Ginkgo B (GB) is an extract from the leaves of Ginkgo biloba, used in the treatment of dementia, cerebral insufficiency or related cognitive decline. In this paper, the main features of the pharmacokinetics of GB emulsion in rats were reviewed and the binding rate of GB to rat plasma and human plasma protein were investigated meanwhile. The concentrations of GB in plasma, tissue, and excretion of rats after i.v. administration of GB were measured using HPLC-ESI-MS. The metabolite was qualitated by LC-MS/MS. Intravenously administered GB was eliminated in a biphasic manner with a prominent initial phase (half-life of 0.3 h) followed by a slower terminal phase (half-life of 1.5 h). After i.v. 4, 12 and 36 mg/kg GB emulsion, the pharmacokinetic parameters from a two compartment model analysis of plasma samples were AUC_0—τ (μg·min/ml): 53.7, 165.5 and 649.7; CL (l/min/kg): 0.07, 0.07 and 0.05; V_C (l/kg): 2.27, 3.27 and 2.76, respectively. Peak concentrations generally occurred at 10 min except brain and fat. Tissue concentration then declined by several-fold during 6 h although still present in most tissues at 6 h. Single intravenous dose was mainly excreted in the urine (40—50%), fece contained less than 30%. The binding rate to rat plasma was little higher than to human plasma, but the difference was negligible. Some metabolites were found in urine and bile through qualitative analysis on the urine and bile by LC-MS/MS.

View full abstract

A simple, specific and reliable high performance liquid chromatography (HPLC) method has been developed and validated for study of fingerprint chromatograms of extracts from the whole plant of Clematis huchouensis TAMURA (CHT) for quality control of a traditional Chinese medicinal (TCM) herb. An Agilent C₁₈ column was used to separate extracts in this protocol and detection was made by ultraviolet absorbance at 340 nm. The column temperature was maintained at 25 °C. A mobile phase consisting of (a) water (with 30 mM KH₂PO₄) and (b) CH₃CN, (c) CH₃OH was found to be suitable for this separation at a flow rate of 0.8 μl min⁻¹ with gradient elution. Under the described conditions, a fingerprint profile of 8 compounds was collected within 35 min, which made the HPLC method unique and interesting. The fingerprint chromatograms had good stability, precision and reproducibility. Moreover, eco-climatic (habitat) effects were studied by comparison of fingerprint chromatograms obtained from extracts of CHT collected from two habitats, with rutin as a reference marker peak. The protocol developed is quite suitable for differentiation of extracts of CHT and can be used as a quality control method for this herb and a model for other herbal drugs.

View full abstract

Recently we showed significance of biosurfactants in the field of non-viral vectors for gene transfection. There, a biosurfactant, mannosylerythritol lipid A (MEL-A), especially increased the efficiency of gene transfection mediated with cationic liposomes. However, the molecular mechanism has not been well-understood yet. Here, through the examination of the ability of cationic liposomes containing an MEL (MEL-A, MEL-B or MEL-C) for important transfectional processes of the DNA capsulation and the membrane fusion with anionic liposomes, we found that MEL-A-containing liposomes increased both processes, but that MEL-B and MEL-C-containing liposomes just increased either of them. The results indicated that these kinds of the physicochemical properties in MEL-A-containing liposomes are able to increase the efficiency of liposome-mediated gene transfection.

View full abstract

Cataractous lenses have an altered distribution of the intracellular ionic environment, and the lens ionic imbalance with increased levels of calcium (Ca²⁺) and sodium (Na⁺), coupled with decreased levels of magnesium (Mg²⁺) and potassium (K⁺), is related to cataract development in human senile cataracts. We previously found that the decrease of ATP in lenses caused lens ionic imbalance, and probably decrease in ATPase function. In this study, we investigated the effect of Mg²⁺ deficiency on cataract progression using human lens epithelial (HLE) cells. Expression levels of inducible nitric oxide synthase (iNOS) mRNA in HLE cells were significantly greater in Mg²⁺-deficient medium (Mg²⁺ 0.021 mM) than in normal Mg²⁺ medium (Mg²⁺ 0.77 mM). The NO release from the HLE cells cultured with Mg²⁺-deficient medium also increased. On the other hand, the ATP levels in HLE cells 24 h after incubation with Mg²⁺-deficient medium were lower than that with normal Mg²⁺ medium. The Ca²⁺- and Na⁺/K⁺-ATPase activities in HLE cells until 24 h incubation with normal Mg²⁺ or Mg²⁺-deficient medium did not change. Both diethyldithiocarbamate 10 μM and aminoguanidine 250 μM attenuated the increase of NO release, and caused an increase in ATP levels in HLE cells 24 h after incubation with Mg²⁺-deficient medium. These results suggest that Mg²⁺ deficiency enhances NO production via iNOS in the lens. It is possible that the excessive production of NO cause the decrease of ATP levels. These results show that Mg²⁺ deficiency in the lens may cause an acceleration of the progression of lens opacification.

View full abstract

A gene (astA) encoding arylsulfate sulfotransferase (ASST), which transfers a sulfate group from phenolic sulfate esters to phenolic acceptors, was cloned from a Eubacterium A-44 genomic library. The probe (1.5 kb fragment) for the astA gene was prepared from the PCR product of the primers produced using two internal amino acid sequences of ASST, which had been purified from Eubacterium A-44. The astA gene was cloned into the pKF3vector. Its sequence revealed a 1863 bp open reading frame (ORF) encoding a protein containing 620 amino acids with a secretary signal peptide, and showed 91% homology (identity) to Eubacterium rectale IIIH previously reported. The cloned astA gene was expressed under the T7 promoter of the expression vectors, pET-39b(+) and pET-26b(+), in Escherichia coli BL21 (DE3), and the expressed ASSTs were purified using His Bind column chromatography. The specific activities of the purified ASSTs were 25.6 μmol/min/mg and 37.1 μmol/min/mg, respectively.

View full abstract

Peroxynitrite, a potent oxidative stress inducer, inhibits the mitochondrial electron transfer, induces cell death, and is considered to be involved in the pathology of various diseases. However, the intracellular mechanisms involved in the cell death process are not fully understood. Here we demonstrate that the enhanced nitration of specific tyrosine residues of cytochrome c, which are induced by continuous peroxynitrite exposure, attenuates cytochrome c-induced caspase-9 activation in vitro. Interestingly, cytochrome c nitrated with a single high dose of peroxynitrite preserved its potency, while this did not occur when cytochrome c was treated with continuous peroxynitrite exposure. Although both of these experiments resulted in cytochrome c nitration at the tyrosine residues, it was found that nitration at specific residues was enhanced only when cytochrome c was exposed to continuous peroxynitrite. This is the first report to demonstrate that cytochrome c nitration affects the apoptotic pathway by means of enhancement of nitration at specific tyrosine residues. This result implies that the nitration pattern of cytochrome c may affect the efficacy of the mitochondrial pathway in apoptotic cell death.

View full abstract

Minoxidil enhances hair growth by prolonging the anagen phase and induces new hair growth in androgenetic alopecia (AGA), whereas retinol significantly improves scalp skin condition and promotes hair growth. We investigated the combined effects of minoxidil and retinol on human hair growth in vitro and on cultured human dermal papilla cells (DPCs) and epidermal keratinocytes (HaCaT). The combination of minoxidil and retinol additively promoted hair growth in hair follicle organ cultures. In addition, minoxidil plus retinol more effectively elevated phosphorylated Erk, phosphorylated Akt levels, and the Bcl-2/Bax ratio than minoxidil alone in DPCs and HaCaT. We found that the significant hair shaft elongation demonstrated after minoxidil plus retinol treatment would depend on the dual kinetics associated with the activations of Erk- and Akt-dependent pathways and the prevention of apoptosis by increasing the Bcl-2/Bax ratio.

View full abstract

The present study was conducted to investigate the effects of Ga-Al-As laser irradiation on the mineralization ability of human dental pulp (HDP) cells. HDP cells in vitro were irradiated once with a Ga-AL-As laser at 0.5 W for 500 s and at 1.0 W for 500 s in order to investigate free radicals as one mechanism for transmission of laser photochemical energy to cells. Production of the hydroxyl radical (·OH) was measured using the ESR spin-trapping method and was found to be increased by laser irradiation. The DMPO-OH was not detected in the presence of dimethyl sulfoxide (DMSO), a ·OH scavenger. The formation of calcification nodule was also investigated by von Kossa staining. The number of calcified nodules was increased by 1.0 W-laser irradiation. Alkaline phosphatase (ALP) activity was higher in the 1.0 W-laser irradiation group. Expression of mRNAs for heat shock protein 27, bone morphogenetic proteins (BMPs) and ALP were greater in the 1.0 W-laser irradiation group. Expression of BMPs in the conditioned medium was also higher in the 1.0 W-laser irradiation group. In particular, DMSO decreased the number of calcified nodule produced by 1.0 W-laser irradiation. These results supposed that the mineralization of HDP cells is stimulated by laser irradiation, and that ·OH generated by laser irradiation is a trigger for promotion of HDP cell mineralization.

View full abstract

Recently, considerable scientific and therapeutic interest has focused on the structure and functions of the flavonoids. In a previous study, we suggested that hydroxyl (OH) substitutions on specific carbons in the skeleton of the flavonoids might significantly affect their apoptosis-modulating properties. Here, to investigate the effect of various OH substitutions on their diphenylpropane (C6C3C6) skeleton carbons, we selected 10 different flavonoids and assessed their role on UV-induced apoptosis of human keratinocytes, the principal cell type of epidermis. The results showed that 5,7,3′,4′-tetrahydroxylflavanone (eriodictyol) and 3,4′-dihydroxy flavone (3,4′-DHF) had a positive effect on cell proliferation of human HaCaT keratinocytes. Treatment with eriodictyol in particular resulted in significant suppression of cell death induced by ultraviolet (UV) light, a major skin-damaging agent. We found that eriodictyol treatment apparently reduced the percentage of apoptotic cells and the cleavage of poly(ADP-ribose) polymerase, concomitant with the repression of caspase-3 activation and reactive oxygen species (ROS) generation. The anti-apoptotic and anti-oxidant effects of eriodictyol were also confirmed in UV-induced cell death of normal human epidermal keratinocyte (NHEK) cells. Taken together, these findings suggest that eriodictyol can be used to protect keratinocytes from UV-induced damage, implying the presence of a complex structure–activity relationship (SAR) in the differential apoptosis-modulating activities of various flavonoids.

View full abstract

In the present study, we examined nuclear DNA sequences in an attempt to reveal the relationships between Pueraria lobata (WILLD). OHWI, P. thomsonii BENTH., and P. montana (LOUR.) MERR. We found that internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA are highly divergent in P. lobata and P. thomsonii, and four types of ITS with different length are found in the two species. On the other hand, DNA sequences of 5S rRNA gene spacer are highly conserved across multiple copies in P. lobata and P. thomsonii, they could be used to identify P. lobata, P. thomsonii, and P. montana of this complex, and may serve as a useful tool in medical authentication of Radix Puerariae Lobatae and Radix Puerariae Thomsonii.

View full abstract

The preventive effect of Anemarrhena asphodeloides BUNGE (Liliaceae), a traditional Chinese medicine, on ischemia-reperfusion-induced brain injury was evaluated in the rat brain. Ischemia was induced by intraluminal occlusion of the right middle cerebral artery for 2 h and reperfusion was continued for 22 h. Water extract of Anemarrhena asphodeloides (WEAA) was orally administered promptly prior to and 2 h after reperfusion. Total infarct volume and edema in the ipsilateral hemispheres of ischemia-reperfusion rats were significantly reduced by treatment with WEAA in a dose-dependent manner (p<0.05). The therapeutic time window of WEAA was 3 h in this ischemia-reperfusion rat model. WEAA also significantly inhibited increased neutrophil infiltration of ischemic brain tissue as estimated by myeloperoxidase (MPO) activity and immunohistochemical analysis. MPO-positive cells were markedly reduced by WEAA administration in striatal and cortical areas. These findings suggest that WEAA plays a crucial protective role in ischemia-induced brain injury, and suggest that WEAA could serve as a lead medicinal herb for the development of neuroprotective agents following transient focal ischemic brain injury.

View full abstract

Previous research has shown that salvianic acid A [2-(3,4-dihydroxyphenyl)-2-hydroxy-propanoic acid, SA] extracted from Salvia miltiorrhiza BGE (Danshen) markedly inhibits lipid peroxidation of mitochondrial membrane of hepatic cells in vitro. The present study was conducted to examine protective effect of SA on liver injury induced by carbon tetrachloride (CCl₄) and its possible mechanism in vivo. Male Sprague-Dowley rats weighing 180—200 g were used in the experiments. Five mmol/kg CCl₄ in olive oil was given to rats i.p. Spectrophotometrical method was used to measure activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum, activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) as well as malondialdehyde (MDA) level in hepatic tissue and the rate of superoxide anion (O₂·⁻) generation in hepatic submitochondrial particles. Hepatic histological structure was observed under light microscopy. CCl₄ caused significant changes of activities of the enzymes, MDA level, and the rate of O₂·⁻ generation and histopathological changes of acute hepatic injury were noted. SA reversed the significant changes induced by CCl₄. These results demonstrate that SA produces protective action on acute hepatic injury induced by CCl₄ via an antioxidative mechanism.

View full abstract

The aim of the present study was to investigate the vasoactive effect of Crotalaria sessiliflora L. extract (CSE) on rats and its mechanism when combining in vivo and in vitro approaches. CSE (0.5—5 mg/ml) induced concentration-dependent relaxation on endothelium-intact thoracic aortic rings precontracted with phenylephrine (PE, 10⁻⁵ M). This effect disappeared with the removal of functional endothelium. Pretreatment of the aortic strips with either N^G-nitro-L-arginine (L-NNA, 10⁻⁵ M) or methylene blue (10⁻⁵ M) significantly reduced the relaxation induced by CSE. The endothelium-dependent relaxation caused by CSE was associated with production of cGMP. CSE (5 mg/ml) increased the production of cGMP in endothelium-intact aortic rings and this effect was significantly attenuated by L-NNA (10⁻⁵ M) and methylene blue (10⁻⁵ M). Relaxation in response to CSE in strips precontracted with PGF_2α (3×10⁻⁵ M) was eliminated by removing extracellular Ca²⁺ and significantly reduced by pretreatment with ruthenium red (10⁻⁵ M). In in vivo tests, injection of 40 mg/kg of CSE induced an increase in plasma NO production, and this effect was blocked by L-NNA. Furthermore, CSE produced dose-dependent and transient decrease in blood pressure in normotensive rats and this effect was blocked by atropine as well as L-NNA. These findings suggest that CSE induces endothelium-dependent relaxation via NO/cGMP signaling by promoting extracellular Ca²⁺ influx and the release of Ca²⁺ from intracellular stores of endothelium, probably due to endothelial muscaric receptor activation.

View full abstract

Solifenacin succinate [YM905; (3R)-1-azabicyclo[2.2.2]oct-3-yl(1S)-1-phenyl-3,4-dihydroisoquinoline-2(1H)-carboxylate monosuccinate] is a new muscarinic receptor antagonist developed for the treatment of overactive bladder. The aim of the present study was to evaluate the antimuscarinic properties of solifenacin and to compare the results with those obtained for tolterodine, oxybutynin, darifenacin, propiverine and atropine. In radioligand receptor binding assay, K_i values of solifenacin for human muscarinic M₁, M₂, M₃, M₄ and M₅ receptors were 26, 170, 12, 110 and 31 nM, respectively. In isolated rat urinary bladder, solifenacin competitively antagonized carbachol-induced contractions, with a pA₂ value of 7.44±0.09. In these in vitro studies, the antimuscarinic action of solifenacin was more potent than that of propiverine and less potent than those of tolterodine, oxybutynin, darifenacin and atropine. In anesthetized rats, solifenacin and oxybutynin increased the maximum bladder capacity in a dose-dependent manner and also decreased the maximum intravesical pressure. The dosages required to produce a 30% increase in maximum bladder capacity (ED₃₀ values) of solifenacin and oxybutynin were 0.35 and 0.30 mg/kg i.v., respectively, indicating approximately equal efficacies. These results support the fact that solifenacin, similarly to currently used antimuscarinic agents, is an effective agent in the treatment of overactive bladder symptoms such as urinary frequency and urge incontinence.

View full abstract

Tetrandrine (TET), a bis-benzylisoquinoline alkaloid isolated from the dried root of Hang-Fang-Chi (Stephania tetrandra S. MOORE), is traditionally used in China for treating inflammation, hypertension and silicosis. In this study, our aim was to examine the anti-inflammatory mechanism of TET through measuring the inducible nitric oxide synthase (iNOS), cyclooxygenase-1, and -2 (COX-1 and COX-2) expression, cytokines (TNF-α, IL-4 and IL-8) formation, nitric oxide (NO) release and prostaglandin E₂ (PGE₂) generation in lipopolysaccharide (LPS)-induced human monocytic (THP-1) cells. Results showed that TET remarkably suppressed the LPS (1 μg/ml) induction of NO release and PGE₂ generation. It also significantly attenuated the LPS-induced transcription of proinflammatory cytokines (TNF-α, IL-4 and IL-8) in a dose-dependent manner. Furthermore, TET at 100 μM significantly blocked the LPS induction of iNOS and COX-2 expression, but not the COX-1. Taken together, these results suggest that TET exerts anti-inflammatory effects probably through the suppression of COX-2 and iNOS expression.

View full abstract

In this study the effects of compound B1, bis(3-aryl-3-oxo-propyl)methylamine hydrochloride, and an anti-inflammatory drug, indomethacin, were tested by carrageenan-induced paw edema and cotton pellet granuloma tests, for effects on acute and chronic phases of inflammation, respectively. Their effects on vascular permeability were also determined by hyaluronidase-induced capillary permeability test. Anti-inflammatory activity of B1 was compared with indomethacin. B1 decreased the carrageenan-induced paw edema by 49%, 35%, and 47% at 50, 100, and 200 mg kg⁻¹ doses, respectively, while this decrease was 82% by indomethacin at 20 mg kg⁻¹ dose. Antiproliferative effects in cotton pellet test of B1 at 50 mg kg⁻¹ and indomethacin at 20 mg kg⁻¹ doses were 44% and 43%, respectively. Indomethacin but not B1 inhibited the hyaluronidase-induced increase in capillary permeability. Our results suggest that B1 inhibits both acute and chronic phases of inflammation probably by an effect not mediated by prevention of increased capillary permeability. Especially, its anti-inflammatory activity against chronic phase of inflammation was comparable with that of indomethacin. Further detailed studies are needed to clarify the mechanism(s) of action responsible for the anti-inflammatory activity of B1.

View full abstract

Overexpression of Candida drug resistance 1 (CDR1) gene in Candida albicans (C. albicans), an efflux pump, is one of the major mechanisms contributing to drug resistance. C. albicans for fluconazole resistance 1 protein (Fcr1p) is a member of the family of zinc cluster proteins homologous to Pdr1p and Pdr3p (pleiotropic drug resistance protein) mediating azole resistance in Saccharomyces cerevisiae (S. cerevisiae) by regulating the expression of pleiotropic drug resistance 5 (PDR5) homologous to C. albicans CDR1. A previous study has showed that for fluconazole resistance 1 (FCR1) could also confer azole resistance in S. cerevisiae pdr1 pdr3 mutant by regulating PDR5. Therefore, we investigated the role of FCR1 in the development of C. albicans azole resistance in vitro and in vivo. Our results showed that Fcr1p inhibited fluconazole (FLC) resistance development in C. albicans through abolishing the induction of CDR1 expression by FLC, and in contrast FLC resistance development was accelerated resulting from the deletion of FCR1.

View full abstract

The antioxidant activities of curcumin, its natural demethoxy derivatives (demethoxycurcumin, Dmc and bisdemethoxycurcumin, Bdmc) and metabolite hydrogenated derivatives (tetrahydrocurcumin, THC; hexahydrocurcumin, HHC; octahydrocurcumin; OHC) were comparatively studied using 2,2-diphenyl-1-picrylhydrazyl (DDPH) radical, 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) induced linoleic oxidation and AAPH induced red blood cell hemolysis assays. Hydrogenated derivatives of curcumin exhibited stronger DPPH scavenging activity compared to curcumin and a reference antioxidant, trolox. The scavenging activity significantly decreased in the order THC>HHC=OHC>trolox>curcumin>Dmc>>.Bdmc. Stronger antioxidant activities toward lipid peroxidation and red blood cell hemolysis were also demonstrated in the hydrogenated derivatives. By the model of AAPH induced linoleic oxidation, the stoichiometric number of peroxyl radical that can be trapped per molecule (n) of hydrogenated derivatives were 3.4, 3.8 and 3.1 for THC, HHC and OHC, respectively. The number (n) of curcumin and Dmc were 2.7 and 2.0, respectively, which are comparable to trolox, while it was 1.4 for Bdmc. The inhibition of AAPH induced red blood cell hemolysis significantly decreased in the order OHC>THC=HHC>trolox>curcumin=Dmc. Results in all models demonstrated the lower antioxidant activity of the demethoxy derivatives, suggesting the ortho-methoxyphenolic groups of curcumin are involved in antioxidant activities. On the other hand, hydrogenation at conjugated double bonds of the central seven carbon chain and β diketone of curcumin to THC, HHC and OHC remarkably enhance antioxidant activity.

View full abstract

In this study, we aimed to investigate the possible protective effect of resveratrol on gentamicin induced nephrotoxicity. Experiments were carried out in male Wistar rats weighing 200—250 g. Gentamicin sulfate (80 mg/kg per day i.p.), resveratrol (10 mg/kg per day i.p.) and gentamicin together with resveratrol were administered for 6 d. The animals were sacrificed 24 h after the last injection. Urine, blood samples and tissue samples were collected from the animals on the seventh day of the treatment before they were sacrificed. Kidneys were collected for histopathological studies and fixed in 10% buffered formalin solution. Tissue samples were stored at −70 °C in liquid nitrogen for the determination of glutathione (GSH), glutathione-S-transferase (GST), malondialdehyde (MDA) and catalase (CAT). Glutathione assay was determined by the method of Beutler et al. GST amounts were measured by the method of Habig et al. Catalase activitiy was tested by Aebi's method and MDA was determined according to Thayer's method. Blood urea level was significantly increased in the gentamicin treated group. The study showed lowered levels of urea and creatinine levels in resveratrol administered groups when compared with gentamicin administered rats, and the difference was statistically significant. It has been determined that resveratrol caused statistically significant decrease in lipid peroxidation and reduced the level of catalase. Histopathological examination showed that resveratrol prevented partly gentamicin induced tubular damage. The results histopathologically demonstrated that resveratrol has a protective effect against gentamicin induced nephrotoxicity, lipid peroxidation and cellular damage in rats.

View full abstract

Single injection of streptozotocin (STZ) resulted diabetes mellitus which was reflected here by the levels of fasting blood glucose and serum insulin. Moreover, this experimental diabetes also resulted testicular dysfunctions evaluated by count, viability and motility of sperm as well as by the activities of key enzymes for androgen synthesis. Diabetes induced testicular oxidative stress has been indicated here by the monitoring of testicular peroxidase and catalase activities as well as by quantification of TBARS and CD of testis. Testicular glucose was increased and leydig cell nuclear area was decreased in STZ induced diabetes. Treatment of herbal formulated drug named as MTEC consist of aqueous-methanol extract of Musa paradisiaca, Tamarindus indica, Eugenia jambolana and Coccinia indica to streptozotocin induced diabetic rat at the ratio of 2 : 2 : 1 : 1 at the dose of 60 mg/d for two times a day for 14 d resulted a significant protection in fasting blood glucose and serum insulin levels (p<0.05) along with correction of testicular above parameters towards the control level (p<0.05). This herbal formulated drug has no general toxic effects on the body weight, as well as on the activities of serum glutamate and pyruvate transaminases in serum. The results support the validity of this herbal drug for the management of testicular disorders noted in diabetic state.

View full abstract

We investigated the effects of intravenous administration of conivaptan hydrochloride, a dual vasopressin V_1A and V₂ receptor antagonist, on blood electrolytes and plasma osmolality in rats with an experimental syndrome of inappropriate secretion of antidiuretic hormone (SIADH). The experimental SIADH rat model was developed by means of continuous administration of arginine vasopressin (AVP) via a subcutaneously implanted osmotic mini pump, and hyponatremia was induced by additional water loading. This model possesses similar characteristics to those observed in patients with SIADH, specifically decreases in blood sodium concentration and plasma osmolality. In this experimental model, intravenous administration of conivaptan (0.1, 1 mg/kg) significantly increased blood sodium concentration and plasma osmolality. On the other hand, intravenous administration of furosemide (10 mg/kg) did not increase either blood sodium concentration or plasma osmolality in the SIADH rats. Moreover, furosemide significantly lowered blood potassium concentration. These results show that conivaptan improves hyponatremia in rats with SIADH, supporting the therapeutic potential of conivaptan in treatment of patients with hyponatremia associated with SIADH.

View full abstract

Randia siamensis is used in Thai folkloric medicine for inducing abortion and controlling blood pressure. The present study investigated the cardiovascular effects of an R. siamensis fruit extract, and mechanisms involved in anesthetized normal and reserpinized rats. R. siamensis (0.4—12 mg/kg) i.v. increased the mean arterial pressure (MAP) and heart rate. Both effects were significantly inhibited by phentolamine (2 mg/kg, i.v.) or propranolol (0.6 mg/kg, i.v.). The combination of phentolamine and propranolol, or reserpine pretreatment, inhibited the positive chronotropic effect with a slight decrease in the MAP. In vitro, R. siamensis (0.001—0.3 mg/ml) increased the rate of beating of the right atrium and the strength of the electrical field-stimulated contraction of the left atrium, both effects were inhibited by propranolol, or with reserpine pretreated rats. R. siamensis (0.01—3 mg/ml) produced a contraction of isolated thoracic aorta, which was potentiated by N^G-nitro-L-arginine (LNA), or by removal of the vascular endothelium, but inhibited by phentolamine, or reserpine. R. siamensis (0.3—3 mg/ml) caused a relaxation of phenylephrine-preconstricted aortic rings, which was potentiated with reserpine pretreatment, and abolished after removal of the vascular endothelium, or in the presence of LNA. These results suggest that R. siamensis extract exerts both hypertensive and positive chronotropic effects via the α- and β-adrenergic receptors of blood vessels and the heart, due to release of endogenous catecholamines, likely from nerve ending and adrenal medulla. The hypotensive activity results from the release of nitric oxide causing dilatation of the blood vessels. The present data support the folkloric therapeutic uses of this plant.

View full abstract

Changes in heat shock protein (Hsp) 60 of the viable left ventricular muscle (viable LV) after myocardial infarction in rats and the effect of the angiotensin I-converting enzyme inhibitor (ACEI) trandolapril were examined. Myocardial infarction was induced in rats by ligation of the left coronary artery. The coronary artery-ligated (CAL) and sham-operated (Sham) rats were orally treated with 3 mg/kg/d trandolapril from the 2nd to 8th week after surgery. Hemodynamic parameters and tissue weights of the left and right ventricles of the animals at the 8th week after CAL (8w-CAL rats) showed signs indicating chronic heart failure. An increase in Hsp60 content, a decrease in mitochondrial oxygen consumption rate (OCR), and an increase in the mitochondrial thiobarbiturate-reacting substance (TRS) of the viable LV were detected. Eight weeks after CAL. Long-term treatment of the CAL rats with trandolapril improved the hemodynamic parameters, attenuated the CAL-induced increase in Hsp60 content, the decrease in mitochondrial OCR, and the increase in the mitochondrial TRS content of the viable LV at the 8th week after myocardial infarction. The increase in Hsp60 content was closely related to the decrease in the mitochondrial OCR and to a rise in the LVEDP of the CAL animal at the 8th week after myocardial infarction. These results suggest that a series of pathophysiological alterations, including a reduction in mitochondrial function, appearance of reactive oxygen stress, and production of Hsp60 is involved in the development of cardiac failure and that trandolapril is beneficial for preventing these alterations.

View full abstract

Rhus verniciflua STOKES (RVS) has traditionally been used for medical purpose, such as healing of inflammatory diseases in South Korea. Glycoprotein (36 kDa) was isolated from RVS fruit, purified and used to evaluate the inhibitory effect on inflammatory-related proteins and nitric oxide (NO) production in lipopolysaccharide (LPS, 200 ng/ml)-stimulated RAW 264.7 (murine macrophage cell line). Our results were showed that RVS glycoprotein has a strong antioxidative activity against lipid peroxyl radicals in cell-free system, and inhibits NO production in LPS-stimulated RAW 264.7 cells. To elucidate the inhibitory effect of RVS glycoprotein on activities of inflammatory-related proteins, we firstly evaluated the amount of intracellular reactive oxygen species (ROS), and expression of intracellular protein kinase C (PKC), nuclear factor (NF)-κB, and activator protein-1 (AP-1). The results in the present study showed that RVS glycoprotein (200 μg/ml) inhibits ROS production and PKCα translocation, and down-regulates the expression of NF-κB and AP-1. Such upstream signals consequently inhibited the levels of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 expression. Therefore, we speculate that RVS glycoprotein inhibits the inflammatory-related protein and can act as an anti-inflammatory agent.

View full abstract

We examined the influence of 8-hydroxy-2-di-n-propylamino tetralin (8-OH-DPAT), a serotonin 1A (5-HT_1A) receptor full agonist, on the wet-dog shake response induced by the (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a 5-HT_2A receptor agonist, in adrenocorticotropic hormone (ACTH)-treated rats. Chronic ACTH (100 μg/rat, s.c.) treatment for 14 d increased the wet-dog shake response induced DOI. The 8-OH-DPAT inhibited the wet-dog shake response induced by DOI in rats with ACTH for 14 d. On the other hand, the 8-OH-DPAT-induced hypothermia and flat body posture were inhibited when ACTH was administered for 14 d. These findings suggest that chronic treatment with ACTH decreased the sensitivity of the 5-HT_1A receptor system; however, the inhibitory effects from the 5-HT_1A receptors to the 5-HT_2A receptor system is not inhibited in ACTH-treated rats.

View full abstract

Atherosclerosis, a disease of the large arteries, is the primary cause of heart disease and stroke. The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial walls is an important pathogenetic factor of vascular disorders like atherosclerosis and restenosis after angioplasty. In the present study, the possible anti-proliferative effect of a synthetic 1,4-naphthoquinone derivative, 2-chloro-3-(4-hexylphenyl)-amino-1,4-naphthoquinone (NQ304) was investigated on rat aortic VSMCs. NQ304 was shown to potently inhibit 5% fetal bovine serum (FBS)-induced the growth of VSMCs. Pre-treatment of VSMCs with NQ304 (1—10 μM) for 24 h resulted in significant cell number decreases, i.e., inhibition percentages were 44.75±10.77, 73.85±6.38 and 89.77±6.52% at NQ304 concentrations of 1, 5 and 10 μM, respectively. NQ304 was also found to significantly inhibit 5% FBS-induced DNA synthesis in a concentration-dependent manner. Furthermore, NQ304 elevated p21^cip1 and p27^kip1 mRNA levels and caused G₀/G₁ phase arrest in cell cycle progression. However, no evidence of NQ304-induced apoptotic or necrotic cell death was obtained, as determined by flow cytometery analysis and DNA fragmentation assays. To investigate the mechanism underlying the anti-proliferative effect of NQ304, we examined the effects of NQ304 on c-fos mRNA expression, activator protein-1 (AP-1) binding activity and extracellular signal-regulated kinase1/2 (ERK1/2) and Akt activation. Pre-treatment of VSMCs with NQ304 (1—10 μM) was found to significantly inhibit the 5% FBS-induced phosphorylations of ERK1/2 and Akt, the activation of AP-1 and the expression of c-fos. These data suggest that the anti-proliferative and cell cycle arresting effects of NQ304 on serum-induced VSMCs may be mediated by AP-1 activation downregulation via the suppression of phosphatidylinositol 3-kinase (PI3K)/Akt and ERK1/2 signaling pathways, and it may contribute to the prevention of atherosclerosis through inhibition of VSMC proliferation.

View full abstract

Nanoscale hydroxyapatite (nano-HAP) has been reported to exhibit anti-cancer effect on several human cancers, but the molecular mechanism of which remains unclear. The aim of this study was to explore the mechanisms by investigating the effects of nano-HAP on human gastric cancer SGC-7901 cells. Our results showed that nano-HAP significantly reduced cell viability, and induced apoptosis in SGC-7901 cells characterized by hypodiploid DNA contents, morphological changes and DNA fragmentation. The increase in apoptosis was accompanied with the increased expression of Bax, a pro-apoptotic protein, and decreased expression of Bcl-2, an anti-apoptotic protein, the decrease of mitochondrial membrane potential and the release of cytochrome c from mitochondria into cytosol. Furthermore, the activation of caspases-3, and -9, but not activation of caspases-8 was induced by nano-HAP. Z-VAD-fmk, a universal caspase inhibitor, dose-dependently inhibited nano-HAP-induced apoptosis. This study demonstrates that nano-HAP inhibits the proliferation of SGC-7901 cells by inducing apoptosis, and the apoptotic pathway of nano-HAP-induced apoptosis is mediated through the mitochondrial-dependent and caspase-dependent pathway.

View full abstract

N^ε>-(Carboxymethyl)lysine (CML) adduct, a major structure of advanced glycation end product, facilitated production of immature microvessels from choroidal explant cultured in fibrin gel. The present study was investigated an action of endogenous CML adduct on neovascularization of cultured choroidal explants of aged Wistar rats with 9 months of age. The number of microvessels budded from explants was counted under optical microscope and used as an index of in vitro neovascularization. Aged choroidal explants increased the neovascularization in an age-dependent manner. Anti-CML antibody decreased age-facilitated neovascularization as well as CML-human serum albumin (HSA)-facilitated neovascularization. Both the aged explant and CML-HSA-treated explant significantly released vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF) α and platelet-derived growth factor (PDGF)-B during the culture period. The release of TNF α and PDGF-B was earlier than that of VEGF from the aged explants. The antibodies against these factors decreased the CML-facilitated and age-facilitated neovascularization in the choroidal explants. The inhibitory capacity of anti-TNF α antibody was greater than those of anti-VEGF and anti-PDGF-B antibodies. In conclusion, endogenous CML adduct overproduced the neovascularization of the aged choroidal explant. The CML adduct releases TNF α which might induce the production and release of VEGF for the abnormal choroidal neovascularization in the patients of age-related macular degeneration.

View full abstract

Asiatic acid, a pentacyclic triterpene, has been reported to induce apoptosis of various human cancer cells. In the present study, we assessed the anti-tumor promoting effect of asiatic acid against 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated skin tumorigenesis in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated ICR mice. Topical application of asiatic acid prior to each application of TPA resulted in a significant reduction in skin tumor formation. We also found that pre-application of asiatic acid alleviated TPA-induced [³H]thymidine incorporation, which is a conventional marker for skin tumor promotion. In addition, asiatic acid inhibited the TPA-induced generation of nitric oxide (NO) and expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), which are known to play important roles in tumor growth, especially in the promotion stage. In addition, topical application of aminoguanidine (AG), a selective iNOS inhibitor, and N^G-nitro-L-arginine-methyl ester (NAME), another iNOS inhibitor, 30 min prior to TPA treatment significantly inhibited the TPA-induced COX-2 expression. These results suggest that asiatic acid may exert anti-tumorigenesis through inhibitory actions in NO and COX-2 signals.

View full abstract

During our on-going attempts to develop a new skin-whitening agent, we identified a novel candidate compound KHG22394, a 2-imino-1,3-thiazoline derivative. Our data show that KHG22394 significantly inhibits melanin production in a dose-dependent manner, but that it does not directly inhibit tyrosinase, the rate limiting melanogenic enzyme. It has been reported that the activation of extracellular signal-regulated kinase (ERK) reduces melanin synthesis by downregulating microphthalmia-associated transcription factor (Mitf). Thus, we examined the effects of KHG22394 on the ERK pathway and found that it induced ERK and 90 kDa ribosomal S6 kinase (RSK-1) activation. Moreover, α-melanocyte-stimulating hormone (α-MSH) is known to increase melanin biosynthesis by increasing tyrosinase production, and here, we found that α-MSH-induced Mitf and tyrosinase increases were inhibited in B16 melanoma cells treated with KHG22394. These findings suggest that the hypopigmentary effect of KHG22394 results from the downregulation of Mitf and subsequently of tyrosinase, although KHG22394 did not inhibit tyrosinase activity directly. Our findings indicate that 2-imino-1,3-thiazoline derivatives are potential skin whitening agents.

View full abstract

Essential oils have been widely used in anti-infectious application. In the present study, we elucidated the antifungal activities of eugenol and nerolidol isolated from Japanese cypress oil in a guinea pig model infected by Microsporum gypseum (M. gypseum). A minimal inhibitory concentration (MIC), skin lesion scoring, hair culture and histopathologic examination of skin tissues were performed to evaluate the antifungal effect of these oils. The MICs of eugenol, nerolidol and econazole (positive control) were 0.01—0.03% and 0.5—2% and 4—16 μg/ml, respectively. Based on these MICs, eugenol and nerolidol were adjusted to 10% concentration with a base of Vaseline^® petroleum jelly and were applied topically to the skin lesion infected with M. gypseum daily for 3 weeks. Both eugenol and nerolidol were clinically effective at improving the lesion during the first week of application, as determined by skin lesion scoring. Nerolidol improved the skin lesions infected by M. gypseum, but eugenol did not, as determined in the hair culture test. Histopathologic examination revealed that the eugenol- and nerolidol-treated groups had a lower degree of hyperkeratosis and inflammatory cell infiltration than the positive control. Taken together, these results suggest that eugenol and nerolidol could apply supplementary antifungal agents.

View full abstract

Resveratrol (trans-3,4′,5-trihydroxystilbene) is a natural phytoalexin found in grape skin, and has been suggested to be an antioxidant agent, an anticancer agent and a cardioprotectic agent. In particular, recent experimental evidence has demonstrated that resveratrol exhibits neuroprotective effects in various assay systems. During the study on the resveratrol derivatives, we found that (4-methoxybenzylidene)-(3-methoxyphenyl)amine (MBMPA), which has blocked free phenolic gruops, strongly protects neuronal cells against ischemic damage on a higher activity than resveratrol. The MBMPA potently reduced the level of neuronal cell death in an oxygen and glucose deprivation-exposed rat organotypic hippocampal slice culture. In addition, ATP depletion following the onset of oxygen and glucose deprivation in an adult hippocampal slice was blocked by the MBMPA treatment. These results suggest that MBMPA has a neuroprotective effect on an in vitro ischemia model, and may be useful for treating stroke.

View full abstract

In the present study, the effects of terpenes (styraxosides A and B) and lignans (egonol, masutakeside I, and styraxlignolide A) isolated from the stem bark of Styrax japonica SIEB. et ZUCC. (styracaceae) were evaluated on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production by the RAW 264.7 macrophage cell line. Of the tested compounds, styraxoside A was found to most potently inhibit the productions of NO and PGE₂, and also significantly reduced the release of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Consistent with these observations, the protein expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the mRNA expression levels of iNOS, COX-2, TNF-α and IL-1β were found to be inhibited by styraxoside A in a concentration-dependent manner. Furthermore, styraxoside A inhibited the LPS-induced DNA binding activity of nuclear factor-κB (NF-κB). Taken together, our data indicate that styraxoside A inhibits LPS-induced iNOS, COX-2, TNF-α, and IL-1β expressions through the down-regulation of NF-κB-DNA binding activity.

View full abstract

Glycyrrhizae Radix is used to treat abdominal pain as a component of Shakuyakukanzoto (Shaoyao-Gancao-tang), a traditional Chinese medicine formulation. Previously, we have reported the isolation of glycycoumarin as a potent antispasmodic with an IC₅₀ value of 2.93±0.94 μM for carbamylcholine (CCh)-induced contraction of mouse jejunum from an aqueous extract of Glycyrrhizae Radix (licorice), and clarified that its mechanism of action involves inhibition of phosphodiesterase 3. The purpose of the present study was to examine an antispasmodic principle of licorice other than glycycoumarin. Isoliquiritigenin was isolated from an aqueous extract of licorice as a potent relaxant, which inhibited the contraction induced by various types of stimulants, such as CCh, KCl, and BaCl₂ with IC₅₀ values of 4.96±1.97 μM, 4.03±1.34 μM and 3.70±0.58 μM, respectively, which are close to those of papaverine. However, the amount of isoliquiritigenin in the aqueous extract of licorice was very small. When the aqueous licorice extract was treated with naringinase, the amounts of glycosides such as isoliquiritin, which were abundant but had much less potent relaxant activity, were decreased while isoliquiritigenin was increased. At the time, the relaxant activity of the treated sample was increased significantly, shifting the IC₅₀ from 358±104 to 150±38 μg/ml for CCh-induced contraction. Isoliquiritigenin also showed the most potent inhibition of mouse rectal contraction induced by CCh with an IC₅₀ value of 1.70±0.07 μM. These results suggest that isoliquiritigenin acts as a potent relaxant in the lower part of the intestine by transformation from its glycosides.

View full abstract

In the course of our screening, we found that the methanolic extract of Sappan Lignum showed strong activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) production by macrophages in vitro. As it was reported that Brazilin inhibited inducible NO gene, we conducted to similar tests for six known compounds isolated from Sappan Lignum, namely, brazilein, sappanchalcone, protosappanin A, protosappanin B, protosappanin C besides brazilin. And six compounds were also subjected to six tests to speculate their properties: (1) inhibition of NO production by cultured J774.1 (macrophage-like) cell line, (2) suppression of inducible NO synthase (iNOS) gene expression, (3) inhibition of NO production by murine peritoneal macrophages, (4) DPPH radical scavenging activity, (5) reduction of ferric ion and (6) antioxidant activity. Brazilein and sappanchalcone showed significant inhibition of lipopolysaccharide (LPS)-induced NO production by J774.1 cell line like Brazilin; 100% inhibition at 30 μM in test (1) and at 10 μM in test (3). The mechanisms underlying the inhibition of NO production by the compounds were investigated in test (2). As a result, brazilin was found to almost completely suppress iNOS gene expression at 100 μM as reported, and brazilein and sappanchalcone also suppressed iNOS gene expression. But strong activities were not observed for protosappanins A, B and C. So, we conducted tests (4), (5) and (6) to investigate other properties about six compounds. Protosappanin A and Brazilin demonstrated high antioxidant activity compared with Vitamin E in tests (4) and (5). Protosappanin A and B inhibited the oxidation of linoleic acid in test (6). Among the dibenzoxocin derivatives, only protosappanin C did not show significant activity in all the tests. We found that sappanchalcone showed same activity as brazilin, and six compounds isolated from Sappan Lignum showed various properties.

View full abstract

The unripe fruit of Evodia rutaecarpa (JUSS) BENTH (ER, Family Rutaceae) has been used frequently as a traditional medicine against inflammatory diseases in Korea, China and Japan. To evaluate antiallergic effect of ER, we isolated its main constituents, evodiamine and rutaecarpine, and evaluated in vivo their inhibitory effects against passive cutaenous anaphylaxis (PCA) reaction induced by IgE-antigen complex and scratching behaviors by compound 48/80. ER and its constituents, evodiamine and rutaecarpine, potently inhibited PCA reaction and scratching behaviors in mice, although ER weakly inhibited scratching behaviors. Evodiamine and rutaecarpine inhibited TNF-α and IL-4 protein expression in RBL-2H3 cells induced by IgE–antigen complex, although these did not inhibit degranulation of RBL-2H3 cells induced by IgE–antigen complex and rat peritoneal mast cells induced by compound 48/80. These findings suggest that ER and its constituents, evodiamine and rutaecarpine, may be effective for IgE-induced allergic diseases such as atopic dermatitis and rhinitis.

View full abstract

We conducted a comparative study of 20 antioxidants including antioxidative vitamins and polyphenols to examine their inhibitory activities against the in vitro invasion, growth and experimental lung metastasis of murine colon 26-L5 carcinoma cells. Among the compounds tested, epigallocatechin gallate (EGCG), gallocatechin gallate and genistein exhibited significant reductions at 77%, 46% and 44% in tumor metastasis by an intraperitoneal administration for 5 d beginning at 3 d before tumor inoculation, respectively. Quercetin also showed a slight but not statistically significant inhibition. α-Tocopherol, β-carotene, ascorbic acid and 2 EGCG-related compounds of epicatechin gallate and epigallocatechin had no effect. EGCG also inhibited tumor metastasis dose-dependently with 98% suppression at 2 μmol; and an almost equivalent inhibition was also produced by only pre-administration of EGCG at the same dose before tumor inoculation. EGCG significantly inhibited tumor cell invasion and proliferation, but its inhibition of these activities was much less effective than that of other compounds which did not show any antimetastatic effect. No statistically significant relationship was observed between the radical scavenging activities of the test compounds and their rates of inhibition of tumor metastasis. The antimetastatic mechanism of EGCG thus seems to be independent of its inhibition of tumor invasion and growth, as well as its radical scavenging activity. Our results suggest that EGCG is potentially beneficial for tumor metastasis inhibition.

View full abstract

Acyl-CoA: cholesterol acyltransferase (ACAT) plays an important role in the esterification of cholesterol with its substrates, cholesterol and fatty acyl coenzyme A, to facilitate both intracellular storage and intercellular transport. ACAT-1 is more involved in macrophage foam cell formation and ACAT-2 plays a critical role in the cholesterol absorption process in intestinal enterocytes. Three aliphatic acid amides, β-sanshool (1), γ-sanshool (2), and hydroxy-β-sanshool (3), were isolated by bioassay-guided fractionation of the ethanolic extracts of Zanthoxylum piperitum DC. Compounds 1 and 2 inhibited human ACAT-1 and -2 activities with IC₅₀ values of 39.0 and 79.7 μM for 1 and of 12.0 and 82.6 μM for 2, respectively. However, the hACAT-1 and -2 inhibitory activities of compound 3 having hydroxyl group were relatively less than those of compounds 1 and 2. A semi-synthetic compound 4, which has acetyl residue at 2′-OH of compound 3, exhibited the increased hACAT-1 and -2 inhibitory activities with IC₅₀ values of 28.1 and 87.5 μM, respectively.

View full abstract

Increasing evidence has suggested that infection with high-risk human papillomavirus (HPVs) is closely associated with esophageal squamous cell carcinoma (ESCC) in China. The E6 and E7 oncoproteins expressed in ESCC are considered as attractive tumor-specific antigen targets for immunotherapy. We have reported that the HPV16 mE6Δ/mE7/TBhsp70Δ fusion protein vaccination induced powerful anti-tumor immunity against TC-1 tumor cells in a C57BL/6 mouse model. In the present study, we further evaluate the protective efficacy of this fusion protein vaccine using an HPV E7-expressing human ESCC cell line (EC9706) and a Hu-PBL-SCID mouse model. We demonstrated that immunization with the fusion protein vaccine caused significant inhibition of tumor growth with the delay time to tumor detection (tests vs. controls, 16 d vs. 9 d, p<0.01) and much smaller tumor size (p<0.01) in vivo. The inhibitory rate was ca. 69.6%, and 25% of the fusion protein vaccinated-mice remained tumor free by the end of the experiment (42 d). Furthermore, the activated lymphocytes (CD8⁺) were capable of infiltrating into the tumor site, and much more apoptotic cells along with activation of caspase-3 were observed in the tumors from vaccinated-mice. Also, high expression levels of human IFN-γ, TNF-α, granzyme B and perforin were detected in the tumors from vaccinated-mice. Therefore, we concluded that the HPV16 mE6Δ/mE7/TBhsp70Δ fusion protein vaccine is able to stimulate cellular-mediated immune response against E7-containing ESCC cells through CD8⁺-dependent CTL-induced apoptosis in Hu-PBL-SCID mice. These findings provide a scientific basis for HPV E7-expressing ESCC active immunotherapy.

View full abstract

Acetaminophen (APAP) is a popular analgesic. In the present study, we characterized the pharmacokinetics and pharmacodynamics of APAP in the Japanese. Five healthy volunteers were administered 1000 mg of APAP orally. Five patients with chronic pain were administered the optimal oral dose of APAP ranging from 600 to 1000 mg to allow for an adequate analgesic effect. Plasma APAP and APAP metabolite concentrations were measured in the volunteers, plasma APAP concentrations and pain scores using a visual analog scale were measured in the patients with chronic pain. Patient data were fitted to a first-order absorption one-compartment model with delayed effects accounted for by an effect compartment. A sigmoid E_max model was used as the pharmacodynamic model. Acetaminophen-cysteine metabolites, which are conjugates of the toxic metabolite N-acetyl-p-benzoquinone-imine, were detected in the plasma at levels lower than 0.2 μg/ml, but no side effects were observed. The pharmacokinetic and pharmacodynamic parameter (mean±S.D.) estimates were as follows: clearance, 18.7±4.7 l/h; distribution volume, 30.9±6.8 l; absorption rate constant, 2.4±1.3 h⁻¹; rate constant for the elimination of APAP from the effect compartment, 1.3±0.5 h⁻¹; maximum pain relief score, 4.6±2.2 units; effect compartment concentration at 50% maximum, 2.0±1.2 μg/ml; and sigmoid factor, 1.3±0.7. These results suggest that these parameters can be used to determine an effective APAP dosage regimen for Japanese patients with chronic pain.

View full abstract

A Diskhaler is a dry powder type of inhaler that utilizes a breath controlled drug delivery system. The inspiratory flow rate of the patient would have a significant influence on the effects of drugs administered by a Diskhaler. Thus, we investigated the relationship between inspiratory flow rate and amount of drug delivered into the lungs when using a fluticasone propionate dry powder inhaler with a Diskhaler (FP-DH). To investigate the amount of drug inhaled, we used an inhalation simulator, which consisted of a flow recorder placed in a plastic air-tight box that covered the FP-DH equipped with a twin impinger and a vacuum pump. Drugs located in a plastic box, as well as the device, throat, and stage 1 and stage 2, were assayed by HPLC-UV, following in vitro inhalation at the various flow rates ranged from 18.7 to 77.3 l/min for 2 s. The relationship between peak inspiratory flow rate and amount of drug released from the device was analyzed. A positive linear correlation between the dose released from the device and amount of drug deposited in stage 2 was observed (r=0.899, p<0.001). The doses deposited in stage 2 were estimated to be 2.9 μg at a flow rate of 20 l/min, 6.6 μg at 30 l/min, 8.4 μg at 40 l/min, 10.1 μg at 60 l/min, and 11.3 μg at 90 l/min. It was suggested that the amount of drug in the lungs decreased along with a decrease in peak inspiratory flow rate when it was lower than 60 l/min. Our results were found to be very useful to estimate lung deposition by using peak inspiratory flow rate for administration planning, especially in patients with a flow rate of less than 60 l/min.

View full abstract

In our previous paper, mixed polyetheleneglycol (PEG) modification of liposomes by a mixture of 1-monomethoxypolyethyleneglycol-2,3-distearoylglycerol (PEG-DSG) with short polyoxyethylene chain and PEG-DSG with long one was shown to increase fixed aqueous layer thickness (FALT) around the liposomal membrane, and this was useful in vivo. In this study, we investigated the characterization of mixed PEG modification of liposomes with different anchors (PEG2000-DSG and PEG2000-cholesterol (CHO)). When the liposomes was modified by a mixture of PEG2000-DSG and PEG2000-CHO, FALT was increased compared to that of each single PEG-lipids modification and the most suitable mix modification (PEG2000-DSG : PEG2000-CHO=3 : 1) showed a maximum FALT. This phenomenon was speculated to be based on the difference in the insertion state of the PEG anchor unit in the liposomal membrane. PEG-CHO-modified liposomes (single or mixed PEG-modified liposomes) were easily incorporated into the liposomal membranes compared with that of single PEG-DSG-modified liposomes. Namely, it was considered that the cholesterol anchor as a single chain was able to be easily introduced, compared with the DSG anchor as two chains, and induced some interaction with both PEG modification. In conclusion, it is expected that novel PEG-modified liposomes with PEG2000-DSG and PEG2000-CHO (3 : 1) had superior physicochemical properties.

View full abstract