Preparation and Evaluation of PEGylated Poly-L-ornithine Complex as a Novel Absorption Enhancer.

Polycationic compounds, such as poly-L-arginine and poly-L-ornithine (PLO), enhance the nasal absorption of hydrophilic macromolecular drugs. However, the bio availability corresponding to the dose of these enhancers has not been obtained in an open system study, where an administered solution is transferred to the pharynx because they do not exhibit mucoadhesion/retention in the nasal cavity. In this study, we prepared PEGylated-poly-L-ornithine (PEG-PLO) and investigated the effects of PEGylation on in vitro adhesion/retention properties, permeation enhancement efficiency, and cytotoxicity. PEG-PLO bearing 3-4 polyethylene glycol (PEG) chains per PLO molecule was more retentive than unmodified PLO on an inclined plate. The permeability of a model drug, FD-4, across Caco-2 cell sheets was enhanced by PEG-PLO as well as by PLO. PLO showed cytotoxicity at high concentrations, whereas PEG-PLO did not decrease cell viability, even above the concentration giving a sufficient enhancement effect. These findings suggest that PEGylation of polycationic absorption enhancers improves their adhesion/retention and decreases their cytotoxicity, which may lead to enhancers with greater utility.

Application of Microneedles to Skin Induces Activation of Epidermal Langerhans Cells and Dermal Dendritic Cells in Mice.

An adequate immune response to percutaneous vaccine application is generated by delivery of sufficient amounts of antigen to skin and by administration of toxin adjuvants or invasive skin abrasion that leads to an adjuvant effect. Microneedles penetrate the stratum corneum, the outermost layer of the skin, and enable direct delivery of vaccines from the surface into the skin, where immunocompetent dendritic cells are densely distributed. However, whether the application of microneedles to the skin activates antigen-presenting cells (APCs) has not been demonstrated. Here we aimed to demonstrate that microneedles may act as a potent physical adjuvant for successful transcutaneous immunization (TCI). We prepared samples of isolated epidermal and dermal cells and analyzed the expression of major histocompatibility complex (MHC) class II and costimulatory molecules on Langerhans or dermal dendritic cells in the prepared samples using flow cytometry. The expression of MHC class II and costimulatory molecules demonstrated an upward trend in APCs in the skin after the application of 500- and 300-µm microneedles. In addition, in the epidermal cells, application of microneedles induced more effective activation of Langerhans cells than did an invasive tape-stripping (positive control). In conclusion, the use of microneedles is likely to have a positive effect not only as an antigen delivery system but also as a physical technique inducing an adjuvant-like effect for TCI.

Characterization of Ocular Iontophoretic Drug Transport of Ionic and Non-ionic Compounds in Isolated Rabbit Cornea and Conjunctiva.

Ocular iontophoresis (IP) in isolated rabbit cornea and conjunctiva was examined in terms of transport enhancement, tissue viability and integrity using electrophysiological parameters by the Ussing-type chamber technique. Lidocaine hydrochloride (LC, a cationic compound), sodium benzoate (BA, anionic compound), and fluorescein isothiocyanate labeled dextran (molecular weight 4400 Da, FD-4, hydrophilic large compound) were used as model permeants. Direct electric current was applied at 0.5-5.0 mA/cm(2) for the cornea and 0.5-20 mA/cm(2) for the conjunctiva for 30 min. LC and BA fluxes across the cornea and conjunctiva were significantly increased by the application of electric current up to 2.3- and 2.5-fold and 4.0- and 3.4-fold, respectively, and returned to their baseline level on stopping the current. Furthermore, a much higher increase by IP application was obtained for the FD-4 transport. The increased FD-4 flux in the conjunctiva returned to baseline on stopping the current, whereas the flux in the cornea was sustained at a higher level after stopping the current. The transepithelial electric resistance of the cornea and conjunctiva was lowered by electric current application but fully recovered after stopping the current up to 2.0 mA/cm(2) for the cornea and 10 mA/cm(2) for the conjunctiva, suggesting that the corneal and conjunctival viability and integrity are maintained even after application of these current densities. These results indicate that ocular IP may be a useful non-invasive technique to achieve drug delivery of hydrophilic large molecules into the eyes.

Development of a Transnasal Delivery System for Recombinant Human Growth Hormone (rhGH): Effects of the Concentration and Molecular Weight of Poly-L-arginine on the Nasal Absorption of rhGH in Rats.

A novel system for delivering recombinant human growth hormone (rhGH) that is noninvasive and has a simple method of administration is strongly desired to improve the compliance of children. The aim of this study was to investigate the potential for the intranasal (i.n.) co-administration of rhGH with poly-L-arginine (PLA) as a novel delivery system by evaluating the effects of the concentration and molecular weight of PLA on the nasal absorption of rhGH. The influence of the formation of insoluble aggregates and a soluble complex in the dosage formulation on nasal rhGH absorption was also evaluated by size-exclusion chromatography and ultrafiltration. PLA enhanced the nasal absorption of rhGH at each concentration and molecular weight examined. Nasal rhGH absorption increased dramatically when the PLA concentration was 1.0 % (w/v) due to the improved solubility of rhGH in the formulation. A delay in rhGH absorption was observed when the molecular weight of PLA was increased. This appeared to be because the increase in molecular weight caused the formation of a soluble complex. It seems that the PLA concentration affects the absorption-enhancing effect on rhGH, while the molecular weight of PLA affects the time when the maximum plasma rhGH concentration was reached (Tmax) of rhGH after i.n. administration, mainly because of the interactions among rhGH, PLA, and additives. Therefore, the transnasal rhGH delivery system using PLA is considered to be a promising alternative to subcutaneous (s.c.) injection if these interactions are sufficiently controlled.

Serotonin regulates ß-casein expression via 5-HT7 receptors in human mammary epithelial MCF-12A cells.

We previously reported that serotonin (5-hydroxytryptamine; 5-HT) suppresses ß-casein expression, a differentiation marker in mammary epithelial cells, via inhibition of the signal transducer and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial cell line, MCF-12A. In this study, we investigated the expression pattern of the different 5-HT receptor subtypes in MCF-12A cells, and identified the receptors involved in 5-HT-mediated suppression of ß-casein protein expression. ß-Casein mRNA expression was inhibited by 30 µM 5-HT in a time-dependent manner. Treatment with 30 µM 5-HT for 72 h decreased ß-casein protein levels and STAT5 phosphorylation (pSTAT5). The cells expressed four 5-HT receptors subtypes (5-HTR1D, 2B, 3A, and 7) at the mRNA and protein level, and their expression was elevated by prolactin (PRL) treatment. Additionally, the mRNA levels of 5-HTR1D and 5-HTR7 were significantly higher than the other 5-HT receptors in the cells. Tryptophan hydroxylase 1 mRNA was detectable in the cells in the absence of PRL, and PRL treatment significantly increased its expression. ß-Casein and pSTAT5/STAT5 levels in the cells co-treated with 5-HT and a selective 5-HTR1D inhibitor, BRL15572, were equal to those observed in cells treated with 5-HT alone. However, in the cells co-treated with 5-HT and a selective 5-HTR7 inhibitor, SB269970, ß-casein and pSTAT5/STAT5 levels increased in a SB269970 concentration-dependent manner. In conclusion, we showed that 5-HT regulates ß-casein expression via 5-HTR7 in MCF-12A human mammary epithelial cells.

A mechanism enhancing macromolecule transport through paracellular spaces induced by Poly-L-Arginine: Poly-L-Arginine induces the internalization of tight junction proteins via clathrin-mediated endocytosis.

PURPOSE: Poly-L-arginine (PLA) enhances the paracellular permeability of the Caco-2 cell monolayer to hydrophilic macromolecules by disappearance of tight junction (TJ) proteins from cell-cell junctions. However, the mechanism of the disappearance of TJ proteins in response to PLA has been unclear. In this study, we investigated the mechanism of disappearance of TJ proteins from cell-cell junctions after the application of PLA to Caco-2 cell monolayers. METHODS: The membrane conductance (Gt), FITC-dextran (FD-4) permeability, and localization of TJ proteins were examined after the treatment of Caco-2 cell monolayers with PLA in the presence of various endocytosis inhibitors. In addition, the localization of endosome marker proteins was also observed. RESULTS: Clathrin-mediated endocytosis inhibitors suppressed the increase in Gt and Papp of FD-4 induced by PLA, and also significantly suppressed the disappearance of TJ proteins induced by PLA. Furthermore, occludin, one of the TJ proteins, colocalized with early endosome and recycling endosomes after the internalization of occludin induced by PLA, and then was recycled to the cell-cell junctions. CONCLUSION: PLA induced the transient internalization of TJ proteins in cell-cell junctions via clathrin-mediated endocytosis, subsequently increasing the permeability of the Caco-2 cell monolayer to FD-4 via a paracellular route.

Serotonin suppresses ß-casein expression via inhibition of the signal transducer and activator of transcription 5 (STAT5) protein phosphorylation in human mammary epithelial cells MCF-12A.

Serotonin (5-hydroxytryptamine; 5-HT) has an important physiological role in controlling lactation, namely, milk volume homeostasis, within mammary glands. The objectives of this study were to evaluate whether exogenous 5-HT can suppress ß-casein expression, a differentiation marker, produced in human mammary epithelial cells, and to determine whether 5-HT can attenuate ß-casein signaling via the prolactin (PRL) receptor (PRLr) and Janus kinase 2/signal transducer and activator of transcription 5 (STAT5) pathway. PRL treatment increased the mRNA level of ß-casein in the MCF-12A human mammary epithelial cell line, and the highest level occurred at days 7 and 14 of culture. In contrast, PRLr expression was not affected significantly by PRL treatment. PRL treatment in MCF-12A cells increased levels of ß-casein and phosphorylated STAT5 (pSTAT5) proteins in a concentration-dependent manner, with a slight increase of STAT5 protein. ß-Casein expression was inhibited by 0.1 mM 5-HT in a time-dependent manner. Additionally, treatment with 0.1 mM 5-HT for 72 h decreased protein levels of ß-casein and pSTAT5, with a slight decrease in STAT5 levels. These results suggest that exogenous 5-HT can inhibit STAT5 phosphorylation, resulting in a decrease in ß-Casein expression. In conclusion, we showed that exogenous 5-HT decreased ß-casein expression in MCF-12A human mammary epithelial cells, and that 5-HT was responsible for inhibiting phosphorylation of STAT5, resulting in a decline in lactational function.

Effect of poly-L-arginine on intestinal absorption of hydrophilic macromolecules in rats.

We have already reported that poly-L-arginine (PLA) remarkably enhanced the in vivo nasal absorption of hydrophilic macromolecules without producing any significant epithelial damage in rats. In the present study, we examined whether PLA could enhance the absorption of a model hydrophilic macromolecule, fluorescein isothiocyanate-dextran (FD-4), across the intestinal mucosa, as well as the nasal mucosa, by an in situ closed-loop method using the rat intestine. PLA was found to enhance the intestinal absorption of FD-4 in a concentration-dependent manner within the concentrations investigated in this study, but segment-specific differences were found to be associated with this effect (ileum>jejunum>duodenum≧colon). The factors responsible for the segment-specific differences were also investigated by intestinal absorption studies using aprotinin, a trypsin inhibitor, and an analysis of the expression of occludin, a tight junction protein. In the small intestine, the differences in the effect of PLA on the absorption of FD-4 may be related to the enzymatic degradation of PLA. In the colon, the reduced effect of PLA on the absorption of FD-4 may be related to the smaller surface area for absorption and the higher expression of occludin compared with other segments.

Poly-L-arginine-Induced internalization of tight junction proteins increases the paracellular permeability of the Caco-2 cell monolayer to hydrophilic macromolecules.

We investigated whether poly-L-arginine (PLA) enhances the paracellular permeability of the Caco-2 monolayer to hydrophilic macromolecules and clarified the disposition of tight junction (TJ) proteins. The transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran (FD-4) permeation were determined after treatment with PLA. TJ proteins were visualized using immunofluorescence microscopy after PLA exposure and depletion, and their expression levels were determined. The barrier function of TJs was also evaluated by measuring the alterations in the TEER and in the localization of TJ proteins. PLA induced an increase in hydrophilic macromolecule, FD-4, permeation through Caco-2 cell monolayers and a decrease in the TEER in a concentration-dependent manner, without any significant impact on the cell viability. This increased paracellular permeability induced by PLA was found to be internalized of claudin-4, ZO-1, tricellulin and mainly occludin from cell-cell junction to the subcellular space. ZO-1 appeared to play an important role in the reconstitution of TJ strand structures following PLA depletion. These results indicate that the PLA led to the internalization of TJ proteins to the subcellular space, subsequently increasing the permeability of the Caco-2 cell monolayer to FD-4 via a paracellular route.

Effect of ultrasound treatment of the skin on activation of Langerhans cells and antibody production in rodents.

In this study, we investigated whether stimulating the skin with ultrasound (US) could activate Langerhans cells (LCs) - antigen-presenting cells in the epidermis and stimulate antibody production following the subcutaneous and intradermal injection of ovalbumin (OVA) in hairless rats and BALB/c mice. Three different US frequencies (20 kHz, 1, and 3 MHz) were used and the expression of langerin was monitored as a marker for the activation of LCs in the epidermal sheet. In hairless rats, the langerin signal peaked at 12 h post-US treatment and returned to control levels at 24 h. Its expression increased with increasing irradiation time, up to 20 min, and 20 kHz US induced the highest langerin expression among the three frequencies tested. These results were reproduced in BALB/c mice. When the skin was pretreated with 20 kHz US at 0.41 W/cm2 for 10 min, the production of OVA-specific immunoglobulin G1 in mice increased by 2.8- and 3.4-fold 28 days after subcutaneous or intradermal OVA injections, respectively. These findings indicate that stimulating the skin with US can trigger skin immune responses, leading to effective antigen-specific antibody production. US-assisted transdermal vaccine delivery delivers antigens to the skin and evokes an immune response, providing an effective noninvasive immunization strategy.

Intranasal administration of milnacipran in rats: evaluation of the transport of drugs to the systemic circulation and central nervous system and the pharmacological effect.

Recently, transnasal drug delivery has attracted a great deal of attention as an administration route to deliver drugs directly to the central nervous systems (CNS) and drug targeting of the CNS is expected to increase. In the present study, we investigated the possibility of using a transnasal delivery system for milnacipran, a serotonin-noradrenaline reuptake inhibitor (SNRI), by evaluating the transport to the systemic circulation and cerebrospinal fluid (CSF) and the pharmacological effect after intranasal (i.n.) administration. Moreover, the effect of chitosan as a bioadhesive material on the transport to the systemic circulation and CSF and the pharmacological effect after i.n. administration were evaluated. As a result, i.n. administration of milnacipran was found to produce a higher direct delivery to the CNS as well as to the systemic circulation, suggesting that this is a promising route of administration and an alternative to peroral (p.o.) administration. Furthermore, the i.n. co-administration with chitosan led to increased plasma and CSF concentrations and an enhanced pharmacological effect, evaluated by means of the forced swimming test. The results suggested that chitosan produced a long residence time of milnacipran in the nasal cavity due to its bioadhesive effect, leading to the enhanced transport of milnacipran from the systemic circulation to the CNS via the blood-brain barrier by an increase in systemic absorption as well as direct transport to the CNS, resulting in a higher antidepressant effect compared to that with p.o. administration.

Characterization of systemic and histologic injury after crush syndrome and intervals of reperfusion in a small animal model.

BACKGROUND: Prolonged compression of limb muscles and subsequent decompression are important in the development of crush syndrome (CS). We applied a simple rubber tourniquet to rat hind limbs to create a CS model. METHODS: Anesthetized rats were subjected to bilateral hind limb compression for 5 hours followed by decompression and reperfusion for 0 hour, 1 hour, 3 hours, and 24 hours under monitoring of arterial blood pressure and electrocardiography. Blood and tissue samples were collected for histology, biochemical analysis, and tissue myeloperoxidase activity assessment. RESULTS: The survival rates of the CS-model groups remained at 100% until 3 hours, however, dropped to 25% at 24 hours after reperfusion mainly because of hyperkalemia and consequent hypotension observed at 1 hour and deteriorated at 3 hours after reperfusion. Rhabdomyolysis evaluated by circulating and histologic markers of injury was found as early as 1 hour and more marked at 3 hours, resulting in impaired renal function 24 hours after reperfusion. Myeloperoxidase activities increased with incremental periods after reperfusion not only in injured limb muscles but also in kidney and lung, suggesting an abnormal interaction between the vascular endothelium and circulating leukocytes after rhabdomyolysis, possibly causing subsequent multiple organ dysfunction frequently encountered in CS. CONCLUSION: The findings from this study demonstrate the feasibility of a novel small animal model of extremity crush injury. By using this model, the impact of incremental periods of reperfusion on mortality and remote organ dysfunctions can be characterized. Future studies are necessary to better define a threshold for this injury pattern and the impact of other factors underlying this syndrome.

Relationships between the particle velocity and introduction of drug-loaded microparticles into the skin in a microparticulate bombardment system.

Recently, it has been suggested that a microparticulate bombardment system would be a very useful tool for the delivery of a variety of powdered drugs as an alternative to parenteral injection via a needle. However the relationship between the particle dynamics and introduction into the skin has not been researched using this system. In the present study, we analyzed the velocity of microparticles bombarded by the Helios(TM) gun system under various conditions using particle image velocimetry (PIV). The particle kinetic energy, which depended on the particle velocity and particle mass, was increased with increasing helium pressure and particle size, decreasing bombardment dose, resulting in the increased percentage introduction and relative bioavailability (F(0-24 h)). The particle velocity had a greater influence than the particle mass. Therefore, in order to be the most effective system for introduction into the skin, it is necessary to use a high helium pressure and microparticles of high density. However, it is also necessary to consider the skin damage after bombardment.

[Effects of sperminated pullulans on the pulmonary absorption of insulin].

Sperminated pullulans (SP) having different molecular weights (MWs) were prepared, and the enhancing effect on the pulmonary absorption of insulin in rats was examined. SP acted as enhancers of insulin absorption when a 0.1% solution was applied with insulin simultaneously and their enhancing effects depended on the MW of the SP; the same solutions exhibited low toxicity in the in vivo LDH leaching test. In the in vitro experiments using Calu-3 cells, tight junction-opening effects and a toxic effect of SP in the MTT assay were observed at lower concentrations compared with the in vivo experiments. A mucus layer might interfere with the interaction between SP and the cell surface and might suppress both these effects and toxicity. SP having a high MW will be useful for preparing safe and efficient formulations of peptide and protein drugs. The change in the localization of the tight junction proteins may be related to the permeation-enhancing mechanism of SP.

Analysis of the rat skin permeation of hydrophilic compounds using the Renkin function.

The Renkin function was applied to characterize the penetration pathways through rat skin following different pretreatments. Nonmetabolic oligosaccharides and sugar alcohols, as model hydrophilic compounds, were applied simultaneously to the excised skin to obtain the equivalent cylindrical pore radius (R) and pore occupancy/length ratio (ε/L) for each skin piece. The R and ε/L values obtained were used to construct the simulation curves of the permeability coefficient (P(a))-molecular weight (MW). In the case of full-stripped skin, the P(a) of the model compounds and separately obtained P(a) of 5(6)-carboxyfluorescein (CF) showed good agreement with the simulation curve based on the Renkin function, suggesting that the viable epidermis and dermis in the full-stripped skin contained permeation pathways for hydrophilic compounds like aqueous channels. On the other hand, there was poor agreement of P(a) with the simulation curve for skin pretreated with an ethanol-menthol mixed enhancer system and the observed P(a) of CF in the pretreated skin was twice that calculated. The enhancer system might not be able to create aqueous channels in the lipid layer of the stratum corneum and could increase the permeation of CF in the layer in a different way. The analysis presented here will be useful not only for quantitative evaluation of drug permeation through aqueous channels in treated skins but also for investigation of the mechanism of skin-permeation enhancing techniques.

[Prevention of irinotecan hydrochloride-induced diarrhea by oral administration of Lactobacillus casei strain Shirota in rats].

Irinotecan hydrochloride is an inhibitor of DNA topoisomerase I enzyme by its main active metabolite SN-38. However, irinotecan-induced severe diarrhea has often limited its more widespread use. We assessed the effect of oral administration of Lactobacillus casei strain Shirota (LcS) on irinotecan-induced diarrhea in rats. Rats in the LcS group were administered LcS (1.64 x 10(11) cfu/0.5 g/3 mL saline) orally for 28 days. Fourteen days later, irinotecan was given for 4 days (100 mg/kg i. p.). Control group rats were administered 3 mL saline orally for 28 days together with irinotecan, as in the LcS group. As a result, LcS significantly inhibited the weight decrease due to irinotecan and the food intake was greater than in the controls. The delayed diarrhea symptoms induced by irinotecan also seemed to be improved. Although we cannot conclude why LcS improved the side effect of irinotecan, LcS might inhibit beta-glucuronidase activity, which is produced by intestinal flora and plays a key role in the development of irinotecan-induced delayed diarrhea. Further investigations including this issue are warranted.

Development of a human mammary epithelial cell culture model for evaluation of drug transfer into milk.

In the present study, a human mammary epithelial cell (HMEC) culture model was developed to evaluate the potential involvement of carrier-mediated transport systems in drug transfer into milk. Trypsin-resistant HMECs were seeded on Matrigel-coated filters to develop monolayers of functionally differentiated HMEC. Expression of the specific function of HMEC monolayers was dependent of the number of trypsin treatments. Among the monolayers with different numbers of treatment (treated 1 to 3 times), the monolayer treated 3 times (3-t-HMEC monolayer) showed the highest maximal transepithelial resistance and expression of beta-casein mRNA as an index of differentiation. Transport of tetraethylammonium (TEA) across the 3-t-HMEC monolayer in the basolateral-to-apical direction was significantly higher than that in the apical-to-basolateral direction (p < 0.05), whereas such directionality was not observed for p-amino-hippurate, suggesting the existence of organic cation transporters, but not organic anion transporters. In fact, expression of mRNAs of human organic cation transporter (OCT) 1 and 3 were detected in the 3-t-HMEC monolayer. These results indicate that the 3-t-HMEC monolayer is potentially useful for the evaluation of carrier-mediated secretion of drugs including organic cations into human milk.

Elucidation of the transport pathway in hairless rat skin enhanced by low-frequency sonophoresis based on the solute-water transport relationship and confocal microscopy.

In this study, we examined a relationship between hydrophilic solute and water (vehicle) transports in the excised hairless rat skin in the presence of ultrasound (41 kHz, 60-300 mW/cm2) irradiation and also conducted skin surface observation using confocal microscopy. When the applied intensity was increased stepwise over the rage of 60-300 mW/cm2, the transport of tritiated water (3H2O) was increased 140-fold in an intensity-dependent manner and this returned to normal on stopping the ultrasound application. The skin permeation clearance (mul/h) of model hydrophilic solutes, calcein (MW 623) and FITC-labeled dextrans [MW 4400 (FD-4) and MW 38000 (FD-40)], across the skin under the influence of ultrasound was plotted against the corresponding 3H2O flux (microl/h) to estimate the potential contribution of convective solvent flow, induced by the ultrasound application, to the solute transport. Good correlations were observed between the 3H2O flux and solute clearances and, unexpectedly, the slope values obtained from linear regression of the plots were consistent for all solutes examined (1.04+/-0.29 for calcein, 1.07+/-0.17 for FD-4, and 1.08+/-0.23 for FD-40, respectively). Transport of intact FD-4 and FD-40 was confirmed by gel permeation chromatography. When the skin surface and deeper regions of the skin after sonophoresis of FD-40 were observed using a confocal microscope, the fluorescence of FD-40 was uniformly distributed in the area under the ultrasound horn and also evident in crack-like structures in the boundary of the horn. On the other hand, a hexagonal structure of horny cells in the stratum corneum (SC) observed by post-staining with rhodamine B was fully conserved in the area under the horn. These findings suggest that 41 kHz ultrasound can increase the transdermal transport of hydrophilic solutes by inducing convective solvent flow probably via both corneocytes and SC lipids as well as newly developed routes. Our observation also suggests that 41 kHz (low-frequency) ultrasound has the potential to deliver hydrophilic large molecules transdermally.

Usefulness of coadministration of bucolome in warfarin therapy: pharmacokinetic and pharmacodynamic analysis using outpatient prescriptions.

Bucolome, a nonsteroidal anti-inflammatory drug, has often been coadministered to patients who take warfarin as an anticoagulant. This combination increases the anticoagulant effect, which is most likely due to the interaction of bucolome with the pharmacokinetics (PK) or pharmacodynamics (PD) of warfarin. More than 30 years ago the mechanism of this interaction was reported to be inhibition of warfarin protein binding by bucolome, and the inhibition of warfarin metabolism by bucolome was also recently reported. Here, we examined daily doses of warfarin and its anticoagulant effect (thrombo-test, TT) in outpatient prescriptions in five hospitals to elucidate the drug interaction and the usefulness of this drug combination. Among the warfarin prescriptions, 78 were for patients also taking bucolome and 99 were for patients not taking bucolome. The daily dose of warfarin in patients taking bucolome was significantly lower than those without bucolome (ca. 40%). TT in patients taking bucolome was significantly lower as compared to those not taking bucolome. Control of the anticoagulant effect was greater with coadministration of bucolome and warfarin than with warfarin alone. PK and PD analysis of our results suggests that the improved therapeutic effect resulting from coadministration of warfarin with bucolome was due to lower and less patient-to-patient variation of intrinsic hepatic clearance (CL(int)) of warfarin, since bucolome decreased the high CL(int) but did not have a great effect on the low CL(int). In conclusion, administration of bucolome in warfarin therapy is useful to control the anticoagulant effect of warfarin. Attention should also be paid to the enzymatic inhibition by bucolome on the PK of coadministered drugs.

Inhibitory effect of fluvoxamine on ß-casein expression via a serotonin-independent mechanism in human mammary epithelial cells.

Selective serotonin reuptake inhibitors (SSRIs) are widely used as a first-line therapy in postpartum depression. The objective of this study was to determine the mechanism underlying the inhibitory effects of the SSRI, fluvoxamine, on ß-casein expression, an indicator of lactation, in MCF-12A human mammary epithelial cells. Expression levels of serotonin (5-hydroxytryptamine; 5-HT) transporter, an SSRI target protein, and tryptophan hydroxylase 1, a rate-limiting enzyme in 5-HT biosynthesis, were increased in MCF-12A cells by prolactin treatment. Treatment with 1 µM fluvoxamine for 72 h significantly decreased protein levels of ß-casein and phosphorylated signal transducer and activator transcription 5 (pSTAT5). Extracellular 5-HT levels were significantly increased after exposure to 1 µM fluvoxamine, in comparison with those of untreated and vehicle-treated cells; however, extracellular 5-HT had little effect on the decrease in ß-casein expression. Expression of glucose-related protein 78/binding immunoglobulin protein, a regulator of endoplasmic reticulum (ER) stress, was significantly increased after treatment with 1 µM fluvoxamine for 48 h. Exposure to tunicamycin, an inducer of ER stress, also decreased expression of ß-casein and pSTAT5 in a manner similar to fluvoxamine. Our results indicate that fluvoxamine suppresses ß-casein expression in MCF-12A cells via inhibition of STAT5 phosphorylation caused by induction of ER stress. Further studies are required to confirm the effect of fluvoxamine on the function of mammary epithelial cells.