1. Background

2. Medical need

3. Existing and emerging

therapies

4. Competitive environment

5. Expert opinion

Review

Emerging therapies andtherapeutic concepts forlysosomal storage diseasesThomas KirkegaardOrphazyme ApS, Copenhagen, Denmark

Introduction: The success of the first enzyme replacement therapy (ERT) for a

lysosomal storage disease (LSD) and the regulatory and commercial incentives

provided by authorities for orphan and rare diseases has spawned a massive

interest for developing drugs for these intriguing but devastating genetic dis-

orders. The potential for new drugs in this arena is vast, as not only a high

number of LSDs have no available therapy, but also alternative therapeutic

approaches for diseases with existing treatment are much needed as a num-

ber of challenges facing the existing therapies have become very obvious.

A significant unmet medical need is therefore apparent for most, if not all

of the LSDs and the development of new therapies based on the increasing

knowledge of the pathophysiological mechanisms involved in these devastat-

ing diseases is therefore anticipated with great interest from all stakeholders.

Areas covered: The reader will be introduced to the intricate biological pro-

cesses involved in lysosomal regulation and how these are exploited for cur-

rent and emerging therapies. Therapies utilizing these processes will be

thoroughly reviewed with regard to their mechanism of action, their clinical

status and the challenges they are faced with and/or are aiming to address.

For this review, a literature research has been undertaken that covers the

years 1955 -- 2012.

Expert opinion: The interest in lysosomal biology and disease has surged over

the past decade not only in the halls of science but also of pharmaceutical

companies. As the complexity of the LSDs increasingly become revealed, so

do novel therapeutic targets continuously nurturing the development of

new candidate drugs for these devastating diseases. Among this multitude

of approaches, the ERTs still account for the vast majority of approved thera-

pies but a number of exciting alternative approaches are emerging targeting

various components of the pathophysiological cascade. This evolution of the

field is much needed as the presently available treatments are unable to

address all clinical aspects of these multifaceted diseases. Future therapy will

most likely consist of combinations of these established and emerging

approaches as well as other yet to be discovered concepts as the complexity

of the diseases demands a certain degree of humbleness to the expectations

for a cure based on a single therapy.

Keywords: chaperone, enzyme replacement, Fabry disease, Gaucher disease, glycosphingolipids,

lysosomal storage disease, lysosomes, substrate optimization, substrate reduction, therapy

Expert Opinion on Orphan Drugs (2013) 1(5):385-404

1. Background

Since the discovery and initial characterization of lysosomes by Francois Apple-mans, Robert Wattiaux and Christian de Duve in 1955 [1-3], the interest for thisstill enigmatic organelle and its close relatives has seen a resurgence in the past

10.1517/21678707.2013.780970 © 2013 Informa UK, Ltd. e-ISSN 2167-8707 385All rights reserved: reproduction in whole or in part not permitted

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two decades spurred by the rediscovered and increasing real-ization of their crucial roles in physiological homeostasis.The interest in lysosomes from not only academia but alsobiotech and pharma has been further kindled by a numberof clinical, regulatory and commercially transforming eventsthat have provided a new paradigm for developing therapiesfor not only lysosome-related diseases but also other rareand orphan diseases.With the introduction of the Orphan Drugs Act in the

USA in 1983, which was followed by similar legislation inSingapore (1991), Australia (1993), Japan (1997) and theEU (1999), a regulatory framework has been laid down forguiding the development of drugs for rare diseases with highlyunmet need. However, the commercial potential of develop-ing drugs for lysosomal storage diseases (LSDs) was not real-ized until the development of Cerezyme� (recombinantglucosylceramidase (glucocerebrosidase) for the treatment oftype I Gaucher disease) by the Boston-based biotechcompany Genzyme.The scientific rationale for the development of lysosome-

targeted therapies however predates the advent of Cerezymeby several decades and driven by the re-kindled interest inlysosomal diseases several novel therapeutic concepts andtherapies are now emerging for these devastating disorders.

1.1 LysosomesAs the main compartment for intracellular degradation andsubsequent recycling of cellular constituents, the lysosomesreceive both hetero- and autophagic cargo, which in thelumen of this multifaceted organelle find their final destina-tion. The degradation is carried out by a number of acidhydrolases (glycosidases, proteases, sulfatases, lipases, etc.)capable of digesting all major cellular macromolecules [4].These acid hydrolases function optimally at the acidic pH ofthe lysosomes (pH 4 -- 5) although several can still functionand have distinct roles at the neutral pH outside the lyso-somes, albeit having decreased stability and/or alteredspecificity [5].Until recently, the function of many of these enzymes was

thought to be limited to intralysosomal macromoleculeturnover. However, from the complex and diverse clinicalpresentation of the diseases originating from lysosomal mal-function and involvement in neoplastic events it is clear thatthe lysosomes have an absolutely critical role in physiologicalhomeostasis [6,7]. As such, the potential impact of therapeuti-cally addressing the lysosomes and their constituents shouldnot be underestimated.Interestingly, recent data suggest that the biogenesis and

functioning of endosomal and autophagosomal pathways ispartially controlled by the transcription factor EB (TFEB),which regulates a coordinated lysosomal expression and regu-lation (CLEAR) gene network [8], a finding which argues foran evolutionary need to intimately control and efficientlyadapt the lysosomal system to rapid changes in the cellsmetabolic state.

1.2 Dynamics of the lysosomal systemThe intracellular trafficking of vesicles involved in, or relatedto, the lysosomal system, serve an essential role in the mam-malian cell through its delivery of membrane components,various solute molecules and receptor-associated ligands to arange of intra- and extracellular compartments. The mainpathways involved in this system are depicted in Figure 1,along with highlights of the various facets of these pathwaysbeing targeted for therapeutic intervention in the LSDs(Figure 1).

As a testament to the importance of this system and its con-stituents, defects in any part of it leads to a number of severediseases or syndromes. Be it defects in lysosomal exocytosis(e.g., Chediak--Higashi syndrome), reduced lysosomal cata-bolic efficacy (e.g., Niemann--Pick disease types A and Band Gaucher disease), lysosomal transport machinery defects(e.g., Griscella syndrome and Charcot--Marie--Tooth disease),lysosomal metabolite efflux impairment (e.g., Niemann--Picktype C and cystinosis/Fanconi syndrome) or dysfunction oflysosomal integral membrane proteins (e.g., Danon disease),the disease most often affect multiple organs and tissues,involves the central nervous system (CNS) and is often fatalat a young age in its aggressive forms [7,9-13].

In order to understand the complexity of the LSDs andwhy defects in this refined machinery can lead to such detri-mental clinical manifestations as well as grant the reader aninitial overview of the possibilities that might exist for thera-peutic intervention, a brief description of the inter-relationsin the lysosomal system is provided below.

1.2.1 Endocytic route to lysosomesThe best understood endocytic pathway, which is also exten-sively exploited in enzyme replacement therapies (ERTs) forthe LSDs, is the receptor-mediated endocytosis of moleculesvia the formation of clathrin-coated pits [14]. In the conven-tional receptor-mediated endocytic pathway, receptors such asthe transferrin receptor, the low-density lipoprotein receptorand the mannose 6-phosphate receptor (M6PR) concentrateinto clathrin-coated pits on the surface of the plasma mem-brane and form early endosomes [15,16]. Although the majorityof lysosomal enzymes are targeted to the lysosomes from thetrans-Golgi network (TGN) through mannose-6-phosphate(M6P)-mediated binding to M6PRs in the medial Golgi andthen released once the Golgi-derived transport vesicles fusewith late endosomes (detailed in Section 1.2.2), some amountsof lysosomal enzymes are destined for secretion and subsequentreuptake from the extracellular space via plasma membranereceptors that are either the same or similar to those involvedin intracellular sorting and the biosynthetic route to the lyso-somes [15]. This concept developed from a number of metaboliccomplementation studies in cells from patients with differentLSDs and is the fundamental principle for enzyme therapiesof LSDs [17].

As the endosome mature, its luminal pH steadily drops,mainly through the action of the vacuolar-type proton

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ATPase (V-ATPase) [18], facilitating the dissociation of recep-tor and ligand while shifts in membrane lipid and proteincomposition also occur as the vesicles mature to form lateendosomes and subsequently lysosomes. The late endosomesand lysosomes differ from endosomes primarily in theirdegree of acidification, higher buoyant density, higher abun-dance of integral lysosome-associated membrane proteins(LAMPs) and enrichment of acidic hydrolases [19].

1.2.2 Biosynthetic route to lysosomesApart from endocytosis, late endosomes and lysosomes alsoreceive cargo via the M6PR pathway from the TGN (thebiosynthetic route). The cation-dependent M6PR and thecation-independent (CI) M6PR/insulin-like growth factor-

II (IGF-II) receptor share the task of delivery of newly synthe-sized acid hydrolases from the TGN to the lysosomes. Therecognition of acid hydrolases by M6PRs requires the addi-tion of carbohydrates in the endoplasmic reticulum (ER)and the subsequent modification and phosphorylation of thecarbohydrate residues to M6P moieties in the cis-Golgi [15,20].The M6PR-bound hydrolases are first delivered to endo-somes, where they dissociate from the receptors due to thedrop in the lumenal pH, hereby allowing the receptors torecycle back to the TGN.

1.2.3 Autophagic routes to lysosomesAutophagy is the third relatively well-characterized route bywhich macromolecules reach the lysosome. Autophagy is an

ECM

Early endosome

Late endosome

Trans golgi network

Lysosome

Hybrid organelles

Secretory lysosome

CYTOSOL

Sorting endosome

Endocytic recycling compartment

Autophagosome

Exosome

Rough endoplasmic reticulum

Golgi secretorypathway

Enzyme

Cis golgi network

Mannose-6-phosphate receptor

Exogenous (ERT) or secretedenzyme (BMT/HSCT)

Alternative receptorsystem: e.g. LRP-1

Molecular chaperone (Hsp70)or 2nd generation ERT

Chemical chaperonetechnology

Figure 1. Lysosomal pathways. The figure provides a general overview of the dynamics of the endolysosomal system. For a

thorough description of the events along the pathways and the therapeutic concepts illustrated in the figure, please refer to

the text.BMT/HSCT: Bone marrow transplantation/hematopoietic stem cell transplantation; ECM: Extracellular matrix; ERT: Enzyme replacement therapy.

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Expert Opinion on Orphan Drugs (2013) 1(5) 387

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evolutionary conserved pathway involved in the turnover oflong-lived proteins and organelles and is essential for main-taining cellular energy and metabolic homeostasis [21-23].There are three main modes of autophagy: macro-, micro-and chaperone-mediated autophagy; macroautophagy ischaracterized by a flat membrane cistern wrapping aroundcytoplasmic organelles and/or a portion of cytosol therebyforming a closed double-membrane bound vacuole, the auto-phagosome. The autophagosome finally fuses with lysosomesforming autophagolysosomes/autolysosomes, where the deg-radation and recycling of the engulfed macromolecules occur.Microautophagy is characterized by engulfment of cytosol bythe lysosomes through invaginations of the lysosomal mem-brane. Besides the macromolecules, which are present in theengulfed cytosol, this process may also involve the uptake oforganelles such as peroxisomes and mitochondria, with theseparticular autophagic processes being coined the terms pexo-and mitophagy, respectively [24,25]. Finally, chaperone-mediated transport of cytosolic proteins into the lysosomallumen presents a more direct and selective form of autophagy.This pathway is dependent on the presence of the consti-tutively expressed member of the heat shock protein70 (Hsp70) family, Hsc70 (HspA8), on both sides of the lyso-somal membrane and its interaction with an isoform of thelysosomal membrane protein, LAMP-2, the protein that isdefective in the LSD Danon disease [10,26].

1.2.4 Reformation of lysosomes and lysosomal

secretion/exocytosisAfter fusion of lysosomes with late endosomes or autophago-somes, the lysosomes are reformed from the resultant hybridorganelles through sequestration of membrane proteins andcondensation of the lumenal content [21]. The lysosomes,however, cannot be seen as the terminal point of the endocyticpathways as they are also able to form secretory lysosomesthrough fusion with secretory granules, a process that is Ca2+-dependent and was first recognized in secretory cells ofhematopoietic origin [27,28]. However, evidence also existsfor a Ca2+-regulated membrane-proximal lysosomal compart-ment responsible for exocytosis in non-secretory cells [29,30], aprocess which is dependent on the protein Rab27a, a memberof the Rab protein family of small GTPases that have key reg-ulatory roles in most membrane-transport steps including ves-icle formation, motility, docking and fusion. At least 13 Rabproteins are utilized in the endocytic pathways in order todetermine the fate of the various endocytosed molecules andtheir vesicles and alterations in the Rab GTPases or associatedregulatory molecules give rise to a number of diseases rangingfrom bleeding and pigmentation disorders (Griscellisyndrome) through mental retardation and neuropathy(Charcot--Marie--Tooth disease) to kidney disease (tuberoussclerosis) and blindness (choroideremia) [11,31].Ultimately, the reformation of lysosomes from autolyso-

somes (autophagosomes fused with lysosomes), endolysosomes(late endosome--lysosome fusion) and other lysosome-hybrid

organelles completes each cycle of lysosomal degradationyielding functional lysosomes that are available for anotherround of macromolecular breakdown.

Importantly, the efficient processing of macromolecularsubstrates in the hybrid organelles are essential for lysosomalreformation, a process which does not occur de novo, butis the result of a reformation/budding from the hybridorganelles [32-34].

Defects in lysosome reformation which is thought to berequired for the exocytosis of lysosomal cargo-containingmembrane vesicles have also been shown to have pathologicalconsequences as evidenced by studies in Niemann--Pick typeC1 and C2 deficient cells (NPC1 and NPC2). These studieshave revealed a significant difference in the lysosomal conse-quences of defects in the NPC1 and NPC2 proteins whichotherwise share virtually indistinguishable clinical pathologyin patients. For NPC1, one of the primary lysosomal conse-quences is a compromised ability to form the initial hybridorganelle, whereas in NPC2 deficient cells the impairmentof lysosome reformation appears to be the primary cellulardefect [7,35-38].

Interestingly, the impaired reformation of lysosomes couldalso constitute a more general molecular pathological featureof LSDs as secondary storage material in other LSDs, arisingas a consequence of the primary genetic deficiency, couldcause abnormalities in the necessary lipid dynamics involvedin not only lysosomal reformation but also vesicle dockingand fusion [39].

1.3 Lysosomal storage diseasesIndividually, the LSDs are ultra-orphan diseases withprevalences ranging from 1/60,000 live births for Gaucherdisease to 1/4.2 million live births for sialidosis. As a grouphowever, the combined prevalence has been estimated to beca. 1/7700 live births [40].

As delineated in the previous section, the interdynamics ofthe lysosomal system is a complex maze of a number of crucialevents that all needs to function properly for the lysosomes tobe the effective multifunctional organelles they are. The LSDsare excellent examples of the importance of these eventsas > 50 LSDs to this day have been characterized, rangingfrom primary defects in membrane proteins, transporters,fusion machinery and of course hydrolytic enzymes with themanifest cellular pathology associated with the diseases (lyso-somal accumulation and storage) coining the name to thesedevastating diseases.

More than 50 LSDs are classified according to the majorstorage material accumulating, although their monogeneticorigin does not always directly predict the affected substratescausing some discrepancies in understanding the molecularetiology of the diseases. In sphingolipidoses for example, themajor storage materials are glycosphingolipids and immediatederivatives thereof, whereas for the neuronal ceroid lipofusci-nosis, lipofuscins are the major storage materials accumulat-ing, but on the background of a genetically and functionally

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mixture of deficient proteases, peptidases, membrane proteinsand other proteins with as yet unknown function.

The mechanisms by which the accumulated substratesimpact cellular function and cause the pathological mani-festations of the primary genetic defects are still not wellunderstood, although several recent advances in the under-standing of these processes are now shedding light into thisdark(er) corner of human biology. These discoveries includemechanisms related to alterations of intracellular calciumhomeostasis, impairment of autophagy, activation of signaltransduction pathways by substrates and their derivatives,inflammation, altered intra- and limiting membrane propertiesand others [7,8,39,41-44].

Earlier, clinical management of LSDs was mainly confinedto treatment of complications, although in some of the sphin-golipidoses such as Gaucher and Fabry diseases attempts weremade to improve the patients’ condition by transplantation ofthe major organs affected (liver and kidney, respectively), butthese interventions did not alter the course of the diseases [45].In mucopolysaccharidosis type I (Hurler syndrome, MPSIH),bone marrow transplantations has shown some benefit, how-ever, provided the intervention is performed early enough [46].The benefit of early intervention is a principle that holds notonly for bone marrow transplantations but for all therapiesapplied to the LSDs, due to the irreversible nature of someof the pathological changes during the course of the diseases.

The breakthrough in the treatment of LSDs beganmodestly > 40 years ago when Neufeld and collaboratorsdemonstrated the principle of metabolic complementationin cell cultures from patients with different LSDs [17,47]. Sub-sequent studies provided insight into the nature of the correc-tive factors (secreted lysosomal enzymes) and that these wereendocytosed by binding to the M6PR [48]. These early studiesshowed that LSDs should be generally amenable to therapyrelying on reconstitution of the deficient enzymes by exogene-ous administration of a functional version, a concept which isknown as ERT. In some cell types, exogenous lysosomalenzymes are not recognized by the M6PR but rather by otherreceptor systems which bind, for example, terminal galactose(hepatocytes) or mannose residues (macrophages) [49]. Thatseveral receptor systems exist, needs careful consideration dur-ing the development of effective ERTs, but also holds oppor-tunities for changing or modifying the targeting signals of agiven enzyme in order to manipulate its pharmacodynamicproperties. A classic example is the modification of glucosyl-ceramidase that in order to be targeted to macrophages inGaucher disease, has to be modified in order to exposemannose residues [49].

The success of ERT in Gaucher disease has made thisapproach the standard for treating lysosomal storage disorders(Table 1), although the first clinical trial of an ERT in1973 (GM2-gangliosidosis) was not a clinical success althoughit did confirm the biochemical principle. In this trial, an infantwas injected with hexosaminidase A that had been purifiedfrom human urine, resulting in a remarkable reduction of

the storage substance in the circulation. However, the patient’sclinical condition remained unchanged [50]. The success oftreating Gaucher and the apparent failure in treatingGM2-gangliosidosis stresses the crucial point, that therapiesshould be developed that affect the primary sites of pathology.Unfortunately for the LSDs, the major organ involved in mostof these diseases is the CNS which by no means is an easyorgan to reach and even less so to rescue.

This realization has prompted the development of a num-ber of therapies aimed not only at addressing the peripheralsymptoms of the LSDs but importantly these novel conceptsoften aim at providing a clinical benefit in terms of mani-festation of CNS-related symptoms, the holy grail ofLSD therapy.

2. Medical need

The LSDs number over 50 diseases with a combined inci-dence of approx. 1/7700 and with only a very limited numberhaving approved therapies available.

The clinical manifestations of these diseases are extremelyvariable ranging from severe debilitating, lethal diseases inearly infancy, to attenuated presentations in late adulthood,often with no clear genotype/phenotype correlation as exem-plified by Niemann--Pick disease type C in which the diseasecan vary from severe, lethal infantile disease to a more psychi-atric symptom-driven disease that gradually manifest itselfduring the later decades [51].

The current standard of treatment has evolved from mainlysupportive care and symptomatic treatment to the standard oftoday, ERT, that became available to patients with Gaucherdisease two decades ago and now has also been developedand approved for other LSDs such as Fabry disease, MPStypes I, II and VI and Pompe disease (Table 1). The efficacyof many of these therapies is limited however, due to thefact that the exogenously provided enzymes do not have effecton all aspects of the diseases. This is caused in part by the irre-versibility of some aspects of these diseases but is also due tothe enzyme formulations’ inherent inability to reach all majortarget organs in therapeutically efficacious amounts. Particu-larly the CNS, but also bone, cartilage, cardiovascular andrenal systems are not necessarily efficiently targeted by enzymereplacement strategies due to the extremely selective perme-ability of the blood--brain barrier (BBB) and restrictedreceptor expression and lack of sufficient blood flow to sup-port the needed doses for efficacy in other peripheral tissuesand organs.

Furthermore, the formation of antibodies against the exog-enously delivered enzyme may have a negative impact on effi-cacy as well as elicit unwanted infusion-related adverseevents [52,53].

It comes as little surprise therefore, that there exists asubstantial unmet need for the development of therapiesaddressing the limitations described above as well as providingalternative treatment regimens that eventually might even

Emerging therapies and therapeutic concepts for lysosomal storage diseases

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supplement each other based on a rational understanding oftheir distinguishing mechanisms of action.

3. Existing and emerging therapies

The currently approved pharmacological therapies for LSDsare summarized in Table 1, while emerging therapies and ther-apeutic concepts are extensively covered in Tables 2 and 3. Ascan be readily seen, both established and emerging therapiesare dominated by ERT or second-generation variants thereof.The following sections provide a comprehensive overview

of current and emerging therapies for the LSDs and will focuson the scientific and medical rationale for these therapeuticconcepts.

3.1 Bone marrow transplantation/hematopoietic

stem cell transplantationBone marrow and hematopoietic stem cell transplantations(BMT/HSCT) can trace the origin of their scientific rationaleback to the same fundamental principle of metabolic cross-correction as the ERTs, that is, the ability of lysosomal enzymesto enter into a secretion-reuptake cycle. The first uses of trans-plantation approaches emerged in the 1980s and have seen itsuse in many of the LSDs including MPS types I, VI and VII,metachromatic leukodystrophy (MLD), alpha-mannosidosis,fucosidosis, Krabbe disease and type III Gaucher disease [54].Despite a rather extensive use of BMT/HSCT in MPS type

I and 1H (Hurler variant) and with some promising results,particularly in terms of reducing visceromegaly, cardiac func-tion and airway obstruction [55], it is unfortunately still hardto conclude decisively on the status of this potentially effective

therapy as most reports on BMT and HSCT are bothanecdotal and/or only encompass a small number of patients.

However, with the advancement of methods for HSCT andwith a more systematic approach in evaluating the therapythere is no scientific reason as to why BMT/HSCT shouldnot be a both viable and a promising therapy for many ofthe LSDs, although several challenges such as the occurrenceof variable musculoskeletal disease progression even after suc-cessful stem cell transplantation (SCT) in MPSIH patientshave to be overcome [56,57].

3.2 Enzyme replacement therapiesAs for bone marrow and hematopoietic SCTs the fundamen-tal principle of ERT is the same: the substitution of the defi-cient enzyme by a functional version hereof.

Provided the enzyme can be manufactured and safelyadministered, the administration of the enzyme usually takesplace through either weekly or biweekly infusions althoughmore frequent administrations are also seen for some ERTssuch as asfotase alfa in development for hypophosphatasia(Clinicaltrials identifier: NCT01176266).

The promise of most ERTs lies in their potential capacityto correct the pathology of non-neural tissue as the enzymesare incapable of traversing the BBB, although many peripheraltissues such as bone, cartilage, cardiovascular and renal sys-tems are not easily reached due to the biology of the receptorsystems needed for the endocytosis of the exogenouslydelivered enzymes.

Tables 1 and 2 summarize the ERTs that have beenapproved for marketing authorization and those that are incurrent development, respectively.

Table 1. Overview of marketed therapies available for LSDs.

LSD Drug Company Status Mechanism Comments

Gaucher disease Cerezyme� (imiglucerase) Genzyme On market ERTGaucher disease VPRIV� (velaglucerase alfa) Shire HGT On market ERTGaucher disease Elelyso� (taliglucerase) Protalix On market ERT Plant cell manufacture,

only approved in the USAGaucher disease Zavesca� (miglustat) Actelion On market SRT Only for Gaucher type I patients

for whom ERT is unsuitableFabry disease Fabrazyme� (agalsidase beta) Genzyme On market ERTFabry disease Replagal� (agalsidase alfa) Shire HGT On market ERT Not approved in the USA,

approved in the EU, Asiaand Canada

Niemann--Picktype C disease

Zavesca (miglustat) Actelion On market SRT Not approved in the USA,approved in the EU, Canada,Switzerland, Brazil, Australia,Turkey and Israel

MPS type I Aldurazyme� (laronidase) Genzyme On market ERTMPS type II Elaprase� (idursulfase) Shire HGT On market ERTMPS type VI Naglazyme� (galsulfase) Biomarin On market ERTPompe disease Myozyme� (alglucosidase alfa) Genzyme On market ERTCystinosis Cystagon� (cysteamine) Various On market Cysteamine suppl. On market 1994Cystinosis Cystaran� (cysteamine) Sigma-Tau

PharmaceuticalsApproved Cysteamine suppl. Cysteamine ophthalmic solution

ERT: Enzyme replacement therapy; LSD: Lysosomal storage disease; MPS: Mucopolysaccharidosis; SRT: Substrate reduction therapy.

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Table

2.Overview

ofERTsandseco

nd-generationvariants

inco

mmercialdevelopmentforLS

Ds.

LSD

Drugcandidate

Company

Status

Mech

anism

Comments

CNStargeting

Alpha-m

annosidosis

Lamazym

�(alpha-D-m

annosidase)

Zym

enex

Phase

IIERT

-Fabry

disease

Migalastat+ERT

Amicus/GSK

Phase

IICCT/ERT

Combinationapproach

-Fabry

disease

PRX-102(a-galactosidase-A)

Protalix

Phase

I/II

ERT

Plantcellmanufacture

-Fabry

disease

JR-051(a-galactosidase

A)

JCRPharm

aceuticals/GSK

Preclinical

ERT

Gaucherdisease

JR-101(glucocerebrosidase)

JCRPharm

aceuticals/GSK

Preclinical

ERT

-Gaucherdisease

Oralglucocerebrosidase

Protalix

Preclinical

ERT

OralERT

-Krabbedisease

Galaczym

�(galactocerebrosidase)

Zym

enex

Preclinical

ERT

-Late

infantile

NCL

(Batten)

BMN-190(TPP1)

Biomarin

Preclinical

ERT

-

MLD

IntrathecalHGT1110(arylsulfatase

A)

ShireHGT

Phase

I/II

ERT

Intrathecaldelivery

+MLD

AGT-183(HIRmAb-arylsulfatase

A)

Arm

agen

Preclinical

Second-generation

ERT

Insulin

receptorantibody

fusedto

arylsulfatase

A+

MPStypeI

AGT-181(HIRmAb-iduronidase)

Arm

agen

Preclinical

Second-generation

ERT

Insulin

receptorantibody

fusedto

iduronidase

+

MPStypeII

IntrathecalElaprase

�

(iduronate-2-sulfatase)

ShireHGT

Phase

I/II

ERT

Intrathecaldelivery

+

MPStypeII

AGT-182(HIRmAb-iduronate-2-sulfatase

Arm

agen

Preclinical

Second-generation

ERT

Insulin

receptorantibody

fusedto

iduronate-2-sulfatase

+

MPStypeII

JR-032(iduronate-2-sulfatase)

JCRPharm

aceuticals/GSK

Preclinical

ERT

-MPStypeIIIA

IntrathecalHGT1410

(heparanN-sulfatase)

ShireHGT

Phase

I/II

ERT

Intrathecaldelivery

+

MPStypeIIIB

IntrathecalHGT3010

ShireHGT

Preclinical

ERT

Intrathecaldelivery

+MPStypeIIIB

SBC-103(a-N-acetyl-glucosaminidase)

Synageva

Preclinical

ERT

-MPStypeIVA

BMN-110GALN

S(N-acetylgalactosamine-6

sulfatase)

Biomarin

Phase

IIIERT

-

MPStypeVII

UX003(b-glucuronidase)

Ultragenyx

Preclinical

ERT

-Niemann--PicktypeB

ASM

Genzyme

Phase

IERT

-Pompedisease

Neo-rhGAA

Genzyme

Preclinical

Second-generation

ERT

Carbohydrate

remodeling

totargetCI-M6PR

-

Pompedisease

BMN-701(IGF2-G

AA)

Biomarin

Phase

ISecond-generation

ERT

Fusionprotein

ofIGF2

peptideandGAA

+

Pompedisease

Duvoglustat+ERT

Amicus

Phase

IICCT/ERT

Combinationapproach

-Pompedisease

OXY2098-85(m

odifiedGAA)

Oxyrane

Preclinical

Second-generation

ERT

Engineered95kDa

precursorto

target

CI-M6PR

-

Wolm

andisease

Sebelipase

alfa(lysosomalacidlipase)

Synageva

Phase

IIERT

-UndisclosedLSDs

p97(m

elanotransferrin)/ERTconjugate

ShireHGT/biOasis

Discovery

Second-generation

ERT

p97to

facilitate

transport

ofERTacross

BBB

+

UndisclosedLSDs

Angiopep-ERTconjugate

GSK/Angiochem

Discovery

Second-generation

ERT

Angiopeptargets

LRP-1

tofacilitate

transport

across

BBB

+

ASM:Acidsphingomyelinase;BBB:Blood--b

rain

barrier;CCT:Chemicalchaperonetechnology;

CI-M6PR:Cation-independentmannose-6-phosphate

receptor;CNS:Centralnervoussystem;ERT:Enzymereplacement

therapy;

GAA:Acidalpha-glucosidase;HIRmAb:Monoclonalantibodyto

thehumaninsulin

receptor;IGF2:Insulin-likegrowth

factor2;LRP-1:Low-density

lipoprotein

receptor-relatedprotein

1;LSD:Lysosomalstorage

disease;MLD

:Metachromaticleukodystrophy;

TPP1:Tripeptidyl

peptidase-1.

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The first marketed ERT was developed for type I Gaucherdisease, a sphingolipid storage disorder, characterized bysplenomegaly, thrombocytopenia and anemia. The first clini-cal trial was performed by Brady and collaborators with anenzyme preparation purified from human placenta, treatedwith specific glycosidases to facilitate uptake by mannosereceptors on the macrophages which is the main cell typeinvolved in the disease [49]. Based on the positive data fromthis trial, the enzyme preparation (Ceredase�, Genzyme)was approved for patients with Gaucher disease and wassome years later replaced by a recombinant form, imiglucerase(Cerezyme, Genzyme). A number of reports and publicationshave confirmed the long-lasting positive effect as well as thesafety and tolerability of imiglucerase in patients sufferingfrom type I Gaucher disease which has led to ERT becomingthe standard of care for these patients [58,59] but does not haveany influence on the CNS symptoms of the disease as seen inthe neuropathic forms of the disease, not even at highdoses [60].The initial success of imiglucerase, has led to the develop-

ment of ERT for other LSDs such as Fabry disease, Pompedisease and several of the MPS. In Fabry disease for example,

two different a-galactosidase enzymes have been developed,but although Fabry disease is a glycosphingolipidosis just asGaucher disease, its pathology is significantly different fromthat of Gaucher, giving rise to a number of challenges mainlyassociated with the proper engagement of the ERT with thetarget organs. In Fabry disease, kidney failure, cardiomyopa-thy and cerebrovascular events are the main complicationswhich give rise to the morbidity and mortality associatedwith this disease while most clinical efficacy measurementshave been based on plasma Gb3 levels (the main accumulat-ing lipid) [61,62]. While plasma Gb3 levels are quite easilyquantifiable and respond readily to intravenous injections ofERT, the main organs giving rise to disease symptomatologyare neither as readily accessible to therapy nor as well investi-gated [61]. This inconsistency in addressing clinically relevantend points has made a clear conclusion on the benefits ofERT for Fabry disease difficult and the experience withERT for Fabry disease has not been as satisfying as that inGaucher disease, in which the pathological storage mainlyinvolves a more easily targetable cell population. Also,recently the central role of Gb3 in Fabry disease and its rele-vance as a surrogate marker has been questioned as the lyso

Table 3. Overview of non-ERT emerging therapies in commercial development for LSDs.

LSD Drug candidate Company Status Mechanism Comments CNS

targeting

Multiple LSDs(sphingolipidosesamongst others (ao))

Orph-001 (Hsp70) Orphazyme Preclinical MCT Main receptor LRP-1,targets BBB

+

Multiple LSDs Orph-002(orally availablesmall molecules)

Orphazyme Preclinical MCT Non-toxic, small moleculemolecular chaperoneinducers, BBB penetrating

+

Multiple LSDs(gangliosidoses)

Not disclosed ZacharonPharmaceuticals

Discovery SRT Ganglioside synthesisinhibition

+

Multiple LSDs(MPS types I, II and III)

Not disclosed ZacharonPharmaceuticals

Preclinical SOT Inhibition of 2-O sulfationof heparan sulfate

+

Gaucher disease AT2101 + ERT,AT3375 + ERT

Amicus Preclinical CCT/ERT Combination approach -

Fabry disease Migalastat Amicus/GSK Phase III CCT Study did not meetprimary end points

-

Fabry disease Migalastat + ERT Amicus/GSK Phase II CCT/ERT Combination approach -MPS type IIIA SAF-301

(AAVrh.10-SGSH- SUMF1)

Lysogene Phase I/II GT rh.10 serotyped adeno-associated virus carryingthe deficient SGSH andSUMF1 gene

+

MPS type IIIB Viral vector carrying a-N-acetylglucosaminidase

UniQure Phase I/II GT +

Niemann--Pick type C HPB-cyclodextrin NICHD Phase I Cholesterol sequesteringagent, ICV administration

+

Pompe disease Duvoglustat + ERT Amicus Phase II CCT/ERT Combination approach -Cystinosis RP103 Raptor

PharmaceuticalsRegistration Slow release

cysteamineReformulation approach -

Combination approaches with ERT have been included.

BBB: Blood--brain barrier; CCT: Chemical chaperone technology; CNS: Central nervous system; ERT: Enzyme replacement therapy; GT: Gene therapy; Hsp70: Heat

shock protein 70; ICV: Intracerebroventricular; LRP-1: Low-density lipoprotein receptor-related protein 1; LSD: Lysosomal storage disease; MCT: Molecular

chaperone therapy; MPS: Mucopolysaccharidosis; NICHD: National Institute of Child Health and Human Development; SOT: Substrate optimization therapy;

SRT: Substrate reduction therapy.

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derivative of Gb3 (globotriaosylsphingosine) may be a morepathologically relevant metabolite [63,64].

The findings in Fabry disease highlights the importance fora given therapy to reach the clinically relevant target organs intherapeutically efficacious doses, and the need to monitor thisrigorously to aid conclusions on the effectiveness of a giventherapy. It also points to the challenge that not only for Fabrydisease, but for many of the LSDs, the current understandingof their molecular pathology is still far from complete.

If one turns to another group of LSDs, the MPS, the pat-tern observed for Fabry disease repeats itself. In MPS, thereare 11 known genetic deficiencies all involving enzymes thatare part of the catabolism of glycosaminoglycans (GAGs), giv-ing rise to seven distinct MPS types. As with Fabry disease, theMPS are multisystemic diseases, affecting a multitude of celltypes in a variety of tissues [65]. For MPSI, II and VI, ERTis now available [66-69], while first- or second-generationERTs are also in development for several of the diseases(Table 2). For all of the approved ERTs, hepatosplenomegalyresponds rapidly to the administration of intravenous enzymewith a concomitant reduction in urinary excretion of GAGsbut as for Fabry disease, the main disease burden lies notwithin the easiest accessible organs, as the functional problemsthe MPS patients suffer are rather due to skeletal dystosis andinvolvement of the soft tissues, heart and lungs. The clinicalefficacy of ERT when measured in terms of joint mobility,vital capacity and walk tests, has not been nearly as clear asthe effects observed on surrogate markers, and while theERTs have evidently not been able to influence the CNSmanifestations of the diseases, they have to some extent beenable to alter the natural history of the disease and can insome cases improve the patient’s quality of life. As for theother LSDs, the earlier the intervention, the better.

The latest ERT marketed for a new LSD indication isalglucosidase alfa for the treatment of Pompe disease (glyco-gen storage disease type II, acid maltase deficiency), which isprimarily affecting the muscle tissue, enabling a better ERTtargeting of the clinically relevant tissue. For this disease, asfor many of the LSDs, there is a broad spectrum of diseasepresentation with the most severe, infantile onset cases pre-senting in the first weeks after birth with both cardiovascularand skeletal myopathy while later onset cases usually sees asparing of the cardiovascular tissue but manifesting itselfwith a progressive proximal myopathy which can lead torespiratory failure, if involving the musculature of the dia-phragm. The clinical efficacy of this ERT has been veryencouraging with early intervention in the infantile casesbeing lifesaving, even in cases of advanced disease [70,71]. Theefficacy of the ERT has furthermore been confirmed by sev-eral clinical trials in patients with different age of onset anddisease severity and alglucosidase alfa has received approvalfor treatment of Pompe disease for all age groups [72].

It is evident that several factors define the clinical success ofa given ERT: the primary challenge for any ERT lies in itsclinical efficacy on the organ systems involved. As described,

these systems vary from disease to disease but are often organswhich are not readily accessible to ERT.

Although there are reports of enzymes successfully crossingthe BBB in animal models of MLD and alpha-mannosido-sis [73,74], clinical data for all commercially available ERTshave not shown any evidence of ERTs being able to penetratethe BBB and provide a therapeutic benefit to the patients. It istherefore of little surprise that all the second-generation ERTs(as well as many of the other emerging therapies) are aiming ateither crossing the BBB and/or increasing the uptake ofenzyme into the relevant peripheral tissue.

Of the latter there are currently several biologically similarapproaches in development for the treatment of Pompe dis-ease in which the primary tissue to target is the muscle tissue.Whether through carbohydrate remodeling of the enzymeitself, its conjugation to a IGF2-derived peptide tag or theengineering of a precursor form of the enzyme, all of thesesecond-generation approaches aim at changing the biodistri-bution toward increased uptake in muscle tissue of the activeenzyme by increasing its affinity to the CI-M6PR [75-77].

When it comes to targeting second-generation ERTs fortransport across the BBB several approaches are in develop-ment. One approach is intrathecal delivery of standard ERTwhich is in clinical development for MPSII, MPSIIIA andMLD. Another and well-characterized alternative approachis the more physiological approach of utilizing endogeneousreceptor systems for enzyme transcytosis across the BBB, anapproach that is considered to have several advantages com-pared with the rather invasive surgical procedure of intrathecalinfusion of enzymes [78]. Although several receptor systemshave been characterized that facilitate transport across theBBB, for the LSDs two main receptor systems are currentlybeing targeted by various companies in the hope that thesewill provide access for the engineered enzymes to the neuronsof the CNS in therapeutically relevant doses. These emergingapproaches are targeting enzymes for the treatment of MPSI,MPSII, MLD and other LSDs to the CNS via either theinsulin receptor or the probably best characterized blood--brain transcytosis receptor, LRP-1 (low-density lipoproteinreceptor-related protein 1) (CD91) [79-84].

For the targeting of the insulin receptor, engineered versionsof the enzyme are fused to the carboxyl terminus of the heavychain of a chimeric monoclonal antibody (mAb) to the humaninsulin receptor (HIR). These HIRmAb--enzyme fusion pro-teins then cross the BBB via the endogenous insulin receptorand acts as a so-called molecular Trojan horse to ferry theenzyme into brain with approximately 2 -- 3% of injecteddose reaching the brain [80,85].

The LRP-1 and -2 have been exploited to target a largevariety of drugs to the brain and LRP is the best characterizedsystem for BBB penetration to date [78]. LRP-1 has a numberof physiological ligands and this has formed the basis for twoalternate fusion protein approaches, one relying on the conju-gation of p97/melanotransferrin to the enzyme of interest, theother on an optimized peptide, a so-called angiopep, with

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increased affinity for the LRP-1 receptor [82,84]. Interestingly,the human molecular chaperone Hsp70 which is being devel-oped for the treatment of a panel of LSDs and covered later inthis review also utilize LRP-1 as one of its primaryreceptors [86].Besides the above, a number of different targeting systems

are being explored academically, such as intercellular adhesionmolecule (ICAM) and apolipoprotein B (ApoB)-mediatedcarriers [87,88] but it is considered beyond this review to coverall early discovery pharmacology not in a company orcompany-related pipeline.Based on the preclinical activity surrounding the second-

generation ERTs, one can only hope that any of theseapproaches will prove successful, but a number of challengesare facing the development of these more extensively engi-neered enzymes: Although promising data have been gener-ated in murine models of the disease for many of thesecompounds, only a marginal ratio of the total injected enzymebecomes available to the CNS and it remains to be seen if thisamount of enzyme can confer the same therapeutic benefit inhuman subjects. This challenge unfortunately only becomesharder when one considers that chronic administration ofthese modified enzymes will be necessary for sustained effectin patients as this will almost certainly give rise to significantantibody responses, as has been seen for all ERTs, albeit tovarious degrees.Also, the use of any receptor system begs the question as to

what effect will be caused by the extra-physiological use ofsuch a system to deliver drugs, that is, could any adverse sig-naling cascades be activated or will the natural receptor half-life, distribution and activity be compromised by this extrautilization. Also, does the receptor actively transport thedrug across the BBB or is the drug just binding to the receptorand sequestered in the BBB endothelium? For LRP-1 forexample, these considerations are not necessarily a majorproblem as the receptor is rather promiscuous with manyligands already using the receptor and as it also has one ofthe fastest transcytosis rates (transfer coefficient/Kin) of anyBBB receptor system [89], adverse receptor signaling, satura-tion and re-distribution as well as drug sequestration in theendothelium should not pose a significant risk for this systemalthough this of course remains to be tested clinically.Given all the challenges that remain to be faced by the

second-generation ERTs, one should bear in mind however,that significant clinical benefit has been achieved for other dis-eases in which receptor systems have been exploited, for exam-ple, in the case for dopamine/L-Dopa for Parkinson’s diseasepatients, in which the large neutral amino acid carrier hasbeen used to deliver L-Dopa, the metabolic precursor of dopa-mine, to the brain resulting in a clear clinical benefit asdopamine in itself is not able to cross the BBB.

3.3 Substrate reduction therapiesWhereas ERTs focus on increasing the catabolism of build-up substrate, the principle in substrate reduction therapy

(SRT) is to limit the production of substrate to the cataboli-cally compromised lysosomes. The first demonstration ofthis principle was done by Platt et al. in 1994 with theimino sugar N-butyldeoxynojirimycin (NB-DNJ, miglustat,Zavesca�) which has the ability to inhibit the enzymatic activ-ity of ceramide glucosyltransferase (glucosylceramide syn-thase) which synthesizes glucosylceramide, the precursor ofseveral glycosphingolids such as the globo- and gangliosides,and which is the main accumulating lipid in Gaucher dis-ease [90]. Miglustat was subsequently tested in a clinical trialwith 28 Gaucher disease patients, who for several reasonsdid not receive ERT and on basis of this trial miglustat gainedmarketing approval in Europe and the USA for the treatmentof adult patients with mild to moderate type 1 Gaucherdisease for whom ERT is not a therapeutic option [91].

An SRT based on the inhibition of ceramide glucosyltrans-ferase holds the potential of being a therapy for all LSDs withglycosphingolipid storage and since miglustat crosses theBBB, this therapy has been evaluated for a number of sphin-golipidoses with prominent neurodegeneration such asTay--Sachs disease, type 3 Gaucher disease, MPSIII, juvenileGM2-gangliosidosis and Niemann--Pick type C disease [92-96].Except for Niemann--Pick type C disease, none of these trialshave shown improvement in the miglustat-treated patientsalthough some of these data should be handled with care asthe studies were done on very limited numbers of patients.For Niemann--Pick type C, miglustat received marketingapproval in Europe in 2009 based on a clinical trial in patientsaged 12 or older, which demonstrated that treatment withmiglustat improved eye movement velocity and swallowingcapacity [95].

Being a small molecule sugar analog, the side-effect profileof miglustat is significantly different from the side-effectprofiles associated with ERTs with miglustat having a broaderarray of side effects including gastrointestinal symptoms,particularly diarrhea.

A conceptually similar, but chemically different, approachfor SRT centered on ceramide-based inhibitors of ceramideglucosyltransferase provides a novel alternative to the iminosugar-based SRT. Based on this approach, a new inhibitorof ceramide glucosyltransferase, eliglustat tartrate (Genz-112638) is currently in development for Gaucher diseasetype I, and has shown promising results in an open-labelPhase II trial, combining a higher specificity for ceramideglucosyltransferase with a more beneficial side-effect profilecompared with miglustat and having a clear effect on severaldisease parameters [97].

For the MPS, the accumulation of GAGs can be inhibitedby genistein, an isoflavone extract from soybeans beingexplored academically. The effect of genistein on urinaryGAG excretion, hair morphology and behavior has beentested in an open-label study of 10 patients suffering fromeither MPSIIIA or IIIB and a 2-year follow-up including eightpatients assessing the cognitive function and general statuswas recently published. Albeit consisting of a small number

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of patients, after 1 year of oral administration of a genistein-rich soy isoflavone extract (5 mg/kg/day), a statistically signif-icant improvement was observed with a larger variance inefficacy being apparent after 2 years [98].

3.4 Chaperone technologiesAs most LSDs are characterized by significantly reducedenzyme activity due to missense mutations rather than acomplete loss of function, the LSDs have long been thoughtamenable to chaperoning by chemical substrate mimicstargeting the active site of the relevant enzyme for increasedstability/folding.

A more recent approach relies on utilizing the already exist-ing molecular chaperone machinery available in the cells inorder to avoid the inherently counterproductive mechanismof enzyme inhibition associated with chemical chaperonetherapies.

The advantages to both approaches compared with ERTinclude better distribution profiles including CNS availabilityas well as easier drug administration as the small moleculeapproaches for both concepts offer the potential of oraladministration rather than the more patient-demandinginfusions of ERT.

3.4.1 Molecular chaperone technologiesThere are currently two approaches in development for utiliz-ing the naturally occurring molecular chaperone machinery,both exploiting the recently discovered mechanism for howthe archetypical molecular chaperone Hsp70 aside from itswell-characterized cytoprotective effects also enhances cell sur-vival and functionality through a direct lysosomal action [43].One approach relies on the receptor-mediated endocyticuptake of a recombinant version of Hsp70 whereas the secondapproach relies on utilizing small molecules capable ofenhancing the endogenous production of heat shock proteins,here amongst Hsp70. Hsp70 is an evolutionarily highly con-served molecular chaperone which has been shown to pro-mote the survival of stressed cells by inhibiting lysosomalmembrane permeabilization [99-101], a hallmark of stress-induced cell death [6,102]. Recently, Kirkegaard et al. describedhow Hsp70 stabilizes lysosomes by binding to the endolysoso-mal anionic phospholipid bis(monoacylglycero)phosphate(BMP), an essential co-factor for lysosomal sphingolipidmetabolism hereby facilitating the BMP binding andincreased activity of acid sphingomyelinase (ASM), theenzyme compromised in Niemann--Pick diseases. Notably,the reduced ASM activity in cells from patients withNiemann--Pick disease A and B was shown to associate witha marked decrease in lysosomal stability, and this phenotypeas well as the pathological accumulation of unstable lysosomescould be effectively corrected by treatment with recombinantHsp70. The mechanism of action of Hsp70 entails the pros-pect of using the protein for the treatment of several LSDs,most notably the sphingolipidoses involving enzymes thatare dependent on interaction with BMP [103] and it is

currently in preclinical development for a number ofthese diseases.

The approach to utilize small molecules to increase theexpression of heat shock proteins during pathological stressconditions and harness this response for therapeutic use, stemsfrom the ability of these molecules to stabilize the transcrip-tion factor for the heat shock proteins, heat shock factor-1 (HSF-1) [104]. Interestingly, this emerging approach forLSDs is also in development for a number of neurodegenera-tive conditions, including amyotrophic lateral sclerosis andhas a well-described safety record with very limited sideeffects, which could possibly accelerate the development ofthis therapeutic concept for LSDs [105].

3.4.2 Chemical chaperone technologiesContrary to the molecular chaperone approach which utilizesthe potentiating effects of endogenous cellular chaperones,chemical chaperone technologies rely on using competitiveinhibitors of lysosomal enzymes at subinhibitory concentra-tions in order to facilitate the transition of poorly foldedlysosomal enzymes otherwise caught in the ER/proteasomaldegradation machinery to the lysosomes as first describedby Fan et al in 1999 [106]. On maturation and entry in thelysosomes, the concept demands that the kinetics of theenzyme/inhibitor interaction are shifted due to for example,the reduced pH of the lysosomes, facilitating the dissociationof the inhibitor and the enzyme, thus finally leaving a largerfraction of the functionally compromised enzyme availablefor increased substrate degradation in the lysosome [106,107].

A number of molecules are in development for LSDs basedon this approach, including combination efforts with ERTs.The most advanced program for a LSD utilizing a chemicalchaperone as stand-alone therapy is deoxygalactonojirimycin(DGJ; migalastat hydrochloride) which is currently inPhase III for Fabry disease. Despite a considerable powerin this study as 67 patients diagnosed with Fabry diseasewith genetic mutations amenable to chaperone monotherapywere enrolled, the study recently reported an initial negativeoutcome, as it did not meet any of its primary end pointsduring its first phase [108].

Although not in formal development programs for LSDs, theFood and Drug Administration (FDA)-approved drugs pyri-methamine and ambroxol have been identified as possiblechemical chaperones for hexosaminidase A and ceramide gluco-syltransferase and both have been tested in cells from patientssuffering from late-onset forms of GM2-gangliosidosis(Tay--Sachs and Sandhoff disease) and Gaucher disease [109,110].Recently, data from a small-scale open-label Phase I/II clinicalstudy of the tolerability and efficacy of pyrimethamine inSandhoff disease patients have been reported [111]. A significantside-effect profile was observed at doses of 75 mg pyrimeth-amine daily, while variable enzyme activity enhancement wasseen at 50 mg/day. Although the design of the study does notallow for proper conclusions, the significant side-effect profilecharacterized by neurological side effects such as ataxia and

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incoordination experienced in all subjects of the study, and thevery narrow window to the dose conferring a seeminglyincreased enzymatic activity presents significant challenges forthe further development of this compound.Combination therapies of chemical chaperones and ERTs

are being pursued for Gaucher, Fabry and Pompe diseasesand are currently in preclinical (Gaucher) and Phase II (Fabryand Pompe) stages of development. Given the recent develop-ment challenges of the chemical chaperones (duvoglustat andmigalastat) for Pompe and Fabry disease, respectively, theevaluation of these inhibitors in ERT combination studiesfor Pompe and Fabry diseases will be interesting.

3.5 Substrate optimizationThe mechanism of action of current therapies targeting thesubstrates accumulating in LSDs such as miglustat and eli-glustat tartrate is focused on the inhibition of enzymes nec-essary for substrate biosynthesis and as such this approachentails the risk of reducing the substrate to a level where itsnormal functions are compromised. As an alternative tothis, a concept has been described in which small moleculesare used, not to prevent synthesis of substrates, but rather tomodify their biosynthesis in order to change the structure ofthe substrate, which then no longer is dependent on the defi-cient enzyme for degradation but can be degraded by alter-native enzymes with normal function [112]. This strategyhas been termed substrate optimization therapy and is cur-rently in development for MPS types I, II and III in whichthe targets for the substrate optimization are glucosamino-glycans (GAGs). By compound library screening, 15 inhibi-tors of GAG synthesis were identified that can be categorizedinto N-, 2-O-, or 6-O sulfation inhibitors. A 2-O sulfationinhibitor, ZP2345, could reduce the levels of 2-O sulfationwith a compensatory increase in 6-O sulfation of heparansulfate with the modified heparan sulfate being more amena-ble to degradation in vitro in fibroblasts from MPS type IIpatients (iduronate sulfatase deficiency) [113]. Albeit stillearly stage, the technology has the potential of being ableto target more than one MPS with the same compound aswell as being able to cross the BBB and other organs thathave proven hard to reach for ERTs.

3.6 Gene therapyA number of gene therapeutic approaches using retro-, lenti- oradeno-associated viral vectors have been evaluated pharmacolog-ically in a comprehensive array of animal models of LSDs andthe general conclusion is that this therapeutic approach hasshown clear disease-modifying capacity in vivo [114]. The mainchallenges that remain to be overcome are the transfer of thesefindings to larger brains (the human brain volume is ca. 2000times that of the mice, making efficient pan-cerebral delivery achallenge even with intracranial injections), the possible immu-nogenic responses to the viral vectors carrying the gene of inter-est as well as the potential need for immune suppression for thepatients who are complete null for the enzyme.

Despite these challenges, a number of clinical trials havebeen initiated for the LSDs, but as is the case for many trialswithin the LSD field the trials are featuring small number ofpatients and solid conclusions are hard to make as only asmall number of studies have been completed. Reports oncompleted studies of gene therapy in Gaucher disease andMPS type II using retroviral vectors showed only low expres-sion of the gene product and no improvement in diseasepathology [115,116]. However, these were the first two clinicaltrials of gene therapy in LSDs and since their initiation morethan a decade ago there has been a marked developmentand improvement of vector design and delivery which willhopefully lead to improved results. Although a recently com-pleted study in Batten disease involving direct injection of arecombinant adeno-associated viral (rAAV) serotype 2-basedvector into the CNS of affected children indicated that pro-gression of disease might have been slowed, a clear therapeu-tic benefit was not established and significant serious adverseevents were also encountered, the causes of which couldnot be identified [117]. However, as for the retroviruses,rAAV-based gene therapy has also evolved substantially sincethe initiation of this study and several studies are underwayutilizing other rAAV serotype vectors in for example, Pompedisease [118] and MPS types IIIA and IIIB, the latter twobeing researched and developed in commercial settings.The ongoing Pompe disease study uses a rAAVserotype 1 (rAAV1) vector encoding acid-a-glucosidase,and recently reported preliminary findings [118]. No adverseevents or systemic toxicities related to vector administrationwere reported and significant elevation in respiratoryparameters was noted for the first cohort of patients on thelower dose of treatment. These findings are encouragingfor the development of gene therapies for LSDs, but thehope that all pathological components of a given LSD canbe corrected by a systemic delivery of a single vector is prob-ably too optimistic as the complex nature of these devasta-ting diseases makes even this approach extremely difficult.A number of factors such as the timing of gene transfer inrelation to diagnosis and symptom onset/aggravation,the variable levels of gene product needed to efficaciouslytreat various organ pathologies and the possible immuneconsequences related to administration procedures, choiceof vector and naivety to gene therapy product will allhave to be addressed in order to bring about the fullpotential of this therapeutic approach.

On top of these scientific and development challenges,challenges regarding regulatory considerations and commer-cial feasibility of developing gene therapies are also consider-able. Very encouragingly however, for gene therapy as afuture therapy for a number of genetic diseases, not only theLSDs, the first European regulatory approval of a gene ther-apy was recently announced, with this therapy also beingbased on an rAAV1 vector, AAV1-LPLS447X (alipogenetiparvovec, Glybera�) targeting lipoprotein lipase deficiency,an ultra-rare genetic disease [119].

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3.7 Other therapies and emerging approachesThe LSD cystinosis involves lysosomal storage of the aminoacid cystine in all organs and tissues due to a defect in thelysosomal membrane transport protein, cystinosin, and wasthe first LSD recognized to be due to defective lysosomalmembrane transport, and thus serves as a prototype for a smallgroup of lysosomal transport disorders. In 1994, a novel ther-apy was approved based on depleting cysteine in the form oforally administered cysteamine bitartrate (Cystagon�), whichhas revolutionized the management and prognosis of nephro-pathic cystinosis [120]. On administration, cysteamine entersthe lysosomes and reacts with cystine, forming the mixeddisulfide of half cystine (cysteine) and cysteamine. This com-plex can then exit the lysosomes via the transport system forcationic amino acids [121]. The efficacy of cysteamine hasbeen validated in a number of studies and cysteamine therapyshould be considered for all affected individuals, regardless ofage and transplantation status [122].

The side-effect profile of cysteamine includes unpleasanttaste, nausea and other digestive issues with the most commonside effect being nausea that can be alleviated with antiemeticsin the early stages of therapy initiation [122]. A different routeof administration targets photophobia associated with the dis-ease as topical cysteamine eye drops, administered every1 -- 2 h, dissolve corneal crystals and ameliorate this part ofthe pathology within a few weeks [123].

As nonsense mutations have been identified in a number ofLSDs, leading to premature translation termination and thesynthesis of truncated protein as in the case of the Arg220Xmutation in Fabry disease, and as there exist evidence thatsmall molecule drugs such as gentamicin can induce the read-though of such premature stop codons, bringing aboutincreases in otherwise null enzymatic activity, the concept ofstop-codon readthrough has been explored in the severeform of MPS type I (Hurler disease). In cell cultures of patientfibroblasts carrying different nonsense mutations, gentamicintreatment increased the a-L-iduronidase activity in all celllines tested except one providing an initial in vitro proof-of-concept for this approach [124]. Further development of ami-noglycoside analogs exhibiting reduced cell toxicity and supe-rior readthrough efficiency compared with gentamicin mighthold hopes for an even better therapeutic future for thisemerging concept [125]. In addition to the aminoglycosides, anovel chemical compound was recently identified, whichselectively induces ribosomal readthrough of premature, butnot normal termination codons [126]. This compound,PCT124 (ataluren), has entered clinical trials and mighthold great potential for genetic disorders such as cystic fibro-sis, for which no other therapeutic options are available [127].

As exemplified with the use of cysteamine for cystinosis, thereduction of storage material might not only be achieved byenhancement of enzymatic activity or inhibition of substratesynthesis as covered in the previous sections. Accumulatingsubstrates might also be eliminated by substances such as2-hydroxy-propyl-b-cyclodextrin, which is capable of binding

unesterified cholesterol and other hydrophobic molecules. Asunesterified cholesterol is one of the major storage com-pounds in Niemann--Pick type C disease, the compound hasbeen tested in both the murine and feline model of the dis-ease. Encouraging data from the most commonly used mousemodel of the disease (the NPCnih model) led to the FDAapproval of a compassionate use trial of cyclodextrin in a smallnumber of patients suffering from advanced Niemann--Picktype C disease [128-130]. As cyclodextrins have been widelyused as formulation vehicles to increase the amount of drug,including hormones and vitamins, which can be solubilizedin aqueous vehicles [131], its use and toxicological profile hasbeen extensively studied in rodents, dogs and monkeys whereit is well tolerated at low doses [131,132]. However, daily i.v.administration of greater than 200 mg/kg caused reducedbody weight, foamy macrophage infiltration of the lungs, ele-vations in hepatic enzymes, increased Kuppfer cells in the liverand renal cortical tubular vacuolization in rodents [131,133,134].

Doses used to reach therapeutic effect in the murine modelof Niemann--Pick type C are several-fold higher than thedoses at which no adverse events are seen (4000 mg/kg usedin the NPC mouse models) and apart from the availabletoxicological data in healthy animals increasing data fromanimal models of LSDs strongly suggest that the use ofthis compound for any LSD should be carefully evaluated asa number of side effects have been seen on treatmentincluding hearing loss and increased cholesterol burden andmacrophage infiltration of the lungs [128,135-137]. Furthermore,as cyclodextrin does not cross the BBB [138] and as its use inother murine models of cholesterol-storing LSDs such asGM1-gangliosidosis and MPSIIIA had no effect on stor-age [130], it is clear that the mechanism of action is not fullyunderstood. Addressing these challenges will hopefully aidthe development and future therapeutic approaches relyingon this class of compounds.

Based on the complex pathology of the LSDs and the variouscellular and biological organelles and processes involved, anumber of experimental strategies, some including commer-cially available compounds, are being researched for their useas disease modifiers. These approaches include calcium modu-lation, enhancing exocytosis, regulation of proteostasis, modu-lation of autophagy and the use of non-steroidal anti-inflammatory drugs [42,139-145]. While most of these approachesare at an early stage and still have a long way ahead to the clinic,it is clear that a multifaceted approach is most likely needed toaddress the complex pathology of LSDs. No doubt, as theunderstanding of disease pathology advance, additional creativeapproaches to treatment will emerge and undergo similar earlydevelopment with the hope that any of these approachesultimately lead to clinical benefit for the patients.

4. Competitive environment

Table 1 summarizes the current status of the competitiveenvironment for LSD therapies with market approval,

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while Tables 2 and 3 summarize the various emerging thera-pies and concepts and provide a thorough overview of the sta-tus of the programs in primarily commercial developmentpipelines for the LSDs.The emerging concepts being explored for LSDs with high

unmet clinical needs or unaddressed pathology such as CNSdeterioration is an exciting field which holds a number ofpromises for complimentary mechanisms of action offering apotentially larger arsenal of drugs for future therapy but inaddition hereto, a number of primarily ERT projects arealso being developed for LSDs with established and effectivetherapy as for example, type 1 Gaucher disease.For type 1 Gaucher disease, three therapies are already

commercially available (two ERTs (imiglucerase and velaglu-cerase alfa) and one SRT (miglustat)), with two ERT pro-grams, a CCT/ERT combination program and an SRT alsocurrently in development. The two ERT programs are basedon alternative manufacturing systems and whether the prod-ucts of these programs have significant differentiating factorswith therapeutic relevance to the established therapies willbe interesting to follow. Interestingly, one of these products,taliglucerase (Elelyso�, Protalix/Pfizer), was recently approvedby the FDA (1 May 2012), but was rejected marketingapproval by the EU commission as velaglucerase alfa(VPRIV�) has 10 years marketing exclusivity under theorphan drug framework.

5. Expert opinion

The current state of the LSD field is both complex andencouraging. Complex as the understanding of the underlyingmolecular pathology of the vast heterogeneity in clinical pre-sentation is still limited and constantly evolves. Encouragingas novel therapeutic approaches evolve from these discoveries,carrying with them the hope that they may eventually impactthe course of these devastating diseases.Among this multitude of approaches, the ERTs still reign

supreme in number of products commercially available, aswell as number of programs in the development pipelines ofpharmaceutical companies, although a number of alternativeapproaches are emerging.Gaucher disease was the first LSD for which ERT became

available and the impact made by this approach not only clin-ically but also commercially subsequently prompted thedevelopment of ERTs for a number of other LSDs. Recently,a 10-year follow-up study of Gaucher disease patients treatedwith ERT reported the lasting impact on patients health bytreatment with imiglucerase but the same degree of successhas unfortunately not been achieved with all ERTs althoughearly intervention in for example, infantile Pompe diseasehas also been a marked success as this has proved life-sav-ing [59,70]. Importantly, lessons learned from the use ofERTs in the clinic have clarified the challenges still facingERTs and these are by no means trivial; adverse immunolog-ical reactions to infusion of enzyme are common and many

sites of pathology are not effectively treated by intravenousadministration of enzyme as these sites are not easily reachedby the infused enzyme. In addition, some manifestations ofdisease has proven very hard to alleviate such as bone diseasein Gaucher disease and the MPS, renal complications in Fabrydisease and the degeneration of the CNS observed in manyLSDs. All of these sites provide therapeutic targets whichhave yet to be efficiently reached by an ERT or therapeuticvariant hereof.

A major therapeutic advancement will therefore be mole-cules which can reach these sites of pathology and a numberof the emerging therapies are indeed aiming at exactly this,with therapies being able to cross the BBB being a particularfocus for many of these development efforts. Until now onlyone drug approved for an LSD has shown indications that itmight affect CNS complications. The orally administeredSRT, miglustat, was first approved for the treatment of adultpatients with mild to moderate type 1 Gaucher disease forwhom ERT is not a therapeutic option [91] and has sincebeen approved in the EU and other countries for the treat-ment of Niemann--Pick type C based on a clinical trial, whichdemonstrated that treatment with miglustat improved eyemovement velocity and swallowing capacity indicating aneffect on CNS pathology [95]. The challenges facing SRTsare based on their inherently unspecific mechanism of actionas the inhibition of ceramide glucosyltransferase (an earlycore component in the glycosphingolipid synthesis pathway)by virtue of the sequential synthesis steps in this pathwayunavoidably affects the synthesis and equilibrium of all down-stream derivatives. Furthermore, miglustat is known to inhibitseveral glycosidases, including a-glucosidase I and II as well assucrase and maltase, which might also explain parts of its side-effect profile [146]. This inherent non-specificity raises con-cerns about long-term toxicities or adverse events for anySRT but to date the side effects such as diarrhea have beencontrollable, although there are still concerns regarding thetremors and paresthesias that develop in some treatedpatients [91]. A novel SRT in development, eliglustat tartrate,has shown promising data including a more benign side-effect profile, probably owing to its higher selectivity, butunfortunately this agent is a target for P-glycoprotein mean-ing that it is efficiently pumped out of the brain and hencewill most likely not have an impact on CNS disease inpatients [97,147,148]. Nevertheless, the higher specificity andmilder adverse events indicate that this compound hold greatpotential for being an efficacious SRT for the treatment ofperipheral disease in Gaucher disease and other sphingolipido-ses, and the promise of SRT still spurs design of new agentscombining increased specificity and brain penetrating proper-ties, providing a continued flow of potentially beneficial drugsfor a large subset of the LSDs [148].

Gene therapy strategies are a possibly very important inter-vention which has seen a breakthrough with the EU approvalof the first gene therapy (Glybera). This approach might offeran alternative to existing therapies as might oral approaches

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and approaches with mechanisms of action across diseasessuch as the SRT and molecular chaperone therapies.

In case of chaperone approaches to treating LSDs, twointriguing concepts with the potential of addressing severalLSDs including diseases with CNS involvement are in devel-opment. These are approaches that employ either endogenousmolecular or chemical chaperones, respectively. Theapproaches are inherently dissimilar as the molecular chaper-one therapy approach utilizes the endogenous chaperonemachinery to not only enhance the activity of compromisedenzymes but also provide the dysfunctional lysosomes andcells with the survival promoting benefits of the heat shockproteins [43,103].

Almost counterintuitively, chemical chaperone technolo-gies use competitive inhibitors to enhance residual enzymeactivity, with the technology having a plausible theoreticalbasis in the conformational memory of proteins. CCT usescompetitive inhibitors of lysosomal enzymes at subinhibi-tory concentrations in order to facilitate the transition ofpoorly folded lysosomal enzymes otherwise caught in theER/proteasomal degradation machinery to the lyso-somes [106]. The technology has seen widespread develop-ment with a number of programs in current clinical trials,but recent clinical data have highlighted the difficulties inusing inhibitors in already severely compromised enzymaticsystems. The most recent setback being reported onlyrecently as initial data from a Phase III randomized,placebo-controlled study of migalastat in Fabry diseaseincluding 67 patients showed that the study did not meetits primary end points [108].

A major aid to any clinical trial and a significant advance-ment to the understanding of LSDs will be the developmentof tests/biomarkers that accurately predict the disease out-comes and aggressiveness. Tools for better and early diagnosisare also needed as the often irreversible degeneration andpathology observed for many of these diseases prompt an earlyand efficient intervention in order to have the highest likeli-hood of significantly improving the clinical outcome. Thedevelopment of these tools will be important not just for theunderstanding of the diseases and their progression but willhopefully allow for better clinical trial designs and a better

definition of subpopulations of patients that will have betteror poorer responses to therapies.

As early intervention is generally considered a prerequisitefor the prevention of irreversible complications in any LSD,the development of suitable biomarkers is of significant impor-tance as their absence makes it difficult to support presymptom-atic pharmacological therapy without the capacity to monitorconsequences of the intervention. Of course, early life-saving interventions are completely unethical to withhold infor example, infantile Pompe, Krabbe and Niemann--Picktype A disease, but when should one intervene in more gradu-ally developing diseases? As the current therapies are not benignand have not only medical but also social and economical impli-cations for the patient in terms of insurability and employabil-ity, the initiation of therapy needs very careful considerationwhich will also be the case for the therapies in development.However, progress in biomarker characterization is beingmade for a number of diseases such as Gaucher disease, Fabrydisease, the MPS and Niemann--Pick type C disease [149-151].

No doubt the number of emerging therapies for LSDs isencouraging, with a lot of exciting biological mechanismsbeing explored as potential drug targets. This is however,also necessary as the complex nature of the diseases almost cer-tainly demands a combined effort targeting not only generalor specific pathology but rather aims at a shot-gun approachfor treatment utilizing a battery of therapeutic modalities todeal with the intricate molecular pathology underlying thesedevastating diseases.

Importantly, the significant cost of therapy for these veryrare diseases has to be considered not only at an individuallevel, but also by physicians and society at large as we seekto improve the life of patients with LSDs. This considerationbecomes of particular relevance in the case of combinationtherapies, which will most likely be the future way to improvethe life of patients who by odd chance have developed suchdevastating diseases.

Declaration of interest

The author is employed by and holds shares in OrphazymeApS, which develops therapies for lysosomal storage diseases.

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BibliographyPapers of special note have been highlighted as

either of interest (�) or of considerable interest(��) to readers.

1. Appelmans F, Wattiaux R, de Duve C.

Tissue fractionation studies 5 The

association of acid phosphatase with a

special class of cytoplasmic granules in

rat liver. Biochem J 1955;59:438-45.. The first paper identifying lysosomes

as a separate functional organelle.

2. de Duve C, Pressman BC, Gianetto R,

et al. Tissue fractionation studies

6 Intracellular distribution patterns of

enzymes in rat-liver tissue. Biochem J

1955;60:604-17

3. de Duve C, Wattiaux R. Functions of

lysosomes. Annu Rev Physiol

1966;28:435-92.. A fantastic review on the emerging

field of lysosomal biology, introducing

concepts that have become

fundamental for the understanding of

the organelle.

4. Bainton DF. The discovery of lysosomes.

J Cell Biol 1981;91:66s-76s

5. Turk V, Turk B, Turk D. Lysosomal

cysteine proteases: facts and

opportunities. Embo J 2001;20:4629-33

6. Kirkegaard T, Jaattela M. Lysosomal

involvement in cell death and cancer.

Biochim Biophys Acta 2009;1793:746-54

7. Platt FM, Boland B, van der Spoel AC.

The cell biology of disease: lysosomal

storage disorders: The cellular impact of

lysosomal dysfunction. J Cell Biol

2012;199:723-34

8. Sardiello M, Palmieri M, di Ronza A,

et al. A gene network regulating

lysosomal biogenesis and function.

Science (New York, N.Y.)

2009;325:473-7. The first report on a coordinated

regulation of lysosomal

gene expression.

9. Huynh C, Roth D, Ward DM, et al.

Defective lysosomal exocytosis and

plasma membrane repair in Chediak-

Higashi/beige cells. Proc Natl Acad

Sci USA 2004;101:16795-800

10. Eskelinen EL, Tanaka Y, Saftig P. At the

acidic edge: emerging functions for

lysosomal membrane proteins.

Trends Cell Biol 2003;13:137-45

11. Stein MP, Dong J, Wandinger-Ness A.

Rab proteins and endocytic trafficking:

potential targets for therapeutic

intervention. Adv Drug Deliv Rev

2003;55:1421-37

12. Xu Y-H, Barnes S, Sun Y,

Grabowski GA. Multi-system disorders

of glycosphingolipid and ganglioside

metabolism. J Lipid Res

2010;51:1643-75. A great review of glycosphingolipid

and ganglioside metabolism and its

involvement in lysosomal

storage diseases.

13. Ruivo R, Anne C, Sagne C, Gasnier B.

Molecular and cellular basis of lysosomal

transmembrane protein dysfunction.

Biochim Biophys Acta 2009;1793:636-49

14. Maxfield FR, McGraw TE. Endocytic

recycling. Nat Rev Mol Cell Biol

2004;5:121-32

15. Ghosh P, Dahms NM, Kornfeld S.

Mannose 6-phosphate receptors: new

twists in the tale. Nat Rev Mol Cell Biol

2003;4:202-12

16. Bonifacino JS, Lippincott-Schwartz J.

Coat proteins: shaping membrane

transport. Nat Rev Mol Cell Biol

2003;4:409-14

17. Neufeld EF. From serendipity to therapy.

Annu Rev Biochem 2011;80:1-15

18. Sun-Wada GH, Wada Y, Futai M.

Lysosome and lysosome-related organelles

responsible for specialized functions in

higher organisms, with special emphasis

on vacuolar-type proton ATPase.

Cell Struct Funct 2003;28:455-63

19. Saftig P, Klumperman J. Lysosome

biogenesis and lysosomal membrane

proteins: trafficking meets function.

Nat Rev Mol Cell Biol 2009;10:623-35

20. Dahms NM, Hancock MK. P-type

lectins. Biochim Biophys Acta

2002;1572:317-40

21. Luzio JP, Pryor PR, Bright NA.

Lysosomes: fusion and function. Nat Rev

Mol Cell Biol 2007;8:622-32

22. Eskelinen E-L, Saftig P. Autophagy:

a lysosomal degradation pathway with a

central role in health and disease.

Biochim Biophys Acta 2009;1793:664-73

23. Shintani T, Klionsky DJ. Autophagy in

health and disease: a double-edged

sword. Science 2004;306:990-5

24. Ogier-Denis E, Codogno P. Autophagy:

a barrier or an adaptive response to

cancer. Biochim Biophys Acta

2003;1603:113-28

25. Deosaran E, Larsen KB, Hua R, et al.

NBR1 acts as an autophagy receptor for

peroxisomes. J Cell Science

2012. [Epub ahead of print]

26. Chiang HL, Terlecky SR, Plant CP,

Dice JF. A role for a 70-kilodalton heat

shock protein in lysosomal degradation

of intracellular proteins. Science

1989;246:382-5

27. Blott EJ, Griffiths GM. Secretory

lysosomes. Nat Rev Mol Cell Biol

2002;3:122-31

28. Stinchcombe J, Bossi G, Griffiths GM.

Linking albinism and immunity: the

secrets of secretory lysosomes. Science

2004;305:55-9

29. Andrews NW. Regulated secretion of

conventional lysosomes. Trends Cell Biol

2000;10:316-21

30. Jaiswal JK, Andrews NW, Simon SM.

Membrane proximal lysosomes are the

major vesicles responsible for

calcium-dependent exocytosis in

nonsecretory cells. J Cell Biol

2002;159:625-35

31. Zerial M, McBride H. Rab proteins as

membrane organizers. Nat Rev Mol

Cell Biol 2001;2:107-17

32. Bright NA, Reaves BJ, Mullock BM,

Luzio JP. Dense core lysosomes can fuse

with late endosomes and are re-formed

from the resultant hybrid organelles.

J Cell Sci 1997;110(Pt 1):2027-40

33. Yu L, McPhee CK, Zheng L, et al.

Termination of autophagy and

reformation of lysosomes regulated by

mTOR. Nature 2010;465:942-6

34. Pryor PR, Luzio JP. Delivery of

endocytosed membrane proteins to the

lysosome. Biochimica et Biophysica Acta

2009;1793:615-24

35. Schmid JA, Mach L, Paschke E, Glossl J.

Accumulation of sialic acid in endocytic

compartments interferes with the

formation of mature lysosomes Impaired

proteolytic processing of cathepsin B in

fibroblasts of patients with lysosomal

sialic acid storage disease. J Biol Chem

1999;274:19063-71

36. Goldman SDB, Krise JP. Niemann-Pick

C1 functions independently of

Niemann-Pick C2 in the initial

stage of retrograde transport of

T. Kirkegaard

400 Expert Opinion on Orphan Drugs (2013) 1(5)

Exp

ert O

pini

on o

n O

rpha

n D

rugs

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

125.

238.

254.

109

on 0

3/03

/14

For

pers

onal

use

onl

y.

membrane-impermeable lysosomal cargo.

J Biol Chem 2010;285:4983-94

37. Vanier MT, Millat G. Niemann--Pick

disease type C. Clin Genet

2003;64(4):269-81

38. Devlin C, Pipalia NH, Liao X, et al.

Improvement in lipid and protein

trafficking in Niemann-Pick C1 cells by

correction of a secondary enzyme defect.

Traffic (Copenhagen, Denmark)

2010;11:601-15

39. Abdul-Hammed M, Breiden B,

Adebayo MA, et al. Role of endosomal

membrane lipids and NPC2 in

cholesterol transfer and membrane

fusion. J Lipid Res 2010;51:1747-60

40. Meikle PJ, Hopwood JJ, Clague AE,

Carey WF. Prevalence of lysosomal

storage disorders. JAMA

1999;281:249-54

41. Cox TM, Cachon-Gonzalez MB. The

cellular pathology of lysosomal diseases.

J Pathol 2012;226:241-54.. An excellent review on the

pathophysiology of lysosomal

storage diseases.

42. Lloyd-Evans E, Morgan AJ, He X, et al.

Niemann-Pick disease type C1 is a

sphingosine storage disease that causes

deregulation of lysosomal calcium.

Nat Med 2008;14:1247-55

43. Kirkegaard T, Roth AG, Petersen NH,

et al. Others, Hsp70 stabilizes lysosomes

and reverts Niemann-Pick disease-

associated lysosomal pathology. Nature

2010;463:549-53. The first report on the use of a

molecular chaperone as a therapeutic

approach for lysosomal

storage diseases.

44. Schuchman EH. Acid sphingomyelinase,

cell membranes and human disease:

lessons from Niemann-Pick disease.

FEBS Lett 2010;584:1895-900

45. Beck M. Therapy for lysosomal storage

disorders. IUBMB Life 2010;62:33-40. The first experiment demonstrating the

principle of lysosomal enzyme

secretion-reuptake cycle.

46. de Ru MH, Boelens JJ, Das AM, et al.

Enzyme replacement therapy and/or

hematopoietic stem cell transplantation at

diagnosis in patients with

mucopolysaccharidosis type I: results of a

European consensus procedure.

Orphanet J Rare Dis 2011;6:55

47. Fratantoni JC, Hall CW, Neufeld EF.

Hurler and Hunter syndromes: mutual

correction of the defect in cultured

fibroblasts. Science (New York, N.Y.)

1968;162:570-2. The first experiments demonstrating

the principle of lysosomal enzyme

secretion-reuptake.

48. Dahms NM, Lobel P, Kornfeld S.

Mannose 6-phosphate receptors and

lysosomal enzyme targeting. J Biol Chem

1989;264:12115-18

49. Barton NW, Brady RO, Dambrosia JM,

et al. Replacement therapy for inherited

enzyme deficiency–macrophage-targeted

glucocerebrosidase for Gaucher’s disease.

N Engl J Med 1991;324:1464-70. The first report on the clinical use of

enzyme replacement therapy in

Gaucher disease.

50. Johnson WG, Desnick RJ, Long DM,

et al. Intravenous injection of purified

hexosaminidase A into a patient with

Tay-Sachs disease. Birth Defects Orig

Artic Ser 1973;9:120-4

51. Vanier MT. Niemann-Pick disease type

C. Orphanet J Rare Diseases 2010;5:16

52. Richards SM, Olson TA,

McPherson JM. Antibody response in

patients with Gaucher disease after

repeated infusion with

macrophage-targeted glucocerebrosidase.

Blood 1993;82:1402-9

53. Banugaria SG, Prater SN, Ng YK, et al.

The impact of antibodies on clinical

outcomes in diseases treated with

therapeutic protein: lessons learned from

infantile Pompe disease. Genet Med

2011;13:729-36

54. Krivit W, Peters C, Shapiro EG. Bone

marrow transplantation as effective

treatment of central nervous system

disease in globoid cell leukodystrophy,

metachromatic leukodystrophy,

adrenoleukodystrophy, mannosidosis,

fucosidosis, aspartylglucosaminuria,

Hurler, Maroteaux-Lamy, and Sly syn.

Curr Opin Neurol 1999;12:167-76

55. Souillet G, Guffon N, Maire I, et al.

Outcome of 27 patients with Hurler’s

syndrome transplanted from either

related or unrelated haematopoietic stem

cell sources. Bone Marrow Transplant

2003;31:1105-17

56. Sauer M, Meissner B, Fuchs D, et al.

Allogeneic blood SCT for children with

Hurler’s syndrome: results from the

German multicenter approach

MPS-HCT.

2005;Bone Marrow Transplant

2009;43:375-81

57. Grigull L, Sykora K-W, Tenger A, et al.

Variable disease progression after

successful stem cell transplantation:

prospective follow-up investigations in

eight patients with Hurler syndrome.

Pediatr Transplant 2011;15:861-9

58. Weinreb NJ. Imiglucerase and its use for

the treatment of Gaucher’s disease.

Expert Opin Pharmacother

2008;9:1987-2000

59. Weinreb NJ, Goldblatt J, Villalobos J,

et al. Long-term clinical outcomes in

type 1 Gaucher disease following

10 years of imiglucerase treatment.

J Inherited Metabolic Disease

2012. [Epub ahead of print]. Long-term follow-up of Gaucher

disease patients receiving imiglucerase.

60. Vellodi A, Tylki-Szymanska A,

Davies EH, et al. Management of

neuronopathic Gaucher disease: revised

recommendations. J Inherit Metab Dis

2009;32:660-4

61. Schaefer RM, Tylki-Szymanska A,

Hilz MJ. Enzyme replacement therapy

for Fabry disease: a systematic review of

available evidence. Drugs

2009;69:2179-205

62. Mehta A, Beck M, Eyskens F, et al.

Fabry disease: a review of current

management strategies. QJM

2010;103:641-59

63. Aerts JM, Groener JE, Kuiper S, et al.

Elevated globotriaosylsphingosine is a

hallmark of Fabry disease. Proc Natl

Acad Sci USA 2008;105:2812-17

64. Rombach SM, Dekker N,

Bouwman MG, et al. Plasma

globotriaosylsphingosine: diagnostic value

and relation to clinical manifestations of

Fabry disease. Biochim Biophys Acta

2010;1802:741-8

65. Muenzer J. Overview of the

mucopolysaccharidoses.

Rheumatology (Oxford, England)

2011;50(Suppl 5):v4-12. A good review of

the mucopolysaccharidoses.

66. Clarke LA, Wraith JE, Beck M, et al.

Long-term efficacy and safety of

laronidase in the treatment of

mucopolysaccharidosis I. Pediatrics

2009;123:229-40

Emerging therapies and therapeutic concepts for lysosomal storage diseases

Expert Opinion on Orphan Drugs (2013) 1(5) 401

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ert O

pini

on o

n O

rpha

n D

rugs

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

125.

238.

254.

109

on 0

3/03

/14

For

pers

onal

use

onl

y.

67. Muenzer J, Beck M, Eng CM, et al.

Long-term, open-labeled extension study

of idursulfase in the treatment of Hunter

syndrome. Genet Med 2011;13:95-101

68. Muenzer J, Beck M, Eng CM, et al.

Multidisciplinary management of Hunter

syndrome. Pediatrics 2009;124:e1228-39

69. Harmatz P, Giugliani R, Schwartz IVD,

et al. Long-term follow-up of endurance

and safety outcomes during enzyme

replacement therapy for

mucopolysaccharidosis VI: final results of

three clinical studies of recombinant

human N-acetylgalactosamine 4-sulfatase.

Mol Genet Metab 2008;94:469-75

70. Kishnani PS, Corzo D, Nicolino M,

et al. Recombinant human acid [alpha]-

glucosidase: major clinical benefits in

infantile-onset Pompe disease. Neurology

2007;68:99-109. The first report on a significant benefit

of enzyme replacement therapy in

infantile Pompe disease.

71. Nicolino M, Byrne B, Wraith JE, et al.

Clinical outcomes after long-term

treatment with alglucosidase alfa in

infants and children with advanced

Pompe disease. Genet Med

2009;11:210-19

72. van der Ploeg AT, Clemens PR,

Corzo D, et al. A randomized study of

alglucosidase alfa in late-onset Pompe’s

disease. N Engl J Med

2010;362:1396-406

73. Matzner U, Herbst E, Hedayati KK,

et al. Enzyme replacement improves

nervous system pathology and function

in a mouse model for metachromatic

leukodystrophy. Hum Mol Genet

2005;14:1139-52

74. Blanz J, Stroobants S,

Lullmann-Rauch R, et al. Reversal of

peripheral and central neural storage and

ataxia after recombinant enzyme

replacement therapy in

alpha-mannosidosis mice.

Hum Mol Genet 2008;17:3437-45

75. Zhu Y, Li X, McVie-Wylie A, et al.

Carbohydrate-remodelled acid

alpha-glucosidase with higher affinity for

the cation-independent mannose

6-phosphate receptor demonstrates

improved delivery to muscles of Pompe

mice. Biochem J 2005;389:619-28

76. Maga JA, Zhou J, Kambampati R, et al.

Glycosylation-independent lysosomal

targeting of acid alpha-glucosidase

enhances muscle glycogen clearance in

Pompe mice. J Biol Chem

2013;288(3):1428-38

77. Moreland RJ, Jin X, Zhang XK, et al.

Lysosomal acid alpha-glucosidase consists

of four different peptides processed from

a single chain precursor. J Biol Chem

2005;280:6780-91

78. Gabathuler R. Approaches to transport

therapeutic drugs across the blood-brain

barrier to treat brain diseases.

Neurobiol Dis 2010;37:48-57. Reviews approaches to target the

blood-brain barrier.

79. Boado RJ, Zhang Y, Zhang Y, et al.

Genetic engineering of a lysosomal

enzyme fusion protein for targeted

delivery across the human blood-brain

barrier. Biotechnol Bioeng

2008;99:475-84

80. Lu JZ, Hui EK-W, Boado RJ,

Pardridge WM. Genetic engineering of a

bifunctional IgG fusion protein with

iduronate-2-sulfatase. Bioconjug Chem

2010;21:151-6

81. Boado RJ, Lu JZ, Hui EK-W, et al.

Pharmacokinetics and brain uptake in

the Rhesus monkey of a fusion protein

of arylsulfatase A and a monoclonal

antibody against the human insulin

receptor. Biotechnol Bioeng

2012. [Epub ahead of print]

82. Demeule M, Poirier J, Jodoin J, et al.

High transcytosis of melanotransferrin

(P97) across the blood-brain barrier.

J Neurochem 2002;83:924-33

83. Demeule M, Regina A, Che C, et al.

Identification and design of peptides as a

new drug delivery system for the brain.

J Pharmacol Exp Ther

2008;324:1064-72

84. Demeule M, Currie J-C, Bertrand Y,

et al. Involvement of the low-density

lipoprotein receptor-related protein in the

transcytosis of the brain delivery vector

angiopep-2. J Neurochem

2008;106:1534-44

85. Pardridge WM, Boado RJ. Reengineering

biopharmaceuticals for targeted delivery

across the blood-brain barrier. 1st

edition. Methods in Enzymology,

Elsevier, Inc; 2012;503:269-92

86. Takemoto S, Nishikawa M, Takakura Y.

Pharmacokinetic and tissue distribution

mechanism of mouse recombinant heat

shock protein 70 in mice. Pharm Res

2005;22:419-26

87. Muro S, Schuchman EH,

Muzykantov VR. Lysosomal enzyme

delivery by ICAM-1-targeted nanocarriers

bypassing glycosylation- and

clathrin-dependent endocytosis.

Mol Ther 2006;13:135-41

88. Spencer BJ, Verma IM. Targeted delivery

of proteins across the blood-brain barrier.

Proc Natl Acad Sci USA

2007;104:7594-9

89. Thomas FC, Taskar K, Rudraraju V,

et al. Uptake of ANG1005, a novel

paclitaxel derivative, through the

blood-brain barrier into brain and

experimental brain metastases of breast

cancer. Pharm Res 2009;26:2486-94

90. Platt FM, Neises GR, Dwek RA,

Butters TD. N-butyldeoxynojirimycin is

a novel inhibitor of glycolipid

biosynthesis. J Biol Chem

1994;269:8362-5. The first report on substrate reduction

therapy as a therapeutic approach for

lysosomal storage diseases.

91. Cox T, Lachmann R, Hollak C, et al.

Novel oral treatment of Gaucher’s disease

with N-butyldeoxynojirimycin (OGT

918) to decrease substrate biosynthesis.

Lancet 2000;355:1481-5. Pivotal trial of substrate reduction

therapy providing proof-of-principle.

92. Bembi B, Marchetti F, Guerci VI, et al.

Substrate reduction therapy in the

infantile form of Tay-Sachs disease.

Neurology 2006;66:278-80

93. Shapiro BE, Pastores GM, Gianutsos J,

et al. Miglustat in late-onset Tay-Sachs

disease: a 12-month, randomized,

controlled clinical study with 24 months

of extended treatment. Genet Med

2009;11:425-33

94. Guffon N, Bin-Dorel S, Decullier E,

et al. Evaluation of miglustat treatment

in patients with type III

mucopolysaccharidosis: a randomized,

double-blind, placebo-controlled study.

J Pediatr 2011;159:838-844.e1

95. Patterson MC, Vecchio D, Prady H,

et al. Miglustat for treatment of

Niemann-Pick C disease: a randomised

controlled study. Lancet Neurol

2007;6:765-72

96. Maegawa GHB, Banwell BL, Blaser S,

et al. Substrate reduction therapy in

juvenile GM2 gangliosidosis.

Mol Genet Metab 2009;98:215-24

T. Kirkegaard

402 Expert Opinion on Orphan Drugs (2013) 1(5)

Exp

ert O

pini

on o

n O

rpha

n D

rugs

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

125.

238.

254.

109

on 0

3/03

/14

For

pers

onal

use

onl

y.

97. Lukina E, Watman N, Arreguin EA,

et al. Improvement in hematological,

visceral, and skeletal manifestations of

Gaucher disease type 1 with oral

eliglustat tartrate (Genz-112638)

treatment: 2-year results of a Phase II

study. Blood 2010;116:4095-8

98. Piotrowska E, Jakobkiewicz-Banecka J,

Maryniak A, et al. Two-year follow-up of

Sanfilippo disease patients treated with a

genistein-rich isoflavone extract:

assessment of effects on cognitive

functions and general status of patients.

Med Sci Monitor 2011;17:CR196-202

99. Nylandsted J, Gyrd-Hansen M,

Danielewicz A, et al. Heat shock protein

70 promotes cell survival by inhibiting

lysosomal membrane permeabilization.

J Exp Med 2004;200:425-35

100. Hwang JH, Ryu JK, Yoon YB, et al.

Spontaneous activation of pancreas

trypsinogen in heat shock protein

701 knock-out mice. Pancreas

2005;31:332-6

101. Bivik C, Rosdahl I, Ollinger K.

Hsp70 protects against UVB induced

apoptosis by preventing release of

cathepsins and cytochrome c in human

melanocytes. Carcinogenesis

2007;28:537-44

102. Leist M, Jaattela M. Four deaths and a

funeral: from caspases to alternative

mechanisms. Nat Rev Mol Cell Biol

2001;2:589-98

103. Petersen NHT, Kirkegaard T.

HSP70 and lysosomal storage disorders:

novel therapeutic opportunities.

Biochem Soc Trans 2010;38:1479-83

104. Hargitai J, Lewis H, Boros I, et al.

Bimoclomol, a heat shock protein

co-inducer, acts by the prolonged

activation of heat shock factor-1.

Biochem Biophys Res Commun

2003;307:689-95

105. Kieran D, Kalmar B, Dick JRT, et al.

Treatment with arimoclomol, a

coinducer of heat shock proteins, delays

disease progression in ALS mice.

Nat Med 2004;10:402-5

106. Fan JQ, Ishii S, Asano N, Suzuki Y.

Accelerated transport and maturation of

lysosomal alpha-galactosidase A in Fabry

lymphoblasts by an enzyme inhibitor.

Nat Med 1999;5:112-15. The first report on chemical chaperone

therapy for a lysosomal storage disease.

107. Fan J-Q, Ishii S. Active-site-specific

chaperone therapy for Fabry disease Yin

and Yang of enzyme inhibitors. FEBS J

2007;274:4962-71

108. Amicus Therapeutics and

GlaxoSmithKline Announce Top Line

6-Month Primary Treatment Period

Results From First Phase III Fabry

Monotherapy Study with migalastat.

2012. Available from: http://ir.

amicustherapeutics.com/releasedetail.cfm?

ReleaseID=727915

109. Maegawa GHB, Tropak M, Buttner J,

et al. Pyrimethamine as a potential

pharmacological chaperone for late-onset

forms of GM2 gangliosidosis.

J Biol Chem 2007;282:9150-61

110. Maegawa GHB, Tropak MB,

Buttner JD, et al. Identification and

characterization of ambroxol as an

enzyme enhancement agent for Gaucher

disease. J Biol Chem 2009;284:23502-16

111. Clarke JTR, Mahuran DJ, Sathe S, et al.

An open-label Phase I/II clinical trial of

pyrimethamine for the treatment of

patients affected with chronic

GM2 gangliosidosis (Tay-Sachs or

Sandhoff variants). Mol Genet Metab

2011;102:6-12

112. Brown JR, Crawford BE, Esko JD.

Glycan antagonists and inhibitors:

a fount for drug discovery. Crit Rev

Biochem Mol Biol 2007;42:481-515. Description of substances for

substrate optimization.

113. Crawford B, Brown J, Tolmie K, et al.

Small molecule inhibitors of

Glycosaminoglycan Biosynthesis as

substrate optimization therapy for the

Mucopolysaccharidoses.

Mol Genet Metab 2011;102:S12-13

114. Byrne BJ, Falk DJ, Clement N, Mah CS.

Gene therapy approaches for lysosomal

storage disease: next-generation

treatment. Hum Gene Ther

2012;23:808-15

115. Dunbar CE, Kohn DB, Schiffmann R,

et al. Retroviral transfer of the

glucocerebrosidase gene into CD34+ cells

from patients with Gaucher disease:

in vivo detection of transduced cells

without myeloablation. Hum Gene Ther

1998;9:2629-40

116. Alexander BL, Ali RR, Alton EWF, et al.

Progress and prospects: gene therapy

clinical trials (part 1). Gene Ther

2007;14:1439-47

117. Worgall S, Sondhi D, Hackett NR, et al.

Treatment of late infantile neuronal

ceroid lipofuscinosis by CNS

administration of a

serotype 2 adeno-associated virus

expressing CLN2 cDNA.

Hum Gene Ther 2008;19:463-74

118. Byrne B, Smith B, Martin A, et al.

Phase I/II Trial of Adeno-associated

Virus Acid-alpha-Glucosidase (AAV-

GAA) Diaphragm Gene Therapy for

Ventilatory Failure in Pompe Disease.

Mol Genet Metab 2012;105:S24

119. Gaudet D, Methot J, Dery S, et al.

Efficacy and long-term safety of

alipogene tiparvovec (AAV1-LPL

(S447X)) gene therapy for lipoprotein

lipase deficiency: an open-label trial.

Genet Therapy 2012.

[Epub ahead of print]

120. Nesterova G, Gahl WA. Cystinosis: the

evolution of a treatable disease.

Pediatric Nephrology (Berlin, Germany)

2012;28:51-9

121. Pisoni RL, Thoene JG, Christensen HN.

Detection and characterization of

carrier-mediated cationic amino acid

transport in lysosomes of normal and

cystinotic human fibroblasts Role in

therapeutic cystine removal? J Biol Chem

1985;260:4791-8. First report on the mechanistic

rationale for cysteamine therapy

for cystinosis.

122. Kleta R, Bernardini I, Ueda M, et al.

Long-term follow-up of well-treated

nephropathic cystinosis patients. J Pediatr

2004;145:555-60

123. Gahl WA, Kuehl EM, Iwata F, et al.

Corneal crystals in nephropathic

cystinosis: natural history and treatment

with cysteamine eye drops.

Mol Genet Metab 2000;71:100-20

124. Hein LK, Bawden M, Muller VJ, et al.

alpha-L-iduronidase premature stop

codons and potential read-through in

mucopolysaccharidosis type I patients.

J Mol Biol 2004;338:453-62

125. Nudelman I, Glikin D, Smolkin B, et al.

Repairing faulty genes by

aminoglycosides: development of new

derivatives of geneticin (G418) with

enhanced suppression of diseases-causing

nonsense mutations.

Bioorgan Med Chem 2010;18:3735-46

126. Welch EM, Barton ER, Zhuo J, et al.

PTC124 targets genetic disorders caused

Emerging therapies and therapeutic concepts for lysosomal storage diseases

Expert Opinion on Orphan Drugs (2013) 1(5) 403

Exp

ert O

pini

on o

n O

rpha

n D

rugs

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

125.

238.

254.

109

on 0

3/03

/14

For

pers

onal

use

onl

y.

by nonsense mutations. Nature

2007;447:87-91

127. Kerem E, Hirawat S, Armoni S, et al.

Effectiveness of PTC124 treatment of

cystic fibrosis caused by nonsense

mutations: a prospective Phase II trial.

Lancet 2008;372:719-27

128. Liu B, Li H, Repa JJ, et al. Genetic

variations and treatments that affect the

lifespan of the NPC1 mouse. J Lipid Res

2008;49:663-9

129. Ramirez CM, Liu B, Taylor AM, et al.

Weekly cyclodextrin administration

normalizes cholesterol metabolism in

nearly every organ of the Niemann-Pick

type C1 mouse and markedly prolongs

life. Pediatr Res 2010;68:309-15

130. Davidson CD, Ali NF, Micsenyi MC,

et al. Chronic cyclodextrin treatment of

murine Niemann-Pick C disease

ameliorates neuronal cholesterol and

glycosphingolipid storage and disease

progression. PLoS ONE 2009;4:e6951

131. Gould S, Scott RC. 2-Hydroxypropyl-

beta-cyclodextrin (HP-beta-CD):

a toxicology review. Food Chem Toxicol

2005;43:1451-9

132. Brewster ME, Anderson WR,

Meinsma D, et al. Intravenous and oral

pharmacokinetic evaluation of a 2-

hydroxypropyl-beta-cyclodextrin-based

formulation of carbamazepine in the dog:

comparison with commercially available

tablets and suspensions. J Pharm Sci

1997;86:335-9

133. Irie T, Uekama K. Pharmaceutical

applications of cyclodextrins III

Toxicological issues and safety evaluation.

J Pharm Sci 1997;86:147-62

134. Stella VJ, He Q. Cyclodextrins.

Toxicol Pathol 2008;36:30-42

135. Ward S, O’Donnell P, Fernandez S,

Vite CH. 2-hydroxypropyl-beta-

cyclodextrin raises hearing threshold in

normal cats and in cats with

Niemann-Pick type C disease.

Pediatr Res 2010;68:52-6

136. Crumling MA, Liu L, Thomas PV, et al.

Hearing loss and hair cell death in mice

given the cholesterol-chelating agent

hydroxypropyl-beta-cyclodextrin.

PLoS ONE 2012;7:e53280

137. Muralidhar A, Borbon IA, Esharif DM,

et al. Pulmonary function and pathology

in hydroxypropyl-beta-cyclodextrin-

treated and untreated Npc1-/- mice.

Mol Genet Metab 2011;103:142-7

138. Pontikis CC, Davidson CD,

Walkley SU, et al. Cyclodextrin alleviates

neuronal storage of cholesterol in

Niemann-Pick C disease without

evidence of detectable blood-brain

barrier permeability. J Inherited

Metabolic Disease

2013. [Epub ahead of print]

139. Balch WE, Morimoto RI, Dillin A,

Kelly JW. Adapting proteostasis for

disease intervention. Science (New York,

N.Y.) 2008;319:916-19

140. Medina DL, Fraldi A, Bouche V, et al.

Transcriptional activation of lysosomal

exocytosis promotes cellular clearance.

Dev Cell 2011;21:421-30

141. Mu T-W, Fowler DM, Kelly JW. Partial

restoration of mutant enzyme

homeostasis in three distinct lysosomal

storage disease cell lines by altering

calcium homeostasis. PLoS Biol

2008;6:e26

142. Strauss K, Goebel C, Runz H, et al.

Exosome secretion ameliorates lysosomal

storage of cholesterol in Niemann-Pick

type C disease. J Biol Chem

2010;285:26279-88

143. Smith D, Wallom K-L, Williams IM,

et al. Beneficial effects of

anti-inflammatory therapy in a mouse

model of Niemann-Pick disease type C1.

Neurobiol Dis 2009;36:242-51

144. Mu T-W, Ong DST, Wang Y-J, et al.

Chemical and biological approaches

synergize to ameliorate protein-folding

diseases. Cell 2008;134:769-81

145. Xu M, Liu K, Swaroop M, et al.

delta-Tocopherol reduces lipid

accumulation in Niemann-Pick type

C1 and Wolman cholesterol storage

disorders. J Biol Chem

2012;287:39349-60

146. Andersson U, Butters TD, Dwek RA,

Platt FM. N-

Butyldeoxygalactonojirimycin: a more

selective inhibitor of glycosphingolipid

biosynthesis than

N-butyldeoxynojirimycin, in vitro and in

vivo. Biochem Pharmacol 2000;59:821-9

147. McEachern KA, Fung J, Komarnitsky S,

et al. A specific and potent inhibitor of

glucosylceramide synthase for substrate

inhibition therapy of Gaucher disease.

Mol Genet Metab 2007;91:259-67

148. Larsen SD, Wilson MW, Abe A, et al.

Property-based design of a

glucosylceramide synthase inhibitor that

reduces glucosylceramide in the brain.

J Lipid Res 2012;53:282-91

149. Porter FD, Scherrer DE, Lanier MH,

et al. Cholesterol oxidation products are

sensitive and specific blood-based

biomarkers for Niemann-Pick C1 disease.

Sci Transl Med 2010;2:56ra81

150. Aerts JM, Kallemeijn WW, Wegdam W,

et al. Biomarkers in the diagnosis of

lysosomal storage disorders: proteins,

lipids, and inhibodies. J Inherit

Metab Dis 2011;34:605-19

151. Clarke LA, Winchester B, Giugliani R,

et al. Biomarkers for the

mucopolysaccharidoses: discovery and

clinical utility. Mol Genet Metab

2012;106:395-402

AffiliationThomas Kirkegaard PhD

Chief Scientific Officer,

Orphazyme ApS, Ole Maaløes Vej 3,

2200 Copenhagen, Denmark

Tel: +45 40532454;

E-mail: tkj@orphazyme.com

T. Kirkegaard

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care

.com

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125.

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on 0

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onal

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