Systemic delivery of a glucosylceramide synthase inhibitor reduces CNS substrates and increases lifespan in a mouse model of type 2 Gaucher disease

Abstract

Neuropathic Gaucher disease (nGD), also known as type 2 or type 3 Gaucher disease, is caused by a deficiency of the enzyme glucocerebrosidase (GC). This deficiency impairs the degradation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph), leading to their accumulation in the brains of patients and mouse models of the disease. These accumulated substrates have been thought to cause the severe neuropathology and early death observed in patients with nGD and mouse models. Substrate accumulation is evident at birth in both nGD mouse models and humans affected with the most severe type of the disease. Current treatment of non-nGD relies on the intravenous delivery of recombinant human glucocerebrosidase to replace the missing enzyme or the administration of glucosylceramide synthase inhibitors to attenuate GluCer production. However, the currently approved drugs that use these mechanisms do not cross the blood brain barrier, and thus are not expected to provide a benefit for the neurological complications in nGD patients. Here we report the successful reduction of substrate accumulation and CNS pathology together with a significant increase in lifespan after systemic administration of a novel glucosylceramide synthase inhibitor to a mouse model of nGD. To our knowledge this is the first compound shown to cross the blood brain barrier and reduce substrates in this animal model while significantly enhancing its lifespan. These results reinforce the concept that systemically administered glucosylceramide synthase inhibitors could hold enhanced therapeutic promise for patients afflicted with neuropathic lysosomal storage diseases.

Figure 1. GluCer and GluSph are significantly elevated in the brains of neonatal K14 mice.

Mass spectrometry analysis of glucosyl- and galactosylceramides shows that (A) GluCer was elevated 10-fold in K14 mice compared to WT mice through the first 2 weeks of life, (B) GalCer levels were similar over time for both K14 and WT mice, (C) GluSph levels were ≥10-fold higher in K14 mice than age-matched WT mice over the first 2 weeks of life; GluSph levels in WT animals were below the level of detection (<0.3 ng/mg). (D) There were no significant differences in brain weights between K14 and WT mice over the first 2 weeks of life. Data points represent mean values and error bars SEM for N = 4.

Figure 2. Systemic administration of GZ 161 reduces GluCer and GluSph levels in the K14 mouse brain.

K14 and WT mice were treated daily (IP) beginning at P4 with vehicle or 5 mg/kg GZ 161, and brains analyzed for GluCer and GluSph at P10. GZ 161-treated animals were asymptomatic at this time. Treatment with GZ 161 reduced K14 (A) GluCer levels by ∼70% and (B) GluSph levels by ∼60%. Post-treatment levels of both glycosphingolipids remained significantly elevated compared to their WT littermates. and genotypes were confirmed by post-mortem DNA analysis. *p<0.05. N = 4/group.

Figure 3. Systemic administration of GZ 161 reduces CD68 staining throughout the brain of K14 mice.

(Upper panels) Representative immunohistochemical CD 68 staining at P10 in the hippocampus, thalamus, brainstem and cerebellum of K14 mice treated daily (IP) beginning at P4 with vehicle or GZ 161 and WT mice treated with vehicle. (Lower panels) Quantitation of staining in the groups shown above, showing that systemic treatment with GZ 161 results in significant reductions the CD68+ cells in all brain regions. Similar reductions were observed in other structures such as the olfactory bulb and frontal cortex (data not shown). **p<0.01. N = 4/group.

Figure 4. Systemic administration of GZ 161 reduces F4/80 staining in some brain regions of K14 mice.

(Upper panels) Representative immunohistochemical F4/80 staining at P10 in the hippocampus, thalamus, brainstem and cerebellum of K14 mice treated daily (IP) beginning at P4 with vehicle or GZ 161, and WT mice treated with vehicle. (Lower panels) Quantitation of staining in the groups shown above, showing that systemic treatment with GZ 161 results in significant reductions the F4/80+ cells in the thalamus and brainstem. Similar reductions were observed in other structures such as the olfactory bulb and frontal cortex; statistical differences were observed in both structures (data not shown). *p<0.05. N = 4/group.

Figure 5. Systemic administration of GZ 161 decreases gliosis in K14 mice.

(Upper panels) Representative immunohistochemical GFAP staining at P10 in the hippocampus, thalamus, brainstem and cerebellum of K14 mice treated daily (IP) beginning at P4 with vehicle or GZ 161, and WT mice treated with vehicle. (Lower panels) Quantitation of staining in the groups shown above, showing that systemic treatment with GZ 161 results in significant reductions the GFAP+ cells in the hippocampus and cerebellum; statistical differences were observed in both structures (data not shown).

Figure 6. Systemic administration of GZ 161 increases the median lifespan of K14 mice.

K14 mice were injected (IP) daily beginning at P4 with vehicle or GZ 161 or given a combined treatment of three intracerebroventricular (ICV) injections of rhGC at P1,2,3 together with daily (IP) injections of GZ 161 beginning at P4. Vehicle treated mice had a 15 day median lifespan (N = 25); GZ 161 treated mice had an 18 day median lifespan (N = 12; p<0.0001 compared to vehicle-treated); mice administered GZ 161 and rhGC had a 26 day median lifespan (N = 13).