Yokukansankachimpihange Is Useful to Treat Behavioral/Psychological Symptoms of Dementia.

Yokukansankachimpihange is a Japanese herbal medicine reported to benefit anxiety and sleep disorders, and it has recently been introduced to treat behavioral and psychological symptoms of dementia. There are no multicenter studies of its effectiveness regarding dementia in Japan, and this study's main objective was to clarify the effects of Yokukansankachimpihange on behavioral and psychological symptoms of dementia in a sample of patients from multiple healthcare centers. Nine facilities affiliated with Osaka Association of Psychiatric Clinics participated in November 2013 through April 2015 and provided 32 Alzheimer's disease patients to whom Yokukansankachimpihange was orally administered for 8 weeks. During the study, the patients continued their regular medication regimens. Behavioral and psychological symptoms of dementia (Behavioral Pathology in Alzheimer's Disease Rating Scale [Behave-AD]), core symptoms [Mini-Mental State Examination (MMSE)], activities of daily living [Nishimura Activity of Daily Living Scale (N-ADL)], and gastrointestinal symptoms (nausea/vomiting, loss of appetite, gastric discomfort, constipation, and diarrhea) were measured at baseline, after 4 weeks of treatment and after 8 weeks of treatment. Yokukansankachimpihange was orally administered at a dosage of 7.5 g twice daily before or between meals for 8 weeks. The Behave-AD mean score significantly improved after 8 weeks of treatment. There were no significant changes in MMSE, N-ADL, or gastrointestinal symptoms; however, decreased gastrointestinal scores were observed after 8 weeks. There were no side effects related to Yokukansankachinpihange. Pharmaceutical treatments are important for treating behavioral and psychological symptoms of dementia, and this study confirmed Yokukansankachimpihange's efficacy for treating Alzheimer's disease. Because the aggressiveness and sleep disorder components of the Behave-AD construct were the symptoms most improved and those symptoms are known to significantly burden dementia patients' caregivers, Yokukansankachimpihange's efficacy might indirectly relieve these caregivers' burden of care.

Inhibitory effect of siRNA complexes with polyamidoamine dendrimer/α-cyclodextrin conjugate (generation 3, G3) on endogenous gene expression.

In the present study, we prepared the small interfering RNA (siRNA) complexes with polyamidoamine (PAMAM) dendrimer (G3) conjugate with α-cyclodextrin (α-CDE (G3)), and examined the physicochemical properties, serum resistance, in vitro RNAi effects on endogenous gene expression, cytotoxicity, interferon response, hemolytic activity, cellular association and intracellular distribution. In addition, these results were compared to the siRNA complexes with the commercial transfection reagents such as linear polyethyleneimine (PEI), Lipofectamine™2000 (L2) and RNAiFect™ (RF). α-CDE (G3) interacted with siRNA, and suppressed siRNA degradation by serum. The siRNA complex with α-CDE (G3) showed the potent RNAi effects against Lamin A/C and Fas expression with negligible cytotoxicity and hemolytic activity, compared to those of the transfection reagents in Colon-26-luc cells and NIH3T3-luc cells. Cell-death patterns induced by siRNA polyplexes with α-CDE (G3) and PEI were different from siRNA lipoplexes with L2 and RF. α-CDE (G3) delivered fluorescent-labeled siRNA to cytoplasm, not nucleus, after transfection in NIH3T3-luc cells. Taken together, α-CDE (G3) could be potentially used as a siRNA carrier to provide the RNAi effect on endogenous gene expression with negligible cytotoxicity.

Potential use of polyamidoamine dendrimer/alpha-cyclodextrin conjugate (generation 3, G3) as a novel carrier for short hairpin RNA-expressing plasmid DNA.

The potential of starburst polyamidoamine dendrimer (dendrimer, generation 3, G3) conjugate with alpha-cyclodextrin (alpha-CyD) having an average degree of substitution of 2.4 (alpha-CDE) as a novel carrier of short hairpin RNA (shRNA) expressing plasmid DNA (shpDNA) was evaluated and the shpDNA transfer activity of alpha-CDE was compared with that of dendrimer (G3). Alpha-CDE formed a stable and condensed complex with shpDNA and induced a conformational transition of shpDNA in solution even in the low charge ratios. In addition, alpha-CDE markedly inhibited the enzymatic degradation of shpDNA by DNase I. The shpDNA complex with alpha-CDE at the charge ratio of 20/1 (alpha-CDE/shpDNA) elicited the most potent RNAi effects in cells transiently and stably expressing the GL3 and GL2 luciferase genes without cytotoxicity among the complexes with the various charge ratios. Besides, the RNAi effects were strikingly enhanced by the further addition of the adequate amounts of siRNA to the shpDNA complex with alpha-CDE. Taken together, the prominent RNAi effects of the shpDNA complex with alpha-CDE could be attributed to the stabilizing effect of alpha-CDE on enzymatic degradation of shpDNA and negligible cytotoxicity. These results suggest that alpha-CDE possesses the potential to be a novel carrier for shpDNA and siRNA.

Evaluation of polyamidoamine dendrimer/alpha-cyclodextrin conjugate (generation 3, G3) as a novel carrier for small interfering RNA (siRNA).

As the first step toward an evaluation of the potential use of the PAMAM dendrimer (G3) conjugate with alpha-cyclodextrin (alpha-CDE) for a small interfering RNA (siRNA) carrier, the ternary complexes of alpha-CDE or the transfection reagents such as Lipofactamine 2000 (L2), TransFast (TF) and Lipofectin (LF) with plasmid DNA (pDNA) and siRNA were prepared, and their RNAi effects, cytotoxicity, physicochemical properties and intracellular distribution were compared. Here the pGL2 control vector (pGL2) and pGL3 control vector (pGL3) encoding the firefly luciferase gene and the two corresponding siRNAs (siGL2 and siGL3) were used. The ternary complexes of pGL3/siGL3/alpha-CDE showed the potent RNAi effects with negligible cytotoxicity compared to those of the transfection reagents in various cells. alpha-CDE strongly interacted with both pDNA and siRNA, and suppressed siRNA degradation by serum, compared to those of the transfection reagents. alpha-CDE allowed fluorescent labeled siRNA to distribute in cytoplasm, whereas the transfection reagents resided in both nucleus and cytoplasm in NIH3T3 cells. Furthermore, the binary complex of siRNA/alpha-CDE provided the significant RNAi effect in NIH3T3 cells transiently and stably expressing luciferase gene. These results suggest that alpha-CDE may be utilized as a novel carrier for siRNA.

Improvement of gene delivery mediated by mannosylated dendrimer/alpha-cyclodextrin conjugates.

The purpose of this study is to evaluate in vitro and in vivo gene delivery efficiency of polyamidoamine (PAMAM) starburst dendrimer (generation 2, G2) conjugate with alpha-cyclodextrin (alpha-CDE conjugate (G2)) bearing mannose (Man-alpha-CDE conjugates) with the various degrees of substitution of the mannose moiety (DSM) as a novel non-viral vector in a variety of cells. Man-alpha-CDE conjugates (DSM 3.3 and 4.9) were found to have much higher gene transfer activity than dendrimer, alpha-CDE conjugate and Man-alpha-CDE conjugates (DSM 1.1 and 8.3) in various cells, which are independent of the expression of cell surface mannose receptors. Cellular association of pDNA complexes with dendrimer, alpha-CDE conjugate and Man-alpha-CDE conjugate (DSM 3.3) and their cytotoxic effects differed only very slightly. Surface plasmon resonance study demonstrated that the specific binding activity of Man-alpha-CDE conjugates to concanavalin A was not very strong. Much more conjugation of the mannose moiety to alpha-CDE conjugates provided unfavorable physicochemical properties of pDNA complexes for gene transfer, e.g. the low interaction with pDNA, the low enzymatic stability of pDNA and the lack of pDNA compaction. Man-alpha-CDE conjugate (DSM 3.3) provided gene transfer activity higher than dendrimer and alpha-CDE conjugate in kidney 12 h after intravenous injection in mice. These results suggest the potential use of Man-alpha-CDE conjugate (DSM 3.3) as a non-viral vector.

Enhancing effects of galactosylated dendrimer/alpha-cyclodextrin conjugates on gene transfer efficiency.

To improve in vitro gene transfer efficiency and/or achieve cell-specific gene delivery of polyamidoamine (PAMAM) starburst dendrimer (generation 2, G2) conjugate with alpha-cyclodextrin (alpha-CDE conjugate (G2)), we prepared alpha-CDE conjugate bearing galactose (Gal-alpha-CDE conjugates) with the various degrees of substitution of the galactose moiety (DSG) as a novel non-viral vector. The agarose gel electrophoretic studies revealed that Gal-alpha-CDE conjugates formed complexes with plasmid DNA (pDNA) and protected the degradation of pDNA by DNase I, but these effects impaired as the DSG value increased. Dendrimer and alpha-CDE conjugate exerted pDNA condensation through the complexation, but Gal-alpha-CDE conjugates did not. Gal-alpha-CDE conjugate (DSG 4) was found to have much higher gene transfer activity than dendrimer, alpha-CDE conjugate and Gal-alpha-CDE conjugates (DSG 8, 15) in HepG2, NIH3T3 and A549 cells, which are independent of the expression of the asialoglycoprotein receptor. Transfection activity of Gal-alpha-CDE conjugate (DSG 4) was insensitive to the existence of competitors (asialofetuin and galactose) and serum. In addition, no cytotoxicity after transfection of the complex of pDNA with Gal-alpha-CDE conjugate (DSG 4) was observed. These results suggest the potential use of Gal-alpha-CDE conjugate (DSG 4) as a non-viral vector in various cells.

In vitro and in vivo gene transfer by an optimized alpha-cyclodextrin conjugate with polyamidoamine dendrimer.

The purpose of the present study is to optimize the structure of the polyamidoamine starburst dendrimer (dendrimer) conjugate with alpha-cyclodextrin (alpha-CDE conjugate) as a nonviral vector. alpha-CDE conjugates of dendrimer (generation 3, G3) with various average degrees of substitution (DS) of alpha-CyD of 1.1, 2.4, and 5.4 were prepared. alpha-CDE conjugates formed the complexes with pDNA, resulting in a change of the particle sizes of pDNA complexes, but the distinction of physicochemical properties among their vector/pDNA complexes was only very slight. The membrane-disruptive ability of alpha-CDE conjugates on liposomes encapsulating calcein and their cytotoxicity to NIH3T3 and HepG2 increased with an increase in the DS value of alpha-CyD. In vitro gene transfer activity of alpha-CDE conjugates in both NIH3T3 and HepG2 cells augmented as the charge ratio (vector/pDNA) increased, and the activity of alpha-CDE conjugate (DS 2.4) was the highest at higher charge ratios among dendrimer (G3), the three alpha-CDE conjugates, and TransFast. After intravenous administration of pDNA complexes in mice, alpha-CDE conjugate (DS 2.4) delivered pDNA more efficiently in spleen, liver, and kidney, compared with dendrimer and other alpha-CDE conjugates (DS 1.1 and 5.4). The potential use of alpha-CDE conjugate (G3, DS 2.4) could be expected as a nonviral vector in vitro and in vivo, and these data may be useful for design of alpha-CyD conjugates with other nonviral vectors.

Effects of structure of polyamidoamine dendrimer on gene transfer efficiency of the dendrimer conjugate with alpha-cyclodextrin.

To improve gene transfer activity of a new nonviral vector, a polyamidoamine dendrimer (G2) conjugate with alpha-cyclodextrin (alpha-CDE conjugate (G2)), we prepared alpha-CDE conjugates with dendrimer having different generations (G3 and G4), and their gene transfer activities were compared with those of alpha-CDE conjugate (G2) and TransFast, a novel transfection reagent. alpha-CDE conjugates (G2, G3, and G4) formed the complexes with pDNA, changing the zeta-potential and particle size of pDNA complexes and the protection of pDNA from DNase I in a charge ratio-dependent manner, although their differences at higher charge ratios (vector/pDNA) were small. The gene transfer activity of alpha-CDE conjugates (G2, G3, and G4) was higher than that of the corresponding dendrimer alone in NIH3T3 and RAW264.7 cells. Of these CDE conjugates, alpha-CDE conjugate (G3) had a superior gene transfer activity which was comparable to that of TransFast in NIH3T3 cells. The intracellular distribution of pDNA after application of the pDNA complex with alpha-CDE conjugate (G3) to NIH3T3 cells was different from that with dendrimer alone (G3), although the cellular association of pDNA was almost comparable among all vectors. alpha-CDE conjugate (G3) strongly interacted with a fluorescence probe, 2-(p-toluidinyl)-naphthalene-6-sulfonate (TNS), suggesting that the conjugate possesses the inclusion ability with biomembrane constituents such as phospholipids after transfection. These results suggest that alpha-CDE conjugates, particularly the G3 conjugate, could be novel nonviral gene transfer agents.