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. 2016 Apr 20;3(5):366-80.
doi: 10.1002/acn3.306. eCollection 2016 May.

Efficacy and ototoxicity of different cyclodextrins in Niemann-Pick C disease

Cristin D Davidson  1 , Yonatan I Fishman  1 , István Puskás  2 , Julianna Szemán  2 , Tamás Sohajda  2 , Leslie A McCauliff  3 , Jakub Sikora  4 , Judith Storch  3 , Marie T Vanier  5 , Lajos Szente  2 , Steven U Walkley  1 , Kostantin Dobrenis  1
Affiliations

Efficacy and ototoxicity of different cyclodextrins in Niemann-Pick C disease

Cristin D Davidson et al. Ann Clin Transl Neurol. .

Abstract

Objective: Niemann-Pick type C (NPC) disease is a fatal, neurodegenerative, lysosomal storage disorder characterized by intracellular accumulation of unesterified cholesterol (UC) and other lipids. While its mechanism of action remains unresolved, administration of 2-hydroxypropyl-β-cyclodextrin (HPβCD) has provided the greatest disease amelioration in animal models but is ototoxic. We evaluated other cyclodextrins (CDs) for treatment outcome and chemical interaction with disease-relevant substrates that could pertain to mechanism.

Methods: NPC disease mice treated for 2 weeks with nine different CDs were evaluated for UC, and GM2 and GM3 ganglioside accumulation using immunohisto/cytochemical and biochemical assays. Auditory brainstem responses were determined in wild-type mice administered CDs. CD complexation with UC, gangliosides, and other lipids was quantified.

Results: Four HPβCDs varying in degrees of substitution, including one currently in clinical trial, showed equivalent storage reduction, while other CDs showed significant differences in relative ototoxicity and efficacy, with reductions similar for the brain and liver. Importantly, HPγCD and two sulfobutylether-CDs showed efficacy with reduced ototoxicity. Complexation studies showed: incomplete correlation between CD efficacy and UC solubilization; an inverse correlation for ganglioside complexation; substantial interaction with several relevant lipids; and association between undesirable increases of UC storage in Kupffer cells and UC solubilization.

Interpretation: CDs other than HPβCD identified here may provide disease amelioration without ototoxicity and merit long-term treatment studies. While direct interactions of CD-UC are thought central to the mechanism of correction, the data show that this does not strictly correlate with complexation ability and suggest interactions with other NPC disease-relevant substrates should be considered.

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Figures

Figure 1
Figure 1
UC, and GM2 and GM3 ganglioside accumulation in the brain cells of 3‐week‐old mice treated with different CDs. Top row: Sample fluorescence photomicrographs of dorsal neocortex from untreated Wt mouse (A), and CD‐treated (B–G) and untreated (H) Npc1 −/− mice, stained with filipin to detect UC. Virtually, all neurons in untreated Npc1 −/− mice show positive cytoplasmic staining of UC (white spots) (H), whereas those in Wt mice are negative (A). Note that HPβCD (Sigma) (B), HPγCD (C), and SBEγCD (D) all show highly effective reduction in UC storage, while some UC remains with SBEβCD treatment (E). HPαCD (F) and SBEαCD (G) show UC storage grossly equivalent to untreated mice (H). Middle row: Sample brightfield photomicrographs of dorsal neocortex stained by immunoperoxidase to detect GM2 ganglioside. Dark brown puncta of GM2 immunoreactivity are evident throughout dorsal neocortical neurons in untreated Npc1 −/− (H) in contrast to Wt (A) mice. The most effective reduction of GM2 in Npc1 −/− mice is seen with HPβCD (B) and HPγCD (C). Noticeably more remaining GM2 is evident in Npc1 −/− mouse treated with SBEγCD (D), and substantially more with SBEβCD and αCD treatments (E–G) which appear equivalent to untreated Npc1 −/− mouse. Bottom row: Sample bright‐field photomicrographs of immunoperoxidase stained dorsal neocortex to detect GM3. Dark brown puncta of GM3 immunoreactivity are evident in neurons of untreated Npc1 −/− mouse (H), though less abundant than GM2, and absent in Wt mouse cortex (A). The relative efficacy of different CDs to reduce GM3 ganglioside parallels UC reduction: HPβCD (B), HPγCD (C), and SBEγCD (D) are nearly indistinguishable from Wt (A); SBEβCD (E) shows an intermediate impact; and αCDs (F–G) show no appreciable reduction. Wt panels for GM2 and GM3 staining are split: Nissl counterstain in left half reveals cortical layers, marked by roman numerals. Scale bars = 50 μm.
Figure 2
Figure 2
Results of scoring of neuronal UC and ganglioside accumulation in stained samples of dorsomediolateral neocortex from 3‐week‐old CD‐treated Npc1 −/− mice. Tissue sections were scored blind by three independent observers on a scale of 0–10 (10 = greatest accumulation, 0 = no accumulation for that stain). Each point represents one observer's score of sections from one mouse, and horizontal lines show mean value. All stains were found to have evidence of significant differences between conditions (nonparametric ANOVA, P < 0.0001). Color‐coded asterisks at top of each graph indicate level of significance found in post hoc pairwise statistical comparisons. (A) UC results based on filipin staining. All HPβCDs as well as HPγCD and SBEγCD treatment groups were significantly different from both vehicle and αCDs, and not significantly different (P ≥ 0.05) from one another. (Note that filipin data for Trappsol treatment was limited to two biological replicates.) SBEβCD produced a range of intermediate values that yielded no significant difference from any group, while αCDs showed no difference from vehicle. (B) GM2 ganglioside scores from immunohistochemical staining. Note that HPβCDs (Sigma, Kleptose HP, Kleptose HPB, and Trappsol) and HPγCD were significantly different from vehicle and αCDs, and these effective CDs showed no difference between them. SBEγCD produced intermediate scores and was not significantly different from any group with the exception of HPγCD. SBEβCD scores were similar to vehicle and statistically different from all HPβCDs and HPγCD. (C) GM3 ganglioside accumulation scores from immunohistochemical staining. The CDs with significantly different results from vehicle were all the HPβCDs, HPγCD, and SBEγCD, with no significant differences among these. SBEβCD produced intermediate values with no significant differences from any groups. Note that GM3 scoring results were remarkably equivalent to UC, with only some differences in confidence (P) level of statistical significance.
Figure 3
Figure 3
Biochemical analysis of GM2 and GM3 ganglioside levels in the brain of mice treated with different CDs. Data are expressed as % of total gangliosides after thin layer chromatographic separation of extracts of cerebral homogenates from mice as indicated. While sample size of some groups was insufficient for statistical analysis, individual values are well clustered and confirm reduction of ganglioside levels in Npc1 −/− mice treated with all the HPβCDs, HPγCD, and SBEγCD to levels approaching that in Wt mice. Ganglioside levels in Npc1 −/− mice treated with SBEβCD showed an intermediate level of reduction, and HPαCD and SBEαCD‐treated Npc1 −/− mice showed no reduction relative to untreated Npc1 −/− mice. Each pip is a biological replicate and horizontal line shows the mean value.
Figure 4
Figure 4
UC accumulation in liver of mice treated with different CDs. First two columns: (A) Filipin labeling of liver from untreated Npc1 −/− mice revealed widespread accumulation of UC in hepatocytes and Kupffer cells while liver from untreated Wt mice exhibited only diffuse filipin labeling. (B–G) Npc1 −/− mice treated with HPβCD, HPγCD, and SBEγCD showed UC reduction within hepatocytes (B, C, D), while SBEβCD, HPαCD, and SBEαCD treatments showed little to no difference from untreated Npc1 −/− mice (E, F, G vs. A). Administration of all CDs to Wt, as well as disease mice, resulted in elevated UC accumulation in presumptive liver macrophages (Kupffer cells), but hepatocytes remained filipin‐negative in Wt mice. Note that the accumulation appeared less in Wt than in disease mice, but the order of impact by different CDs was similar for the two, for example, in both cases, SBEβCD showed the highest and HPβCD the second highest accumulation in Kupffer cells. Second two columns: Confocal images of liver sections double‐labeled with anti‐CD68 (magenta), to unambiguously delineate lysosomal membranes of Kupffer cells, and filipin (green). (A) CD68 + Kupffer cells exhibited conspicuous vesicular/vacuolar‐like filipin+ labeling in untreated Npc1 −/− but not Wt mice. (B–G) CDs tested in Npc1 −/− instigated varying degrees of increased UC accumulation in Kupffer cells. Wt mice administered different CDs also produced some UC accumulation specifically within Kupffer cells. Scale bars = 50 μm (first two columns), 5 μm (second two columns).
Figure 5
Figure 5
Ototoxicity of different CDs as assessed by auditory brainstem responses (ABR) recordings at 12 weeks of age. (A) Representative ABR recordings from Wt mice administered different CDs are depicted. Several stereotypical ABR waveform components are evident, especially at the higher SPLs tested. Differences in waveform morphologies can be attributed to individual differences across mice and to slight variations in the placement of the subcutaneous needle electrodes. Waveforms obtained at threshold are plotted in red. HPβCD and HPγCD traces show pronounced hearing loss while SBEγCD, SBEβCD, and HPαCD traces demonstrate hearing thresholds equivalent to those of vehicle‐treated mice (~ 40 dB). (B) Plot of hearing thresholds for individual mice reveals minute variability across mice treated with a particular CD with the exception of HPγCD, in which hearing thresholds were more variable. (C) Two‐tailed t‐tests comparing mean thresholds for each pair of treatment groups revealed statistically significant differences in threshold only between HPβCD or HPγCD and all other treatment groups.
Figure 6
Figure 6
Membrane–membrane interaction of different CDs. Absolute change in absorbance after 30 min incubation period of 100 μmol/L Large unilamellar vesicles (LUVs) with 1 mmol/L CD provides a measure of LUV aggregation (mean ± SE, N = 3 experiments). A highly significant statistical difference was found among samples (ANOVA, P < 0.0001). Post hoc analysis (Tukey's multiple comparison test) showed that each of the HPβCDs and MβCD were significantly different from control (LUVs with buffer only) (P < 0.01; asterisks), and there was no significant difference (P > 0.05) among these CDs in pairwise comparisons. While the values for other CDs variably trended above background levels, this aggregation was not found statistically significantly different from control, nor between these other CDs. Similar results were obtained with CDs at 10 μmol/L and 100 μmol/L in three independent experiments (data not shown).
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References

    1. Vanier MT. Complex lipid trafficking in Niemann‐Pick disease type C. J Inherit Metab Dis 2015;38:187–199. - PubMed
    1. Infante RE, Wang ML, Radhakrishnan A, et al. NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes. Proc Natl Acad Sci USA 2008;105:15287–15292. - PMC - PubMed
    1. Rosenbaum AI, Maxfield FR. Niemann‐Pick type C disease: molecular mechanisms and potential therapeutic approaches. J Neurochem 2011;116:789–795. - PMC - PubMed
    1. Davidson CD, Ali NF, Micsenyi MC, et al. Chronic cyclodextrin treatment of murine Niemann‐Pick C disease ameliorates neuronal cholesterol and glycosphingolipid storage and disease progression. PLoS ONE 2009;4:e6951. - PMC - PubMed
    1. Liu B, Li H, Repa JJ, et al. Genetic variations and treatments that affect the lifespan of the NPC1 mouse. J Lipid Res 2008;3:663–669. - PubMed