Hitoshi Yamauchi

Intra-articular penetration of ketoprofen and analgesic effects after topical patch application in rats

The purpose of this study was to evaluate percutaneous penetration and pharmacological effects of ketoprofen after transdermal administration, compared to the oral route. Skin and knee joint penetration of ketoprofen was tested by a microdialysis technique in rats and in vivo recovery was determined by retrodialysis. After oral and transdermal administration of ketoprofen, dialysate was sampled at 60 min intervals up to 360 min, for determination of concentrations of ketoprofen and prostaglandin E2. Analgesic effects of ketoprofen in iodoacetate and adjuvant-induced arthritis models were evaluated using the weight bearing method. The average recoveries of ketoprofen over 360 min in the skin and knee joint were 60.2+/-3 and 15.8+/-9%, respectively. Cmax values for ketoprofen absorbed within the skin after oral and transdermal administration were 20.1+/-5 and 297.5+/-478 ng/mL, respectively, and within the knee joint, 4.4+/-0.4 and 2.7+/-0.9 ng/mL. The Cmax value for the plasma concentration of ketoprofen after oral administration was approximately 80 times higher than with the transdermal route. Both transdermal and oral administration of ketoprofen significantly decreased PGE2 production in the skin and knee joint and improved weight bearing after exposure to iodoacetate and adjuvant. These results indicate that the transdermal ketoprofen patch is a useful formulation that can deliver the drug in sufficient amounts to inhibit prostaglandin E2 production in the skin and knee joint.

Photoinflammatory responses to UV-irradiated ketoprofen mediated by the induction of ROS generation, enhancement of cyclooxygenase-2 expression, and regulation of multiple signaling pathways.

Ketoprofen (KP) is photolabile and undergoes degradation when irradiated by sunlight, causing the development of various skin diseases. In this study, we found that UVB-irradiated KP can lead to inflammatory responses mediated by the induction of COX-2 and production of PGE(2). The ability of cells to repair UVB-induced cyclobutane pyrimidine dimers was impaired by UVB-irradiated KP, which consequently facilitated UVB-induced DNA damage to keratinocytes. The reactive oxygen species (ROS) generated by the photodegradation of KP facilitate UVB-induced inflammation and apoptosis in HaCaT cells. Elevation of the COX-2 levels was inhibited by an NADPH oxidase inhibitor and an NF-kappaB inhibitor but was largely enhanced after glutathione depletion by buthionine sulfoximine. Inhibition of ERK1/2, p38, and PI3K signaling attenuated the induction of COX-2, whereas inhibition of JNK signaling by SP600125 had very little effect. UVB-irradiated KP provoked an appreciable accumulation of pSer(15)-p53/COX-2 complexes, but this nuclear association of complexes was partially inhibited by PD98059. Silencing of COX-2 with siRNA was associated with reduced p53 phosphorylation and enhanced KP-photoinduced loss of mitochondrial membrane potential and cleavage of caspase 3 and PARP. This induction of apoptosis was prevented by N-acetylcysteine. In conclusion, this study highlights the particular inflammatory response to a photooxidative drug and suggests that KP-photoinduced inflammatory responses are predominantly attributable to induction of ROS generation and directly impair DNA repair.

Camptothecin disrupts androgen receptor signaling and suppresses prostate cancer cell growth

The androgen receptor (AR) is the main therapeutic target for treatment of metastatic prostate cancers. The present study demonstrates that the topoisomerase I inhibitor camptothecin selectively inhibits androgen-responsive growth of prostate cancer cells. Camptothecin strikingly inhibited mutated and wild-type AR protein expression in LNCaP and PC-3/AR cells. This inhibition coincided with decreased androgen-mediated AR phosphorylation at Ser(81) and reduced androgen-mediated AR transcriptional activity in a dose-dependent manner. Additionally, camptothecin disrupted the association between AR and heat shock protein 90 and impeded binding of the synthetic androgen [3H]R1881 to AR in LNCaP cells. Camptothecin also blocked androgen-induced AR nuclear translocation, leading to downregulation of the AR target gene PSA. In addition to decreasing the intracellular and secreted prostate-specific antigen (PSA) levels, camptothecin markedly inhibited androgen-stimulated PSA promoter activity. Collectively, our data reveal that camptothecin not only serves as a traditional genotoxic agent but, by virtue of its ability to target and disrupt AR, may also be a novel candidate for the treatment of prostate cancer.

Oligoarginine-linked polymers as a new class of penetration enhancers

Oligoarginines, which are known as cell-penetrating peptides, enhance the cellular uptake of poorly membrane-permeable bioactive molecules that are chemically conjugated to them. We designed a novel polymer: oligoarginine-linked poly(N-vinylacetamide-co-acrylic acid), with the expectation that the polymers will enhance the cellular uptake of the bioactive molecules that are physically mixed with them. Oligoarginines were grafted onto the polymer backbone through the chemical reaction with acrylic acid functional groups. The changes in the blood glucose concentration after nasal administration of insulin with and without the polymer were monitored in mice. The blood glucose concentration was slightly reduced when insulin was given solely at a dose of 10IU/kg. A D-octaarginine-linked poly(N-vinylacetamide-co-acrylic acid) with a grafting degree of 2% significantly enhanced the insulin-induced hypoglycemic effect. A similar enhancement was not observed when the polymer was substituted with intact D-octaarginine. The penetration-enhancing function of D-octaarginine-linked poly(N-vinylacetamide-co-acrylic acid) increased dramatically with an increase in the grafting degree of D-octaarginine. Substitution of D-octaarginine with the corresponding optical isomer and an increase in the number of arginine residues rather reduced the penetration-enhancing function. In vitro cell studies also indicated that a D-octaarginine-linked poly(N-vinylacetamide-co-acrylic acid) with a grafting degree of 17% enabled fluorescein isothiocyanate-dextran to effectively penetrate the cell membrane. Results demonstrated that our oligoarginine-linked polymer has a potential to provide a new class of penetration enhancers.

Percutaneous penetration of felbinac after application of transdermal patches: relationship with pharmacological effects in rats.

We have evaluated the percutaneous penetration of felbinac following application of topical patches using a microdialysis technique, and have examined correlations with pharmacological effects. A linear microdialysis probe with a 20‐mm dialysis fibre was inserted into the skin of anaesthetized rats. Probe perfusion was started at 2.0 μL min−1 with physiological saline and after a 60‐min baseline sampling of dialysate, 0.1 mL croton oil was applied to the skin surface at a concentration of 8%, v/v. A felbinac patch was then applied to the same point 60 min thereafter and dialysate was sampled at 60‐min intervals up to 300 min after patch application, for determination of concentrations of felbinac and prostaglandin (PG) E2. Analgesic effects of felbinac patches in an iodoacetate‐induced osteoarthritis model and an incisional pain model were evaluated using the weight bearing method. After application of patches, felbinac penetration into the skin was rapid, maximum concentrations in the dialysates with 0.07, 0.5 and 3.5% w/w felbinac patches being 0.046 ± 0.02, 0.104 ± 0.06 and 0.244 ± 0.2 μg mL−1, respectively. Dermal administration of croton oil caused an increment in PGE2 levels, which was significantly decreased by 0.5 and 3.5% felbinac patches 2–5 h after application. In pharmacological studies, 3.5% felbinac patches suppressed pain‐associated behaviour induced by iodoacetate injection and plantar incision. These results suggested that the transdermal patch containing 3.5% felbinac may become a useful formulation.

Performance of cell-penetrating peptide-linked polymers physically mixed with poorly membrane-permeable molecules on cell membranes

We are investigating a new class of penetration enhancers that enable poorly membrane-permeable molecules physically mixed with them to effectively penetrate cell membranes without their concomitant cellular uptake. Since we previously revealed that poly(N-vinylacetamide-co-acrylic acid) modified with d-octaarginine, which is a typical cell-penetrating peptide, significantly enhanced the nasal absorption of insulin, we examined the performance of the polymers on cell membranes. When Caco-2 cells were incubated with 5(6)-carboxyfluorescein (CF) for 30 min, approximately 0.1% of applied CF was internalized into the cells. This poor membrane permeability was dramatically enhanced by d-octaarginine-linked polymers; a 25-fold increase in the cellular uptake of CF was observed when the polymer concentration was adjusted to 0.2mg/mL. None of the individual components, for example, d-octaarginine, had any influence on CF uptake, demonstrating that only d-octaarginine anchored chemically to the polymeric platform enhanced the membrane permeation of CF. The polymer-induced CF uptake was consistently high even when the incubation time was extended to 120 min. Confocal laser scanning microphotographs of cells incubated with d-octaarginine-linked polymers bearing rhodamine red demonstrated that the cell outline was stained with red fluorescence. The polymer-induced CF uptake was significantly suppressed by 5-(N-ethyl-N-isopropyl)amiloride, which is an inhibitor of macropinocytosis. Results indicated that d-octaarginine-linked polymers remained on the cell membrane and poorly membrane-permeable CF was continuously internalized into cells mainly via macropinocytosis repeated for the individual peptidyl branches in the polymer backbone.

Automated analysis of fluvoxamine in rat plasma using a column-switching system and ion-pair high-performance liquid chromatography.

We have established a robust, fully automated analytical method for the analysis of fluvoxamine in rat plasma using a column-switching ion-pair high-performance chromatography system. The plasma sample was injected onto a precolumn packed with Shim-pack MAYI-ODS (50 microm), where the drug was automatically purified and enriched by on-line solid-phase extraction. After elution of the plasma proteins, the analyte was back-flushed from the precolumn and then separated isocratically on a reversed-phase C18 column (L-column ODS) with a mobile phase (acetonitrile-0.1% phosphoric acid, 36:64, v/v) containing 2 mM sodium 1-octanesulfonate. The analyte was monitored by a UV detector at a wavelength of 254 nm. The calibration line for fluvoxamine showed good linearity in the range of 5-5000 ng/mL (r > 0.999) with the limit of quantification of 5 ng/mL (RSD = 6.51%). Accuracy ranged from -2.94 to 4.82%, and the within- and between-day precision of the assay was better than 8% across the calibration range. The analytical sensitivity and accuracy of this assay is suitable for characterization of the pharmacokinetics of orally-administered fluvoxamine in rats.