Successful Treatment for Hepatoblastoma in Trisomy 18: A Case Report.

Children with trisomy 18 tend to develop hepatoblastoma. Since the introduction of appropriate management for organ malfunction, individuals with trisomy 18 have come to have a longer life expectancy. However, the predisposition to hepatoblastoma becomes a significant issue for the quality of a case. Here, we present a rare multifocal hepatoblastoma involving predominantly Couinaud segments 5 and 7 in a 10-month-old boy with trisomy 18. Though the first-line cisplatin monotherapy resulted in unsatisfactory tumor shrinkage, the second-line neoadjuvant chemotherapy administrating irinotecan and vincristine gave rise to significant tumor reduction in volume, leading to the completion of partial resection of the liver without the microscopic residual disease. The patient has been free from recurrence for 44 months. Because anatomical right hepatectomy can cause circulatory instability, including acute onset of pulmonary hypertension in trisomy 18 patients, physicians should balance treatment benefits and potential adverse effects. Our successful experience utilizing a combination of efficacious and less cardiotoxic neoadjuvant chemotherapy followed by the partial hepatectomy encourages physicians to treat a patient with trisomy 18 and tackle hepatoblastoma with a genetic background.

Sustained Release Formulation of Hydroxypropyl-ß-cyclodextrin Eye Drops Using Xanthan Gum.

Bietti's crystalline dystrophy (BCD) is an autosomal recessive chorioretinal degeneration caused by mutations in the CYP4V2 gene. It is characterized by cholesterol accumulation and crystal-like deposits in the retinas. Hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) exerts therapeutic effects against BCD by reducing lysosomal dysfunction and inhibiting cytotoxicity in induced pluripotent stem cell (iPSC)-RPE cells established from patient-derived iPS cells. However, the ocular retention of HP-ß-CyD is low and needs to be improved. Therefore, this study used a viscous agent to develop a sustained-release ophthalmic formulation containing HP-ß-CyD. Our results suggest that HP-ß-CyD-containing xanthan gum has a considerably higher sustained release capacity than other viscous agents, such as methylcellulose and sodium alginate. In addition, the HP-ß-CyD-containing xanthan gum exhibited pseudoplastic behavior. It was less cytotoxic to human retinal pigment epithelial cells compared with HP-ß-CyD alone. Furthermore, the slow release of HP-ß-CyD from xanthan gum caused a sustained decrease in free intracellular cholesterol. These results suggest that xanthan gum is a useful substrate for the sustained release formulation of HP-ß-CyD, and that HP-ß-CyD-containing xanthan gum has potential as an eye drop for BCD treatment.

Folate-Appended Hydroxypropyl-ß-Cyclodextrin Induces Autophagic Cell Death in Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a heterogenous myeloid neoplasm that remains challenging to treat. Because intensive conventional chemotherapy reduces survival rates in elderly patients, drugs with lower toxicity and fewer side effects are needed urgently. 2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) is used clinically as a pharmaceutical excipient for poorly water-soluble drugs. Previously, we showed that HP-ß-CyD exerts antitumor activity by disrupting cholesterol homeostasis. Recently, we developed folate-conjugated HP-ß-CyD (FA-HP-ß-CyD) and demonstrated its potential as a new antitumor agent that induces not only apoptosis, but also autophagic cell death; however, we do not know whether FA-HP-ß-CyD exerts these effects against AML. Here, we investigated the effects of FA-HP-ß-CyD on folate receptor (FR)-expressing AML cells. We found that the cytotoxic activity of FA-HP-ß-CyD against AML cells was stronger than that of HP-ß-CyD. Also, FA-HP-CyD induced the formation of autophagosomes in AML cell lines. FA-HP-ß-CyD increased the inhibitory effects of cytarabine and a BCL-2-selective inhibitor, Venetoclax, which are commonly used treat elderly AML patients. Notably, FA-HP-ß-CyD suppressed the proliferation of AML cells in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double-deficient mice with AML. These results suggest that FA-HP-ß-CyD acts as a potent anticancer agent for AML chemotherapy by regulating autophagy.

Colletofragarone A2 Inhibits Cancer Cell Growth In Vivo and Leads to the Degradation and Aggregation of Mutant p53.

Mutant p53 not only loses its original tumor suppressor function but also acquires new abilities regarding oncogenic progression. Therefore, the strategy of targeting mutant p53 has attracted attention for cancer therapy. We isolated colletofragarone A2 (CF) from the fungus Colletotrichum sp. (13S020), which decreases mutant p53 levels in cells, and herein examine its effect on mutant p53. CF showed more potent cytotoxic activities on cells with p53R175H structural mutants than those with different p53 statuses such as a DNA-contact mutant, wild-type, and null cells. CF markedly decreased tumor cell growth in vivo using a mouse xenograft model with HuCCT1 (p53R175H) cells. Cotreatment of SK-BR-3 (p53R175H) cells with CF and cycloheximide decreased mutant p53 levels by promoting p53 degradation. In the presence of MG-132, CF induced the accumulation of the aggregated mutant p53. These results suggest that CF inhibits the function of molecular chaperones such as HSP90.

The Effects of Sacran, a Sulfated Polysaccharide, on Gut Microbiota Using Chronic Kidney Disease Model Rats.

The aim of this study was to investigate the beneficial effects of sacran, a sulfated polysaccharide, on renal damage and intestinal microflora, in 5/6 nephrectomy rats as a model for chronic kidney disease (CKD). 5/6 Nephrectomy rats were divided into sacran treated and non-treated groups and examined for lethality after 4 weeks. The 5/6 nephrectomy rats were also divided into three groups: sacran treated, non-treated and AST-120 treated groups, and treated orally in a concentration-dependent manner for 4 weeks. Renal function was estimated by biochemical and histopathological analyses. Metagenomic analysis of feces from each group after 4 weeks was also performed and changes in intestinal microflora were compared. The administration of sacran to CKD rats at ≥19 mg/d increased their survival. In addition, the sacran-treated group improved CKD-related parameters in a concentration-dependent manner, and the inhibitory effect of 40 mg/d of sacran was comparable to that of AST-120. The changes in the intestinal microflora of the sacran treated group were positively correlated with an increase in the number of Lactobacillus species, which are known to be rich in beneficial bacteria, and the increment of this beneficial bacteria was negatively correlated with the concentration of indoxyl sulfate, a uremic toxin, in plasma. These results strongly suggest that the oral administration of sacran could contribute to the stabilization of intestinal microflora in CKD rats and to the reduction of oxidative stress as well as the inhibition of progression of CKD.

Feasibility Study of Dendrimer-Based TTR-CRISPR pDNA Polyplex for Ocular Amyloidosis in Vitro.

Hereditary amyloidgenic transthyretin (ATTR) amyloidosis is caused by a genetic point-mutated transthyretin such as TTR Val30Met (TTR V30M), since it forms protein aggregates called amyloid resulting in the tissue accumulation and functional disorders. In particular, ATTR produced by retinal pigment epithelial cells often causes ATTR ocular amyloidosis, which elicits deterioration of ocular function and ultimately blindness. Therefore, development of novel therapeutic agents is urgently needed. Genome-editing technology using Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas9) system is expected to be a therapeutic approach to treat genetic diseases, such as ATTR amyloidosis caused by a point mutation in TTR gene. Previously, we reported that glucuronylglucosyl-ß-cyclodextrin conjugated with a polyamidoamine dendrimer (CDE) had excellent gene transfer ability and that underlying dendrimer inhibited TTR aggregation. Conversely, folate receptors are known to be highly expressed in retina; thus, folate has potential as a retinal target ligand. In this study, we prepared a novel folate-modified CDE (FP-CDE) and investigated its potential as a carrier for the retinal delivery of TTR-CRISPR plasmid DNA (pDNA). The results suggested that FP-CDE/TTR-CRISPR pDNA could be taken up by retinal pigment epithelial cells via folate receptors, exhibited TTR V30M amyloid inhibitory effect, and suppressed TTR production via the genome editing effect (knockout of TTR gene). Thus, FP-CDE may be useful as a novel therapeutic TTR-CRISPR pDNA carrier in the treatment of ATTR ocular amyloidosis.

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.

Hyaluronic Acid-Decorated Glycol Chitosan Nanoparticles for pH-Sensitive Controlled Release of Doxorubicin and Celecoxib in Nonsmall Cell Lung Cancer.

Nonsmall cell lung cancer (NSCLC) is a leading cause of global cancer mortality. Recently, combinatorial treatment approaches have shown promise as they better address tumor heterogeneity. However, drug pharmacokinetics and tissue distribution differences remain problematic. To overcome these issues and improve therapeutic efficacies, the use of nanomedicines has been suggested. We devised a CD44 receptor target hyaluronic acid (HA)-decorated glycol chitosan (GC) nanoparticle which is conjugated to doxorubicin (DOX) by a pH-sensitive linker and coloaded celecoxib (CXB; HA-GC-DOX/CXB). Successful chemical conjugation of GC to DOX was confirmed and HA-GC-DOX/CXB showed ∼150 nm of uniform spherical shape. HA-GC-DOX/CXB were stable at pH 7.4 but steadily increased in size and released drugs at pH 6.0 and 4.0. In vitro NSCLC cells showed that DOX and CXB combination therapy has synergism in both free drug and nanoparticle formulation. In vivo NSCLC xenograft mice showed DOX and CXB exhibited a synergistic tumor suppressive effect in HA-GC-DOX/CXB. Furthermore, HA-GC-DOX/CXB dramatically inhibited tumor growth compared to other treatments as well as suppressed inflammation and metastasis-related gene/protein in the tumor tissues. Our findings demonstrate HA-GC-DOX/CXB is a potential anticancer therapy that controlled release under acidic tumor microenvironments and enhanced CD44 overexpressed tumor target efficacy.

Improvement of Pharmaceutical Properties of Zerumbone, a Multifunctional Compound, Using Cyclodextrin Derivatives.

Zerumbone is a multifunctional compound which shows various biological activities, such as antitumor activity, anti-inflammatory activity, antiulcer activity, etc. However, to use Zerumbone as functional foods or medicines, its pharmaceutical properties such as solubility should be improved. In the present study, we prepared its inclusion complexes with various cyclodextrin (CyD) derivatives, and evaluated their solubility, release profile of the drug and cytotoxic activity. Among 11 CyDs, sulfobutylether (SBE)-ß-CyD showed the highest solubilizing effect for Zerumbone. Phase solubility diagrams of SBE-ß-CyD/Zerumbone in 10% methanol solution showed AL type, and the stability constant was 756 M-1. SBE-ß-CyD also formed the solid complex with Zerumbone by kneading for 90 min. Importantly, the dissolution rate of Zerumbone was improved by complexation with SBE-ß- and hydroxypropyl (HP)-ß-CyDs, and its supersaturation was maintained for several hours. The solubilizing effects by SBE-ß-CyD was greater than that of HP-ß-CyD. Moreover, SBE-ß-CyD/Zerumbone complex also retained the cytotoxic activity of Zerumbone. These results suggest that CyDs, especially SBE-ß-CyD, were useful to improve the solubility of Zerumbone.

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.

Hepatocyte-Targeted Delivery of siRNA Polyplex with PEG-Modified Lactosylated Dendrimer/Cyclodextrin Conjugates for Transthyretin-Related Amyloidosis Therapy.

Targeted drug delivery system (DDS) is required for RNA interference (RNAi) therapy to increase the therapeutic effect and to reduce the adverse effect. Especially in transthyretin (TTR)-related amyloidosis, hepatocyte specific delivery is desired because TTR mainly expresses in hepatocyte. Herein, we report on a hepatocyte-specific small interfering RNA (siRNA) delivery system using polyethylene glycol (PEG)-modified lactosylated dendrimer (generation 3; G3) conjugates with α-cyclodextrin (PEG-LαCs (G3)) for TTR-related amyloidosis therapy, and investigated the in vitro and in vivo gene silencing effect of PEG-LαCs (G3)/siRNA polyplexes. PEG-LαC (G3, average degree of substitution of PEG (DSP) 2)/TTR siRNA (siTTR) polyplex exhibited the asialoglycoprotein receptor (ASGPR)-mediated cellular uptake, high endosomal escaping ability and localization of the siRNA in cytoplasm, resulting in significant TTR silencing in HepG2 cells. In vivo studies showed that PEG-LαC (G3, DSP2)/siTTR polyplex led to a significant TTR silencing effect in liver after systemic administration to mice. Furthermore, safety evaluation revealed that PEG-LαC (G3, DSP2)/siTTR polyplex had no significant toxicity both in vitro and in vivo. These findings suggest the utility of PEG-LαC (G3, DSP2) as a promising hepatocyte-specific siRNA delivery system both in vitro and in vivo, and as a therapeutic approach for TTR-related amyloidosis.

A comprehensive understanding of lowly-hydrolyzed polyvinyl alcohol-based ternary solid dispersions with the use of a combined mixture-process design.

We recently reported lowly hydrolyzed polyvinyl alcohol (L-PVA, 70-74% hydrolyzed, about 580 polymerized, JR-05) as a promising matrix for hot-melt extrusion (HME) due to its unique micelle formation ability compared to the most commonly used PVA (87-89% hydrolyzed, about 580 polymerized). In the present study, we focused on the effect of composition [indomethacin (IND), L-PVA, sorbitol] and process parameters (temperature and screw speed) on each response, i.e. processing torque, and physicochemical properties such as residual crystallinity, residual ratio, and area under the dissolution curve (AUDC) in supersaturated solution using a HME by applying the design of experiment (DoE) approach. To overcome the poor processability of L-PVA, given its semicrystalline nature, we applied sorbitol as a plasticizer and systematically and simultaneously evaluated its influence on the outputs based on the mixture design combined with process factors. Few studies have focused on comprehensive evaluation of the composition and HME process conditions because obtaining a design space requires numerous experiments. We found that incorporating sorbitol into the L-PVA greatly improved the processing torque. However, sorbitol negatively influenced the degree of residual crystallinity and the AUDC of IND. Lastly, we established a laboratory-scale design space that could achieve high supersaturation and ensure adequate miscibility between each component, using an acceptable processing torque for HME, by applying the minimum amount of sorbitol. These fundamental results suggest that sorbitol maximizes the potency of L-PVA as a carrier in HME.

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.

Supramolecular Pharmaceutical Sciences: A Novel Concept Combining Pharmaceutical Sciences and Supramolecular Chemistry with a Focus on Cyclodextrin-Based Supermolecules.

Supramolecular chemistry is an extremely useful and important domain for understanding pharmaceutical sciences because various physiological reactions and drug activities are based on supramolecular chemistry. However, it is not a major domain in the pharmaceutical field. In this review, we propose a new concept in pharmaceutical sciences termed "supramolecular pharmaceutical sciences," which combines pharmaceutical sciences and supramolecular chemistry. This concept could be useful for developing new ideas, methods, hypotheses, strategies, materials, and mechanisms in pharmaceutical sciences. Herein, we focus on cyclodextrin (CyD)-based supermolecules, because CyDs have been used not only as pharmaceutical excipients or active pharmaceutical ingredients but also as components of supermolecules.

Supramolecular Complex of Methyl-ß-cyclodextrin with Adamantane-Grafted Hyaluronic Acid as a Novel Antitumor Agent.

Methyl-ß-cyclodextrin (M-ß-CyD) exhibits cytotoxic activity, and has the potentials as an antitumor agent. However, a tumor-selectivity of M-ß-CyD is low, leading to low antitumor activity and the adverse effects. Meanwhile, hyaluronic acid (HA) is known as a promising tumor targeting ligand, because various cancer cells overexpress CD44, a HA-binding glycoprotein. In the present study, to develop a tumor-selective delivery system for M-ß-CyD, we designed a supramolecular complex of M-ß-CyD with adamantane-grafted HA (Ad-HA/M-ß-CyD) and evaluated it as a tumor-selective antitumor agent. M-ß-CyD formed a stable complex with Ad-HA (Kc>104 M-1). In addition, Ad-HA/M-ß-CyD formed slightly a negative-charged nanoparticle with ca. 140 nm of a particle size, indicating the favorable physicochemical properties for antitumor agents. Ad-HA/M-ß-CyD showed the superior cytotoxic activity via CD44-mediated endosomal pathways in HCT116 cells (CD44(+)), a human colon cancer cell line. In addition, cytotoxic activity of Ad-HA/M-ß-CyD was induced by apoptosis. These results suggest that Ad-HA/M-ß-CyD has the potentials as a tumor-selective supramolecular antitumor agent.

Self-Assembly PEGylation Retaining Activity (SPRA) Technology via a Host-Guest Interaction Surpassing Conventional PEGylation Methods of Proteins.

Polyethylene glycol (PEG) modification (PEGylation) is one of the best approaches to improve the stabilities and blood half-lives of protein drugs; however, PEGylation dramatically reduces the bioactivities of protein drugs. Here, we present "self-assembly PEGylation retaining activity" (SPRA) technology via a host-guest interaction between PEGylated ß-cyclodextrin (PEG-ß-CyD) and adamantane-appended (Ad) proteins. PEG-ß-CyD formed stable complexes with Ad-insulin and Ad-lysozyme to yield SPRA-insulin and SPRA-lysozyme, respectively. Both SPRA-proteins showed high stability against heat and trypsin digest, comparable with that of covalently PEGylated protein equivalents. Importantly, the SPRA-lysozyme possessed ca. 100% lytic activity, whereas the activity of the covalently PEGylated lysozyme was ca. 23%. Additionally, SPRA-insulin provided a prolonged and peakless blood glucose profile when compared with insulin glargine. It also showed no loss of activity. In contrast, the covalently PEGylated insulin showed a negligible hypoglycemic effect. These findings indicate that SPRA technology has potential as a generic method, surpassing conventional PEGylation methods for proteins.

Potential Use of Cyclodextrins as Drug Carriers and Active Pharmaceutical Ingredients.

Cyclodextrins (CyDs) are extensively used in various fields, and especially have been widely utilized as pharmaceutical excipients and drug carriers in the pharmaceutical field. Owing to the multi-functional and biocompatible characteristics, CyDs can improve the undesirable properties of drug molecules. This review outlines the current application of CyDs in pharmaceutical formulations, focusing on their use as CyD-based drug carriers for several kinds of drugs. Additionally, CyDs have great potential as active pharmaceutical ingredients against various diseases with few side effects.

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.

In vitro evaluation of 2-hydroxyalkylated ß-cyclodextrins as potential therapeutic agents for Niemann-Pick Type C disease.

This study was conducted to evaluate the attenuating potential of 2-hydroxypropyl-ß-cyclodextrin (HPBCD) against Niemann-Pick Type C (NPC) disease, as well as the physical and chemical properties, particularly the cholesterol-solubilizing ability, in an NPC disease model in vitro. As parameters of NPC abnormalities, intracellular free and esterified cholesterol levels and lysosome volume were measured in Npc1 null Chinese hamster ovary cells. HPBCD showed dose-dependent effects against dysfunctional intracellular cholesterol trafficking, such as the accumulation and shortage of free and esterified cholesterols, respectively, in Npc1 null cells. However, the effectiveness was gradually offset by exposure to ≥8mM HPBCD. The same effect was also observed for increasing lysosome volume in Npc1 null cells. The degree of substitution of the hydroxypropyl group had little influence on the attenuating effects of HPBCD against the NPC abnormalities, at least in the range between 2.8 and 7.4. Next, we compared the effects of other hydroxyalkylated ß-cyclodextrin derivatives with different cholesterol-solubilizing abilities, such as 2-hydroxyethyl-ß-cyclodextrin (HEBCD) and 2-hydroxybutyl-ß-cyclodextrin (HBBCD). The cholesterol solubilizing potential, attenuating effects against NPC abnormalities and cytotoxicity induction were HBBCD≫HPBCD>HEBCD, HBBCD=HPBCD>HEBCD and HBBCD≫HPBCD=HEBCD, respectively. HPBCD may be superior in terms of safety and efficacy in Npc1 null cells compared with HEBCD and HBBCD. The results of this study will provide a rationale for the optimization of HPBCD therapy for NPC disease.

In Vitro and In Vivo Tumor-Targeting siRNA Delivery Using Folate-PEG-appended Dendrimer (G4)/α-Cyclodextrin Conjugates.

We previously reported that folate-polyethylene glycol (PEG)-appended dendrimer (generation 3)/α-cyclodextrin conjugate (Fol-PαC (G3)) shows folate receptor-α (FR-α)-overexpressing tumor cell-selective in vitro siRNA transfer activity. However, Fol-PαC (G3)/siRNA complex did not induce a significant in vivo RNAi effect after intravenous administration to tumor-bearing mice, possibly resulting from immediate dissociation of the complex in blood. Herein, to develop the novel siRNA carrier having high blood circulating ability, high in vivo siRNA transfer activity, and high safety profile, we newly prepared Fol-PαCs with higher generation (G4) and evaluated their potential as tumor-targeting siRNA carriers in vitro and in vivo. Fol-PαC (G4, average degree of substitution of α-cyclodextrin (DSC) 2.9, average degree of substitution of folate-PEG (DSF) 2)/siRNA complex had the prominent RNAi effect through adequate physicochemical properties, FR-α-mediated endocytosis, efficient endosomal escape, and siRNA delivery to cytoplasm with negligible cytotoxicity. Importantly, Fol-PαC (G4, DSC2.9, DSF2) improved the serum stability, blood circulating ability, and in vivo RNAi effects of siRNA, compared to Fol-PαC (G3). Furthermore, Fol-PαC (G4, DSC2.9, DSF2) complex with siRNA against Polo-like kinase 1 (siPLK1) suppressed the tumor growth compared to control siRNA complex. These results suggest that Fol-PαC (G4, DSC2.9, DSF2) has the potential as a novel tumor-targeting siRNA carrier in vitro and in vivo.