Journal: Annals of Clinical and Translational Neurology

Article Title: Efficacy and ototoxicity of different cyclodextrins in Niemann–Pick C disease

doi: 10.1002/acn3.306

Figure Lengend Snippet: 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 HP β CD (Sigma) (B), HP γ CD (C), and SBE γ CD (D) all show highly effective reduction in UC storage, while some UC remains with SBE β CD treatment (E). HP α CD (F) and SBE α CD (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 HP β CD (B) and HP γ CD (C). Noticeably more remaining GM2 is evident in Npc1 −/− mouse treated with SBE γ CD (D), and substantially more with SBE β CD and α CD 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: HP β CD (B), HP γ CD (C), and SBE γ CD (D) are nearly indistinguishable from Wt (A); SBE β CD (E) shows an intermediate impact; and α CDs (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 μ m.

Article Snippet: 2‐Hydroxypropyl‐ β ‐CD (Trappsol) , Trappsol , CTD, Inc. (THPB‐EC) , 1541 , 7.0.

Techniques: Fluorescence, Staining

Journal: Annals of Clinical and Translational Neurology

Article Title: Efficacy and ototoxicity of different cyclodextrins in Niemann–Pick C disease

doi: 10.1002/acn3.306

Figure Lengend Snippet: 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 HP β CDs as well as HP γ CD and SBE γ CD treatment groups were significantly different from both vehicle and α CDs, and not significantly different ( P ≥ 0.05) from one another. (Note that filipin data for Trappsol treatment was limited to two biological replicates.) SBE β CD produced a range of intermediate values that yielded no significant difference from any group, while α CDs showed no difference from vehicle. (B) GM2 ganglioside scores from immunohistochemical staining. Note that HP β CDs (Sigma, Kleptose HP, Kleptose HPB, and Trappsol) and HP γ CD were significantly different from vehicle and α CDs, and these effective CDs showed no difference between them. SBE γ CD produced intermediate scores and was not significantly different from any group with the exception of HP γ CD. SBE β CD scores were similar to vehicle and statistically different from all HP β CDs and HP γ CD. (C) GM3 ganglioside accumulation scores from immunohistochemical staining. The CDs with significantly different results from vehicle were all the HP β CDs, HP γ CD, and SBE γ CD, with no significant differences among these. SBE β CD 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.

Article Snippet: 2‐Hydroxypropyl‐ β ‐CD (Trappsol) , Trappsol , CTD, Inc. (THPB‐EC) , 1541 , 7.0.

Techniques: Staining, Produced, Immunohistochemical staining

Journal: Annals of Clinical and Translational Neurology

Article Title: Efficacy and ototoxicity of different cyclodextrins in Niemann–Pick C disease

doi: 10.1002/acn3.306

Figure Lengend Snippet: 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 HP β CDs, HP γ CD, and SBE γ CD to levels approaching that in Wt mice. Ganglioside levels in Npc1 −/− mice treated with SBE β CD showed an intermediate level of reduction, and HP α CD and SBE α CD‐treated Npc1 −/− mice showed no reduction relative to untreated Npc1 −/− mice. Each pip is a biological replicate and horizontal line shows the mean value.

Article Snippet: 2‐Hydroxypropyl‐ β ‐CD (Trappsol) , Trappsol , CTD, Inc. (THPB‐EC) , 1541 , 7.0.

Techniques:

Journal: Annals of Clinical and Translational Neurology

Article Title: Efficacy and ototoxicity of different cyclodextrins in Niemann–Pick C disease

doi: 10.1002/acn3.306

Figure Lengend Snippet: 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 HP β CD, HP γ CD, and SBE γ CD showed UC reduction within hepatocytes (B, C, D), while SBE β CD, HP α CD, and SBE α CD 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, SBE β CD showed the highest and HP β CD 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 μ m (first two columns), 5 μ m (second two columns).

Article Snippet: 2‐Hydroxypropyl‐ β ‐CD (Trappsol) , Trappsol , CTD, Inc. (THPB‐EC) , 1541 , 7.0.

Techniques: Labeling, Produced

Journal: Annals of Clinical and Translational Neurology

Article Title: Efficacy and ototoxicity of different cyclodextrins in Niemann–Pick C disease

doi: 10.1002/acn3.306

Figure Lengend Snippet: 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. HP β CD and HP γ CD traces show pronounced hearing loss while SBE γ CD, SBE β CD, and HP α CD 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 HP γ CD, 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 HP β CD or HP γ CD and all other treatment groups.

Article Snippet: 2‐Hydroxypropyl‐ β ‐CD (Trappsol) , Trappsol , CTD, Inc. (THPB‐EC) , 1541 , 7.0.

Techniques: Two Tailed Test

Journal: Annals of Clinical and Translational Neurology

Article Title: Efficacy and ototoxicity of different cyclodextrins in Niemann–Pick C disease

doi: 10.1002/acn3.306

Figure Lengend Snippet: Membrane–membrane interaction of different CDs. Absolute change in absorbance after 30 min incubation period of 100 μ mol/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 HP β CDs and M β CD 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 μ mol/L and 100 μ mol/L in three independent experiments (data not shown).

Article Snippet: 2‐Hydroxypropyl‐ β ‐CD (Trappsol) , Trappsol , CTD, Inc. (THPB‐EC) , 1541 , 7.0.

Techniques: Membrane, Incubation, Comparison, Control

Journal: Annals of Clinical and Translational Neurology

Article Title: Efficacy and ototoxicity of different cyclodextrins in Niemann–Pick C disease

doi: 10.1002/acn3.306

Figure Lengend Snippet: Cyclodextrins used

Article Snippet: 2‐Hydroxypropyl‐ β ‐CD (Trappsol) , Trappsol , CTD, Inc. (THPB‐EC) , 1541 , 7.0.

Techniques: Molecular Weight

Journal: Annals of Clinical and Translational Neurology

Article Title: Efficacy and ototoxicity of different cyclodextrins in Niemann–Pick C disease

doi: 10.1002/acn3.306

Figure Lengend Snippet: Binding constants (M ‐1 ) for CD interaction with Niemann‐Pick type C disease related compounds: unesterified cholesterol, brain‐relevant oxysterols, glucosylceramide, lactosylceramide, BMP, sphingosine, and oleic acid

Article Snippet: 2‐Hydroxypropyl‐ β ‐CD (Trappsol) , Trappsol , CTD, Inc. (THPB‐EC) , 1541 , 7.0.

Techniques: Binding Assay

Journal: Annals of Clinical and Translational Neurology

Article Title: Efficacy and ototoxicity of different cyclodextrins in Niemann–Pick C disease

doi: 10.1002/acn3.306

Figure Lengend Snippet: a Complexation values of CDs with Niemann‐Pick type C disease related compounds: unesterified cholesterol, brain‐relevant oxysterols, glucosylceramide, lactosylceramide, BMP, sphingosine, and oleic acid

Article Snippet: 2‐Hydroxypropyl‐ β ‐CD (Trappsol) , Trappsol , CTD, Inc. (THPB‐EC) , 1541 , 7.0.

Techniques: