Systemic administration of 2-hydroxypropyl-β-cyclodextrin to symptomatic Npc1-deficient mice slows cholesterol sequestration in the major organs and improves liver function

Abstract

In Niemann-Pick type C (NPC) disease, loss-of-function mutations in either NPC1 or NPC2 result in progressive accumulation of unesterified cholesterol (UC) and glycosphingolipids in all organs, leading to neurodegeneration, pulmonary dysfunction and sometimes liver failure. There is no cure for this disorder. Studies using primarily NPC mouse models have shown that systemic administration of 2-hydroxypropyl-β-cyclodextrin (2HPβCD), starting in early neonatal life, diminishes UC accumulation in most organs, slows disease progression and extends lifespan. The key question now is whether delaying the start of 2HPβCD treatment until early adulthood, when the amount of entrapped UC throughout the body is markedly elevated, has any of the benefits found when treatment begins at 7 days of age. In the present study, Npc1(-/-) and Npc1(+/+) mice were given saline or 2HPβCD subcutaneously at 49, 56, 63 and 70 days of age, with measurements of organ weights, liver function tests and tissue cholesterol levels performed at 77 days. In Npc1(-/-) mice, treatment with 2HPβCD from 49 days reduced whole-liver cholesterol content at 77 days from 33.0 ± 1.0 to 9.1 ± 0.5 mg/organ. Comparable improvements were seen in other organs, such as the spleen, and in the animal as a whole. There was a transient increase in biliary cholesterol concentration in Npc1(-/-) mice after 2HPβCD. Plasma alanine aminotransferase and aspartate aminotransferase activities in 77-day-old 2HPβCD-treated Npc1(-/-) mice were reduced compared with saline-treated controls. The lifespan of Npc1(-/-) mice given 2HPβCD marginally exceeded that of the saline-treated controls (99 ± 1.1 vs 94 ± 1.4 days, respectively; P < 0.05). Thus, 2HPβCD is effective in mobilizing entrapped cholesterol in late-stage NPC disease leading to improved liver function.

Keywords: biliary cholesterol; brain; hepatosplenomegaly; lifespan; lung; lysosomal storage disease; relative organ weight.

Fig. 1

Final body weights of Npc1^+/+ and Npc1^−/− mice studied at baseline (49 days) or after 4 weeks of treatment with 2HPβCD. This experiment was carried out as described in “study design” in Methods. One set of mice was studied at 49 days without any earlier treatment whereas two other sets received subcutaneous injections of either normal saline only (−) or 2HPβCD in saline (+) starting at 49 days of age and then again at 56, 63, and 70 days before study at 77 days. All values are the mean±1 SEM of data from 7 mice in each group. Different letters (a or b) denote statistically significant differences between values (P<0.05) as determined by two-way ANOVA with genotype, treatment, and age as variables.

Fig. 2

Relative weight (A–C) and total cholesterol content (D–F) of multiple organs in 77-day old Npc1^+/+ and Npc1^−/− mice treated weekly with either saline only (−) or 2HPβCD (+) from 49 days of age. Values are the mean±1 SEM of data from 7 animals in each group. Different letters (a–d) denote statistically significant differences between values (P<0.05) as determined by two-way ANOVA with genotype, treatment, and age as variables.

Fig. 3

Cholesterol concentration in the gallbladder bile of Npc1^+/+ and Npc1^−/− mice at varying times following a single treatment with either saline or 2HPBCD. Groups of Npc1^+/+ and Npc1^−/− mice, all in the age range of 49 to 53 days, either remained untreated, or received a single sc injection of either saline (−) or 2HPBCD (+). At 24, 48 and 120 h later, gallbladder bile was obtained from several mice in each group for the measurement of cholesterol concentration. Values represent the mean±1 SEM of measurements in 5 to 10 mice in each group. Different letters (a–c) denote statistically significant differences between values (P<0.05) as determined by two-way ANOVA, with genotype, treatment, and time after treatment as variables.

Fig. 4

Plasma transaminase activities and representative liver histology in Npc1^+/+ and Npc1^−/− mice given either saline or 2HPBCD at weekly intervals starting at 49 days of age. In one experiment, plasma ALT (A) and AST (B) activities were determined in 77-day old Npc1^+/+ and Npc1^−/− mice that had received either saline (−) or 2HPBCD (+) injections at 49, 56, 63 and 70 days of age. Liver histology, by hematoxylin and eosin staining, (C, D and E) was carried out in a smaller number of 91-day old mice that had received treatment at 49, 56, 63 70, 77, and 84 days of age. In all panels photos are at the same magnification (×200). The values for plasma ALT and AST activity are the mean±1 SEM of data from 4 to 7 animals in each group. Different letters (a–c) denote statistically significant differences between values (P<0.05) as determined by two-way ANOVA, with genotype, age and treatment as variables.

Fig. 5

Lifespan of Npc1^−/− mice treated systemically with either saline or 2HPβCD at weekly intervals starting at 49 days of age until death. This study utilized 22 Npc1^−/− mice (10 males, 12 females) and 12 matching Npc1^+/+ controls (6 males and 6 females). In each case, half of the males and females were given 2HPβCD and the remaining mice received only saline. Neurological scores were used to determine when euthanasia of mice was warranted. There were no gender related differences in the neurological scores so the values for the male and female mutants within each treatment group were combined (A). This score, which broadly measures neurological function, rises as the animal’s condition deteriorates. All of the Npc1^−/− mice used for these measurements were symptomatic at 49 days and were assigned a score of 1.0 at that time. For the Npc1^+/+ controls, this score remained at 0 throughout the study and so are not shown (A). Values in A are the mean±1 SEM of data from the specified number of animals, up until some of the Npc1^−/− mice given saline or 2HPβCD reached the end of their lifespan. Nearly all of the Npc1^−/− mice given saline only had died by 98 days whereas the majority of those receiving 2HPβCD died in the week following (B). The statistical analyses for the lifespan data were performed using the Gehan-Breslow-Wilcoxon test and log-rank analyses. The asterisk indicates a statistically significant difference (P<0.05) in age at death.