Jiro Takata

Hot spots in prion protein for pathogenic conversion

Prion proteins are key molecules in transmissible spongiform encephalopathies (TSEs), but the precise mechanism of the conversion from the cellular form (PrPC) to the scrapie form (PrPSc) is still unknown. Here we discovered a chemical chaperone to stabilize the PrPC conformation and identified the hot spots to stop the pathogenic conversion. We conducted in silico screening to find compounds that fitted into a “pocket” created by residues undergoing the conformational rearrangements between the native and the sparsely populated high-energy states (PrP*) and that directly bind to those residues. Forty-four selected compounds were tested in a TSE-infected cell culture model, among which one, 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide, termed GN8, efficiently reduced PrPSc. Subsequently, administration of GN8 was found to prolong the survival of TSE-infected mice. Heteronuclear NMR and computer simulation showed that the specific binding sites are the A-S2 loop (N159) and the region from helix B (V189, T192, and K194) to B-C loop (E196), indicating that the intercalation of these distant regions (hot spots) hampers the pathogenic conversion process. Dynamics-based drug discovery strategy, demonstrated here focusing on the hot spots of PrPC, will open the way to the development of novel anti-prion drugs.

Vitamin E isoforms α-tocotrienol and γ-tocopherol prevent cerebral infarction in mice

Abstract α-tocopherol and its derivatives have been shown to be effective in reducing cerebral ischemia-induced brain damage. However, the effects of other vitamin E isoforms have not been characterized. In the present study, we investigated the effects of six different isoforms of vitamin E on the ischemic brain damage in the mice middle cerebral artery (MCA) occlusion model. All vitamin E isoforms were injected i.v., twice, immediately before and 3 h after the occlusion. α-tocopherol (2 mM), α-tocotrienol (0.2 and 2 mM) and γ-tocopherol (0.2 and 2 mM) significantly decreased the size of the cerebral infarcts 1 day after the MCA occlusion, while γ-tocotrienol, δ-tocopherol and δ-tocotrienol showed no effect on the cerebral infarcts. These results suggest that α-tocotrienol and γ-tocopherol are potent and effective agents for preventing cerebral infarction induced by MCA occlusion.

Orally administered sphingomyelin in bovine milk is incorporated into skin sphingolipids and is involved in the water-holding capacity of hairless mice.

BACKGROUND We previously reported that dietary sphingomyelin (SM) concentrate from bovine milk improves epidermal functions. SM is a known precursor of ceramide (Cer) in the stratum corneum (SC). Neither the uptake nor distribution of orally administered SM nor its effects on epidermal functions have been demonstrated. OBJECTIVE We evaluated the effects of dietary SM on epidermal functions, and the distribution and fate of its radiolabeled metabolites in mice orally administered [4,5-(3)H-sphinganyl] sphingomyelin ((3)H-SM). METHODS Bovine milk SM (98% purity) was administered orally to 13-week-old hairless mice at 142 mg/kg per day for eight weeks. Their SC hydration, transepidermal water loss (TEWL), and SC Cer content were measured. (3)H-SM was then administered orally to 10-week-old hairless mice. Its distribution and metabolites in the skin were evaluated with whole-body autoradiography, liquid scintillation counting, and thin-layer chromatography. RESULTS SC hydration in the SM-administered mice was higher than that in control mice, whereas their TEWL and Cer contents did not differ. Radioactivity was distributed extensively in the bodies of the experimental mice and decreased gradually with time. In contrast, the radioactivity in the SC remained constant after its administration, and radiolabeled SM and Cer were detected in the skin. This suggests that dietary SM is transferred to the skin and then converted to Cer in the SC. CONCLUSIONS Orally administered SM is incorporated into skin SM and converted to SC Cer, which is involved in the water-holding capacity of the SC.

Preparation and In Vivo Evaluation of a Water-Soluble Prodrug for 2R-γ-Tocotrienol and as a Two-Step Prodrug for 2,7,8-Trimethyl-2S-(β-carboxyethyl)-6-hydroxychroman (S-γ-CEHC) in Rat

2R-γ-Tocotrienol (γ-T3) is currently receiving attention because it has beneficial effects not observed with α-tocopherol. To achieve the effective delivery of γ-T3, we synthesized three kinds of ester derivatives of γ-T3 and evaluated their use as hydrophilic prodrugs for γ-T3 in vitro and in vivo. 2R-γ-Tocotrienyl N,N-dimethylamino-acetate hydrochloride (compound 3) was a solid compound, with high solubility and stability in water, and was converted to γ-T3 by esterases in rat and human liver. Intravenous administration of 3 in rats led to a rapid increase in the plasma, liver, heart, and kidney levels of γ-T3. The bioavailability (plasma level) after intravenous administration was 82.5 ± 13.4% and 100 ± 11.3% for 3 and γ-T3 in surfactant, respectively, and the availability in liver was 213 ± 47.6% and 100 ± 4.8% for 3 and γ-T3 in surfactant, respectively. Furthermore, the systemic availability of 2,7,8-trimethyl-2S-(β-carboxyethyl)-6-hydroxychroman (S-γ-CEHC), a metabolite of γ-T3, was 78.6% for compound 3, 47.1% for γ-T3 in surfactant, and 100% for racemic γ-CEHC. Based on these results, we identified compound 3 as the most promising water-soluble prodrug of γ-T3 and two-step prodrug of S-γ-CEHC.

Delivery systems for antioxidant nutrients

The novel role of oxidants and antioxidants as part of cell signaling cascades has opened new areas of research in several disease states and their therapeutic strategies. For successful therapeutic manipulation of reactive oxygen species (ROS)-mediated cellular signaling pathways, it would necessitate control of the critical balance of oxidants/antioxidants in the target site by the antioxidant. Another way of controlling the critical balance is to avoid excessive generation of ROS from nutrients and/or drugs. From the viewpoint of controlling the balance between the oxidant and antioxidant status, this review focuses on the prodrug approach for delivery systems of vitamin E, a major antioxidant nutrient in the membrane, and on the reductive activation-independent delivery system of vitamin K hydroquinone by a prodrug approach, which can avoid excessive generation of ROS synchronized with the activation process of vitamin K.

Vitamin K Prodrugs: 1. Synthesis of Amino Acid Esters of Menahydroquinone-4 and Enzymatic Reconversion to an Active Form

The efficacy and toxicity of vitamin K depends on the pathway and the extent of enzymatic reductive activation to vitamin K hydroquinone, which is an essential cofactor for the synthesis of clotting factors. Parenteral use of vitamin K is impaired by its water insolubility. With the aim to improve delivery problems associated with menahydroquinone-4 (MKH, 2), an active form of menaquinone-4, N,N-dimethylglycine esters of 2 (1-mono, 4-mono, and 1,4-bis) were synthesized and assessed as potential water-soluble prodrugs for parenteral use. The esters can deliver the hydroquinone to its active site without a quinone reductive activation step. The hydrochloride salts of the esters were found to be quite soluble in water. The hydrolysis of the esters in 20% rat liver homogenate 9000 × g supernatant, rat plasma and phosphate buffer, pH 7.4, at 37°C was kinetically studied in the presence and absence of an esterase inhibitor. The hydrolysis was catalyzed by esterases located in the rat liver and rat plasma and quantitatively yielded 2. These results suggest that esterification of 2 with N,N-dimethylglycine is a promising way for obtaining water-soluble prodrug forms of 2. Based on the high susceptibility to liver esterase, the esters are potential prodrugs for achieving the site-specific delivery of 2.

Topical Application of a Novel, Water-soluble γ-Tocopherol Derivative Prevents UV-induced Skin Damage in Mice¶

Abstract We investigated whether the topical application of a novel, water-soluble γ-tocopherol (γ-Toc) derivative, γ-tocopherol-N,N-dimethylglycinate hydrochloride (γ-TDMG), could protect against UV-induced skin damage in hairless mice. Topical pre- or post-application of a 5% (93 mM) γ-TDMG solution in water/propylene glycol/ethanol (2:1:2) significantly prevented sunburn cell formation, lipid peroxidation and edema/inflammation that were induced by exposure to a single dose of UV irradiation of 5 kJ/m2 (290–380 nm, maximum 312 nm). This effect was greater than that seen with two α-Toc derivatives, α-tocopherol acetate (α-TA) and α-tocopherol-N,N-dimethylglycinate (α-TDMG). When a 5% solution of γ-TDMG was applied to mouse skin for 1 h, cutaneous γ-Toc increased by 25-fold after 24 h; levels of cutaneous α-Toc increased by only two- and eight-fold in α-TDMG and α-TA treated skins, respectively. These findings indicated that γ-TDMG immediately converted to γ-Toc in the skin and suggest that ability of γ-TDMG to protect the skin from the damaging effects of irradiation was due to its conversion to γ-Toc. When a 5% solution of γ-Toc was applied to mouse skin for 1 h, cutaneous γ-Toc rapidly increased by 25-fold, but fell to baseline levels by 24 h. In contrast, the concentration of γ-Toc in skin that was treated with γ-TDMG similarly increased, but these high levels were maintained after 24 h. These results suggest that γ-TDMG may be a more effective source of γ-Toc in skin. Thus, the topical application of γ-TDMG may be efficacious for the prevention of UV-B-induced skin damage.

Vitamin K Prodrugs: 2. Water-Soluble Prodrugs of Menahydroquinone-4 for Systemic Site-Specific Delivery

AbstractPurpose. The hydrochloride salts of the N, N-dimethylglycine esters of menahydroquinone-4 (1-mono, 1; 4-mono, 2; and 1,4-bis, 3) were assessed in vivo as prodrug for the systemic site-specific delivery system of menahydroquinone-4 (MKH), the active form of menaquinone-4 (MK-4, vitamin K2(20)). Methods. The disposition of MK-4 and menaquinone-4 epoxide (MKO) following the intravenous administration of the prodrugs and MK-4 preparation solubilized with surfactant (H-MK-4) were studied in vitamin K cycle inhibited rats. The relative bioavailability of MKH after the administration of the prodrugs was assessed from the area under the plasma concentration of MKO vs. time curve (AUCMKO). The specific delivery of MKH to its active site (liver) and coagulation activity after the administration of selected prodrug 1 were then compared with those of H-MK-4 in warfarin poisoned rats. Results. All compounds showed linear pharmacokinetics, and significant bioavailability of MKH was also observed following the administration of 1 (188%), 2 (87%) and 3(135%). Prodrug 1 caused the following increases; AUCliver of MKO from 70.7 ± 5.77 (H-MK-4) to 167 ± 7.89 nmol · h/g, MRTliver of MKO, from 3.87 ± 0.307 to 8.57 ± 0.432 h. The liver accumulation of intrinsic 1 reached a maximum (88% of dose) by 0.25 h. The rapid and liver-selective uptake and liver esterase mediated MKH regeneration characteristics of 1 enhanced the delivery of MKH to its active site and the selective advantage was increased 5.7 fold. The coagulation activity was extended 1.9 fold by 1 administration. Conclusions. The results indicated that these highly water-soluble and liver-esterase hydrolyzable ester derivatives of MKH are potential candidates for parenteral prodrugs which can thus achieve the systemic site-specific delivery of MKH. Such effective and selective delivery of MKH to its active site can therefore lead to enhanced pharmacological efficacy and can also avoid the toxicity induced by the solubilizing agent used in the H-MK-4 preparation.

Relationship between the acyl chain length of paradol analogues and their antiobesity activity following oral ingestion.

6-Paradol is known to activate thermogenesis in brown adipose tissue (BAT), and paradol analogues with different acyl chain lengths possess different pungency thresholds. In this study, the influence of the acyl chain length on the antiobesity activity of the paradol analogues was investigated. The antiobesity activity of 6-paradol in mice fed a high-fat diet for 8 weeks was greater than that of dihydrocapsiate. A comparison of the antiobesity activities of zingerone and 6-paradol showed that the length of the acyl chain in the paradol analogue was important for strong activity. Furthermore, the antiobesity activities of 6-, 8-, and 12-paradol appeared to decrease in an acyl chain length-dependent manner. The mechanism of the antiobesity activity of 6-paradol was enhanced by increasing levels of energy metabolism in the BAT, as well as an increase in the expression of uncoupling proteins 1 via the activation of sympathetic nerve activity.

Tumor-specific accumulation of 125I-labeled mouse-human chimeric anti-CEA antibody in a xenografted human cancer model demonstrated by whole-body autoradiography and immunostaining

Whole-body autoradiography (WBAR) was used to study the biodistribution of 125I-labeled mouse-human chimeric antibody (Ch F11-39) to carcinoembryonic antigen (CEA) in athymic nude mice bearing the CEA-producing MKN-45 human gastric carcinoma xenografts. Significantly high uptake of 125I-Ch F11-39 in the tumors obtained by tissue-counting technique was confirmed by WBAR of mice of 12, 24, 48, and 96 h postinjection of 125I-Ch F11-39. When compared with histochemical or immunohistochemical staining results of the tumor tissue sections, imaging profiles of 125I-Ch F11-39 obtained by WBARs were topographically correlated with histopathological findings of tissues and immunohistochemical localization of CEA in the tumor tissues, indicating that the accumulation of 125I-Ch F11-39 at the tumor site is based on its specificity for CEA. These results demonstrate that this chimeric antibody may serve as a potential useful diagnostic and/or therapeutic reagent for human CEA-producing cancers.