**2. Preclinical studies**

#### **2.1.** *In vitro* **experience**

The efflux of cholesterol from cells in culture to cyclodextrin acceptors has been reported to be substantially more rapid than efflux induced by other known acceptors of cholesterol. A comparison of the time course of cellular [3H]cholesterol efflux mediated by HDL3 or by various concentrations of cyclodextrins showed the release of 50–90% of L cell [3H]cholesterol after 8 hours of incubation with HP-β-CD and methyl-β-cyclodextrin (M-β-CD) at 10 mM. The order of efficiency in accepting cholesterol was found to be M-β-CD > HP-β-CD > beta-cyclodextrin. The kinetics of the cholesterol efflux time course studies suggested that incubation of L cells with cyclodextrin resulted in the rapid equilibration of labeled cholesterol between cells and medium. [12].

Several studies have shown that cholesterol released from late endosomes/lysosomes of NPCproteins deficient cells by HP-β-CD reaches the cytosolic compartment and is accessible to the endoplasmic reticulum (ER). In cultured cerebellar neurons, astrocytes, and microglia from NPC1-deficient mice, the sequestered cholesterol was mobilized to the ER by low concentrations (0.1–1.0 mM) of HP-β-CD [13].

In murine models of NPC1, cell culture studies have shown that M-β-CD is more potent in exchanging cholesterol than HP-β-CD. Efficacy comparison of M-β-CD and HP-β-CD in reducing cholesterol accumulation in late endosome/lysosome in human fibroblasts NPC1/ NPC2 deficient after treatment with 300 μM M-β-CD and HP-β-CD for 1 day have shown to reduce the cholesterol accumulation detected by filipin labeling. After 2 and 3 days after treatment, cholesterol accumulation started to increase, but there was still a significant reduction. In concordance with animal studies, M-β-CD produces effects equivalent to those of HP-β-CD at lower concentrations [14].There are several theories about the mechanism by which cyclodextrins affect cholesterol homeostasis but none have been confirmed.

*In vitro* beta-cyclodextrins showed a high affinity for sterols as compared to other lipid and, because of the relatively high specificity of this substance for cholesterol, it was suggested that beta-cyclodextrins might be effective in modifying cholesterol metabolism in vivo.
