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Niemann-Pick disease Type C (NPC) is a lysosomal storage disorder caused by mutation of the NPC1/NPC2 genes, which ultimately results in the accumulation of unesterified cholesterol (UEC) in lysosomes, thereby inducing symptoms such as progressive neurodegeneration and hepatosplenomegaly. This study determines the effects of 6-O-α-maltosyl-β cyclodextrin (Mal-βCD) on lipid levels and synthesis in Npc1-deficient (Npc1-KO cells) and vehicle CHO cells. Compared to vehicle cells, Npc1-KO cells exhibited high level of UEC, and low levels of esterified cholesterols (ECs) and long-chain fatty acids (LCFAs). The difference in lipid levels between Npc1-KO and CHO cells was largely ameliorated by Mal-βCD administration. Moreover, the effects of Mal-βCD were reproduced in the lysosomes prepared from Npc1-KO cells. Stable isotope tracer analysis with extracellular addition of D4-deuterated palmitic acid (D4-PA) to Npc1-KO cells increased the synthesis of D4-deuterated LCFAs (D4-LCFAs) and D4-deuterated ECs (D4-ECs) in a Mal-βCD-dependent manner. Simultaneous addition of D6-deuterated UEC (D6-UEC) and D4-PA promoted the Mal-βCD-dependent synthesis of D6-/D4-ECs, consisting of D6-UEC and D4-PA, D4-deuterated stearic acid, or D4-deuterated myristic acid, in Npc1-KO cells. These results suggest that Mal-βCD helps to maintain normal lipid metabolism by restoring balance among UEC, ECs, and LCFAs through acting on behalf of NPC1 in Npc1-KO cells and may therefore be useful in designing effective therapies for NPC., Competing Interests: Conflicts of interest The authors declare no conflicts of interest. The funding sponsors had no role in the design of the study; collection, analyses, or interpretation of data; writing of the manuscript; or decision to publish the results. Mal-βCD was a gift from Ensuiko Sugar Refining Co. Ltd. This company played no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Niemann-Pick disease Type C (NPC) is a rare lysosomal storage disease characterized by the dysfunction of intracellular cholesterol trafficking with progressive neurodegeneration and hepatomegaly. We evaluated the potential of 6- O -α-maltosyl-β-cyclodextrin (G2-β-CD) as a drug candidate against NPC. The physicochemical properties of G2-β-CD as an injectable agent were assessed, and molecular interactions between G2-β-CD and free cholesterol were studied by solubility analysis and two-dimensional proton nuclear magnetic resonance spectroscopy. The efficacy of G2-β-CD against NPC was evaluated using Npc1 deficient Chinese hamster ovary (CHO) cells and Npc1 deficient mice. G2-β-CD in aqueous solution showed relatively low viscosity and surface activity; characteristics suitable for developing injectable formulations. G2-β-CD formed higher-order inclusion complexes with free cholesterol. G2-β-CD attenuated dysfunction of intercellular cholesterol trafficking and lysosome volume in Npc1 deficient CHO cells in a concentration dependent manner. Weekly subcutaneous injections of G2-β-CD (2.9 mmol/kg) ameliorated abnormal cholesterol metabolism, hepatocytomegaly, and elevated serum transaminases in Npc1 deficient mice. In addition, a single cerebroventricular injection of G2-β-CD (21.4 μmol/kg) prevented Purkinje cell loss in the cerebellum, body weight loss, and motor dysfunction in Npc1 deficient mice. In summary, G2-β-CD possesses characteristics favorable for injectable formulations and has therapeutic potential against in vitro and in vivo NPC models., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, and in the decision to publish the results. G2-β-CD and HP-β-CD were gifts from Ensuiko Sugar Refining Co., Ltd. and Nihon Shokuhin Kako Co., Ltd., respectively. These companies have not imposed a role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

We aimed to investigate whether 6-O-α-maltosyl-β-cyclodextrin (Mal-βCD) is incorporated into cells and lysosomes during the release of unesterified cholesterol in Npc1-deficient Chinese hamster ovary (CHO) cells (Npc1 KO cells) and CHO-JP17 cells (JP17 cells). Internalization of Mal-βCD in cells and lysosomes and extracellular release of lysosomal unesterified cholesterol were demonstrated by LC/MS/MS and LC/MS, respectively. Internalization of Mal-βCD was greater in Npc1 KO cells than in JP17 cells. The majority of internalized Mal-βCD in both cell types was metabolized by lysosomal α-glucosidase to 6-O-α-D-glucosyl-β-cyclodextrin (Glc-βCD). However, Mal-βCD did not directly enter the lysosomes prepared from cell homogenates. Mal-βCD-treated Npc1 KO cells and JP17 cells both released Mal-βCD and Glc-βCD, together with unesterified cholesterol, out of cells. The release of unesterified cholesterol by Mal-βCD in Npc1 KO cells was much greater than that in JP17 cells. This study is the first to report the influx of Mal-βCD into the lysosomes of Npc1 KO cells and JP17 cells, followed by generation of Glc-βCD, and the efflux of Mal-βCD/Glc-βCD and unesterified cholesterol to the extracellular fluid, based on the quantitative LC/MS analysis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)