Summary /

To demonstrate chaperone activity, the capacity of ML156 and ML155 to increase the translocation of GC to the lysosome was measured (8, 16, 30, 31). In this experiment, wildtype and mutant fibroblasts were incubated for five days with compound 8i, followed by cell fixation and staining with a selective fluorescent GC antibody. Compounds able to promote trafficking from the ER to the lysosome increased the fluorescent lysosomal signal. DMSO and isofagomine were used as negative and positive controls. Figure 2 shows the increment of signal in both cell lines that resulted from treatment, confirming the chaperone capacity of these compounds.

Figure 2: Chaperone activity of compound 13, NCGC00182292 (CID 40225210), and others using wildtype and homozygous, mutant N370S GC fibroblasts. Two genotypes of fibroblasts, fibroblasts homozygous for wildtype GC (top) and fibroblasts homozygous for N370S GC (bottom) were stained both with a Cy3-labeled antibody for GC protein content (first row) and a FITC-labeled antibody specific for lysosomal compartments (LAMP1; second row) after treatment with (1) DMSO vehicle, (2) 10μM Isofagomine (CID 447607), (3) 10μM compound 13, NCGC00182292 (CID 40225210), and (4) 1μM NCGC00159568 (CID 9893924). Several compounds, including isofagomine (CID 447607) and compound 13, NCGC00182292 (CID 40225210), show increased lysosomal GC protein after treatment.

Summary /

The result of a microsomal stability study of ML156 (CID9893924) is shown in Table 1. After incubation of the molecule in mouse liver microsomes for 60 minutes in the present of NADPH, 58% of the molecule remained, this suggested that ML156 has a reasonable microsomal stability. In addition,  the pharmacokinetic profiles of ML156 in male Swiss Albino mice after intraperitoneal administration twice at a 12 hour interval of the probe at 20mg/kg dose are shown in Table 2.  This probe has demonstrated chaperone activity, and preliminary pharmacokinetic studies have demonstrated that the molecule has good exposure in plasma, brain and liver upon IP administration. Exposure in brain is especially significant, as the current standard-of-care, enzyme replacement therapy, has no effect on the neurological component of the disease. In addition, at this high dose, the compound did not exhibit any acute toxicity in initial testing.

Table 1. Mouse microsomal stability assay at 60 minutes.

Table 2. Concentrations of ML156 / CID9893924 in plasma, brain, liver and tail; intraperitoneal administration twice at a 12 hour interval of  ML156 / CID9893924 at 20mg/kg dose in male Swiss Albino Mice (mean and SD).