Ascorbate-assisted nitric oxide release from photocontrollable nitrosonium ion releasers for potent ex vivo photovasodilation.

We found that N-nitrosoaminoanisole derivatives tethered to dyes work as photocontrollable nitrosonium cation releasers and are converted to potent nitric oxide releasers in the presence of sodium ascorbate. The N-nitrosoaminoanisole derivative 2 worked as a more potent photovasodilating reagent ex vivo than previously reported nitric oxide releasers.

Influx and Efflux Transporters Contribute to the Increased Dermal Exposure to Active Metabolite of Regorafenib After Repeated Oral Administration in Mice.

The multikinase inhibitor regorafenib, which is a standard treatment for certain cancer patients after disease progression following other approved therapies, exhibits delayed-onset dermal toxicity. Here, we aimed to clarify the mechanisms that contribute to the increased dermal exposure to active metabolite M-5 of regorafenib after repeated oral administration. The dermal concentration of M-5 at 24 h after the last 5 oral administrations of regorafenib in mdr1a/1b/bcrp-/- mice was more than 190 times that in wild-type mice. The skin-to-plasma concentration ratio of M-5 in mdr1a/1b/bcrp-/- was also higher than in wild-type mice, suggesting possible involvement of P-glycoprotein and breast cancer resistance protein in regulating the dermal distribution. The area under the plasma concentration-time curve values of M-5 and its precursor M-2 in plasma of mdr1a/1b/bcrp-/- were at most 26 and 3 times those in wild-type mice, respectively. Interestingly, repeated administration of regorafenib markedly increased the area under the plasma concentration-time curve of M-5 in plasma, but not liver, compared with a single dose. Intravenous administration of M-5 dose-dependently reduced the liver-to-plasma concentration ratio. Our results indicate that hepatic uptake of M-5 may partially explain the accumulation of M-5 in the systemic circulation, but multiple factors, including influx and efflux transporters, are involved in determining dermal exposure to M-5.

[Use of Transdermal Fentanyl in a Hospital].

Recently, the use of transdermal fentanyl (TDF) has been increasing at our hospital owing to its convenience. Furthermore, TDF tends to be increasingly used for patients who have never used opioids. However, the appropriate criteria for indicating TDF have not been established yet. Therefore, we examined how TDF was prescribed in practice and determined its effective dosage. In 43 cases, the reasons, effects, and side effects of TDF were investigated retrospectively. Of the patients, 60% continued using TDF for 30 days. Meanwhile, approximately 25% of them terminated TDF therapy within 8 days. Of those who discontinued TDF therapy, some entirely stopped taking TDF and others chose other opioids instead because of poor pain control. Before receiving TDF therapy, 17 patients (45%) used oxycodone and 14 (37%) never used opioids. In addition, the main reason for starting TDF in opioid-naive patients was gastrointestinal condition. Between opioid-naive and opioid-using groups, no significant differences were observed in usage duration and incidence of side effects. The side effects included somnolence in 6 patients, delirium in 2 patients, and nausea and vomiting, constipation, and breathing restraint in 1 patient each. TDF was considered as an effective treatment regardless of the previous use of opioids. Nonetheless, careful deliberation is necessary because of the slow effects and difficulty with dosage adjustment.

Impact of platelet reactivity on long-term clinical outcomes and bleeding events in Japanese patients receiving antiplatelet therapy with aspirin.

AIM: Aspirin is an antiplatelet drug widely used for the prevention of cardiovascular disease; however, it is known to increase bleeding events. A low response to aspirin was reported to correlate with poor prognosis in patients undergoing antiplatelet therapy with aspirin. The aim of this study was to evaluate the impact of the antiplatelet activity of aspirin on cardiovascular and bleeding events in Japanese patients. METHODS: We analyzed the clinical course of 239 Japanese patients undergoing antiplatelet therapy with aspirin for a median of 64 months in this study. Their residual platelet reactivity was examined at enrollment and after 2 years. The co-primary endpoints were the occurrence of major adverse cardiac and cerebrovascular events (MACCEs) and bleeding events. RESULTS: The annual incidence of MACCEs and major bleeding events was 3.7% and 0.48%, respectively. With defined criteria, 67 patients (28%) were classified as low responders based on the platelet aggregability measured at enrollment. Low response to aspirin was not associated with increased MACCEs, while it clearly increased MACCEs in patients less than 70 years old (low responders 36.9% vs. responders 14.8%, log rank p=0.008). Five major types of bleeding occurred in the responders, but not in low responders, although the difference was not statistically significant (p= 0.07). CONCLUSION: Low response to aspirin was not associated with the increase of long-term MACCEs, while it increased MACCEs in patients less than 70 years old; however, it tended to decrease major bleeding events in Japanese patients.

Characterization of the antiplatelet effect of aspirin at enrollment and after 2-year follow-up in a real clinical setting in Japan.

BACKGROUND: Aspirin is an antiplatelet drug widely used for the prevention of cardiovascular diseases. It has been reported that some patients who exhibit a reduced antiplatelet effect of aspirin have higher cardiovascular risk. It is still controversial whether the antiplatelet effect of aspirin diminishes after a few years of treatment. This study aimed to evaluate the antiplatelet effect of aspirin and its 2-year change in Japanese patients. METHODS AND RESULTS: Collagen-induced platelet-aggregability was measured at enrollment by conventional optical aggregometer in 239 patients undergoing antiplatelet therapy with aspirin alone. Among them, 167 patients were evaluated after 2 years. Whole blood aggregability based on the screen-filtration method was also evaluated. Optical aggregometer studies showed that 27% of patients were low-responders. Multivariate analyses revealed that female sex and non-use of calcium-channel blockers were associated with low responsiveness. The antiplatelet effect of aspirin did not decrease after 2 years. Similar data were obtained with the whole blood aggregometer. CONCLUSIONS: In this Japanese patient group, 27% were low-responders to aspirin, and the antiplatelet effect of aspirin did not decrease after a 2-year interval.

Clopidogrel resistance in Japanese patients scheduled for percutaneous coronary intervention.

BACKGROUND: Dual antiplatelet therapy with acetylsalicylic acid (ASA) and a P2Y(12) ADP-receptor blocker is standard for prevention of coronary stent thrombosis. Clopidogrel, a 2(nd)-generation P2Y(12) blocker, has recently become available in Japan and this study aimed to evaluate its antiplatelet effects in Japanese patients. METHODS AND RESULTS: Thirty Japanese patients scheduled for elective coronary stent implantation were enrolled. Under low-dose ASA therapy, 300 mg clopidogrel was loaded on the 1(st) day and a daily 75-mg dose was administered on the following days. Assessed by optical aggregometer, rapid inhibition occurred at 4 h, when the inhibition of platelet aggregation rate (IPA) was 16.4+/-12.8% using 5 mumol/L ADP as the stimulus. The antiplatelet efficacy of clopidogrel was reasonably constant in each patient throughout the study period, although there was a broad inter-individual variation. At 48 h after clopidogrel loading, the ratios of responders (IPA > or =30%), hypo-responders (10%< or =IPA<30%), and non-responders (IPA <10%) were 36%, 50%, and 14%, respectively. CONCLUSIONS: The antiplatelet effectiveness of clopidogrel appeared individual-specific with wide inter-individual variation. The rate of clopidogrel non-responders was 14% among the examined Japanese patients.

Evaluation of the antiplatelet effects of cilostazol, a phosphodiesterase 3 inhibitor, by VASP phosphorylation and platelet aggregation.

BACKGROUND: Cilostazol, a phosphodiesterase 3 inhibitor, is an antiplatelet drug that is widely used for preventing cardiovascular events, although, to date, there are few methods for evaluating its effects. METHODS AND RESULTS: Blood samples were taken at baseline and at 3 and 12 h in 10 healthy male subjects after 100 mg cilostazol intake. Each sample was examined by Western blot for phosphorylation levels of vasodilator-stimulated phosphoprotein (VASP), an abundant cAMP-dependent kinase substrate in platelets, and by the optical aggregometer for ADP- and collagen-induced aggregation, before and after 8 nmol/L prostaglandin E(1) (PGE(1)) treatment. Cilostazol intake did not affect VASP phosphorylation levels or the maximal aggregation rates without PGE(1) treatment. However, cilostazol intake apparently enhanced PGE(1)-induced VASP phosphorylation and PGE(1)-mediated reduction of ADP-and collagen-induced maximal aggregation rates. Levels of VASP phosphorylated at Ser157 were correlated and the maximal aggregation rates induced by ADP were inversely correlated with cilostazol concentrations in the plasma. CONCLUSION: The antiplatelet effects of cilostazol intake could be evaluated by measuring VASP phosphorylation levels and maximal aggregation rates in platelets by ex vivo treatment with a low concentration of PGE(1).

Structural basis of the initial binding of tRNA(Ile) lysidine synthetase TilS with ATP and L-lysine.

In the bacterial genetic-code system, the codon AUA is decoded as isoleucine by tRNA(Ile)(2) with the lysidine residue at the wobble position. Lysidine is derived from cytidine, with ATP and L-lysine, by tRNA(Ile) lysidine synthetase (TilS), which is an N-type ATP pyrophosphatase. In this study, we determined the crystal structure of Aquifex aeolicus TilS, complexed with ATP, Mg2+, and L-lysine, at 2.5 A resolution. The presence of the TilS-specific subdomain causes the active site to have two separate gateways, a large hole and a narrow tunnel on the opposite side. ATP is bound inside the hole, and L-lysine is bound at the entrance of the tunnel. The conserved Asp36 in the PP-motif coordinates Mg2+. In these initial binding modes, the ATP, Mg2+, and L-lysine are held far apart from each other, but they seem to be brought together for the reaction upon cytidine binding, with putative structural changes of the complex.

Synergistic effect of fosfomycin and arbekacin on a methicillin-resistant Staphylococcus aureus-induced biofilm in a rat model.

Biofilms are a major concern for clinicians in the treatment of infectious disease because of the resistance to a wide range of antibiotics. Using a rat air pouch model, methicillin-resistant Staphylococcus aureus (MRSA) growing as a biofilm was treated with a combination of fosfomycin (FOM) and arbekacin (ABK) or by the agents alone. This model has the advantage of permitting frequent sampling of exudates for bacterial counts and anti-bacterial activity, and morphological examination of the biofilm structure and inflammatory process in the pouch tissues. A clear synergistic effect was observed in the rats treated with a combination of fosfomycin and arbekacin. Morphological studies using scanning electron microscopy and histological staining showed dramatic changes of the biofilm structure as well as the inflammatory response in the rats. These results suggested an enhancement of bactericidal activity of arbekacin penetrating through the biofilm layer by virtue of fosfomycin. A possible mechanism of the synergistic effect is discussed.

Effect of arbekacin on a methicillin-resistant Staphylococcus aureus-induced biofilm in a rat model.

Biofilms are a major concern for clinicians in the treatment of infectious disease because of their resistance to a wide range of antibiotics. Arbekacin, an aminoglycoside antibiotic, is the drug of choice for the treatment of infection caused by methicillin-resistant Staphylococcus aureus (MRSA). However, it has not yet been defined whether arbekacin tends to penetrate into the biofilm structure induced by MRSA infection. In this study, we treated a biofilm mode of MRSA growth with arbekacin, using a rat air-pouch model. The model has the advantage of permitting frequent sampling of exudates for bacterial counts and antibacterial activity. A clear dose-dependent bactericidal effect was detected in rats treated with arbekacin at concentrations between 0.3 and 10 mg/kg, but 0.1 mg/kg of arbekacin was ineffective against the experimental MRSA infection in rats. Morphological studies using scanning electron microscopy and histochemical staining demonstrated that an effective dosage of arbekacin induced dramatic changes in the biofilm membranous structure as well as in the inflammatory response, resulting in eradication of the biofilm structure and resolution of inflammation.