In vivo Efficacy and Safety Evaluation of Lactosyl-ß-cyclodextrin as a Therapeutic Agent for Hepatomegaly in Niemann-Pick Type C Disease.

Niemann-Pick type C disease (NPC) is a fatal, autosomal recessive disorder, which causes excessive accumulation of free cholesterol in endolysosomes, resulting in progressive hepatomegaly and neurodegeneration. Currently, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) is used at a high dose for the treatment of NPC, risking lung toxicity and hearing loss during treatment. One method to reduce the required dose of HP-ß-CyD for the treatment of hepatomegaly is to actively deliver ß-cyclodextrin (ß-CyD) to hepatocytes. Previously, we synthesized lactosyl-ß-CyD (Lac-ß-CyD) and demonstrated that it lowers cholesterol in NPC model liver cells. In the present study, we studied the efficacy and safety of Lac-ß-CyD treatment of hepatomegaly in Npc1-/- mice. After subcutaneous administration, Lac-ß-CyD accumulated in the liver and reduced hepatomegaly with greater efficacy than HP-ß-CyD. In addition, subcutaneous administration of a very high dose of Lac-ß-CyD was less toxic to the lungs than HP-ß-CyD. Notably, the accumulation of intracellular free cholesterol in endolysosomes of NPC-like liver cells was significantly lower after administration of Lac-ß-CyD than after treatment with HP-ß-CyD. In conclusion, these results suggest that Lac-ß-CyD is a candidate for the effective treatment of hepatomegaly in NPC.

Synthesis of multi-lactose-appended ß-cyclodextrin and its cholesterol-lowering effects in Niemann-Pick type C disease-like HepG2 cells.

Niemann-Pick type C (NPC) disease, characterized by intracellular accumulation of unesterified cholesterol and other lipids owing to defects in two proteins NPC1 and NPC2, causes neurodegeneration and other fatal neurovisceral symptoms. Currently, treatment of NPC involves the use of 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD). HP-ß-CD is effective in the treatment of hepatosplenomegaly in NPC disease, albeit at a very high dose. One of the methods to reduce the required dose of HP-ß-CD for treatment of NPC is to actively targeting hepatocytes with ß-cyclodextrin (ß-CD). The aim of the present study was to synthesize a novel multi-lactose-appended ß-CD (multi-Lac-ß-CD) and to evaluate its cholesterol-lowering effect in U18666A-treated HepG2 (NPC-like HepG2) cells. Further, the study aimed at delivering ß-CD to hepatocytes via cholesterol-accumulated HepG2 cells, and indicated that the newly synthesized multi-Lac-ß-CD had an average degree of substitution of lactose (DSL) of 5.6. This newly synthesized multi-Lac-ß-CD was found to significantly decrease the concentration of intracellular cholesterol with negligible cytotoxicity as compared to HP-ß-CD. An increased internalization of TRITC-multi-Lac-ß-CD (DSL 5.6) as compared to TRITC-HP-ß-CD was observed in NPC-like HepG2 cells. Further, the dissociation constant of peanut lectin with multi-Lac-ß-CD (DSL5.6) was found to be extremely low (2.5 × 10-8 M). These results indicate that multi-Lac-ß-CD (DSL5.6) diminished intracellular cholesterol levels in NPC-like HepG2 cells via asialoglycoprotein receptor (ASGPR)-mediated endocytosis.

Cholesterol-Lowering Effect of Octaarginine-Appended ß-Cyclodextrin in Npc1-Trap-CHO Cells.

Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal storage disorder, which is an inherited disease characterized by the accumulation of unesterified cholesterol in endolysosomes. Recently, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) has been used for the treatment of NPC, and ameliorated a hepatosplenomegaly in the patients. However, to obtain the treatment efficacy, a high dose of HP-ß-CyD was necessary. Therefore, the decrease in dose by using active intracellular delivery system of ß-CyD to NPC cells is expected. In this study, to efficiently deliver ß-CyD to NPC-like cells, we newly synthesized octaarginine (R8)-appended ß-CyD with a spacer of γ-aminobutyric acid (R8-ß-CyD) and evaluated its cytotoxicity, intracellular distribution, endocytosis pathway and cholesterol-lowering effect in Npc1-trap-Chinese hamster ovary (CHO) cells, cholesterol-accumulated cells through the impairment of NPC1 function. R8-ß-CyD did not show cytotoxicity in the cells. In addition, Alexa568-labeled R8-ß-CyD was actively internalized into Npc1-trap-CHO cells, possibly through micropinocytosis. Notably, R8-ß-CyD significantly decreased intracellular cholesterol content compared with HP-ß-CyD. These results suggest that R8-ß-CyD may be a promising therapeutic agent for ameliorating cholesterol accumulation in NPC.

Cholesterol lowering effects of mono-lactose-appended ß-cyclodextrin in Niemann-Pick type C disease-like HepG2 cells.

The Niemann-Pick type C disease (NPC) is one of inherited lysosomal storage disorders, emerges the accumulation of unesterified cholesterol in endolysosomes. Currently, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) has been applied for the treatment of NPC. HP-ß-CyD improved hepatosplenomegaly in NPC patients, however, a high dose of HP-ß-CyD was necessary. Therefore, the decrease in dose by actively targeted-ß-CyD to hepatocytes is expected. In the present study, to deliver ß-CyD selectively to hepatocytes, we newly fabricated mono-lactose-appended ß-CyD (Lac-ß-CyD) and evaluated its cholesterol lowering effects in NPC-like HepG2 cells, cholesterol accumulated HepG2 cells induced by treatment with U18666A. Lac-ß-CyD (degree of substitution of lactose (DSL) 1) significantly decreased the intracellular cholesterol content in a concentration-dependent manner. TRITC-Lac-ß-CyD was associated with NPC-like HepG2 cells higher than TRITC-ß-CyD. In addition, TRITC-Lac-ß-CyD was partially localized with endolysosomes after endocytosis. Thus, Lac-ß-CyD entered NPC-like HepG2 cells via asialoglycoprotein receptor (ASGPR)-mediated endocytosis and decreased the accumulation of intracellular cholesterol in NPC-like HepG2 cells. These results suggest that Lac-ß-CyD may have the potential as a drug for the treatment of hepatosplenomegaly in NPC disease.

2-Hydroxypropyl-ß-Cyclodextrin Acts as a Novel Anticancer Agent.

2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) is a cyclic oligosaccharide that is widely used as an enabling excipient in pharmaceutical formulations, but also as a cholesterol modifier. HP-ß-CyD has recently been approved for the treatment of Niemann-Pick Type C disease, a lysosomal lipid storage disorder, and is used in clinical practice. Since cholesterol accumulation and/or dysregulated cholesterol metabolism has been described in various malignancies, including leukemia, we hypothesized that HP-ß-CyD itself might have anticancer effects. This study provides evidence that HP-ß-CyD inhibits leukemic cell proliferation at physiologically available doses. First, we identified the potency of HP-ß-CyD in vitro against various leukemic cell lines derived from acute myeloid leukemia (AML), acute lymphoblastic leukemia and chronic myeloid leukemia (CML). HP-ß-CyD treatment reduced intracellular cholesterol resulting in significant leukemic cell growth inhibition through G2/M cell-cycle arrest and apoptosis. Intraperitoneal injection of HP-ß-CyD significantly improved survival in leukemia mouse models. Importantly, HP-ß-CyD also showed anticancer effects against CML cells expressing a T315I BCR-ABL mutation (that confers resistance to most ABL tyrosine kinase inhibitors), and hypoxia-adapted CML cells that have characteristics of leukemic stem cells. In addition, colony forming ability of human primary AML and CML cells was inhibited by HP-ß-CyD. Systemic administration of HP-ß-CyD to mice had no significant adverse effects. These data suggest that HP-ß-CyD is a promising anticancer agent regardless of disease or cellular characteristics.