Intracisternal cyclodextrin prevents cerebellar dysfunction and Purkinje cell death in feline Niemann-Pick type C1 disease

Abstract

Niemann-Pick type C1 (NPC) disease is a lysosomal storage disease caused by mutations in the NPC1 gene, leading to an increase in unesterified cholesterol and several sphingolipids, and resulting in hepatic disease and progressive neurological disease. We show that subcutaneous administration of the pharmaceutical excipient 2-hydroxypropyl-β-cyclodextrin (HPβCD) to cats with NPC disease ameliorated hepatic disease, but doses sufficient to reduce neurological disease resulted in pulmonary toxicity. However, direct administration of HPβCD into the cisterna magna of presymptomatic cats with NPC disease prevented the onset of cerebellar dysfunction for greater than a year and resulted in a reduction in Purkinje cell loss and near-normal concentrations of cholesterol and sphingolipids. Moreover, administration of intracisternal HPβCD to NPC cats with ongoing cerebellar dysfunction slowed disease progression, increased survival time, and decreased the accumulation of brain gangliosides. An increase in hearing threshold was identified as a potential adverse effect. These studies in a feline animal model have provided critical data on efficacy and safety of drug administration directly into the central nervous system that will be important for advancing HPβCD into clinical trials.

Conflict of interest statement

Competing Interests:

Vite and Ory have received honoraria from Actelion and Biomarin.

Vanier has received travel expenses, consulting fees and presentation honoraria from Actelion Pharmaceuticals, Ltd.

Patents: Ory – Disease specific biomarkers for Niemann-Pick C disease (US Patent 8,497,122

Walkley – Methods for therapeutic use of glucosylceramide synthesis inhibitors and composition thereof (US Patent 6,683,076)

Copyright © 2015, American Association for the Advancement of Science.

Figures

Fig 1. Age of onset and severity of ataxia in untreated NPC cats and NPC cats administered IC HPβCD

(A) A dose-related effect on the age of onset of ataxia was noted in NPC cats receiving IC HPβCD compared to untreated NPC cats (†p

Fig 2. Liver histology and biochemistry in NPC cats administered SC or IC HPβCD

(A) Untreated NPC cats showed severe vacuolization of hepatocyte and Kupffer cell cytoplasm that was ameliorated by 1000 mg/kg HPβCD but was unaffected by 120 mg IC HPβCD. All cats were 24 weeks of age; scale bar = 100 microns. (B) HPTLC of total lipids in 2 mg tissue samples. Migration in chloroform-methanol-H2O 65:25:4, anisaldehyde spray. The prominent storage of cholesterol and sphingomyelin was greatly reduced in all SC-treated cats but remained unchanged with IC administration. (C) HPTLC showing a similar effect of treatment on neutral glycosphingolipids from 5 mg tissue samples after saponification of total lipids. Migration as in B; stained with orcinol-H2SO4 spray. (D) Free sphingosine concentrations (measured by HPLC) that were increased ~50-fold in untreated NPC cats, decreased ~5 times after SC-HPβCD treatment. For the SC graph, both cats received 8000 mg/kg HPβCD. For the IC graph, data from cats receiving 15 mg, 30 mg, and 60 mg were included as there was no dose effect. Normal values: 37±15 pmol/mg protein (mean ± SD, n=7). Abbreviations: Untr’d: untreated; Norm: normal; SC8, SC4, SC1: subcutaneous, 8000, 4000 or 1000 mg/kg; IC120, IC60: intracisternal, 120 or 60 mg; Chol: unesterified cholesterol; BMP: bis(monoacylglycero)phosphate; Sph: sphingomyelin; GlcCer: glucosylceramide; Lac Cer: lactosylceramide; Gb3: globotriaosylceramide; HPTLC, high performance thin-layer chromatography; HPLC, high pressure liquid chromatography.

Fig 3. Calbindin and GM2 immunohistochemical staining and filipin staining of cerebellum from normal control, untreated NPC, and HPβCD-treated NPC cats

Top row depicts immunohistochemical calbindin labeling of Purkinje cells (PCs), whose cell body and dendritic arbor are highlighted in brown. Middle row shows filipin labeling of unesterified cholesterol, accumulation of which is noted by white cytoplasmic staining. Bottom row demonstrates GM2 accumulation (dark brown punctae). Normal control column depicts a full complement of PCs lacking cholesterol and GM2 storage, typical of a normal cat at 25 wks of age. Untreated NPC column shows a large axonal spheroid (red arrow) in one of the remaining PCs within this 25-wk-old NPC cat as well as abundant storage of cholesterol and GM2 gangliosides present in all remaining PCs. Treatment of an NPC1 cat with 120 mg IC HPβCD (third column) resulted in substantial PC rescue as well as decreased cholesterol and GM2 ganglioside accumulation within PCs of this 29-wk-old cat. Abbreviations: MC, molecular cell layer; PC: Purkinje cell layer; GC: granular cell layer; scale bar = 50 microns.

Fig 4. Cerebral gray matter lipids in normal control, untreated NPC, and NPC cats administered IC HPβCD

(A) Upper panel: HPTLC of total gangliosides (from 3 mg tissue samples), showing striking and selective reduction of GM3 and GM2 in NPC cats administered IC HPβCD; the gangliosides patterns remained essentially unchanged in NPC cats administered SC HPβCD (migration in chloroform-methanol-0.2% CaCl2 55:45:10, resorcinol-HCl spray). Lower panel: Quantitative data (24–26 week-old cats; GM3 and GM2 expressed as % of total gangliosides). IC treatment at 7.5 and 3.5 mg doses appeared less efficient at reducing GM2 and GM3 than 15 mg or higher doses. (B) Lactosylceramide concentrations (HPTLC from 10 mg tissue samples) were reduced in cats treated with IC HPβCD. (C) Free sphingosine concentrations (measured by HPLC, expressed as pmol/mg protein) were normalized in all IC treated cats. (D) HPTLC of total gangliosides (from 3 mg tissue samples) of untreated NPC cats at various ages (left), and developmental profiles of GM2 and GM3 storage (right). Normal proportions (% of total gangliosides) are 2.0 ± 0.5 % for GM2, and 2.5 ± 0.6% for GM3 (Mean ± SD, n=5). Abbreviations: Untr’d: untreated; Norm: normal; SC8, SC4, SC1: subcutaneous, 8000, 4000 or 1000 mg/kg; IC120, IC60: intracisternal, 120 or 60 mg; HPTLC, high performance thin-layer chromatography; HPLC, high pressure liquid chromatography.

Fig 5. Pulmonary histology from 6-month-old untreated NPC cats and NPC cats administered 8000 mg/kg SC HPβCD

(A) In untreated NPC cats, the alveolar septa were expanded by foam cells (black arrowheads) as well as by macrophages containing larger irregular clear vacuoles. Alveolar spaces similarly contained foam cells (arrows). (B) Cats given 8000 mg/kg SC HPβCD had evidence of acute to subacute diffuse alveolar damage. Alveolar spaces contained abundant proteinaceous fluid with wispy strands of fibrin, foamy macrophages and neutrophils. The alveolar septa were lined by hypertrophied type II pneumocytes with multifocal hyaline membrane formation. The septa were congested and contained foamy macrophages (black arrows), neutrophils and lymphocytes. Arrowheads denote thickened alveolar septae. No evidence of alveolar damage was seen in the other treatment groups. (C) Lung from a normal control cat; scale bar = 100μm.

Fig 6. Filipin-staining of marginal gyrus of cerebral cortex

Untreated 24-week-old NPC cats (B) showed storage of cholesterol (visualized as white cytoplasmic labeling within cells) in the marginal gyrus of the cerebral cortex; no cholesterol storage was observed in healthy control animals (A). Cats receiving 1000 mg/kg SC HPβCD showed no reduction in cholesterol staining (C). Cats receiving 8000 mg/kg SC HPβCD (D) showed substantially less cholesterol staining than did untreated NPC cats. Indeed, the example shown is from a 37-week-old cat that had received SC HPβCD until 24 weeks of age at which time it was discontinued. 24-week-old cats receiving 30 mg or greater IC HPβCD presymptomatically showed the greatest amelioration of the cholesterol storage defect (E, H). In contrast, treatment of NPC cats with 3.8 mg IC HPβCD (G) or 120 mg IC HPβCD administered post-symptomatically (F) revealed little effect on stored cholesterol in cats at 24 weeks of age, while both 30 mg and 120 mg HPβCD administered presymptomatically showed substantial reductions in cholesterol storage. Scale bar = 100 microns.

Fig 7. Calbindin immunofluorescence of cerebellar paravermis in 6-month-old cats

Untreated NPC cats (B) showed a severe loss of Purkinje cells compared to normal control cats (A). Subcutaneous (SC) HPβCD administration at a dose of 8000 mg/kg resulted in increased Purkinje cell survival (D), whereas cats receiving 1000 mg/kg SC HPβCD were indistinguishable from untreated NPC cats (C). Cats treated with IC HPβCD at 30 mg or greater (H) resulted in substantial Purkinje cell survival. In contrast, 3.8 mg IC HPβCD (G) or 120 mg IC HPβCD given either presymptomatically (E) or postsymptomatically (120 mg IC late) (F) resulted in small but non-significant increases in Purkinje cell number compared to untreated NPC cats. Scale bar = 200 microns.

Fig 8. Purkinje cell quantification and hearing threshold in NPC cats administered IC HPβCD

(A) Untreated 24-week-old NPC cats showed significantly fewer Purkinje cells per unit length compared to age-matched normal control cats (*p