LC/ESI/MS method was employed for the pharmacokinetic evaluation of total panax notoginsenoside (TPNS) in rats. After oral or intravenous administration of TPNS at the dosage of 300.0 or 10.0 mg kg−1 to rats respectively, panax notoginsenoside R1, ginsenoside Rg1, Rd, Re and Rb1 were simultaneous determined in rat plasma. Pharmacokinetic parameters and absolute bioavailability of panax notoginsenoside R1, ginsenoside Rg1, Rd, Re and Rb1 were obtained by the Drug And Statistics for windows (DAS) pharmacokinetic software. The pharmacokinetic parameters of all analytes were different form each other. T1/2 were changed from 0.72 to 22.16 h and AUC were changed from 1.03 to 98.94 mg/l·h after oral or intravenous administration TPNS or Xuesaitong (TPNS) injection. The absolute bioavailability of R1, Rg1, Rd, Re and Rb1 were of 9.29%, 6.06%, 2.36%, 7.06% and 1.18%, respectively.
The present study investigated the potential cytoprotective properties of a combination of plant extracts (KIOM-79) obtained from Magnolia officinalis, Pueraria lobata, Glycyrrhiza uralensis, and Euphorbia pekinensis, against the oxidative stresses induced by streptozotocin (STZ) in a rat pancreatic β-cells (RINm5F). KIOM-79 was found to scavenge intracellular reactive oxygen species (ROS), thereby preventing DNA damage and lipid peroxidation. The KIOM-79 inhibited apoptosis of the β-cells exposed to STZ via radical scavenging activity and activation of antioxidant enzymes. KIOM-79 inhibited activation of extracellular regulated kinase (ERK) induced by STZ and inhibited DNA binding activity of an activator protein-1 (AP-1), a downstream transcription factor of ERK. Taken together, these findings suggest that KIOM-79 protects against STZ induced cell death in RINm5F cells by inhibiting ROS generation and the ERK pathway.
Recent studies have shown that MCF-7 cells undergo autophagy under some conditions, such as tamoxifen treatment and starvation. In this study, we investigated autophagy in MCF-7 cells under oridonin treatment and further examined the relationship between autophagy and apoptosis. After 3-MA (the specific inhibitor of autophagy) pre-culture, MCF-7 cells were exposed to oridonin, and the growth inhibitory ratio, morphologic changes, DNA fragmentation, proteins expression, autophagic ratio and apoptotic ratio were evaluated. Oridonin inhibited the proliferation of MCF-7 cells and induced autophagy in vitro. MDC (a specific dye for autophagosome) recruitment and typical apoptotic features, including apoptotic bodies, membrane blebbing as well as nuclear condensation, were induced by oridonin. Oridonin downregulated the phosphorylation of ERK, whereas those of JNK and P38 kinase were upregulated. In the condition of oridonin treatment, 3-MA significantly reduced the autophagic level, and the apoptotic cell ratio was also declined. Furthermore, combined treatment with oridonin and 3-MA upregulated ERK phosphorylation and downregulated JNK and P38 kinases phosphorylation compared with oridonin alone treatment groups, indicating that autophagy facilitated apoptosis in oridonin-induced MCF-7 cells. In addition, 3-MA application downregulated DNA ladder and Bax expression but upregulated Bcl-2 expression, compared with oridonin alone treatment. Taken together, oridonin simultaneously induced MCF-7 cells both apoptosis and autophagy, and in this settings, inhibition of autophagy induced lowered apoptotic level, therefore, autophagy participated in upregulation of apoptosis.
To explore their genomic and functional characteristics, 292 human testis-specific genes were obtained from a UniGene library and a full-scale analysis was made. Various bioinformatics tools were applied to analyze the gene ontology and chromosome location, and the expression profiles of eight selected candidates from the 292 genes were analyzed using RT-PCR for 12 adult human tissues. The results showed that of the total 292 genes, 153 were known (114 assigned genes and 39 named genes), and 139 were unknown. Of the 114 assigned genes, 63 were labeled to molecular function, 28 to cellular component, 23 to biological process. All 292 genes are distributed on human chromosomes at different gene density, lower gene density appears on chromosomes 21 (R=0.22), X (R=0.33), 14 (R=0.39), 10 (R=0.61), 8 (R=0.63), and 18 (R=0.67) and higher density on chromosomes 19 (R=3.65), 20 (R=1.83), 16 (R=1.74), and 17 (R=1.64). The expression profile of the eight selected genes in the 12 human tissues showed that five candidate genes: Hs.443729, Hs.115366, Hs.558087, Hs.534501, and Hs.132104 were expressed exclusively in human testis; Hs.132310, Hs.443299 were expressed highly in testis and also expressed weakly in human heart; Hs.160370 was expressed in human testis, ovary, uterus, and not expressed in other tissues. Our study can be a basis for characterization of the function of human testis-specific genes during male mammalian spermatogenesis.
We isolated a novel inhibitor of melanin biosynthesis from the flowers of Arnica montana L. (Compositae), and identified it as a traxastane-type triterpene (3β,16β-dihydroxy-21α-hydroperoxy-20(30)-taraxastene)  by means of 1D or 2D-NMR and liquid chromatography/high-resolution mass spectrometry (LC-HR-MS). Compound  at the concentration of 0.53 μM completely inhibited melanin accumulation in cultured B16 melanoma cells. It is one of the most potent among known plant inhibitors of melanin biosynthesis in cultured cells, being 50 times more potent than 4-methoxyphenol, which is used as an anti-pigmentation agent. Its mechanism of action is considered to involve inhibition of transcriptional factor MITF-M (melanocyte-type isoform of microphthalmia-associated transcription factor), which would lead to a decrease of tyrosinase and related genes. We confirmed that compound  decreased the protein levels of tyrosinase and its related proteins in B16 melanoma cells. Further study revealed that a similar hydroperoxy triterpene also suppressed the melanin pigment accumulation of B16 melanoma cells. These results indicate that the hydroperoxy group may play an important role in the suppression of the melanin accumulation by compound .
To elucidate the mechanism of induction of apoptosis by geranylgeraniol (GGO), which is a potent inducer of apoptosis in various lines of human cancer cells, we examined the role of intracellular acidification during GGO-induced apoptosis using human leukemia HL60 cells. Flow cytometry analysis revealed that apoptosis induced in human leukemia HL60 cells by GGO was associated with intracellular acidification. Both GGO-induced intracellular acidification and apoptosis as analyzed by DNA fragmentation were inhibited by phorbol myristate acetate (TPA) and O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), an intracellular Ca2+ chelator, but not by ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). These results suggest that the early concentration change of intracellular Ca2+ and the corresponding decrease in intracellular pH are required for the induction of apoptosis in HL60 cells by GGO.
The mitochondrial ADP/ATP carrier (AAC) has 6 transmembrane regions and 3 matrix loops. Our previous mutational study on the Cys residue in the LM1s of chimeric bovine type 1 AAC (yN-bAAC1), in which the N-terminal 11 amino acids of bovine type 1 AAC are substituted with the corresponding 26 amino acids of yeast type 2 AAC (yAAC2), and yAAC2 in the yeast expression system suggested the possibility of a different structural feature between their LM1s. In the present study, we compared the effects of the SH cross-linking reagent copper-o-phenanthroline (Cu(OP)2) on yN-bAAC1 and yAAC2 in order to study the difference between these LM1s of the 2 carriers. Cu(OP)2 is known to cross-link 2 AAC molecules in a functional dimer via a Cys residue in each first matrix loop (LM1). yN-bAAC1 exhibited intra- and inter-molecular cross-linking, in agreement with the results of a previous study on the native bovine carrier and suggesting that yN-bAAC1 had the same structure as the native carrier. yAAC2 also showed intra- and inter-molecular cross-linking. However, the speed of formation of the inter-molecular cross-linking of yN-bAAC1 was faster than that of yAAC2, suggesting that the conformational state of the LM1 was different between the 2 carriers. In addition, we also studied the effects of AAC-specific inhibitors and solubilization with Triton X-100 on the cross-linking.
We have found that fibronectin (FN) has a functional cryptic site opposing cell adhesion to extracellular matrix (ECM): a synthetic FN peptide derived from the 14th FN type III-like (FN-III) repeat, termed peptide FNIII14, inhibits cell adhesion to the FN without binding to β1 integrins. This antiadhesive activity of peptide FNIII14 depends on its C-terminal amino acid sequence YTIYVIAL. A 50-kDa membrane protein (p50) has been detected as a specific binding protein of peptide FNIII14. Here we showed that antiadhesive activity of peptide FNIII14 was depedent upon the presence of p50 on cell surfaces. Furthermore, we found that there exists a sequence, analogous to the YTIYVIAL, in the 10th FN-III repeat of the FN molecule and that a FN peptide containing this analogous sequence, termed peptide FNIII10, inhibited cell adhesion to the FN. Peptide FNIII10 appeared to share p50 with peptide FNIII14 in expressing the antiadhesive activity. As a physiological consequence of decreased adhesion, peptides FNIII10 and FNIII14 accelerated the anoikis-like apoptosis of normal fibroblasts by down-regulating Bcl-2 expression through blocking the FAK/PI3K/Akt signaling pathway. Thus, the YTIYVIAL-related sequences of the FN molecule may be involved in cell regulation by modulating negatively cell adhesion to the ECM, in which p50 probably serves as a membrane receptor.
Several mammalian nucleoside transporters have been identified at the molecular level. Human and rat equilibrative nucleoside transporter 2 (hENT2 and rENT2, respectively) was previously reported to have the dual ability of transporting both nucleosides and nucleobases. In the present study, we characterized the transport of a variety of nucleosides and nucleobases via recombinant mouse ENT2 (mENT2). Cloned mENT2 mediated the uptake of nucleosides and purine nucleobases, but not pyrimidine nucleobases. The mENT2-mediated uptake of adenosine was significantly inhibited by nucleosides and nucleobases, irrespective of purine and pyrimidine. The Km values for the uptake of nucleosides and purine nucleobases mediated by mENT2 varied between 1.24 and 16.3 μM, and the transport clearances of adenosine and hypoxanthine via the transporter were greater than those of other substrates. Therefore, we concluded that mENT2 is nucleoside and purine nucleobase transporter, and pyrimidine nucleobases are blockers for the transporter, differing from hENT2 and rENT2 that were reported to also transport pyrimidine nucleobases.
To determine the short- to mid-term effects of ovariectomy on bone turnover, bone mass and bone strength in rats. SD rats aged 12 weeks were randomly divided into No-treatment, Sham and OVX groups. The rats were sacrificed for sample collection at week 0, week 4 and week 18 after surgical operation. Chemistries in serum and urine were measured by standard colorimetric methods and bone turnover markers were measured by ELISA kits. Bone mass and bone strength were determined using pQCT system and three-point bending tests, respectively. At week 4, OVX rats showed drastic increase in the level of urine Ca, P and DPD. At week 18, in OVX rats the levels of serum ALP, urine DPD and Ca were much higher and the level of serum Ca was much lower when comparing with Sham rats. Ovariectomy produced significant reduction in cancellous BMD, total BMD and SSI of proximal tibial metaphysis rapidly at week 4 and continuously at week 18 after surgical operation. However, no marked changes of bone mass and bone strength were found in the diaphysis of tibia and femur, respectively. The current study concluded that ovariectomy induced the uncoupling of bone turnover, and the proximal metaphysis of long bone was the sensitive site for the short- to mid-term effect of ovariectomy, demonstrated as the markers of bone mass and stress strain index.
Plant lignans, such as pinoresinol diglucoside, secoisolariciresinol diglucoside and arctiin, are metabolized to mammalian lignans, enterolactone or enterodiol, by human intestinal bacteria. Their metabolic processes include deglucosylation, ring cleavage, demethylation, dehydroxylation and oxidation. Here we isolated an intestinal bacterium capable of demethylating arctigenin, an aglycone of arctiin, to 2,3-bis(3,4-dihydroxybenzyl)butyrolactone (1) from human feces, and identified as an Eubacterium species (E. sp. ARC-2), which is similar to Eubacterium limosum on the basis of morphological and biochemical properties and 16S rRNA gene sequencing. By incubating with E. sp. ARC-2, arctigenin was converted to 1 through stepwise demethylation. Demethylation of arctigenin by E. sp. ARC-2 was tetrahydrofolate- and ATP-dependent, indicating that the reaction was catalyzed by methyltransferase. Moreover, E. sp. ARC-2 transformed secoisolariciresinol to 2,3-bis(3,4-dihydroxybenzyl)-1,4-butanediol by demethylation.
New guidelines suggest that HIV-infected pregnant women should be offered combination antiretroviral therapy (zidovudine and protease inhibitors) to prevent fetal HIV infection but concerns remain about potential adverse effects for the infant. Prior small case series have suggested an increased risk for hemangioma. In this study we used zidovudine and indinavir, alone or in combination, to assess the effect on an in vitro angiogenesis system for endothelial cells. The increase in capillary tube formation, was associated with a significant increase in vascular endothelial growth factor (VEGF) production. Zidovudine and indinavir used in combination do not further strengthen both endothelial cell tubes formation and VEGF secretion. We conclude that zidovudine and indinavir may induce angiogenesis in an in vitro model.
We investigated the vasodilator responses of retinal arterioles induced by stimulating corticotropin-releasing factor receptors in non-diabetic and diabetic rats. Male Wistar rats were treated with streptozotocin (65 mg/kg, i.v.) and experiments were performed 6—8 weeks later. Rats were treated with tetrodotoxin (50 μg/kg, i.v.) to eliminate any nerve activity and prevent movement of the eye and infused with a mixture of norepinephrine and epinephrine to maintain adequate systemic circulation under artificial ventilation. Fundus images were captured with an original high-resolution digital fundus camera system. The vasodilator responses of retinal arterioles were assessed by measuring changes in diameters of retinal arterioles in response to urocortin and urocortin 2. Both urocortin (0.03—1.0 μmol/kg, i.v.) and urocortin 2 (0.1—3.0 μmol/kg, i.v.) increased diameters of retinal arterioles and decreased systemic blood pressure in a dose-dependent manner. The responses to urocortins were reduced in diabetic rats. These results suggest that urocortin and urocortin 2 play as vasodilators in retinal and peripheral resistance arterioles. The impairment of vasodilation mediated by the corticotropin-releasing factor receptors may contribute to the alteration of retinal and systemic circulation in the diabetic state.
In this paper, we directly demonstrate, for the first time, the activation of Ca2+-dependent protein kinase C (PKC) in the spinal cord of diabetic mice. In streptozotocin (STZ)-treated (200 mg/kg, i.v.) diabetic mice, hypersensitivity (allodynia) to mechanical stimulation appeared 7 d after STZ injection. This mechanical allodynia was inhibited by intrathecal injection of the PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and calphostin C, but not the protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89). The activity of membrane-associated Ca2+-dependent PKC in the spinal cords of STZ-induced diabetic mice was significantly higher than that observed in non-diabetic mice. These results suggest that activation of Ca2+-dependent PKC in the spinal cord, contributes to the mechanical allodynia in the pain associated with diabetic neuropathy.
Trimidox (3,4,5-trihydroxybenzamidoxime) is one of the most potent ribonucleotide reductase inhibitors, revealing an antitumor effect in several experimental studies. We have examined the effect of trimidox on the induction of cytotoxicity and apoptosis via oxidative stress by typical free radical inducers, hydrogen peroxide (H2O2), tert-butylhydroperoxide (tBuOOH) or ultraviolet (UV) irradiation in a human diffuse histiocytic lymphoma U937 cell line. Trimidox showed strong radical scavenging activity by the DPPH reduction assay. The 50% rate inhibited the DPPH reduction concentration of trimidox, and its derivates didox, or gallic acid were 8.8 μM, 117.5 μM, or 41.8 μM, respectively. Induction of cytotoxicity by H2O2 (500 μM) or tBuOOH (100 μM) was concentration-dependently attenuated by incubation with Trimidox (10—150 μM). Trimidox also prevented the effect of UV-induced apoptosis estimated by both nuclear morphological change and DNA fragmentation. This effect was due to inhibition of the production of reactive oxygen species. Moreover, the activity and mRNA expression of catalase, an antioxidant enzyme, was significantly increased by trimidox. These results indicate that trimidox has radical scavenging activity and prevents exogenous oxidative stress and increase in catalase; therefore, trimidox is suggested as an anticancer agent exhibiting potent antioxidant properties in this study.
Moutan cortex (MC) is one of the most widely used Oriental herbal medicines for treating inflammatory diseases. In this study, the effect of MC on lipopolysaccharide (LPS) and recombinant interferon-gamma (rIFN-γ)-induced production of nitric oxide (NO) and tumor necrosis factor (TNF)-alpha were examined using mouse peritoneal macrophages. MC inhibited the LPS/rIFN-γ-induced expression of inducible nitric oxide synthase (iNOS) and TNF-alpha release. To clarify the mechanism involved, the effect of MC on the activation of nuclear factor (NF)-kappaB was examined. The LPS/rIFN-γ-induced activation of NF-kappaB was almost completely blocked by MC at 0.5 mg/ml. These findings demonstrate that the inhibition of the LPS/rIFN-γ-induced production of NO and TNF-alpha by MC is due to the inhibition of NF-kappaB activation.
Ginsenosides have been regarded as the main active components of Panax ginseng C. A. MEYER, and the antioxidant activity of ginsenosides in vivo is well described. However, there has been virtually no report describing the direct free radical-scavenging activities of ginsenosides through the long history of ginseng research. The hydroxyl radical (·OH)-scavenging activity test using an electron spin resonance spectrometer (ESR) is suggested to be the most appropriate to measure the antioxidant activities of ginsenosides. Therefore, we investigated the ·OH-scavenging and ferrous metal ion-chelating activities of several ginsenosides of Panax ginseng using ESR for the identification of active ginsenosides and its structure and activity relationships. As a result, 20(S)-Rg3 showed the strongest activity, and the next were in the decreasing order of Rb1, Rg1, Rc, Rb2, and Rd when dissolved with water. The ·OH-scavenging activities of ginsenosides were related to the ferrous metal ion-chelating activities of their aglycone, 20(S)-protopanaxadiol. In addition, the ferrous metal ion-chelating activities of ginsenosides were thought to be influenced by their types of hydrophilic sugar moieties.
In this study, we investigated the effects of an aqueous extract of peanut (Arachis hypogaea L.) seed skin (PSE) and its main constituent procyanidin A1 (PA) on the allergic response to allergen ovalbumin (OVA) in a mouse model. Mice immunized interaperitoneally with OVA dramatically increased anti-OVA IgE and total IgG1 levels in serum compared with non-treated control mice. Oral injection of PSE at doses ranging from 10 to 100 mg/kg/d (for 21 consecutive days) decreased anti-OVA IgE and IgG1 levels 21 d after OVA-immunization. OVA-induced increments in spleen weight and peripheral white blood cell count were also suppressed by this PSE administration. Polyphenol-enriched fractions from apple (30 mg/kg) and grape seed (30 mg/kg) also decreased anti-OVA IgE level but did not affect total IgG1 levels. Oral injection of PA (1 to 10 mg/kg/d) purified from PSE resulted in a suppression of IgE and total IgG1 levels in serum. An increment of serum interleukin-4 level in mice that were immunized with OVA was reduced by all tested samples, whereas PSE and PA were the only compounds that could reverse the reduced interferon-gamma level by OVA. These findings suggest that intake of PSE or its main active constituent PA may prevent an allergic reaction by inhibiting immunoglobulin synthesis, and the mechanism of this action of PSE and PA is in part due to their regulation of T helper cytokine production.
UVB irradiation is an important inducer of biological changes in skin and can activate inflammatory reactions and apoptotic pathways, leading to skin damage. A root extract of Lithospermum erythrorhizon (SK), which has naphthoquinone pigments containing shikonin and shikonin derivatives, is known for its anti-inflammatory, anti-bacterial, and anti-tumor activity, and for its scavenging of reactive oxygen species. However, the effect of SK against UV damage is not clear. The aim of this study was to evaluate the efficacy of SK against UVB induced damage in normal human epidermal keratinocytes (NHEK). UVB-irradiated NHEK showed decreased cell viability, increased production of interleukin (IL)-1α, IL-6, IL-8, and tumor necrosis factor-α, and induced apoptosis. In an apoptosis pathway assay, UVB-irradiated NHEK showed increased caspase-3 activity, p53 and its phosphorylation at serine 15 compared with non-irradiated cells. All these effects induced by UVB irradiation were clearly inhibited by treatment with SK before and after UVB irradiation for 24 h. It is suggested that SK can protect epidermal cells against harmful effects of UVB irradiation and that SK treatment is probably beneficial for photoprotection of the skin.
The in vitro effects of a methanol extract from the aerial parts of Centella asiatica on shear-induced platelet activation and coagulation were assessed after oral administration to rats, by subjecting non-anticoagulated blood to haemostatometry. 3,5-Di-O-caffeoyl quinic acid, 1,5-di-O-caffeoyl quinic acid, 3,4-di-O-caffeoyl quinic acid, 4,5-di-O-caffeoyl quinic acid, and chlorogenic acid, together with asiaticoside, kaempferol, quercetine, kaempferol-3-O-β-D-glucoside and quercetin-3-O-β-D-glucoside were all isolated from the methanol extract. Amongst these, only 3,5-di-O-caffeoylquinic acid showed significant inhibition of shear-induced platelet activation and dynamic coagulation. The reactive curve of the inhibitory effect on the platelet reaction and the dynamic coagulation showed a bell-shape.
The Rubus species has been used in folk medicine to treat several ailments, including infectious and dolorous diseases. In this work we evaluate the phytochemical and analgesic activity of hydroalcoholic extract (HE), some fractions (hexane, dichloromethane, ethyl acetate and butanolic), as well as a pure compound denoted as 28-methoxytormentic acid (1) obtained from aerial parts of R. rosaefolius. The compounds were isolated and identified by chromatographic and spectroscopic analysis. The antinociceptive action was evaluated by two well know models of pain in mice: writhing and formalin induced-pain. The results showed that the HE, fractions and compound (1), exhibits potent and dose-related analgesic activity when evaluated in both models of pain. Compound (1), which seems to be the main active principle, showed promising analgesic effects, being several times more potent than aspirin and paracetamol, two well-known analgesic and antiinflammatory drugs used as reference. In the writhing test, it showed an ID50 of 5.10 (3.64—7.14) mg kg−1 and maximum inhibition (MI) of 64.22%. When analyzed by formalin induced-pain test, this compound showed ID50 values of 9.98 (8.08—12.31) and 6.31 (5.07—7.98) mg kg−1 and MI of 59.37 and 90.37% for the first and second phases, respectively. The results justify, at least partially the popular use of this plant for the treatment of dolorous processes, suggesting that 1 is one of the active principles of this plant.
The purpose of present study was to examine spleen-selective gene transfer following the administration of naked plasmid DNA (pDNA) onto the spleen surface in mice. Gene expression in the spleen and other tissues was evaluated based on firefly luciferase activity. Six hours after spleen surface instillation of naked pDNA, high gene expression in the spleen was observed. On the contrary, intravenous and intraperitoneal administration of naked pDNA resulted in no detectable gene expression. After instilling naked pDNA onto the spleen surface, gene expression in the spleen was significantly higher than those in other tissues. Six hours after instillation of naked pDNA onto the spleen surface, gene expression in the spleen reached the peak value, and thereafter decreased gradually. By utilizing a glass-made diffusion cell that is able to limit the contact dimension between the spleen surface and naked pDNA solution administered, site-specific gene expression in the spleen was found. This novel gene transfer method is expected to be a safe and effective strategy for DNA vaccine against serious infectious diseases and cancers.
The radioprotective effects of propolis and polyphenolic compounds from propolis on the radiation-induced mortality of mice exposed to 9 Gy of γ-irradiation were studied. Intraperitoneal (i.p.) treatment of mice at doses of 100 mg kg−1 body weight of propolis (water or ethanolic extract; WSDP or EEP) or its polyphenolic compounds (quercetin, naringin caffeic acid, chrysin) consecutively for 3 d before irradiation, delayed the onset of mortality and reduced the symptoms of radiation sickness. All test compounds provided protection against hematopoietic death (death within 30 d after irradiation). The greatest protection was achieved with quercetin; the number of survivors at the termination of the experiment was 63%. According to statistical analyses by the Kaplan–Meier method and the log-rank test, a significant difference between test components and control was found (p<0.001). Treatment with test components after lethal irradiation was ineffective. These results suggest that propolis and its polyphenolic compounds given to mice before irradiation protect mice from the lethal effects of whole-body irradiation.
Teicoplanin is a glycopeptide antibiotic comprising six closely related major components whose activities against specific microbial species differ. In order to clarify the significance of monitoring these components separately for determining the therapeutic effectiveness of teicoplanin, we measured the total and unbound concentrations of the main teicoplanin components in plasma and the unbound fractions in patients. Teicoplanin components in plasma were determined separately by high-performance liquid chromatography following a co-extractive clean-up procedure. The concentrations of unbound teicoplanin components were estimated after plasma ultrafiltration. The plasma concentrations of the main components of teicoplanin were strongly correlated with each other. The apparent elimination rate constants of total bound and unbound teicoplanin calculated by population pharmacokinetic parameters were almost same among the components. Furthermore, the mean population unbound clearance corrected by the unbound fraction was almost the same among the components. These results suggest that monitoring the individual components of teicoplanin has no clinical significance based on the pharmacokinetics of teicoplanin.
To elucidate the relationships between the pharmacokinetics and pharmacological effects of oxybutynin ((R/S)-OXY), the micturition pressure and the plasma concentration profiles of (R)-OXY and (R)-N-desethyloxybutynin ((R)-DEOB), a pharmacologically active metabolite, after administration by three different routes (i.v., p.o. and transdermal) in rats were measured and analyzed using an inhibitory effect Emax model with their in vitro pharmacological effects. The plasma exposure ratios of (R)-DEOB to (R)-OXY calculated from the AUCs were somewhat different among the routes administered. (R)-OXY and (R)-DEOB equally inhibited the acetylcholine-induced contractions in vitro. The micturition pressure, measured using the cystometric method in vivo, exhibited saturation against the dose administered. The inhibitory effect Emax model well described the relationship between the micturition pressure and the receptor occupancy calculated from the plasma concentrations and pA2 values and resulted in an extremely small receptor occupancy (0.206%) to exhibit half of the maximum effect. The estimated receptor occupancy profiles suggested a sufficient and long-lasting receptor occupation after transdermal administration of (R/S)-OXY, while the receptor occupancy diminished rapidly after the i.v. and p.o. administration. These data indicate that transdermal administration of (R/S)-OXY would be useful to achieve suitable pharmacological effects without excess plasma concentrations.
The efficacy of many drugs is improved by liposomal formulations. The greatest improvements in therapeutic benefits are achieved if the drug is retained in the liposomes for several hours after administration. Many basic drugs can be concentrated efficiently into liposomes in response to a transmembrane pH gradient. However, the rate of release from liposomal formulations is drug-dependent; for example, doxorubicin is released slowly from liposomes whereas vincristine leaks out rapidly. The aim of this study was to identify the causes of the rapid release of drugs from liposomes and then to apply this knowledge to the development of more stable formulations. Our initial focus was to explore the influence of liposomal size on the rate of release of drugs. The retention of doxorubicin within liposomes was independent of the particle size as far as this experimental condition was concerned. However, the rate of release of vincristine varied in relation to the particle size of the liposomes; vincristine was retained more effectively in larger liposomes. Experimental data generated using 31P-NMR analysis and trap volume measurements, indicated that the number of lipid bilayers in liposomes increased as the particle size was increased. Additional lipid bilayers are likely to present a more effective barrier thereby slowing the release of drugs.
1-Furan-2-yl-3-pyridin-2-yl-propenone (FPP-3) is an anti-inflammatory agent with a propenone moiety. Following a single intravenous injection of male Sprague-Dawley rats with 4 mg/kg of FPP-3, three different metabolites of FPP-3 were identified as M1 (1-furan-2-yl-3-pyridin-2-yl-propan-1-one), M2 (1-furan-2-yl-3-pyridin-2-yl-propan-1-ol) and M3 (a glucuronide conjugate of M2) in rat urine by a liquid chromatography-electrospray tandem mass spectrometry. The structures of M1 and M2 were the same as observed previously following the incubation of rat liver microsomes with FPP-3 in the presence of NADPH. Although all metabolites of FPP-3 were identified in rat urine, only M1 and M2 were observed in the bile and feces. In addition, FPP-3 and its metabolites were mostly excreted into the urine. The M3 was identified as a glucuronide conjugate of M2 because of the addition of 176 Da from the protonated molecular ion of M2 in MS2 and because of the production of free M2 following an incubation of urine with β-glucuronidase. From these studies, a possible metabolic fate of FPP-3 could be proposed in vivo.
The poor selective cytotoxicity of anticancer drugs lead to dose-limiting adverse effects which compromise the clinical outcome. Solid tumors recruit new blood vessels to support their growth, and epitopes that are uniquely expressed on tumor cells and tumor endothelial cells (ECs) can function as targets for immunoliposomal anticancer drugs. Membrane type 1 matrix metalloproteinase (MT1-MMP), an important protein related to tumor growth and angiogenesis, is expressed on malignant tumor cells and is activated ECs. Selective delivery could be achieved by targeting MT1-MMP, as well as other angiogenic ECs. In this regard, an anti-MT1-MMP Fab′ antibody was used to prepare a MT1-MMP targeted sterically stabilized immunoliposomes (SIL[anti-MT1-MMP(Fab′)]). The binding and intracellular distribution of SIL[anti-MT1-MMP(Fab′)] and a non-targeted sterically stabilized liposomes (SL) were examined using human fibrosarcoma HT-1080 cells. SIL[anti-MT1-MMP(Fab′)] was taken up by the cells in a lipid concentration, temperature, and time dependent manner, ultimately accumulating in the lysosomes. The cytotoxicity of doxorubicin (DXR)-containing SIL[anti-MT1-MMP(Fab′)] (DXR-SIL[anti-MT1-MMP(Fab′)]) was significantly higher than that of DXR-containing SL. The cellular internalization of SIL[anti-MT1-MMP(Fab′)] was inhibited by endocytosis inhibitors, suggesting that their internalization was mediated via clathrin- or caveolae-dependent endocytosis. Furthermore, the efficient binding of SIL[anti-MT1-MMP(Fab′)] was observed on human umbilical vein endothelial cells (HUVEC). Based on these results, it would be expected that DXR-SIL[anti-MT1-MMP(Fab′)] may achieve direct tumor cell kill and indirect tumor cell kill via the destruction of the tumor endothelium in vivo. This strategy may have the potential for overcoming some major limitations in conventional chemotherapy in vivo.
Lafutidine, a histamine H2-receptor antagonist, inhibits gastric acid secretion during the daytime, however, the relationship between the plasma concentration and the drug response remains unclear. The aim of this study was to compare the pharmacokinetic and pharmacodynamic properties of lafutidine and famotidine following postprandial oral administration. After a lafutidine tablet (10 mg), famotidine tablet (20 mg), or water only (control) was administered, blood samples were taken and intragastric pH was measured. The plasma concentrations of lafutidine and famotidine were determined by HPLC, and the median intragastric pH values per 30 min were used as the degrees of gastric acid suppression. Data were analyzed based on a one-compartment pharmacokinetic model and a sigmoid Emax pharmacodynamic model. Lafutidine plasma concentrations rapidly increased after administration; famotidine required some time to increase the plasma concentrations, requiring an absorption lag time in the pharmacokinetic model. Between the plasma concentration and ΔpH (the difference in intragastric pH by the drug vs. control), lafutidine showed an anticlockwise hysteresis loop which indicated equilibration delay between the plasma concentration and effect site, requiring an effect site compartment in the pharmacodynamic model; famotidine showed more parallel relationship. These results indicated that the pharmacokinetic and pharmacodynamic properties of lafutidine after postprandial oral administration were different from those of famotidine at least 4.5 h after dosing.
The purpose of this study is to propose a kinetic model to predict the absorption of nasally applied drugs from their permeability to the Caco-2 monolayer (PCaco-2). Since a drug applied to the nose in an in vivo physiologic condition is translocated to the gastrointestinal (GI) tract by coordinated beats of cilia (mucociliary clearance, MC), the drug undergoes absorption both from the nasal cavity and from the GI tract. The detailed MC of the rat was examined, using inulin as a marker of the applied solution. Inulin disappeared monoexponentially from the nasal cavity, indicating that the MC can be assumed to follow first-order kinetics. From the disappearance of inulin, the first order rate constant for MC (kMC) was calculated as 0.0145 min−1. In the proposed kinetic model, the fractional absorption of the drug following nasal application is predicted as the sum of FNC (fractional absorption from the nasal cavity) and FGI (fractional absorption from the GI tract), both of which are estimated indirectly from PCaco-2. FNC is calculated according to the equation, ka/(ka+kMC), where ka is the absorption rate constant. Nasal drug absorption is assumed to follow first order kinetics. The ka of four drugs was initially calculated from kMC and their FNC; thereafter, the linear relationship between ka and PCaco-2, from which ka is predicted, was determined. FGI is calculated as Fp.o.(1−FNC), where Fp.o. is fractional absorption after oral administration. Fp.o. was predicted from the previously determined sigmoid curve between Fp.o. and PCaco-2. The proposed kinetic model is the first estimation system for nasal drug absorption based on drug disposition after nasal application and is useful for the development of nasal dosage forms.
As 67Ga is injected into the blood, 67Ga is immediately bound to transferrin (Tf) and transported to various tissues and the Tf–67Ga complex binds to Tf receptor on various tissues. In partial hepatectomy (PH) a part of blood in circulation is lost together with removed liver tissues, consequently the amounts of blood cells and Tf in circulation decrease. In order to investigate the effect of those decreases on 67Ga uptake by the liver, we compared the uptake in partially hepatectomized rats with that in venesectioned rats in which only a part of blood in circulation decreased. A two-thirds PH was performed. Two milliliters of blood was venesectioned. Each treated rat was intraveneously injected with 67Ga. The changes of erythrocyte and reticulocyte contents after PH did not differ from those after venesection (VS) at all. But 67Ga uptake by reticulocytes significantly increased after VS but did not after PH. On the other hand, 67Ga uptake by the liver significantly increased after PH but did not after VS. These differences must be related to the different expression of Tf receptors on the liver after PH and VS.
The present study investigated the effects of Hachimi-jio-gan (HJ) on diabetic hyperglycemia in streptozotocin (STZ)-induced diabetic rats. After STZ administration, rats had free access to pellets containing 1% HJ extract powder for four weeks. HJ markedly suppressed hyperglycemia in STZ-induced diabetic rats at three and four weeks after the start of administration. There were also significant increases in serum and pancreatic immunoreactive insulin levels in STZ and HJ co-administering rats. However, in the present study, the number of β cells in the pancreatic Langerhans' islets did not increase. Next, in order to investigate the action mechanism besides the glycemic control action of insulin, the expression of glucose transporter 2 (GLUT2) protein, which is involved in glucose uptake and release in the liver, was investigated. GLUT2 protein expression was increased by STZ administration but was normalized after four weeks of HJ administration. Therefore, irrespective of the structural changes in pancreatic β-cells due to STZ, HJ increased insulin production and secretion by the pancreas and significantly suppressed GLUT2 synthesis in the liver. Amylase secretion from the pancreas was measured to assess pancreatic secretion. Amylase activity was decreased by STZ but was increased by HJ. Therefore, the effects of HJ on STZ-induced hyperglycemia in rats could be summarized as follows: besides increasing insulin synthesis and release, HJ normalizes GLUT2 protein expression in the liver to suppress hyperglycemia. Hence, the results of the present study suggest for the first time that HJ affects not only the production and secretion of insulin, but also the release of glucose from the liver.
In vitro liver microsomal stability, permeability, pharmacokinetics (PK) and oral bioavailability of SB639, a novel HDACi (Histone Deacetylase inhibitor), were determined. The in vitro metabolism was examined in mouse, rat, dog and human liver microsomes. The permeability and efflux potential of SB639 were determined using Caco-2 cell monolayers. To determine pharmacokinetics and oral bioavailability, blood samples were drawn at pre-determined intervals up to 24 h post-dose after single intravenous (i.v.) or oral (p.o.) administration of SB639 to mouse or rat. The concentrations of SB639 in plasma samples were determined by a validated LC-MS/MS method. In vitro liver microsomal stability data revealed that SB639 was stable in human and dog liver microsomes, unstable in mouse and rat liver microsomes. The Caco-2 data has shown that SB639 is highly permeable with an apparent permeability of 3.01·10−6 cm/s at 10 μM. After oral administration, maximum concentrations of SB639 were achieved within 0.5 h of post dose. Following i.v. administration, the concentration of SB639 declined in a bi-exponential fashion with terminal elimination half-life of 1.67 h for mice and 1.12 h for rats. The systemic clearance and volume of distribution of SB639 in mice were 15.8 l/h/kg and 38 l/kg, respectively, while the respective values in rats were 3.84 l/h/kg and 3.67 l/kg. Elimination half-life in rats ranged between 1.12—2.26 h. Absolute oral bioavailability of SB639 in mouse and rat was 13% and 10%, respectively. In conclusion, the superior potency, physicochemical and PK properties of SB639 compared to the recently FDA approved drug Zolinza (Suberoylanilide hydroxamic acid or Vorinostat) in the preclinical setting makes it a potential clinical candidate.