Accepted Manuscript Novel treatment approaches for lysosomal disorders Timothy M. Cox, MA MSc MD FRCP FMedSci, Professor of Medicine
PII:
S1521-690X(15)00013-5
DOI:
10.1016/j.beem.2015.01.001
Reference:
YBEEM 1011
To appear in:
Best Practice & Research Clinical Endocrinology & Metabolism
Please cite this article as: Cox TM, Novel treatment approaches for lysosomal disorders, Best Practice & Research Clinical Endocrinology & Metabolism (2015), doi: 10.1016/j.beem.2015.01.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Best Practice and Research in
Volume edited by
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Professor Carla E M Hollak
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Clinical Endocrinology and Metabolism
Amsterdam Medical Centre
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University of Amsterdam
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The Netherlands
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Publisher: Elsevier, North Holland
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Novel Treatment Approaches for Lysosomal Disorders
Timothy M Cox MA MSc MD FRCP FMedSci Professor of Medicine Department of Medicine University of Cambridge UK
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Abstract
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Striking therapeutic advances for lysosomal diseases have harnessed the biology of this organelle and illustrate its central rôle in the dynamic economy of the cell. Further Innovation will require improved protein-targetting or realization of therapeutic gene- and cell transfer stratagems. Rescuing function before irreversible injury, mandates a deep knowledge of clinical behaviour as well as molecular pathology – and frequently requires an understanding of neuropathology.
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Whether addressing primary causes, or rebalanceing the effects of disordered cell function, true therapeutic innovation depends on continuing scientific exploration of the lysosome. Genuine partnerships between biotech and the patients affected by this extraordinary family of disorders continue to drive productive pharmaceutical discovery.
Key words:
Practice Points
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Lysosome, organelle, protein targeting, sphingolipids, inhibitors of biosynthesis, substrate reduction; chaperone, therapeutic, innovation, gene transfer
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• Although lysosomal diseases are rare and often clinically severe, several are treatable and the field remains an area of very active therapeutic research with considerable biopharmaceutical development of orphan medicinal products.
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• Treatments are principally based on improving the biochemical abnormality in metabolic clearance of pathological cellular molecules that would normally be digested in the lysosomal compartment. The following specific stratagems are being explored: (i) chemical storage dissolution (ii) stimulating lysosomal exocytosis by heat-shock proteins (iii) cellular complementation, especially haematopoietic stem-cell transplantation (iv) gene transfer, including genetic transduction of autologous stem cells for re-infusion as well as direct approaches, mainly with viral-based vectors (v) enzyme therapy based on harnessing cell-surface receptors that serve as recognition markers for lysosomal proteins (vi) pharmacological chaperones to stabilize mutant lysosomal proteins (vii) strategems based on control of cognate substrate biosynthesis; slowing endogenous formation of the substrate class whose degradation is impaired to match residual
digestive activity in the lysosomal compartment - the intention is to rebalance ACCEPTED MANUSCRIPT synthesis and degradation and thus correct the primary defect. Treatments in sections (i), (ii) (vi) and (vii) may involve small molecules that may have the potential to cross the blood-brain barrier; treatments (iii), (iv) and (v) rely principally on biologics of high molecular weight and/or cellular complexity.
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Research agenda
• Completing the molecular understanding of lysosomal disesases as a means to understanding their severity determinants and design highly specific molecular probes
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• Completing the developmental and surveillance programme for eliglustat and its congeners in Gaucher disease and related sphingolipidoses – especially exploring potential benefit for the neurological complications with the newly introduced Genz 161 agent.
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• Determining the value of dissolution therapy in Niemann-Pick disease type C (cyclodextrin and possibly the value of ariclomolol in this disease). If active, determining the long term outcomes. Cyclodextrin may cause deafness and haver other cranial effects.
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• Gene transfer – this holds much promise for lysosomal diseases - beyond Sanfilippo types A and B as well as LINCL 2 (late-infantile) neuronal ceroid lipofuscinosis wider clinical development is entering clinical application - especially, but not exclusively, for those diseases with a devastating neurological component.
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These developments will mandate research into improved methods to monitor outcomes of interventions using biomarkers as well as exploring neuropsychiatric disease-scoring techniques.
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• Further understanding of the clinical practicability of gene transfer, including genetic transduction of autologous stem cells for re-infusion, and approaches directed to the most critical tissues and regions of the brain. Case selection and intensive clinical engagement will be important matters to develop for enrolment in future advanced therapy research in the lysosomal diseases.
The Lysosome as a Gateway to Treatment
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The lysosome and assembly of its components during organelle biogenesis have, from the time of its discovery by Chrisitan de Duve, incited interest for intervention. With the recognition of inborn errors of lysosomal function as single-gene defects causing multisystem diseases [1], the appetite for therapeutic exploration has burgeoned. Painstaking research to determine the nature of the substrates, which contribute to the
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phenotype of intracellular storage was simply a first step in understanding lysosomal disorders caused by deficiency of acid hydrolases; this classical approach provided critical insight into the central role of the organelle in macromolecular recycling for the economy of
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the cell.
Subsequent molecular characterization of the genetic basis and protein defect(s) implicated
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in each disease revealed fundamental precepts and possibilities for definitive treatment – namely, replacing the primarily deficient factor required for normal lysosomal function. While much remains to be learnt about molecular pathogenesis of the individual conditions – many of which exhibit features of inflammation and other, compensatory, tissue reactions that await exploration – approaches based on complementing the function of proteins
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which are defective are fundamental to future therapeutic development.
De Duve shared the Nobel Prize for Physiology or Medicine in 1974 with Albert Claude and George Palade – an event which heralded Cell Biology as a radically new and
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interdisciplinary science [2]. With his colleagues, de Duve rapidly promoted the concept of a dynamic and interactive entity defined by a distinct acidic microenvironment within the
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living cell [3]. This functional definition immediately suggested that the lysosome was part of an interlinked membrane system in contact with the plasma membrane and its invaginations at the cell surface - thus participating in the processes of fluid-phase endocytosis, pinocytosis and receptor-mediated uptake, as well as autophagy. De Duve quickly realised that the lysosomal compartment would be continually exposed to the external environment and thus provide a direct access route for potential therapies [4].
Lysosomal diseases Henri-Gery Hers, a former colleague of de Duve, pursued research into glycogen storage diseases – thereby encountering Pompe disease, in which all the known enzymes of
glycogen breakdown and synthesis functioned normally. In a brilliant series of
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investigations, Hers ultimately identified the deficient enzyme which proved to be an acid αglucosidase (maltase); and thus a lysosomal enzyme previously unconnected to glycogen metabolism. Other colleagues, notably François Van Hoof, joined to define the concept of inborn lysosomal diseases [1]; and thus numerous disorders representing defects in the recycling of cellular macromolecules were classified as ‘lysosomal storage diseases’. More than 70 heritable diseases of lysosomes and lysosome-related organelles are now
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recognized - each best considered as a unique pathological domain [5, 6]. Given the potential for access of proteins to the lysosomal compartment, and numerous enzyme defects responsible for lysosomal diseases, de Duve realised that by a succession of chance
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events: “thus was elucidated, with important consequences for diagnosis, prevention and therapy, a vast chapter of pathology that had remained totally mysterious …” [7].
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Specific complementation of deficient lysosomal function
The dominant principle of functional complementation utilizing specialised pathways for protein delivery to the organelle, owes its origin to the discovery of the lysosome, and especially its dynamic rôle in cellular recycling. This led to an early prediction that, beyond its critical role in autophagy and renewal, a substantial network of membranes and
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compartments could be accessed by molecules presented at the cell surface [4].
De Duve had no concept of the mechanistic link of lysosomal protein targeting in biogenesis of the organelle – but remarkable experiments carried out by Elizabeth Neufeld and
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colleagues at the National Institutes of Health provided direct experimental support for this supposition [8]. Studies of fibroblasts cultured from genetically distinct forms of the human
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mucopolysaccharidoses ultimately led to an understanding of how nascent lysosomal enzymes were trafficked to the lysosome. With this came the realization that restoring function by enzymatic complementation of lysosomal defects might indeed be clinically tractable [8].
The complementing factors proved to be high molecular weight forms of the soluble lysosomal hydrolases deficient in each genetically distinct disorder; these glycoproteins are decorated by the critical mannose 6-phosphate moiety – and recognition signal which serves as a ligand for cell-surface receptors that mediate uptake and sorting to the lysosomal compartment after endocytosis. Contemporaneous studies of I-cell disease
provided reciprocal information: in this disease, genetic defects in the formation of the
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recognition signal, leading to multiple deficiencies of acid hydrolases in the lysosome but elevated activities in plasma and other extracellular fluids [9, 10]. These studies also confirmed the importance of receptor-mediated intracellular trafficking and co-translational processing for delivery of nascent enzymes to the lysosome.
While this receptor system is of critical importance, it is not used by all lysosomal proteins
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[11]. The topic has been extensively reviewed [10, 12]; but at first it was disappointing that early attempts to restore lysosomal activities in patients with mucopolysaccharidoses by cell and tissue transplantation were unsuccessful. Delivery of the appropriate disease-specific
took many years to be realized [13, 14].
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‘corrective factors’, principally as recombinant genetically engineered therapeutic products,
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Glycoprotein uptake by macrophages and therapy of Gaucher disease
A radical alternative, and, as it turns out, crucial development in the development of therapy for lysosomal diseases also came about fortuitously at the National Institutes of Health, Maryland. Gilbert Ashwell and his colleagues characterized the uptake of plasma glycoproteins by the liver, leading amongst other things, to identification of the
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asialoglycoprotein receptor [15]. In relation to lysosomal diseases, the studies by Ashwell and Morrell were an inspiration for pioneering work by John Barranger and Scott Furbish, in the laboratory of Roscoe Brady, which had been dedicated since the mid-1960s to the development of enzyme therapy for Gaucher disease – a relatively frequent sphingolipidosis
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due to deficiency of the acid β-glucosidase, lysosomal glucocerebrosidase [16]. After many false leads, the first successful outcome of enzyme therapy occurred in a single
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patient, a boy aged four years, in clinical studies conducted by Norman Barton with John Barranger and colleagues, who led the clinical programme in the Brady laboratory at the NIH [17]. The investigational agent used human glucocerebrosidase purified from placentae and remodelled to display terminal mannose sugars – in this way serving as a ligand for mannose receptors on macrophages [18]. The spectacular therapeutic success of enzyme therapy for Gaucher disease was again attributed to serendipity; but chance was combined with unremitting investigative work over several decades [19,20]. Successful complementation here had depended on the identification [18-22] of the mannose receptor system – the principal receptor that mediates the high-capacity uptake system for mannose-terminated glycoproteins into cells of the mononuclear-macrophage system and the preferential in vivo
target for therapeutic correction of the peripheral manifestations of Gaucher disease in the
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bone marrow and viscera [23].
Subsequently, the basis for the mannose 6-phosphate receptor-independent lysosomal targeting of glucocerebrosidase to the lysosomal compartment (in non-haematological cells) has been shown to be directed by the integral membrane protein, LIMP-2 [24]. Careful structural studies have moreover shown how, mediated by a histidine residue, LIMP-2 binds
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and releases the enzyme according to changes in pH; the finding suggest that LIMP-2 localizes to the ceramide portion of the substrate adjacent to the catalytic site [25]. A phosphomannose residue linked to N-acetylglucosamine on LIMP-2 is covalently attached to
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an asparagine residue, so that LIMP-2 binds to the mannose 6-phopsphate receptor, via mannose 6-phosphate [25].
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After decades of painstaking research and clinical development, it is now clear that diseases of the lysosome offer unrivalled therapeutic opportunities. The clinical outcomes and burgeoning commercial success of enzyme therapy for Gaucher disease serves as a paradigmatic example of fruitful application of molecular cell biology to human disease [1314, 19-20, 26]. Not only were the innermost recesses of the diseased cell opened up to
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salutary intervention, but the stimulus for commercial investment has enhanced interest far beyond the expected market for this group of individually rare diseases; several biopharmaceutical companies have developed enzyme therapies and other agents now
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approved for lysosomal diseases.
Treating fundamental causes of lysosomal disease
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The relationship between macromolecular storage and complex human disease phenotypes has been difficult to understand in molecular terms that allow prediction of either the evolution of disease, or indeed its responses at different stages to intervention at the primary ‘replacement’ level. Characterization of the principal macromolecular substrates that accumulate in the lysosomal compartment has been largely completed but how these contribute to the individual so-called storage phenotypes is unknown. Even in the case of enzyme therapy for Gaucher disease, which has been spectacularly successful since it directs the replacement factor specifically to the principal cellular population which most evidently manifests the glycosphingolipid “storage” [1], we are left still mystified by comorbidities such as Parkinson’s disease, intractable skeletal manifestations, progressive neuronopathic
features and the unexpected development of myeloma, B-cell lymphoma, and other
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haematopoietic malignancies [27-29].
Treating lysosomal diseases which affect the nervous system Most lysosomal disorders affect nervous tissue and cause neurodegeneration that is often a dominant clinical feature; moreover, the progressive nature of the process is a source of much human distress – and genuine unmet clinical need that mandates persistent scientific
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investment and exploration of imaginative remedies. While this time-honoured aspect remains a challenging, if not almost intractable aspect of the care currently available for patients with lysosomal diseases, new opportunities arise in the use of pharmaceutical
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drugs (‘small molecules’) as well as biological approaches (stratagems for cell- and gene therapies as well as ‘biologics’) to overcome the formidable barriers to therapeutic development for lysosomal diseases of the brain. Thus, running throughout the conspectus
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of emerging treatments is the perennial challenge of securing a safe and effective method for therapeutic delivery across the blood-brain barrier.
Therapeutic stratagems specifically for lysosomal disease include: (a) Dissolution - and removal - of intraluminal storage material; (b) Enhanced exocytosis of pathological intralysosomal content; (c) Modulating substrate biosynthesis (substrate-reduction
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therapy); (d) Complementation stratagems - (i) Haematopoietic stem-cell (previously bonemarrow) allo-transplantation
(ii)
Reinfusion of vector-transduced autologous
haematopoietic stem cells (iii) Direct gene transfer (iv) Enzymatic augmentation (v)
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Enzymatic enhancement and stabilization (pharmacological chaperone therapy).
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(A) Dissolution - and removal - of intraluminal storage material An often-forgotten stratagem, successfully used as a remedy for a lysosomal disease with prominent intra-compartmental metabolic storage, is the direct dissolution of the putative storage molecules held to be toxic. The first example of this approach is given by as in the membrane transport disorder, cystinosis – a disease which remains a focus of therapeutic development.
Unwanted macromolecular storage in the lysosomal compartment Since it is founded on a simple notion of pathogenesis – ie. that lysosomal diseases result from intra-lysosomal “storage” of unwanted substrates – here we review the effects of
substrate dissolution. This therapeutic concept has in fact long been applied successfully to
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a transport disorder in which the egress of lysosomal digestion products is impaired. While it is tempting to reject this notion as an outmoded and simplistic concept for understanding pathogenesis, its demonstrable efficacy in cystinosis, and now extending to the otherwise intractable neurodegenerative disorder, Niemann-Pick disease type C, cannot be denied. Moreover, the stratagem remains an active area of clinical innovation and with several
approved (cysteamine bitartrate).
Storage dissolution (cystinosis) – new developments
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investigational new drugs in late clinical development (eg, cyclodextrins) or recently
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It should not be forgotten that the pathological storage concept has been explored effectively in at least one lysosomal disease – employing a direct biochemical approach to reduce the storage of the putative toxin by promoting its dissolution and egress from the
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lysosomal compartment. The paradigmatic example of this approach is provided by the use of cysteamine in patients with cystinosis [30]. Cystinosis is caused by genetic defects in the lysosomal cystine/proton symporter, cystinosin. Systemic accumulation of cystine causes widespread injury, including renal failure. Cysteamine is an aminothiol which interacts with the crystalline aggregates of cystine (oxidised cysteine dimers) converting lysosomal free
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cystine to cysteine and the mixed disulphide of cysteine-cysteamine, which leaves the lysosome via the recently characterized cationic amino acid transporter system, which also transports lysine [31, 32].
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This simple intervention is demonstrably effective in terms of clinical outcome and disease survival since, when high doses are given early in the disease, cysteamine delays the
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progression to renal failure [30, 33]; moreover, topical therapy directly prevents loss of vision and painful corneal injury due to local deposits of cystine [34]. The approach is significant because it supports the classical view that at least in some instances, the causal pathological effect of storage in the genesis of at least one lysosomal disease and points to the potential value of substrate dissolution as a direct therapeutic avenue. Cysteamine, a thiol agent, is a malodorous and unappetising compound and its use carries with it a huge burden for patients with cystinosis, especially in terms of social acceptability and gastric tolerance. The recent exploration of delayed-release preparations of the newly available cysteamine bitartrate appear to offer large practical advantages for patients in terms of acceptability and with better compliance due to improved pharmacodynamics
profile and other properties - and hence practical efficacy [30, 33]. These are early days in
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the studies of this modestly innovative agent for patients with cystinosis but the potential effect on patients’ life quality is too important to be discounted.
Storage dissolution (Niemann-Pick disease type C) An emerging and intriguing application of the concept of substrate dissolution in the treatment of lysosomal diseases, is given by Niemann-Pick disease Type C. While the
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primary nature of this condition is known in genetic terms, (mutations in the NPC1 and NPC2 genes), the exact nature of the defect and its pathogenesis, principally in the brain, liver and gastrointestinal system, is less clear. The cognate protein products, NPC1, a
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polytopic membrane protein, and NPC2, a matrix hydrolase, collaborate to bring about the export of free cholesterol and other lipid molecules from the endolysosomal system [35]. Lately, increased lysosomal sphingosine concentrations have been implicated in the
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pathogenesis; these shift the acid-base balance of the lysosome and apparently lead to harmful sequestration of free calcium ions [36].
The sole approved treatment for Niemann-Pick disease Type C, at least in European countries, is miglustat – an imino sugar (N-butyl deoxynojirimycin, originally OGT918) which
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retards the onset of symptoms and signs of disease and prolongs survival in naturallyoccurring Niemann-Pick disease in mice and cats. Miglustat is approved as Zavesca™ for treating the neurological manifestations of this condition in the European Union, Brazil, Canada and Australia, but is hardly a definitive treatment for this devastating
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neurodegenerative disorder. While the mode of action of miglustat may be to reduce biosynthesis of sphingosine, the numerous “off-target” effects of this agent render it
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difficult to be sure that this is the true mode of action.
Cyclodextrins are cyclic oligosaccharide molecules with amphipathic properties: and are able to bind steroids and reduce the cellular content of cholesterol. The potential application of cyclodextrins to Niemann-Pick disease Type C emerged serendipitously [35]. Niemann-Pick disease Type C mice lived longer when cyclodextrin was present as a contaminant vehicle of a steroid compound under investigation.
The drug was found to delay the onset of
neurological manifestations and prolong survival in this genetically coherent and natural model [37]. Soon after these findings were appreciated, in 2009, the US Food & Drug Administration approved the use of intravenous preparations of hydroxypropyl β-
cyclodextrin as a new investigational agent in clinical trials of young patients with Niemann-
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Pick disease Type C, and the drug was subsequently awarded orphan medicinal status as a branded preparation as designated for the treatment of this condition [38].
Following an experimental programme in feline Niemann-Pick disease, the results of which are in press, further trials of this drug given intrathecally for patients with the disease are awaiting approval [39].
Cyclodextrin may indeed represent an important departure as a
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treatment for patients with Niemann-Pick disease Type C and a therapeutic development of value in this intractable condition; unfortunately there are reports of nerve deafness in cats which constrains unconditional enthusiasm and has mandated intensive study during the
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conduct of human clinical trials. Its mode of action is believed to be that of an agent which restores the defective function of the NPC1 and NPC2 gene products in mediating the egress of cholesterol from the lysosomal space in neurons and it has hitherto been presumed that
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the effect depends on the drug entering the brain to facilitate removal of excess cholesterol from the endolysosomal compartment of neurones and other cells. Recently however, it has been convincingly shown that hydroxypropyl-β-cyclodextrin does not significantly traverse the blood-brain barrier in either adult or neonatal mice [40]. At the same time, the volume of distribution available to the agent greatly exceeds the predicted plasma and vascular
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volume of the brain, suggesting that cyclodextrin binds to the endothelial cell surface of the cerebral vessels and clears intracellular cholesterol indirectly. However the exact mechanism by which the apparent therapeutic effect is brought about is unclear.
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B. Enhanced exocytosis
As above, the concept of removing unwanted, unmetabolized and toxic storage molecules
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by harnessing the physiological process of exocytosis from the lysosomal space is consistent with the notion of substrate dissolution therapy and harnesses one pathway of lysosomal membrane recycling. Hitherto this stratagem remains entirely experimental and has not been attempted in patients with lysosomal diseases but the notion has had appreciable support in animal disease models [41].
A provocative report using the molecular chaperone, heat-shock protein 70, has excited interest in this manoeuvre. As part of the cell death response, the integrity of lysosomal membranes is impaired and the organelle becomes generally more permeable to proteins, allowing leakage of the contents; heat-shock proteins, are known to prolong the survival of
stressed cells by stabilizing lysosomal membranes. It was moreover reported that a
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fragment of the heat-shock protein 70 (HSP70) translocates to the mammalian compartment under severe stress conditions [42]. Kirkegaard and colleagues later found that Hsp70 stabilizes lysosomes by binding to the abundant endolysosomal anionic phospholipid, bis(monoacylglycero)phosphate, which is required for
sphingomyelin
breakdown by serving as a cofactor of acid sphingomyelinase in the lysosome. It was further reported that inhibition of the hydrolase and its binding prevents the stabilization of
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lysosomes by HSP70 – a feature of fibroblasts from patients with Niemann-Pick diseases types A and B due to natural mutations in the SMPD1 gene which encodes acid sphingomyelinase. Notably, the cellular phenotype could be corrected by simple addition of
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recombinant HSP70 to the cultures: this led to a striking collapse of the distended lysosomal compartment and correction of the storage phenotype as a result of enhanced exocytosis of
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organellar contents.
Therapeutic exploration of the heat-shock response in a lysosomal disorder These above findings were taken to suggest new possibilities for developing of treatments for lysosomal diseases with compounds that enter the lysosomal lumen by the endocytic delivery pathway to promote exocytosis of content and improve membrane stability [42].
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The agent, arimoclomol citrate (BRX-345) is an orally active analogue of bimoclomol, a hydroxylamine co-inducer of the heat-shock response. The agent activates heat shock factor-1 which in the context of cellular stress associated with ischaemia or heat shock, is a transcriptional regulator of expression of inducible molecular chaperones such as HSP70.
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Heat shock factor-1 is a member of a versatile family of transcription factors that, in addition to protecting cells against proteotoxicity due to aggregation and the unfolded
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protein response, are essential for physiological functions, particularly during development [42]. Arimoclomol™ is already in clinical phase 2/3 trials for various indications in humans with diseases, such a amyotrophic lateral sclerosis (specifically the hereditary variant due to mutations in superoxide dismutase) associated with protein aggregation in neurones. Now licensed to Orphazyme, arimoclomol™ is under clinical investigation in a neurodegenerative lysosomal disease: the EU Committee for Orphan Medicinal Products has recently adopted a positive opinion on the orphan drug application for arimoclomol citrate as a treatment of Niemann-Pick disease type C. Clearly the results of this bold initiative are eagerly awaited.
Promoting exocytosis by dynamic control of lysosomal biogenesis and autophagy
In a brilliant series of studies, Ballabio and colleagues identified a transcriptional mechanism
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that regulates the biogenesis of lysosomes and autophagy. In most lysosomal diseases, the lysosomal network is expanded and lysosomal enzyme activities are enhanced [43].
While a regulatory mechanism with enhanced transcriptional expression was suspected, Sardiello et al used pattern discovery analysis of promoters where the cognate gene encoded lysosomal proteins, to identify a common palindromic motif (coordinated
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lysosomal expression and regulation, CLEAR), which mediates transcriptional activation, [44]. The CLEAR element binds to TFEB, a master regulator of lysosomal biogenesis and a nuclear transcription factor. In cultured cells, TFEB was shown to activate transcription of
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many lysosomal genes [45].
TFEB coordinates the functions of two organelle systems in cellular recycling and autophagy
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and stimulates formation of autophagosomes and their fusion with lysosomes. Phosphorylation of a critical residue (serine 142) mediated by the extracellular signalregulated kinase 2 (ERK2) prevents nuclear translocation; this phosphorylation is sensitive to nutrient activation in the mitogen-activated protein kinase pathway as well as the multiprotein mTOR (mammalian target of rapamycin) complex. In nutrient-rich conditions,
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ERK2 is activated and stimulates growth while inhibiting autophagy; reciprocal effects pertain when the supply of nutrients is poor and these induce autophagy. Moreover, TFEB over-expression stimulated biogenesis of the organelle with expansion of the lysosomal compartment. Expression studies in stable transfectants showed that TFEB enhanced the
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degradation of glycosaminoglycans, as well as the breakdown of mutant polyQ expanded huntingtin polymers in an inducible model of Huntington disease [46]. To relieve the burden
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of intralysosomal as well as extralysosomal glycogen burden in Pompe disease, lysosomal exocytosis was stimulated by upregulating TFEB expression in mice with Pompe disease. Overexpression of TFEB in muscle cells removed enlarged lysosomes, reduced pathological glycogen deposits, and restored autophagy, arrest of which is a prominent feature of the disorder. Enhanced exocytosis involved autophagosomes and lysosomes, indicating that TFEB induces the exocytosis of both compartments [47].
Hitherto, stimulating exocytosis has a strong theoretical basis for the relief of lysosomal disorders in which there is a prominent accumulation of debris in autophagic vacuoles and represents a completely novel avenue for therapeutic exploration in man. Identification of
the regulatory molecules implicated in the transcriptional control of lysosomal biogenesis,
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autophagy and nutrient sensing through TFEB and its functional partners, undoubtedly suggests therapeutic opportunities for storage clearance and stabilization of diseased organelles in a range of diseases. Although the stratagem is not yet undergoing substantive clinical development, it represents a promising field of endeavour.
C. Modulating substrate biosynthesis (‘substrate-reduction therapy’)
adequately
be
degraded
has
general
precedents
in
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The intriguing therapeutic idea of limiting formation of excess metabolites which cannot clinical
practice:
gout,
hypercholesterolaemia, the acute porphyrias and hereditary tyrosinaemia - metabolic
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diseases which are readily identified as ‘overproduction diseases’. Thus, while not strictly original in concept, restricting substrate biosynthesis for lysosomal diseases, has been fully realized in at least one sphingolipid disorder – non-neuronopathic Gaucher disease.
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In respect of these conditions which are not only biochemically complex but often associated with otherwise intractable neurological manifestations, such therapeutic initiatives have distinct advantages. The stratagem remains actively under clinical development in Fabry disease, chronic neuronopathic Gaucher disease and other glycosphingolipidoses such a Niemann-Pick type C and GM2 gangliosidosis; there is
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moreover an attempt to investigate this approach in clinical studies of disordered glycosaminoglycan formation in mucopolysaccharidoses. The range of inhibitory compounds with improved pharmaceutical properties has been expanded – and, with the notable convenience of being orally active, several of these small molecules have shown to traverse
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the blood-brain barrier.
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Most lysosomal disorders are understood principally in terms of excess ‘storage’ of inefficiently recycled cellular debris: attenuating the rate of synthesis of the specific precursors of incompletely degraded macromolecules is thus an approach not only consistent with elementary ideas about pathogenesis but also has parallels in diseases caused by toxic metabolites. Use of the statins to limit hepatic cholesterol biosynthesis in familial hypercholesterolaemia by inhibiting hydroxymethylglutaryl coenzyme A reductase and of allopurinol to suppress uric acid formation at the level of xanthine oxidase in gout are familiar examples of this application. Indeed allopurinol was first explored in patients with gout fifty years ago [48] and statins in hypercholesterolaemia in 1971 [49].
A frequent operational characteristic of successful substrate inhibitors, is that they act on
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the first, energetically committed, biosynthetic reaction. In this way, overproduction of intermediary molecules or collateral metabolites with the potential for toxicity shared with the excess primary product, is avoided. On the other hand, as in the pharmaceutical use of statins in the biosynthesis of essential sterols, care must be taken to attenuate, rather than abrogate formation of the critical biosynthetic product on which life often depends. In the case of glycosphingolipids, the importance of not disturbing the delicate homeostasis of
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bioactive sphingoid bases cannot be overestimated. These molecules are imbued with critical functions: ceramides stimulate apoptosis and inhibit cell growth as well as differentiation; the cognate sphingols stimulate proliferation, control cell migration,
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angiogenesis as well as inflammatory responses.
If attainable, development of selective inhibitors of the thermodynamically defining first
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step in a complex biosynthetic pathway is likely to be rewarded by fewer ‘off-target’ actions with deleterious effects. As noted, the basic premise is to prevent the clinical consequences of a build-up of noxious metabolites: in the case of Gaucher disease, the putative toxins are the many variant N-acyl-sphingosyl-1-O-β-D glucosides with differing acyl and sphingosine moieties and their unacylated analogues, the ß-glucosylsphingosines or ‘psychosines’ [50].
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These metabolites arise because a key enzyme responsible for sphingolipid recycling in the lysosome, acid β-glucosylceramidase (D-glucosyl-N- acylsphingosine glucohydrolase, EC 3.2.1.45), encoded by the GBA1 gene is impaired. In milder clinical forms of Gaucher disease, the ß-glucosylceramides are derived from exogenous membrane components after
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phagocytosis of effete cells, particularly by macrophages. In those forms of Gaucher disease where acid ß-glucosylceramidase deficiency is more profound, lysosomal recycling of
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endogenous cellular glycosphingolipids becomes limiting: under these circumstances, cells, such as neurones, whose membranes are enriched with gangliosides and other cognate lipids undergoing dynamic recycling, are more prominently affected.
Formation of most complex glycosphingolipids is initiated by formation of ßglucosylceramides. De novo synthesis occurs on the cytosolic leaflet of the Golgi complex but the products are translocated to the luminal surface of this apparatus, where they serve as core structures for building hundreds of higher-order sphingolipids, including complex gangliosides with multifunctional roles in cellular processes [51,52].
It is noteworthy that
the accumulating sphingolipids in the lysosomal compartment of patients with Fabry
disease; GM1 gangliosidosis; GM2 gangliosidosis (Tay-Sachs, Sandoff diseases and GM2
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activator deficiency) as well as Niemann-Pick disease type C, are all generated biosynthetically from the ß-glucosylceramide scaffold; it is this precursor glycosphingolipid which accumulates in the tissues of patients with all sub-types of Gaucher disease.
The biosynthetic reaction is thermodynamically favoured by the energized glycosyl donor a sugar nucleotide - and catalysed by UDP-glucose: N-acylsphingosine D-glucosyltransferase
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(UDP-glucosylceramide transferase). In Gaucher disease, unhydrolysed ß-glucosylceramides are principally sequestered in the lysosome; after their formation de novo, glucosylceramides would normally pass to other cellular compartments and undergo further
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glycosylation reactions. Norman Radin perceived this first committed step of glycosphingolipid biosynthesis as a target for innovative drugs in Gaucher disease and related glycosphingolipidoses [53]. Indeed, he had earlier sought selective inhibitors of the
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analogous UDP-galactosylceramide transferase for Krabbe disease [54]. Given residual lysosomal ß-glucosylceramidase activity, Radin argued that attenuating glucosylceramide biosynthesis would allow steady-state substrate concentrations to be re-equilibrated so that the remaining hydrolytic activity would match the rate of incoming substrate - thus
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balancing glycosphingolipid metabolism and avoiding the consequences of disease.
At present, two principal chemical classes of inhibitor are undergoing comprehensive therapeutic exploration in the sphingolipidoses:
iminosugars, originally derived from
naturally-occurring plant products [55] and another class of synthetic or semi-synthetic
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compounds originally made by Radin, Shayman and colleagues [56,57]. These latter pyrrolidine compounds serve as potent ceramide mimetics and bind to the transition-state
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of UDP-glucose: N-acylsphingosine D-glucosyltransferase, the key therapeutic target.
Iminosugars
Gaucher disease
It was the iminosugars, in particular N-butyldeoxynojirimycin, previously explored for an unrelated application in HIV infection, that were chosen for clinical development in the sphingolipid diseases by Frances Platt and Terry Butters in the University of Oxford [55]. The original compounds synthesized by Radin and colleagues were useful experimental inhibitors of the UDP-glucosylceramide synthase, but because of their cellular toxicity, they were not developed clinically for many years [58]. Platt and Butters had found that
micromolar concentrations of N-butyldeoxynojirimycin inhibited the biosynthesis of
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glucosylceramide in cultures of a murine macrophages treated with an irreversible inhibitor of acid-β-glucosylceramidase, conduritol-β-epoxide. This agent induces lysosomal abnormalities accompanied by an accumulation of glucosylceramide but co-addition of Nbutyldeoxynojirimycin abrogated the lysosomal storage. Subsequent animal studies were conducted in knock-out mice lacking β-hexosaminidases A and B, with accumulation of GM2 ganglioside in the brain.
The Sandhoff mouse has a shortened life expectancy with
throughout the central neuraxis.
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neurological manifestations, accompanied by progressive storage of GM2 ganglioside Administration of N-butyldeoxynojirimycin reduced
ganglioside storage in peripheral organs and brain of these animals and extended survival by
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approximately 40% [59]. N-butyldeoxynojirimycin, (known as miglustat or Zavesca™) had been administered to humans as a potential inhibitor of the viral α-mannosidases involved in the synthesis of the HIV glycoprotein coat: this greatly facilitated introduction of a clinical
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trial of a repurposed agent in Gaucher disease. At a dose of 100mg thrice daily, the agent reduced visceral enlargement and slowly improved haematological parameters, as well as plasma chitotriosidase, in patients with Type I Gaucher disease [60,61]. An unwanted effect of the iminosugar treatment is diarrhoea, caused by an inhibition of intestinal disaccharidases. Some patients also develop tremor and an axonal peripheral
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neuropathy has been reported. The drug is approved in the United States and Europe as a second-line treatment for patients with mild to moderate Type I Gaucher disease. In some mouse strains the agent causes non-genotoxic male sterility, and contraception is advised; the drug is not licensed for use in children. Despite these drawbacks, at the time of its
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development and licensing, miglustat as an orally available small molecule offered the first credible opportunity to explore the treatment of Gaucher disease and related
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glycosphingolipidoses, including their neurological manifestations, which remain refractory to protein-based therapies. With the single endpoint chosen, in the minority of patients with Gaucher disease able tolerate the agent and remained on the trial, it proved in a oneyear study not to be inferior in patients who transferred to the agent after control of their disease by enzyme therapy [62].
Given the potential for the small iminosugar molecule to penetrate the blood-brain barrier, a further open-label randomised trial using a novel design was conducted over 24 months in 30 children with chronic neuronopathic Gaucher disease receiving enzyme therapy [63]. While this trial failed to meet its clinical endpoint (vertical saccadic eye movement velocity),
notably the drug improved pulmonary function and decreased the plasma chitotriosidase
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biomarker activity in the period of miglustat exposure compared with enzyme therapy alone. Despite this, the drug is not recommended for neurological manifestations in Gaucher disease and is not licensed for use in children.
Miglustat is a powerful inhibitor of the neutral glucosylceramidase isozyme 2 [64], and this may contribute to its clinical effects as has been suggested. The cognate gene, encoding the
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enzyme assigned the term, GBA-2, has now been identified. Almost simultaneously, males in some strains of mice deficient in this enzyme as a result of homozygosity for an inactivated GBA-2 locus, were found to be infertile. Mild glycolipid excess was detected in the tissues
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but the mice of both sexes had a normal life span and were without overt disease [65]. The molecular basis for this strain and species variation is unclear but it seems probable that the enzyme is implicated in the trafficking of glycolipids from the endoplasmic reticulum to the
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cell membrane and acrosome -at least of murine spermatozoa.
Although miglustat is known to reduce the rate of formation of glycosphingolipids as predicted from its proposed action as a substrate-inhibitor for Gaucher and related diseases at the level of UDP glucosylceramide transferase, its powerful effect on the non-lysosomal
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glucocerebrosidase, GBA-2, contributes to its therapeutic efficacy as indicated by genetic crosses of GBA-2 deficient mice onto an inducible Gaucher disease strain [66]. However, with the recent description of severe neurological abnormalities in patients harbouring mutations in GBA-2 [67, 68], there are theoretically uncomfortable aspects to the clinical
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development of non-selective hydrophobic iminosugars such as N-butyldeoxynojirimycin
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(miglustat) for patients with non-neurological manifestations of Gaucher disease.
Niemann-Pick disease type C (innovative therapy for a neurodegenerative disease) Of note, N-butyldeoxynojirimycin (miglustat, ‘Zavesca™‘) has recently been approved by the European Medicines agency for Niemann-Pick disease Type C, another lysosomal disease affecting the brain in which disturbed cholesterol trafficking to lysosomes and its defective esterification is associated with neuronal accumulation of glycosphingolipids. Inhibition of glycosphingolipid synthesis by miglustat was found to delay onset and prolong survival in experimental (genetic) models of this disease in mice and cats [69,70]. The agent also corrected the lysosomal trafficking defect using cultured B lymphocytes obtained from patients with the disease after exposure to the drug [71].
A prospective clinical trial appeared to show neurological stabilization or even benefit in
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some individuals with improvement in supranuclear gaze palsy and dysphagia [72]. Since then, long-term clinical findings supportive of miglustat for stabilizing Niemann-Pick C disease have been reported [73]. The agent, which may serve to reduce the intralysosomal pool of calcium-sequestrating sphingosine in this disorder [74], has been approved for in 43 countries, including the European Union since 2009 and Japan since 2012 for the treatment of progressive neurological manifestations in adult patients and paediatric patients with
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Niemann-Pick type C disease. However approval of this innovative agent has not, at the time of writing, been granted by the Food and Drug Administration (USA). Since there is no definitive treatment for this neurodegenerative disorder, miglustat delivers, in part at least,
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relief from accelerated clinical decline in some patients with this cruel dementing illness who otherwise would be without hope. It moreover opens up other avenues for laboratory
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investigation and clinical research in Niemann-Pick type C disease.
Iminosugars for potential development as substrate-biosynthesis inhibitors Medicinal use of the imino sugar miglustat, (N-butyldeoxynojirimycin) is restricted by its non-ideal inhibitory activity directed against its therapeutic target (IC50 for UDP-glucose: Nacylsphingosine D-glucosyltransferase ≈ 10μM) and its limited specificity. This explains the
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untoward clinical effects, including diarrhoea and weight loss, which are in part consequences of inhibition of off-target glycosidases. Moreover, the pharmaceutical action and some unwanted effects of miglustat may be attributable to its inhibitory effect on the neutral glucocerebrosidase (GBA-2). Despite these manifest difficulties, however, it was
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realized from the time of first use, that the ability of the substituted iminosugars to traverse the blood-brain barrier and distribute in nervous tissue had distinct potential for
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pharmaceutical exploitation, which was lacking in the competing prototypic inhibitors based on pyrrolidone structures synthesised by Radin and colleagues. This characteristic has drawn attention to the glycosphingolipidoses with manifestations in the central nervous system as a justifiable and important focus of continuing therapeutic research.
The existence of a promising congener of miglustat, the galactose analogue (N-butyl galactodeoxynojirimycin, OGT928 – now also owned by Actelion) has improved properties and would appear to hold a high priority for therapeutic exploration [75]. Like miglustat, the agent is excreted unchanged principally by the kidney and its imino ring cannot readily be cleaved in mammalian tissues. It does not inhibit many intestinal disaccharidases and unlike
miglustat in experimental animals does not induce weight loss – neither does it inhibit
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alpha-mannosidases involved in N-linked oligosaccharide processing. Platt and colleagues have shown no toxicity in high doses given to mice (4.8g per kilogram body weight daily)[76]. Phase 0/I studies have been conducted in healthy persons at a single dose in the early and further studies and dose-escalation studies have conducted in volunteers; the outcome of long-term exposure in animals are awaited. While, the inhibitory concentration of this galactose-based analogue for the target enzyme
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for sphingolipid biosynthesis, UDP-glucosylceramide transferase, is rather high at ≈ 10μM, its potential therapeutic ratio appears to be far more favourable. This compound enters the brain and distributes readily in peripheral tissues. With these properties in mind,
glycosphingolipidoses must surely be encouraged [77].
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enthusiastic exploration of its potential value in Niemann-Pick type C and other
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Other iminosugars have been studied intensively, including N-(5’-adamantane-1’-ylmethoxy)-pentyldeoxynojirimycin (AMP-DNM).This latter compound is a 100-fold more powerful inhibitor of glycosphingolipid biosynthesis compared with miglustat (Nbutyldeoxynojirimycin); it is moreover freely bioavailable and apparently well tolerated [78]. Pharmacological reduction of glycosphingolipid abundance by AMP-DNM, did not materially
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change concentrations of ceramide (see below).
GM2 gangliosidosis (Tay-Sachs and Sandhoff diseases)
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Given the relative clinical success in mild-to-moderate Gaucher disease, two clinical trials of miglustat have been carried out in attenuated forms of GM2 gangliosidosis: in 30 late-onset
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patients with Tay-Sachs disease [79] and in juvenile Sandhoff disease [80,81]. To evaluate the safety and efficacy of miglustat in patients with Tay-Sachs disease, a randomized multicentre study was conducted over 12 months with a further 24 months extension [82]. Primary efficacy endpoints were change in eight measures of isometric muscle strength in the limbs and isometric grip strength; other endpoints included gait, balance, disability, and other neurological assessments.
No differences attributable to the study drug were
observed in any measure of efficacy, during the entire study period. The most common treatment-related adverse events were decrease in weight and diarrhoea. Minor indications of possible benefit in swallowing were observed in one small study but cognition deteriorated [80]. In another open-label and single-centre study of five Sandhoff patients
treated with miglustat, all showed neurological deterioration over the period of the study,
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with worsening of gait, speech and coordination [81]. It seems that despite other isolated reports of slight benefit, miglustat does not ameliorate progressive deterioration in most neurological domains, including cognition.
The prototypic substrate biosynthesis inhibitor in this field, miglustat, is a weak inhibitor of UDP-glucosylceramide transferase, but to some extent traverses the blood-brain barrier;
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while it appears that achievable concentrations in the nervous system are insufficient to improve neurological injury in Gaucher disease, the agent may have useful effects on pulmonary manifestations in patients with chronic neuronopathic variants [63]. While the
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drug is useful in some patients with Niemann-Pick type C disease, it has proved disappointing in GM2 gangliosidosis – with progressive neurodegeneration related to impaired degradation of higher-order glycosphingolipids that are several biosynthetic
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reactions removed from glucosylceramide
Innovative synthetic ceramide analogues as treatment for glycosphingolipidoses Synthetic molecules which serve as powerful inhibitors of the reaction target, UDP-glucose: N-acylsphingosine D-glucosyltransferase (UDP-glucosylceramide transferase) have been
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identified. After many years, the lead agent has finally been introduced as a second-in-class substrate biosynthesis inhibitor, and other molecules are in late-phase clinical development for the sphingolipidoses. Already there is convincing evidence of therapeutic success and clinical utility with the lead compound, which in 2014, has gained regulatory approvals for
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Eliglustat
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first-line use in Gaucher disease, internationally.
Eliglustat (Genz-112638) is a distant derivative of a series of inhibitors of UDPglucosylceramide transferase originally designed by Vunnam and Radin [53]. However, the prototype molecule not only depleted glucosylceramide-based sphingolipids but inhibited cell growth and induced apoptosis. Investigations revealed elevated cellular ceramide concentrations, which were at first attributed to the build-up of a precursor pool of ceramide that had not been utilized in the UDP-glucose: N-acylsphingosine Dglucosyltransferase reaction. It was later realized that the erythro-enantiomers of pyrrolidino-substituted molecules independently increased ceramide concentrations [57]. Derivatives of the inhibitors with aliphatic substitutions were subsequently found to have
enhanced potency with respect to glucosylceramide biosynthesis, as well as lesser effects on
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intracellular concentrations ceramide [58]. The ‘off-target’ activity of the whole series of agents was eventually localised to an unknown cellular pathway involving acylation of ceramide at the 1-hydroxyl position and catalysed by a previously unknown 1-Oacylceramide synthase (phospholipase A2) [83]. In the presence of ceramide as an acceptor, 1-O-acylceramide synthase transacylates ceramide, utilizing the sn-2-fatty acid of
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phosphatidylethanolamine or phosphatidylcholine.
The lead inhibitor for preclinical development, D-threo-1-ethylenedioxyphenyl-2-palmitoyl3-pyrrolidino-propanol had proven activity in the nanomolar range towards UDP-
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glucosylceramide transferase and all the threo-diastereomers of the pharmacophore demonstrated this desired activity at concentrations not inhibitory towards 1-Oacylceramide synthase – a matter of critical importance for subsequent pharmaceutical
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development [84]. Substitutions of the acyl group in amide linkage influenced potency but the final agent, Genz-112638, with an octanoyl substituted (eight-carbon fatty acid) analogue had the optimal half-life and pharmaceutical properties chosen for joint clinical development with the Genzyme corporation as Genz-112638, eliglustat, (N-((1R,2R)-1-(2,3 dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)octanamide
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(2R,3R)-2,3-dihydroxysuccinate) [56, 85, 86].
After the pivotal trial of miglustat in patients with Gaucher disease [60], the eliglustat clinical platform was accelerated. The enhanced enthusiasm for clinical development
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resulted in a good effect, albeit nearly 15 years later: eliglustat has recently been approved (2014) as Cerdelga™ for the first-line oral therapy of type 1 Gaucher disease in adults by the
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US Food and Drug Administration, as well as the European Medicines Agency.
Unlike miglustat, eliglustat tartrate and its congeners are potent, highly specific inhibitors of UDP glucosylceramide transferase at ≈25nM; the inhibition is non-competitive with respect to the nucleotide sugar substrate. As a transition-state analogue rather than a sugar analogue, eliglustat does not inhibit intestinal disaccharidases at concentrations in the high micromolar range; the molecule has likewise no inhibitory effect on the acid glycosidase βglucosylceramidase, which is itself deficient in Gaucher disease [85]. Eliglustat tartrate (Genz-112638) and its free base (Genz-99067) have undergone comprehensive exploration of its phamacodynamic and pharmacokinetic properties, as well as preclinical toxicology
[85,86,87]. After exposure in healthy volunteers, eliglustat has been evaluated over the last
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decade in phase II and III clinical trials involving nearly 400 patients with type 1 Gaucher disease in nearly 20 countries.
Recently published four-year results of an open-label phase II study of nineteen previously untreated type 1 adult patients of mean age 31 years (range 18-55) with splenomegaly complicated by thrombocytopenia and/or anaemia as entry criteria showed: that
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haemoglobin concentration increased by 2.3 ± 1.5 (baseline: 11.3 ± 1.5) g/dL and the platelet count increased by a mean of 95% (baseline: 68.7 ± 21.2) x 109/ L , respectively. Mean spleen and liver volumes decreased by 63% (baseline: 17.3 ± 9.5) and 28% (baseline:
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1.7 ± 0.4) multiples of normal, respectively. The surrogate markers of Gaucher disease activity, plasma chitotriosidase and chemokine CCL-18 each decreased by 82%; plasma glucosylceramide and GM3 concentrations rapidly diminished into the healthy reference
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range after drug exposure. Unexpectedly, the medicinal effects of this oral drug were found to be far-reaching in the skeleton with mean bone mineral density T-score for the lumbar spine significantly increased by 0.8 (60%) (baseline: − 1.6 ± 1.1). Skeletal magne\c resonance T1w imaging showed stable or diminished femoral ‘dark’ marrow signals in 17/18 patients. There was a significant decrease in the semi-quantitative bone marrow burden
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score. These latter findings are attributable to improved radiological features of Gaucher cell infiltration; notably, no clinical bone crisis events occurred during the entire study period. [88,89].
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In the long-term phase II cohort, exposure to eliglustat improved the femoral marrow parameters in 28 % of the patients in the first year and 56 % by the fourth year. The
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response was qualitatively similar to that observed with imiglucerase, in which marrow improvements occur within the first year in a minority with more patients showing improvement with prolonged treatment - although up to one third remain unresponsive [90].
The results of controlled clinical trials in adult patients with non-neuronopathic Gaucher disease naϊve to specific treatment compared with placebo (‘ENCORE’) and in another noninferiority trial (‘ENCORE’), changing to eliglustat after control of their disease with enzyme therapy (principally imiglucerase) and stable for at least three years, also confirm that eliglustat has a therapeutic effect which rivals that of enzyme therapy. These two pivotal
phase III clinical trials have been completed and are now in their extension phases [91].
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Two-year data are soon to be released.
Eliglustat, although a potent and selective inhibitor of a key biosynthetic reaction, appears to have few serious unwanted effects. Less than 5% of patients stopped the drug. Hitherto with almost a decade of clinical exposure, including about 400 patients, most adverse events appear to be mild and in the trials, mainly unrelated to treatment. An analysis 535
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patient-years of drug exposure involving 393 trial subjects, indicates that it was welltolerated: most adverse events were mild to moderate and not considered to be related to eliglustat. These included headaches, arthralgia, and transient diarrhoea. Most serious
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adverse events were unrelated to eliglustat tartrate: pregnancy, myocardial infarction (three patients with risk factors), and manifestations attributable to Gaucher disease (fracture, joint dislocation, a liver carcinoma which was in retrospect present at the time of
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enrolment, and cholecystitis). Adverse events probably related to eliglustat occurred in two patients (abdominal pain, lethargy, and skin eruption); one patent had signs of a mild peripheral neuropathy not attributed to eliglustat.
Eliglustat is principally metabolised by the liver and this is dependent on the cytochrome
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P450 CYP2D6 isozyme -genetic variation of which determines metaboliser status as well as the need to scrutinize potential drug-drug interactions with co-medication [92]. Unless there is scrupulous attention to sampling, plasma concentrations are generally not reliable for determining metaboliser status. Thus molecular analysis of genetic CYP2D6 variants is
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required to guide dosing of eliglustat, which is normally correlated with CYP2D6 genotype and body weight. Adjustments are needed in this context and in relation to the choice of
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concomitant medication. At present, patients who are CYP2D6 ultra-rapid metabolisers may not achieve adequate concentrations of the drug to achieve a therapeutic effect; further, a specific dosage cannot be recommended for those patients whose CYP2D6 genotype is indeterminate. Preclinical studies showed that eliglustat, like other drugs in clinical use, can at high concentrations affect cardiac conduction. Thus there is a risk that eliglustat will prolong cardiac PR, QTc, and QRS intervals at substantially raised plasma concentrations; for these reasons, the drug should be avoided in patients with pre-existing cardiac disease or in combination with cardioactive drugs in Class IA (e.g. quinidine, procainamide) and Class III (e.g. amiodarone, sotalol). However, with rigorous monitoring throughout the clinical programme, no convincing evidence for harmful effects on the heart and its conducting
system were reported. The behaviour of the drug in patients with renal or hepatic
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impairment is unknown and these conditions should contraindicate its use; similarly in young women who are breast-feeding or may become pregnant and are not using contraception, the drug should be avoided.
Therapeutic position of eliglustat
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The therapeutic properties of eliglustat offer the prospect of a unique first-line oral agent and as a medication for maintenance in adult patients with non-neuronopathic Gaucher disease who have already received targeted enzyme treatment. Registration will be
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accompanied by a regulatory commitment to exploration for paediatric use through the conduct of additional clinical trials in children. However, while obviating the need for infusions may be particularly welcome by children, eliglustat is in one sense a
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disappointment - for it cannot be expected to alleviate the neurological consequences of Gaucher disease in the brain. This disadvantage is poignant in the severe variants of Gaucher disease in which the neuronopathic effects are invariably manifest in childhood.
Anderson-Fabry disease
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Extended use of eliglustat and its congeners for the treatment of glycosphingolipid diseases will be restricted to those disorders such as Anderson-Fabry disease, where the condition principally affects peripheral tissues and where already there is experimental evidence of the predicted therapeutic efficacy of this inhibitor class [93].
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While infusions of α-galactosidase help to clear endothelial storage of globosides associated with the lethal cardiac, renal and cerebrovascular disease, variability is seen in the clearance
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from several other cell types and the therapeutic effects are relatively poor compared with enzyme therapy in Gaucher disease. Thus it is possible that further clinical developments for single or combined treatments will be undertaken for Fabry disease (see below).
Use of eliglustat for neuronopathic variants of Gaucher disease and the GM2 gangliosidoses is out of the question but as discussed in relation to the iminosugars, given the massive human need for credible innovation in specific medical care for these progressive diseases, there is a strong case for further scientific advancement. The likely explanation for the poor distribution of eliglustat in the brain is that the compound is transported by the pglycoprotein, multidrug-resistance (MDR1) molecule which is expressed at the site of the
blood-brain barrier tight junctions between the cerebral endothelial cells, which form the
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diffusion barrier selectively excluding most blood-borne substances and xenobiotics from entering the brain. Recently, Shayman and colleagues have confirmed that eliglustat is a substrate for the MDR system and have designed new inhibitors of UDP-glucosylceramide transferase which are neither identified by the p-glycoprotein nor transported; one of these was shown to be effective in reducing brain glucosylceramide concentrations in a short-term in vivo experiment carried out in the murine model of acute neuronopathic Gaucher
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disease. The authors have moreover set out a useful general strategy for testing compounds that lack affinity for MDR1 but retain their potency as well as critical selectivity
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towards UDP-glucosylceramide transferase [94].
Other innovative glycosphingolipid biosynthesis inhibitors
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Aware of the perennial need for improvement and innovation, particularly in relation to the optimal pharmacological properties required – including specificity towards the therapeutic target , metabolic disposal, lack of toxicity and pharmacokinetics as well as the capacity for distribution into the central nervous system, Genzyme (a Sanofi company) appears to have made a decision to ‘go it alone’. Recently the company has filed a US patent for chemically
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novel glucosylceramide synthase inhibitors [US 20140255381 A1]. The application is described as relating to new inhibitors with radically distinct chemical structures that will be useful for the treatment of metabolic conditions, ‘such as lysosomal storage diseases, either
cancer’.
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alone or in combination with enzyme replacement therapy, and for the treatment of
It is not relevant to discuss this development in detail, but it is clear that the protean
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involvement of glycosphingolipids beyond disorders affecting the lysosome extends our knowledge of intriguing biological science to become highly attractive for investment by Biotech. The application is expansive and describes many potential uses; diseases that cause renal hypertrophy or hyperplasia such as diabetic nephropathy; diseases that cause hyperglycemia or hyperinsulinemia; cancers in which glycolipid synthesis is abnormal, infectious diseases caused by organisms which use cell surface glycolipids as receptors, diseases in which synthesis of glucosylceramide is essential or important, diseases in which excessive glycolipid synthesis occurs (e.g., atherosclerosis, polycystic kidney disease, and renal hypertrophy), neuronal disorders, neuronal injury, inflammatory diseases or disorders associated with macrophage recruitment and activation (e.g., rheumatoid arthritis, Crohn's
disease, asthma and sepsis) and diabetes mellitus and obesity. Particular mention is made of
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the observation that in acute myeloid leukemia cells P-glycoprotein confers resistance to ceramide-induced apoptosis and that the inhibitors can be useful for treatment of proliferative disorders by inducing apoptosis.
The innovative lead molecule, Genz 161 ((S)-Quinuclidin-3-yl (2-(2-(4-fluorophenyl) thiazol4-yl)propan-2-yl)carbamate), is chemically distinct from the prototypic inhibitors of UDP-
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glucosylceramide transferase first developed by Radin, Shayman and their colleagues. Genz 161 has been successfully tested pre-clinically in the K14 mouse, a severe murine model of acute neuronopathic Gaucher disease [95]. Using this stringent model with complete loss of
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glucosylceramidase activity, the authors showed that after systemic administration of the inhibitor (which crosses the blood-brain barrier), accumulation of glucosylsphingosine and glucosyceramide was diminished; gliosis with signs of neuro-inflammation and infiltration of
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macrophages and microglia were ameliorated and survival was prolonged. These findings, based on an in-house discovery programme strengthen the belief that potent systemically administered glucosylceramide synthase inhibitors that traverse the blood-brain barrier are likely to be developed by Genzyme; they ultimately hold out considerable hope of benefit
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for patients afflicted by neuronopathic lysosomal diseases.
Substrate inhibitors for lysosomal diseases other than glycosphingolipidoses Genistein
The mucopolysaccharide diseases have a common cause in impaired breakdown of
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glycosaminoglycans, the undegraded complexes of which are considered to play an important part in cellular pathogenesis. While enzyme therapy is at various stages of clinical
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development for several of the mucopolysaccharide diseases (MPS I, II, IVA, VI and VII), improved treatments are needed – not least to address the frequent neurological manifestations which complicate most of these conditions and which are likely to remain beyond the reach of enzyme therapy. Hitherto, the search for tractable biochemical means to interfere with the biosynthesis of complex glycosaminoglycans in patients with mucopolysaccharidosis has been disappointing.
Genistein, an isoflavone found in legumes particularly soybean, is a potent phytoestrogen with wide-ranging biological effects, several of which are promulgated in the sphere of alternative therapeutics for combating oxidative stress, cancer, diabetes, obesity,
inflammation, osteoporosis, neuropathy - and others. However, Wegrzyn and colleagues
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indicate a more specific action by investigating the effects of specific components of genistein preparations on the incorporation of radioactive sulphate into glycosaminoglycans by cultured fibroblasts from healthy subjects and patients with several different mucopolysaccharidoses [8,14]. This research had been suggested by reports showing effects on tyrosine kinase receptors and glycosaminoglycan synthesis [96, 97]. At low micromolar (≈10μM) concentrations, the agent was found to inhibit incorporation of radioactive
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sulphate by about 90% in all samples; prolonged incubation in MPS type 1 fibroblasts showed that total cellular glycosaminoglycans were reduced to a level comparable to that induced by in vitro exposure to recombinant α-L-iduronidase (laronidase, Aldurazyme™)
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with partial restoration of the ultrastructural appearances of pathological lysosomes to normal [98].
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Later preclinical studies of the effects of oral genistein preparations have been conducted in mouse models of MPS II (Hunter) and Sanfilippo B (MPS IIIB) diseases [99, 100]. High doses (160 mg/kg/day) of genistein aglycone (4,5,7-trihydroexyisoflavone) either partially corrected the storage of glycosaminoglycans [99] or when given continuously over a 9 month period to MPS IIIB mice significantly reduced lysosomal storage of heparan sulphate s
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and neuroinflammation in the cerebral cortex and hippocampus while improving the behavioural defects [101]. Enhanced synaptic vesicle protein expression and lysosomal accumulation of glycosaminoglycans in the cerebral cortex were also noted.
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In an open-label study of 10 patients given genistein for 12 months, there was a significant reduction in urinary excretion of heparan sulphate with improved hair morphology and
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cognitive function; no adverse reactions were observed. [102].
While several studies have reported treatment of MPS III patients with low dose genistein (5-15mg/kg/day) with no serious adverse effects and variable neurocognitive outcomes, controlled clinical trials of various doses and genistein preparations with variable compositional efficacy have been examined principally in Sanfilippo disease [102]. The effects at low dose (10mg/Kg/day) in a blinded cross-over trial were to induce modest reductions in the excretion of glycosaminoglycans without overt clinical benefit [103]. However, while apart from inducing gynaecomastia in young males, attributable to the phytooestrogenic properties of the agent, higher doses (150mg/Kg/day) appear to be
tolerable, although at the time of writing, biochemical and clinical efficacy outcomes appear to be less certain [104,105].
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Those mucopolysaccharide diseases in which there is a neurological component are immensely challenging; and compared with the sphingolipidoses, they are far less well understood in biochemical terms. With this comes a more limited understanding of molecular pathogenesis and a limited range of techniques materially to correct the toxic
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effects of harmful metabolites, especially in the nervous system. The rôle of genistein is not yet fully worked out but even if ultimately it is found to be a disappointing starting point for innovative treatment, it provides a focus of clinical interest in a condition where the
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ultimate rewards evidently attract courageous scientific effort.
D. Functional Complementation
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Complementation stratagems
These established stratagems depend principally upon therapeutic complementation, either of whole cell function, as in haematopoietic stem-cell transplantation, which may or may not provide additional complementation at a distance through the process of secretion and recapture of lysosomal matrix enzymes, or in the provision of the missing protein, for
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example in macrophage-targeted enzyme therapy in Gaucher disease. Here the ‘patch-up’ concept of organ replacement (eg renal, cardiac or hepatic transplantation, jointreplacement surgery) is excluded, since these tactics have been used for many decades and are now incorporated into the mainstream of contemporary medical practice in developed Also
important
for
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countries.
functional
complementation
is
consideration
of
pharmacological chaperone molecules to enhance the activity of residual mutant proteins,
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and gene therapy, which is now on the threshold of clinical practice after many challenging years of disappointment in the context of failed clinical trials and premature trumpeting of its potential benefits. Finally, it is important to realise that each of these different therapeutic categories have in their sights the possibility of addressing the challenging neurological manifestations of many lysosomal diseases.
(i) Haematopoietic stem-cell (previously bone-marrow) allo-transplantation (ii) Reinfusion of ex-vivo vector-transduced autologous haematopoietic stem cells (iii) Direct gene transfer
(iv) Enzymatic augmentation
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(v) Enzymatic enhancement and stabilization (pharmacological chaperone therapy)
(i) Stem-cell therapy Induced pluripotential stem cells have excited much interest and principally are of use for in vitro modelling of human disorders. The highly attractive and futuristic concept of stem-cell
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therapy, based on reinfusion of genetically corrected autologous pluripotential stem cells, derived from somatic cells after a simple biopsy procedure, is firmly in this category. While rapid advances are underway, at present, safe means for inducing such stem cells for donorrecipient transfer and much-awaited therapeutic correction [106] have yet to be introduced
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for clinical application and will not be discussed further here.
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Haematopoietic stem-cell therapy
At present, two main stem cell complementation systems are of interest for development in the lysosomal diseases: (a) further exploration of haematopoietic stem-cell therapy – one of the first potentially curative treatments used in lysosomal diseases with early experience in the 1980’s for the treatment of mucopolysaccharidoses [107] (principally MPS I and II in the
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first instance) and Gaucher disease, in which it was first successfully introduced in 1982 [108]; (b) ex-vivo gene transduction of autologous haematopoietic stem cells using lentviral vectors; these cells are then re-infused for engraftment under myelo-ablative conditions
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[109,110].
Stem cell therapy (without gene transduction)
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Haematopoietic stem cell transplantation has been used in attempts to treat lysosomal diseases with some success over three decades. The haematopoietic stem cells from suitable donors allow the generation of progeny that are competent in lysosomal functions and can migrate in small numbers over time to the brain by traversing the physical bloodbrain barrier. As occurs in the bone marrow, and other niches of destination, the donor stem cells generate a steady supply of self-renewing blood cell lineages that are competent in autonomous functions (such as phagocytosis and immune reactions); and in the brain migrate to sites of disease where they can undertake scavenging activity as healthy counterparts of the diseased microglia and other cells with impaired lysosomal function. In the case of diseases such as Krabbe, the Sanfilippo syndromes, as well as metachromatic leukodystrophy, the donor cells are also able to supply enzymatic complementation to
neighbouring tissue by means of the secretion-recapture process for soluble matrix acid
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hydrolases [8, 14, 111].
The outcomes of transplantation in more than 60 European patients were earlier reported [114] and in Gaucher disease, particularly, the earliest reports clearly show that timely replacement of the diseased macrophage population and complementation of other haematopoietic lineages can secure dramatic improvement in outcome and almost
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complete correction (arrest) of somatic disease [112,113,114]. Contemporary stepwise improvements in conditioning, clinical selection, source of stem cells and nature of the donor as well as timing of the procedure have led to greatly improved safety and remain a
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focus for continued refinement and innovation in current practice.
Haematopoietic stem cell therapy has been extended to the lysosomal diseases with rapid
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loss of faculties and neurodegeneration such as metachromatic leukodystrophy and Krabbe disease - largely based on the advocacy of early investigators [115, 116,117]. Current developments in this demanding clinical field mainly focus on ensuring the indications for the intervention are tailored to the stage of disease and likely response; they also relate to
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improved techniques and regimens for conditioning to ensure safe engraftment.
At present, while haematopoietic stem-cell therapy offers an incontrovertible therapeutic effect in several disorders, particularly the MPS disorders and possibly in severe chronic neuronopathic Gaucher disease (type 3), it must be recognized that numerous practical
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limitations restrict its use. Typically, a suitable HLA-matched sibling donor is not available, and despite the best efforts of charitable or state-funded agencies to search for matched
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unrelated donors, their identification and confirmation of compatibility can be a lengthy process; moreover, the success of the procedure mandates early diagnosis in the recipient – a persistent challenge in all therapeutic settings for rare disorders. In the last 15 years, the use of umbilical cord blood donors as a rich source of unrelated donor stem cells for suitable recipients and the safety of the procedure - with clearer case selection and optimized conditioning and myeloablative techniques – has led to stepwise improvement in outcomes for some lysosomal diseases.
Despite these improvements, persistent morbidity and mortality is associated with the procedure and the decision to use it must as always be balanced carefully against the
severity of the disease and the likely therapeutic response [118,119,120]. Hitherto its use,
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with the typical exception of MPS I Hurler has usually been deferred in favour of enzyme therapy - whenever this is available. Recent evidence suggests that while enzyme therapy may improve engraftment, the use of haematopoietic stem cell transplantation improves the high incidence of neutralizing allo-antibodies observed in MPS I after pharmacological enzyme replacement therapy [121]. It has to be recognised however, that in countries where government support for expensive treatments is limited or cannot be underwritten
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for long periods due to political and budgetary uncertainties, the challenging but single large cost of haematopoietic stem cell transplantation may be the only solution.
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There are numerous contemporary examples of patients with Gaucher disease in such healthcare settings where the procedure has been carried out: in several Middle-Eastern and Asia countries this option has been the only one available. However, given that severe
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neuronopathic Gaucher disease is greatly over-represented in these populations, and that some patients may secure greater benefit from the procedure than enzyme therapy, one cannot be sure that they have always had the inferior choice of primary treatment. This issue and support for haematopoietic stem-cell therapy for Gaucher disease has very recently been revisited by Ito and Barrett [122]. While their thesis is marred by some
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inaccuracies and limited citation of contemporary therapeutic developments in the treatment of Gaucher disease [123], the authors raise an argument that merits fuller discussion for future developments in Gaucher disease and other lysosomal diseases, such as MPS II (Hunter disease), in the context of global medical realpolitik. A patient advocacy
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group ensured that a global moratorium to prevent bone marrow transplantation in this disease was put in place in the treating community. However, recent data from Japan cast
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doubt on this extreme view [124,125] and if future development is to proceed scientifically for the benefit of patients, a re-evaluation of the potential efficacy and indications for haematopoietic stem-cell therapy would be advisable.
(ii) Ex vivo haematopoietic stem-cell gene (reinfusion) therapy This stratagem represents an imaginative extension to the use of haematopoietic stem cell therapy: while it offers to overcome some of the difficulties associated with donor-derived cells, it also seeks to enhance the weak therapeutic effect of cellular complementation delivered into the peripheral circulation and able to correct many of the peripheral manifestations of disease but with limited salutary effects on the brain.
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Bigger and Wynn [126] provide an excellent contemporary review of the development of this technique and clinical extension from haematological diseases and immune deficiency syndromes such as adenosine deaminase deficiency [127] and X-linked severe combined immunodeficiency [128] to the metabolic leukodystrophies, adrenoleukodystrophy, an Xlinked peroxisomal defect [129] and latterly, as already set out, metachromatic leukodystrophy [110].
Over three decades since the first clinical attempts at retroviral
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transfer of genetic material into haematopoietic progenitor cells, it has been shown that gene therapy can be curative, while obviating the need for suitably HLA-matched allogeneic donors and avoiding the risk of graft-versus-host disease. Improvement in the efficiency and
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nature of self-inactivating lentiviral vectors also avoids mutagenesis with oncogenic effects due to biased integration events at key loci in the host genome. Third-generation vectors with refined protocols for myeloablation and engraftment, as well as improved
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manufacturing procedures are already opening up this stratagem for therapeutic gene transfer [130] and, sooner or later, much more extensive application in the lysosomal diseases.
It is clear that despite their often severe nature and multisystem effects and distribution,
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the lysosomal diseases are attractive therapeutic targets for the application of gene transfer techniques. Correction by gene transfer of a small focus of living cells within an affected tissue or organ would be expected to induce secretion and distribution of the cognate processed glycoprotein harbouring the appropriate carbohydrate recognition signals locally
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into extravascular space and circulation for capture and uptake with correction of the
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lysosomal defect in diseased cells at a distance.
Ex vivo gene therapy utilizing haematopoietic stem cells harnesses the concept of a therapeutic Trojan horse. The application has principally been directed to the delivery of the competent cells to brain, and with the aim of enhancing efficacy as a consequence of increased expression of the corrective lysosomal protein brought about by genetic transduction of the stem cell population. The use of autologous stem cells harvested for transduction ex vivo has the merit of ready availability and lack of immune incompatibility.
Of particular interest have been the heroic efforts and emerging clinical success of the research group led by Naldini and colleagues in the San Raffaele University in Milan. After
considerable efforts to perfect the techniques in the somewhat unsatisfactory clinical model
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of metachromatic leukodystrophy in mice [131], Biffi et al [109] recently reported a phase I/II clinical trial employing ex vivo transfer of the wild type human arylsulphatase A gene into marrow-derived CD34+ haematopoietic progenitor cells harvested from three unrelated infants diagnosed with infantile metachromatic leukodystrophy in the pre-symptomatic phase as a consequence of disease in older siblings. The trial subjects received the intervention 2 to 12 months before the reported age of onset of the disease in their affected
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siblings. Transduction was optimized to reach at least two vector copies per genome to ensure optimal enzymatic overexpression for therapeutic efficacy previously determined in experimental studies. These genetically engineered CD34+ cells were stimulated ex vivo with
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early-acting cytokines and transduced with purified third-generation lentiviral vectors expressing the human cDNA under the control of the human phosphoglycerate kinase. After expansion, the transduced progenitor cells were infused into the recipients who had
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undergone myeloablation with intravenous busulphan, given every 6 hours for three days beforehand. After a busulphan-free interval of at least 24 hours, the engineered CD34+ cell product was infused as a suspension; no immunosuppression was given to the recipient.
Biffi et al confirmed stable engraftment of the transduced stem cells in the bone marrow
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and peripheral blood of all patients at all times tested, with 45 to 80% of bone marrow– derived haematopoietic colonies harbouring the vector. Arylsulphatase A activity was expressed above the healthy reference range in the haematopoietic cell lineages and in cerebrospinal fluid. Safety monitoring required repeated analysis of lentiviral integration
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sites: this showed that the high gene expression was associated with polyclonal engraftment of transduced cells; no aberrant clonal behaviour was identified. Several integration sites
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were shared among mature blood elements as well as progenitors and mature blood elements, indicating efficient transduction and engraftment.
The authors provide evidence for a clear therapeutic benefit of this intervention initially in three trial subjects followed for at least two years after the intervention. Neurological and motor evaluation using standard tests and neuropsychological assessment was used to assess the presence of disease; neurological imaging by MR was also conducted. The disease did not progress in any of those treated patients, even beyond the time of onset of disease predicted and that projected from the behaviour of the condition and their previously affected siblings. While formally no absolute comparison was made between this
intervention and conventional haematopoietic stem-cell transplantation in early selected
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presymptomatic patients with this form of metachromatic leukodystrophy, on the balance of probabilities, the safe and tolerated therapeutic effect was gratifying and sufficiently convincing for the programme to secure pharmaceutical investment and further continuation and expanded recruitment [131]. While this laborious ex vivo stratagem is invasive and in this group of devastating neurodegenerative lysosomal diseases, not readily applicable to the many patients who are
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first affected without systematic population-based screening procedures in place, the therapeutic concept has to a large extent been convincingly demonstrated in human medicine. However, as in the case of conventional transplantation therapy which has
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already proved to be highly controversial in the context of the specific neonatal screening programme for Krabbe disease in New York State [133, 134], the practicalities of introducing neonatal screening require careful thought – even though this programme has expanded to
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several other states [135]. Simply linked to a potentially beneficial but innovative and heroic therapy with potentially fatal effects related to myeloablation the debate cannot be ignored, the mortality remains even for the best centres at 5-10%. When these considerations relate to gene therapy, realization of the therapeutic potential and crystallization of the innovation in the ‘real world’ of clinical practice will require further
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planning and review.
Extension to other lysosomal diseases, cystinosis – a cell-autonomous disease
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An additional aspect here with potential for innovation, is the exploratory extension of haematopoietic stem-cell therapy to other lysosomal diseases that have not been in the first
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ranks of priority. In the case of cystinosis, management of the consequences of the Fanconi syndrome with progressive interstitial nephritis placed emphasis on renal transplantation and later, as the systemic nature of the disease rapidly became apparent, the early introduction of cysteamine therapy has become a dominant priority for a disease caused by defects in the lysosomal membrane protein, cystinosin. It is notable that latterly, there is mounting evidence that this membrane transport disorder of the lysosome may also show a salutary response to haematopoietic stem cell therapy – even though, as with Gaucher disease, there is no possibility of functional complementation via the secretion-recapture pathway that is envisaged to occur in vivo in the case of soluble matrix enzyme of the lysosome.
Cherqui and colleagues [136] have carried out syngeneic bone-marrow transplantation from
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wild-type donor mice to mice lacking cystinosin. These mice serve as a useful model of human cystinosis and develop pathological systemic cystine crystal deposits, with tubulopathy and renal failure accompanied by bone and muscular defects; characteristic cystinotic ocular changes also occur. Transplantation of stem cells from wild-type donors prevents kidney injury and corneal deposition in the mutant recipients. These investigators showed that full engraftment of marrow stem cells expressing functional cystinosin was
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required to arrest the disease. Moreover, fastidious quantitative analysis of the expression of functional protein and tissue colonization by donor stem cells was shown to correlate with significant (57-94%) reduction of cystine concentrations. While these findings appear
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counterintuitive and given some discrepancies between the mouse and human disease, direct extrapolation of these results to patients with cystinosis will be challenging, the
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results are tantalizing.
One speculative possibility is that phagocytosis of circulating leukocytes harbouring excess cystin and fluid-phase endocytosis contribute to the dynamic fluxes of the metabolite; in this way, a donor population of healthy haematopoietic stem cells could partially ameliorate the cystin load at peripheral sites. Indirect support for this possibility emerges from reports
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of increased plasma chitotriosidase activity in patients with cystinosis – chitotriosidase is a classical protein marker of the macrophage [137]. The possibility of cell-cell transfer of the corrective transmembrane factor (cystinosin) is also a possibility but the scale of complementation appears to be implausibly generous for such a process. This stratagem is
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now actively under investigation and represents another innovative application of therapeutic potential in contemporary medicine for the challenging specialist domain of
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lysosomal diseases [138] (see below).
Gene therapy
The principle here is to transfer human cDNA encoding the cognate wild type sequence corresponding to the appropriate protein or proteins that are malfunctioning as a result of genetic mutation in the patient with a lysosomal disorder. In this instance, the wild-type genes are transferred with viral or other vectors directly to the appropriate tissues in the host. Lysosomal diseases have the theoretical potential for successful correction by this stratagem, since the secretion-recapture system allows for extensive functional
complementation in diseased tissues; the uptake of corrective protein (almost invariably a
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soluble acid hydrolase of the lysosomal matrix) is designed to restore healthy activity and thus amelliorate, arrest or reverse the disease. In most instances where this has been successful in experimental animals, viral vector systems have been utilized; the transduced cells to act as a source of functioning enzyme which is released to effect the therapeutic complementation after uptake by the diseased cells [139]. In principle, the greater amount of enzyme that is generated and released, the more will be available for receptor-mediated
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uptake and extent of correction.
Criticial rôle of animal models for clinical development of gene therapy
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The single-gene disorders lend themselves readily to gene transfer stratagems: a large therapeutic effect is predicted after successful transduction of healthy copies of the cognate gene which when mutated has highly penetrant effects. Emerging clinical data show that
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this technology is rapidy entering a fullblown clinical phase and biotech commerical interest is burgeoning [140]. Numerous naturally-occurring and transgenic models of lysosomal diseases have been reported: not only do these usually serve as useful experimental models of the related human diseases, they are coherent biochemically and genetically – even allowing in mice for valid simulacra of the effects of particular mutations which can be
Contemporary progress
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introduced through ‘knock-in’ genetic technologies [141, 142].
Many lysosomal diseases affect the nervous system as well as peripheral tissues and are
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thus particularly challenging to treat. Nonetheless untreated neurodegeneration has extreme human and medical costs. Thus with encouraging signs of effective experimental
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gene transfer in valid animal models, expectations that clinical gene therapy will be introduced in the field as a credible option for many lysosomal diseases where effective therapy is otherwise unavailing, seem justified. With appropriate safeguards, scrupulous choice of biological target, a careful enrolment policy and evidence of efficacy based on clinically articulate criteria, direct gene transfer technology offers a vision of definitive and affordable treatment. The resurgence of contemporary interest in gene therapy has resulted from a fortunate conflation of: (i) the extraordinary therapeutic accessibility of inborn lysosomal diseases to enzymatic complementation; (ii) the success of innovative biotech investment in niche ultra-orphan therapies, where clinical need can be addressed by efficacious therapies developed in the context of anti-competitive legislation for orphan
medicinal products; and last, but dominantly (iii) the continued development of safe, simple
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but powerfully expressing vectors for gene transfer. These include small vectors derived from non-pathogenic, non-integrating adeno-associated viruses, ideally suited for safe and sustained transduction of non-mitotic cells – and which can harbour cDNA inserts encoding constitutively expressed polypeptides (for example those that assemble to generate lysosomal acid hydrolases).
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In relation to those lysosomal diseases affecting the nervous system, all therapies must take account of the blood-brain barrier and the physical blocks to generalized correction of disease at privilged sites (see chapter 3). While some innovative viral-derived vector systems
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that can traverse the blood-brain barrier after administration to the perpheral organs or circulation in adult recipients are in active development [143] in most instances where clinical stratagems are in progress, the vector delivery of the corrective lysosomal protein
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occurs after a physical breach of the barrier using direct administration at the time of a neurosurgical procedure [144]. In future innovations, however, it seems likely that rAAVrh.8, rh.10 and 9, will offer hope for treating neurological diseases in the adult patients; systemically delivered rAAVrh.10 efficiently transduces the nervous system but at least in primates, transgene expression can be limited peripherally by endogenous microRNA
Direct gene transfer
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sequences.
Here follows a brief review of gene transfer for lysosomal diseases, particularly to address
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those with an important neurological component, and with experimental development that is at, or close to clinical application. Latterly advances in viral and non-viral vectors and
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methods for their delivery have explored the correction of central nervous and peripheral tissues; this research has largely been predicated on methods to evaluate neurological outcomes in animal models.
The main challenges for gene transfer are (i) generating sufficient delivery and distribution of the therapeutic gene product in all at-risk tissues with injurious pathology such as the brain (ii) sustaining expression of the corrective gene(s) (iii) ensuring safe transduction and protein expression without harmful effects on recipient and transduced tissues. Here various stratagems are exemplified.
Intracerebral injection of recombinant adeno-associated viral vectors (rAAV) expressing
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human lysosomal proteins into white matter appears to be safe in primate brain; the vectors are capable of generating long-term sustained expression of lysosmal hydrolases at activity levels predicted to have useful therapeutic effects in late infantile neuronal lipofuscinosis type 2, (NCL2)[145]. [145]. In a clinical phase I/II study using rAAV-2 serotype vector, neurosurgical techniques and experience were reported in human subjects subjected to intensive radiological scrutiny [146]. Ten patients with late infantile neuronal
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ceroid lipofuscinosis disease each underwent infusion of AAV2 (3 x 1012 particle units) at 12 distinct cerebral locations (twodepths/burr hole, 75 minutes/infusion at 2 µL/minute). Techniques were developed to overcome obstacles for which there are few established
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procedures. The surgeon relied on preoperative stereotactic planning to optimize a parenchymal target and diffuse administration. Six entry sites, with injection at two depths each were used to reduce operative time and enhance distribution. A low-profile rigid
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fixation system with 6 integrated holding arms was utilized for simultaneous infusions in an expeditious manner and without ill-effect. These studies emboldened later trials using more developed high-expression vector serotypes for direct administration in this fatal neurodegenerative disease.
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As a prelude to clinical trials using the recombinant adeno-associated viral vector (rAAV) serotype rh.10 (rAAVrh.10) planned in 2015 for patients with the devastating disorder, metachromatic leukodystrophy, Aubourg and colleagues investigated the optimal delivery route of the vector. They used rAAV encoding human arylsulphatase A in the primate brain
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and each animal received a total dose of 1.5 × 1012 genome copies of AAVrh.10hARSA-FLAG at: (1) delivery to white matter centrum ovale; (2) deep gray matter delivery (putamen,
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thalamus, and caudate) plus overlying white matter; (3) convection-enhanced delivery to same deep gray matter locations; (4) lateral cerebral ventricle; and (5) intraarterial delivery with hyperosmotic mannitol to the middle cerebral artery. After 13 weeks, the distribution of enzymatic activity subsequent to each of the three direct intraparenchymal administration routes was significantly higher than in phosphate-buffered salineadministered controls, but administration by the intraventricular and intraarterial routes failed to demonstrate measurable levels above controls. Immunohistochemical staining in the cortex, white matter, deep gray matter of the striatum, thalamus, choroid plexus, and spinal cord dorsal root ganglions confirmed these results. Of the five routes studied, administration to the white matter led to the broadest distribution of corrective enzyme
with 80% of the brain displaying more than a therapeutic (10%) increase in arylsulphatase A
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activity above control injections. No significant toxicity was observed with any delivery route as measured by safety parameters, although microscopic inflammatory changes were observed. The author concluded that AAVrh.10-mediated delivery by direct administration into white matter is likely to be safe and yield the widest distribution of corrective enzyme;
Efficacy in human gene therapy trials for lysosomal diseases
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they have selected this asthe most suitable route for clinical vector delivery [147].
At the time of writing, investigators clearly stand at the threshold of effective clinical application of gene therapy for lysosomal diseases. From the success of treatments for
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inherited retinal disease and haemophilia [148,149]], suitable vectors and stratagems that avoid immune shut-down are becoming available. At the same time, financial ‘models’ to support largely on-off costs for their ultimate testing in phase III clinical trials are being
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negotiated with biotechnology companies.
At present, the clinical outcome of executed [144, 145] in progress [150], and planned [147] phase I/II gene therapy trials, respectively in late-infantile neuronal ceroid lipofuscinosis, Sanfilippo A , Sanfilippo B and Metachromatic Leukodystrophy are awaited. All are
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predicated on the outcomes of cogent studies in genetically and clinical coherent animal models. Additional trials in, for example, GM2 gangliosidosis [151] and other very severe near-exclusive neurological diseases of the lysosome are also at a promissory stage or active
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consideration.
After early sensational interest several decades previously, Biotech has latterly been
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disappointingly ‘risk-adverse’ towards gene transfer technologies. As a consequence, until now, the cadre of competitive interest external to academic intitutions, which is the essential catalyst for development, has been lacking. Although in many cases breakthrough treatments have hitherto proved elusive, the technologies for therapeutic exploration in humans are now safer and possess demonstrably greater efficacy for challenging lysosomal diseases such as the mucopolysaccharidoses, in which mixed systemic and neurological manifestations occur and where very costly enzyme therapy is inadequate materially to prevent decline or restore function [152]. Currently perhaps the most promising vectors are based on rAAV vectors; but the safety of non-integrating, self-annealing 3rd and 4th generation retroviral systems now render them suitable candidates for transducing of
slowly-dividing cells in the diseased tissues of patients with non-lethal chronic disorders [153]. Previously,
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although
retroviruses were
successfully introduced for fatal
haematopoietic and immunological diseases in which great experience has been gained [154], commercialization through phase III clinical trial development remained tardy.
Practical issues in relation to trials of gene therapy in lysosomal diseases While gene therapy is in its clinical infancy, it has nonetheless been delivered – hitherto
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safely - as a regulated and approved clinical entity in several severe lysosomal diseases. Several phase I/II clinical studies are underway with the main regulatory focus on safety and tolerability; but a crucial aim is to discover an effective therapy for humans, principally
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young children suffering from these ultra-rare and fatal disorders.. Ultimately the viability of gene transfer technologies will depend upon careful design, selection, conduct and analysis of heroically complex experimental procedures carried out in only a few trial Gene therapy offers the prospect of large therapeutic effects but , robust
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subjects.
information about the natural course of the disease will assist objective determination of clinical efficacy and possibly indicate whether the intervention rescues disease, reduces the rate of decline or restores neurological function. In this respect, selection of presymptomatic subjects with previously affected first-degree relatives offers the best chance
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of determining whether the intervention will indeed offer clinically articulate benefit.
The prospect of direct adminstration of vectors to the brain may ultimately prove unneccesary in some cases since it is now clear that members of the AAV9 and, possibly,
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rh.10 vector families have blood-brain barrier penetrating properties even in adult mammals. They thus can transduce the nervous system via a single peripheral injection not
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only when administered intravenously and into cerebrospinal fluid. While the safety and tantalizing efficacy of such technologies are being evaluated [155], the recent report of a high proportion of liver and lung tumours in mice with GM2 gangliosidosis that received systemic rAAV9 in the neonatal period raises concerns about oncogenicity [156] but the long-term experience with successful human rAVV therapy in haemophilia B is reassuring.
(iv) Enzymatic augmentation As demonstrated in other sections of this volume (chapters 3, 4, 5, 6 and 7), systemic administration by parenteral infusion of lysosomal proteins targeted to affected tissues by exposure of cell-surface recognition signals has had a distinguished history and continues to
hold promise for the treatment of inborn errors affecting matrix enzymes of the lysosomal
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compartment. While the stratagem is no longer new, with advances in the manufacture of stable and active recombinant therapeutic proteins in a form suitable for administration, innovative methodological adjustments continue.
Delivery of therapeutic proteins to the central nervous system depends upon biological therapies and these ‘biologics’ are in effect high molecular weight glycoproteins to which
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the mammalian blood-brain barrier is impermeable. Much effort in the competitive niche area of lysosomal diseases is thus being expended on overcoming the blood-brain barrier in practical and commercially applicable ways. Fortunately, and an advance on the position at
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the time of the development of systemic enzyme therapy for Gaucher disease, the existence of naturally-occurring and transgenic animal models, facilitates these endeavours. Proof-ofconcept studies carried out in animal models of several important lysosomal diseases has
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allowed direct intrathecal or intra-cerebro-ventricular routes for delivery of therapeutic proteins to be explored as a prelude to clinical trials. Novel and courageous applications for those diseases which affect the central nervous system have recently taken hold: intrathecal and intraventricular administration for metachromatic leukodystrophy, Sanfilippo A, Sanfilippo B and even Krabbe disease are, despite the harsh odds against sustained
blood-brain barrier.
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therapeutic success, under development as the most direct means to breach the physical
For completeness, the reader is also directed to new therapeutic enzymes in late-stage
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development for systemic treatment of Niemann-Pick disease type B, discussed in chapter 9, alpha mannosidosis, mucopolysaccharidosis type 1V A (Morquio-Brailsford disease), and
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acid lipase deficiency (Wolman and cholesterol-ester disease). Finally, mention should be made of an extraordinary phenomenon in the commercial arena of ultra-rare disorders and orphan medicinal products: the innovative emergence of exceptional competition. Apart from the two enzyme therapies simultaneously approved in Europe for Fabry disease (TKT [Shire] and Genzyme [Sanofi]), and three enzyme preparations available in the United States (two in Europe) for Gaucher disease, generic biosimilar alternatives are emerging. The first successful small biotech company in South Korea, for example, now has about 40% of the market share: ISU ABXIS have Abcertin for Gaucher disease and FABAGAL for Fabry disease. While these initiatives indicate that currently the field is attractive for technological
investment and new/old products, the changing entrepreneurial equilibrium may ultimately
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signal the end of an era of burgeoning research into innovative enzyme therapies.
(v) Pharmacological Chaperones Definition
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Chaperones interact with polypeptides to generate functionally active proteins; they inhibit formation of misfolded or aggregated structures [157]. Chemical chaperones influence the intracellular environment in which protein folding occurs and stabilize proteins against thermal and chemical denaturation. These agents are often required at very high
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concentrations and are thus never going to be appropriate for in vivo therapeutic use; however, they possess properties of utility in the manufacture of recombinant proteins. In
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this sense, they are valuable as important components for enzyme therapies. Molecular chaperones are typically endogenous heat-shock proteins which recognise misfolded proteins and bind to their hydrophobic surfaces, thus preventing denaturation. At the same time, the process known as chaperone-mediated autophagy ensures that a particular pool of cytosolic proteins is delivered to lysosomes for destruction. Proteins bearing a specific amino acid sequence, related to KFERQ, are recognized by the heat shock
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cognate protein of 70kDa, the chaperone that mediates their delivery to lysosomes for degradation – about one third of soluble cytosolic proteins contain the motif [158]. Heat shock proteins have a rôle in exocytosis and the maintenance of lysosomal integrity and are
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discussed in detail with respect another innovative treatment for lysosomal diseases that is
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in clinical development [in section B] above.
Clinical use of pharmacological chaperones Pharmacological chaperones bind to specific polypeptide conformations and thereby stabilise proteins and prevent their degradation in the proteasome and lysosomal compartment. Use of pharmacological chaperones to rescue critical functions of proteins that are misrouted because of misfolding and even aggregation in the cell is an intriguing stratagem with a long but until recently, a substantially neglected, developmental history in therapeutics.
The greatest experience of this approach has emerged from clinical experimentation in inborn errors of metabolism, many of which are have been treated empirically by supplying
large doses of relevant cofactors enzyme known to be essential for the normal function of
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the cognate protein in which the functional deficiency is identified. A long-standing classical example is the use of pyridoxine supplements in approximately half of the adult patients with homocystinuria due to mutations in cystathionine ß- synthase who can be shown to be responsive to the agent [159]. A vivid and recent example is provided by the important disorder, phenylketonuria, in which most mutations in the deficient enzyme, phenylalanine hydroxylase, cause misfolding and instability. In about one third of patients
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supplementation with natural tetrahydrobiopterin cofactor has been shown usefully to improve phenylalanine intolerance and metabolic control – leading to marketing approval of the synthetic factor sapropterin dihydrochloride as a chaperone therapy in 2007
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[www.drugs.com › FDA History]. Since the catalytic mechanism, regulation by substrate and cofactor in the context of intensive biochemical and structural studies of the enzyme have been solved, it is clear that the therapy represents a striking example of a pharmacological
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chaperone effect enhanced by positive allosteric interactions between the cofactor and common aberrant enzyme variants [160].
Thus as in this case, small molecules which serve as specific ligands for aberrant proteins are clearly attractive for exploration as pharmacological chaperones with the potential for
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clinical use. While there are few known cofactors for lysosomal enzymes, the frequent identification of unstable and misfolding variants associated with lysosomal diseases suggests that this stratagem deserves close examination.
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Protein conformation diseases and the unfolded protein response The concept of protein conformational diseases can be traced to classical studies of
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amyloidosis by Virchow and Rokitansky in the nineteenth century. Amyloid deposits were found originally to contain aggregates of natural polypeptides that were overproduced in inflammatory diseases. However, it was later shown that certain proteins such as immunoglobulin light chains and mutant polypeptides were predisposed to misfold and aggregate in amyloidosis and related diseases. It was also realized that protein aggregation was a hallmark of Alzheimer and Parkinson disease with Lewy body dementia (and thus a strong link to lysosomal disorders); protein aggregation is central to prion diseases and other neurodegenerative diseases such the Huntington disease and the tauopathies.
Polypeptides harbouring pathological mutations frequently misfold and thereby escape
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normal processing; they overwhelm the surveillance and quality-control systems of the cell and are often retained in the endoplasmic reticulum (ER). The unfolded protein response is an adaptive signalling pathway which supports the homeostasis of the endoplasmic reticulum and responds to metabolic and oxidative stress as well as inflammatory reactions. The two main aspects of the ER stress response involve decreased protein flux and reduced protein translation via PERK-mediated phosphorylation of the eukaryotic translation factor
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eIF-2α and transcriptional upregulation of ER folding capacity via cleavage and activation of X-box binding protein-1 [161]. Degradation of misfolded polypeptides initiated by the protein quality control system involves chaperones but ultimately ubiquitination and
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delivery to proteasomes.
While some misfolded proteins may retain their functions, their destination is inappropriate,
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or even injurious to the cell. As a result, the outcome of many inherited defects is the consequence of impaired function combined with the toxic effects of aggregation and deleterious mistargeting. In the face of sustained activation due to uncleared misfolded polypeptides in the dynamic environment of the endoplasmic reticulum, the unfolded protein response induces apoptosis via phosphorylation through the PERK signalling
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pathway. The consequences of this and related pathways orchestrated by the unfolded protein response are observed frequently in human diseases.
Not only do the nascent lysosomal matrix polypeptides undergo extensive co-translational
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and other processing as part of their intracellular itinerary, they also interact naturally with numerous molecules which ensure appropriate trafficking to their destination in the
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organelle; these involve the mannose-6-phosphate-dependent and independent pathways [162,163,164]. Moreover, as set out above [Section B], a large family of naturally occurring and readily inducible heat-shock proteins also contribute to lysosomal integrity and health; these too serve as endogenous molecular chaperones to restore the function of misfolded proteins under stress conditions and where those proteins are disrupted and aggregated as a consequence of structural mutations. Here we survey pharmacological chaperones for lysosomal diseases: small molecules that penetrate cells to bind, stabilize and restore the three-dimensional and quaternary structure of proteins and also secure their function at their site of action.
Pharmacological chaperones for lysosomal diseases
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The specialised pathways of biosynthesis and trafficking of nascent lysosomal hydrolases are susceptible to interdiction by the pharmacological chaperones. Here I introduce the recent exploration of pharmacological chaperones as a therapeutic stratagem for lysosomal diseases. But for the remarkable clinical triumph of other initiatives such as enzyme therapy, this approach would not be practicable, but the approach has attracted considerable
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scientific investment and continues to hold promise [165].
Translation in the rough endoplasmic reticulum occurs in a neutral environment but delivery of the mature processed glycoproteins to the lysosome involves a radical pH transition in
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which the hydrogen-ion concentration increases about one thousand-fold. At the destination, the reaction conditions are most favourable for acid hydrolysis and the stability of the mature enzyme glycoform; furthermore, the concentration of the presented natural
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substrate is at its greatest. Small molecules with an affinity for the protein in the endoplasmic reticulum might, as with many chaperones, bind to its active site. Under these circumstances, the putative chaperone molecule would stabilize the lysosomal enzyme during trafficking and co-translational, as well as post-translational processing, in the endoplasmic reticulum and as the nascent glycoprotein traverses the Golgi stack. On
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delivery by fusion with the lysosome, the binding affinity of the molecular ligand on the protein will change but the stabilized and fully processed enzyme will be now be able to interact with its natural substrates at its physiological site of action [166,167]. The ideal pharmacological molecule will be a weak inhibitor at acid pH, so that once its rôle as
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chaperone is complete it will be readily displaced from the active site of the specific
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therapeutic target.
Clinical application of pharmacological chaperones in lysosomal diseases Fabry disease
An early example of the application of the pharmacological chaperone principle in the lysosomal disease is provided by the use of D-galactose in a patient with clinically attenuated Fabry disease [168]. Experimental studies showing its capacity to enhance the enzyme activity in lymphocytes and myocardial biopsy specimens, infusions of 1 g/kg galactose over four hours on a daily basis significantly enhanced α-galactosidase A activity, in the case of the heart, from
PII:
S1521-690X(15)00013-5
DOI:
10.1016/j.beem.2015.01.001
Reference:
YBEEM 1011
To appear in:
Best Practice & Research Clinical Endocrinology & Metabolism
Please cite this article as: Cox TM, Novel treatment approaches for lysosomal disorders, Best Practice & Research Clinical Endocrinology & Metabolism (2015), doi: 10.1016/j.beem.2015.01.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Best Practice and Research in
Volume edited by
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Professor Carla E M Hollak
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Clinical Endocrinology and Metabolism
Amsterdam Medical Centre
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University of Amsterdam
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The Netherlands
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Publisher: Elsevier, North Holland
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Novel Treatment Approaches for Lysosomal Disorders
Timothy M Cox MA MSc MD FRCP FMedSci Professor of Medicine Department of Medicine University of Cambridge UK
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Abstract
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Striking therapeutic advances for lysosomal diseases have harnessed the biology of this organelle and illustrate its central rôle in the dynamic economy of the cell. Further Innovation will require improved protein-targetting or realization of therapeutic gene- and cell transfer stratagems. Rescuing function before irreversible injury, mandates a deep knowledge of clinical behaviour as well as molecular pathology – and frequently requires an understanding of neuropathology.
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Whether addressing primary causes, or rebalanceing the effects of disordered cell function, true therapeutic innovation depends on continuing scientific exploration of the lysosome. Genuine partnerships between biotech and the patients affected by this extraordinary family of disorders continue to drive productive pharmaceutical discovery.
Key words:
Practice Points
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Lysosome, organelle, protein targeting, sphingolipids, inhibitors of biosynthesis, substrate reduction; chaperone, therapeutic, innovation, gene transfer
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• Although lysosomal diseases are rare and often clinically severe, several are treatable and the field remains an area of very active therapeutic research with considerable biopharmaceutical development of orphan medicinal products.
•
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• Treatments are principally based on improving the biochemical abnormality in metabolic clearance of pathological cellular molecules that would normally be digested in the lysosomal compartment. The following specific stratagems are being explored: (i) chemical storage dissolution (ii) stimulating lysosomal exocytosis by heat-shock proteins (iii) cellular complementation, especially haematopoietic stem-cell transplantation (iv) gene transfer, including genetic transduction of autologous stem cells for re-infusion as well as direct approaches, mainly with viral-based vectors (v) enzyme therapy based on harnessing cell-surface receptors that serve as recognition markers for lysosomal proteins (vi) pharmacological chaperones to stabilize mutant lysosomal proteins (vii) strategems based on control of cognate substrate biosynthesis; slowing endogenous formation of the substrate class whose degradation is impaired to match residual
digestive activity in the lysosomal compartment - the intention is to rebalance ACCEPTED MANUSCRIPT synthesis and degradation and thus correct the primary defect. Treatments in sections (i), (ii) (vi) and (vii) may involve small molecules that may have the potential to cross the blood-brain barrier; treatments (iii), (iv) and (v) rely principally on biologics of high molecular weight and/or cellular complexity.
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Research agenda
• Completing the molecular understanding of lysosomal disesases as a means to understanding their severity determinants and design highly specific molecular probes
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• Completing the developmental and surveillance programme for eliglustat and its congeners in Gaucher disease and related sphingolipidoses – especially exploring potential benefit for the neurological complications with the newly introduced Genz 161 agent.
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• Determining the value of dissolution therapy in Niemann-Pick disease type C (cyclodextrin and possibly the value of ariclomolol in this disease). If active, determining the long term outcomes. Cyclodextrin may cause deafness and haver other cranial effects.
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• Gene transfer – this holds much promise for lysosomal diseases - beyond Sanfilippo types A and B as well as LINCL 2 (late-infantile) neuronal ceroid lipofuscinosis wider clinical development is entering clinical application - especially, but not exclusively, for those diseases with a devastating neurological component.
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These developments will mandate research into improved methods to monitor outcomes of interventions using biomarkers as well as exploring neuropsychiatric disease-scoring techniques.
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• Further understanding of the clinical practicability of gene transfer, including genetic transduction of autologous stem cells for re-infusion, and approaches directed to the most critical tissues and regions of the brain. Case selection and intensive clinical engagement will be important matters to develop for enrolment in future advanced therapy research in the lysosomal diseases.
The Lysosome as a Gateway to Treatment
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The lysosome and assembly of its components during organelle biogenesis have, from the time of its discovery by Chrisitan de Duve, incited interest for intervention. With the recognition of inborn errors of lysosomal function as single-gene defects causing multisystem diseases [1], the appetite for therapeutic exploration has burgeoned. Painstaking research to determine the nature of the substrates, which contribute to the
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phenotype of intracellular storage was simply a first step in understanding lysosomal disorders caused by deficiency of acid hydrolases; this classical approach provided critical insight into the central role of the organelle in macromolecular recycling for the economy of
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the cell.
Subsequent molecular characterization of the genetic basis and protein defect(s) implicated
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in each disease revealed fundamental precepts and possibilities for definitive treatment – namely, replacing the primarily deficient factor required for normal lysosomal function. While much remains to be learnt about molecular pathogenesis of the individual conditions – many of which exhibit features of inflammation and other, compensatory, tissue reactions that await exploration – approaches based on complementing the function of proteins
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which are defective are fundamental to future therapeutic development.
De Duve shared the Nobel Prize for Physiology or Medicine in 1974 with Albert Claude and George Palade – an event which heralded Cell Biology as a radically new and
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interdisciplinary science [2]. With his colleagues, de Duve rapidly promoted the concept of a dynamic and interactive entity defined by a distinct acidic microenvironment within the
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living cell [3]. This functional definition immediately suggested that the lysosome was part of an interlinked membrane system in contact with the plasma membrane and its invaginations at the cell surface - thus participating in the processes of fluid-phase endocytosis, pinocytosis and receptor-mediated uptake, as well as autophagy. De Duve quickly realised that the lysosomal compartment would be continually exposed to the external environment and thus provide a direct access route for potential therapies [4].
Lysosomal diseases Henri-Gery Hers, a former colleague of de Duve, pursued research into glycogen storage diseases – thereby encountering Pompe disease, in which all the known enzymes of
glycogen breakdown and synthesis functioned normally. In a brilliant series of
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investigations, Hers ultimately identified the deficient enzyme which proved to be an acid αglucosidase (maltase); and thus a lysosomal enzyme previously unconnected to glycogen metabolism. Other colleagues, notably François Van Hoof, joined to define the concept of inborn lysosomal diseases [1]; and thus numerous disorders representing defects in the recycling of cellular macromolecules were classified as ‘lysosomal storage diseases’. More than 70 heritable diseases of lysosomes and lysosome-related organelles are now
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recognized - each best considered as a unique pathological domain [5, 6]. Given the potential for access of proteins to the lysosomal compartment, and numerous enzyme defects responsible for lysosomal diseases, de Duve realised that by a succession of chance
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events: “thus was elucidated, with important consequences for diagnosis, prevention and therapy, a vast chapter of pathology that had remained totally mysterious …” [7].
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Specific complementation of deficient lysosomal function
The dominant principle of functional complementation utilizing specialised pathways for protein delivery to the organelle, owes its origin to the discovery of the lysosome, and especially its dynamic rôle in cellular recycling. This led to an early prediction that, beyond its critical role in autophagy and renewal, a substantial network of membranes and
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compartments could be accessed by molecules presented at the cell surface [4].
De Duve had no concept of the mechanistic link of lysosomal protein targeting in biogenesis of the organelle – but remarkable experiments carried out by Elizabeth Neufeld and
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colleagues at the National Institutes of Health provided direct experimental support for this supposition [8]. Studies of fibroblasts cultured from genetically distinct forms of the human
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mucopolysaccharidoses ultimately led to an understanding of how nascent lysosomal enzymes were trafficked to the lysosome. With this came the realization that restoring function by enzymatic complementation of lysosomal defects might indeed be clinically tractable [8].
The complementing factors proved to be high molecular weight forms of the soluble lysosomal hydrolases deficient in each genetically distinct disorder; these glycoproteins are decorated by the critical mannose 6-phosphate moiety – and recognition signal which serves as a ligand for cell-surface receptors that mediate uptake and sorting to the lysosomal compartment after endocytosis. Contemporaneous studies of I-cell disease
provided reciprocal information: in this disease, genetic defects in the formation of the
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recognition signal, leading to multiple deficiencies of acid hydrolases in the lysosome but elevated activities in plasma and other extracellular fluids [9, 10]. These studies also confirmed the importance of receptor-mediated intracellular trafficking and co-translational processing for delivery of nascent enzymes to the lysosome.
While this receptor system is of critical importance, it is not used by all lysosomal proteins
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[11]. The topic has been extensively reviewed [10, 12]; but at first it was disappointing that early attempts to restore lysosomal activities in patients with mucopolysaccharidoses by cell and tissue transplantation were unsuccessful. Delivery of the appropriate disease-specific
took many years to be realized [13, 14].
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‘corrective factors’, principally as recombinant genetically engineered therapeutic products,
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Glycoprotein uptake by macrophages and therapy of Gaucher disease
A radical alternative, and, as it turns out, crucial development in the development of therapy for lysosomal diseases also came about fortuitously at the National Institutes of Health, Maryland. Gilbert Ashwell and his colleagues characterized the uptake of plasma glycoproteins by the liver, leading amongst other things, to identification of the
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asialoglycoprotein receptor [15]. In relation to lysosomal diseases, the studies by Ashwell and Morrell were an inspiration for pioneering work by John Barranger and Scott Furbish, in the laboratory of Roscoe Brady, which had been dedicated since the mid-1960s to the development of enzyme therapy for Gaucher disease – a relatively frequent sphingolipidosis
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due to deficiency of the acid β-glucosidase, lysosomal glucocerebrosidase [16]. After many false leads, the first successful outcome of enzyme therapy occurred in a single
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patient, a boy aged four years, in clinical studies conducted by Norman Barton with John Barranger and colleagues, who led the clinical programme in the Brady laboratory at the NIH [17]. The investigational agent used human glucocerebrosidase purified from placentae and remodelled to display terminal mannose sugars – in this way serving as a ligand for mannose receptors on macrophages [18]. The spectacular therapeutic success of enzyme therapy for Gaucher disease was again attributed to serendipity; but chance was combined with unremitting investigative work over several decades [19,20]. Successful complementation here had depended on the identification [18-22] of the mannose receptor system – the principal receptor that mediates the high-capacity uptake system for mannose-terminated glycoproteins into cells of the mononuclear-macrophage system and the preferential in vivo
target for therapeutic correction of the peripheral manifestations of Gaucher disease in the
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bone marrow and viscera [23].
Subsequently, the basis for the mannose 6-phosphate receptor-independent lysosomal targeting of glucocerebrosidase to the lysosomal compartment (in non-haematological cells) has been shown to be directed by the integral membrane protein, LIMP-2 [24]. Careful structural studies have moreover shown how, mediated by a histidine residue, LIMP-2 binds
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and releases the enzyme according to changes in pH; the finding suggest that LIMP-2 localizes to the ceramide portion of the substrate adjacent to the catalytic site [25]. A phosphomannose residue linked to N-acetylglucosamine on LIMP-2 is covalently attached to
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an asparagine residue, so that LIMP-2 binds to the mannose 6-phopsphate receptor, via mannose 6-phosphate [25].
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After decades of painstaking research and clinical development, it is now clear that diseases of the lysosome offer unrivalled therapeutic opportunities. The clinical outcomes and burgeoning commercial success of enzyme therapy for Gaucher disease serves as a paradigmatic example of fruitful application of molecular cell biology to human disease [1314, 19-20, 26]. Not only were the innermost recesses of the diseased cell opened up to
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salutary intervention, but the stimulus for commercial investment has enhanced interest far beyond the expected market for this group of individually rare diseases; several biopharmaceutical companies have developed enzyme therapies and other agents now
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approved for lysosomal diseases.
Treating fundamental causes of lysosomal disease
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The relationship between macromolecular storage and complex human disease phenotypes has been difficult to understand in molecular terms that allow prediction of either the evolution of disease, or indeed its responses at different stages to intervention at the primary ‘replacement’ level. Characterization of the principal macromolecular substrates that accumulate in the lysosomal compartment has been largely completed but how these contribute to the individual so-called storage phenotypes is unknown. Even in the case of enzyme therapy for Gaucher disease, which has been spectacularly successful since it directs the replacement factor specifically to the principal cellular population which most evidently manifests the glycosphingolipid “storage” [1], we are left still mystified by comorbidities such as Parkinson’s disease, intractable skeletal manifestations, progressive neuronopathic
features and the unexpected development of myeloma, B-cell lymphoma, and other
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haematopoietic malignancies [27-29].
Treating lysosomal diseases which affect the nervous system Most lysosomal disorders affect nervous tissue and cause neurodegeneration that is often a dominant clinical feature; moreover, the progressive nature of the process is a source of much human distress – and genuine unmet clinical need that mandates persistent scientific
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investment and exploration of imaginative remedies. While this time-honoured aspect remains a challenging, if not almost intractable aspect of the care currently available for patients with lysosomal diseases, new opportunities arise in the use of pharmaceutical
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drugs (‘small molecules’) as well as biological approaches (stratagems for cell- and gene therapies as well as ‘biologics’) to overcome the formidable barriers to therapeutic development for lysosomal diseases of the brain. Thus, running throughout the conspectus
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of emerging treatments is the perennial challenge of securing a safe and effective method for therapeutic delivery across the blood-brain barrier.
Therapeutic stratagems specifically for lysosomal disease include: (a) Dissolution - and removal - of intraluminal storage material; (b) Enhanced exocytosis of pathological intralysosomal content; (c) Modulating substrate biosynthesis (substrate-reduction
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therapy); (d) Complementation stratagems - (i) Haematopoietic stem-cell (previously bonemarrow) allo-transplantation
(ii)
Reinfusion of vector-transduced autologous
haematopoietic stem cells (iii) Direct gene transfer (iv) Enzymatic augmentation (v)
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Enzymatic enhancement and stabilization (pharmacological chaperone therapy).
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(A) Dissolution - and removal - of intraluminal storage material An often-forgotten stratagem, successfully used as a remedy for a lysosomal disease with prominent intra-compartmental metabolic storage, is the direct dissolution of the putative storage molecules held to be toxic. The first example of this approach is given by as in the membrane transport disorder, cystinosis – a disease which remains a focus of therapeutic development.
Unwanted macromolecular storage in the lysosomal compartment Since it is founded on a simple notion of pathogenesis – ie. that lysosomal diseases result from intra-lysosomal “storage” of unwanted substrates – here we review the effects of
substrate dissolution. This therapeutic concept has in fact long been applied successfully to
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a transport disorder in which the egress of lysosomal digestion products is impaired. While it is tempting to reject this notion as an outmoded and simplistic concept for understanding pathogenesis, its demonstrable efficacy in cystinosis, and now extending to the otherwise intractable neurodegenerative disorder, Niemann-Pick disease type C, cannot be denied. Moreover, the stratagem remains an active area of clinical innovation and with several
approved (cysteamine bitartrate).
Storage dissolution (cystinosis) – new developments
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investigational new drugs in late clinical development (eg, cyclodextrins) or recently
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It should not be forgotten that the pathological storage concept has been explored effectively in at least one lysosomal disease – employing a direct biochemical approach to reduce the storage of the putative toxin by promoting its dissolution and egress from the
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lysosomal compartment. The paradigmatic example of this approach is provided by the use of cysteamine in patients with cystinosis [30]. Cystinosis is caused by genetic defects in the lysosomal cystine/proton symporter, cystinosin. Systemic accumulation of cystine causes widespread injury, including renal failure. Cysteamine is an aminothiol which interacts with the crystalline aggregates of cystine (oxidised cysteine dimers) converting lysosomal free
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cystine to cysteine and the mixed disulphide of cysteine-cysteamine, which leaves the lysosome via the recently characterized cationic amino acid transporter system, which also transports lysine [31, 32].
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This simple intervention is demonstrably effective in terms of clinical outcome and disease survival since, when high doses are given early in the disease, cysteamine delays the
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progression to renal failure [30, 33]; moreover, topical therapy directly prevents loss of vision and painful corneal injury due to local deposits of cystine [34]. The approach is significant because it supports the classical view that at least in some instances, the causal pathological effect of storage in the genesis of at least one lysosomal disease and points to the potential value of substrate dissolution as a direct therapeutic avenue. Cysteamine, a thiol agent, is a malodorous and unappetising compound and its use carries with it a huge burden for patients with cystinosis, especially in terms of social acceptability and gastric tolerance. The recent exploration of delayed-release preparations of the newly available cysteamine bitartrate appear to offer large practical advantages for patients in terms of acceptability and with better compliance due to improved pharmacodynamics
profile and other properties - and hence practical efficacy [30, 33]. These are early days in
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the studies of this modestly innovative agent for patients with cystinosis but the potential effect on patients’ life quality is too important to be discounted.
Storage dissolution (Niemann-Pick disease type C) An emerging and intriguing application of the concept of substrate dissolution in the treatment of lysosomal diseases, is given by Niemann-Pick disease Type C. While the
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primary nature of this condition is known in genetic terms, (mutations in the NPC1 and NPC2 genes), the exact nature of the defect and its pathogenesis, principally in the brain, liver and gastrointestinal system, is less clear. The cognate protein products, NPC1, a
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polytopic membrane protein, and NPC2, a matrix hydrolase, collaborate to bring about the export of free cholesterol and other lipid molecules from the endolysosomal system [35]. Lately, increased lysosomal sphingosine concentrations have been implicated in the
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pathogenesis; these shift the acid-base balance of the lysosome and apparently lead to harmful sequestration of free calcium ions [36].
The sole approved treatment for Niemann-Pick disease Type C, at least in European countries, is miglustat – an imino sugar (N-butyl deoxynojirimycin, originally OGT918) which
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retards the onset of symptoms and signs of disease and prolongs survival in naturallyoccurring Niemann-Pick disease in mice and cats. Miglustat is approved as Zavesca™ for treating the neurological manifestations of this condition in the European Union, Brazil, Canada and Australia, but is hardly a definitive treatment for this devastating
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neurodegenerative disorder. While the mode of action of miglustat may be to reduce biosynthesis of sphingosine, the numerous “off-target” effects of this agent render it
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difficult to be sure that this is the true mode of action.
Cyclodextrins are cyclic oligosaccharide molecules with amphipathic properties: and are able to bind steroids and reduce the cellular content of cholesterol. The potential application of cyclodextrins to Niemann-Pick disease Type C emerged serendipitously [35]. Niemann-Pick disease Type C mice lived longer when cyclodextrin was present as a contaminant vehicle of a steroid compound under investigation.
The drug was found to delay the onset of
neurological manifestations and prolong survival in this genetically coherent and natural model [37]. Soon after these findings were appreciated, in 2009, the US Food & Drug Administration approved the use of intravenous preparations of hydroxypropyl β-
cyclodextrin as a new investigational agent in clinical trials of young patients with Niemann-
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Pick disease Type C, and the drug was subsequently awarded orphan medicinal status as a branded preparation as designated for the treatment of this condition [38].
Following an experimental programme in feline Niemann-Pick disease, the results of which are in press, further trials of this drug given intrathecally for patients with the disease are awaiting approval [39].
Cyclodextrin may indeed represent an important departure as a
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treatment for patients with Niemann-Pick disease Type C and a therapeutic development of value in this intractable condition; unfortunately there are reports of nerve deafness in cats which constrains unconditional enthusiasm and has mandated intensive study during the
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conduct of human clinical trials. Its mode of action is believed to be that of an agent which restores the defective function of the NPC1 and NPC2 gene products in mediating the egress of cholesterol from the lysosomal space in neurons and it has hitherto been presumed that
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the effect depends on the drug entering the brain to facilitate removal of excess cholesterol from the endolysosomal compartment of neurones and other cells. Recently however, it has been convincingly shown that hydroxypropyl-β-cyclodextrin does not significantly traverse the blood-brain barrier in either adult or neonatal mice [40]. At the same time, the volume of distribution available to the agent greatly exceeds the predicted plasma and vascular
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volume of the brain, suggesting that cyclodextrin binds to the endothelial cell surface of the cerebral vessels and clears intracellular cholesterol indirectly. However the exact mechanism by which the apparent therapeutic effect is brought about is unclear.
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B. Enhanced exocytosis
As above, the concept of removing unwanted, unmetabolized and toxic storage molecules
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by harnessing the physiological process of exocytosis from the lysosomal space is consistent with the notion of substrate dissolution therapy and harnesses one pathway of lysosomal membrane recycling. Hitherto this stratagem remains entirely experimental and has not been attempted in patients with lysosomal diseases but the notion has had appreciable support in animal disease models [41].
A provocative report using the molecular chaperone, heat-shock protein 70, has excited interest in this manoeuvre. As part of the cell death response, the integrity of lysosomal membranes is impaired and the organelle becomes generally more permeable to proteins, allowing leakage of the contents; heat-shock proteins, are known to prolong the survival of
stressed cells by stabilizing lysosomal membranes. It was moreover reported that a
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fragment of the heat-shock protein 70 (HSP70) translocates to the mammalian compartment under severe stress conditions [42]. Kirkegaard and colleagues later found that Hsp70 stabilizes lysosomes by binding to the abundant endolysosomal anionic phospholipid, bis(monoacylglycero)phosphate, which is required for
sphingomyelin
breakdown by serving as a cofactor of acid sphingomyelinase in the lysosome. It was further reported that inhibition of the hydrolase and its binding prevents the stabilization of
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lysosomes by HSP70 – a feature of fibroblasts from patients with Niemann-Pick diseases types A and B due to natural mutations in the SMPD1 gene which encodes acid sphingomyelinase. Notably, the cellular phenotype could be corrected by simple addition of
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recombinant HSP70 to the cultures: this led to a striking collapse of the distended lysosomal compartment and correction of the storage phenotype as a result of enhanced exocytosis of
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organellar contents.
Therapeutic exploration of the heat-shock response in a lysosomal disorder These above findings were taken to suggest new possibilities for developing of treatments for lysosomal diseases with compounds that enter the lysosomal lumen by the endocytic delivery pathway to promote exocytosis of content and improve membrane stability [42].
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The agent, arimoclomol citrate (BRX-345) is an orally active analogue of bimoclomol, a hydroxylamine co-inducer of the heat-shock response. The agent activates heat shock factor-1 which in the context of cellular stress associated with ischaemia or heat shock, is a transcriptional regulator of expression of inducible molecular chaperones such as HSP70.
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Heat shock factor-1 is a member of a versatile family of transcription factors that, in addition to protecting cells against proteotoxicity due to aggregation and the unfolded
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protein response, are essential for physiological functions, particularly during development [42]. Arimoclomol™ is already in clinical phase 2/3 trials for various indications in humans with diseases, such a amyotrophic lateral sclerosis (specifically the hereditary variant due to mutations in superoxide dismutase) associated with protein aggregation in neurones. Now licensed to Orphazyme, arimoclomol™ is under clinical investigation in a neurodegenerative lysosomal disease: the EU Committee for Orphan Medicinal Products has recently adopted a positive opinion on the orphan drug application for arimoclomol citrate as a treatment of Niemann-Pick disease type C. Clearly the results of this bold initiative are eagerly awaited.
Promoting exocytosis by dynamic control of lysosomal biogenesis and autophagy
In a brilliant series of studies, Ballabio and colleagues identified a transcriptional mechanism
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that regulates the biogenesis of lysosomes and autophagy. In most lysosomal diseases, the lysosomal network is expanded and lysosomal enzyme activities are enhanced [43].
While a regulatory mechanism with enhanced transcriptional expression was suspected, Sardiello et al used pattern discovery analysis of promoters where the cognate gene encoded lysosomal proteins, to identify a common palindromic motif (coordinated
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lysosomal expression and regulation, CLEAR), which mediates transcriptional activation, [44]. The CLEAR element binds to TFEB, a master regulator of lysosomal biogenesis and a nuclear transcription factor. In cultured cells, TFEB was shown to activate transcription of
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many lysosomal genes [45].
TFEB coordinates the functions of two organelle systems in cellular recycling and autophagy
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and stimulates formation of autophagosomes and their fusion with lysosomes. Phosphorylation of a critical residue (serine 142) mediated by the extracellular signalregulated kinase 2 (ERK2) prevents nuclear translocation; this phosphorylation is sensitive to nutrient activation in the mitogen-activated protein kinase pathway as well as the multiprotein mTOR (mammalian target of rapamycin) complex. In nutrient-rich conditions,
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ERK2 is activated and stimulates growth while inhibiting autophagy; reciprocal effects pertain when the supply of nutrients is poor and these induce autophagy. Moreover, TFEB over-expression stimulated biogenesis of the organelle with expansion of the lysosomal compartment. Expression studies in stable transfectants showed that TFEB enhanced the
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degradation of glycosaminoglycans, as well as the breakdown of mutant polyQ expanded huntingtin polymers in an inducible model of Huntington disease [46]. To relieve the burden
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of intralysosomal as well as extralysosomal glycogen burden in Pompe disease, lysosomal exocytosis was stimulated by upregulating TFEB expression in mice with Pompe disease. Overexpression of TFEB in muscle cells removed enlarged lysosomes, reduced pathological glycogen deposits, and restored autophagy, arrest of which is a prominent feature of the disorder. Enhanced exocytosis involved autophagosomes and lysosomes, indicating that TFEB induces the exocytosis of both compartments [47].
Hitherto, stimulating exocytosis has a strong theoretical basis for the relief of lysosomal disorders in which there is a prominent accumulation of debris in autophagic vacuoles and represents a completely novel avenue for therapeutic exploration in man. Identification of
the regulatory molecules implicated in the transcriptional control of lysosomal biogenesis,
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autophagy and nutrient sensing through TFEB and its functional partners, undoubtedly suggests therapeutic opportunities for storage clearance and stabilization of diseased organelles in a range of diseases. Although the stratagem is not yet undergoing substantive clinical development, it represents a promising field of endeavour.
C. Modulating substrate biosynthesis (‘substrate-reduction therapy’)
adequately
be
degraded
has
general
precedents
in
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The intriguing therapeutic idea of limiting formation of excess metabolites which cannot clinical
practice:
gout,
hypercholesterolaemia, the acute porphyrias and hereditary tyrosinaemia - metabolic
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diseases which are readily identified as ‘overproduction diseases’. Thus, while not strictly original in concept, restricting substrate biosynthesis for lysosomal diseases, has been fully realized in at least one sphingolipid disorder – non-neuronopathic Gaucher disease.
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In respect of these conditions which are not only biochemically complex but often associated with otherwise intractable neurological manifestations, such therapeutic initiatives have distinct advantages. The stratagem remains actively under clinical development in Fabry disease, chronic neuronopathic Gaucher disease and other glycosphingolipidoses such a Niemann-Pick type C and GM2 gangliosidosis; there is
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moreover an attempt to investigate this approach in clinical studies of disordered glycosaminoglycan formation in mucopolysaccharidoses. The range of inhibitory compounds with improved pharmaceutical properties has been expanded – and, with the notable convenience of being orally active, several of these small molecules have shown to traverse
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the blood-brain barrier.
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Most lysosomal disorders are understood principally in terms of excess ‘storage’ of inefficiently recycled cellular debris: attenuating the rate of synthesis of the specific precursors of incompletely degraded macromolecules is thus an approach not only consistent with elementary ideas about pathogenesis but also has parallels in diseases caused by toxic metabolites. Use of the statins to limit hepatic cholesterol biosynthesis in familial hypercholesterolaemia by inhibiting hydroxymethylglutaryl coenzyme A reductase and of allopurinol to suppress uric acid formation at the level of xanthine oxidase in gout are familiar examples of this application. Indeed allopurinol was first explored in patients with gout fifty years ago [48] and statins in hypercholesterolaemia in 1971 [49].
A frequent operational characteristic of successful substrate inhibitors, is that they act on
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the first, energetically committed, biosynthetic reaction. In this way, overproduction of intermediary molecules or collateral metabolites with the potential for toxicity shared with the excess primary product, is avoided. On the other hand, as in the pharmaceutical use of statins in the biosynthesis of essential sterols, care must be taken to attenuate, rather than abrogate formation of the critical biosynthetic product on which life often depends. In the case of glycosphingolipids, the importance of not disturbing the delicate homeostasis of
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bioactive sphingoid bases cannot be overestimated. These molecules are imbued with critical functions: ceramides stimulate apoptosis and inhibit cell growth as well as differentiation; the cognate sphingols stimulate proliferation, control cell migration,
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angiogenesis as well as inflammatory responses.
If attainable, development of selective inhibitors of the thermodynamically defining first
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step in a complex biosynthetic pathway is likely to be rewarded by fewer ‘off-target’ actions with deleterious effects. As noted, the basic premise is to prevent the clinical consequences of a build-up of noxious metabolites: in the case of Gaucher disease, the putative toxins are the many variant N-acyl-sphingosyl-1-O-β-D glucosides with differing acyl and sphingosine moieties and their unacylated analogues, the ß-glucosylsphingosines or ‘psychosines’ [50].
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These metabolites arise because a key enzyme responsible for sphingolipid recycling in the lysosome, acid β-glucosylceramidase (D-glucosyl-N- acylsphingosine glucohydrolase, EC 3.2.1.45), encoded by the GBA1 gene is impaired. In milder clinical forms of Gaucher disease, the ß-glucosylceramides are derived from exogenous membrane components after
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phagocytosis of effete cells, particularly by macrophages. In those forms of Gaucher disease where acid ß-glucosylceramidase deficiency is more profound, lysosomal recycling of
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endogenous cellular glycosphingolipids becomes limiting: under these circumstances, cells, such as neurones, whose membranes are enriched with gangliosides and other cognate lipids undergoing dynamic recycling, are more prominently affected.
Formation of most complex glycosphingolipids is initiated by formation of ßglucosylceramides. De novo synthesis occurs on the cytosolic leaflet of the Golgi complex but the products are translocated to the luminal surface of this apparatus, where they serve as core structures for building hundreds of higher-order sphingolipids, including complex gangliosides with multifunctional roles in cellular processes [51,52].
It is noteworthy that
the accumulating sphingolipids in the lysosomal compartment of patients with Fabry
disease; GM1 gangliosidosis; GM2 gangliosidosis (Tay-Sachs, Sandoff diseases and GM2
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activator deficiency) as well as Niemann-Pick disease type C, are all generated biosynthetically from the ß-glucosylceramide scaffold; it is this precursor glycosphingolipid which accumulates in the tissues of patients with all sub-types of Gaucher disease.
The biosynthetic reaction is thermodynamically favoured by the energized glycosyl donor a sugar nucleotide - and catalysed by UDP-glucose: N-acylsphingosine D-glucosyltransferase
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(UDP-glucosylceramide transferase). In Gaucher disease, unhydrolysed ß-glucosylceramides are principally sequestered in the lysosome; after their formation de novo, glucosylceramides would normally pass to other cellular compartments and undergo further
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glycosylation reactions. Norman Radin perceived this first committed step of glycosphingolipid biosynthesis as a target for innovative drugs in Gaucher disease and related glycosphingolipidoses [53]. Indeed, he had earlier sought selective inhibitors of the
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analogous UDP-galactosylceramide transferase for Krabbe disease [54]. Given residual lysosomal ß-glucosylceramidase activity, Radin argued that attenuating glucosylceramide biosynthesis would allow steady-state substrate concentrations to be re-equilibrated so that the remaining hydrolytic activity would match the rate of incoming substrate - thus
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balancing glycosphingolipid metabolism and avoiding the consequences of disease.
At present, two principal chemical classes of inhibitor are undergoing comprehensive therapeutic exploration in the sphingolipidoses:
iminosugars, originally derived from
naturally-occurring plant products [55] and another class of synthetic or semi-synthetic
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compounds originally made by Radin, Shayman and colleagues [56,57]. These latter pyrrolidine compounds serve as potent ceramide mimetics and bind to the transition-state
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of UDP-glucose: N-acylsphingosine D-glucosyltransferase, the key therapeutic target.
Iminosugars
Gaucher disease
It was the iminosugars, in particular N-butyldeoxynojirimycin, previously explored for an unrelated application in HIV infection, that were chosen for clinical development in the sphingolipid diseases by Frances Platt and Terry Butters in the University of Oxford [55]. The original compounds synthesized by Radin and colleagues were useful experimental inhibitors of the UDP-glucosylceramide synthase, but because of their cellular toxicity, they were not developed clinically for many years [58]. Platt and Butters had found that
micromolar concentrations of N-butyldeoxynojirimycin inhibited the biosynthesis of
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glucosylceramide in cultures of a murine macrophages treated with an irreversible inhibitor of acid-β-glucosylceramidase, conduritol-β-epoxide. This agent induces lysosomal abnormalities accompanied by an accumulation of glucosylceramide but co-addition of Nbutyldeoxynojirimycin abrogated the lysosomal storage. Subsequent animal studies were conducted in knock-out mice lacking β-hexosaminidases A and B, with accumulation of GM2 ganglioside in the brain.
The Sandhoff mouse has a shortened life expectancy with
throughout the central neuraxis.
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neurological manifestations, accompanied by progressive storage of GM2 ganglioside Administration of N-butyldeoxynojirimycin reduced
ganglioside storage in peripheral organs and brain of these animals and extended survival by
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approximately 40% [59]. N-butyldeoxynojirimycin, (known as miglustat or Zavesca™) had been administered to humans as a potential inhibitor of the viral α-mannosidases involved in the synthesis of the HIV glycoprotein coat: this greatly facilitated introduction of a clinical
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trial of a repurposed agent in Gaucher disease. At a dose of 100mg thrice daily, the agent reduced visceral enlargement and slowly improved haematological parameters, as well as plasma chitotriosidase, in patients with Type I Gaucher disease [60,61]. An unwanted effect of the iminosugar treatment is diarrhoea, caused by an inhibition of intestinal disaccharidases. Some patients also develop tremor and an axonal peripheral
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neuropathy has been reported. The drug is approved in the United States and Europe as a second-line treatment for patients with mild to moderate Type I Gaucher disease. In some mouse strains the agent causes non-genotoxic male sterility, and contraception is advised; the drug is not licensed for use in children. Despite these drawbacks, at the time of its
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development and licensing, miglustat as an orally available small molecule offered the first credible opportunity to explore the treatment of Gaucher disease and related
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glycosphingolipidoses, including their neurological manifestations, which remain refractory to protein-based therapies. With the single endpoint chosen, in the minority of patients with Gaucher disease able tolerate the agent and remained on the trial, it proved in a oneyear study not to be inferior in patients who transferred to the agent after control of their disease by enzyme therapy [62].
Given the potential for the small iminosugar molecule to penetrate the blood-brain barrier, a further open-label randomised trial using a novel design was conducted over 24 months in 30 children with chronic neuronopathic Gaucher disease receiving enzyme therapy [63]. While this trial failed to meet its clinical endpoint (vertical saccadic eye movement velocity),
notably the drug improved pulmonary function and decreased the plasma chitotriosidase
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biomarker activity in the period of miglustat exposure compared with enzyme therapy alone. Despite this, the drug is not recommended for neurological manifestations in Gaucher disease and is not licensed for use in children.
Miglustat is a powerful inhibitor of the neutral glucosylceramidase isozyme 2 [64], and this may contribute to its clinical effects as has been suggested. The cognate gene, encoding the
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enzyme assigned the term, GBA-2, has now been identified. Almost simultaneously, males in some strains of mice deficient in this enzyme as a result of homozygosity for an inactivated GBA-2 locus, were found to be infertile. Mild glycolipid excess was detected in the tissues
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but the mice of both sexes had a normal life span and were without overt disease [65]. The molecular basis for this strain and species variation is unclear but it seems probable that the enzyme is implicated in the trafficking of glycolipids from the endoplasmic reticulum to the
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cell membrane and acrosome -at least of murine spermatozoa.
Although miglustat is known to reduce the rate of formation of glycosphingolipids as predicted from its proposed action as a substrate-inhibitor for Gaucher and related diseases at the level of UDP glucosylceramide transferase, its powerful effect on the non-lysosomal
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glucocerebrosidase, GBA-2, contributes to its therapeutic efficacy as indicated by genetic crosses of GBA-2 deficient mice onto an inducible Gaucher disease strain [66]. However, with the recent description of severe neurological abnormalities in patients harbouring mutations in GBA-2 [67, 68], there are theoretically uncomfortable aspects to the clinical
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development of non-selective hydrophobic iminosugars such as N-butyldeoxynojirimycin
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(miglustat) for patients with non-neurological manifestations of Gaucher disease.
Niemann-Pick disease type C (innovative therapy for a neurodegenerative disease) Of note, N-butyldeoxynojirimycin (miglustat, ‘Zavesca™‘) has recently been approved by the European Medicines agency for Niemann-Pick disease Type C, another lysosomal disease affecting the brain in which disturbed cholesterol trafficking to lysosomes and its defective esterification is associated with neuronal accumulation of glycosphingolipids. Inhibition of glycosphingolipid synthesis by miglustat was found to delay onset and prolong survival in experimental (genetic) models of this disease in mice and cats [69,70]. The agent also corrected the lysosomal trafficking defect using cultured B lymphocytes obtained from patients with the disease after exposure to the drug [71].
A prospective clinical trial appeared to show neurological stabilization or even benefit in
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some individuals with improvement in supranuclear gaze palsy and dysphagia [72]. Since then, long-term clinical findings supportive of miglustat for stabilizing Niemann-Pick C disease have been reported [73]. The agent, which may serve to reduce the intralysosomal pool of calcium-sequestrating sphingosine in this disorder [74], has been approved for in 43 countries, including the European Union since 2009 and Japan since 2012 for the treatment of progressive neurological manifestations in adult patients and paediatric patients with
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Niemann-Pick type C disease. However approval of this innovative agent has not, at the time of writing, been granted by the Food and Drug Administration (USA). Since there is no definitive treatment for this neurodegenerative disorder, miglustat delivers, in part at least,
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relief from accelerated clinical decline in some patients with this cruel dementing illness who otherwise would be without hope. It moreover opens up other avenues for laboratory
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investigation and clinical research in Niemann-Pick type C disease.
Iminosugars for potential development as substrate-biosynthesis inhibitors Medicinal use of the imino sugar miglustat, (N-butyldeoxynojirimycin) is restricted by its non-ideal inhibitory activity directed against its therapeutic target (IC50 for UDP-glucose: Nacylsphingosine D-glucosyltransferase ≈ 10μM) and its limited specificity. This explains the
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untoward clinical effects, including diarrhoea and weight loss, which are in part consequences of inhibition of off-target glycosidases. Moreover, the pharmaceutical action and some unwanted effects of miglustat may be attributable to its inhibitory effect on the neutral glucocerebrosidase (GBA-2). Despite these manifest difficulties, however, it was
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realized from the time of first use, that the ability of the substituted iminosugars to traverse the blood-brain barrier and distribute in nervous tissue had distinct potential for
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pharmaceutical exploitation, which was lacking in the competing prototypic inhibitors based on pyrrolidone structures synthesised by Radin and colleagues. This characteristic has drawn attention to the glycosphingolipidoses with manifestations in the central nervous system as a justifiable and important focus of continuing therapeutic research.
The existence of a promising congener of miglustat, the galactose analogue (N-butyl galactodeoxynojirimycin, OGT928 – now also owned by Actelion) has improved properties and would appear to hold a high priority for therapeutic exploration [75]. Like miglustat, the agent is excreted unchanged principally by the kidney and its imino ring cannot readily be cleaved in mammalian tissues. It does not inhibit many intestinal disaccharidases and unlike
miglustat in experimental animals does not induce weight loss – neither does it inhibit
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alpha-mannosidases involved in N-linked oligosaccharide processing. Platt and colleagues have shown no toxicity in high doses given to mice (4.8g per kilogram body weight daily)[76]. Phase 0/I studies have been conducted in healthy persons at a single dose in the early and further studies and dose-escalation studies have conducted in volunteers; the outcome of long-term exposure in animals are awaited. While, the inhibitory concentration of this galactose-based analogue for the target enzyme
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for sphingolipid biosynthesis, UDP-glucosylceramide transferase, is rather high at ≈ 10μM, its potential therapeutic ratio appears to be far more favourable. This compound enters the brain and distributes readily in peripheral tissues. With these properties in mind,
glycosphingolipidoses must surely be encouraged [77].
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enthusiastic exploration of its potential value in Niemann-Pick type C and other
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Other iminosugars have been studied intensively, including N-(5’-adamantane-1’-ylmethoxy)-pentyldeoxynojirimycin (AMP-DNM).This latter compound is a 100-fold more powerful inhibitor of glycosphingolipid biosynthesis compared with miglustat (Nbutyldeoxynojirimycin); it is moreover freely bioavailable and apparently well tolerated [78]. Pharmacological reduction of glycosphingolipid abundance by AMP-DNM, did not materially
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change concentrations of ceramide (see below).
GM2 gangliosidosis (Tay-Sachs and Sandhoff diseases)
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Given the relative clinical success in mild-to-moderate Gaucher disease, two clinical trials of miglustat have been carried out in attenuated forms of GM2 gangliosidosis: in 30 late-onset
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patients with Tay-Sachs disease [79] and in juvenile Sandhoff disease [80,81]. To evaluate the safety and efficacy of miglustat in patients with Tay-Sachs disease, a randomized multicentre study was conducted over 12 months with a further 24 months extension [82]. Primary efficacy endpoints were change in eight measures of isometric muscle strength in the limbs and isometric grip strength; other endpoints included gait, balance, disability, and other neurological assessments.
No differences attributable to the study drug were
observed in any measure of efficacy, during the entire study period. The most common treatment-related adverse events were decrease in weight and diarrhoea. Minor indications of possible benefit in swallowing were observed in one small study but cognition deteriorated [80]. In another open-label and single-centre study of five Sandhoff patients
treated with miglustat, all showed neurological deterioration over the period of the study,
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with worsening of gait, speech and coordination [81]. It seems that despite other isolated reports of slight benefit, miglustat does not ameliorate progressive deterioration in most neurological domains, including cognition.
The prototypic substrate biosynthesis inhibitor in this field, miglustat, is a weak inhibitor of UDP-glucosylceramide transferase, but to some extent traverses the blood-brain barrier;
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while it appears that achievable concentrations in the nervous system are insufficient to improve neurological injury in Gaucher disease, the agent may have useful effects on pulmonary manifestations in patients with chronic neuronopathic variants [63]. While the
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drug is useful in some patients with Niemann-Pick type C disease, it has proved disappointing in GM2 gangliosidosis – with progressive neurodegeneration related to impaired degradation of higher-order glycosphingolipids that are several biosynthetic
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reactions removed from glucosylceramide
Innovative synthetic ceramide analogues as treatment for glycosphingolipidoses Synthetic molecules which serve as powerful inhibitors of the reaction target, UDP-glucose: N-acylsphingosine D-glucosyltransferase (UDP-glucosylceramide transferase) have been
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identified. After many years, the lead agent has finally been introduced as a second-in-class substrate biosynthesis inhibitor, and other molecules are in late-phase clinical development for the sphingolipidoses. Already there is convincing evidence of therapeutic success and clinical utility with the lead compound, which in 2014, has gained regulatory approvals for
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Eliglustat
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first-line use in Gaucher disease, internationally.
Eliglustat (Genz-112638) is a distant derivative of a series of inhibitors of UDPglucosylceramide transferase originally designed by Vunnam and Radin [53]. However, the prototype molecule not only depleted glucosylceramide-based sphingolipids but inhibited cell growth and induced apoptosis. Investigations revealed elevated cellular ceramide concentrations, which were at first attributed to the build-up of a precursor pool of ceramide that had not been utilized in the UDP-glucose: N-acylsphingosine Dglucosyltransferase reaction. It was later realized that the erythro-enantiomers of pyrrolidino-substituted molecules independently increased ceramide concentrations [57]. Derivatives of the inhibitors with aliphatic substitutions were subsequently found to have
enhanced potency with respect to glucosylceramide biosynthesis, as well as lesser effects on
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intracellular concentrations ceramide [58]. The ‘off-target’ activity of the whole series of agents was eventually localised to an unknown cellular pathway involving acylation of ceramide at the 1-hydroxyl position and catalysed by a previously unknown 1-Oacylceramide synthase (phospholipase A2) [83]. In the presence of ceramide as an acceptor, 1-O-acylceramide synthase transacylates ceramide, utilizing the sn-2-fatty acid of
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phosphatidylethanolamine or phosphatidylcholine.
The lead inhibitor for preclinical development, D-threo-1-ethylenedioxyphenyl-2-palmitoyl3-pyrrolidino-propanol had proven activity in the nanomolar range towards UDP-
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glucosylceramide transferase and all the threo-diastereomers of the pharmacophore demonstrated this desired activity at concentrations not inhibitory towards 1-Oacylceramide synthase – a matter of critical importance for subsequent pharmaceutical
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development [84]. Substitutions of the acyl group in amide linkage influenced potency but the final agent, Genz-112638, with an octanoyl substituted (eight-carbon fatty acid) analogue had the optimal half-life and pharmaceutical properties chosen for joint clinical development with the Genzyme corporation as Genz-112638, eliglustat, (N-((1R,2R)-1-(2,3 dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)octanamide
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(2R,3R)-2,3-dihydroxysuccinate) [56, 85, 86].
After the pivotal trial of miglustat in patients with Gaucher disease [60], the eliglustat clinical platform was accelerated. The enhanced enthusiasm for clinical development
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resulted in a good effect, albeit nearly 15 years later: eliglustat has recently been approved (2014) as Cerdelga™ for the first-line oral therapy of type 1 Gaucher disease in adults by the
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US Food and Drug Administration, as well as the European Medicines Agency.
Unlike miglustat, eliglustat tartrate and its congeners are potent, highly specific inhibitors of UDP glucosylceramide transferase at ≈25nM; the inhibition is non-competitive with respect to the nucleotide sugar substrate. As a transition-state analogue rather than a sugar analogue, eliglustat does not inhibit intestinal disaccharidases at concentrations in the high micromolar range; the molecule has likewise no inhibitory effect on the acid glycosidase βglucosylceramidase, which is itself deficient in Gaucher disease [85]. Eliglustat tartrate (Genz-112638) and its free base (Genz-99067) have undergone comprehensive exploration of its phamacodynamic and pharmacokinetic properties, as well as preclinical toxicology
[85,86,87]. After exposure in healthy volunteers, eliglustat has been evaluated over the last
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decade in phase II and III clinical trials involving nearly 400 patients with type 1 Gaucher disease in nearly 20 countries.
Recently published four-year results of an open-label phase II study of nineteen previously untreated type 1 adult patients of mean age 31 years (range 18-55) with splenomegaly complicated by thrombocytopenia and/or anaemia as entry criteria showed: that
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haemoglobin concentration increased by 2.3 ± 1.5 (baseline: 11.3 ± 1.5) g/dL and the platelet count increased by a mean of 95% (baseline: 68.7 ± 21.2) x 109/ L , respectively. Mean spleen and liver volumes decreased by 63% (baseline: 17.3 ± 9.5) and 28% (baseline:
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1.7 ± 0.4) multiples of normal, respectively. The surrogate markers of Gaucher disease activity, plasma chitotriosidase and chemokine CCL-18 each decreased by 82%; plasma glucosylceramide and GM3 concentrations rapidly diminished into the healthy reference
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range after drug exposure. Unexpectedly, the medicinal effects of this oral drug were found to be far-reaching in the skeleton with mean bone mineral density T-score for the lumbar spine significantly increased by 0.8 (60%) (baseline: − 1.6 ± 1.1). Skeletal magne\c resonance T1w imaging showed stable or diminished femoral ‘dark’ marrow signals in 17/18 patients. There was a significant decrease in the semi-quantitative bone marrow burden
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score. These latter findings are attributable to improved radiological features of Gaucher cell infiltration; notably, no clinical bone crisis events occurred during the entire study period. [88,89].
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In the long-term phase II cohort, exposure to eliglustat improved the femoral marrow parameters in 28 % of the patients in the first year and 56 % by the fourth year. The
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response was qualitatively similar to that observed with imiglucerase, in which marrow improvements occur within the first year in a minority with more patients showing improvement with prolonged treatment - although up to one third remain unresponsive [90].
The results of controlled clinical trials in adult patients with non-neuronopathic Gaucher disease naϊve to specific treatment compared with placebo (‘ENCORE’) and in another noninferiority trial (‘ENCORE’), changing to eliglustat after control of their disease with enzyme therapy (principally imiglucerase) and stable for at least three years, also confirm that eliglustat has a therapeutic effect which rivals that of enzyme therapy. These two pivotal
phase III clinical trials have been completed and are now in their extension phases [91].
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Two-year data are soon to be released.
Eliglustat, although a potent and selective inhibitor of a key biosynthetic reaction, appears to have few serious unwanted effects. Less than 5% of patients stopped the drug. Hitherto with almost a decade of clinical exposure, including about 400 patients, most adverse events appear to be mild and in the trials, mainly unrelated to treatment. An analysis 535
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patient-years of drug exposure involving 393 trial subjects, indicates that it was welltolerated: most adverse events were mild to moderate and not considered to be related to eliglustat. These included headaches, arthralgia, and transient diarrhoea. Most serious
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adverse events were unrelated to eliglustat tartrate: pregnancy, myocardial infarction (three patients with risk factors), and manifestations attributable to Gaucher disease (fracture, joint dislocation, a liver carcinoma which was in retrospect present at the time of
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enrolment, and cholecystitis). Adverse events probably related to eliglustat occurred in two patients (abdominal pain, lethargy, and skin eruption); one patent had signs of a mild peripheral neuropathy not attributed to eliglustat.
Eliglustat is principally metabolised by the liver and this is dependent on the cytochrome
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P450 CYP2D6 isozyme -genetic variation of which determines metaboliser status as well as the need to scrutinize potential drug-drug interactions with co-medication [92]. Unless there is scrupulous attention to sampling, plasma concentrations are generally not reliable for determining metaboliser status. Thus molecular analysis of genetic CYP2D6 variants is
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required to guide dosing of eliglustat, which is normally correlated with CYP2D6 genotype and body weight. Adjustments are needed in this context and in relation to the choice of
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concomitant medication. At present, patients who are CYP2D6 ultra-rapid metabolisers may not achieve adequate concentrations of the drug to achieve a therapeutic effect; further, a specific dosage cannot be recommended for those patients whose CYP2D6 genotype is indeterminate. Preclinical studies showed that eliglustat, like other drugs in clinical use, can at high concentrations affect cardiac conduction. Thus there is a risk that eliglustat will prolong cardiac PR, QTc, and QRS intervals at substantially raised plasma concentrations; for these reasons, the drug should be avoided in patients with pre-existing cardiac disease or in combination with cardioactive drugs in Class IA (e.g. quinidine, procainamide) and Class III (e.g. amiodarone, sotalol). However, with rigorous monitoring throughout the clinical programme, no convincing evidence for harmful effects on the heart and its conducting
system were reported. The behaviour of the drug in patients with renal or hepatic
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impairment is unknown and these conditions should contraindicate its use; similarly in young women who are breast-feeding or may become pregnant and are not using contraception, the drug should be avoided.
Therapeutic position of eliglustat
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The therapeutic properties of eliglustat offer the prospect of a unique first-line oral agent and as a medication for maintenance in adult patients with non-neuronopathic Gaucher disease who have already received targeted enzyme treatment. Registration will be
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accompanied by a regulatory commitment to exploration for paediatric use through the conduct of additional clinical trials in children. However, while obviating the need for infusions may be particularly welcome by children, eliglustat is in one sense a
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disappointment - for it cannot be expected to alleviate the neurological consequences of Gaucher disease in the brain. This disadvantage is poignant in the severe variants of Gaucher disease in which the neuronopathic effects are invariably manifest in childhood.
Anderson-Fabry disease
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Extended use of eliglustat and its congeners for the treatment of glycosphingolipid diseases will be restricted to those disorders such as Anderson-Fabry disease, where the condition principally affects peripheral tissues and where already there is experimental evidence of the predicted therapeutic efficacy of this inhibitor class [93].
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While infusions of α-galactosidase help to clear endothelial storage of globosides associated with the lethal cardiac, renal and cerebrovascular disease, variability is seen in the clearance
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from several other cell types and the therapeutic effects are relatively poor compared with enzyme therapy in Gaucher disease. Thus it is possible that further clinical developments for single or combined treatments will be undertaken for Fabry disease (see below).
Use of eliglustat for neuronopathic variants of Gaucher disease and the GM2 gangliosidoses is out of the question but as discussed in relation to the iminosugars, given the massive human need for credible innovation in specific medical care for these progressive diseases, there is a strong case for further scientific advancement. The likely explanation for the poor distribution of eliglustat in the brain is that the compound is transported by the pglycoprotein, multidrug-resistance (MDR1) molecule which is expressed at the site of the
blood-brain barrier tight junctions between the cerebral endothelial cells, which form the
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diffusion barrier selectively excluding most blood-borne substances and xenobiotics from entering the brain. Recently, Shayman and colleagues have confirmed that eliglustat is a substrate for the MDR system and have designed new inhibitors of UDP-glucosylceramide transferase which are neither identified by the p-glycoprotein nor transported; one of these was shown to be effective in reducing brain glucosylceramide concentrations in a short-term in vivo experiment carried out in the murine model of acute neuronopathic Gaucher
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disease. The authors have moreover set out a useful general strategy for testing compounds that lack affinity for MDR1 but retain their potency as well as critical selectivity
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towards UDP-glucosylceramide transferase [94].
Other innovative glycosphingolipid biosynthesis inhibitors
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Aware of the perennial need for improvement and innovation, particularly in relation to the optimal pharmacological properties required – including specificity towards the therapeutic target , metabolic disposal, lack of toxicity and pharmacokinetics as well as the capacity for distribution into the central nervous system, Genzyme (a Sanofi company) appears to have made a decision to ‘go it alone’. Recently the company has filed a US patent for chemically
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novel glucosylceramide synthase inhibitors [US 20140255381 A1]. The application is described as relating to new inhibitors with radically distinct chemical structures that will be useful for the treatment of metabolic conditions, ‘such as lysosomal storage diseases, either
cancer’.
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alone or in combination with enzyme replacement therapy, and for the treatment of
It is not relevant to discuss this development in detail, but it is clear that the protean
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involvement of glycosphingolipids beyond disorders affecting the lysosome extends our knowledge of intriguing biological science to become highly attractive for investment by Biotech. The application is expansive and describes many potential uses; diseases that cause renal hypertrophy or hyperplasia such as diabetic nephropathy; diseases that cause hyperglycemia or hyperinsulinemia; cancers in which glycolipid synthesis is abnormal, infectious diseases caused by organisms which use cell surface glycolipids as receptors, diseases in which synthesis of glucosylceramide is essential or important, diseases in which excessive glycolipid synthesis occurs (e.g., atherosclerosis, polycystic kidney disease, and renal hypertrophy), neuronal disorders, neuronal injury, inflammatory diseases or disorders associated with macrophage recruitment and activation (e.g., rheumatoid arthritis, Crohn's
disease, asthma and sepsis) and diabetes mellitus and obesity. Particular mention is made of
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the observation that in acute myeloid leukemia cells P-glycoprotein confers resistance to ceramide-induced apoptosis and that the inhibitors can be useful for treatment of proliferative disorders by inducing apoptosis.
The innovative lead molecule, Genz 161 ((S)-Quinuclidin-3-yl (2-(2-(4-fluorophenyl) thiazol4-yl)propan-2-yl)carbamate), is chemically distinct from the prototypic inhibitors of UDP-
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glucosylceramide transferase first developed by Radin, Shayman and their colleagues. Genz 161 has been successfully tested pre-clinically in the K14 mouse, a severe murine model of acute neuronopathic Gaucher disease [95]. Using this stringent model with complete loss of
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glucosylceramidase activity, the authors showed that after systemic administration of the inhibitor (which crosses the blood-brain barrier), accumulation of glucosylsphingosine and glucosyceramide was diminished; gliosis with signs of neuro-inflammation and infiltration of
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macrophages and microglia were ameliorated and survival was prolonged. These findings, based on an in-house discovery programme strengthen the belief that potent systemically administered glucosylceramide synthase inhibitors that traverse the blood-brain barrier are likely to be developed by Genzyme; they ultimately hold out considerable hope of benefit
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for patients afflicted by neuronopathic lysosomal diseases.
Substrate inhibitors for lysosomal diseases other than glycosphingolipidoses Genistein
The mucopolysaccharide diseases have a common cause in impaired breakdown of
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glycosaminoglycans, the undegraded complexes of which are considered to play an important part in cellular pathogenesis. While enzyme therapy is at various stages of clinical
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development for several of the mucopolysaccharide diseases (MPS I, II, IVA, VI and VII), improved treatments are needed – not least to address the frequent neurological manifestations which complicate most of these conditions and which are likely to remain beyond the reach of enzyme therapy. Hitherto, the search for tractable biochemical means to interfere with the biosynthesis of complex glycosaminoglycans in patients with mucopolysaccharidosis has been disappointing.
Genistein, an isoflavone found in legumes particularly soybean, is a potent phytoestrogen with wide-ranging biological effects, several of which are promulgated in the sphere of alternative therapeutics for combating oxidative stress, cancer, diabetes, obesity,
inflammation, osteoporosis, neuropathy - and others. However, Wegrzyn and colleagues
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indicate a more specific action by investigating the effects of specific components of genistein preparations on the incorporation of radioactive sulphate into glycosaminoglycans by cultured fibroblasts from healthy subjects and patients with several different mucopolysaccharidoses [8,14]. This research had been suggested by reports showing effects on tyrosine kinase receptors and glycosaminoglycan synthesis [96, 97]. At low micromolar (≈10μM) concentrations, the agent was found to inhibit incorporation of radioactive
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sulphate by about 90% in all samples; prolonged incubation in MPS type 1 fibroblasts showed that total cellular glycosaminoglycans were reduced to a level comparable to that induced by in vitro exposure to recombinant α-L-iduronidase (laronidase, Aldurazyme™)
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with partial restoration of the ultrastructural appearances of pathological lysosomes to normal [98].
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Later preclinical studies of the effects of oral genistein preparations have been conducted in mouse models of MPS II (Hunter) and Sanfilippo B (MPS IIIB) diseases [99, 100]. High doses (160 mg/kg/day) of genistein aglycone (4,5,7-trihydroexyisoflavone) either partially corrected the storage of glycosaminoglycans [99] or when given continuously over a 9 month period to MPS IIIB mice significantly reduced lysosomal storage of heparan sulphate s
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and neuroinflammation in the cerebral cortex and hippocampus while improving the behavioural defects [101]. Enhanced synaptic vesicle protein expression and lysosomal accumulation of glycosaminoglycans in the cerebral cortex were also noted.
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In an open-label study of 10 patients given genistein for 12 months, there was a significant reduction in urinary excretion of heparan sulphate with improved hair morphology and
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cognitive function; no adverse reactions were observed. [102].
While several studies have reported treatment of MPS III patients with low dose genistein (5-15mg/kg/day) with no serious adverse effects and variable neurocognitive outcomes, controlled clinical trials of various doses and genistein preparations with variable compositional efficacy have been examined principally in Sanfilippo disease [102]. The effects at low dose (10mg/Kg/day) in a blinded cross-over trial were to induce modest reductions in the excretion of glycosaminoglycans without overt clinical benefit [103]. However, while apart from inducing gynaecomastia in young males, attributable to the phytooestrogenic properties of the agent, higher doses (150mg/Kg/day) appear to be
tolerable, although at the time of writing, biochemical and clinical efficacy outcomes appear to be less certain [104,105].
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Those mucopolysaccharide diseases in which there is a neurological component are immensely challenging; and compared with the sphingolipidoses, they are far less well understood in biochemical terms. With this comes a more limited understanding of molecular pathogenesis and a limited range of techniques materially to correct the toxic
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effects of harmful metabolites, especially in the nervous system. The rôle of genistein is not yet fully worked out but even if ultimately it is found to be a disappointing starting point for innovative treatment, it provides a focus of clinical interest in a condition where the
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ultimate rewards evidently attract courageous scientific effort.
D. Functional Complementation
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Complementation stratagems
These established stratagems depend principally upon therapeutic complementation, either of whole cell function, as in haematopoietic stem-cell transplantation, which may or may not provide additional complementation at a distance through the process of secretion and recapture of lysosomal matrix enzymes, or in the provision of the missing protein, for
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example in macrophage-targeted enzyme therapy in Gaucher disease. Here the ‘patch-up’ concept of organ replacement (eg renal, cardiac or hepatic transplantation, jointreplacement surgery) is excluded, since these tactics have been used for many decades and are now incorporated into the mainstream of contemporary medical practice in developed Also
important
for
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countries.
functional
complementation
is
consideration
of
pharmacological chaperone molecules to enhance the activity of residual mutant proteins,
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and gene therapy, which is now on the threshold of clinical practice after many challenging years of disappointment in the context of failed clinical trials and premature trumpeting of its potential benefits. Finally, it is important to realise that each of these different therapeutic categories have in their sights the possibility of addressing the challenging neurological manifestations of many lysosomal diseases.
(i) Haematopoietic stem-cell (previously bone-marrow) allo-transplantation (ii) Reinfusion of ex-vivo vector-transduced autologous haematopoietic stem cells (iii) Direct gene transfer
(iv) Enzymatic augmentation
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(v) Enzymatic enhancement and stabilization (pharmacological chaperone therapy)
(i) Stem-cell therapy Induced pluripotential stem cells have excited much interest and principally are of use for in vitro modelling of human disorders. The highly attractive and futuristic concept of stem-cell
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therapy, based on reinfusion of genetically corrected autologous pluripotential stem cells, derived from somatic cells after a simple biopsy procedure, is firmly in this category. While rapid advances are underway, at present, safe means for inducing such stem cells for donorrecipient transfer and much-awaited therapeutic correction [106] have yet to be introduced
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for clinical application and will not be discussed further here.
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Haematopoietic stem-cell therapy
At present, two main stem cell complementation systems are of interest for development in the lysosomal diseases: (a) further exploration of haematopoietic stem-cell therapy – one of the first potentially curative treatments used in lysosomal diseases with early experience in the 1980’s for the treatment of mucopolysaccharidoses [107] (principally MPS I and II in the
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first instance) and Gaucher disease, in which it was first successfully introduced in 1982 [108]; (b) ex-vivo gene transduction of autologous haematopoietic stem cells using lentviral vectors; these cells are then re-infused for engraftment under myelo-ablative conditions
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[109,110].
Stem cell therapy (without gene transduction)
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Haematopoietic stem cell transplantation has been used in attempts to treat lysosomal diseases with some success over three decades. The haematopoietic stem cells from suitable donors allow the generation of progeny that are competent in lysosomal functions and can migrate in small numbers over time to the brain by traversing the physical bloodbrain barrier. As occurs in the bone marrow, and other niches of destination, the donor stem cells generate a steady supply of self-renewing blood cell lineages that are competent in autonomous functions (such as phagocytosis and immune reactions); and in the brain migrate to sites of disease where they can undertake scavenging activity as healthy counterparts of the diseased microglia and other cells with impaired lysosomal function. In the case of diseases such as Krabbe, the Sanfilippo syndromes, as well as metachromatic leukodystrophy, the donor cells are also able to supply enzymatic complementation to
neighbouring tissue by means of the secretion-recapture process for soluble matrix acid
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hydrolases [8, 14, 111].
The outcomes of transplantation in more than 60 European patients were earlier reported [114] and in Gaucher disease, particularly, the earliest reports clearly show that timely replacement of the diseased macrophage population and complementation of other haematopoietic lineages can secure dramatic improvement in outcome and almost
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complete correction (arrest) of somatic disease [112,113,114]. Contemporary stepwise improvements in conditioning, clinical selection, source of stem cells and nature of the donor as well as timing of the procedure have led to greatly improved safety and remain a
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focus for continued refinement and innovation in current practice.
Haematopoietic stem cell therapy has been extended to the lysosomal diseases with rapid
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loss of faculties and neurodegeneration such as metachromatic leukodystrophy and Krabbe disease - largely based on the advocacy of early investigators [115, 116,117]. Current developments in this demanding clinical field mainly focus on ensuring the indications for the intervention are tailored to the stage of disease and likely response; they also relate to
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improved techniques and regimens for conditioning to ensure safe engraftment.
At present, while haematopoietic stem-cell therapy offers an incontrovertible therapeutic effect in several disorders, particularly the MPS disorders and possibly in severe chronic neuronopathic Gaucher disease (type 3), it must be recognized that numerous practical
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limitations restrict its use. Typically, a suitable HLA-matched sibling donor is not available, and despite the best efforts of charitable or state-funded agencies to search for matched
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unrelated donors, their identification and confirmation of compatibility can be a lengthy process; moreover, the success of the procedure mandates early diagnosis in the recipient – a persistent challenge in all therapeutic settings for rare disorders. In the last 15 years, the use of umbilical cord blood donors as a rich source of unrelated donor stem cells for suitable recipients and the safety of the procedure - with clearer case selection and optimized conditioning and myeloablative techniques – has led to stepwise improvement in outcomes for some lysosomal diseases.
Despite these improvements, persistent morbidity and mortality is associated with the procedure and the decision to use it must as always be balanced carefully against the
severity of the disease and the likely therapeutic response [118,119,120]. Hitherto its use,
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with the typical exception of MPS I Hurler has usually been deferred in favour of enzyme therapy - whenever this is available. Recent evidence suggests that while enzyme therapy may improve engraftment, the use of haematopoietic stem cell transplantation improves the high incidence of neutralizing allo-antibodies observed in MPS I after pharmacological enzyme replacement therapy [121]. It has to be recognised however, that in countries where government support for expensive treatments is limited or cannot be underwritten
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for long periods due to political and budgetary uncertainties, the challenging but single large cost of haematopoietic stem cell transplantation may be the only solution.
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There are numerous contemporary examples of patients with Gaucher disease in such healthcare settings where the procedure has been carried out: in several Middle-Eastern and Asia countries this option has been the only one available. However, given that severe
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neuronopathic Gaucher disease is greatly over-represented in these populations, and that some patients may secure greater benefit from the procedure than enzyme therapy, one cannot be sure that they have always had the inferior choice of primary treatment. This issue and support for haematopoietic stem-cell therapy for Gaucher disease has very recently been revisited by Ito and Barrett [122]. While their thesis is marred by some
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inaccuracies and limited citation of contemporary therapeutic developments in the treatment of Gaucher disease [123], the authors raise an argument that merits fuller discussion for future developments in Gaucher disease and other lysosomal diseases, such as MPS II (Hunter disease), in the context of global medical realpolitik. A patient advocacy
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group ensured that a global moratorium to prevent bone marrow transplantation in this disease was put in place in the treating community. However, recent data from Japan cast
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doubt on this extreme view [124,125] and if future development is to proceed scientifically for the benefit of patients, a re-evaluation of the potential efficacy and indications for haematopoietic stem-cell therapy would be advisable.
(ii) Ex vivo haematopoietic stem-cell gene (reinfusion) therapy This stratagem represents an imaginative extension to the use of haematopoietic stem cell therapy: while it offers to overcome some of the difficulties associated with donor-derived cells, it also seeks to enhance the weak therapeutic effect of cellular complementation delivered into the peripheral circulation and able to correct many of the peripheral manifestations of disease but with limited salutary effects on the brain.
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Bigger and Wynn [126] provide an excellent contemporary review of the development of this technique and clinical extension from haematological diseases and immune deficiency syndromes such as adenosine deaminase deficiency [127] and X-linked severe combined immunodeficiency [128] to the metabolic leukodystrophies, adrenoleukodystrophy, an Xlinked peroxisomal defect [129] and latterly, as already set out, metachromatic leukodystrophy [110].
Over three decades since the first clinical attempts at retroviral
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transfer of genetic material into haematopoietic progenitor cells, it has been shown that gene therapy can be curative, while obviating the need for suitably HLA-matched allogeneic donors and avoiding the risk of graft-versus-host disease. Improvement in the efficiency and
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nature of self-inactivating lentiviral vectors also avoids mutagenesis with oncogenic effects due to biased integration events at key loci in the host genome. Third-generation vectors with refined protocols for myeloablation and engraftment, as well as improved
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manufacturing procedures are already opening up this stratagem for therapeutic gene transfer [130] and, sooner or later, much more extensive application in the lysosomal diseases.
It is clear that despite their often severe nature and multisystem effects and distribution,
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the lysosomal diseases are attractive therapeutic targets for the application of gene transfer techniques. Correction by gene transfer of a small focus of living cells within an affected tissue or organ would be expected to induce secretion and distribution of the cognate processed glycoprotein harbouring the appropriate carbohydrate recognition signals locally
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into extravascular space and circulation for capture and uptake with correction of the
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lysosomal defect in diseased cells at a distance.
Ex vivo gene therapy utilizing haematopoietic stem cells harnesses the concept of a therapeutic Trojan horse. The application has principally been directed to the delivery of the competent cells to brain, and with the aim of enhancing efficacy as a consequence of increased expression of the corrective lysosomal protein brought about by genetic transduction of the stem cell population. The use of autologous stem cells harvested for transduction ex vivo has the merit of ready availability and lack of immune incompatibility.
Of particular interest have been the heroic efforts and emerging clinical success of the research group led by Naldini and colleagues in the San Raffaele University in Milan. After
considerable efforts to perfect the techniques in the somewhat unsatisfactory clinical model
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of metachromatic leukodystrophy in mice [131], Biffi et al [109] recently reported a phase I/II clinical trial employing ex vivo transfer of the wild type human arylsulphatase A gene into marrow-derived CD34+ haematopoietic progenitor cells harvested from three unrelated infants diagnosed with infantile metachromatic leukodystrophy in the pre-symptomatic phase as a consequence of disease in older siblings. The trial subjects received the intervention 2 to 12 months before the reported age of onset of the disease in their affected
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siblings. Transduction was optimized to reach at least two vector copies per genome to ensure optimal enzymatic overexpression for therapeutic efficacy previously determined in experimental studies. These genetically engineered CD34+ cells were stimulated ex vivo with
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early-acting cytokines and transduced with purified third-generation lentiviral vectors expressing the human cDNA under the control of the human phosphoglycerate kinase. After expansion, the transduced progenitor cells were infused into the recipients who had
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undergone myeloablation with intravenous busulphan, given every 6 hours for three days beforehand. After a busulphan-free interval of at least 24 hours, the engineered CD34+ cell product was infused as a suspension; no immunosuppression was given to the recipient.
Biffi et al confirmed stable engraftment of the transduced stem cells in the bone marrow
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and peripheral blood of all patients at all times tested, with 45 to 80% of bone marrow– derived haematopoietic colonies harbouring the vector. Arylsulphatase A activity was expressed above the healthy reference range in the haematopoietic cell lineages and in cerebrospinal fluid. Safety monitoring required repeated analysis of lentiviral integration
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sites: this showed that the high gene expression was associated with polyclonal engraftment of transduced cells; no aberrant clonal behaviour was identified. Several integration sites
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were shared among mature blood elements as well as progenitors and mature blood elements, indicating efficient transduction and engraftment.
The authors provide evidence for a clear therapeutic benefit of this intervention initially in three trial subjects followed for at least two years after the intervention. Neurological and motor evaluation using standard tests and neuropsychological assessment was used to assess the presence of disease; neurological imaging by MR was also conducted. The disease did not progress in any of those treated patients, even beyond the time of onset of disease predicted and that projected from the behaviour of the condition and their previously affected siblings. While formally no absolute comparison was made between this
intervention and conventional haematopoietic stem-cell transplantation in early selected
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presymptomatic patients with this form of metachromatic leukodystrophy, on the balance of probabilities, the safe and tolerated therapeutic effect was gratifying and sufficiently convincing for the programme to secure pharmaceutical investment and further continuation and expanded recruitment [131]. While this laborious ex vivo stratagem is invasive and in this group of devastating neurodegenerative lysosomal diseases, not readily applicable to the many patients who are
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first affected without systematic population-based screening procedures in place, the therapeutic concept has to a large extent been convincingly demonstrated in human medicine. However, as in the case of conventional transplantation therapy which has
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already proved to be highly controversial in the context of the specific neonatal screening programme for Krabbe disease in New York State [133, 134], the practicalities of introducing neonatal screening require careful thought – even though this programme has expanded to
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several other states [135]. Simply linked to a potentially beneficial but innovative and heroic therapy with potentially fatal effects related to myeloablation the debate cannot be ignored, the mortality remains even for the best centres at 5-10%. When these considerations relate to gene therapy, realization of the therapeutic potential and crystallization of the innovation in the ‘real world’ of clinical practice will require further
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planning and review.
Extension to other lysosomal diseases, cystinosis – a cell-autonomous disease
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An additional aspect here with potential for innovation, is the exploratory extension of haematopoietic stem-cell therapy to other lysosomal diseases that have not been in the first
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ranks of priority. In the case of cystinosis, management of the consequences of the Fanconi syndrome with progressive interstitial nephritis placed emphasis on renal transplantation and later, as the systemic nature of the disease rapidly became apparent, the early introduction of cysteamine therapy has become a dominant priority for a disease caused by defects in the lysosomal membrane protein, cystinosin. It is notable that latterly, there is mounting evidence that this membrane transport disorder of the lysosome may also show a salutary response to haematopoietic stem cell therapy – even though, as with Gaucher disease, there is no possibility of functional complementation via the secretion-recapture pathway that is envisaged to occur in vivo in the case of soluble matrix enzyme of the lysosome.
Cherqui and colleagues [136] have carried out syngeneic bone-marrow transplantation from
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wild-type donor mice to mice lacking cystinosin. These mice serve as a useful model of human cystinosis and develop pathological systemic cystine crystal deposits, with tubulopathy and renal failure accompanied by bone and muscular defects; characteristic cystinotic ocular changes also occur. Transplantation of stem cells from wild-type donors prevents kidney injury and corneal deposition in the mutant recipients. These investigators showed that full engraftment of marrow stem cells expressing functional cystinosin was
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required to arrest the disease. Moreover, fastidious quantitative analysis of the expression of functional protein and tissue colonization by donor stem cells was shown to correlate with significant (57-94%) reduction of cystine concentrations. While these findings appear
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counterintuitive and given some discrepancies between the mouse and human disease, direct extrapolation of these results to patients with cystinosis will be challenging, the
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results are tantalizing.
One speculative possibility is that phagocytosis of circulating leukocytes harbouring excess cystin and fluid-phase endocytosis contribute to the dynamic fluxes of the metabolite; in this way, a donor population of healthy haematopoietic stem cells could partially ameliorate the cystin load at peripheral sites. Indirect support for this possibility emerges from reports
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of increased plasma chitotriosidase activity in patients with cystinosis – chitotriosidase is a classical protein marker of the macrophage [137]. The possibility of cell-cell transfer of the corrective transmembrane factor (cystinosin) is also a possibility but the scale of complementation appears to be implausibly generous for such a process. This stratagem is
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now actively under investigation and represents another innovative application of therapeutic potential in contemporary medicine for the challenging specialist domain of
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lysosomal diseases [138] (see below).
Gene therapy
The principle here is to transfer human cDNA encoding the cognate wild type sequence corresponding to the appropriate protein or proteins that are malfunctioning as a result of genetic mutation in the patient with a lysosomal disorder. In this instance, the wild-type genes are transferred with viral or other vectors directly to the appropriate tissues in the host. Lysosomal diseases have the theoretical potential for successful correction by this stratagem, since the secretion-recapture system allows for extensive functional
complementation in diseased tissues; the uptake of corrective protein (almost invariably a
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soluble acid hydrolase of the lysosomal matrix) is designed to restore healthy activity and thus amelliorate, arrest or reverse the disease. In most instances where this has been successful in experimental animals, viral vector systems have been utilized; the transduced cells to act as a source of functioning enzyme which is released to effect the therapeutic complementation after uptake by the diseased cells [139]. In principle, the greater amount of enzyme that is generated and released, the more will be available for receptor-mediated
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uptake and extent of correction.
Criticial rôle of animal models for clinical development of gene therapy
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The single-gene disorders lend themselves readily to gene transfer stratagems: a large therapeutic effect is predicted after successful transduction of healthy copies of the cognate gene which when mutated has highly penetrant effects. Emerging clinical data show that
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this technology is rapidy entering a fullblown clinical phase and biotech commerical interest is burgeoning [140]. Numerous naturally-occurring and transgenic models of lysosomal diseases have been reported: not only do these usually serve as useful experimental models of the related human diseases, they are coherent biochemically and genetically – even allowing in mice for valid simulacra of the effects of particular mutations which can be
Contemporary progress
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introduced through ‘knock-in’ genetic technologies [141, 142].
Many lysosomal diseases affect the nervous system as well as peripheral tissues and are
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thus particularly challenging to treat. Nonetheless untreated neurodegeneration has extreme human and medical costs. Thus with encouraging signs of effective experimental
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gene transfer in valid animal models, expectations that clinical gene therapy will be introduced in the field as a credible option for many lysosomal diseases where effective therapy is otherwise unavailing, seem justified. With appropriate safeguards, scrupulous choice of biological target, a careful enrolment policy and evidence of efficacy based on clinically articulate criteria, direct gene transfer technology offers a vision of definitive and affordable treatment. The resurgence of contemporary interest in gene therapy has resulted from a fortunate conflation of: (i) the extraordinary therapeutic accessibility of inborn lysosomal diseases to enzymatic complementation; (ii) the success of innovative biotech investment in niche ultra-orphan therapies, where clinical need can be addressed by efficacious therapies developed in the context of anti-competitive legislation for orphan
medicinal products; and last, but dominantly (iii) the continued development of safe, simple
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but powerfully expressing vectors for gene transfer. These include small vectors derived from non-pathogenic, non-integrating adeno-associated viruses, ideally suited for safe and sustained transduction of non-mitotic cells – and which can harbour cDNA inserts encoding constitutively expressed polypeptides (for example those that assemble to generate lysosomal acid hydrolases).
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In relation to those lysosomal diseases affecting the nervous system, all therapies must take account of the blood-brain barrier and the physical blocks to generalized correction of disease at privilged sites (see chapter 3). While some innovative viral-derived vector systems
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that can traverse the blood-brain barrier after administration to the perpheral organs or circulation in adult recipients are in active development [143] in most instances where clinical stratagems are in progress, the vector delivery of the corrective lysosomal protein
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occurs after a physical breach of the barrier using direct administration at the time of a neurosurgical procedure [144]. In future innovations, however, it seems likely that rAAVrh.8, rh.10 and 9, will offer hope for treating neurological diseases in the adult patients; systemically delivered rAAVrh.10 efficiently transduces the nervous system but at least in primates, transgene expression can be limited peripherally by endogenous microRNA
Direct gene transfer
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sequences.
Here follows a brief review of gene transfer for lysosomal diseases, particularly to address
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those with an important neurological component, and with experimental development that is at, or close to clinical application. Latterly advances in viral and non-viral vectors and
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methods for their delivery have explored the correction of central nervous and peripheral tissues; this research has largely been predicated on methods to evaluate neurological outcomes in animal models.
The main challenges for gene transfer are (i) generating sufficient delivery and distribution of the therapeutic gene product in all at-risk tissues with injurious pathology such as the brain (ii) sustaining expression of the corrective gene(s) (iii) ensuring safe transduction and protein expression without harmful effects on recipient and transduced tissues. Here various stratagems are exemplified.
Intracerebral injection of recombinant adeno-associated viral vectors (rAAV) expressing
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human lysosomal proteins into white matter appears to be safe in primate brain; the vectors are capable of generating long-term sustained expression of lysosmal hydrolases at activity levels predicted to have useful therapeutic effects in late infantile neuronal lipofuscinosis type 2, (NCL2)[145]. [145]. In a clinical phase I/II study using rAAV-2 serotype vector, neurosurgical techniques and experience were reported in human subjects subjected to intensive radiological scrutiny [146]. Ten patients with late infantile neuronal
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ceroid lipofuscinosis disease each underwent infusion of AAV2 (3 x 1012 particle units) at 12 distinct cerebral locations (twodepths/burr hole, 75 minutes/infusion at 2 µL/minute). Techniques were developed to overcome obstacles for which there are few established
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procedures. The surgeon relied on preoperative stereotactic planning to optimize a parenchymal target and diffuse administration. Six entry sites, with injection at two depths each were used to reduce operative time and enhance distribution. A low-profile rigid
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fixation system with 6 integrated holding arms was utilized for simultaneous infusions in an expeditious manner and without ill-effect. These studies emboldened later trials using more developed high-expression vector serotypes for direct administration in this fatal neurodegenerative disease.
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As a prelude to clinical trials using the recombinant adeno-associated viral vector (rAAV) serotype rh.10 (rAAVrh.10) planned in 2015 for patients with the devastating disorder, metachromatic leukodystrophy, Aubourg and colleagues investigated the optimal delivery route of the vector. They used rAAV encoding human arylsulphatase A in the primate brain
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and each animal received a total dose of 1.5 × 1012 genome copies of AAVrh.10hARSA-FLAG at: (1) delivery to white matter centrum ovale; (2) deep gray matter delivery (putamen,
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thalamus, and caudate) plus overlying white matter; (3) convection-enhanced delivery to same deep gray matter locations; (4) lateral cerebral ventricle; and (5) intraarterial delivery with hyperosmotic mannitol to the middle cerebral artery. After 13 weeks, the distribution of enzymatic activity subsequent to each of the three direct intraparenchymal administration routes was significantly higher than in phosphate-buffered salineadministered controls, but administration by the intraventricular and intraarterial routes failed to demonstrate measurable levels above controls. Immunohistochemical staining in the cortex, white matter, deep gray matter of the striatum, thalamus, choroid plexus, and spinal cord dorsal root ganglions confirmed these results. Of the five routes studied, administration to the white matter led to the broadest distribution of corrective enzyme
with 80% of the brain displaying more than a therapeutic (10%) increase in arylsulphatase A
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activity above control injections. No significant toxicity was observed with any delivery route as measured by safety parameters, although microscopic inflammatory changes were observed. The author concluded that AAVrh.10-mediated delivery by direct administration into white matter is likely to be safe and yield the widest distribution of corrective enzyme;
Efficacy in human gene therapy trials for lysosomal diseases
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they have selected this asthe most suitable route for clinical vector delivery [147].
At the time of writing, investigators clearly stand at the threshold of effective clinical application of gene therapy for lysosomal diseases. From the success of treatments for
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inherited retinal disease and haemophilia [148,149]], suitable vectors and stratagems that avoid immune shut-down are becoming available. At the same time, financial ‘models’ to support largely on-off costs for their ultimate testing in phase III clinical trials are being
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negotiated with biotechnology companies.
At present, the clinical outcome of executed [144, 145] in progress [150], and planned [147] phase I/II gene therapy trials, respectively in late-infantile neuronal ceroid lipofuscinosis, Sanfilippo A , Sanfilippo B and Metachromatic Leukodystrophy are awaited. All are
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predicated on the outcomes of cogent studies in genetically and clinical coherent animal models. Additional trials in, for example, GM2 gangliosidosis [151] and other very severe near-exclusive neurological diseases of the lysosome are also at a promissory stage or active
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consideration.
After early sensational interest several decades previously, Biotech has latterly been
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disappointingly ‘risk-adverse’ towards gene transfer technologies. As a consequence, until now, the cadre of competitive interest external to academic intitutions, which is the essential catalyst for development, has been lacking. Although in many cases breakthrough treatments have hitherto proved elusive, the technologies for therapeutic exploration in humans are now safer and possess demonstrably greater efficacy for challenging lysosomal diseases such as the mucopolysaccharidoses, in which mixed systemic and neurological manifestations occur and where very costly enzyme therapy is inadequate materially to prevent decline or restore function [152]. Currently perhaps the most promising vectors are based on rAAV vectors; but the safety of non-integrating, self-annealing 3rd and 4th generation retroviral systems now render them suitable candidates for transducing of
slowly-dividing cells in the diseased tissues of patients with non-lethal chronic disorders [153]. Previously,
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although
retroviruses were
successfully introduced for fatal
haematopoietic and immunological diseases in which great experience has been gained [154], commercialization through phase III clinical trial development remained tardy.
Practical issues in relation to trials of gene therapy in lysosomal diseases While gene therapy is in its clinical infancy, it has nonetheless been delivered – hitherto
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safely - as a regulated and approved clinical entity in several severe lysosomal diseases. Several phase I/II clinical studies are underway with the main regulatory focus on safety and tolerability; but a crucial aim is to discover an effective therapy for humans, principally
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young children suffering from these ultra-rare and fatal disorders.. Ultimately the viability of gene transfer technologies will depend upon careful design, selection, conduct and analysis of heroically complex experimental procedures carried out in only a few trial Gene therapy offers the prospect of large therapeutic effects but , robust
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subjects.
information about the natural course of the disease will assist objective determination of clinical efficacy and possibly indicate whether the intervention rescues disease, reduces the rate of decline or restores neurological function. In this respect, selection of presymptomatic subjects with previously affected first-degree relatives offers the best chance
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of determining whether the intervention will indeed offer clinically articulate benefit.
The prospect of direct adminstration of vectors to the brain may ultimately prove unneccesary in some cases since it is now clear that members of the AAV9 and, possibly,
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rh.10 vector families have blood-brain barrier penetrating properties even in adult mammals. They thus can transduce the nervous system via a single peripheral injection not
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only when administered intravenously and into cerebrospinal fluid. While the safety and tantalizing efficacy of such technologies are being evaluated [155], the recent report of a high proportion of liver and lung tumours in mice with GM2 gangliosidosis that received systemic rAAV9 in the neonatal period raises concerns about oncogenicity [156] but the long-term experience with successful human rAVV therapy in haemophilia B is reassuring.
(iv) Enzymatic augmentation As demonstrated in other sections of this volume (chapters 3, 4, 5, 6 and 7), systemic administration by parenteral infusion of lysosomal proteins targeted to affected tissues by exposure of cell-surface recognition signals has had a distinguished history and continues to
hold promise for the treatment of inborn errors affecting matrix enzymes of the lysosomal
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compartment. While the stratagem is no longer new, with advances in the manufacture of stable and active recombinant therapeutic proteins in a form suitable for administration, innovative methodological adjustments continue.
Delivery of therapeutic proteins to the central nervous system depends upon biological therapies and these ‘biologics’ are in effect high molecular weight glycoproteins to which
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the mammalian blood-brain barrier is impermeable. Much effort in the competitive niche area of lysosomal diseases is thus being expended on overcoming the blood-brain barrier in practical and commercially applicable ways. Fortunately, and an advance on the position at
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the time of the development of systemic enzyme therapy for Gaucher disease, the existence of naturally-occurring and transgenic animal models, facilitates these endeavours. Proof-ofconcept studies carried out in animal models of several important lysosomal diseases has
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allowed direct intrathecal or intra-cerebro-ventricular routes for delivery of therapeutic proteins to be explored as a prelude to clinical trials. Novel and courageous applications for those diseases which affect the central nervous system have recently taken hold: intrathecal and intraventricular administration for metachromatic leukodystrophy, Sanfilippo A, Sanfilippo B and even Krabbe disease are, despite the harsh odds against sustained
blood-brain barrier.
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therapeutic success, under development as the most direct means to breach the physical
For completeness, the reader is also directed to new therapeutic enzymes in late-stage
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development for systemic treatment of Niemann-Pick disease type B, discussed in chapter 9, alpha mannosidosis, mucopolysaccharidosis type 1V A (Morquio-Brailsford disease), and
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acid lipase deficiency (Wolman and cholesterol-ester disease). Finally, mention should be made of an extraordinary phenomenon in the commercial arena of ultra-rare disorders and orphan medicinal products: the innovative emergence of exceptional competition. Apart from the two enzyme therapies simultaneously approved in Europe for Fabry disease (TKT [Shire] and Genzyme [Sanofi]), and three enzyme preparations available in the United States (two in Europe) for Gaucher disease, generic biosimilar alternatives are emerging. The first successful small biotech company in South Korea, for example, now has about 40% of the market share: ISU ABXIS have Abcertin for Gaucher disease and FABAGAL for Fabry disease. While these initiatives indicate that currently the field is attractive for technological
investment and new/old products, the changing entrepreneurial equilibrium may ultimately
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signal the end of an era of burgeoning research into innovative enzyme therapies.
(v) Pharmacological Chaperones Definition
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Chaperones interact with polypeptides to generate functionally active proteins; they inhibit formation of misfolded or aggregated structures [157]. Chemical chaperones influence the intracellular environment in which protein folding occurs and stabilize proteins against thermal and chemical denaturation. These agents are often required at very high
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concentrations and are thus never going to be appropriate for in vivo therapeutic use; however, they possess properties of utility in the manufacture of recombinant proteins. In
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this sense, they are valuable as important components for enzyme therapies. Molecular chaperones are typically endogenous heat-shock proteins which recognise misfolded proteins and bind to their hydrophobic surfaces, thus preventing denaturation. At the same time, the process known as chaperone-mediated autophagy ensures that a particular pool of cytosolic proteins is delivered to lysosomes for destruction. Proteins bearing a specific amino acid sequence, related to KFERQ, are recognized by the heat shock
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cognate protein of 70kDa, the chaperone that mediates their delivery to lysosomes for degradation – about one third of soluble cytosolic proteins contain the motif [158]. Heat shock proteins have a rôle in exocytosis and the maintenance of lysosomal integrity and are
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discussed in detail with respect another innovative treatment for lysosomal diseases that is
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in clinical development [in section B] above.
Clinical use of pharmacological chaperones Pharmacological chaperones bind to specific polypeptide conformations and thereby stabilise proteins and prevent their degradation in the proteasome and lysosomal compartment. Use of pharmacological chaperones to rescue critical functions of proteins that are misrouted because of misfolding and even aggregation in the cell is an intriguing stratagem with a long but until recently, a substantially neglected, developmental history in therapeutics.
The greatest experience of this approach has emerged from clinical experimentation in inborn errors of metabolism, many of which are have been treated empirically by supplying
large doses of relevant cofactors enzyme known to be essential for the normal function of
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the cognate protein in which the functional deficiency is identified. A long-standing classical example is the use of pyridoxine supplements in approximately half of the adult patients with homocystinuria due to mutations in cystathionine ß- synthase who can be shown to be responsive to the agent [159]. A vivid and recent example is provided by the important disorder, phenylketonuria, in which most mutations in the deficient enzyme, phenylalanine hydroxylase, cause misfolding and instability. In about one third of patients
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supplementation with natural tetrahydrobiopterin cofactor has been shown usefully to improve phenylalanine intolerance and metabolic control – leading to marketing approval of the synthetic factor sapropterin dihydrochloride as a chaperone therapy in 2007
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[www.drugs.com › FDA History]. Since the catalytic mechanism, regulation by substrate and cofactor in the context of intensive biochemical and structural studies of the enzyme have been solved, it is clear that the therapy represents a striking example of a pharmacological
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chaperone effect enhanced by positive allosteric interactions between the cofactor and common aberrant enzyme variants [160].
Thus as in this case, small molecules which serve as specific ligands for aberrant proteins are clearly attractive for exploration as pharmacological chaperones with the potential for
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clinical use. While there are few known cofactors for lysosomal enzymes, the frequent identification of unstable and misfolding variants associated with lysosomal diseases suggests that this stratagem deserves close examination.
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Protein conformation diseases and the unfolded protein response The concept of protein conformational diseases can be traced to classical studies of
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amyloidosis by Virchow and Rokitansky in the nineteenth century. Amyloid deposits were found originally to contain aggregates of natural polypeptides that were overproduced in inflammatory diseases. However, it was later shown that certain proteins such as immunoglobulin light chains and mutant polypeptides were predisposed to misfold and aggregate in amyloidosis and related diseases. It was also realized that protein aggregation was a hallmark of Alzheimer and Parkinson disease with Lewy body dementia (and thus a strong link to lysosomal disorders); protein aggregation is central to prion diseases and other neurodegenerative diseases such the Huntington disease and the tauopathies.
Polypeptides harbouring pathological mutations frequently misfold and thereby escape
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normal processing; they overwhelm the surveillance and quality-control systems of the cell and are often retained in the endoplasmic reticulum (ER). The unfolded protein response is an adaptive signalling pathway which supports the homeostasis of the endoplasmic reticulum and responds to metabolic and oxidative stress as well as inflammatory reactions. The two main aspects of the ER stress response involve decreased protein flux and reduced protein translation via PERK-mediated phosphorylation of the eukaryotic translation factor
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eIF-2α and transcriptional upregulation of ER folding capacity via cleavage and activation of X-box binding protein-1 [161]. Degradation of misfolded polypeptides initiated by the protein quality control system involves chaperones but ultimately ubiquitination and
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delivery to proteasomes.
While some misfolded proteins may retain their functions, their destination is inappropriate,
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or even injurious to the cell. As a result, the outcome of many inherited defects is the consequence of impaired function combined with the toxic effects of aggregation and deleterious mistargeting. In the face of sustained activation due to uncleared misfolded polypeptides in the dynamic environment of the endoplasmic reticulum, the unfolded protein response induces apoptosis via phosphorylation through the PERK signalling
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pathway. The consequences of this and related pathways orchestrated by the unfolded protein response are observed frequently in human diseases.
Not only do the nascent lysosomal matrix polypeptides undergo extensive co-translational
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and other processing as part of their intracellular itinerary, they also interact naturally with numerous molecules which ensure appropriate trafficking to their destination in the
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organelle; these involve the mannose-6-phosphate-dependent and independent pathways [162,163,164]. Moreover, as set out above [Section B], a large family of naturally occurring and readily inducible heat-shock proteins also contribute to lysosomal integrity and health; these too serve as endogenous molecular chaperones to restore the function of misfolded proteins under stress conditions and where those proteins are disrupted and aggregated as a consequence of structural mutations. Here we survey pharmacological chaperones for lysosomal diseases: small molecules that penetrate cells to bind, stabilize and restore the three-dimensional and quaternary structure of proteins and also secure their function at their site of action.
Pharmacological chaperones for lysosomal diseases
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The specialised pathways of biosynthesis and trafficking of nascent lysosomal hydrolases are susceptible to interdiction by the pharmacological chaperones. Here I introduce the recent exploration of pharmacological chaperones as a therapeutic stratagem for lysosomal diseases. But for the remarkable clinical triumph of other initiatives such as enzyme therapy, this approach would not be practicable, but the approach has attracted considerable
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scientific investment and continues to hold promise [165].
Translation in the rough endoplasmic reticulum occurs in a neutral environment but delivery of the mature processed glycoproteins to the lysosome involves a radical pH transition in
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which the hydrogen-ion concentration increases about one thousand-fold. At the destination, the reaction conditions are most favourable for acid hydrolysis and the stability of the mature enzyme glycoform; furthermore, the concentration of the presented natural
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substrate is at its greatest. Small molecules with an affinity for the protein in the endoplasmic reticulum might, as with many chaperones, bind to its active site. Under these circumstances, the putative chaperone molecule would stabilize the lysosomal enzyme during trafficking and co-translational, as well as post-translational processing, in the endoplasmic reticulum and as the nascent glycoprotein traverses the Golgi stack. On
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delivery by fusion with the lysosome, the binding affinity of the molecular ligand on the protein will change but the stabilized and fully processed enzyme will be now be able to interact with its natural substrates at its physiological site of action [166,167]. The ideal pharmacological molecule will be a weak inhibitor at acid pH, so that once its rôle as
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chaperone is complete it will be readily displaced from the active site of the specific
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therapeutic target.
Clinical application of pharmacological chaperones in lysosomal diseases Fabry disease
An early example of the application of the pharmacological chaperone principle in the lysosomal disease is provided by the use of D-galactose in a patient with clinically attenuated Fabry disease [168]. Experimental studies showing its capacity to enhance the enzyme activity in lymphocytes and myocardial biopsy specimens, infusions of 1 g/kg galactose over four hours on a daily basis significantly enhanced α-galactosidase A activity, in the case of the heart, from
