Validation of the

The cluster structure of the neutron-rich isotope ^{10}Be has been probed via the (p,pα) reaction at 150 MeV/nucleon in inverse kinematics and in quasifree conditions. The populated states of ^{6}He residues were investigated through missing mass spectroscopy. The triple differential cross section for the ground-state transition was extracted for quasifree angle pairs (θ_{p},θ_{α}) and compared to distorted-wave impulse approximation reaction calculations performed in a microscopic framework using successively the Tohsaki-Horiuchi-Schuck-Röpke product wave function and the wave function deduced from antisymmetrized molecular dynamics calculations. The remarkable agreement between calculated and measured cross sections in both shape and magnitude validates the molecular structure description of the ^{10}Be ground-state, configured as an α-α core with two valence neutrons occupying π-type molecular orbitals.

Pharmacokinetics of loxoprofen and its active metabolite after dermal application of loxoprofen gel to rats.

This study was conducted to evaluate the pharmacokinetics of loxoprofen (LX) and its active metabolite (trans-OH form) after a single dermal application of LX gel (LX-G) to rats. In the skin at the treated site, generation of the trans-OH form was detected and both LX and the trans-OH form remained at high concentrations for 24 h after dermal application. Furthermore, both LX and the trans-OH form also remained in the skeletal muscle over 24 h after the single dermal application, while they eliminated rapidly after the single oral administration. The area under the curve up to the last measurable point (AUC(0-t)) for plasma concentrations of LX or the trans-OH form after dermal application of LX-G was less than 11% of that after oral administration of LX. In addition, C(max) and AUC(0-t) increased in a saturable manner while increasing the dose. Overall, these results demonstrated that the trans-OH form was generated at the treated site with the process of dermal absorption of LX and it remained at the target site for a long period with low systemic exposure compared to oral administration.

Three-dimensional evaluation of upper airway in patients with different anteroposterior skeletal patterns.

OBJECTIVES: To investigate variability in the upper airway of subjects with different anteroposterior skeletal patterns by evaluating the volume and the most constricted cross-sectional area of the pharyngeal airway and defining correlations between the different variables. MATERIAL AND METHODS: The study sample consisted of 60 patients (29 boys, 31 girls) divided into three groups: Class I (1 ≤ ANB ≤ 3), Class II (ANB>3), and Class III (ANB<1), to evaluate how the jaw relationship affects the airway volume and the most constricted cross-sectional area (Min-CSA). Differences between groups were determined using the Tukey-Kramer test. Correlations between variables were tested using Pearson's correlation coefficient. RESULTS: The volume and the Min-CSA of the pharyngeal airway (PA) were significantly related to anteroposterior skeletal patterns (p < 0.05). The nasopharyngeal airway (NA) volume of Class I and Class III subjects was significantly larger than that of Class II subjects (p < 0.05). The Min-CSA and the length of PA were significantly related to the volume of PA (p < 0.05). The site and the size of the Min-CSA varied among the three groups. CONCLUSIONS: The volume and the most constricted cross-sectional area of the airway varied with different anteroposterior skeletal patterns. The NA volume of Class I and Class III subjects was significantly larger than that of patients with a Class II skeletal pattern.

Prediction of fluoroquinolone-induced elevation in serum creatinine levels: a case of drug-endogenous substance interaction involving the inhibition of renal secretion.

The aim of this study was to examine the mechanism underlying the elevation in serum creatinine levels caused by a novel des-fluoro(6)-quinolone antibacterial agent, DX-619, in healthy subjects. hOCT2 showed a prominent uptake of creatinine (K(m) = 56.4 mmol/l) among renal organic ion transporters. DX-619 is a potent inhibitor of hOCT2 (K(i) = 0.94 micromol/l), hMATE1 (0.82 µmol/l), and hMATE2-K (0.10 micromol/l). The pharmacokinetic model involving the inhibition of hOCT2 (model 1), hOCT2, and MATE1 or MATE2-K (model 2) could predict the elevation in serum creatinine levels in individual subjects receiving DX-619. This assumes that a significant contribution of tubular secretion (59, 38, and 31%) and reabsorption ranged from 3-50, 4-30, and 5-21% in model 1, -2a (hOCT2/hMATE1), and -2b (hOCT2/hMATE2-K), respectively, for creatinine. In conclusion, DX-619, at its therapeutic dose, is able to inhibit hOCT2, hMATE1, and hMATE2-K, leading to a significant inhibition of tubular secretion of creatinine and consequently to elevation of serum creatinine levels.

Comparison of mechanism-based inhibition of human cytochrome P450 2C19 by ticlopidine, clopidogrel, and prasugrel.

Mechanism-based inhibition of CYP2C19 in human liver microsomes by the thienopyridine antiplatelet agents clopidogrel, prasugrel and their thiolactone metabolites was investigated by determining the time- and concentration-dependent inhibition of the activity of S-mephenytoin 4'-hydroxylase as typical CYP2C19 activity and compared with ticlopidine and its metabolite. Clopidogrel was shown to be a mechanism-based inhibitor of CYP2C19 with the inactivation kinetic parameters, k(inact) and K(I), equal to 0.0557 min(-1) and 14.3 microM, respectively, as well as ticlopidine (0.0739 min(-1) and 3.32 microM, respectively). The thiolactone metabolite of ticlopidine and clopidogrel inhibited CYP2C19 only in a concentration-dependent manner. In contrast, neither prasugrel nor its thiolactone metabolite inhibited CYP2C19 at concentrations up to 100 microM. The oxidation of the thiophene moiety of clopidogrel to form their respective thiolactones was found to be the critical reaction that produces the chemically reactive metabolites which cause the mechanism-based inhibition of CYP2C19. Estimation of in vivo drug-drug interaction using in vitro parameters predicted clinically observed data. For clopidogrel, there was no increase in the area under the curve (AUC) at its clinical dose level as predicted by the in vitro parameters, and for ticlopidine the prediction agreed with the clinically observed AUC increase. In conclusion, clopidogrel is potent mechanism-based inhibitors of CYP2C19 as well as ticlopidine, whereas prasugrel did not inactivate CYP2C19. Administration of prasugrel would not cause a clinically relevant interaction with CYP2C19.

Highly frequent anti-idiotype antibody in cynomolgus monkeys developed against mouse-derived regions of anti-Fas antibody humanized by complementarity determining region grafting.

BACKGROUND AND PURPOSE: We investigated the immunogenicity of a humanized anti-human Fas monoclonal antibody, R-125224, in cynomolgus monkeys to estimate its efficacy, as well as its toxicity in clinical situations. EXPERIMENTAL APPROACH: R-125224 was intravenously administered to cynomolgus monkeys at single doses of 0.4, 1.2, 6 and 30 mg kg(-1), and the plasma concentrations of R-125224 and anti-R-125224 antibody (ARA) were measured. We conducted a competitive enzyme-linked immunosorbent assay to determine which part of R-125224 was recognized by ARA. We also examined the retention of radioactivity in mononuclear cells and granulocytes after the injection of [(125)I]-R-125224 to a collagen-induced arthritis monkey model. KEY RESULTS: After i.v. administration of R-125224, the elimination of the plasma R-125224 concentrations was accelerated at around 10 days post-dose, and 10 of 12 monkeys were ARA positive. From an epitope analysis of ARA, the ARA produced in monkeys recognized the mouse-derived regions located in complementarity determining regions, but could not recognize the human IgG. After the injection of [(125)I]-R-125224 to a collagen-induced arthritis monkey model, a significantly longer retention of the radioactivity in mononuclear cells compared to granulocytes was observed. CONCLUSIONS AND IMPLICATIONS: In monkeys, the development of antibodies against R-125224 is rapid and highly frequent. Our hypothesis is that this highly frequent development of ARA might be due to the binding of R-125224 to immune cells, and its circulation in monkey blood might contribute to an increase in its chances of being recognized as an immunogen.

Pharmacokinetics and disposition of recombinant human osteoprotegerin (rhOPG) after intravenous administration in female fischer rats.

1. Osteoprotegerin (OPG) is a secreted member of the tumour necrosis factor receptor (TNFR) family that leads to the suppression of the differentiation, activation and survival of osteoclasts. The objective was to investigate the in vivo pharmacokinetics and tissue distribution of full-length recombinant human OPG (rhOPG) as well as its clearance mechanism using (125)I-labelled protein ((125)I-rhOPG) after intravenous administration to female Fischer rats. 2. (125)I-rhOPG was rapidly and predominantly distributed to the liver after dosing (3 mg kg(-1)). Immunohistochemical analysis indicated that rhOPG was located in the sinusoids of the liver. 3. The hepatic uptake of (125)I-rhOPG (0.01 mg kg(-1)) was partly regulated under a saturable process. Pre-dosing of some sulfated glycans (20 mg kg(-1)), especially dextran sulfate, heparin and fucoidan, markedly inhibited the hepatic uptake of (125)I-rhOPG. The clearance of (125)I-rhOPG was markedly reduced by the conjugation of dextran sulfate. 4. The results suggested that the hepatic clearance of (125)I-rhOPG was mainly mediated by the interaction with glycosaminoglycans.

Comparison of formation of thiolactones and active metabolites of prasugrel and clopidogrel in rats and dogs.

Prasugrel and clopidogrel are antiplatelet prodrugs that are converted to their respective active metabolites through thiolactone intermediates. Prasugrel is rapidly hydrolysed by esterases to its thiolactone intermediate, while clopidogrel is oxidized by cytochrome P450 (CYP) isoforms to its thiolactone. The conversion of both thiolactones to the active metabolites is CYP mediated. This study compared the efficiency, in vivo, of the formation of prasugrel and clopidogrel thiolactones and their active metabolites. The areas under the plasma concentration versus time curve (AUC) of the thiolactone intermediates in the portal vein plasma after an oral dose of prasugrel (1 mg kg(-1)) and clopidogrel (0.77 mg kg(-1)) were 15.8 +/- 15.9 ng h ml(-1) and 0.113 +/- 0.226 ng h ml(-1), respectively, in rats, and 454 +/- 104 ng h ml(-1) and 23.3 +/- 4.3 ng h ml(-1), respectively, in dogs, indicating efficient hydrolysis of prasugrel and little metabolism of clopidogrel to their thiolactones in the intestine. The relative bioavailability of the active metabolites of prasugrel and clopidogrel calculated by the ratio of active metabolite AUC (prodrug oral administration/active metabolite intravenous administration) were 25% and 7%, respectively, in rats, and 25% and 10%, respectively, in dogs. Single intraduodenal administration of prasugrel showed complete conversion of prasugrel, resulting in high concentrations of the thiolactone and active metabolite of prasugrel in rat portal vein plasma, which demonstrates that these products are generated in the intestine during the absorption process. In conclusion, the extent of in vivo formation of the thiolactone and the active metabolite of prasugrel was greater than for clopidogrel's thiolactone and active metabolite.

p51/p63 Inhibits ultraviolet B-induced apoptosis via Akt activation.

The epidermis must be protected against excess apoptotic cell death in response to ultraviolet-B (UV-B) irradiation. p53 is known to be critical for this protection. Although the p53 family member DeltaNp51B/DeltaNp63alpha (an N terminal-deleted form of p51/p63) is abundantly expressed in keratinocytes, its contribution to UV-B-dependent apoptosis is largely unknown. We found that, after a transient increase, DeltaNp51B is downregulated in UV-B-irradiated keratinocytes undergoing apoptosis, whereas p53 is upregulated with delayed kinetics. Furthermore, the reduction of DeltaNp51B by small interfering RNAs augmented UV-B-dependent apoptosis in keratinocytes, indicating that DeltaNp51B blocks keratinocyte apoptosis. Although the exogenous expression of DeltaNp51B in keratinocytes did not further block the UV-B-dependent apoptosis, to our surprise the expression of TAp51B (an isoform with a full NH(2)-terminal transactivation domain that is structurally and functionally similar to p53) decreased apoptosis significantly. The blockade of keratinocyte apoptosis by the p51 was dependent on the phosphorylation of Akt, resulting in the activation of a survival pathway. Thus, in addition to its indispensable roles in epithelial development, p51 acts in adult cells to protect the epidermis against UV-B irradiation by preventing excess depletion of keratinocytes.

Disposition and metabolic fate of prasugrel in mice, rats, and dogs.

The disposition and metabolism of prasugrel, a thienopyridine prodrug and a potent inhibitor of platelet aggregation in vivo, were investigated in mice, rats, and dogs. Prasugrel was rapidly absorbed and extensively metabolized. In the mouse and dog, maximum plasma concentration of radioactivity was observed in less than 1 h after an oral [14C]prasugrel dose. Most of the administered prasugrel dose was recovered in the faeces of rats and dogs (72% and 52-73%, respectively), and in mice urine (54%). Prasugrel is hydrolysed by esterases to a thiolactone, which is subsequently metabolized to thiol-containing metabolites. The main circulating thiol-containing metabolite in the three animal species is the pharmacologically active metabolite, R-138727. The thiol-containing metabolites are further metabolized by S-methylation and conjugation with cysteine.

Absorption, distribution and excretion of the new thienopyridine agent prasugrel in rats.

Prasugrel is converted to the pharmacologically active metabolite after oral dosing in vivo. In this study, (14)C-prasugrel or prasugrel was administered to rats at a dose of 5 mg kg(-1). After oral and intravenous dosing, the values of AUC(0-infinity) of total radioactivity were 36.2 and 47.1 microg eqx h ml(-1), respectively. Oral dosing of unlabeled prasugrel showed the second highest AUC(0-8) of the active metabolite of six metabolites analyzed. Quantitative whole body autoradiography showed high radioactivity concentrations in tissues for absorption and excretion at 1 h after oral administration, and were low at 72 h. The excretion of radioactivity in the urine and feces were 20.2% and 78.7%, respectively, after oral dosing. Most radioactivity after oral dosing was excreted in bile (90.1%), which was reabsorbed moderately (62.4%). The results showed that orally administered prasugrel was rapidly and fully absorbed and efficiently converted to the active metabolite with no marked distribution in a particular tissue.

The greater in vivo antiplatelet effects of prasugrel as compared to clopidogrel reflect more efficient generation of its active metabolite with similar antiplatelet activity to that of clopidogrel's active metabolite.

BACKGROUND AND METHODS: Prasugrel is a novel orally active thienopyridine prodrug with potent and long-lasting antiplatelet effects. Platelet inhibition reflects inhibition of P2Y(12) receptors by its active metabolite (AM). Previous studies have shown that the antiplatelet potency of prasugrel is at least 10 times higher than that of clopidogrel in rats and humans, but the mechanism of its higher potency has not yet been fully elucidated. RESULTS: Oral administration of prasugrel to rats resulted in dose-related and time-related inhibition of ex vivo platelet aggregation, and its effect was about 10 times more potent than that of clopidogrel. The plasma concentration of prasugrel AM was higher than that of clopidogrel AM despite tenfold higher doses of clopidogrel, indicating more efficient in vivo production of prasugrel AM than of clopidogrel AM. In rat platelets, prasugrel AM inhibited in vitro platelet aggregation induced by adenosine 5'-diphosphate (ADP) (10 microm) with an IC(50) value of 1.8 microm. Clopidogrel AM similarly inhibited platelet aggregation with an IC(50) value of 2.4 microm. Similar results were also observed for ADP-induced (10 microm) decreases in prostaglandin E(1)-stimulated rat platelet cAMP levels. These results indicate that both AMs have similar in vitro antiplatelet activities. CONCLUSIONS: The greater in vivo antiplatelet potency of prasugrel as compared to clopidogrel reflects more efficient in vivo generation of its AM, which demonstrates similar in vitro activity to clopidogrel AM.

p53 homologue, p51/p63, maintains the immaturity of keratinocyte stem cells by inhibiting Notch1 activity.

p53 homologue, p51/p63, predominantly expressed in keratinocyte stem cells, is indispensable for the formation of epidermis. Notch1, another such gene indispensable for the process, induces growth arrest and differentiation in keratinocytes. We found that exogenous expression of DeltaNp51B (DeltaNp63alpha), one of the isoforms of p51 specifically expressed in basal keratinocytes, blocked Notch 1-dependent growth arrest and differentiation in mouse keratinocytes by inhibiting p21 expression and maintaining integrins expression. Furthermore, DeltaNp51B by itself was found to have ability to induce expression of integrin alpha6beta4, which promotes attachment of basal cells to basal membrane thereby keeping the cells in immature state. Therefore, we conclude that DeltaNp51B expression warrants integrin expression even under the influence of Notch1 and that DeltaNp51B is a long-sought factor required to maintain basal cell keratinocytes immaturity by inhibiting Notch1 activity. We will postulate a plausible model explaining the maintenance of the squamous epithelium architectures as well as offering mechanistic explanations for pathological features of skin diseases, including cancers, psoriasis along with physiological wound healings.

Prediction of pharmacokinetics of CS-023 (RO4908463), a novel parenteral carbapenem antibiotic, in humans using animal data.

The pharmacokinetics of CS-023 (RO4908463, formerly R-115685), a novel parenteral carbapenem antibiotic, in humans was successfully predicted using the data collected from mice, rats, rabbits, and dogs; while inclusion of the monkey data led to a significant underestimation of the total plasma clearance (CL). Double logarithmic plots of CL and distribution volume at the steady-state (Vss) vs. body weight in four animal species were linear with high correlation coefficients; and the predicted CL and Vss values in humans agreed well with the observed values after administration of CS-023 by an intravenous drip infusion for 30 min. The plasma concentration-time profile in humans, which was predicted using a bi-exponential equation fitted to a complex Dedrick plot of the animal data, approximated the observed profile. An underestimation of CL caused by including the monkey data in a prediction is quite likely due to the net tubular reabsorption in monkeys, but not at least in rabbits, dogs, and humans.

Distinctly abnormal brain metabolism in late-onset ornithine transcarbamylase deficiency.

OBJECTIVE: To assess alterations in brain metabolites in patients with late-onset ornithine transcarbamylase deficiency (OTCD). METHODS: Six unrelated, asymptomatic Japanese late-onset OTCD patients were analyzed by proton MRS ((1)HMRS) using a point-resolved spectroscopy technique (repetition and echo times, 5000 and 30 ms). Localized spectra for the centrum semiovale were acquired and absolute metabolite concentrations were calculated using an LCModel. RESULTS: Compared with age-matched controls, N-acetylaspartate and creatine concentrations were normal in all patients. The glutamine (Gln) plus glutamate concentration was increased in four patients, which progressed in proportion to the clinical stage. myo-inositol (mI) could not be detected in five symptomatic patients. A decreased choline (Cho) concentration was detected in two clinically severe patients. (1)HMRS after liver transplantation in one patient revealed the normalization of all metabolites. CONCLUSION: These findings suggest progression of neurochemical events in OTCD, i.e., mI depletion and Gln accumulation followed by Cho depletion, which is reverse of that in hepatic encephalopathy, i.e., Cho depletion followed by mI depletion and Gln accumulation.

Brain N-acetylaspartate is elevated in Pelizaeus-Merzbacher disease with PLP1 duplication.

OBJECTIVE: To assess alterations in brain metabolites of patients with Pelizaeus-Merzbacher disease (PMD) with the proteolipid protein gene 1 (PLP1) duplications using quantitative proton MRS. METHODS: Five unrelated male Japanese patients with PMD with PLP1 duplications were analyzed using automated proton brain examination with the point resolved spectroscopy technique (repetition and echo time of 5,000 and 30 msec). Localized spectra in the posterior portion of the centrum semiovale were acquired, and absolute metabolite concentrations were calculated using the LCModel. RESULTS: Absolute concentrations of N-acetylaspartate (NAA), creatine (Cr), and myoinositol (MI) were increased by 16% (p < 0.01), 43% (p < 0.001), and 31% (p < 0.01) in patients with PMD as compared with age-matched controls. There was no statistical difference in choline concentration. CONCLUSION: The increased concentration of NAA, which could not be detected by previous relative quantitation methods, suggests two possibilities: axonal involvement secondary to dysmyelination, or increased cell population of oligodendrocyte progenitors. Elevated Cr and MI concentrations may reflect the reactive astrocytic gliosis. Our study thus emphasizes the importance of absolute quantitation of metabolites to investigate the disease mechanism of the dysmyelinating disorders of the CNS.

[Percutaneous cryosurgery for liver tumors].

A new cryoprobe developed in our department makes it possible to perform percutaneous cryoablation for small liver tumors. This cryoprobe is placed into each lesion using an ultrasonic guidance technique. In this study there were 10 patients with hepatocellular carcinoma and 5 patients with liver metastases from colorectal carcinoma. In 6 cases the tumor size was below 2 cm, in 6 cases between 2 and 3 cm, and in 3 cases over 3 cm. Using a cryoprobe of 3 mm in diameter, each lesion was frozen using liquid nitrogen for 15 or 20 minutes then thawed for 10 minutes, and repeated. The ice ball was found to enlarge to 3 cm in 20 minutes and 5 cm in 60 minutes experimentally. Six cases were CR, 5 PR, 2 NC and 2 PD. After cryoablation, liver function did not change and there were no complications. However, as the size of the ice ball was small (about 3 cm), this method was insufficiently effective in some cases. Percutaneous cryoablation using this probe should be performed for tumors smaller than 3 cm.

[Four cases of reversible posterior leukoencephalopathy syndrome].

We reported four children cases with reversible posterior leukoencephalopathy syndrome (RPLS). Magnetic resonance imaging (MRI) of the brain demonstrated reversible multiple cortical and subcortical lesions predominant in the occipital region. All patients presented with neurological symptoms associated with hypertension, such as headache, seizures and visual disturbances, which were successfully treated with antihypertensive therapy. Although RPLS is rare in childhood, characteristic lesions on MRI in the hypertensive children should be recognized as manifestations of RPLS. Subsequent clinical management should focus on the treatment of the hypertension and/or its underlying causes.

Adverse effect of dentine bonding agent on the oral mucosa of guinea pigs.

The purpose of present study was to determine the possibility of nebulous discoloration of the oral mucosa of guinea pigs as a result of the application of dentine bonding agents. The materials used were the Clearfil Photo Bond (CPB) system's mixture agent, universal agent and catalyst agent. Four types of experimental catalysts were used. In male albino guinea pigs weighing 300-500 g, oral mucosa irritation tests were performed in accordance with the guidelines of the Cosmetic, Toiletry and Fragrance Association (CTFA). Macroscopical findings on the oral mucosa showed that there was strong nebulous discoloration immediately after application of CPB mix and CPB catalyst and throughout the experimental periods. However, CPB without 10-methacryloxdecyl dihydrogen phosphate (MDP) did not produce noticeable nebulous discoloration and inflammation. The CPB mix caused pathological changes, such as paraketosis and hyperkeratosis on the endepidermis of the mucosa, immediately after its application.

Investigation of usefulness of sperm analyses in dogs for male fertility study.

The usefulness of sperm analyses in dogs, including sperm motion analysis, was investigated. Sperm motion analysis was performed with the CellSoft-4000 computer-assisted sperm analysis (CASA) system. First, we examined the conditions for preservation of optimal semen quality. We found that sperm retained more of their motility at 4 degrees C than at 37 degrees C. Secondly, we observed sperm motion, concentration and morphology in dog semen continuously for 11 weeks. We collected semen samples during the test period, and the samples retained sperm motion, concentration and morphology. Finally, we administrated alpha-chlorohydrin, which decreases rodent sperm motion, at a single oral dose of 100 mg/kg or 150 mg/kg to dogs. Sperm motion was inhibited immediately after alpha-chlorohydrin treatment, and recovered after 2 weeks. None of the experimental animals were sacrificed in the above-mentioned examinations. We thus confirmed that sperm analyses including motion analysis in dogs are useful in male fertility studies.