biotin increase ATP production by mitochondria in chronic MS lesions? This might be assessed in In summary, we hypothesize that high-dose biotin treatment relevant in vitro and in vivo models. is targeting two key pathophysiological mechanisms in progressive MS: (1) by triggering myelin synthesis by oligodendrocytes and (2) by replenishing ATP in hypoxic neurons (Fig. 4). Targeting both demyelination and neurodegeneration is likely to be Author contributions important in the treatment of progressive MS. Although several other therapeutics that target one of these processes are currently All authors contributed to the writing of this paper. under investigation in patients with MS, high-dose biotin might be simultaneously preventing the hypoxia-driven neuro- Conﬂict of interest degeneration of neurons and promoting the remyelination of axons. Authors FS and DB are employees of MedDay Pharmaceuticals;

Table 1 Ongoing phase IIB/III randomized, double-blind, placebo-controlled trials of MD1003 (300 mg/day).

Study Primary objective Primary efﬁcacy endpoint Study design/population Anticipated EudraCT no. name completion date

MS-SPI To demonstrate the superiority of Proportion of patients with decreased 12 months' treatment followed by Jan 2016 2013-002113-35 MD1003 at 300 mg/day over placebo in EDSSa or 20% improvement in TW25 at 12-month open-label extension phase clinical improvement of patients with month 9 (conﬁrmed at month 12) (105 patients randomized 2:1) spinal progressive MS compared with best baseline values MS-ON To demonstrate the superiority of Mean change in best corrected visual 6 months' treatment followed by Jan 2016 2013-002112-27 MD1003 at 300 mg/day over placebo in acuity (logMAR) at 100% contrast between 12-month open-label extension phase the visual improvement of patients baseline and month 6 of the diseased eyeb (105 patients randomized 2:1) suffering from chronic visual loss after optic neuritis related to MS MD1003- To demonstrate the superiority of Mean change of 2MWT time between 12 months' treatment followed by 2016 2014-000698-38 AMN MD1003 at 300 mg/day over placebo in month 12 and baseline 12-month open-label extension phase the clinical improvement of patients (60 patients randomized 2:1) with AMN

2MWT, 2 min walking test; AMN, adrenomyeloneuropathy; EDSS, Expanded Disability Status Scale; EudraCT, European Clinical Trials Database; MS, multiple sclerosis; TW25, Timed 25-Foot Walk. a Defined as a decrease of 0.5 point if initial EDSS from 6 to 7 and a decrease of 1 point if initial EDSS from 4.5 to 5.5. b Defined as the eye with the worst visual acuity (<5/10) at baseline and with evidence of worsening during the past three years. F. Sedel et al. / Neuropharmacology 110 (2016) 644e653 651 author DM has served as a consultant for MedDay Pharmaceuticals; Magnetization transfer ratio evolution with demyelination and remyelination in e author AT has received in the last year, consulting and lecturing multiple sclerosis lesions. Ann. Neurol. 63, 254 262. Chiang, G.S., Mistry, S.P., 1974. 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