Summary /

To measure the capacity of our compounds to activate ER-lysosome translocation of GAA, we treated Pompe patient-derived fibroblasts with compound for five days, followed by immunostaining (GAA and Cathepsin D as lysosomal marker), wash, and image analysis (Parenti 2007, Okumiya 2007). Pompe fibroblasts are difficult to obtain, and in general, they grow slowly; they can only be expanded by a few passages. Two cell cultures derived from a single patient with the mutations p.Y455C/p.G638W were obtained. The residual specific activity of these patient-derived fibroblasts is around 3% of the activity displayed by fibroblast with wildtype GAA. Although the specific activity of GAA in fibroblasts (skin biopsies) is definitive for a Pompe disease diagnosis, fibroblasts do not express GAA in abundant amounts, and that expression decreases with increasing passage number, even for wildtype cells. Nevertheless, the present patient-derived cells had sufficient protein expression to enable us to characterize the chaperone activity of this series, which is done by examining the co-localization of GAA staining with lysosome marker staining (CathD). No lysosomal GAA staining can be seen in DMOS vehicle treated Pompe cells. As a reference, in our hands, the GAA signal in wildtype fibroblast by passage 8 could be observed by staining in 10% of the cells. The inhibitory chaperone duvoglustat is a non-selective iminosugar currently in clinical trials for Pompe disease. The chaperone activity of duvoglustat varies by mutation, and there is no previous literature indicating whether duvoglustat is active against p.Y455C/p.G638W GAA mutations. No chaperone activity was observed at compound concentrations of 1, 5, and 15 μM. At 20 μM, translocation of GAA to the lysosome occurred in 3% of the cells. These values are in the same range as those reported for other GAA mutants, where duvoglustat increased GAA translocation at 20 μM. Duvoglustat does not increase the translocation of wildtype GAA. In contrast, probe molecule ML 247 and some of its analogs increases translocation of GAA to lysosomes in patient-derived fibroblasts. ML247 gave the most robust responses, with around 10% translocation of mutant GAA to the lysosomes at 20 μM. Translocation could still be observed at 15 and 10 μM, but not at 5 μM (Figure 1). In addition, profound restoration of lysosomal GAAA in patient-derived fibroblast is observed after 5 day incubation with 20 uM ML247 (Figure 2). Comparison between the results obtained with duvoglustat vs. ML247 show that at the same concentration, the probe molecule is superior than the prior art molecule duvoglustat.

Figure 1. Probe compound ML247 restores GAA translocation to the lysosome (blue arrows). False-colored images of fibroblast cells show DAPI nuclear stain (blue), the lysosomal marker Cathepsin D (red) and GAA (green) in cells from (A) an unaffected donor with wildtype GAA, and (B-E) Pompe patient-derived cells with p.Y455C/p.G638W mutations. Cells were cultured for five days with compound that was dispensed using a DMSO vehicle. Yellow color (overlapping green and red) indicates co-localized lysosomal GAA protein, typically present in (A) DMSO vehicle treated wildtype cells, but absent in (B) DMSO vehicle treated patient-derived cells, and partially restored with (C) 20 μM duvoglustat, (D) 15μM compound 1, and more strongly with (E) 15 μM ML247.

Figure 2. Two representative fields showing profound restoration of lysosomal GAA in patient-derived fibroblasts (blue arrows), upon five day incubation with 20 μM probe compound ML247. Coloring scheme is the same as Figure 1.

Summary /

ML247, has excellent Caco-2 permeability (> 5cm-8/s) and robust stability, with >60% remaining after a 60 minute incubation with mouse liver microsomes. The addition of NADPH also had little effect on compound stability, indicating only very modest P450 mediated metabolism (Table 1). ML247 was further studied in a single-dose pharmacokinetics experiment. At 50 mg/kg IP dosing, no acute toxicity was observed (Figure 3). Compound half-life was 2.75 hours in plasma, with concentrations in plasma, liver and heart all exceeding 10 μM at one hour. Pharmacokinetic properties and therapeutic window considerations place ML247 as an excellent candidate for further advancement studies toward  in vivo efficacy model and eventually in the treatment of Pompe disease.

Table 1. In vitro ADME profile of ML247.

Figure 3. In vivo exposure by ML247 following a single 50 mg/kg IP dose in mice.