RESUMO
Niemann-Pick disease type C (NPC) is a lethal genetic disease with mutations in NPC1 or NPC2 gene. Npc1-deficient (Npc1-/-) mice have been used as a model for NPC pathogenesis to develop novel therapies for NPC. However, Npc1-/- mice are infertile; thus, securing sufficient numbers for translational research is difficult. Hence, we attempted reproductive engineering techniques such as in vitro fertilization (IVF) and sperm cryopreservation. For the first time, we succeeded in producing fertilized oocytes via IVF using male and female Npc1-/- mice. Fertilized oocytes were also obtained via IVF using cryopreserved sperm from Npc1-/- mice. The obtained fertilized oocytes normally developed into live pups via embryo transfer, and they eventually exhibited NPC pathogenesis. These findings are useful for generating an efficient breeding system that overcomes the reproductive challenges of Npc1-/- mice and will contribute to developing novel therapeutic methods using NPC model mice.
RESUMO
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.
Assuntos
Modelos Animais de Doenças , Fígado , Doença de Niemann-Pick Tipo C , gama-Ciclodextrinas , Animais , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/patologia , Camundongos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , gama-Ciclodextrinas/farmacologia , Distribuição Tecidual , Colesterol/metabolismo , Masculino , Camundongos Endogâmicos BALB CRESUMO
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.
Assuntos
Ciclodextrinas , Doença de Niemann-Pick Tipo C , Ototoxicidade , Humanos , Ciclodextrinas/farmacologia , Simulação de Acoplamento Molecular , Doença de Niemann-Pick Tipo C/tratamento farmacológico , ColesterolRESUMO
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.