Intracerebroventricular 2-hydroxypropyl-γ-cyclodextrin alleviates hepatic manifestations without distributing to the liver in a murine model of Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is a lysosomal lipid storage disorder characterized by progressive neurodegeneration and hepatic dysfunction. A cyclic heptasaccharide, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), is currently under clinical investigation for NPC, but its adverse events remain problematic. We previously identified that a cyclic octasaccharide, 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD), also ameliorated NPC manifestations with higher biocompatibility than HP-ß-CD. However, preclinical studies describing the associations between the biodistribution and pharmacodynamics of these compounds, which are essential for clinical application, are still lacking. Here, we investigated these properties of HP-γ-CD by measuring its organ biodistribution and therapeutic effect after systemic and central administration. The effect of HP-γ-CD on disturbed cholesterol homeostasis appeared within several hours after exposure and persisted for several days in NPC model cells and mice. Tissue distribution indicated that only a small fraction of subcutaneously administered HP-γ-CD rapidly distributed to peripheral organs and contributed to disease amelioration. We found that a subcutaneous dose of HP-γ-CD negligibly ameliorated neurological characteristics because it has limited penetration of the blood-brain barrier; however, an intracerebroventricular microdose unexpectedly attenuated hepatic dysfunction without the detection of HP-γ-CD in the liver. These results demonstrate that central administration of HP-γ-CD can indirectly attenuate peripheral manifestations of NPC.

Different solubilizing ability of cyclodextrin derivatives for cholesterol in Niemann-Pick disease type C treatment.

BACKGROUND: Niemann-Pick disease type C (NPC) is a fatal neurodegenerative disorder caused by abnormal intracellular cholesterol trafficking. Cyclodextrins (CDs), the most promising therapeutic candidates for NPC, but with concerns about ototoxicity, are cyclic oligosaccharides with dual functions of unesterified cholesterol (UC) shuttle and sink that catalytically enhance the bidirectional flux and net efflux of UC, respectively, between the cell membrane and the extracellular acceptors. However, the properties of CDs that regulate these functions and how they could be used to improve treatments for NPC are unclear. METHODS: We estimated CD-UC complexation for nine CD derivatives derived from native α-, ß-, and γ-CD with different cavity sizes, using solubility and molecular docking analyses. The stoichiometry and complexation ability of the resulting complexes were investigated in relation to the therapeutic effectiveness and toxicity of each CD derivative in NPC experimental models. FINDINGS: We found that shuttle and sink activities of CDs are dependent on cavity size-dependent stoichiometry and substituent-associated stability of CD-UC complexation. The ability of CD derivatives to form 1:1 and 2:1 complexes with UC were correlated with their ability to normalize intracellular cholesterol trafficking serving as shuttle and with their cytotoxicity associated with cellular UC efflux acting as sink, respectively, in NPC model cells. Notably, the ability of CD derivatives to form an inclusion complex with UC was responsible for not only efficacy but ototoxicity, while a representative derivative without this ability negligibly affected auditory function, underscoring its preventability. CONCLUSIONS: Our findings highlight the importance of strategies for optimizing the molecular structure of CDs to overcome this functional dilemma in the treatment of NPC.

Fine-tuned cholesterol solubilizer, mono-6-O-α-D-maltosyl-γ-cyclodextrin, ameliorates experimental Niemann-Pick disease type C without hearing loss.

Niemann-Pick disease type C (NPC) is a fatal disorder with abnormal intracellular cholesterol trafficking resulting in neurodegeneration and hepatosplenomegaly. A cyclic heptasaccharide with different degrees of substitution of 2-hydroxypropyl groups, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), acts as a strong cholesterol solubilizer and is under investigation for treating this disease in clinical trials, but its physicochemical properties and ototoxicity remain a concern. Here, we evaluated the potential of mono-6-O-α-maltosyl-γ-CD (G2-γ-CD), a single-maltose-branched cyclic octasaccharide with a larger cavity than HP-ß-CD, for treating NPC. We identified that G2-γ-CD ameliorated NPC manifestations in model mice and showed lower ototoxicity in mice than HP-ß-CD. To investigate the molecular mechanisms of action behind the differential ototoxicity of these CDs, we performed cholesterol solubility analysis, proton nuclear magnetic resonance spectroscopy, and molecular modeling, and estimated that the cholesterol inclusion mode of G2-γ-CD maintained solely the 1:1 inclusion complex, whereas that of HP-ß-CD shifted to the highly-soluble 2:1 complex at higher concentrations. We predicted the associations of these differential complexations of CDs with cholesterol with the profile of disease attenuation and of the auditory cell toxicity using specific cell models. We proposed that G2-γ-CD can serve as a fine-tuned cholesterol solubilizer for treating NPC, being highly biocompatible and physicochemically suitable for clinical application.

Development and Validation of an HPLC Method for Analysis of Topotecan in Human Cerebrospinal Fluid and Its Application in Elimination Evaluation of Topotecan after Intraventricular Injection.

Intrathecal administration of anticancer drugs is an effective dosage strategy, but the elimination of intraventricular drugs is not uniform in all patients. For safety, a system to evaluate local pharmacokinetics in the ventricles after administration is desired. In this study, we developed a simple and reproducible method to measure topotecan concentration in the cerebrospinal fluid (CSF) and confirmed its clinical applicability. High-performance liquid chromatography (HPLC) analysis was performed using a C18 column to measure the total topotecan concentration in the CSF. Clinical CSF samples were obtained from a 1-year old child with poor CSF absorption and stagnation. The patient received topotecan via an intraventricular subcutaneous reservoir. The HPLC method complied with the validation criteria. The lower limit of quantitation of this method was 0.04 µM. Using the developed method, we could determine the difference in topotecan CSF concentrations at 24 and 48 h after administration. The patient's topotecan elimination rate was extremely low, and signs of adverse effects were observed at high CSF concentration of topotecan. The developed method could detect the delay in topotecan elimination after intrathecal injection. The findings of this study are valuable for the development of personalized treatments for the intrathecal administration of anticancer drugs.

Differential mode of cholesterol inclusion with 2-hydroxypropyl-cyclodextrins increases safety margin in treatment of Niemann-Pick disease type C.

BACKGROUND AND PURPOSE: Niemann-Pick disease type C (NPC) is a lysosomal storage disorder with disrupted intracellular cholesterol trafficking. A cyclic heptasaccharide, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), is a cholesterol solubilizer that is being developed to treat NPC, but its ototoxicity and pulmonary toxicity remain important issues. We have characterized 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD), a cyclic octasaccharide with a larger cavity than HP-ß-CD, as a candidate drug to treat NPC. However, the molecular target of HP-γ-CD with respect to NPC and its potential for clinical application are still unclear. EXPERIMENTAL APPROACH: We investigated the mode of interaction between HP-γ-CD and cholesterol by phase-solubility analysis, proton NMR spectroscopy and molecular dynamics simulations. We then evaluated the therapeutic effects of HP-γ-CD compared with HP-ß-CD using cellular and murine NPC models. Mouse auditory and pulmonary function tests were also conducted. KEY RESULTS: HP-γ-CD solely formed a 1:1 inclusion complex with cholesterol with an affinity similar to that of HP-ß-CD. In vitro, HP-γ-CD and HP-ß-CD amelioration of NPC-related manifestations was almost equivalent at lower concentrations. However, at higher concentrations, the cholesterol inclusion mode of HP-ß-CD shifted to the highly soluble 2:1 complex whereas that of HP-γ-CD maintained solely the 1:1 complex. The constant lower cholesterol solubilizing ability of HP-γ-CD conferred it with significantly reduced toxicity compared with HP-ß-CD, but equal efficacy in treating a mouse model of NPC. CONCLUSIONS AND IMPLICATIONS: HP-γ-CD can serve as a fine-tuned cholesterol solubilizer for the treatment of NPC with a wider safety margin than HP-ß-CD in terms of ototoxicity and pulmonary toxicity.

Intracerebroventricular Treatment with 2-Hydroxypropyl-ß-Cyclodextrin Decreased Cerebellar and Hepatic Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) Expression in Niemann-Pick Disease Type C Model Mice.

Niemann-Pick disease type C (NPC) is a recessive hereditary disease caused by mutation of the NPC1 or NPC2 gene. It is characterized by abnormality of cellular cholesterol trafficking with severe neuronal and hepatic injury. In this study, we investigated the potential of glycoprotein nonmetastatic melanoma protein B (GPNMB) to act as a biomarker reflecting the therapeutic effect of 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) in an NPC mouse model. We measured serum, brain, and liver expression levels of GPNMB, and evaluated their therapeutic effects on NPC manifestations in the brain and liver after the intracerebroventricular administration of HP-ß-CD in Npc1 gene-deficient (Npc1-/-) mice. Intracerebroventricular HP-ß-CD inhibited cerebellar Purkinje cell damage in Npc1-/- mice and significantly reduced serum and cerebellar GPNMB levels. Interestingly, we also observed that the intracerebral administration significantly reduced hepatic GPNMB expression and elevated serum ALT in Npc1-/- mice. Repeated doses of intracerebroventricular HP-ß-CD (30 mg/kg, started at 4 weeks of age and repeated every 2 weeks) drastically extended the lifespan of Npc1-/- mice compared with saline treatment. In summary, our results suggest that GPNMB level in serum is a potential biomarker for evaluating the attenuation of NPC pathophysiology by intracerebroventricular HP-ß-CD treatment.

Differential Effects of 2-Hydroxypropyl-Cyclodextrins on Lipid Accumulation in Npc1-Null Cells.

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder characterized by abnormal accumulation of free cholesterol and sphingolipids in lysosomes. The iminosugar miglustat, which inhibits hexosylceramide synthesis, is used for NPC treatment, and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a cyclic oligosaccharide derivative, is being developed to treat NPC. Moreover, therapeutic potential of 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD) was shown in NPC models, although its mechanism of action remains unclear. Here, we investigated the effects of HP-ß-CD, HP-γ-CD, and their homolog 2-hydroxypropyl-α-cyclodextrin (HP-α-CD) on lipid accumulation in Npc1-null Chinese hamster ovary (CHO) cells compared with those of miglustat. HP-ß-CD and HP-γ-CD, unlike HP-α-CD, reduced intracellular free cholesterol levels and normalized the lysosome changes in Npc1-null cells but not in wild-type CHO cells. In contrast, miglustat did not normalize intracellular free cholesterol accumulation or lysosome changes in Npc1-null cells. However, miglustat decreased the levels of hexosylceramide and tended to increase those of sphingomyelins in line with its action as a glucosylceramide synthase inhibitor in both Npc1-null and wild-type CHO cells. Interestingly, HP-ß-CD and HP-γ-CD, unlike HP-α-CD, reduced sphingomyelins in Npc1-null, but not wild-type, cells. In conclusion, HP-ß-CD and HP-γ-CD reduce the accumulation of sphingolipids, mainly sphingomyelins, and free cholesterol as well as lysosome changes in Npc1-null, but not in wild-type, CHO cells.

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.

In Vitro and In Vivo Evaluation of 6-O-α-Maltosyl-ß-Cyclodextrin as a Potential Therapeutic Agent Against Niemann-Pick Disease Type C.

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-ß-cyclodextrin (G2-ß-CD) as a drug candidate against NPC. The physicochemical properties of G2-ß-CD as an injectable agent were assessed, and molecular interactions between G2-ß-CD and free cholesterol were studied by solubility analysis and two-dimensional proton nuclear magnetic resonance spectroscopy. The efficacy of G2-ß-CD against NPC was evaluated using Npc1 deficient Chinese hamster ovary (CHO) cells and Npc1 deficient mice. G2-ß-CD in aqueous solution showed relatively low viscosity and surface activity; characteristics suitable for developing injectable formulations. G2-ß-CD formed higher-order inclusion complexes with free cholesterol. G2-ß-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-ß-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-ß-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-ß-CD possesses characteristics favorable for injectable formulations and has therapeutic potential against in vitro and in vivo NPC models.

Efficacy of 2-Hydroxypropyl-ß-cyclodextrin in Niemann-Pick Disease Type C Model Mice and Its Pharmacokinetic Analysis in a Patient with the Disease.

Niemann-Pick type C disease (NPC), an autosomal recessive lysosomal storage disorder, is an inherited disease characterized by the accumulation of intracellular unesterified cholesterol. A solubilizing agent of lipophilic compounds, 2-hydroxypropyl-ß-cyclodextrin (HPBCD), is an attractive drug candidate against NPC disease. However, establishment of the optimum dosage of HPBCD remains to be determined. In this study, we evaluated the effective dosage of HPBCD in NPC model (Npc1(-/-)) mice, and determined serum HPBCD concentrations. Subcutaneous injection of 1000-4000 mg/kg HPBCD improved the lifespan of Npc1(-/-) mice. In addition, liver injury and cholesterol sequestration were significantly prevented by 4000 mg/kg HPBCD in Npc1(-/-) mice. Serum HPBCD concentrations, when treated at the effective dosages (1000-4000 mg/kg), were approximately 1200-2500 µg/mL at 0.5 h after subcutaneous injection, and blood HPBCD concentrations were immediately eliminated in Npc1(-/-) mice. Furthermore, we examined serum HPBCD concentrations when treated at 40000 mg (approximately 2500 mg/kg) in a patient with NPC. We observed that the effective concentration in the in vivo study using Npc1(-/-) mice was similar to that in the patient. In the patient, systemic clearance and the volume of distribution of HPBCD were in accordance with the glomerular filtration rate and extracellular fluid volume, respectively. These results could provide useful information for developing the optimal dosage regimen for HPBCD therapy when administered intravenously to NPC patients.

The exacerbating roles of CCAAT/enhancer-binding protein homologous protein (CHOP) in the development of bleomycin-induced pulmonary fibrosis and the preventive effects of tauroursodeoxycholic acid (TUDCA) against pulmonary fibrosis in mice.

The purpose of this study was to evaluate the role of CCAAT/enhancer-binding protein homologous protein (CHOP), an important transcription factor that regulates the inflammatory reaction during the endoplasmic reticulum (ER) stress response, in the development of pulmonary fibrosis induced by bleomycin (BLM) in mice. An intratracheal injection of BLM transiently increased the expression of CHOP mRNA and protein in an early phase (days 1 and 3) in mice lungs. BLM-induced pulmonary fibrosis was significantly attenuated in Chop gene deficient (Chop KO) mice, compared with wild-type (WT) mice. Furthermore, the inflammatory reactions evaluated by protein concentration, the total number of leucocytes and neutrophils in the bronchoalveolar lavage fluid (BALF), the mRNA expression of interleukin 1b and caspase 11, and the apoptotic cell death were suppressed in Chop KO mice compared with those in WT mice. In addition, administration of tauroursodeoxycholic acid (TUDCA), a pharmacological agent that can inhibit CHOP expression, inhibited the BLM-induced pulmonary fibrosis and inflammation, and the increase in Chop mRNA expression in WT mice in a dose-dependent manner. These results suggest that the ER stress-induced transcription factor, CHOP, at least in part, plays an important role in the development of BLM-induced pulmonary fibrosis in mice, and that the inhibition of CHOP expression by a pharmacological agent, such as TUDCA, may be a promising strategy for the prevention of pulmonary fibrosis.

Influence of Npc1 genotype on the toxicity of hydroxypropyl-ß-cyclodextrin, a potentially therapeutic agent, in Niemann-Pick Type C disease models.

Hydroxypropyl-ß-cyclodextrin (HPBCD) is an attractive drug candidate against Niemann-Pick Type C (NPC) disease. However, the safety of HPBCD treatment for NPC patients remains to be elucidated. In this study, we examined the acute toxicity of HPBCD in Npc1-deficient mice. When treated with HPBCD (20,000 mg/kg, subcutaneously), over half of the wild-type (Npc1+/+) or Npc1+/- mice died by 72 h after the injection. In contrast, all of the Npc1-/- mice survived. Marked pathophysiological changes, such as an elevation in serum transaminase and creatinine levels, hepatocellular necrosis, renal tubular damage, interstitial thickening, and hemorrhages in lungs, were induced by the HPBCD treatment in Npc1+/+ or Npc1+/- mice. However, these pathophysiological changes were significantly alleviated in Npc1-/- mice. In addition, in vitro analysis showed that the Npc1 gene deficiency and treatment with U18666A, an Npc1 inhibitor, remarkably attenuated the cytotoxicity of HPBCD in Chinese hamster ovary cells. These results suggest that the NPC1 genotype exacerbates the cytotoxicity of HPBCD and Npc1-/- mice have substantial resistance to the lethality and the organ injury induced by HPBCD injection compared with Npc1+/+ or Npc1+/- mice. We suggest that the Npc1 genotype should be considered in the safety evaluation of HPBCD using experimental animals and cells.