Takayuki Anno

Potential use of γ-cyclodextrin polypseudorotaxane hydrogels as an injectable sustained release system for insulin

The development of injectable hydrogels for protein delivery is a major challenge. In this study, insulin/alpha-cyclodextrin (alpha-CyD) and gamma-CyD polypseudorotaxane (PPRX) hydrogels were prepared through inclusion complexation between high molecular weight poly(ethylene glycol) (PEG) and CyDs. The alpha-CyD and gamma-CyD PPRX hydrogels were formed by inserting one PEG chain in the alpha-CyD cavity and two PEG chains in the gamma-CyD cavity. Insulin/CyD PPRX hydrogel formation was based on physical crosslinking induced by self-assembling without chemical crosslinking reagent. The supramolecular structures of insulin/CyD PPRX hydrogels were confirmed with (1)H nuclear magnetic resonance ((1)H NMR), X-ray diffraction, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The in vitro release study showed that the release rate of insulin from the CyDs PPRX hydrogels decreased in the order of gamma-CyD PPRX hydrogel>alpha-CyD PPRX hydrogel. This decrease was controlled by the addition of CyDs to the medium. The serum insulin level after subcutaneous administration of gamma-CyD PPRX hydrogel to rats was significantly prolonged, accompanying with an increase in the area under serum concentration-time curve, which was clearly reflected in the prolonged hypoglycemic effect. In conclusion, these results suggest the potential use of gamma-CyD PPRX hydrogel as an injectable sustained release system for insulin.

Effect of sulfobutyl ether-β-cyclodextrin on bioavailability of insulin glargine and blood glucose level after subcutaneous injection to rats

Insulin glargine is the first long-acting basal insulin analogue used for subcutaneous administration once daily in patients with type 1 or type 2 diabetes mellitus. To obtain the further bioavailability and the sustained glucose lowering effect of insulin glargine, in the present study, we investigated the effect of sulfobutyl ether-β-cyclodextrin (SBE4-β-CyD), with the degree of substitution of sulfobutyl ether group of 3.9, on pharmaceutical properties of insulin glargine and the release of insulin glargine after subcutaneous injection to rats. SBE4-β-CyD increased the solubility and suppressed aggregation of insulin glargine in phosphate buffer at pH 9.5, probably due to the interaction of SBE4-β-CyD with aromatic amino acid residues such as tyrosine of insulin glargine. In addition, SBE4-β-CyD accelerated the dissolution rate of insulin glargine from its precipitates, compared to that of insulin glargine alone. Furthermore, we revealed that subcutaneous administration of an insulin glargine solution with SBE4-β-CyD to rats enhanced the bioavailability of insulin glargine and sustained the glucose lowering effect, possibly due to the inhibitory effects of SBE4-β-CyD on the enzymatic degradation at the injection site. These results suggest that SBE4-β-CyD can be a useful excipient for sustained release of insulin glargine.

Potential use of glucuronylglucosyl-β-cyclodextrin/dendrimer conjugate (G2) as a DNA carrier in vitro and in vivo

In this study, we evaluated the polyamidoamine starburst dendrimer (dendrimer, generation 2: G2) conjugate with 6-O-α-(4-O-α-D-glucuronyl)-D-glucosyl-β-cyclodextrin (GUG-β-CDE (G2)) as a gene transfer carrier. The in vitro gene transfer activity of GUG-β-CDE (G2, degree of substitution (DS) of cyclodextrin (CyD) 1.8) was remarkably higher than that of dendrimer (G2) conjugate with α-CyD (α-CDE (G2, DS 1.2)) and that with β-CyD(β-CDE (G2, DS 1.3)) in A549 and RAW264.7 cells. The particle size, ζ-potential, DNase I-catalyzed degradation, and cellular association of plasmid DNA (pDNA) complex with GUG-β-CDE (G2, DS 1.8) were almost the same as those of the other CDEs. Fluorescent-labeled GUG-β-CDE (G2, DS 1.8) localized in the nucleus 6 h after transfection of its pDNA complex in A549 cells, suggesting that nuclear localization of pDNA complex with GUG-β-CDE (G2, DS 1.8), at least in part, contributes to its high gene transfer activity. GUG-β-CDE (G2, DS 1.8) provided higher gene transfer activity than α-CDE (G2, DS 1.2) and β-CDE (G2, DS 1.3) in kidney with negligible changes in blood chemistry values 12 h after intravenous injection of pDNA complexes with GUG-β-CDE (G2, DS 1.8) in mice. In conclusion, the present findings suggest that GUG-β-CDE (G2, DS 1.8) has the potential for a novel polymeric pDNA carrier in vitro and in vivo.

Cyclodextrin, a novel therapeutic tool for suppressing amyloidogenic transthyretin misfolding in transthyretin-related amyloidosis

TTR (transthyretin), a β-sheet-rich protein, is the precursor protein of familial amyloidotic polyneuropathy and senile systemic amyloidosis. Although it has been widely accepted that protein misfolding of the monomeric form of TTR is a rate-limiting step for amyloid formation, no effective therapy targeting this misfolding step is available. In the present study, we focused on CyDs (cyclodextrins), cyclic oligosaccharides composed of glucose units, and reported the inhibitory effect of CyDs on TTR amyloid formation. Of various branched β-CyDs, GUG-β-CyD [6-O-α-(4-O-α-D-glucuronyl)-D-glucosyl-β-CyD] showed potent inhibition of TTR amyloid formation. Far-UV CD spectra analysis showed that GUG-β-CyD reduced the conformational change of TTR in the process of amyloid formation. In addition, tryptophan fluorescence and 1H-NMR spectroscopy analyses indicated that GUG-β-CyD stabilized the TTR conformation via interaction with the hydrophobic amino acids of TTR, especially tryptophan. Moreover, GUG-β-CyD exerted its inhibitory effect by reducing TTR deposition in transgenic rats possessing a human variant TTR gene in vivo. Collectively, these results indicate that GUG-β-CyD may inhibit TTR misfolding by stabilizing its conformation, which, in turn, suppresses TTR amyloid formation.

Possible enhancing mechanisms for gene transfer activity of glucuronylglucosyl-β-cyclodextrin/dendrimer conjugate.

We previously reported that glucuronylglucosyl-β-cyclodextrin (GUG-β-CyD) conjugate with polyamidoamine starburst dendrimer (GUG-β-CDE conjugate) with the average degree of substitution (DS) of cyclodextrin (CyD) of 1.8 (GUG-β-CDE conjugate (DS 1.8)), showed remarkably higher gene transfer activity than α-CyD/dendrimer conjugate (α-CDE conjugate (DS 1.2)) and β-CyD/dendrimer conjugate (β-CDE conjugate (DS 1.3)) in vitro and in vivo. In this study, to clarify the enhancing mechanism for high gene transfer activity of GUG-β-CDE conjugate (DS 1.8), we investigated the physicochemical properties, cellular uptake, endosomal escape and nuclear translocation of the plasmid DNA (pDNA) complexes as well as pDNA release from the complexes. The particle size, ζ-potential and cellular uptake of GUG-β-CDE conjugate (DS 1.8)/pDNA complex were mostly comparable to those of α-CDE conjugate (DS 1.2) and β-CDE conjugate (DS 1.3). Meanwhile, GUG-β-CDE conjugate (DS 1.8)/pDNA complex was likely to have high endosomal escaping ability and nuclear localization ability in A549 and RAW264.7 cells. In addition, the pDNA condensation and decondensation abilities of GUG-β-CDE conjugate (DS 1.8) were lower and higher than that of α-CDE conjugate (DS 1.2) or β-CDE conjugate (DS 1.3), respectively. These results suggest that high gene transfer activity of GUG-β-CDE conjugate (DS 1.8) could be, at least in part, attributed to high endosomal escaping ability, nuclear localization ability and suitable pDNA release from its complex.

Potential use of glucuronylglucosyl-β-cyclodextrin/dendrimer conjugate (G2) as a siRNA carrier for the treatment of familial amyloidotic polyneuropathy

Abstract We previously reported that 6-O-α-(4-O-α-d-glucuronyl)-d-glucosyl-β-cyclodextrin (GUG-β-CyD) conjugate with polyamidoamine dendrimer (dendrimer, generation 2; G2) (GUG-β-CDE (G2)) is useful as a gene transfer carrier. In the present study, to investigate the potentials of GUG-β-CDE (G2) as a siRNA carrier, we evaluated the RNAi effect of its complex with siRNA against transthyretin (TTR) mRNA (siTTR) for the treatment of familial amyloidotic polyneuropathy (FAP). Among the various GUG-β-CDEs (G2) having the different degrees of substitution of GUG-β-CyD (degree of substation (DS) 1.8, 2.5, 3.0 and 5.0), GUG-β-CDE (G2, DS 1.8) showed the highest siTTR transfer activity. GUG-β-CDE (G2, DS 1.8)/siTTR complex showed no cytotoxicity in HepG2 cells. After intravenous administration of GUG-β-CDE (G2, DS 1.8)/siTTR complex to BALB/c mice, TTR mRNA expression was tended to reduce with negligible change of blood chemistry data. Particle size, ζ-potential and cellular association of the GUG-β-CDE (G2, DS 1.8) complex were almost the same as those of the other CDEs complexes. Meanwhile, GUG-β-CDE (G2, DS 1.8)/siTTR complex showed high endosomal escaping ability of siTTR in cytoplasm. These findings suggest the potential of GUG-β-CDE (G2, DS 1.8) as a siRNA carrier for the FAP treatment.

Peak-less hypoglycemic effect of insulin glargine by complexation with maltosyl-β-cyclodextrin.

Long-acting insulin products are desired that provide sustained blood glucose lowering without blood glucose level peaks. In the present study, to obtain the more desirable blood glucose lowering effect of long-acting insulin products, we investigated the effect of maltosyl-β-cyclodextrin (G(2)-β-CyD) on physicochemical properties and pharmacokinetics/pharmacodynamics of insulin glargine, which is the one of the most widely used insulin analog. G(2)-β-CyD increased the solubility and suppressed the aggregation of insulin glargine in phosphate buffer at 9.5, probably due to the interaction of G(2)-β-CyD with aromatic residues of the insulin glargine such as tyrosine. In addition, the dissolution rates of insulin glargine from its precipitates were increased by a complexation with G(2)-β-CyD. Subcutaneous administration of an insulin glargine solution with G(2)-β-CyD to rats gradually decreased blood glucose levels and provided a sustained blood glucose lowering effect without showing the glucose level peaks. These results suggest that G(2)-β-CyD can be a useful excipient for sustained release and a truly peak-less formulation of insulin glargine.

Effects of Selected Anionic β-Cyclodextrins on Persistence of Blood Glucose Lowering by Insulin Glargine after Subcutaneous Injection to Rats

Insulin glargine is a synthetic long-acting insulin product used for patients with diabetes mellitus. In this study, to obtain the further desirable blood-glucose lowering profile of insulin glargine, we investigated the effects of β-cyclodextrin sulfate (Sul-β-CyD) and sulfobutylether β-cyclodextrin (SBE7-β-CyD) on physicochemical properties of insulin glargine and pharmacokinetics/pharmacodynamics of insulin glargine after subcutaneous injection to rats. Sul-β-CyD and SBE7-β-CyD increased solubility of insulin glargine. SBE7-β-CyD suppressed the formation of oligomer and enhanced the dissolution rate of insulin glargine from its precipitate, compared to that of Sul-β-CyD. Additionally, we revealed that after subcutaneous administration of an insulin glargine solution, SBE7-β-CyD, but not Sul-β-CyD, increased bioavailability and sustained the blood-glucose lowering effect, possibly due to the inhibitory effects of SBE7-β-CyD on the enzymatic degradation at the injection site. These results suggest that SBE7-β-CyD could be a useful excipient for sustained release of insulin glargine.

Potential use of glucuronylglucosyl-β-cyclodextrin as a novel therapeutic tool for familial amyloidotic polyneuropathy

Transthyretin (TTR)-related familial amyloidotic polyneuropathy, which is induced by amyloidogenic transthyretin (ATTR), is characterized by systemic accumulation of amyloid fibrils. Although it is believed that protein misfolding of monomeric form of TTR is a rate-limiting step for TTR amyloid formation, no effective therapy targeting this misfolding step is available. Our recent studies revealed that cyclodextrins (CyDs), cyclic oligosaccharides composed of glucose units, might interact with TTR and prevent the protein misfolding. In this study, we focused on and elucidated the inhibitory effect of 6-O-α-(4-O-α-d-Glucuronyl)-d-glucosyl-β-CyD (GUG-β-CyD) on TTR amyloid formation. Tryptophan (Trp) fluorescence and 1H-NMR spectroscopy analyses indicated that GUG-β-CyD stabilized TTR conformation via interaction with the hydrophobic amino acids of TTR. Moreover, GUG-β-CyD suppressed TTR deposition in transgenic rats possessing a human ATTR V30M gene in vivo. Collectively, these data indicate that GUG-β-CyD may inhibit TTR misfolding by stabilizing its conformation, which, in turn, suppresses TTR amyloid formation.

Effect of cyclodextrins on transthyretin amyloid formation in transthyretin-related amyloidosis.

Transthyretin (TTR), a beta-sheet rich protein, is a precursor protein of familial amyloidotic polyneuropathy (FAP). Although it has been widely accepted that protein misfolding of monomeric form of TTR is rate limiting for amyloid formation, no effective therapy targeting this misfolding step is available. In this study, we focused on cyclodextrins (CyDs), cyclic oligosaccharides composed of glucose units, and reported the inhibitory effect of CyDs on TTR amyloid formation. Of various b-CyDs, GUGb-CyD showed potent inhibition of TTR amyloid formation. In the presence of GUG-b-CyD, no significant TTR amyloid fibrils were detected by electron microscopic analysis. Moreover, far-UV circular dichroism spectra analysis showed that GUG-b-CyD reduced the conformational change of TTR in the process of amyloid formation. Taken together, these data suggest that GUG-b-CyD may modify the stability of TTR conformation, which, in turn, leads to the suppression of TTR amyloid formation. Introduction: Transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP), which is induced by amyloidogenic TTR (ATTR), is characterized by systemic accumulation of amyloid fibrils [1]. It has been proposed that tetrameric TTR is not itself amyloidogenic, but dissociation of the tetramer into a non-native monomer with low conformational stability can lead to amyloid fibril formation [2]. Previous works have also shown that further structural change within the monomer caused by protein misfolding is a rate-limiting step to form TTR amyloid fibril aggregation [3]. However, no effective therapy targeting this misfolding step is available as of this moment. Cyclodextrins (CyDs), cyclic oligosaccharides composed of 6–8 glucose units, are widely used as prospective drug carriers in the pharmaceutical field [4]. There are three common types of natural CyDs depending on how many glucose units are present: aCyD (6), b-CyD (7), and g-CyD (8). Since CyD contains a central hydrophobic cavity and this cavity can serve as an inclusion site for hydrophobic molecules, CyDs are mainly used as multi-functional drug carriers by enhancing the bioavailability of lipophilic drugs, improving efficacy of drugs, and reducing side effects [5]. In addition, it has been proposed that b-CyDs improve the bioavailability of protein drug formulation. Recent studies revealed that b-CyDs bound to a hydrophobic part of the protein surface and increased its stability, which, in turn, led to prevent the protein misfolding and aggregation [6–8]. Because it is well documented that multiple hydrophobic regions of TTR are exposed in the process of TTR amyloid formation, these evidences suggest that b-CyDs may have potential to prevent TTR amyloid formation by inhibiting the misfolding of monomeric form of TTR. In this study, we elucidated the inhibitory effect of CyDs on TTR amyloid formation. Materials and methods: Both WT-TTR and ATTR-V30M were purified from serum samples obtained from healthy volunteers and homozygotic FAP ATTR V30M patients, respectively. b-CyD, hydroxpropyl-b-CyD (HP-b-CyD), 6-O-a-(4-O-a-DGlucuronyl)-D-glucosyl-b-CyD (GUG-b-CyD), and 6-O-a-maltosyl-b-CyD (G2-b-CyD) were examined in this study. To assess the effect of CyDs on TTR amyloid formation in vitro, thioflavin T-based fluorimetric assay was performed. The presence of TTR amyloid fibril was confirmed by electron microscopic analysis as described previously [9]. To evaluate the effect of CyDs on the conformational change of TTR, far-UV circular dichroism spectra analysis and fluorescent spectrum analysis were performed [10]. Results and discussion: Thioflavin T-based fluorimetric assay was first performed to assess the effect of possible b-CyDs on TTR amyloid formation. Of various b-CyDs, GUG-b-CyD showed potent inhibition of TTR amyloid formation, compared with b-CyD, HP-b-CyD, and G2-b-CyD. The amyloid formation of both WT-TTR and ATTR-V30M was suppressed by GUG-b-CyD in a dose-dependent manner. The effect of GUG-b-CyD was sustained and increased in a time-dependent manner, and the significant inhibition of TTR amyloid formation was observed even at 14 days after GUG-b-CyD administration. To confirm the inhibitory effect of GUG-bCyD further, electron microscopic analysis was performed. In agreement with the results described above, TTR amyloid formation was significantly suppressed in the presence of GUG-b-CyD. From these data, it is suggested that GUG-b-CyD indeed has potential to suppress TTR amyloid formation. To investigate the precise mechanism to how GUG-b-CyD inhibits TTR amyloid formation, the conformational change of TTR by GUG-b-CyD treatment was examined by far-UV CD spectra analysis. CD spectra of TTR were shifted and 58