Targeting protein homeostasis in sporadic inclusion body myositis · MYOPATHY Targeting protein homeostasis in sporadic inclusion body myositis Mhoriam Ahmed,1,2* Pedro M. Machado,1*

R E S EARCH ART I C L E

MYOPATHY

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Targeting protein homeostasis in sporadic inclusionbody myositisMhoriam Ahmed,1,2* Pedro M. Machado,1* Adrian Miller,1,2* Charlotte Spicer,1,2* Laura Herbelin,3

Jianghua He,4 Janelle Noel,4 Yunxia Wang,3 April L. McVey,3 Mamatha Pasnoor,3

Philip Gallagher,5 Jeffrey Statland,3 Ching-Hua Lu,1,2 Bernadett Kalmar,1,2 Stefen Brady,1

Huma Sethi,6 George Samandouras,6 Matt Parton,1 Janice L. Holton,1 Anne Weston,7

Lucy Collinson,7 J. Paul Taylor,8 Giampietro Schiavo,1,2 Michael G. Hanna,1† Richard J. Barohn,3†

Mazen M. Dimachkie,3†‡ Linda Greensmith1,2†‡

Sporadic inclusion body myositis (sIBM) is the commonest severe myopathy in patients more than 50 years ofage. Previous therapeutic trials have targeted the inflammatory features of sIBM but all have failed. Becauseprotein dyshomeostasis may also play a role in sIBM, we tested the effects of targeting this feature of thedisease. Using rat myoblast cultures, we found that up-regulation of the heat shock response with arimoclomolreduced key pathological markers of sIBM in vitro. Furthermore, in mutant valosin-containing protein (VCP)mice, which develop an inclusion body myopathy, treatment with arimoclomol ameliorated disease pathologyand improved muscle function. We therefore evaluated arimoclomol in an investigator-led, randomized, double-blind, placebo-controlled, proof-of-concept trial in sIBM patients and showed that arimoclomol was safe and welltolerated. Although arimoclomol improved some IBM-like pathology in the mutant VCP mouse, we did not seestatistically significant evidence of efficacy in the proof-of-concept patient trial.

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INTRODUCTION

Sporadic inclusion body myositis (sIBM) is the commonest idiopathicinflammatory myopathy (IIM) occurring in patients more than 50 yearsof age (1–7), but no treatment is available. The prevalence of sIBMdiffers between different populations and ranges between 1 and 71 in-dividuals per million (8–13). sIBM is distinguished from other IIMs byearly asymmetric finger flexor and knee extensor weakness, whichleads to loss of hand function and propensity to fall, and resistance toimmunosuppressive therapy. However, any skeletal muscle may beaffected including esophageal and pharyngeal muscles. Late-stage diseaseis characterized by significantmorbidity, includingmotor disability, swal-lowing failure, and poor quality of life. Death in IBM is related to mal-nutrition, cachexia, aspiration, and respiratory failure (1–7).

Although the etiology of sIBM remains uncertain, the varied path-ological findings observed in patient muscles have driven a number ofhypotheses, including viral infection, accumulation of toxic proteins,autoimmune attack, myonuclear degeneration, endoplasmic reticulum(ER) stress, and impairment of autophagy and proteasome function(14–18). Muscle biopsies from sIBM patients typically show several

1Medical Research Council (MRC) Centre for Neuromuscular Diseases, University Col-lege London (UCL) Institute of Neurology, Queen Square, London WC1N 3BG, UK.2Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute ofNeurology, Queen Square, London WC1N 3BG, UK. 3Department of Neurology, Universityof Kansas Medical Center, 3901 Rainbow Boulevard, Mail Stop 2012, Kansas City, KS 66160,USA. 4Department of Biostatistics, University of Kansas Medical Center, Kansas City, KS66160, USA. 5Department of Health, Sport, and Exercise Science, The University of Kansas,Lawrence, KS 66045–7567, USA. 6Victor Horsley Department of Neurosurgery, NationalHospital for Neurology and Neurosurgery, UCL Hospitals, Queen Square, LondonWC1N 3BG, UK. 7The Francis Crick Institute, Lincoln’s Inn Fields Laboratory, 44 Lincoln’sInn Fields, London WC2A 3LY, UK. 8Department of Cell & Molecular Biology, St. Jude Chil-dren’s Research Hospital, Memphis, TN 38105–3678, USA.*These authors contributed equally to this work.†Co-senior authors.‡Corresponding author. E-mail: [email protected] (L.G.); [email protected](M.M.D.)

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pathological features broadly described as either inflammatory or de-generative. Inflammatory features include endomysial infiltration bymononuclear cells, which surround and invade nonnecrotic musclefibers and overexpression of major histocompatibility complex classI (MHC-I), which is not constitutively expressed by skeletal muscle.The degenerative features of sIBM include the formation of rimmedvacuoles and inclusion bodies containing a range of proteins includingb-amyloid precursor protein (b-APP), heat shock proteins (HSPs),phosphorylated tau (p-Tau), p62, and the cytoplasmic mislocalizationof RNA-binding proteins including transactive response DNA bindingprotein 43 (TDP-43), heterogeneous nuclear ribonucleoprotein A1(hnRNPA1), and hnRNPA2B1 (19–22).

Despite lack of any experimental evidence, protein accumulationcould theoretically play a role in triggering inflammation in IBMmuscle. For example, amyloid oligomers can induce key componentsof sIBM pathology, at least in vitro, so that b-APP overexpression andconsequent accumulation impairs innervation by cocultured neurons(23), causes mitochondrial dysfunction (24), and in vivo, results in calciumdyshomeostasis (25). Conversely, inflammation may induce the onset ofdegenerative features through exposure to inflammatory cytokines andincreased nitric oxide production (26). In muscle biopsies of sIBM pa-tients, expression of APP mRNA correlates with inflammation andexpression of mRNAs of chemokines and interferon-g (IFN-g). Further-more, unlike muscles of inflammatory myopathies, in sIBM muscles,inflammatory mediators colocalize with b-amyloid deposits withinmyofibers. In vitro, exposure of human myotubes to interleukin-1b (IL-1b) causes up-regulation of APP with subsequent aggregationof b-amyloid. These findings suggest that there is a link between theproduction of proinflammatory mediators and b-amyloid–associateddegeneration in sIBM muscle (27).

Previous clinical trials in sIBM have only tested agents directed atthe inflammatory component of pathology, and all were ineffective(3, 4, 28–31). Whether the degenerative aspect of IBM is primary to

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the pathogenesis or not, it very likely plays a role in the deleteriouseffects in muscle and may be a potential therapeutic target. Proteinhomeostasis (proteostasis) is essential for normal cellular functions(32), and under conditions of cellular stress, proteins can become un-folded or misfolded, leading to their aberrant aggregation (33). Proteinmisfolding is normally controlled by endogenous chaperone proteins(34) that prevent aberrant protein-protein interactions and promotecorrect protein folding. HSPs are a family of ubiquitously expressedprotein chaperones, which are up-regulated after stress-induced acti-vation of the heat shock response, an endogenous cytoprotectivemechanism. Because the heat shock response declines with advancedage (35), up-regulation of the heat shock response in disorders inwhich there is evidence of protein mishandling, such as sIBM, maybe of therapeutic value.

Arimoclomol is a pharmacological agent that co-induces the heatshock response (36) by prolonging the activation of heat shockfactor 1 (HSF-1) (32), the main transcription factor that controlsHSP expression, thus augmenting HSP levels (36–39). Arimoclomolonly acts on cells under stress in which HSF-1 is already activatedand does not induce the heat shock response in unstressed cells(40), thereby avoiding the side effects associated with widespread,nontargeted heat shock response activation. Indeed, previous exami-nation of the effects of arimoclomol in mice has shown that treatmentwith arimoclomol has no detectable effect on nonstressed cells andthere was no increase in HSP expression in any tissue studied (includ-ing spleen, heart, brain, spinal cord, nerve, and muscle).

Here, we took a three-step translational approach to test whetherheat shock response augmentation may be a potential therapy forsIBM. We first established robust in vitro models representing the de-generative and inflammatory components of sIBM. Primary rodentmyotube cultures were induced to overexpress human b-APP bytransfection of the wild-type human gene, or cultures were exposedto the inflammatory cytokines IL-1b and tumor necrosis factor–a(TNFa). In these models, muscle cells developed several key IBM-likepathological features, which were used as outcome measures to exam-ine the therapeutic potential of arimoclomol in IBM. Having estab-lished the beneficial effects of arimoclomol in vitro, we next testedits ability to ameliorate sIBM-like pathology in mice overexpressingmutant valosin-containing protein (VCP; p97 in mouse). These trans-genic mice model the degenerative disorder multisystem proteinopa-thy (MSP), which has a phenotypic spectrum that includes inclusionbody myopathy, Paget’s disease of the bone, and frontotemporal de-mentia (41). Although these mice are a model of the genetic ratherthan sporadic form of IBM, they do develop progressive muscle weak-ness and pathological hallmarks of inclusion body myopathy. Inaddition, we also undertook a randomized, double-blind, placebo-controlled, proof-of-concept trial of arimoclomol versus placebo forthe treatment of sIBM in human patients. The aim of this trial wasto evaluate the safety and tolerability of arimoclomol and to gatherexploratory efficacy data.

RESULTS

Arimoclomol improves sIBM-like pathology inmyoblast culturesWe first established accurate in vitro models of sIBM-like pathology inwhich primary rat myoblast cultures were either transfected with

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b-APP or exposed to inflammatory cytokines. For both cell culturemodels, we first undertook a series of titration experiments to deter-mine the optimal conditions; the effects of different DNA and Lipo-fectamine ratios or different cytokine concentrations on b-APP andAb40 and Ab42 expression were examined at different stages in vitro.

Transfection of primary rat myocytes with full-length humanb-APP increased the expression of b-APP and its toxic cleavage pro-duct amyloid b-42 (Ab42) and led to the formation of cytoplasmicinclusion bodies, a hallmark of sIBM, containing b-APP, ubiquitin,and TDP-43 among other proteins (Fig. 1, A to C, and fig. S1, A toE). Treatment with arimoclomol after transfection significantly re-duced the formation of cytoplasmic inclusions (P < 0.01; n = 3; Fig.1C). Additionally, b-APP overexpression increased the expressionof MHC-I, a characteristic feature of sIBM muscle, and the pro-apoptotic protein caspase-3 (fig. S1, F and G). Exposure of culturedrat myocytes to IL-1b and TNFa was also associated with elevatedexpression of b-APP and Ab42, although in the absence of inclusionbody formation. Increased MHC-I expression was also reproduced inthe cytokine-exposed cultures (fig. S1G). Similar results were obtainedwith IFN-g.

Overexpression of b-APP or exposure to inflammatory mediatorsresulted in cytoplasmic mislocalization of the C terminus of TDP-43from the nucleus (Fig. 1, D to G), which is a key pathological featureof sIBM patient muscle. This mislocalization was almost completelyprevented by treatment with arimoclomol (Fig. 1, D to G). Thus, al-though only 1.4 ± 0.6% of rat myocytes showed mislocalized TDP-43in control cultures, overexpression of b-APP induced TDP-43 mis-localization in 52.2 ± 5.3% of myocytes, which was reduced to only2.4 ± 0.53% after treatment with arimoclomol for 24 hours after trans-fection (P < 0.001; n = 3; Fig. 1, D and E).

Treatment with inflammatory mediators also induced TDP-43mislocalization in rat myocytes, which was reduced by arimoclomoltreatment, from 47.4 ± 2.8% to 24.8 ± 2.0% in myocyte cultures exposedto IL-1b (P < 0.05) and from 59.9 ± 9.0% to 29.8 ± 2.1% in TNFa-exposed cultures (P < 0.05; Fig. 1, F and G). In addition, exposureto inflammatory cytokines also increased overall TDP-43 expression,and this was also reduced by treatment with arimoclomol (Fig. 1H).The N terminus of TDP-43 remains in its nuclear location under allconditions studied (fig. S1H).

Although the extent of TDP-43 mislocalization induced by b-APPoverexpression and exposure to inflammatory mediators was similar,arimoclomol was more effective in preventing TDP-43 mislocalizationin the b-APP overexpression model. This difference most likely re-flects the underlying cause of TDP-43 mislocalization in the twomodels, where stress induced by protein misfolding is the key patho-logical trigger in b-APP overexpressing cells. Arimoclomol directly re-duces the levels of misfolded proteins and cell stress by up-regulatingthe heat shock response. The mode of action of arimoclomol in theinflammatory models is less direct and may involve a general reductionin cell stress and consequently nuclear factor kB (NF-kB) activation, asshown in Fig. 1 (I to K).

b-APP overexpression also activated the NF-kB cascade, most likelyas part of a nonspecific stress response, as shown by nuclear trans-location of the NF-kB subunit p65, observed in 43.1 ± 6.2% of myocytesin b-APP–transfected cultures compared to 8.8 ± 1.2% in controlcultures (P < 0.01; Fig. 1, I and K). Treatment with arimoclomol re-duced this to 23.6 ± 4.2% (P < 0.05). Unsurprisingly, exposure to inflam-matory cytokines also activated the NF-kB cascade, from 4.6 ± 1.3%




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Fig. 1. b-APP overexpression or exposureto inflammatory mediators induces sIBM-like pathology in cultured rat myocytesthat is abrogated by arimoclomol. (A andB) Cytoplasmic inclusion bodies (white ar-rows) in rat myocytes transfected withfull-length human b-APP that are immuno-reactive for b-APP and ubiquitin, and TDP-43

and ubiquitin. (C) The number of rat myocytes containing ubiquitinated in-clusion bodies as a percentage of the total number of myocytes present[n = 3; *P < 0.05, one-way analysis of variance (ANOVA)]. Ari, arimoclomol.(D and E) Expression of TDP-43 (green) after empty vector (EV) or b-APPtransfection and arimoclomol treatment and the number of rat myocyteswith cytoplasmic mislocalization of TDP-43 (n = 3; *P < 0.001, unpaired t test).(F and G) TDP-43 expression (green) after exposure to inflammatory media-tors and arimoclomol and quantification of TDP-43mislocalization in cytokine-treated cultures (n = 3; *P < 0.05, unpaired t test). (H) Western blot analysisof TDP-43 expression in rat myocyte cultures exposed to cytokines in thepresence and absence of arimoclomol. (I and J) Images show theexpressionof

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NF-kB subunit p65 (green) in b-APP–transfected cultures [4′,6-diamidino-2-
henylindole (DAPI)–labeled nuclei in blue] and cultures exposed to cytokines in the presence and absence of arimoclomol. (K) The number of rat myocytesith the nuclear subunit of NF-kB, p65 as a percentage of the total number ofmyocytes present (n= 3; *P< 0.05, one-way ANOVA). Error bars represent SEM.
Scale bars, 10 mm (A and B); 20 mm (D, F, I, and J).



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and 9.1 ± 1.4% in control cultures to 88.1 ± 6.3% and 38.9 ± 2.9% (P <0.001) in IL-1b–treated and TNFa-treated cultures, respectively (Fig.1, J and K). Arimoclomol reduced this effect to 41.2 ± 6.8% (P < 0.05)and 24.3 ± 3.1% (P < 0.05), respectively.

Arimoclomol augments HSP70 expression and improvescell survivalOverexpression of b-APP and exposure to inflammatory mediatorsresulted in a significant increase in HSP70 expression, which increasedby 3.7- and 2.3-fold of control after b-APP overexpression and treat-ment with IL-1b, respectively (P < 0.05; n = 3). Arimoclomol furtherincreased HSP70 expression by 2.4- and 2.1-fold in b-APP and IL-1b–treated cultures, respectively (Fig. 2, A to C). There was no differencein HSP70 levels in untreated controls and empty vector–treatedcultures (fig. S1I). b-APP overexpression and exposure to inflammatorymediators also resulted in significant cell death, which was reduced bytreatment with arimoclomol (P < 0.05; n = 3; Fig. 2, D and E).

Arimoclomol attenuates ER stress and protein mishandlingb-APP overexpression and exposure to inflammatory mediatorsincreased ER stress in cultured rat myocytes, as determined by mea-surement of intracellular calcium ions [Ca2+], where reduced cytosolic[Ca2+] was indicative of ER stress. In b-APP–overexpressing cells ex-posed to the ER stressor thapsigargin, cytosolic [Ca2+] was significantlylower than in controls (P < 0.05; n = 3; Fig. 3A). b-APP overexpres-sion also induced up-regulation of expression of the ER stress medi-ator CHOP (Fig. 3B). This b-APP–induced disruption in ER calciumion handling was prevented by treatment with arimoclomol, wherecytosolic [Ca2+] was significantly increased to the levels observed incontrol cultures (P < 0.05; n = 3; Fig. 3A), accompanied by a decreasein CHOP expression (Fig. 3B).

Exposure to the inflammatory cytokines IL-1b and TNFa also re-sulted in a significant reduction in cytosolic [Ca2+] (Fig. 3A) and anincrease in the expression of CHOP (Fig. 3B). Treatment with arimo-clomol restored cytosolic [Ca2+] to control levels and reduced the ex-pression of CHOP (P < 0.05; n = 3; Fig. 3, A and B).

Aberrant expression of the shuttle protein p62 (sequestosome 1)was observed in b-APP–overexpressing cells (Fig. 3C). This proteintargets misfolded proteins for degradation and was observed in cyto-plasmic aggregates in cultured rat myocytes, suggesting altered pro-tein handling. Assessment of proteasome function demonstrated asignificant decrease in proteasome activity after b-APP overexpres-sion (P < 0.05; n = 3); however, this was not improved by treatmentwith arimoclomol (Fig. 3E). Autophagic degradation was assessedby examination of the autophagosome marker LC3-II, which increasedby 1.28-fold in b-APP–overexpressing cells compared to controls. How-ever, this was reduced to 0.74-fold the expression level of controls inarimoclomol-treated cultures, indicating a reduction in autophagicdegradation, possibly due to a reduction in misfolded proteins (P <0.05; n = 3; Fig. 3, D and F).

Arimoclomol ameliorates IBM-like pathology in mutantVCP miceArimoclomol has clear beneficial effects in cellular models of sIBM.We therefore next tested the efficacy of arimoclomol in vivo in mutantVCP mice. These mice model MSP and develop characteristic hall-marks of inclusion body myopathy (IBM). Mutant VCP mice weretreated with arimoclomol from 4 to 14 months of age. By 14 months,

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there was a significant decrease in muscle force in mutant VCP mice(P < 0.0001; n = 10), as assessed by longitudinal analysis of gripstrength as well as acute in vivo physiological assessment of isometricmuscle force (Fig. 4, A to C). The loss of muscle force observed at14 months was prevented by treatment with arimoclomol. Gripstrength was significantly greater in treated mutant VCP mice than

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Fig. 2. ArimoclomolaugmentsHSP70expressionand improves survivalof culturedratmyocytes. (A) HSP70 expression (red) in cultured ratmyocytes
after empty vector (EV) transfection (left-hand panel), b-APP overexpression(middle panel), or b-APP overexpression plus treatment with arimoclomol(right-hand panel). (B) HSP70 expression (red) in cultured rat myocytesthat were untreated (control; left-hand panel) or exposed to IL-1b alone(middle panel) or IL-1b plus arimoclomol (right-hand panel). (C) Westernblot analysis of HSP70 expression in rat myocyte cultures exposed to in-flammatory mediators or after b-APP overexpression in the presence or ab-sence of arimoclomol. (D) Cytotoxicity in b-APP–overexpressing rat myocytecultures as a percentage of that in control cultures (n = 3; *P < 0.02, one-wayANOVA), as assessed by a lactate dehydrogenase (LDH) assay. (E) Cyto-toxicity after exposure to inflammatory mediators in the presence or ab-sence of arimoclomol (n = 3; *P < 0.05, one-way ANOVA), as assessed byanMTT [3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide] assay.Error bars represent SEM. Scale bars, 10 mm.



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untreated mice (P = 0.0175; n = 10; Fig. 4, A to C). There was also animprovement in the toe-spreading reflex observed in arimoclomol-treatedmutant VCP mice (fig. S2, A and B). However, arimoclomol had noeffect on the abnormal increase in body weight observed in mutantVCP mice (fig. S2C). Histopathological analysis of hindlimb musclesof mutant VCP mice confirmed the presence of significant IBM-likepathology including myofiber atrophy, increased endomysial connec-tive tissue, and the presence of degenerating fibers and vacuoles as wellas hypertrophic fibers (Fig. 4D). All of these pathological findings werereduced in arimoclomol-treated mutant VCP mice (Fig. 4, D to I, andmovie S1). In addition, muscles of mutant VCP mice showed evidenceof inflammatory cell infiltration as well as up-regulation of MHC-I mol-ecules and the phosphorylated formof theNF-kB substrate, inhibitor ofnuclear factor kB a (IkBa) (Fig. 4, E to G). In contrast, there was noevidence of inflammatory infiltrates in muscles of arimoclomol-treatedmutant VCP mice, and there was a reduction in expression of MHC-Iand IkBa (Fig. 4, E to G). In particular, treatment with arimoclomolmarkedly improvedmitochondrialmorphology and reduced sarcoplas-mic reticulum swelling and vacuole number in mutant VCP mousemuscle fibers compared to controls (Fig. 4H andmovies S2 to S4). Fur-thermore, the macrophage infiltration observed in mutant VCPmouse muscle was also reduced in arimoclomol-treated mice (fig. S2,

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D to F). Arimoclomol-treatedmutant VCPmousemuscles also showeda decrease in muscle fiber diameter compared to untreated mutantmouse muscle (P < 0.0001; n = 3; Fig. 4I). These beneficial effects ofarimoclomol were accompanied by an increase in HSP70 expression inmutant VCPmouse muscles (Fig. 4J), a decrease in the expression of ubi-quitin (Fig. 5, A toC), and a reduction in cytoplasmicmislocalization ofTDP-43 (P = 0.01; n = 3; Fig. 5, D to F).

Clinical trial of safety and tolerability of arimoclomol for thetreatment of sIBMWe next undertook a randomized, double-blind, placebo-controlled,proof-of-concept trial of arimoclomol versus placebo for the treatmentof sIBM. The primary aim of the trial was to evaluate the safety andtolerability of arimoclomol, but exploratory efficacy data were alsogathered. Sixteen sIBM patients were randomized to receive arimoclo-mol and eight to receive placebo. The duration of the treatment periodwas 4 months, and the follow-up continued for a further 8 monthsafter treatment ceased, with an overall trial duration of 12 months.At 4 months (end of the treatment phase), all sIBM patients werestill participating in the trial. At 8 months, two patients had discon-tinued the study (because of travel difficulties), but one of these pa-tients returned for the final assessment at 12 months (fig. S3). Baseline

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sion of the autophagic protein LC3-II (red) in b-APP–transfected desmin-positive rat myocytes after empty vector transfection (left-hand panel),

by the ER stressor thapsigargin (an indicator of ER stress) in b-APP–overexpressingrat myocyte cultures and in cell cultures exposed to inflammatory mediators(n = 3; *P < 0.05, one-way ANOVA). (B) Expression of the ER stress mediatorCHOP determined by Western blot analysis in rat myocyte cultures over-expressing b-APP or exposed to inflammatory mediators, in the presenceor absence of arimoclomol. GAPDH, glyceraldehyde-3-phosphate de-hydrogenase. (C) The images show the presence of a cytoplasmicaggregate immunoreactive for p62 (red; white arrow) in a desmin-positiverat myocyte after b-APP transfection. (D) The images show the expres-

b-APP overexpression (middle panel), or b-APP overexpression plustreatment with arimoclomol (right-hand panel). The white arrow indi-cates punctate LC3-II staining of autophagosomes. (E) The chymotrypsin-like proteasome activity assessed 48 hours after b-APP transfection inthe presence or absence of arimoclomol (n = 3; *P < 0.05, one-wayANOVA). (F) The expression of the autophagosome marker LC3-II inb-APP–transfected rat myocytes with or without arimoclomol (n = 3; *P <0.05, one-way ANOVA). EV, empty vector. Error bars represent SEM. Scalebars, 10 mm.




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Fig. 4. Arimoclomol treatment improvesmusclestrength,musclecontract-ile characteristics, and IBM-like pathology in mutant VCP mice. (A) The

black arrowheads, hypertrophic fibers). (E) H&E staining showing clear in-flammatory cell infiltration in mutant VCP tibialis anterior muscle. (F and G)

change in grip strength in wild-type VCP (WT-VCP), mutant VCP (mVCP),and arimoclomol-treated mutant VCP (mVCP + Ari) mice at 4 and 14 months(M) of age (n = 10; *P < 0.0001, unpaired t test). (B) Typical traces of muscletwitch andmaximum tetanic force of extensor digitorum longus (EDL) musclesin untreated and arimoclomol-treated mutant VCP mice. (C) Mean maximumforce of extensor digitorum longus muscles of WT-VCP, mutant VCP, andarimoclomol-treatedmutant VCP mice (n = 10; *P < 0.04, one-way ANOVA).(D) Hematoxylin and eosin (H&E) staining of tibialis anterior (TA) mus-cles of mice in each experimental group (white arrow, atrophied fiber;

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Western blots showMHC-I and phospho-IkBa expression in tibialis anteriormuscles of mice in each experimental group. (H) Transmission electron mi-croscopy of tibialis anterior muscles of mice in each experimental group. (I)Muscle fiber diameter in tibialis anterior muscles from untreated andarimoclomol-treated mutant VCP mice compared to WT-VCP mice (n = 3;*P < 0.0001, one-way ANOVA). (J) Western blot analysis of HSP70 expres-sion in tibialis anterior muscles from mice in each experimental group. Barchart shows mean relative optical density (n = 3; *P = 0.01, unpaired t test).Error bars represent SEM. Scale bars, 50 mm (D and E); 2 mm (H).




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clinical and demographic characteristics were similar between groups(table S1).

There were no significant differences between treatment groups re-garding the rate, type, and severity of adverse events (table S2). Therewere 8 adverse events in the placebo group and 14 in the arimoclomolgroup, the most common being gastrointestinal (see table S2 for de-tails). In the arimoclomol group, one serious adverse event was reportedas a result of persistent high blood pressure requiring overnight hos-pitalization in a patient with known poorly controlled hypertension, inwhom the first trial muscle biopsy was identified as a stressful event.Blood pressure normalized after adjustment of the patient’s anti-hypertensive medication and remained within the normal rangethroughout the remainder of the trial. Hypertensive episodes were alsoobserved in two placebo patients, under similar circumstances, al-though these cases did not require hospitalization. Two cases of hypo-natremia and one case of high thyroxine levels were observed in thearimoclomol group; however, these were transient, asymptomatic, anddid not require treatment. The episode of hematuria in the arimoclo-mol group was also limited and did not require treatment. All infec-tions resolved with standard treatments, with or without antibiotics,and did not require hospitalization. Ocular toxicity and arrhythmiawere not observed in any study subjects.

Clinical trial secondary outcomesOverall, there was no statistically significant difference in thesecondary outcome measures favoring arimoclomol. However, therewere trends in favor of arimoclomol, but these will require a formallarge-scale patient trial powered for efficacy to be assessed further.Physical function and muscle strength decline rates over time werenumerically higher in the placebo group compared to the arimoclomolgroup (Fig. 6, A to C, and table S3). At 8 months, there was a trendfavoring the arimoclomol group, with P values of 0.055 for change inthe IBM functional rating scale (IBMFRS) score (−0.68 ± 1.58 versus

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−2.50 ± 3.31; mean ± SD), 0.147 for change in the average manualmuscle testing (MMT) score (−0.12 ± 0.22 versus −0.26 ± 0.27), and0.064 for change in the right-grip maximum voluntary isometriccontraction testing (MVICT) score (1.26 ± 2.63 versus −0.54 ± 1.86).No differences were seen for changes in the other MVICT scores,changes in dual-energy x-ray absorptiometry (DEXA) fat-free masspercentage, or changes inmyosin-adjustedHSP70 expression inmuscle(table S3).

DISCUSSION

Here, we examined the effects of targeting the heat shock response insIBM. In sIBM patients, arimoclomol was found to be safe and welltolerated, with a trend of a slower decline in muscle strength andphysical function compared with placebo-controlled sIBM patients, al-though the trend was not significant. Although we did not observestatistically significant clinical efficacy or significant morphologicalchanges in the repeat muscle biopsies taken from arimoclomol-treatedpatients, studies both in cellular models in vitro and in an in vivomouse model, which recapitulates many features of sIBM in muscle,showed that the pathological and functional deficits associated withsIBM were ameliorated by arimoclomol in these model systems.

In vitro, both b-APP overexpression and exposure to inflammatorycytokines induced degenerative sIBM-like pathology in rat myocytes,including an increase in the formation of ubiquitinated inclusionbodies. Treatment with arimoclomol reduced inclusion body forma-tion, indicating an improvement in protein handling. This was mostlikely due to up-regulation of the heat shock response, in particularenhanced HSP70 expression. Both models also recapitulated the in-crease in mislocalized TDP-43 observed in sIBM patient myofibers.TDP-43 is cleaved by caspase-3 (42), allowing the C terminus to leavethe nucleus, thus linking its translocation to increased cell stress.

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Fig. 5. Arimoclomol abrogates IBM-like pathology in mutant VCP mouse muscle. (A to F) Cross sections of tibialis anterior muscle fromWT-VCP, mutant VCP, andarimoclomol-treated mutant VCP mice immunostained for ubiquitin (red) and TDP-43 (green); nuclei stained with DAPI (blue). Scale bars, 50 mm; 25 mm (insets).




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Arimoclomol also reduced TDP-43 mislocalization and TDP-43 expres-sion in cultures exposed to inflammatory mediators, suggesting that thedrug reduced levels of cell stress. Indeed, both b-APP overexpressionand inflammatory mediators induced cell death, which was reducedby arimoclomol.

Nuclear translocation of the NF-kB subunit p65, which was ob-served after exposure to IL-1b and TNFa, was examined as an indica-tion of NF-kB activation. b-APP overexpression also activated NF-kB

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as has been observed in cellular models of Alzheimer’s disease inwhich Ab42 peptide activates the inflammatory cascade (43), mostlikely as part of a nonspecific stress response. Arimoclomol had aninhibitory effect on NF-kB activation in both the b-APP and the in-flammatory cell models, which likely reflects up-regulation of the heatshock response and down-regulation of the NF-kB signaling cascadeby HSPs (44).

Arimoclomol also decreased the disruption in ER calcium ion ho-meostasis induced in both the b-APP and inflammatory cellularmodels of sIBM and consequently reduced the ER stress response, akey mechanism triggered by aberrantly folded proteins. This findingwas reflected by restoration of cytosolic calcium ion concentrations.Indeed, in TNFa-treated rat myotube cultures, the reduction in cyto-solic calcium ion concentration was prevented by arimoclomol, as wasthe expression of the ER stress mediator CHOP. This protective effectof arimoclomol may reflect, at least in part, the chaperone activity dueto augmented expression of HSP70, which serves to improve the effi-ciency of handling and degradation of misfolded proteins.

Examination of the two major protein degradation pathways in thecell culture models revealed disruption in both proteasome functionand increased autophagy. However, arimoclomol had no effect onproteasome function, although formation of mature autophagosomeswas reduced, suggesting a reduced misfolded protein load in the lyso-somal pathway.

In vivo, treatment of mutant VCP mice with arimoclomol improvedthe pathological features and functional deficits characteristic of the in-clusion body myopathy associated with MSP. Thus, in arimoclomol-treated mutant VCP mice, we observed an increase in muscle strengthand a reduction in key pathological hallmarks of IBM, including de-creased ubiquitin expression, a reduction in cytoplasmic mislocalizationof TDP-43, and a marked improvement in mitochondrial morphologyaccompanied by an increase in HSP expression.

These findings provide evidence for the beneficial effects of arimo-clomol in experimental models of sIBM and support clinical assess-ment of the effects of arimoclomol in sIBM patients. The primaryend point of our randomized, double-blind, placebo-controlled,proof-of-concept trial in sIBM patients was met, with results showingthat arimoclomol was both safe and well tolerated by sIBM patients.Regarding the secondary end points (efficacy measures), there were nostatistically significant differences between the treatment groups. How-ever, this finding was not surprising because this first experimentalstudy of a compound targeting the heat shock response in sIBMhad several limitations. These included the small sample size, whichhad been advised by our ethics committees and the U.S. Food andDrug Administration (FDA) because arimoclomol had never beengiven to sIBM patients before and this study was intended as a proof-of-concept/safety study that was not powered for efficacy. The shortduration of treatment (only 4 months) was also mandated by regula-tory agencies. In a slowly progressing disease like sIBM, longer treat-ment periods are likely to be required to be able to detect changes inefficacy outcome measures. Whether arimoclomol can amelioratesIBM pathology will only be determined by performing adequatelypowered clinical trials of longer duration. A 12-month study poweredfor efficacy that will enroll 150 IBM patients has now been approvedand fully funded and enrollment will commence in late 2016.

Finally, regarding the measurement of muscle HSP70 expression insIBM patients, this readout also had several limitations, namely, thefact that muscle HSP70 expression is extremely sensitive to multiple

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at 4, 8, and 12 months (means ± SEM) in sIBM patients treated with arimo-clomol for 4 months. (A to C) IBMFRS score, MMT average score, and right-hand grip (HGR) MVICT score. The IBMFRS is a disease-specific functionalquestionnaire for patientswith sIBMandmeasuresphysical function/disability.MMT is ameasure ofmuscle strength scored by the physician on the basis ofthe clinical assessment. MVICT is a measure of muscle strength performedusing a quantitative muscle assessment (QMA) system that uses an adjust-able cuff to attach the patient’s arm or leg to an inelastic strap that isconnected to a force transducer. Error bars represent SEM. No statisticallysignificant clinical efficacy measures were observed.



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factors, including disease stage, physical activity, age, and gender(45–47), and the fact that muscles on the opposite sides of the bodyare biopsied at baseline and after treatment.

Arimoclomol has previously been shown to be safe in both animalmodels and humans (36, 39, 48) and has been indicated to be ofpotential therapeutic benefit in several neurological disorders includ-ing diabetic peripheral neuropathy and retinopathy (49). Arimoclomolhas also been found to be beneficial in animal models of neurodegen-eration, including models of acute injury–induced neuronal death (37)and has been shown to have therapeutic value in mouse models ofmotor neuron disease, including amyotrophic lateral sclerosis (ALS)(36, 50) and Kennedy’s disease (51). Arimoclomol has been throughseven phase 1 clinical trials in healthy volunteers to assess its safety,tolerability, and pharmacokinetic properties, as well as a small-scalephase 2 trial in ALS patients (48). A phase 2a dose-ranging trial inALS has shown arimoclomol to be safe and well tolerated up to100 mg three times daily (52), and a phase 2/3 randomized, double-blind, placebo-controlled trial is currently under way in familial super-oxide dismutase 1 (SOD1) ALS patients (NCT00706147).

Our results show that arimoclomol is safe and well tolerated insIBM patients and ameliorates key degenerative and inflammatoryfeatures of IBM pathology in experimental cellular and animal models.Together, these findings support further investigation of arimoclomolfor the treatment of sIBM.

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MATERIALS AND METHODS

Study designHere, we took a three-step translational approach to test whether aug-mentation of the heat shock response may be a potential therapy forsIBM, by examining the effects of arimoclomol in rat muscle cells invitro, in a preclinical study in mutant VCP mice, and in sIBM patientsin a safety and tolerability trial. In vitro, the effects of arimoclomolwere examined in primary rat muscle cultures overexpressing b-APPor treated with inflammatory cytokines by assessing the effects onIBM-relevant histopathological characteristics. All experiments wererepeated in at least three independent cultures. In the preclinical effi-cacy study, mutant VCP mice were randomized to arimoclomol orvehicle treatment arms, and the effects on muscle force and histo-pathological characteristics were set as the study end points. Theexperimenter was blind to genotype and treatment group through-out. In sIBM patients, we undertook a randomized, double-blind,placebo-controlled, proof-of-concept trial of arimoclomol versus place-bo to evaluate the safety and tolerability of arimoclomol and to gatherexploratory efficacy data by testing muscle strength and HSP70 expres-sion. This trial was an exploratory study conducted without priorknowledge of effect size of arimoclomol in sIBM. The sample sizewas chosen on the basis of feasibility. Randomization was performedcentrally for both study sites.

In vitro model of IBMPrimary muscle cultures were used as an in vitro model of IBM byextracting satellite cells that lie under the basal lamina of myofibers.Muscle cells were induced to model either the degenerative features ofsIBM by overexpression of b-APP or the inflammatory characteristicsby treatment with inflammatory mediators (see SupplementaryMethods for details of muscle culture preparation and treatment).

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In these in vitro models of sIBM, the following features of sIBMpathology were examined and the effects of treatment with 10 mMarimoclomol investigated: (i) inclusion body formation, (ii) HSP ex-pression, (iii) cytoplasmic translocation of TDP-43, (iv) cell survival,(v) NF-kB activation, and (vi) ER stress (for details, see the Supple-mentary Materials).

Breeding and maintenance of mutant VCP miceAll experimental procedures were carried out under license from the UKHome Office (Scientific Procedures Act 1986) and after approval by theUniversity College London (UCL) Institute of Neurology’s AnimalWelfare and Ethical Review Board. Mice overexpressing the wild-typeor mutant (A232E) human VCP gene under the cytomegalovirus-enhanced chicken b-actin promoter [see Custer et al. (41)] were bredand maintained at the UCL Institute of Neurology. Transgenic femalemice carrying the wild-type or mutant gene were mated with wild-type C57BL/6J male mice to generate transgenic and nontransgeniclittermates. Only male offspring were used in this study to preventgender differences. The mice were genotyped by polymerase chain re-action (PCR) amplification of ear notches and analyzed using agarosegel electrophoresis and visualized using GelRed stain (Sigma-Aldrich).All mice used in this study were housed in a controlled temperatureand humidity environment with a 12-hour light/dark cycle and hadaccess to drinking water and food ad libitum.

Treatment of mutant VCP mice with arimoclomolAfter genotyping, male mice were randomly assigned to a treatmentor vehicle arm of the study. From 4 months of age to the time of ex-amination at 14 months, mutant VCP mice were treated with arimo-clomol (120 mg/kg per day) dissolved in drinking water (n = 10) orwater alone (vehicle; n = 10). The body weight of all mice was recordedfortnightly. Arimoclomol was obtained from Orphazyme ApS. Allexperiments were undertaken blinded to genotype and treatment.

Longitudinal assessment of grip strength and body weightGrip strength was assessed fortnightly in all mice from 4 to 14 monthsof age using a Bioseb force gauge according to the manufacturer’s in-structions. An average of four maximum readings was obtained. Bodymass was recorded at the same time as grip strength. The ratio of gripstrength to body mass was determined for individual animals and waspooled by each genotype.

In vivo analysis of isometric muscle forceThe mice were prepared for in vivo assessment of muscle function [seeKieran et al. (36)]. Briefly, mice were deeply anesthetized [inhalationof 1.5 to 2.0% isoflurane in oxygen delivered through a Fortec vapor-izer (Vet Tech Solutions Ltd.)]. The distal tendons of the tibialis an-terior and extensor digitorum longus muscles in both hindlimbs weredissected free and attached to isometric force transducers (DynamometerUFI Devices). The sciatic nerve was exposed and sectioned, and allbranches were cut except for the deep peroneal nerve that innervatesthe tibialis anterior and extensor digitorum longus muscles. Musclelength was adjusted for maximum twitch tension. Isometric contrac-tions were elicited by stimulating the nerve to the muscles usingsquare-wave pulses of 0.02-ms duration at supramaximal intensityusing silver wire electrodes. Contractions were elicited by trains ofstimuli at frequencies of 40, 80, and 100 Hz for 450 ms, and the max-imum twitch and tetanic tension were measured using force transducers




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connected to a PicoScope 3423 oscilloscope (Pico Technology) and sub-sequently analyzed using PicoScope software v5.16.2 (Pico Technology).Muscle histology, immunohistochemistry, Western blot, and electronmicroscopy protocols are described in the Supplementary Materials.

Clinical trial design and patient populationIn this investigator-initiated, double-blind, placebo-controlled study, 24patients meeting the Griggs diagnostic criteria for definite or probablesIBM (53, 54) were randomized to arimoclomol (100mg) or placebo (2:1),three times a day for 4months (treatment phase). This restricted 4-monthtreatment period, mandated by the FDA, was followed by an 8-monthblinded assessment phase. The study was conducted from August 2008to May 2012, at two centers in two countries (12 patients per center):University of Kansas Medical Center (KUMC) and Medical ResearchCouncil (MRC) Centre for Neuromuscular Diseases. Detailed inclusionand exclusion criteria can be found in Supplementary Methods.

Randomization was performed centrally for both study sites atKUMC. This was done by a General Clinical Research Center (GCRC)statistician, who sent the randomization codes, created using a ran-dom number generator table, to the respective research pharmacies.All personnel involved in the conduct of the trial were blind to theidentity of the treatment assignments, except for the unblinded statis-tician and the pharmacist at each site who labeled the study medica-tion using codes provided by the unblinded statistician. Theappearance of the placebo was identical to that of arimoclomol.

The study was conducted according to the ethical principles of theDeclaration of Helsinki and approved by the Independent Ethics Com-mittee or Institutional Review Board for each center. Informed consentwas obtained from each patient before randomization. The study wasregistered with ClinicalTrials.gov (NCT00769860) and with Interna-tional Standard Randomized Controlled Trial Number Register(ISRCTN80057573).

Clinical trial: Primary outcomesThe safety and tolerability of arimoclomol compared to placebo werethe primary outcome of the trial. Participants were seen for assessmentof adverse events at every study visit. Unscheduled visits to evaluatepotential adverse events could occur at any time. All serious adverseevents were reported to the sponsor and regulatory authoritiesaccording to standard operating procedures. The trial was monitoredby an Independent Safety Monitoring Committee.

During the first 4 months (treatment phase), all participants com-pleted a study medication diary. Pill bottles were brought to each visitfor a count by a research team member to check on whether partici-pants were taking the study medication in the appropriate dosages. Atscreening and months 1, 2, 3, 4, and 12, the study participants had fullsafety laboratory analyses done, which included full blood count withdifferential, prothrombin time, activated partial thromboplastin time,urea, creatinine, electrolytes, glucose, phosphate and calcium, alaninetransaminase, aspartate transaminase, total bilirubin, albumin, fullurine analysis, and 24-hour urine protein content and creatinine clear-ance. At months 0.5, 1.5, 2.5, and 3.5, participants had partial safetylaboratory analyses done including serum creatinine, urea, electrolytes,glucose, phosphate, calcium, and a full urine analysis. An electro-cardiogramwasperformedat screening andmonths 1 and3.Anophthal-mic examination was performed at screening and month 10. Theelectrocardiogram and ophthalmic examination were introduced assafetymeasures following results from an animal model study that raised

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concerns of potentially accelerated cataract formation and apparent riskof sudden, unexplained death with arimoclomol at very high doses andwith arimoclomol used in conjunction with riluzole (CytRx Corpora-tion; arimoclomol Investigator’s Brochure version 7.0, September 2010).

Clinical trial: Secondary outcomesPhysical function was measured using the IBMFRS that is intendedonly for patients with sIBM. It includes 10 measures (swallowing, hand-writing, cutting food and handling utensils, fine motor tasks, dressing,hygiene, turning in bed and adjusting covers, changing position fromsitting to standing, walking, and climbing stairs) graded on a Likertscale from 0 (being unable to perform) to 4 (normal). The sum ofthe 10 items gives a value between 0 and 40, with a higher score repre-senting less functional limitation. The IBMFRS is a sensitive and reli-able tool for assessing activities of daily living in patients with sIBMand is quickly administered (see SupplementaryMethods) (7, 30, 55–57).

Muscle strength was assessed by MMT and MVICT using theQMA system designed by Computer Source (30, 57) (see Supplemen-tary Methods). Each muscle was tested twice, and the maximum forcegenerated by the patient from the two trials was recorded for eachmuscle group. The total summed score of strength in kilograms wascomputed for each patient. MVICT has been shown to be reliable andvalid in several neuromuscular disorders, including sIBM (30, 57, 58).Body composition was obtained using a standard DEXA whole-bodyscan to assess total body fat-free mass. DEXA has been used to measurelean body mass in previous neuromuscular disease studies, includingsIBM (30, 57). Patient muscle HSP70 expression was determined asdescribed in Supplementary Methods.

Statistical methodsIn the preclinical cellular and mutant VCP mouse models, analysis ofnormally distributed results was performed using an unpaired t test or,for comparison of greater than two groups, a one-way ANOVA. Other-wise, the nonparametric Mann-Whitney U test was used.

Datamanagement and statistical analysis were performed by GCRCinformatics staff and aGCRC statistician. Data were entered blindly to aGCRC password-protected electronic database, and analyses wereperformed after database lock. Descriptive statistics were used tosummarize subject disposition and adverse events by treatment group.To reduce measurement error, baseline scores were computed bycalculating the average of visit 1 (screening visit) and visit 2 (baseline vis-it), which had to be less than 21 days apart. Continuous variables werecompared between treatment groups using the Mann-Whitney U test.Categorical variables were compared between treatment groups usingc2 or Fisher’s exact test, as appropriate. Treatment groups were com-pared at baseline as well as regarding changes in several outcome mea-sures at 4months (IBMFRS,MMT,MVICT,DEXA, andHSP70content),8 months (IBMFRS, MMT, and MVICT), and 12 months (IBMFRS,MMT, MVICT, and DEXA). Statistical analyses were performed usingSTATAv10 and in all analyses, statistical significancewas set at the two-sided 5% level. Error bars represent SEM unless otherwise stated.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/331/331ra41/DC1Materials and MethodsFig. S1. Overexpression of b-APP and exposure to inflammatory mediators induce sIBM-likepathology in cultured myocytes.

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Fig. S2. Mutant VCP mice show further signs of pathology.Fig. S3. Consort diagram of the patients participating in the clinical trial.Table S1. Baseline characteristics of the study population.Table S2. Summary of all adverse events over the course of 1 year.Table S3. Mean changes (±SD) in secondary outcome measures throughout the study period.Movie S1. Low-magnification serial block-face scanning electron microscopy of arimoclomol-treated and untreated mutant VCP mouse muscle.Movie S2. High-magnification serial block-face scanning electron microscopy of wild-type VCPmouse muscle.Movie S3. High-magnification serial block-face scanning electron microscopy of untreated mu-tant VCP mouse muscle.Movie S4. High-magnification serial block-face scanning electron microscopy of arimoclomol-treated mutant VCP mouse muscle.References (59, 60)

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Acknowledgments: We thank the participants who enrolled in this intensive study and theKIT (Keep In Touch) and Myositis UK support groups. The following clinicians, research nurses,coordinators, and technicians contributed substantially to the trial: M. Walsh, M. Michaels, F. Raja,

www.Scienc

A. Dick, K. Latinis, L. Dewar, G. Barreto, and I. Skorupinska. We thank Orphazyme ApS and CytRxfor providing drug and placebo. Funding: This work was funded by Arthritis Research UK (ref.no. 19255), MRC Centre for Neuromuscular Diseases grant (G0601943), Kansas University Neu-rology Department Ziegler grant, Kansas University GCRC CReFF (General Clinical ResearchCentre Clinical Research Feasibility Funding) grant, and the Wellcome Trust (107116/Z/15/Z)(G. Schiavo). L.G. is the Graham Watts Senior Research Fellow, supported by the Brain ResearchTrust, Rosetrees Trust, The Stoneygate Trust, and funded by the European Community’s Sev-enth Framework Programme (FP7/2007–2013). C.S. is the recipient of an MRC Studentship. L.C.and A.W. are funded by Cancer Research UK, the MRC, BBSRC (UK Biotechnology and BiologicalSciences Research Council), and EPSRC (Engineering and Physical Sciences Research Council)under grant award MR/K01580X/1 to L.C. and P. O’Toole (York University). P.M.M. reportsfunding from the National Institute for Health Research (NIHR) Rare Diseases Translational Re-search Collaboration (RD TRC) and from the NIHR University College London Hospitals (UCLH)Biomedical Research Centre (BRC). The views expressed are those of the author and not nec-essarily those of the UK National Health Service (NHS), the NIHR, or the Department of Health.This project was also supported by an Institutional Clinical and Translational Science Award andNIH/NCATS (National Center for Advancing Translational Sciences) grant (UL1TR000001). Itscontents are solely the responsibility of the authors and do not necessarily represent the officialviews of the NIH. Author contributions:M.A., A.M., and L.G. designed the in vitro experiments.M.A. and L.G. designed the in vivo experiments, andM.A. and C.S. performed the experiments andthe data analyses. J.P.T. generated and characterized the VCPmice. P.M.M., A.M., L.H., Y.W., A.L.M.,M.P., P.G., J.S., S.B., M.P., J.H., M.G.H., R.J.B., M.M.D., and L.G. designed the clinical trial. P.M.M. per-formed the clinical trial assessments in the UK. L.H., Y.W., A.L.M., M.P., R.J.B., and M.M.D. performed theclinical trial assessments in the United States. J.H. and J.N. conducted data analyses of the trialdata. M.M.D., A.L.M., H.S., and G. Samandouras performed the trial muscle biopsies. J.L.H. performedmuscle histopathology of patient biopsies from the trial. C.-H.L. and B.K. performed the HSP mea-surements in the trial muscle tissue. A.W. and L.C. carried out the electron microscopy analyses, andG. Schiavo undertook their interpretation. M.G.H., R.J.B., and M.M.D. supervised the clinical trial. L.G.supervised the in vitro cell models and in vivo preclinical trial experiments and HSPmeasurementsin the muscle tissue. M.A., P.M.M., C.S., and L.G. wrote the drafts of the manuscript and all authorsprovided scientific input to the manuscript. All authors read and approved the final version of themanuscript. Competing interests: L.G. became an unpaid consultant to Orphazyme ApS (theowner of arimoclomol) after completing this study. The other authors declare that they have nocompeting interests.

Submitted 17 September 2015Accepted 4 March 2016Published 23 March 201610.1126/scitranslmed.aad4583

Citation: M. Ahmed, P. M. Machado, A. Miller, C. Spicer, L. Herbelin, J. He, J. Noel, Y. Wang,A. L. McVey, M. Pasnoor, P. Gallagher, J. Statland, C.-H. Lu, B. Kalmar, S. Brady, H. Sethi,G. Samandouras, M. Parton, J. L. Holton, A. Weston, L. Collinson, J. Paul Taylor, G. Schiavo,M. G. Hanna, R. J. Barohn, M. M. Dimachkie, L. Greensmith, Targeting protein homeostasis insporadic inclusion body myositis. Sci. Transl. Med. 8, 331ra41 (2016).

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Targeting protein homeostasis in sporadic inclusion body myositis

Giampietro Schiavo, Michael G. Hanna, Richard J. Barohn, Mazen M. Dimachkie and Linda GreensmithBrady, Huma Sethi, George Samandouras, Matt Parton, Janice L. Holton, Anne Weston, Lucy Collinson, J. Paul Taylor,Wang, April L. McVey, Mamatha Pasnoor, Philip Gallagher, Jeffrey Statland, Ching-Hua Lu, Bernadett Kalmar, Stefen Mhoriam Ahmed, Pedro M. Machado, Adrian Miller, Charlotte Spicer, Laura Herbelin, Jianghua He, Janelle Noel, Yunxia

DOI: 10.1126/scitranslmed.aad4583, 331ra41331ra41.8Sci Transl Med

arimoclomol and showed that it was safe and well tolerated.pathology and improved muscle function. The authors then treated a small number of sIBM patients with(VCP) mice, which develop an inclusion body myopathy, treatment with arimoclomol ameliorated disease myoblast cell culture, arimoclomol reduced key pathological features of IBM. In mutant valosin-containing proteineffects of targeting protein dyshomeostasis using arimoclomol, a co-inducer of the heat shock response. In rat

. tested theet altargeted the inflammatory component, and all have failed in clinical trials. In a new study, Ahmed both inflammation and protein dyshomeostasis have been implicated in sIBM pathogenesis, treatments have only

IBM) is a debilitating adult myopathy that is difficult to treat. AlthoughsSporadic inclusion body myositis (Targeting protein dyshomeostasis in myopathy

ARTICLE TOOLS http://stm.sciencemag.org/content/8/331/331ra41

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Targeting protein homeostasis in sporadic inclusion body myositis · MYOPATHY Targeting protein homeostasis in sporadic inclusion body myositis Mhoriam Ahmed,1,2* Pedro M. Machado,1*