Duchenne/Becker muscular dystrophy (DMD/BMD) is caused by a defective expression of the dystrophin gene resulting in the absence of the dystrophin protein in muscle fibers.1,2) Approximately 60% of DMD/BMD patients have deletions in the dystrophin gene itself, [3][4][5] while the remaining 40% have small deletions or point mutations in the region that encodes the gene. Furthermore, nonsense mutations located within the gene account for approximately 5-13% of the muscular dystrophies. 6,7)Aminoglycoside antibiotics such as gentamicin (GM) had the ability to allow the ribosome to read through a premature-termination codon of the dystrophin gene, which prevented normal translation of dystrophin protein.8,9) BartonDavis et al. demonstrated the possibility of treating X chromosome-linked muscular distrophy (mdx) mouse, which was an animal model for DMD that possessed a nonsense mutation in the dystrophin gene, with GM in vivo. 10) They used GM to suppress the nonsense mutations and could restore dystrophin expression successfully in mdx mouse. However, the GM therapy has been hindered by several problems such as severe side effects of GM, especially nephrotoxicity and ototoxicity, the poor delivery profile to muscle tissue, and short half-life in blood. Recently, the phase 2b clinical trial of PTC 124 (3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid), 11) which is a new drug to induce reading through a premature-termination codon without clear side-effects, showed that the primary endpoint of the change in 6 min walk distance tests did not reach any statistical significance within the 48 weeks duration of the study according to Genzyme corporation announcement. 12)Therefore, to overcome these inadequacies of GM therapy for DMD, we encapsulated GM in hybrid liposomes (HL) for the delivery system. HL can be prepared by just the sonication of vesicular and micellar molecules in a buffer solution.13,14) HL are free from any contamination with organic solvents and remain stable for longer periods. The physical properties of these liposomes such as size, membrane fluidity, phase transition temperature, and hydrophobicity can be controlled by changing the constituents and compositional ratios.In the course of our study for HL, the following interesting results have been obtained. 23) and hydrophobic agents in the drug delivery system (DDS).In this study, we reported the therapeutic effects of HL including GM (GM-HL) on the mdx mice in vivo. The reduction of side effects of GM-HL is also discussed on the basis of the results from auditory brainstem response (ABR) tests and biodistribution analysis of HL. Kumamoto 860-0811, Japan. Received December 14, 2010; accepted January 28, 2011; published online February 7, 2011 It is known that gentamicin (GM) could be a possible treatment for Duchenne Muscular Dystrophy (DMD). However, GM therapy has been hindered by several problems such as severe side effects of GM. In order to resolve these problems, we developed the drug delivery system (DDS) of GM using hybrid lip...

In general, chemotherapeutic eŠects were low for non-small cell lung cancer (NSCLC) in the lung tumor. We examined the accumulation and antitumor eŠects of hybrid liposomes (HL-23) composed of phospholipid (L-adimyristoylphosphatidylcholine: DMPC) and PEG surfactant [polyoxyethylene (23)

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Stereoselective hydrolysis of amino acid esters have attracted considerable attention in connection with understanding the origins of the stereoselectivity observed with proteolytic enzymes. In the course of our study on the enantioselective hydrolysis (deacylation) of amino acid esters with the functional molecular assemblies composed of surfactants and reactive species, we emphasized that the stereochemical control could be attained by changing the composition of the coaggregates 1-4) and regulating ionic strength 5,6) and temperature. 7,8) Furthermore, since cyclodextrins (CyDs) have been noted as useful enzyme mimics, 9-12) we employed CyDs as host molecules and a markedly high stereoselectivity was attained in the diastereoselective deacylation of dipeptide esters.13) On the other hand, Ueno reported the high D-enantiomer-selective deacylation of amino acid esters mediated by modified b-CyDs.14) However, little has been known about the significant enantioselectivity for the deacylation of amino acid esters with unmodified CyDs. 15) In this study, we report the successful experimental results with marked enantioselectivity for the deacylation of Z-D(L)-amino acid esters (N-(benzyloxycarbonyl)-D(L)-amino acid pnitrophenyl esters) as mediated by unmodified CyDs, and the computer modeling (molecular mechanics) studies on the inclusion complexes of g-CyD with the specific substrates are also presented. Results and DiscussionWith respect to the enantioselective hydrolysis (deacyla-Leu, Phe and Trp-PNP) mediated by unmodified a-, b-and g-CyDs, the kinetic parameters obtained on the basis of the MichaelisMenten principle, the binding constant (K b ) for the formation of CyD-substrate complex and the rate constant (k 2 ) for the deacylation of substrate by CyD, are summarized in Table 1-3. The noteworthy aspects are as follows: (a) Overall, the deacylation mediated by a-CyD was favorable for all of the L-enantiomeric substrates (reflected in k 2 K b , L/Dϭ1.5-1.7), though the K b for D-isomer of Phe substrate and k 2 values for D-isomer of Ala substrate were larger than those for the corresponding L-isomers. (b) In the case of the reaction by bCyD, L-enantiomer-selectivity was also observed for the deacylation of Ala, Leu and Phe substrates (L/Dϭ1.5-3.2), while D-enantiomeric selectivity (D/Lϭ1.4) was observed for the deacylation of Trp substrate. (c) Relatively higher L-enantioselectivity was obtained in the deacylation mediated by gCyD (L/Dϭ2.6-9.0). Most remarkably, the highest enantioselectivity (L/Dϭ9.0) was attained for the deacylation of Ala substrate. Furthermore, it was also attractive that this fairly high enantioselectivity (L/Dϭ9.0) could be mainly originated in the deacylation process of substrates, because it was reflected by a fairly large k 2 L /k 2 D value (7.4) and a smallOn the other hand, Table 4 shows the results of energies for the inclusion complexes on Z-D(L)-Ala-PNP having different shapes with g-CyD calculated by molecular mechanics with water solvent effects. It was suggested that the con...

The development of effective utilization methods of Shochu distillation remnants is desirable from the viewpoint of environmental protection and biomass. In this study, we examined inhibitory effects of the powder (barleypowder) obtained from the barley-Shochu distillation remnants on the various tumor cells in vitro and immunostimulation effect in vivo. Remarkable inhibitory effects on the growth of human lung tumor cells along with the induction of apoptosis were obtained for barley-powder in vitro. On the other hand, the barley-powder induced the production of serum Interferon (IFN)-γ for normal rats in vivo, and increased natural killer (NK) activity. Furthermore, the barley-powder showed no acute toxicity in the tail vein administration to normal rats. It was found that the barley-powder had not only an antitumor effect but also an immunostimulation effect for the first time.

The diastereoselectivity for the hydrolysis of p-methoxycarbonylphenyl N-(benzyloxycarbonyl)-D(L)-phenylalanyl-L-leucinate in aqueous solution was inverted by changing concentrations of the substrates or the reaction temperature. The monomer-aggregate transition operated on the LL-substrate, which was suggested by the spectroscopic measurements, seems to be responsible for such behavior.

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Stereoselective reactions have attracted considerable attention in connection with understanding the origins of the stereoselectivity observed with proteolytic enzymes. [1][2][3][4][5] In the course of our study on the stereoselective hydrolysis (deacylation) of amino acid and dipeptide esters with the functional molecular assemblies composed of synthetic surfactants and reactive species, we emphasized that the stereochemical (enantiomeric and diastereomeric substrates) control could be attained by changing the composition of the coaggregates [6][7][8] and regulating ionic strength 9) and temperature.10,11) Especially, an extremely high enantioselectivity was attained at the optimum composition of coaggregate. 6)However, the values of k t and k s (the first-order rate constants with and without the peptide catalyst, respectively) are fairly close for the enantioselective hydrolysis in the aggregate systems of synthetic surfactants. Then, it is impossible to disregard the errors of k t and k s values when we discuss catalytic activities of the artificial membranes bound enzyme on the basis of k a,obsd values.In this study, we employed native phospholipids instead of synthetic surfactants for the hydrolysis of amino acid esters12) with tripeptide (Z-PheHisLeu) 12) in the coaggregate systems. The advantage of this experiment should enable to evaluate of the catalytic activities directly on the basis of k a,obsd values because of the large difference of the k t and k s values, that is, the k t /k s ratios were above 30 and 4 folds for the L-and D-substrates, respectively.Recently, in the course of our study on artificial membranes composed of native phospholipids, very useful knowledges for medical engineering applications in vitro and in vivo have been obtained. For example, artificial lipid membranes were used as a drug carrier in drug delivery systems, such as 1,3-bis(2-chloroethyl)-1-nitrosourea encapsulated in hybrid liposomes (artificial membranes) which should be effective against malignant glioma in vitro and in vivo. 13)Moreover, the artificial membranes themselves were found to be useful as a new type of anticancer drug without side effects. 14,15) In this study, we report on the remarkable effects (composition of hybrid membranes and reaction temperature) on the enantioselective hydrolysis of C 12 -D(L)-Phe-PNP catalyzed by Z-PheHisLeu in the hybrid membranes composed of phospholipid (DMPC) and nonionic surfactant (C 12 (EO) 8 ) at pH 7.4. The relations between the enantioselectivity and the microenvironment of the reaction fields are discussed. Results and DiscussionThe composition dependence of coaggregates on the enantioselective hydrolysis of C 12 -D(L)-Phe-PNP catalyzed by Z-PheHisLeu was investigated in hybrid membranes of DMPC/C 12 (EO) 8 , as shown in Fig. 1. The concentration of C 12 (EO) 8 is expressed as mol% of total lipids. Both the second-order rate constants (k a,obsd ) and enantioselectivity (k ; 4-22-1 Ikeda, Kumamoto 860-0082, Japan. Received November 5, 2002; accepted November 26, 20...