Improved management of lysosomal glucosylceramide levels in a mouse model of type 1 Gaucher disease using enzyme and substrate reduction therapy

Abstract

Gaucher disease is caused by a deficiency of the lysosomal enzyme glucocerebrosidase (acid beta-glucosidase), with consequent cellular accumulation of glucosylceramide (GL-1). The disease is managed by intravenous administrations of recombinant glucocerebrosidase (imiglucerase), although symptomatic patients with mild to moderate type 1 Gaucher disease for whom enzyme replacement therapy (ERT) is not an option may also be treated by substrate reduction therapy (SRT) with miglustat. To determine whether the sequential use of both ERT and SRT may provide additional benefits, we compared the relative pharmacodynamic efficacies of separate and sequential therapies in a murine model of Gaucher disease (D409V/null). As expected, ERT with recombinant glucocerebrosidase was effective in reducing the burden of GL-1 storage in the liver, spleen, and lung of 3-month-old Gaucher mice. SRT using a novel inhibitor of glucosylceramide synthase (Genz-112638) was also effective, albeit to a lesser degree than ERT. Animals administered recombinant glucocerebrosidase and then Genz-112638 showed the lowest levels of GL-1 in all the visceral organs and a reduced number of Gaucher cells in the liver. This was likely because the additional deployment of SRT following enzyme therapy slowed the rate of reaccumulation of GL-1 in the affected organs. Hence, in patients whose disease has been stabilized by intravenously administered recombinant glucocerebrosidase, orally administered SRT with Genz-112638 could potentially be used as a convenient maintenance therapy. In patients naïve to treatment, ERT followed by SRT could potentially accelerate clearance of the offending substrate.

Fig. 1

Effect of administering exogenous glucocerebrosidase on glucosylceramide (GL-1) levels in liver, spleen and lung. Three-month-old Gaucher mice were administered 10 mg/kg recombinant glucocerebrosidase intravenously two, four, or eight times. The two- and four-dose cohorts received the enzyme 3 days apart, and the group treated with eight doses was injected 2 days apart. GL-1 levels in the liver (squares), spleen (triangles), and lung (circle) were measured 7 days after the last injection. Data are expressed as means ± standard error of the mean (SEM) (n = 5).

Fig. 2

Efficacy of enzyme and substrate reduction therapies at lowering glucosylceramide (GL-1) levels in the liver of Gaucher mice. Liver GL-1 levels were measured in untreated 3-month-old Gaucher mice (A) and following 2 weeks of treatment with recombinant glucocerebrosidase (B). Mice treated with recombinant glucocerebrosidase were analyzed 10 weeks later without further treatment (C) or after therapy with glucosylceramide synthase inhibitor (Genz-112638) (D). GL-1 levels in the liver of mice administered Genz-112638 alone for the entire study period (E) and in untreated, age-matched controls (F) are also shown. Data are expressed as means ± standard error of the mean (SEM) (n = 5). Statistical significance was determined using the unpaired t test.

Fig. 3

Efficacy of enzyme and substrate reduction therapies at lowering glucosylceramide (GL-1) levels in the spleen of Gaucher mice. Spleen GL-1 levels were measured in untreated 3-month-old Gaucher mice (A) and following 2 weeks of treatment with recombinant glucocerebrosidase (B). Mice treated with recombinant glucocerebrosidase were analyzed 10 weeks later without further treatment (C) or after therapy with glucosylceramide synthase inhibitor (Genz-112638) (D). GL-1 levels in the spleen of mice administered Genz-112638 alone for the entire period of study (E) and in untreated, age-matched controls (F) are also shown. Data are expressed as means ± standard error of the mean (SEM) (n = 5). Statistical significance was determined using the unpaired t test.

Fig. 4

Efficacy of enzyme and substrate reduction therapies at lowering glucosylceramide levels in the lung of Gaucher mice. Lung glucosylceramide (GL-1) levels were measured in untreated 3-month-old Gaucher mice (A) and following 2 weeks of treatment with recombinant glucocerebrosidase (B). Mice treated with recombinant glucocerebrosidase were analyzed 10 weeks later without further treatment (C) or after therapy with glucosylceramide synthase inhibitor (Genz-112638) (D). GL-1 levels in the lung of mice administered Genz-112638 alone for the entire study period (E) and in untreated, age-matched controls (F) are also shown. Data are expressed as means ± standard error of the mean (SEM) (n = 5). Statistical significance was determined using the unpaired t test.

Fig. 5

Immunohistochemical staining of liver of Gaucher mice following treatment with enzyme (ERT) and substrate reduction therapy (SRT). Liver sections were stained with an anti-CD68 antibody to visualize macrophages. Sections shown were from untreated 3-month-old Gaucher mice (A) , 3-month-old Gaucher mice dosed with glucocerebrosidase and analyzed after 2 weeks of treatment (B), or 10 weeks later (C) . Liver of Gaucher mice administered enzyme followed by glucosylceramide synthase (Genz-112638) (D) , and from those that received Genz-112638 alone (E). Liver of untreated Gaucher mice at the end of the study (12 weeks later) (F).

Fig. 6

The extent of CD68-positive staining on liver sections was quantified using MetaMorph software. Levels in untreated 3-month-old Gaucher liver (A) or following treatment with glucocerebrosidase (B). Mice treated with enzyme and then analyzed 10 weeks later without further therapeutic intervention (C) or after therapy with glucosylceramide synthase inhibitor (Genz-112638) (D). Extent of staining in the liver of Gaucher mice administered Genz-112638 alone (E) and in untreated, age-matched control mice (F). The data was collated from an analysis of ten 400× images per section from each mouse. Statistical significance was determined using the unpaired t test.