Mutations in proteins that reduce stability or lead to protein misfolding are common causes of lysosomal storage diseases. Unstable or misfolded mutant enzymes are recognized by the cellular endoplasmic reticulum (ER) quality control system and prematurely degraded before reaching lysosomes. Some iminosugars have a high affinity for the active site of lysosomal enzymes, and can reversibly bind, stabilize and act as specific pharmacological chaperones [1, 5, 16, 17]. Stabilized mutant can pass ER quality control more efficiently and traffic to lysosomes . Once in lysosomes, the non-covalently bound chaperones can dissociate, freeing the enzyme to bind its substrate .
In FD, this approach was first demonstrated in patients’ lymphoblasts using 1-deoxygalactonojirimycin (migalastat HCl) . Migalastat HCl mimics the terminal α-galactose of GL-3 and binds to the active site of α-Gal A with high affinity and specificity . The binding increases the stability of the enzyme, shifts the folding in favor of the proper conformation and allows traffic to lysosomes .
The objectives of the current studies were to explore the safety and pharmacodynamics of migalastat HCl in 9 male FD patients given 150 mg orally every other day.
Results from a 12-week and a 24-week study with similar design were combined. There was a consistent increase in α-Gal A activity in PBMCs, skin and kidney in patients carrying responsive GLA mutations. In PBMCs, increases were rapid (week 4) and sustained over the duration of treatment. One patient, with a low baseline α-Gal A activity, showed a progressive increase with a maximum only reached at week 24. Some patients reached an enzyme activity in PBMCs of 30% to 50% of normal. Increase in α-Gal A activity were associated with substrate reduction, as demonstrated by a decrease in urinary GL-3 and GL-3 inclusions in renal PTCs.
These results indicate that the activity of mutated α-Gal A can be increased in vivo following administration of migalastat HCl. The binding of the drug to α-Gal A is reversible and is of lower affinity in the acidic environment of the lysosome. Furthermore, migalastat HCl is rapidly cleared from plasma (half-life 3–4 hours), whereas the lysosomal half-life of the enzyme is significantly longer (around 110–120 hours) . This allows the enzyme to bind and turn over the GL-3 substrate without inhibition by the small molecule. The every-other-day regimen allows additional time for the chaperone to dissociate from the enzyme.
The in vitro HEK-293 cell-based assay  appears to predict the clinical pharmacodynamic response. Of note, both patients with the same p.P259R mutation showed similar pharmacodynamic response. As these studies included only 9 patients carrying 8 different missense mutations, results should be interpreted with caution. The predictive value of the assay will have to be confirmed in larger numbers of FD patients with additional mutations. This assay is currently used to select patients for phase 3 clinical studies.
FD is a rare, lifelong devastating disease, punctuated by acute complications that take many clinical forms . FD phenotypic expression varies from one patient to the next, even within families harboring the same genotype. It is thus a challenge to select clinical outcome measures that can consistently demonstrate therapeutic efficacy . Because GL-3 is the primary lysosomal substrate of α-Gal A, the deficient enzyme in FD, demonstrating a decrease in GL-3 could reflect treatment efficacy.
While GL-3 deposition in renal PTCs has been used as the primary outcome measure of efficacy for the approval of agalsidase beta , this choice may not be appropriate. Despite an overt clinical expression of the disease, our patients had limited amounts of GL-3 in capillary cells of the kidney and skin, and normal levels of GL-3 in plasma. Kidney histology revealed that GL-3 deposition was extensive in podocytes and collecting duct cells, but minimal in PTCs. A previously used histological method for quantifying GL-3 in PTCs  was not sensitive enough to evaluate the low levels of inclusions observed. Thus, a more sensitive method to quantify GL-3 PTC inclusions was developed . Interestingly, the shedding into the urinary tract of tubular cells, and potentially podocytes, accounts for most of the urinary GL-3 in FD , especially when plasma GL-3 levels are low and glomerular filtration of GL-3 negligible. A decrease in u-GL-3 was observed in our subjects and has been advocated as a marker of efficacy in FD, however the clinical relevance remains controversial [13, 20]. Some of the controversies stem from a lack of a reliable method for collection of samples and analysis [13, 21]. To address these concerns, a new GLP assay for urine GL-3 was developed and validated, and will be used in ongoing migalastat HCl phase 3 studies.
In contrast to ERT, migalastat HCl is a small molecule that is excreted unchanged in the urine and can potentially reach podocytes. These cells are central to the renal pathophysiology of FD . Migalastat HCl was generally well tolerated. However, these studies only enrolled 9 subjects and this should be interpreted with evident caution. Long-term extension data from phase 2 studies also indicate that the drug is well tolerated and, as of this writing, no severe adverse reactions related to treatment have been identified after four years of treatment.
A key issue for physicians treating FD patients is the selection of candidates for pharmacological chaperone therapy. Severe GLA defects that result in no residual α-Gal A activity are probably not addressable with chaperone therapy, and only patients who express missense mutants with low levels of activity were included. Different methods were explored to predict which patients would respond to migalastat HCl [8, 11]. We initially recruited patients with missense mutations who had at least 3% of normal activity that increased by at least 20% when lymphocytes were incubated with migalastat HCl. However, the ex vivo lymphocyte assay is complex and cannot be readily performed under GLP conditions. Moreover, the lymphocyte assay did not always correlate with the in vivo PBMC assay. Ultimately, lymphocyte and PBMC assays are not useful in heterozygous FD females. In an X-linked disease, females are mosaics and isolated cells are a mix of cells with normal α-Gal A and ones with mutated α-Gal A. In females, an increase in activity with migalastat HCl might reflect the chaperoning of the wild-type enzyme. It has been increasingly recognized that females with FD can have significant clinical manifestations [23, 24]. For these reasons, an in vitro assay was developed that could be used irrespective of sex .
In summary, migalastat HCl is a candidate pharmacological chaperone that provides a genotype-specific treatment for FD. When administered at an oral dose of 150 mg on an every-other-day regimen, it was well tolerated, increased α-Gal A activity in patients with responsive GLA mutations, and resulted in GL-3 substrate reduction. Phase 3 studies of migalastat HCl for FD are ongoing.
This study describes the first use in patients of an oral small molecule pharmacological chaperone, rather than using enzyme replacement therapy, to treat a lysosomal storage disorder. It shows for the first time in medicine that such a drug increases the activity, or effectively rescues the mutated and dysfunctional enzyme that patients with Fabry disease have expressed their entire lives. In addition to being a novel approach for the treatment of Fabry disease, stabilization of target proteins using small cell-permeable pharmacological chaperones, may represent a generally applicable rescue strategy for other diseases that result from improper protein folding and inefficient cellular targeting [25–27].