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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-β-cyclodextrin (HP-β-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-β-CyD is low and needs to be improved. Therefore, this study used a viscous agent to develop a sustained-release ophthalmic formulation containing HP-β-CyD. Our results suggest that HP-β-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-β-CyD-containing xanthan gum exhibited pseudoplastic behavior. It was less cytotoxic to human retinal pigment epithelial cells compared with HP-β-CyD alone. Furthermore, the slow release of HP-β-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-β-CyD, and that HP-β-CyD-containing xanthan gum has potential as an eye drop for BCD treatment.

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-β-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.

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.

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)-β-CyD showed the highest solubilizing effect for Zerumbone. Phase solubility diagrams of SBE-β-CyD/Zerumbone in 10% methanol solution showed A L type, and the stability constant was 756 M -1 . SBE-β-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-β- and hydroxypropyl (HP)-β-CyDs, and its supersaturation was maintained for several hours. The solubilizing effects by SBE-β-CyD was greater than that of HP-β-CyD. Moreover, SBE-β-CyD/Zerumbone complex also retained the cytotoxic activity of Zerumbone. These results suggest that CyDs, especially SBE-β-CyD, were useful to improve the solubility of Zerumbone.

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.

Methyl-β-cyclodextrin (M-β-CyD) exhibits cytotoxic activity, and has the potentials as an antitumor agent. However, a tumor-selectivity of M-β-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-β-CyD, we designed a supramolecular complex of M-β-CyD with adamantane-grafted HA (Ad-HA/M-β-CyD) and evaluated it as a tumor-selective antitumor agent. M-β-CyD formed a stable complex with Ad-HA (K c >10 4  M -1 ). In addition, Ad-HA/M-β-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-β-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-β-CyD was induced by apoptosis. These results suggest that Ad-HA/M-β-CyD has the potentials as a tumor-selective supramolecular antitumor agent.

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.

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.

Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal storage disorder, which is an inherited disease characterized by the accumulation of unesterified cholesterol in endolysosomes. Recently, 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) has been used for the treatment of NPC, and ameliorated a hepatosplenomegaly in the patients. However, to obtain the treatment efficacy, a high dose of HP-β-CyD was necessary. Therefore, the decrease in dose by using active intracellular delivery system of β-CyD to NPC cells is expected. In this study, to efficiently deliver β-CyD to NPC-like cells, we newly synthesized octaarginine (R8)-appended β-CyD with a spacer of γ-aminobutyric acid (R8-β-CyD) and evaluated its cytotoxicity, intracellular distribution, endocytosis pathway and cholesterol-lowering effect in Npc1-trap-Chinese hamster ovary (CHO) cells, cholesterol-accumulated cells through the impairment of NPC1 function. R8-β-CyD did not show cytotoxicity in the cells. In addition, Alexa568-labeled R8-β-CyD was actively internalized into Npc1-trap-CHO cells, possibly through micropinocytosis. Notably, R8-β-CyD significantly decreased intracellular cholesterol content compared with HP-β-CyD. These results suggest that R8-β-CyD may be a promising therapeutic agent for ameliorating cholesterol accumulation in NPC.

The goal of this study was to investigate the topical anti-inflammatory effects of the megamolecular polysaccharide sacran extracted from cyanobacterium Aphanothece sacrum using various inflammatory animal models. Sacran showed potent anti-inflammatory effects with optimum effective concentrations at 0.01 and 0.05% (w/v). Sacran markedly inhibited paw swelling and neutrophil infiltration in carrageenan-induced rat paw edema. Additionally, 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionyl-carboxylic acid (DMEQ)-labeled sacran had the ability to penetrate carrageenan-induced rat paw skin rather than normal skin. Also, sacran significantly suppressed kaolin-induced and dextran-induced rat paw edema throughout the duration of the study. Furthermore, sacran significantly suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema and mRNA expression levels of cyclooxygenase (COX)-2 as well as pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. Safety of sacran solution was verified by negligible cytotoxicity in HaCaT cells. These results suggest that sacran may be useful as a therapeutic agent against inflammatory skin diseases with no life-threatening adverse effects.

The purpose of this study was to design cyclodextrin (CyD)-based pseudorotaxane-like supramolecular complexes with various isoprenoid compounds, such as reduced coenzyme Q10 (R-CoQ10), squalene, tocotrienol, and teprenone, and to evaluate their pharmaceutical properties. Squalene, tocotrienol, and teprenone formed precipitates with β-CyD and γ-CyD in aqueous solution, whereas R-CoQ10 formed precipitates with γ-CyD aqueous solution. The results of powder X-ray diffraction and (1)H-NMR analyses indicated that these precipitates are derived from pseudorotaxane-like supramolecular complexes. The photostability of teprenone was markedly improved by complexation with CyDs, especially in the γ-CyD system. In addition, the dispersion rates of teprenone in the γ-CyD system were higher than those in the β-CyD system, compared with the corresponding physical mixtures. In conclusion, pharmaceutical properties such as photostability and dispersion rates of isoprenoid compounds were improved by the formation of pseudorotaxane-like supramolecular complexes with β-CyD and/or γ-CyD.

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-β-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.

Melanoma is a life-threatening disorder and its incidence is increasing gradually. Despite the numerous treatment approaches, conventional systemic chemotherapy has not reduced the mortality rate among melanoma patients, probably due to the induction of toxicity to normal tissues. Recently, we have developed folate-conjugated methyl-β-cyclodextrin (FA-M-β-CyD) and clarified its potential as a new antitumor agent involved in autophagic cell death. However, it remains uncertain whether FA-M-β-CyD exerts anticancer effects against melanomas. Therefore, in this study, we investigated the effects of FA-M-β-CyD on the folate receptor-α (FR-α)-expressing melanoma cell-selective cytotoxic effect. FA-M-β-CyD showed cytotoxic effects in Ihara cells, a human melanoma cell line expressing FR-α. In sharp contrast to methyl-β-cyclodextrin, FA-M-β-CyD entered Ihara cells [FR-α(+)] through FR-α-mediated endocytosis. Additionally, FA-M-β-CyD elicited the formation of autophagosomes in Ihara cells. Notably, FA-M-β-CyD suppressed melanoma growth in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double deficient mice bearing Ihara cells. Therefore, these results suggest that FA-M-β-CyD could be utilized as a potent anticancer agent for melanoma chemotherapy by regulating autophagy.

A megamolecular polysaccharide sacran was newly extracted from cyanobacterium Aphanothece sacrum. Sacran has many preferable properties for transdermal application, e.g. a safe biomaterial, a high moisturizing effect, a formation of film and hydrogel. Additionally, it was recently discovered that sacran has an anti-inflammatory effect for atopic dermatitis model mice. In this study, in order to evaluate the feasibility of sacran-hydrogel as a novel sustained release system, we prepared a sacran-hydrogel containing 4-biphenyl acetic acid (BPAA, an acidic drug), prednisolone (PD, a neutral drug) or chlorpheniramine maleate (CPM, a basic drug), and performed the in vitro release studies. The sacran-hydrogel containing BPAA, PD or CPM provided a sustained release profile in accordance with a quasi-Fickian diffusion model. Furthermore, the release rate of drugs from sacran-hydrogels can be controlled by adjusting the concentration of aluminum chloride as a cross linker. These results suggest the potential use of sacran-hydrogel as a sustained release system for drugs.

In this study, we demonstrated the potential use of polypseudorotaxanes (PPRXs) of polyethylene glycol (PEG, molecular weight: 2000)-grafted polyamidoamine dendrimer (PEG-dendrimer) with cyclodextrins (CyDs) as novel sustained release systems for plasmid DNA (pDNA). PEG-dendrimer/pDNA complex formed PPRXs with α-CyD and γ-CyD solutions, but not with β-CyD solution. In the PEG-dendrimer/CyDs PPRXs systems, 17.9 mol of α-CyD and 8.8 mol of γ-CyD were involved in the PPRXs formation with one PEG chain by α-CyD and γ-CyD, respectively. In addition, the CyDs PPRX formation provided the sustained release of pDNA from PEG-dendrimer complex with pDNA at least 72 h in vitro. In addition, the release of pDNA from CyDs PPRX retarded as the dissolution medium volume decreased. These results suggest that the PEG-dendrimer/CyD PPRX systems can work as a sustained DNA release system, and the PPRX formation with CyDs may be useful as a sustained drug delivery technique for other pegylated polymers.

To expand the application of a drug in orally disintegrating tablets, the potential use of beta-cyclodextrin (beta-CyD) and 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) as excipients for the tablets containing dl-alpha-tocopheryl acetate (VE), an oily drug, was evaluated. HP-beta-CyD, not beta-CyD, solubilized VE in water through the formation of higher order of complexes at the molar ratio of 1 : 2 (VE : HP-beta-CyD). When prepared under the optimal preparation conditions, the VE tablets containing lactose and 5% (w/w) of HP-beta-CyD, not beta-CyD, had high hardness more than 4 kg and rapid disintegration within 100 s both in vitro and in vivo. In addition, VE tablets containing lactose and 5% (w/w) of HP-beta-CyD, not beta-CyD, maintained the high hardness and rapid disintegration under the accelerated stability test using different conditions for 4 weeks. Therefore, these results suggest the potential use of HP-beta-CyD, not beta-CyD, as an excipient for orally disintegrating tablets containing VE, an oily drug, in the molding method.

In this study, we prepared four kinds of complexes of Coenzyme Q10 (CoQ10) with gamma-cyclodextrin (gamma-CyD) by the kneading methods and the solubility methods with or without heating, and compared their pharmaceutical properties. Differential scanning calorimetrical curves and powder X-ray diffraction patterns showed that the complexes formed pseudorotaxane-like supramolecular structure, although included free gamma-CyD and CoQ10, when prepared by the kneading method and solubility method without heating. At the preparation process, a heating improved the complexation of CoQ10 with gamma-CyD in the both methods. The dispersion rate of CoQ10 in water increased in the order of CoQ10 alone approximately physical mixture with gamma-CyD<

Pegylation technology has been widely used to improve therapeutic efficacies of protein drugs and a number of selective- or randomly-substituted pegylated proteins are on the market. In this study, we prepared a insulin derivative substituted randomly with poly(ethylene glycol) (PEG, MW about 2200) and its polypseudorotaxanes with cyclodextrins (CyDs). The pegylated insulin formed polypseudorotaxanes with alpha- and gamma-CyDs, by inserting one PEG chain in the alpha-CyD cavity and two PEG chains in the gamma-CyD cavity. The pegylated insulin/CyD polypseudorotaxanes were less soluble in water. The release rate of the pegylated protein from its polypseudorotaxanes decreased in the order of drug alone>the gamma-CyD polypseudorotaxane>the alpha-CyD polypseudorotaxane. The pegylated insulin/gamma-CyD polypseudorotaxane displayed a significantly higher resistance to proteolysis. The results indicated that the CyD polypseudorotaxanes could be formed with randomly-pegylated insulin and work not only as a sustained release system, but also as a stabilizing agent to enzymatic degradations of pegylated insulin.

Lipid rafts on cell membranes have heterogeneity such as cholesterol-rich microdomains and sphingolipids-rich microdomains. We previously reported that beta-cyclodextrin (beta-CyD) induced morphological changes of red blood cells (RBC) from discocyte to stomatocyte, possibly due to extraction of cholesterol from cholesterol-rich lipid rafts of RBC membranes. In this study, the effects of methyl-beta-cyclodextrin (M-beta-CyD) and 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CyD) on lipid rafts and morphological changes in rabbit RBC (RRBC) were examined, compared to those of beta-CyD. In sharp contrast to beta-CyD, M-beta-CyD and DM-beta-CyD induced morphological changes of RRBC from discocyte to echinocyte. At pre-hemolytic concentrations of beta-CyDs, M-beta-CyD and DM-beta-CyD strongly released cholesterol from cholesterol-rich lipid rafts, compared to beta-CyD. Meanwhile, the lowering effects of DM-beta-CyD on fluorescent sphingomyelin analogue in sphingolipids-rich lipid rafts were more potent than those of beta-CyD and M-beta-CyD. The magnitude of the abilities of M-beta-CyD and DM-beta-CyD to extract membrane constituents was higher than that of beta-CyD, consistent with that of hemolytic activity. Furthermore, DM-beta-CyD and M-beta-CyD, not beta-CyD, lowered the amount of proteins in cholesterol-rich lipid rafts of RRBC. These results suggest that higher hemolytic activity and morphological changes from discocyte to echinocyte in RRBC induced by M-beta-CyD and DM-beta-CyD may be due to the extraction of both cholesterol and proteins from cholesterol-rich lipid rafts of RRBC, although DM-beta-CyD may interact with sphingolipids-rich lipid rafts on RRBC membranes only slightly.