1. Introduction
Lysosomal storage diseases are congenital metabolic disorders caused by an absence or abnormality of lysosomal enzymes or proteins [
1]. These diseases are inherited in an autosomal-recessive fashion and are characterized by the abnormal accumulation of lipids, glycoproteins, and mucopolysaccharides in lysosomes [
2]. The disorders are classified into seven types according to the accumulated substrates: (1) glycolipid metabolism disorder (sphingolipidosis); (2) mucopolysaccharide metabolism disorder (mucopolysaccharidosis); (3) glycoprotein metabolism disorder; (4) mucolipidosis; (5) glycogen storage disease type II; (6) acid lipase deficiency; and (7) lysosomal membrane protein disorder [
3,
4].
Niemann-Pick disease type C (NPC) is a lysosomal storage disease that is characterized by the accumulation of free cholesterol and glycosphingolipids in endolysosomes due to the loss of membrane protein, NPC1, or soluble protein, NPC2. Mutations in the
NPC1 gene are observed in approximately 95% of NPC patients, with the remainder having mutations in the NPC2 gene [
5]. The incidence of NPC is approximately 1 in 150,000 live births. Enlargement of the liver (hepatomegaly) or spleen (splenomegaly) is observed in around 85% of NPC patients in childhood to early adolescence, accompanied by abdominal and lower back pressure and pain. In addition, the abnormal accumulation of free cholesterol and glycosphingolipids in brain tissue of NPC patients leads to damage of brain tissue and causes various neurological symptoms. The majority of cases (60–70%) are classic NPC (late infantile and juvenile), and death generally occurs between 7 and 12 years of age [
6]. Several approaches for the treatment of NPC have been proposed, such as glucosylceramide synthase inhibitors (i.e. miglustat) [
7], histone deacetylase (HDAC) inhibitors (i.e. vorinostat) [
8], curcumin [
9], and 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) [
10,
11]. Unfortunately, the efficacies of miglustat, vorinostat, and curcumin are so limited that patients eagerly await new therapies for NPC. Hence, HP-β-CyD has received tremendous attention as a potential NPC therapeutic agent due to its high efficacy, although a high dose is required.
Cyclodextrins (CyDs) are cyclic oligosaccharides with a hydrophobic cavity that can form inclusion complexes with various guest molecules. HP-β-CyD has been most widely used in the pharmaceutical field to improve drug solubility and bioavailability. It has been reported that HP-β-CyD removes cholesterol from lipid rafts on the plasma membrane surface and destroys the structures [
12,
13]. In addition, subcutaneous administration of HP-β-CyD to NPC model mice decreased free cholesterol levels, ameliorated hepatomegaly, and delayed the progression of neurological symptoms (10). Phase I/II and phase IIb/III clinical trials of HP-β-CyD administered intravenously (CTD holdings, Inc.) and intrathecally (Mallinkrodt plc.), respectively, are ongoing. In particular, the administration into the space under the arachnoid membrane of the brain (intrathecal administration) of HP-β-CyD has shown excellent therapeutic effects. However, to obtain efficacy in vivo, high-dose administration of HP-β-CyD is necessary, as HP-β-CyD enter cells only very slightly, owing to its hydrophilicity and relatively high molecular weight. Indeed, Matsuo et al. reported that an NPC patient exhibited fever and transient cloudiness of the lungs after two years of treatment with HP-β-CyD by intravenous infusion. Therefore, the administration route was altered to intraventricular administration [
14].
Although HP-β-CyD is clinically applied to NPC treatment, HP-β-CyD has no cell or tissue selectivity. Therefore, the application of CyD having liver selectivity is expected for the treatment of hepatosplenomegaly in NPC. Meanwhile, asialoglycoprotein receptor (ASGPR), a hepatic galactose and N-acetylglucosamine (GlcNAc) receptor, is responsible for the binding, internalization, and subsequent clearance of glycoproteins containing terminal galactose or GlcNAc residues from the circulation [
15]. Hence, galactosylated nanocarriers have been used for selective delivery of drugs to the liver via ASGPR-mediated endocytosis [
16]. In fact, ASGPR-mediated endocytosis is one of the most promising approaches for delivery of CyDs into hepatocytes for the treatment of hepatosplenomegaly in NPC disease. In our previous report, mono-lactosyl β-CyD (mono-Lac-β-CyD) diminished cholesterol accumulation as efficiently as HP-β-CyD in our model of NPC hepatocytes, U18666A-treated HepG2 liver cells (NPC-like HepG2 cells: U18666A ((3β)-3-[2-(Diethylamino)ethoxy]androst-5-en-17-one hydrochloride) is an inhibitor of lysosomal cholesterol export [
17]. ASGPR recognizes the galactose moiety at three points, collectively known as the “golden triangle” [
18,
19]. However, as mono-Lac-β-CyD can bind to only one point of the golden triangle of ASGPR, it has insufficient binding affinity for ASGPR-mediated internalization. Recently, we created a novel multi-lactosyl β-CyD (Lac-β-CyD) to improve targeting to ASGPR, and evaluated its cholesterol-lowering effect in NPC-like HepG2 cells [
20]. Lac-β-CyD was internalized into hepatocytes via ASGPR-mediated endocytosis and ameliorated excessive accumulation of free cholesterol in NPC-like HepG2 cells. However, its therapeutic effect on hepatomegaly in vivo has not yet been reported.
Based on the above considerations, we aimed to examine the therapeutic effect of Lac-β-CyD on hepatomegaly in the NPC model mice (Npc1−/− mice), and to evaluate its safety in wild type (WT) mice. Initially, we used fluorescently-labeled Lac-β-CyD and HP-β-CyD to investigate their accumulation in the liver after subcutaneous administration to WT mice. Secondly, we examined the effects of Lac-β-CyD on vacuolar degeneration of the liver, liver inflammation, and tissue cholesterol accumulation in Npc1−/− mice after single or weekly administration of Lac-β-CyD. Finally, we analyzed plasma biochemistry and studied the lung toxicity of high acute doses of Lac-β-CyD and HP-β-CyD in WT mice. The results suggested that Lac-β-CyD has great potential for safer and effective treatment of hepatomegaly in NPC.
2. Materials and Methods
2.1. Materials
Lac-β-CyD with average degree of substitution of lactose of 5.6 was fabricated as reported previously [
20]. HP-β-CyD was kindly donated by Nihon Shokuhin Kako (Tokyo, Japan). HyLite
TM Fluor 647 (HF647) acid, SE, was obtained from Anaspec, Inc. (Fremont, CA, USA). Asialofetuin (AF) from fetal calf serum Type I and bovine serum albumin (BSA) Fraction V were purchased from Sigma-Aldrich (St. Louis, MO, USA). The BioRad protein assay kit, CellLight
® ER-GFP BacMam 2.0, LysoTracker
® Green DND-26 (LysoTracker
®), and LysoTracker
® Red DND-99 (LysoTracker
®) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). U18666A, maltoheptaose, miglustat hydrochloride, and Cholesterol E-test Wako
® were purchased from Wako Pure Chemical Industries (Osaka, Japan). Filipin III was purchased from Cayman Chemical (Ann Arbor, MI, USA). Hoechst 33342, and secondary antibodies, goat anti-mouse IgG Alexa Fluor
® 488 (A11029) and goat anti-rabbit IgG Alexa Fluor
® 594 (A11037), were obtained from Invitrogen (Carlsbad, CA, USA). Cyto-ID
TM Autophagy Detection Kit was obtained from Enzo Life Sciences (Farmingdale, NY, USA). Anti-TFE3 antibody (#4240) was obtained from Cell Signaling Technology (Danvers, MA, Japan). SQSTM1 (sc-28359) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The cell-counting kit-8 was obtained from Dojindo Laboratories (Kumamoto, Japan). Other chemicals and solvents were of analytical reagent grade.
2.2. Synthesis of HF647-Labeled β-CyDs
As we previously reported, we synthesized and characterized fluorescently-labeled β-CyDs [
17,
20]. Briefly, to introduce amino groups into HP-β-CyD for the synthesis of fluorescently-labeled HP-β-CyD, HP-β-CyD (100 mg, 71.9 μmol), 1,1-carbonyldiimidazol (98 mg, 604 μmol), and triethylamine (20 μL, 0.14 μmol) were dissolved in 1.2 mL of dehydrated DMSO and stirred for 1.5 h at room temperature. Ethylenediamine (6.8 μL, 1.0 μmol) was added to the reaction mixture and the solution was stirred for a further 5 h at room temperature under a nitrogen atmosphere. After the reaction, NH
2-HP-β-CyD was purified by the acetone precipitation method: the sample was gradually dropped into 50 mL of acetone. After centrifugation (10,000 rpm, 10 min), the precipitant was collected and dissolved in water. The recovered solution was freeze-dried to obtain NH
2-HP-β-CyD as powder. To synthesize HF647-labeled β-CyDs, Lac-β-CyD (100 mg, 34 μmol) and NH
2-HP-β-CyD (100 mg, 72 μmol) were dissolved in 10 mL and 1.5 mL of dehydrated DMSO, respectively. HF647 acid (500 μg, 384 nmol) was added to each reaction mixture and the solutions were stirred in the dark for 24 h at room temperature. HF647-labeled Lac-β-CyD and HF647-labeled HP-β-CyD were purified by the acetone precipitation method.
2.3. Cell Culture
The human hepatocellular carcinoma cell line, HepG2, was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Nissui Pharmaceuticals, Tokyo, Japan), containing penicillin (1 × 105 mU/mL) and streptomycin (0.1 mg/mL), and supplemented with 10% fetal bovine serum (FBS), in a humidified incubator at 5% CO2. NPC-like HepG2 cells containing high amounts of accumulated cholesterol were obtained after 48 h incubation with DMEM containing 1.25 μM U18666A.
2.4. Mice
Male and female homozygous (Npc1−/−) mutant (BALB/cNctr-Npc1m1N) mice were kindly donated by Dr. Kosaku Ohno and Dr. Katsumi Higaki. Wild-type (WT) BALB/c mice were obtained from Japan SLC, Inc. (Shizuoka, Japan). The mice were bred and kept in specific pathogen-free conditions in the Center for Animal Resources and Development, Kumamoto University. All animal experiments in this manuscript were carried out in accordance with the guidelines approved by the Ethics Committee for Animal Care and Use of Kumamoto University composed of a third party (Approval ID: A29-125).
2.5. In Vivo Fluorescence Imaging
The concentration of each of the HF647-β-CyDs in the serum and various organs, including the heart, lung, liver, kidney and spleen, was measured to evaluate the pharmacokinetics of Lac-β-CyD and HP-β-CyD after subcutaneous administration to Npc1−/− mice. Briefly, HF647-Lac-β-CyD or HF647-HP-β-CyD in PBS was administered to 8-week-old WT mice by subcutaneous injection, at a dose of 20 mg/kg or 10 mg/kg, respectively. To study the effect of ASGPR on the distribution of HF647-Lac-β-CyD, 500 mg/kg AF, a competitive inhibitor of ASGPR, was intraperitoneally administered to the mice, 1 min before and 5 min after administration of the labeled β-CyDs. An hour after administration of the test compounds, the mice were sacrificed and perfused with PBS. The organs were harvested, and fluorescent images of the organs were acquired using an IVIS Lumina XRMS in vivo Imaging System (Perkin Elmer Inc., Waltham, MA, USA).
2.6. Treatment of NPC Model Mice with CyDs
Lac-β-CyD or HP-β-CyD was dissolved in PBS to 38 mM, and the solution was filtered using DISMIC®-13HP 0.20 μm (Advantec, Toyo Roshi, Tokyo, Japan). For the acute study, either Lac-β-CyD (0.7 mmol/kg, 2098 mg/kg) or HP-β-CyD (0.7 mmol/kg, 1000 mg/kg) was administered to WT and Npc1−/− mice (8 week old) by subcutaneous injection (injection volume: 20 μL/g of body weight). The mice were anesthetized 24 h after dosing, and the blood and organs were collected. To study the effect of repeated dosing of the animals, Lac-β-CyD (0.7 mmol/kg, 2098 mg/kg) or HP-β-CyD (0.7 mmol/kg, 1000 mg/kg) was administered by subcutaneous injection at the same site to WT and Npc1−/− mice once a week from 4 to 8 weeks of age (injection volume: 20 μL/g of body weight). Blood and various organs were collected as described above 72 h after the final dose.
2.7. Biochemical and Histological Analysisβ
Levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in blood were determined using a FUJI DRI-CHEM 7000 automated clinical chemistry analyzer (FUJIFILM, Tokyo, Japan). After collection of the blood, the mice were infused with saline, and the brain, lungs, liver, spleen, and kidneys were harvested. The tissues were fixed with 10% neutral buffered formalin before being embedded in paraffin. The paraffin blocks were sectioned and stained with hematoxylin and eosin (H&E). For histological analysis, the sections were analyzed using a Biorevo BZ-9000 (Keyence, Osaka, Japan). To evaluate cholesterol levels in liver tissue, the liver samples were fixed in 4% paraformaldehyde (PFA) solution for 48 h at 4 °C. After 3 washes in PBS, the liver samples were incubated overnight in 30% sucrose in PBS, sectioned, and embedded in Tissue-Tek® O.T.C. Compound (Sakura Finetek, Tokyo, Japan) before freezing. The frozen sections were incubated in 1% BSA for 1 h at room temperature, and stained with 50 μg/mL of Filipin III solution (freshly dissolved in ethanol to 5 mg/mL and diluted in PBS) for 1 h at room temperature. After washing in PBS, the fluorescence derived from Filipin III in the liver sections was detected using confocal laser scanning microscopy (Leica TCS SP8, Leica, Wetzlar, Germany).
2.8. In Vivo Safety Evaluation of CyDs in WT Mice
Lac-β-CyD (8 mmol/kg, 23,632 mg/kg) or HP-β-CyD (8 mmol/kg, 11,120 mg/kg) was administered subcutaneously to WT mice (8 weeks old). The lungs, liver, and kidneys were collected 8 h later. To study the toxicity of the compounds to the lungs, the number of inflammatory cells and total protein concentration in the bronchoalveolar lavage fluid (BALF) were determined. Histological analysis of H&E-stained lung, liver, and renal tissues was performed to study the effects of the compounds on these tissues. In addition, the amounts of AST, ALT, blood urea nitrogen (BUN), and lactate dehydrogenase (LDH) in the blood were determined using a FUJI DRI-CHEM 7000 automated clinical chemistry analyzer (FUJIFILM, Tokyo, Japan).
2.9. Cellular Uptake Analysis
U18666A-treated HepG2 cells (5 × 104 cells/35 mm glass bottom dish) were transfected with ER-GFP BacMam 2.0 reagent, to label endoplasmic reticulum (ER) with green fluorescent protein (GFP), for 16 h. After 3 washes in PBS, the cells were exposed to culture medium containing HF647-β-CyDs (100 μM), in the presence or absence of AF (1.0 mg/mL), for 24 h. After washing in PBS, the cells were stained with 50 nM LysoTracker® Red DND-99 (to label acidic organelles) for 30 min at 37 °C. After washing in PBS, the fluorescence derived from HF647, ER-GFP, and LysoTracker® in U18666A-treated HepG2 cells was detected using confocal laser scanning microscopy (Leica TCS SP8, Leica, Wetzlar, Germany). The fluorescence intensity was quantified using ImageJ software.
2.10. Free Cholesterol Levels in Endolysosomes
U18666A-treated HepG2 cells (5 × 104 cells/35 mm glass bottom dish) were incubated in culture medium containing 1 mM β-CyDs, miglustat, or maltoheptaose in the presence or absence of AF (1.0 mg/mL), for 24 h. After washing in PBS, the cells were stained using 50 nM LysoTracker® Green DND-26 (to stain acidic cellular compartments) for 30 min at 37 °C. After washing in PBS, the cells were fixed in 4% PFA for 10 min at room temperature and stained with Filipin III (50 μg/mL), a fluorescent cholesterol-binding molecule, for 1 h at room temperature. After washing in PBS, the fluorescence derived from Filipin III and LysoTracker® in the cells was detected using confocal laser scanning microscopy (Leica TCS SP8, Leica, Wetzlar, Germany). The fluorescence intensity was quantified using ImageJ software.
2.11. Analysis of Cholesterol Solubilization by CyDs
In a phase solubility study, volumes of 0.2 mL of Lac-β-CyD or HP-β-CyD solution (0, 0.5, 1.0, 2.0, 2.5, 4.0, 5.0, 7.5, and 10 mM) were added to glass vials containing approximately 2 mg of cholesterol. The suspensions were vigorously shaken for 72 h at room temperature and filtered through a 0.20 μm DISMIC®-13HP to remove undissolved cholesterol. The cholesterol concentration was determined using a Cholesterol E-test Wako® (Osaka, Japan).
2.12. Hemolytic Activity of CyDs in Rabbit Red Blood Cells
Rabbit red blood cells (RBCs) were isolated from Japanese white male rabbits (Kyudo, Tosu, Japan) as described previously [
21,
22]. Isolated RBCs were centrifuged at 1000 g for 5 min and washed 3 times in PBS. Aliquots of RBC suspension in PBS were incubated in 1 mL of Lac-β-CyD or HP-β-CyD (0.1 and 1.0 mM in PBS) for 30 min at 37 °C. After centrifugation at 1000 g for 10 min, the optical density of the supernatant was measured at 543 nm. The results were expressed as a percentage of the total hemolysis obtained when the RBCs were incubated in water only.
2.13. Cytotoxicity of CyDs
Mitochondrial dehydrogenase activity was used to assess cell viability, and was measured using a water-soluble tetrazolium salt (WST-8) Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol. U18666A-treated HepG2 cells (5 × 104 cells/96 well plate) were treated in culture medium containing Lac-β-CyD or HP-β-CyD (at 10, 20, 30, and 40 mM) for 24 h, and then incubated in the WST-8 solution for 1 h at 37 °C. The absorbance at 450 nm was measured against a reference wavelength of 630 nm using a microplate reader (Bio-Rad iMark, Tokyo, Japan).
2.14. Data Analysis
Quantitative data are expressed as the mean ± standard error of the mean (S.E.M.), while the statistical comparisons were made using the Scheffe’s test. A p-value < 0.05 was considered statistically significant.
4. Discussion
In this study, the therapeutic effects of Lac-β-CyD on hepatomegaly in NPC model mice, and its safety in WT mice, were evaluated. Our in vivo fluorescence imaging analysis revealed that the accumulation of HF647-Lac-β-CyD in liver tissue after subcutaneous administration was significantly higher than that of HF647-HP-β-CyD (
Figure 1a). In addition, the accumulation of HF647-Lac-β-CyD was inhibited in the presence of AF, which is a competitive inhibitor of ASGPR, suggesting that Lac-β-CyD might accumulate in liver tissue through its interaction with ASGPR.
HF647-Lac-β-CyD also accumulated in the kidney. In general, water-soluble molecules, with a molecular weight <45,000 Da which are not bound to plasma protein are freely filtered by the glomerulus, resulting in renal excretion. Previously, Frijlink et al. reported that HP-β-CyD accumulates mainly in the kidney after subcutaneous injection, because of rapid renal clearance [
32]. Therefore, accumulation of Lac-β-CyD in the kidney may also be due to renal clearance. Histological analysis and determination of BUN levels in
Npc1−/− mice and WT mice after subcutaneous administration of Lac-β-CyD (
Figure 2a,
Figure 3a and
Figure 4g) indicated that Lac-β-CyD did not cause any significant toxicological effects on the kidney. Furthermore, Lac-β-CyD had a weak interaction with cholesterol (
Figure 7a) and low cytotoxicity in NPC-like liver cells (
Figure 7c), suggesting that Lac-β-CyD is a safer drug candidate for NPC treatment. Unfortunately, no studies have been conducted on the integrity and functional status of the regional lymph nodes following the subcutaneous injection of Lac-β-CyD, nor on Kupffer cell and splenic macrophage function. Further elaborate studies on these biological functions are necessary to expand the application of Lac-β-CyD to clinical stage.
The
Npc1−/− mice exhibit various phenotypes, such as vacuolization of several tissues and elevation of ALT as hepatic injury markers. In our study, a number of vacuoles were observed in histological sections of the liver tissue of
Npc1−/− mice, probably due to free cholesterol accumulation in hepatocytes (
Figure 2 and
Figure 3). Lac-β-CyD lowered free cholesterol accumulation in the hepatocytes (
Figure 2d and
Figure 3d), resulting in a decreased number of vacuoles in the liver tissue (
Figure 2a and
Figure 3a) and an improvement in the hepatic injury markers of ALT (
Figure 2b,c and
Figure 3b,c). In addition, we demonstrated that Lac-β-CyD accumulated in the liver after subcutaneous administration (
Figure 1). Furthermore, our in vitro cellular uptake study suggests that Lac-β-CyD can enter the hepatocytes through its interaction with ASGPR, specifically expressed on hepatocytes (
Figure 5). We postulate that Lac-β-CyD reduces vacuolization in the liver tissue of
Npc1−/− mice due to its cholesterol lowering effect on hepatocytes after uptake into the cells. Meanwhile, these results may correlate with toxicity reported in
Figure 7. It can be thought that 8 mmol/kg of HP-β-CyD is more toxic than Lac-β-CyD but maybe HP-β-CyD is more efficient to trap the membrane cholesterol.
This hepatocyte-selective cellular uptake may enable a reduction in the dose of Lac-β-CyD required for effective treatment of hepatomegaly in NPC. We demonstrated that Lac-β-CyD enters hepatocytes by clathrin-mediated endocytosis via ASGPR, and is promptly internalized into endolysosomes in NPC-like liver cells, in contrast to HP-β-CyD (
Figure 5). This suggests that a lower dose of Lac-β-CyD than that of HP-β-CyD may be possible for the treatment of hepatomegaly in NPC. However, the mechanism underlying the free cholesterol lowering effect of Lac-β-CyD is still unclear. In general, ligands internalized via ASGPR or LDL receptor-mediated endocytosis are dissociated when protons flow into the endosomes at a pH range of 6.0 to 6.5. The dissociated receptors are recycled and the ligands reach the endolysosomes [
33,
34]. Furthermore, it has been reported that 6-
O-α-maltosyl-β-CyD is slightly internalized into endolysosomes in NPC model cells, and is released to the extracellular fluid as a complex of 6-
O-α-maltosyl-β-CyD and free cholesterol [
30]. Therefore, it may be possible that after internalization of Lac-β-CyD into hepatocytes by ASGPR-mediated endocytosis, Lac-β-CyD may dissociate from ASGPR and solubilize cholesterol in endolysosomes through complex formation, then finally exit from endolysosomes as a complex with cholesterol. Notably, we found that Lac-β-CyD was partially co-localized with both endolysosomes and the endoplasmic reticulum (ER) (
Figure 5). Recently, Kant et al. demonstrated that cholesterol transfer from endosomes to the ER and other organelles is predominantly mediated by lipid-binding proteins that localize at membrane contact sites [
35]. In addition, it is reported that free cholesterol in endolysosomes is transferred to the ER via membrane contact sites and is esterified by acyl-CoA cholesterol acyltransferase (ACAT) [
36,
37]. However, in NPC bearing a spontaneous mutation of the
Npc1 or
Npc2 gene, there is a defect in cholesterol trafficking from the endolysosomes to the ER. Therefore, Lac-β-CyD may improve cholesterol transfer from the endolysosomes to the ER via membrane contact sites. Further elaborate studies are necessary to elucidate not only the role of Lac-β-CyD in cholesterol transfer in cells but also the ability of the Lac-β-CyD to trap the cellular cholesterol located to the membrane.