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European Biopharmaceutical Review

A Rare Approach

In Europe, rare diseases are defined as those which affect one patient or less out of 2,000 individuals in the whole population. While in the US, a disease is said to be rare when it affects less than 200,000 patients in the whole population. Today, about 7,000 of these diseases have been described, and new pathologies still continue to be discovered.

Despite each of these diseases being uncommon, together they affect 8% of the whole population, meaning that at least 55 million people are afflicted by rare diseases in Europe and the US combined. A large fraction of these are genetic in nature, and are caused by some alterations in the genetic material – for example, mutations, deletions and translocations – rather than due to infectious agents or environmental factors. In a third of these cases, children are afflicted, in many cases dying before reaching adulthood.

By definition, orphan diseases are those for which no treatment is available or whose treatment would require substantial efforts to be developed. At present, there are 80 marketed drugs for rare diseases, meaning that out of the 7,000 rare diseases currently known, more than 90% of them remain orphan.

Lysosomal Storage Diseases

Lysosomal storage diseases are a group of over 50 rare genetic conditions caused, in most cases, by defective lysosomal enzymes. The enzymatic deficiencies result in the accumulation of cell constituents – such as glycolipids, polysaccharides and glycosaminoglycans – in the lysosomes, leading to cell dysfunctions and, ultimately, disease symptoms. Many lysosomal storage diseases are characterised by neurologic damage early in life, leading to death within a few years.

The mucopolysaccharidoses (MPS) are a sub-group of 11 lysosomal storage diseases caused by defective enzymes involved in the degradation of glycosaminoglycans, such as heparan sulfate and keratan sulfate. Two types of MPS can be distinguished on the basis of the symptoms they cause. Some of these syndromes predominantly impact the central nervous system (CNS) – for instance MPSIII, also named Sanfilippo disease – whereas others mainly affect development of peripheral organs, such as the skeleton and the heart – for example, MPSIV, also known as Morquio disease. Many of these diseases are severe and affect children early in life, leading to death before their twenties.

Several approaches are being followed in a bid to treat the different MPS. In particular, there are six treatment options:

Enzyme Replacement Therapy
This consists of intravenous administration with a recombinant enzyme and is best-suited for diseases affecting organs, such as the liver, spleen and kidneys, which are readily accessible to protein administered by intravenous injections. Three MPS are now successfully treated by enzyme replacement. However, in the MPS for which CNS impairment predominates, passage through the blood-brain barrier by the recombinant therapeutic protein limits the enzyme replacement therapy option or renders the administration of the drug cumbersome.

Gene Therapy
This treatment option holds big promises but still remains unproven. It proposes to replace the defective gene with a functional one, using recombinant viruses, usually adenoviruses. Here, adenoviruses carrying a functional version of the defective gene are applied directly to the CNS or peripherally. Alternatively, autologous haematopoietic stem cells are transduced with a functional gene and reintroduced in patients following ablative myeloid cell treatment.

Haematopoietic Stem Cell Transplantation
In this procedure, patients are deprived from their haematopoietic cells by chemotherapeutic treatment, before the cells are then reconstituted by autologous or allogeneic haematopoietic stem cells. Albeit successful for MPSI (Hurler syndrome), this treatment modality was not found to be therapeutically beneficial in other MPS – in particular, regarding improvement of cognitive functions. Alternative transplantation regimens tested in animal models also did not result in preventing the course of the diseases.

Substrate Reduction Therapy
This treatment aims to reduce accumulation of cell constituents due to their impaired degradation, by preventing their synthesis in the first place. It has also been used with some success in treating Gaucher disease. The drug Zavesca® is an inhibitor of the enzyme responsible for the synthesis of the cell constituent, whose buildup causes Gaucher disease. It was established in genetically manipulated MPSIIIA mice and found that limiting heparan sulfate synthesis resulted in the reduction of disease severity (1). Furthermore, in some disease contexts, degradation of glycosaminoglycans with slightly altered structures was restored.

Alteration of Substrate Degradation

The degradation pathway of the proteoglycans is still not known in detail. It is thought that the glycosaminoglycan chains are first cleaved by heparanase and are then internalised into the lysosomal compartment forfurther breakdown. As the catabolism of glycosaminoglycans involves several enzymatic steps occurring in different cellular compartments, it certainly offers opportunities for therapeutic intervention.

Molecular Chaperones
Mutated enzymes, in many cases, fail to properly fold in the endoplasmic reticulum and, as a result, are degraded. The principle here is that exposure to inhibitors would encourage the enzymes to adopt a native-like conformation, enabling them to travel to the lysosomes where they would perform their functions.

This treatment modality, initially described in the late 1990s and then championed by Amicus Therapeutics, is in Phase 3 for the treatment of Fabry disease (migalastat/Amigal) (2). The company has also formulated a compound (duvoglustat/AT2220), up to a Phase 2 trial, in combination with Myozyme (recombinant alphaglucosidase) for the treatment of Pompe disease. This is currently in preclinical development, in combination with another form of recombinant – alpha-glucosidase, presented as being more efficiently targeted to the lysosomes.

Iminosugars as Drug Candidates

Iminosugars constitute a class of chemical entities in which the oxygen atom involved in the sugar cycle is replaced by a nitrogen atom. These compounds have been exploited as glycosidase inhibitors and, more recently, for the development of drug candidates (3). Several iminosugarbased drugs are marketed or in clinical evaluation – miglustat/ Zavesca, as a substrate reduction agent for the treatment of Gaucher disease, and migalastat/Amigal, as a chaperone compound for the treatment of Fabry disease – amply establishing that this chemical scaffold is suitable for drug development.

The benefits of iminosugar chaperone molecules as drug candidates include:

  • Iminosugar scaffold has already been explored by several marketed and investigational drugs, with no reported unacceptable toxicology liabilities
  • Passage of the blood-brain barrier for some of these compounds has made them suitable for the treatment of diseases affecting the CNS
  • As small molecules, they do not elicit immune reactions, whereas enzyme replacement therapies frequently do
  • Oral availability for some of these compounds, a feature which simplifies their administration, lowers cost of treatment and improves compliance
  • Cost of manufacturing is lower than for recombinant proteins, in spite of complex syntheses, which counts up to 15 steps 

Enzyme Inhibitors

Iminosugar-based chaperone compounds are designed to mimic the substrate of the target enzyme and, in this sense, they are usually competitive inhibitors. It has been argued that the enzyme, following its transport to the lysosomes, would be prevented from exerting its activity by the compound still occupying its catalytic site. However, this does not seem to be the case in some instances – for certain enzyme and iminosugar-based inhibitor combinations, while a tight binding is observed at a neutral pH, it is later reduced at an acidic pH. Studies have shown that potency of iminosugar-based inhibitors of the betagalactosidase were indeed reduced by 1,000-fold between pH8 and pH4, revealing that the chaperone binds tightly to the mutant enzyme in the neutral environment of the endoplasmic reticulum (4,5). This interaction prompts the newly synthesised protein to adopt a native-like conformation, resulting in the traffic of the enzyme to the lysosomes where the activity of the inhibitors is much reduced.

Alternatively, another option, which avoids the potential drawback associated with the use of inhibitors, consists of the identification of compounds which would convey their chaperone activity through the binding to the target enzyme in another location other than the catalytic site. This has been successfully achieved by several investigators (6). However, it should be kept in mind that, in most cases, the catalytic sites are especially suited to tightly binding small molecules. A strong binding in other locations of the enzyme might be more problematic to achieve. Flat and hydrophilic surfaces, as often encountered at the surface of proteins, are less adequate for interactions with small molecules.

Therapeutic Potential

Animal studies aimed at developing gene therapies in MPS indicated that restoring enzyme levels to 10% of the wild-type level brought about a positive change (7). Similarly, in many in vitro studies, it was observed that exposure of cells from MPS patients to chaperone compounds led to an increase of the residual enzymatic activity, which might be sufficient to bring therapeutic benefit (8).

In many lysosomal storage diseases – and, in particular, in MPS – a large variety of mutations leading to enzymatic deficiency have been identified. This raises the question that the chaperone compounds might be therapeutically active in only a small fraction of patients. Considering the fact that more than two-thirds of the deficiencies are due to point mutations and that many of them result in some residual enzymatic activity, it is anticipated that a sizable fraction of these patients might respond to chaperone compounds. Certainly, in vitro studies conducted on a large number of patient cell lines have indicated that at least 20-40% of them showed an increase in the deficient enzymatic activity (9).

Chaperones compounds could be used in combination with almost any other treatment modalities, such as enzyme replacement therapies, substrate reduction and gene therapy. It has been observed that the coadministration of a recombinant enzyme and a corresponding chaperone compound ameliorated the efficacy of the treatment. Meanwhile, clinical trials combining recombinant enzymes and chaperone compounds were conducted for Fabry and Pompe diseases (10). Whether this strategy has the potential to bring about therapeutic benefit remains to be established.

The seminal article by Fan and colleagues published in 1999 reported about the chaperone activity of iminosugars and their potential use for the treatment of lysosomal storage diseases (2). Fifteen years later, no iminosugar-based chaperone compound is yet to be approved for the treatment of this kind of disease. One reason for this might reside in the complex chemistry of iminosugars. The synthesis of iminosugar variants is a labour-intensive undertaking and, for almost every new molecule, development of a synthetic route is required. This situation might have prevented the optimisation of the iminosugar-based chaperone compounds being selected to enter clinical programmes and contributed to the mixed results obtained thus far.

Based on our own experiences, extensive medicinal programmes conducted on chaperone molecules for two glycosidases – N-acetylalpha- D-glucosaminidase and betagalactosidase, which, when defective, cause MPSIIIB and MPSIVB/GM1- gangliosidosis, respectively – have resulted in compounds with optimised pharmacological characteristics, such as chaperone activity, glycosidase specificity, pharmacokinetics, tissue distribution and, in particular, brain penetration. It is believed that a careful selection of the drug candidates would decrease the risk of failure during clinical evaluation.

Complex Chemistry

Many of the lysosomal storage diseases are caused by deficiencies in glycosidases or enzymes attacking glycosidic substrates. Iminosugar-based chaperone compounds constitute a promising chemical family for the advancement of drugs to treat these severe, and often fatal diseases. It is thought that the successful development of a first efficacious drug of this kind, treating a lysosomal storage disease, will be followed with many more – offering treatment options for these pathologies, many of them still without a cure.


1. Lamanna WC et al, A genetic model of substrate reduction therapy for mucopolysaccharidoses, J Biol Chem 287: pp 36,283-36,290, 2012
2. Fan JQ, Ishii S, Asano N and Suzuki Y, Accelerated transport and maturation of lysosomal alpha-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor, Nat Med 5: pp112-115, 1999
3. Winchester BG, Iminosugars: From botanical curiosities to licensed drugs, Tetrahedron: Asymmetry 20: pp645-651, 2009
4. Valenzano KJ et al, Identification and characterization of pharmacological chaperones to correct enzyme deficiencies in lysosomal storage disorders, Assay Drug Dev Technol 9: pp213-235, 2011
5. Rigat BA et al, Evaluation of N-nonyl-deoxygalactonojirimycin as a pharmacological chaperone for human GM1 gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy, Mol Genet Metab 107: pp203-212, 2012
6. Patnaik S et al, Discovery, structureactivity relationship, and biological evaluation of non-inhibitory small molecule chaperones of glucocerebrosidase, J Med Chem 55: pp5,734-5,748, 2012
7. Langford-Smith A et al, Hematopoietic stem cell and gene therapy corrects primary neuropathology and behavior in mucopolysaccharidosis IIIA mice, Mol Ther 20: pp1,610-1,621, 2012
8. Takai T et al, A bicyclic 1-deoxygalactonojirimycin derivative as a novel pharmacological chaperone for GM1 gangliosidosis, Mol Ther 21: pp526-532, 2013
9. Iwasaki H et al, Fibroblast screening for chaperone therapy in betagalactosidosis, Brain Dev 28: pp482- 486, 2006
10. Benjamin ER et al, Co-administration with the pharmacological chaperone AT1001 increases recombinant human-galactosidase, A tissue uptake and improves substrate reduction in Fabry mice, Mol Ther 20: pp717-726, 2012

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Dr Julie Charollais-Thoenig studied Biology at the University Paris VI, and obtained her PhD in Genetics and Molecular Biology in 2004. She has over 10 years of experience in basic research and in preclinical and clinical drug development, gained in academic institutions and clinical research organisations. In 2012, Julie joined DORPHAN SA as Senior Scientist/Project Manager, where she is in charge of the development of chaperone compounds as drug candidates for the treatment of MPS.

Dr Stéphane Demotz studied Biology at the University of Lausanne, before conducting research in immunology in various organisations. He has more than 15 years of experience in drug development, gained in pharmaceutical companies and start-ups. In 2008, Stéphane co-founded Edimer Pharmaceuticals, which is currently conducting Phase 2 clinical trials of a drug candidate for the rare genetic disease, X-linked hypohidrotic ectodermal dysplasia. In 2012, he co-founded DORPHAN SA.
Julie Charollais-Thoenig
Stéphane Demotz
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