We have developed an enzyme-linked immunosorbent assay (ELISA) for serum 11-dehydrocorticosterone (4-pregnen-21-ol-3,11,20-trione). The antiserum against 11-dehydrocorticosterone 21-hemisuccinate-conjugated bovine serum albumin was raised in rabbits. As an enzyme-labeled antigen, 11-dehydrocorticosterone 21-hemisuccinate was conjugated to horseradish peroxidase. Two ELISA systems were established: one without the extraction of steroids from serum (direct method), and another that used an HPLC purification step (HPLC method). The cross-reactivity of all steroids tested against the antibody was low except cortisone (92%); however, since cortisone levels in rats and mice are negligible, cortisone does not interfere with this direct ELISA. The measurable range of serum 11-dehydrocortiocosterone in both the direct and HPLC methods was 0.3—250 ng/ml and 0.78—400 ng/ml, respectively. Both methods displayed satisfactory parallel dilution, recovery and reproducibility; moreover, the values obtained with each method significantly correlated with the alternate method. To evaluate the two ELISA systems, the serum concentrations of 11-dehydrocorticosterone in normal rats and mice were determined by these two systems. The levels in Wistar rats fluctuated from 3 to 14 weeks of age (7.8±2.6 ng/ml) but at 1 week (1.7±1.2 ng/ml) were significantly low compared to other ages. No sex differences were found in rats and mice. Further, using the proposed direct method, chronological changes of rat serum 11-dehydrocorticosterone levels after 11-dehydrocorticosterone administration have been investigated together with corticosterone levels. These results verify that the proposed ELISA for 11-dehydrocorticosterone is useful for measuring 11β-HSD activities in combination with the determination of serum corticosterone in rats and mice.
The aim of this study was to explore the relationship and interpret the clinical importance of acute physiology and chronic health evaluation III (APACHE III) and levels of cytokines in patients with systemic inflammatory response syndrome (SIRS) after coronary artery bypass grafting (CABG) with or without cardio-pulmonary bypass (CPB) to see if they are beneficial for evaluating the seriousness of SIRS. The data suggested that the APACHE III score and levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and soluble interleukin-2 receptor (sIL-2R) were significantly higher after conventional CABG (CCABG) than after off-pump coronary artery bypass grafting (OPCAB) (p<0.05). With an increase in the APACHE III score, the levels of IL6, IL8, TNF-α, IL-1β, and sIL-2R and the morbidity of multiple organ dysfunction syndrome (MODS) increased gradually (p<0.01), while the level of IL2 decreased (p<0.01). Stepwise regression analysis showed that IL-1β, IL6, IL8, and sIL-2R levels had significant influences on the APACHE III score (p<0.05). The APACHE III score and levels of IL6, IL8, TNF-α, IL-1β, and sIL-2R were significantly higher in the MODS group than in the non-MODS group (p<0.05), but the level of IL2 was significantly lower in the MODS group (p=0.04). In conclusion, despite comparable surgical trauma, we believe that CPB is one of the most important factors responsible for stimulating an inflammatory response. SIRS after OPCAB was clearly mitigated compared with CCABG. Determination of the APACHE III score and plasma IL-1β, IL6, IL8 and sIL-2R concentrations might be helpful for evaluating the severity of SIRS following CABG and making a prognosis.
LAT1 (L-type amino acid transporter 1), one of the L-type amino acid transporters, transports the branched and aromatic amino acids. LAT1 requires the heavy chain of 4F2 antigen (4F2hc) for the functional expression as an amino acid transporter. The expression of this transporter is up-regulated in tumor cells and rapidly-growing cells to support their proliferation. Here, we studied the expression of LAT1 and 4F2hc in human cultured cells by real-time PCR and Western blot, and found that human brain astrocytomas, U343MGa, highly expressed LAT1 and 4F2hc mRNAs and proteins. The uptake of [14C]leucine by U343MGa cells is Na+-independent and inhibited by BCH (2-amino-2-norbornane carboxylic acid), and branched and aromatic amino acids, indicating that the LAT1 is expressed at the cell surface. Pulse chase labeling and surface labeling experiments of this cell line indicate that the protein synthesis of LAT1 and 4F2hc is slow, however, the heterodimeric complex assembled in the cells is very stable, and that the disulfide bond between the LAT1 and 4F2hc is not directly involved in the stability of the heterodimer.
As superoxide (·O2−) and hydroxyl radical (·OH) have been implicated in the pathogenesis of Parkinson disease, free radical scavenging and antioxidants have attracted attention as way to prevent progression of this disease. We examined the effects of eugenol, an essential oil extracted from cloves, on 6-hydroxydopamine (6-OHDA)-induced dopamine (DA) reduction in the mouse striatum. Eugenol administration 3 d before and 7 more days following one intracerebroventricular 6-OHDA injection prevented the reduction of striatal DA and its metabolites. Eugenol administration for 3 d reduced the increase of thiobarbituric acid-reactive substances (an indicator of lipid peroxidation) induced by ferric ion and increased glutathione (GSH) and L-ascorbate (Asc) in the striatum. Eugenol did not change the levels of catalase, glutathione peroxidase, or superoxide dismutase-like activities. Eugenol is known to have ·O2− and ·OH scavenging activities in vitro. These results suggest that eugenol prevents 6-OHDA-induced DA depression by preventing lipid peroxidation directly and indirectly (via stimulation of GSH and Asc generating systems). Furthermore, increased GSH may protect cell death by conjugating with p-quinone produced in 6-OHDA auto-oxidation. The effects of eugenol treatment in this model suggest its possible usefulness for the treatment of Parkinson disease.
Cochinin B, a novel ribosome-inactivating protein (RIP) with a molecular weight of 28 kDa, was purified from the seeds of Momordica cochinchinensis (Cucurbitaceae). The isolation procedure entailed ammonium sulfate precipitation, cation-exchange chromatography on SP Sepharose column and size-exclusion chromatography on Superdex 75 column with a fast protein liquid chromatography (FPLC) system. The first twenty N-terminal amino acid residues of Cochinin B showed homology to type I RIPs from other Momordica species. The purified Cochinin B displayed a strong inhibitory activity on protein synthesis in the cell-free rabbit reticulocyte lysate system with IC50 of 0.36 nM. Furthermore, it exhibited N-glycosidase activity and cytotoxicity against Vero cell line with IC50 higher than 1540 nM. Interestingly, Cochinin B manifested strong anti-tumor activities on human cervical epithelial carcinoma (HeLa), human embryonic kidney (HEK293) and human small cell lung cancer (NCI-H187) cell lines with IC50 of 16.9, 114 and 574 nM, respectively.
In a previous report, we had demonstrated the antioxidant activity of the sialic acid N-acetylneuraminic acid (Neu5Ac, NANA). This activity can counteract the cytotoxicity of hydrogen peroxide (H2O2), and the antitoxicity is a result of a direct chemical reaction, whereby NANA reduces H2O2 in the culture media. The influence of the potential of hydrogen (pH) and temperature in this reaction was investigated. The reaction velocity is remarkably less at low pH and/or low temperature, but it increases with these parameters. Furthermore, the reaction product generated in the slow reaction under acidic conditions (pH 3.1) was analyzed. We detected 4-(acetylamino)-2,4-dideoxy-D-glycero-D-galacto-octonic acid (ADOA) as the decarboxylation product of NANA; this is the same product we previously obtained in a faster reaction at neutral pH (pH 7.5). Furthermore, ADOA was generated not only from the reaction with the NANA monomer but also from that with α(2→8) homodimer of NANA (DP2). Thus, it can be considered that the reaction between NANA and H2O2 can occur under various pH conditions and for NANA residues in a glycochain.
Osteoarthritis (OA) is a slowly progressing chronic joint disease. Glucosamine (GlcN) is a saccharide that is widely used to relieve symptoms associated with OA. However, the mechanism of the effects of GlcN on articular cartilage remains unclear. We studied the effects of GlcN and its analogues, including chitin derivatives included in health supplements containing GlcN, on a chondrogenic cell line, ATDC5. We examined the effects of these saccharides on the proliferation and differentiation of ATDC5 cells. Glucosamine analogues, such as N-acetyl glucosamine and chitobiose, did not affect the proliferation or differentiation of ATDC5 cells. While GlcN did not affect the proliferation of ATDC5 cells, it inhibited their differentiation. Next, we examined whether GlcN affects mineralization and glycosaminoglycan (GAG) production by ATDC5 cells. Mineralization was markedly inhibited by addition of GlcN to the cell culture medium. Moreover, GlcN induced the formation of sulfated GAG in ATDC5. We also analyzed the mRNA levels in ATDC5 cells. GlcN reduced the mRNA levels of Smad2, Smad4 and MGP. GlcN might inhibit expression of MGP mRNA and induce the production of chondroitin sulfate in ATDC5 cells. The mechanism by which GlcN inhibits mineralization may be by regulating the expression of mRNA for the Smad2 and Smad4 chondrogenic master genes.
Hairy roots induced by Agrobacterium rhizogenes grow faster, and are considered as genetically stable. These hairy roots can be used as an interesting material for the production of secondary metabolites of pharmaceutical value. Salidroside has been identified as the major compounds from the roots of Rhodiola sachalinensis A. BOR. Here, we provide an update that adds new perspectives on the prospects and challenges of producing Salidroside from hairy roots induced by Agrobacterium rhizogene in Rhodiola sachalinensis A. BOR. For high salidroside production, the optimal concentration for precursor (Tyrosol, Tyrosine, and Phenylalanine) and elicitor (Aspergillus niger, Coriolus versicolor, and Ganoderma lucidum) was added in the LB liquid medium, respectively. The addition of elicitor in the liquid MS medium and the utilization of precursor from chemical feeding enhanced biomass accumulation and salidroside production. The optimal concentration for elicitor and precursor in the liquid medium was 0.05 mg/l and 1 mmol/l, respectively.
Heme oxygenase (HO)-1, which is a rate-limiting enzyme involved in the catabolism of heme, is upregulated by a variety of stresses including oxidative stresses and inflammatory cytokines, in many cell types. Recent studies have suggested that upregulation of HO-1 might provide cytoprotection and immunomodulatory functions in addition to its obvious role in heme metabolism. In this study, we examined whether HO-1 was upregulated following degranulation in mast cells that initiate vigorous immunity reactions. To trigger degranulation, rat basophilic leukemia (RBL)-2H3 cells were passively sensitized using an antiserum collected from ovalbumin (OA) immunized-Brown Norway rats, and the cells were stimulated by treatment with OA. Degranulation was confirmed by measuring the release of β-hexosaminidase. HO-1 mRNA and presence of HO-1 protein were detected using Northern blot and Western blot analyses, respectively. The effect of the antioxidant N-acetyl-L-cysteine (NAC) on HO-1 expression was also tested. HO-1 mRNA transiently increased at 1—2 h after RBL-2H3 cells were stimulated to degranulate. Its mRNA increases were dependent on the extent of degranulation. Following the upregulation of HO-1 mRNA, HO-1 protein was also increased. We also detected intracellular production of reactive oxygen species following degranulation in RBL-2H3 cells. NAC attenuated the HO-1 expression in a dose-dependent manner. This is the first report to reveal induction of both HO-1 mRNA and protein by degranulation in RBL-2H3 cells. We showed that NAC inhibited HO-1 upregulation. These results suggest that oxidative stress in activated RBL-2H3 cells results in the upregulation of HO-1.
The formation of ATP produced from poly(ADP-ribose) [(ADP-R)n] has been suggested to be required to repair damaged DNA. Here we investigate whether this ATP is involved in DNA replication processes during DNA repair. Poly(ADP-ribosyl)ated mid-S phase cell nuclei, which were isolated from synchronized HeLa S3 cells followed by the treatment with a DNA damaging agent, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), were revealed to retain DNA replication synthesizing activity during preincubation for de-poly(ADP-ribosyl)ation only in the presence of pyrophosphate (PPi) before DNA synthesis was started by adding 3 mM ATP. This DNA replication activity was not maintained in the presence of a potent and specific inhibitor of poly(ADP-ribose) glycohydrolase (PARG), Oenothein B (Oen B) during the preincubation with PPi. In the preincubation with PPi, μM orders of ATP was produced from (ADP-R)n. These results point to an important function of ATP generated from (ADP-R)n in nuclei for the maintenance of replication apparatus during DNA repair.
Candida albicans generally grows in the hyphae form in RPMI1640 medium. Chitohexose (COS-6) induced the yeast form of C. albicans dose-dependently under this condition. When COS-6 was exposed to C. albicans, yeast proliferation was observed 6 h after starting the incubation. When we observed the growth form of C. albicans cultured with COS-6, yeast proliferation was observed at log-phase. These results showed that COS-6 was useful for the yeast form inducer of C. albicans.
Minimum bactericidal concentrations (MBCs) of seven biocides for 42 methicillin-resistant Staphylococcus aureus (MRSA) isolates at 5, 30, or 180 min, for hand scrubs or soaks, isolated in 2003 in Japan were determined. The MBC values of glutaraldehyde, povidone iodine, and ethanol were lower than the user concentrations in all exposure times. However, at 5 min exposure of sodium hypochlorite, benzalkonium chloride, alkyldiaminoethylglycine hydrochloride, and chlorhexidine digluconate some strains showed higher MBC values than the user concentrations. These results indicated the possibility that MRSA survived under proper user concentration conditions and exposure time.
The adherence to HeLa cells by the yeast-type cells of the Candida albicans NIH A-207 strain cultivated for 2 d at 27 °C in the yeast extract-added Sabouraud liquid medium (YSLM) and the 500 mM galactose-added yeast nitrogen base medium (YNB-Gal) was compared. The yeast cells cultured in the YNB-Gal clearly increased the adherence to the HeLa cells compared to the YSLM. The adherence drastically decreased by pronase treatment of the yeast cells. Next, to define the sugar receptors of the yeast cells, lactosylceramide (LC)-, the H type 1 antigen (HA)-, Lewisa antigen (Lea)-, mannan- and BSA-coated polystyrene beads were used for the adherence to the yeast cells. Only the LC- and HA-beads were bound to the yeast cells. The adherence of the two beads to the yeast cells cultured in the YNB-Gal was higher than that to the yeast cells cultured in the YSLM. The yeast cell wall mannan-coated polystyrene beads did not adhere at all to the Hela cells. Based on these results, it is evident that the protein parts involving the LC and HA receptors on the surface of the yeast cells correlate with the adherence to the HeLa cells.
Alchornea glandulosa (Euphorbiaceae) is a plant used in folk medicine as an antiulcer agent. Rats pretreated with methanolic extract obtained from the leaves of A. glandulosa (AG) showed a dose-dependent effect and significant reduction of gastric ulcers induced by absolute ethanol at the doses of 500 (57%) and 1000 mg/kg (85%) in relation to the control group. Pretreatment of mice with AG (500, 1000 mg/kg, p.o.) showed dose-dependent activity and significantly decreased the severity of lesions caused by HCl/ethanol and by non steroidal anti inflammatory drug-induced gastric lesions. Pretreatment with AG also induced antisecretory action via local and systemic routes and a significant decrease in the total gastric acid content. The gastroprotective effects of AG involved the participation of nitric oxide and increased levels of endogenous sulfhydryl compounds, which are defensive mechanisms of the gastrointestinal mucosa against aggressive factors. The ability of AG to heal gastric ulcers was evaluated after 14 consecutive days of treatment. The results showed that single oral administrations of AG (250 mg/kg/once daily) potently stimulates gastric epithelial cell proliferation that contributes to the accelerated healing of gastric ulcers induced by acetic acid. In addition, no subacute toxicity (body weight gain, vital organs, and serum biochemical parameters) was observed during treatment with AG. Phytochemical investigation of AG led to the isolation of myricetin-3-O-α-L-rhamnopyranoside, quercetin-3-O-α-L-arabinopyranoside, quercetin-3-O-β-D-galactopyranoside, quercetin, amentoflavone, methyl gallate, gallic acid, and pterogynidine. We also established the phytochemical profile of AG with the quantification of total phenolic compounds. These compounds may contribute to the observed antiulcerogenic effects of AG.
Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) has potent effects in the brain as a free radical scavenger in ischemia-reperfusion (IR) injuries. However, whether this free radical scavenger can prevent myocardial injury after cerebral IR is not clear. The aim of the present study was to investigate the effect of edaravone against oxidative damage in brain-to-heart signaling triggered by IR injury and its possible mechanism. In this study, the expression of glutathione peroxidase (GSHPx) and protein carbonyl content was examined to evaluate oxidative stress. The activation of mitogen-activated protein kinases (MAPKs) was also examined. Terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) analysis was performed to estimate cardiomyocytes cell death. After edaravone treatment there was a mild increase in activities of GSHPx in cardiomyocytes; however, there was a decrease in protein carbonyl content. p38 MAPK activity was inhibited by edaravone treatment in comparison with the vehicle group in myocardium. These results were further complemented by a significant reduction of TUNEL-positive cells in the heart sections. Our results demonstrate that edaravone provides ameliorative effects in the myocardium after cerebral IR injury by differentially modulating MAPK's activity, thus reducing the oxidative stress state.
The analgesic and anti-inflammatory activities of a salicylate derivatives fraction (SDF) isolated from Gaultheria yunnanensis (FRANCH.) REHDER and the mechanisms of actions were investigated in the present study. The major constituent of SDF, which represented around 50% of this fraction, was a methyl salicylate diglycoside named gaultherin. SDF showed a significant inhibition on the hind paw edema in rats (200, 400 mg/kg body wt., p.o.) and ear swelling in mice (200, 400, 800 mg/kg body wt., p.o.) caused by carrageenin and croton oil, respectively. In addition, SDF (400, 800 mg/kg body wt., p.o.) inhibited only the second phase (inflammatory) in the formalin test, and showed no effect in the hot-plate test in mice. The antinociceptive activity of SDF was predominantly peripheral and independent of the opioid system. These findings demonstrate that SDF from Gaultheria yunnanensis (FRANCH.) REHDER possesses analgesic and anti-inflammatory activities, which may be mediated, at least partly, through the suppression of inflammatory mediators or their release suggested by the animal experiment. The observed effects of SDF are probably due to the presence of high content of salicylate derivatives (80%), including gaultherin, MSTG-A and MSTG-B.
The present study investigated whether Astragalus polysaccharide (APS) possessed immunotherapeutic effects on type 1 diabetes mellitus. Diabetic mice induced by multiple low dose streptozotocin (MLD-STZ) were administered either APS (100, 200, 400 mg/kg body weight) or saline intraperitoneally daily, and sacrificed after 15 or 30 d of treatment. Meanwhile normal mice not treated with STZ nor with APS were offered into non-diabetic group. Blood glucose and serum insulin levels were measured, histologic and morphometric analyses of the pancreas were performed to determine the effect of APS on pancreatic islets. Further investigations on immune changes in spleens were tested by ELISA, semi-quantitative RT-PCR and Western blot. Downregulated blood glucose level, upregulated serum insulin concentration, increased β cell mass, decreased apoptotic β cell percentage, downregulation of Th1/Th2 cytokine ratio and upregulation of peroxisome proliferator-activated receptor gamma (PPARγ) gene expression in spleens were significantly time- and dose-dependent on APS treatment, when compared to saline controls. These results show that APS seems to be helpful to attenuate insulitis and preserve β cells from apoptosis, but it can't entirely rescue type 1 diabetes mellitus. APS ameliorates both the clinical and histological parameters of the MLD-STZ induced diabetic mice in a long-lasting fashion, most likely through immunoregulatory actions on Th1/Th2 cytokine ratio, strongly associated with PPARγ gene expression in spleens.
The influences of histamine H1 receptor antagonists on maximal electroshock seizure were studied using infant rats. In this study, electroconvulsion was induced by stimulating rats using ear-clip electrodes, and the durations of electroencephalogram (EEG) seizure, tonic extensor (TE) seizure and clonic (CL) seizure induced by maximal electroshock were measured. Diphenhydramine, chlorpheniramine, cyproheptadine and ketotifen caused a dose-dependent and significant prolongation of both EEG seizure and TE seizure induced by maximal electroshock. On the other hand, epinastine and fexofenadine caused no such effects, even at a dose of 50 mg/kg. All drugs used in this study showed no significant effect on CL seizure induced by maximal electroshock. From these findings, it is suggested that epinastine and fexofenadine may cause no harmful influence on epilepsy, even when used in a little child.
We investigated the relationship between the pharmacological effect of tamsulosin and its concentrations in plasma and several lower urinary tract (LUT) and arterial tissues in conscious male dogs. Oral administration of tamsulosin (30 and 100 μg/kg) inhibited phenylephrine-induced intraurethral pressure (IUP) elevation. Inhibition peaked at 1, 2 h after dosing and lasted up to 6—8 h. Basal mean blood pressure did not significantly change throughout the observation period. Plasma concentration reached maximum within 0.5 h after dosing, whereas that in LUT tissues (prostate, urethra and bladder) reached maximum at 1, 2 h, and prostatic and urethral concentrations remained higher than those in plasma and arterial tissues at almost all observation points. Prostatic concentrations of tamsulosin at individual time points were 2.2- to 18.0-fold higher than plasma and 3.7- to 12.3-fold higher than mesenteric artery concentrations. Urethral concentrations of tamsulosin were also higher than those in both plasma and mesenteric artery. The prostatic and urethral concentrations of tamsulosin correlated well with its effect on IUP response [r2=0.98 (p<0.01) and r2=0.99 (p<0.01), respectively]. Our data demonstrate that tamsulosin is selectively retained in LUT tissues compared with plasma and arterial tissues and that its sustained effect on IUP response appears to be related to the prostatic and urethral retention of tamsulosin.
Breath test using 13C-labeled compound has been used as a convenient method to evaluate gastric emptying. 13C-Labeled acetic acid or octanoic acid has been used in clinic. However, there is few report comparing two compounds. This study aimed to compare 13C-acetic acid and 13C-octanoic acid in newly-constituted breath test for monitoring gastric emptying in conscious rats. After fasting, rats were orally administered Racol (liquid enteral nutrient formula) containing 13C-labeled compound (same molar of 13C-acetic acid or 13C-octanoic acid) and housed in a chamber. The expired air in the chamber was collected in a breath-sampling bag using a tube and aspiration pump. The level of 13CO2 in the expired air was measured using an infrared spectrometer at appropriate intervals for 120 min. Expired 13CO2 air from 13C-acetic acid changed at significantly higher levels than that from 13C-octanoic acid. Cmax and AUC120 min values of expired 13CO2 from 13C-acetic acid were significantly higher than those from 13C-octanoic acid, but Tmax was not different between them. These results show that 13C-acetic acid is more sensitive for monitoring gastric emptying than 13C-octanoic acid in liquid test meal although both acids clearly monitored gastric emptying.
Recent studies showed that oxidative stress could be an important component of the mechanism of organophosphate (OP) compounds toxicity. The aim of present study was to investigate either prophylactic and therapeutic effects of N-acetylcysteine (NAC) against fenthion-induced oxidative stress in mice. Additionally, the effects on survival rates were investigated. Therefore, we determined the changes of the blood levels of glutathione (GSH), malondialdehyde (MDA), nitrite, and nitrate in blood or serum. Additionally, all animals were observed for 6 h and the survival rates were recorded. It was found that fenthion administration increased the levels of MDA, and decreased the levels of GSH, nitrite and nitrate. On the other hand, both prophylactic and therapeutic NAC treatment decreased the levels of MDA, and increased the levels of GSH, nitrite, and nitrate. The results showed that NAC is able to attenuate the fenthion-induced oxidative stres whereby NAC has not only prophylactic but also therapeutic activity in fenthion poisoning. On the other hand, we found that NAC can clearly improve survival rates in mice administered with an acute high dose of fenthion poisoning. In conclusion, NAC can decrease OP-induced oxidative stress and mortality rate, but the exact mechanism of its NAC protective effect needs to be explored further.
Prostaglandin E2 (PGE2) is a key regulator of gastrointestinal, immunological, and mucosal homeostasis. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the prostaglandin-producing enzyme cyclooxygenases (COXs), and can induce serious complications, such as gastrointestinal damage, with long-term treatment. Orengedokuto (OGT), a Japanese traditional herbal medicine (Kampo medicine), is effective in various animal models of enteropathy. In the present study we examined whether OGT prevents enteropathy induced by NSAIDs in mice. Ulceration in the small intestine was induced with 2 subcutaneous injections of indomethacin (20 mg/kg body weight). Orally administered OGT prevented or reduced lethality, intestinal lesions, bleeding, increased serum nitrate/nitrite levels, and reduction of mucosal PGE2 induced by indomethacin. These beneficial effects of OGT were accompanied by increased production of PGE2 and interleukin 10 by isolated lamina propria mononuclear cells; COX-2 in these cells may be a major source of PGE2 in normal intestines. These findings suggest that OGT could be an effective therapeutic agent for the treatment of inflammatory bowel disease and adverse reactions to NSAIDs.
Cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated chloride channel that has been proposed as a pharmacological target to reduce intestinal fluid loss in cholera. The aim of this study was to identify new CFTR inhibitors by high-throughput screening. Screening of 50,000 drug-like small molecules was performed using a cell-based assay of iodide influx in Fisher rat thyroid (FRT) cells co-expressing human CFTR and halide-sensitive yellow fluorescent protein (YFP-H148Q). Two new CFTR inhibitors, 2-[N-(3-hydroxy-4-carboxyphenyl) amino]-4-(4-methylphenyl)-thiazole (INH 1) and 1-acetyl-5-bromo-2,3-dihydro-N-(1,2,3,4-tetrahydro-1-naphthalenyl)-1H-Indole-7-sulfonamide (INH 2), were identified. They were then determined for potency, reversibility and specificity by electrophysiological methods, and for in vivo efficacy in mouse model of cholera toxin-induced intestinal fluid secretion. INH 1 and INH 2 reversibly inhibited cAMP-activated apical chloride current in FRT cells with Kis of 15 and 20 μM, respectively. Similarly, in short-circuit current analysis in human colonic epithelial cell lines (T84 cells), cAMP-activated chloride secretion was inhibited by INH 1 and INH 2 with Kis of 24.5 and 25.3 μM, respectively. Calcium-activated chloride secretion in the T84 cells was markedly inhibited by 100 μM of INH 1, but was unaffected by 100 μM of INH 2. In vivo studies in mice showed that a single intraperitoneal injection of INH 1 (3 mg/kg) reduced cholera toxin-induced intestinal fluid secretion by 40%, whereas INH 2 produced no effect. Our results indicate that INH 1 could be a new class candidate for a blocker of cholera toxin-induced intestinal fluid secretion as well as a CFTR inhibitor.
5-Hydroxytryptamine (5-HT) modulates gastric motility and gastric emptying via a variety of 5-HT receptor subtypes. However, regional and functional differences among 5-HT receptor subtypes in the rat stomach are not fully investigated. Thus, we aimed to characterize 5-HT receptor subtypes involved in the 5-HT-induced contractions in the isolated antral, corporal and fundic circular muscles of the rat stomach by measuring the contractile force. 5-HT induced concentration-dependent contractions in the antrum, corpus and fundus. 5-HT-induced antral contractions were partly blocked by atropine and enhanced by tetrodotoxin (TTX). Neither atropine nor TTX affected the corporal or the fundic contractions to 5-HT. In the antrum, 5-HT-induced contractions were inhibited by methysergide, tended to be inhibited by ketanserin, enhanced by SB-203186, but were not affected by WAY-100635, GR127935, RS-127445, ondansetron, or SB-269970. In the corpus, 5-HT-induced contractions were inhibited by ketanserin or methysergide. In the fundus, 5-HT-induced contractions were blocked by methysergide or RS-127445, but were enhanced by cinanserin or SB-203186. It is thus concluded that contractile responses to 5-HT in the antrum are mediated by 5-HT receptors on both smooth muscle and neurons whilst in the corpus and fundus responses are mainly mediated by 5-HT receptors on smooth muscle. Moreover, the antrum presents the contractile 5-HT2A and 5-HT2B receptors and the relaxant 5-HT4 receptors. The corpus presents the contractile 5-HT2A receptors, and the fundus presents the contractile 5-HT2B receptors and the relaxant 5-HT2A and 5-HT4 receptors.
We previously demonstrated that tenuifoliside B and 3,6′-disinapoylsucrose in Polygalae Radix, the root of Polygala tenuifolia WILLDENOW, inhibited potassium cyanide (KCN)-induced hypoxia and scopolamine-induced memory impairment in mice. Because both ingredients have a common sinapoyl moiety in their structure, we inferred that the sinapoyl moiety could inhibit hypoxia and memory impairment. In the present study to clarify the hypothesis, sinapic acid inhibited KCN-induced hypoxia and scopolamine-induced memory impairment as well as tenuifoliside B and 3,6′-disinapoylsucrose did. In addition, sinapic acid inhibited decompression- or bilateral carotid artery ligation-induced hypoxia (or mortality) and CO2-induced impairment in mice, and basal forebrain lesion-induced cerebral cholinergic dysfunction (decreases in acetylcholine concentration and choline acetyltransferase activity) in rats. These results, taken together, suggest the possibilities that sinapic acid is not only a very important moiety in the pharmacological activities of tenuifoliside B and 3,6′-disinapoylsucrose but also a candidate for a cerebral protective and cognition-improving medicine.
Angiotensin-converting enzyme (ACE) inhibitor enhances the liver regeneration in rats after partial hepatectomy (PH), though the precise mechanisms are unknown. To determine the roles of bradykinin and angiotensin II in the ACE inhibitor-induced enhancement of liver regeneration, we investingated effects of lisinopril (ACE inhibitor), candesartan and losartan (angiotensin II type 1 (AT1) receptor antagonists) and icatibant (bradykinin B2 receptor antagonist) on the hepatic regenerative response to 70% PH in the rat. The liver regeneration was evaluated by measuring the frequency of 5-bromo-2′-deoxyuridine (BrdU) incorporation into hepatocyte nuclei 48 h after PH. We found that administration of candesartan or losartan, as well as lisinopril, enhanced BrdU incorporation after PH, and the lisinopril-induced enhancement was inhibited in part (40%) by icatibant. PH induced the expression of hepatocyte growth factor (HGF) mRNA in remnant liver, and this PH-induced up-regulation of HGF mRNA was further enhanced not only by lisinopril but also by candesartan and losartan. Administration of icatibant inhibited up to 40% of the lisinopril-induced up-regulation of HGF mRNA. These results suggest that the blockade of the renin-angiotensin system by either ACE inhibitor or AT1 receptor antagonist enhances the hepatic regenerative response to PH, probably through an augmentation of hepatic HGF production. In addition to this mechanism, the activation of B2 receptors may also be involved in the ACE inhibitor-induced enhancement of hepatic regenerative response.
A tyrosinase inhibitor was isolated from the sprout of Polygonum hydropiper L. (Benitade) by activity-guided fractionation and identified as (2R,3R)-(+)-taxifolin (1) by spectroscopic means. Compound 1 inhibited 70% of tyrosinase activity at a concentration of 0.50 mM. ID50 (50% inhibition dose) value of compound 1 was 0.24 mM. As compared with tyrosinase inhibitor known cosmetic agent such as arbutin and kojic acid, compound 1 was more inhibited than the former and showed inhibitory effect equal to that of the latter. To study the inhibitory effect of (2R,3R)-(+)-taxifolin derivatives against tyrosinase activity, 3,7,3′,4′-taxifolin tetraacetate (2) and 5,7,3′,4′-taxifolin teramethyl ether (3) were also assayed together with compound 1.
Previous investigations have demonstrated that Hachimi-jio-gan, a Chinese prescription consisting of eight crude drugs, has a therapeutic potential in diabetes and diabetic nephropathy, using these model rats. To add to these findings, we performed this study to assess whether one of the crude drugs, Corni Fructus (Cornus officinalis SIEB. et ZUCC.), had an effect on streptozotocin-induced diabetic rats as a major active constituent, compared with an inhibitor of advanced glycation end-product (AGE) formation, aminoguanidine. Diabetic rats were orally administrated Corni Fructus extract (50, 100, 200 mg/kg body weight/d) or aminoguanidine (100 mg/kg body weight/d). Treatment with Corni Fructus for 10 d suppressed hyperglycemia, proteinuria, renal AGE formation, and related protein expressions, i.e., receptor for AGEs, nuclear factor-κB, transforming growth factor-β1, and Nε-(carboxymethyl)lysine, in the same way as with aminoguanidine. However, improvement of renal function, shown via serum creatinine (Cr) and Cr clearance, was superior to aminoguanidine treatment. In conclusion, the present study supported the hypothesis that Corni Fructus plays an important role against diabetic pathogenesis, i.e., reducing glucose toxicities, up-regulating renal function, and consequently ameliorating glycation-associated renal damage; thus, this study may provide a new recognition of crude drugs to clarify the mechanisms of Chinese prescriptions.
Chloroplast trnK gene sequences of Cnidium officinale and Ligusticum chuanxiong were determined to establish an effective method for identifying Japanese Senkyu and Chinese Chuanxiong, the two which have the same drug name in Chinese characters, similar external feature, but different botanical origins. Three sites of nucleotide differences were found between these 2 species at positions 767, 924 and 964 from upstream in trnK gene sequence, allowing molecular identification of the two plants and crude drugs. Further, three kinds of specific primers of 14 mer, 23 mer and 30 mer long were designed to detect these 3 sites of marker nucleotides. By using multiplex single base extension (MSBE) analysis with the 3 specific primers, C. officinale and L. chuanxiong could be distinguished clearly by the electrophoretograms, where 3 peaks with different color of ddTMP, ddCMP and ddTMP were observed in case of C. officinale and those of ddGMP, ddAMP and ddGMP in L. chuanxiong. Moreover, trnK gene sequence of “Dongxiong,” a kind of Chuanxiong cultivated in Northeast China, suggested that its botanical origin was C. officinale.
In the course of search for potent chitin synthase inhibitors from natural resources, a novel chitin synthases inhibitor, 2′-benzoyloxycinnamaldehyde (2′-BCA) (I), was isolated from the aerial parts of Pleuropterus ciliinervis NAKAI. 2′-BCA inhibited chitin synthase 1 and 2 of Saccharomyces cerevisiae with the IC50s of 54.9 and 70.8 μg/ml, respectively, whereas it exhibited no inhibitory activity for chitin synthase 3 up to 280 μg/ml. Its derivatives, 2′-chloro- (V) and 2(-bromo-cinnamaldehyde (VI), each showed 1.9 and 2.7-fold stronger inhibitory activities than 2′-BCA, with the IC50s of 37.2 and 26.6 μg/ml, respectively. Especially, the IC50 of compound VI against chitin synthase 2 represented 1.7-fold more potent inhibitory activity than polyoxin D, a well-known chitin synthase inhibitor. Furthermore, compounds V and VI showed potent antifungal activities against various fungi including human pathogenic fungi, with a particularly strong inhibitory activity against Cryptococcus neoformans (MIC=16 μg/ml). Although the chemical synthesis of this compound has been reported, the present study is the first report to describe the isolation of 2′-BCA from natural resources and chitin synthases inhibitory activities of its derivatives. These results suggested that 2′-BCA and its derivatives can potentially serve as useful lead compounds for development of antifungal agents.
There is increasing evidence that epidermal carboxylesterase may be involved in the stereoselective hydrolysis of prodrugs in percutaneous absorption. The present study was designed to evaluate the stereoselective characteristics and mechanisms of ketoprofen ethyl ester hydrolysis by epidermal carboxylesterase expressed in HaCaT keratinocytes. Ketoprofen ethyl ester was mainly hydrolyzed to R-ketoprofen by carboxylesterase of human HaCaT keratinocytes. Human carboxylesterase-1 (hCE-1) and human carboxylesterase-2 (hCE-2) were intensively detected in L02 hepatocytes, hCE-2 was also intensively detected in HaCaT keratinocytes, but hCE-1 was not detected in HaCaT keratinocytes. hCE-2 is thus an abundant carboxylesterase in HaCaT keratinocytes which may be responsible for stereoselective hydrolysis of ketoprofen ethyl ester.
In our previous study it was observed that the frequencies of UGT1A1*6, UGT2B7*3 and CYP2D6*10 in patients who have a low level ability of glucuronidation were significantly higher than those in patients with a high level of ability of glucuronidation. The same tendency was found in the frequency of CYP2D6*5, though there was no significant difference. The purpose of this study was to evaluate the effects of the polymorphism on pharmacokinetics of carvedilol by population pharmacokinetic analysis. Population pharmacokinetic analysis was performed using 373 plasma concentrations from 41 patients with chronic heart failure or angina pectoris. A one compartment pharmacokinetic model with first-order absorption (for oral dosing) was used to describe the concentration-versus-time data for carvedilol. We examined the effects of various clinical and genetic covariables in the regression models for clearance and volume of distribution. The results suggested that the factors of interindividual variation for carvedilol clearance were creatinine clearance and polymorphisms of UGT2B7 and CYP2D6 in the Japanese population with heart disease. It was estimated that UGT2B7*3 decreased the clearance of carvedilol by 37%, but UGT2B7*2 did not show any effect. Clearance in the patients who have intermediate activity of CYP2D6 was decreased by 39%.
Microspheres containing theophylline (TH) were prepared from a hydrophobic dextran derivative by emulsion solvent evaporation method. The objective of this study was to evaluate the effects of poor solvent in dispersed phase on the particle properties and drug release characteristics of the microspheres. Mixtures of acetone and water were used as the dispersed phase and liquid paraffin as the continuous phase. The amount of water (poor solvent for polymer) was varied from 0.5 to 2 ml in 15 ml of dispersed phase. Drug release from the microspheres was examined using JPXIV 1st Fluid (pH 1.2) containing 0.02% Tween 20, and their structure was analyzed by scanning electron microscopy (SEM). The drug release behaviors were greatly affected by the amount of water. The percentage released until 8 h were 89% and 23% for 0.5 and 2.0 ml of water, respectively. The release mechanism shifted from Fickian diffusion to zero-order transport as the amount of water increased. According to SEM observations, TH was uniformly distributed over the entire microsphere prepared using 0.5 ml of water, and existed in the center of the microsphere, having a core-shell structure, when prepared using 2 ml of water. The amount of poor solvent in the dispersed phase was found to be a crucial factor determining the internal structure of microspheres and drug release characteristics.
The effects of capsaicin on intestinal cephalexin absorption were investigated by means of in situ single pass perfusion in rats to clarify whether this pungent compound present in spice is a potential factor altering the intestinal drug absorption processes. Under the control condition, cephalexin was absorbed at a rate of 1.16±0.08 and 0.90±0.06 nmol/min/cm in the jejunum and ileum, respectively. The intestinal cephalexin absorption rate was decreased when capsaicin was dissolved in the perfusate at a concentration of 400 μM, being 0.54±0.07 and 0.46±0.10 nmol/min/cm in the jejunum and ileum, respectively. The inhibitive effect of capsaicin on intestinal cephalexin absorption was diminished when ruthenium red, a non-selective inhibitor of the transient receptor potential (TRP) cation channels, was intravenously infused into the rat during the experiment. Moreover, when we evaluated the paracellular permeability of cephalexin by utilizing a competitive inhibitor, glycylsarcosine, it was demonstrated that glycylsarcosine-insensitive intestinal cephalexin absorption in the jejunum was increased by 4.5 times in the presence of 400 μM capsaicin. These findings indicate that capsaicin affects both transcellular and paracellular pathways of intestinal cephalexin absorption by interacting with the TRP cation channels in intestinal tissues, in which capsaicin seems to change the transport activity of H+/peptide co-transporter 1 (PEPT1), and to a lesser degree, it seems to alter the paracellular permeability of the intestinal epithelia.
The aim of the present study was to evaluate the effect of glycerol-induced acute renal failure (ARF) on the pharmacokinetics and hepatic extraction of metoprolol in rats. Experimental ARF in rats was induced by injections of 50% glycerol into the leg muscle (10 ml/kg). Pharmacokinetics and hepatic extraction of metoprolol was evaluated by means of intravenous, intra-intestinal, and intra-portal administration of the drug. The blood metoprolol concentration following intravenous infusion in ARF rats was similar to that in control rats. On the other hand, the blood metoprolol concentration at 5—10 min after intra-intestinal administration in ARF rats was significantly higher than that in control rats, and the oral clearance (CL/F) of the drug was significantly decreased in ARF rats. Hepatic extraction following intra-portal infusion was not altered by glycerol-induced ARF; however, hepatic first-pass extraction of metoprolol was dose-dependent and saturable in both ARF and control rats. These results suggested that the decreased CL/F of metoprolol in rats with glycerol-induced ARF is mainly a result of the increased initial absorption rate in the intestine followed by partial saturation of hepatic first-pass metabolism.
Previous reports have demonstrated that an intestinal injury causes hypofunctions of the liver associated with down-regulations of cytochrome P450, but an influence on hepatic transporters remains unclear. Here, we tested hepatic transporter functions in a rat model of bowel injury using indomethacin (IDM). After administration of IDM (8.5 mg/kg, i.p., 3 d), the rats suffered the intestinal impairment indicated by a reduction of alkaline phosphatase activity in mucosa. In vivo pharmacokinetic experiments of bromosulfophthalein (BSP) showed that there was a reduction in its plasma elimination rate and cumulative biliary excretion in IDM-treated rats and systemic and biliary clearances reduced to nearly 50% of the control group. Protein expressions in plasma membrane and mRNA levels of organic anion transporting polypeptide 1b2 (Oatp1b2) and multidrug resistance-associated protein 2 (Mrp2), which play hepatic BSP uptake and biliary excretion, respectively, in the liver were significantly reduced following the IDM treatment. In portal plasma, the levels of proinflammatory cytokines were unchanged, while the level of nitric oxide metabolites (NO2−+NO3−) increased to 6.5-fold that of the control. The time-course on IDM treatment indicated that, firstly, intestinal injury was induced, the NO level increased, and the hepatic Oatp1b2 and Mrp2 expression began to fall followed by an increase in plasma ALT. In conclusion, IDM-induced injury to the small intestine causes the hypofunction of hepatic Oatp1b2 and Mrp2 independently on the hepatic impairment, and NO arising from bowel injury may be one of key factors for it through the remote effect.
There is an interesting clinical report indicating that aciclovir, which is mainly excreted into urine, decreases the systemic clearance of theophylline by inhibiting cytochrome P450 (CYP) 1A2-mediated metabolism. In this study, we investigated the effect of aciclovir on the metabolism of theophylline, and on the activity and expression of hepatic CYP1A2 in rats. Theophylline (10 mg/kg) was injected intravenously into rats treated with two different dosages of aciclovir. When theophylline was simultaneously administered with aciclovir (50 mg/kg), the systemic clearance of theophylline and metabolic clearance of its major metabolites, 1-methyluric acid and 1,3-dimethyluric acid, were unchanged. In place of theophylline, when 1-methyl-3-propylxanthine (2.5 mg/kg), which is almost metabolized by CYP1A2 in rats, was coadministered intravenously with aciclovir (50 mg/kg), the pharmacokinetics of 1-methyl-3-propylxanthine was also unchanged. When theophylline was administered to rats pretreated with repeated intraperitoneal injections of aciclovir (25 mg/kg twice daily for 3 d), no significant differences in the systemic clearance of theophylline and its metabolic clearance to 1-methyluric acid and 1,3-dimethyluric acid were observed between the control and aciclovir-treated rats. This dosage of aciclovir did not change the activity of 7-ethoxyresorufin O-dealkylation, which is represented as CYP1A2 activity. In Western blot analysis, no significant change in the protein levels of hepatic CYP1A2 was observed between the control and aciclovir-treated rats. The present study suggests that aciclovir has no effect on the pharmacokinetics and metabolism of theophylline and on the activity and expression of hepatic CYP1A2 in rats.
Previously we have described the development and applications of lipid-based nanoparticles for gene delivery vector. In an attempt to improve transfection efficiency using the cell adhesion of extracellular matrix (ECM) to DNA/lipid complex (nanoplex), the mRNA expression of integrin α2β1 and CD44 in prostate cancer cells was detected as adhesion molecules for fibronectin (Fn), collagen I (Col) and laminin (Lam) using a commercially available cDNA array (GEArrayTM) system. These ECM proteins could enhance DNA transfection activity in cells when coated on the nanoplex. Among the ECM proteins, Fn-coating nanoplexes significantly increased transfection activity 2-fold in prostate cancer PC-3 cells, and exhibited higher DNA transfection activities to PC-3 xenografts, compared with commercially available cationic polymer in vivo jetPEI. These results indicated that Fn-coating nanoplexes could facilitate efficient transfection of prostate tumor cells.
Drugs applied to the nose in in vivo physiologic condition undergo absorption from the nasal cavity and the gastrointestinal (GI) tract because drug solution in the nasal cavity, together with mucus layer, is cleared to pharynx and then to the GI tract by coordinated beat of the cilia on nasal epithelial cells. The purpose of this study was to develop evaluate the contribution of the nasal cavity and the GI tract to drug absorption following nasal application and to clarify the relation to the transepithelial permeability of the drug (the permeability to Caco-2 monolayer, PCaco-2). Male Wistar rats received intravenous, nasal, and oral drug administration and drug concentration–time profiles in plasma were determined. Fractional absorption after nasal application (Fn) and oral administration (Fpo) were calculated from the area under the curve following intravenous injection (AUCiv), nasal application (AUCn), and oral administration (AUCpo) as AUCn/AUCiv and AUCpo/AUCiv, respectively. Fractional absorption from the nasal cavity (FNC) and the GI tract (FGI) following nasal application was calculated as (Fn−Fpo)/(1−Fpo) and Fpo(1−FNC), respectively. The shape of the curve between FNC and PCaco-2 was similar with the one observed in the case of oral bioavailability except the curve shifted right. It is noteworthy that the relation between FGI and PCaco-2 showed a bell-shaped curve with peak at 10−6 cm/s of PCaco-2. Highly permeable drug is primarily absorbed through the nasal mucosa before it is cleared to the GI tract. With the decrease in PCaco-2, the larger amount of the drug is cleared to the GI tract and absorption from the GI tract is increased. Poorly permeable drug, on the other hand, was absorbed neither from the nasal was nor the GI tract. These findings suggest that the primary absorption site of drug after nasal application is decided by mucociliary clearance and absorption through the nasal mucosa.
The transdermal delivery system (TDS) of oxybutynin (OXY) is widely used for the treatment of overactive bladder patients. This report aimed to assess the effect of external heating on the pharmacokinetics of OXY after transdermal administration. Franz-type diffusion cell experiments at room temperature, 36 and 45 °C were performed to estimate the permeations of OXY through human epidermal membrane with an isosorbide dinitrate (ISDN) formulation as a reference. The fluxes of OXY at 36 and 45 °C were only approximately 1.2-fold higher than that at room temperature, while those of ISDN with heating were more than 2.5 times as high as that without heating (p<0.001). These results indicate that the influence of heating on the permeation of OXY from the TDS of OXY may be minimal.
Bacillus polyfermenticus has been used in an effective treatment for long-term intestinal disorders, as live strains in the form of active endospores have been shown to reach the target intestine successfully. In this study, we have assessed the effects of B. polyfermenticus on the antioxidant system and the process of colon carcinogenesis in male F344 rats. The rats were divided into three groups after a 1-week adaptation period, and were then fed on either a high-fat and low-fiber diet (control and DMH groups), or a high-fat and low-fiber diet supplemented with B. polyfermenticus (3.1×108 cfu/d) (DMH+B. polyfermenticus group). One week after beginning the diets, the rats were subjected to 6 weeks of treatment with 1,2-dimethylhydrazine (DMH, 30 mg/kg/week, s.c.). The dietary treatments continued over the entirety of the experimental period. Nine weeks after the initial DMH injection, the rats supplemented with B. polyfermenticus evidenced significantly lower numbers of aberrant crypt foci than were observed in the DMH group. Injections with DMH resulted in significantly higher leukocytic DNA damage and plasma lipid peroxidation levels, as well as a lower plasma total antioxidant potential, and these factors recovered as the result of supplementation with B. polyfermenticus. These data indicate that B. polyfermenticus exerts a protective effect on the antioxidant system and the process of colon carcinogenesis, thereby suppressing the development of preneoplastic lesions.
CW-270033, an injectable carbapenem, is a novel, synthesized pyrrolidinyl-thio carbapenem. In the present study, the in-vitro and in-vivo antibacterial activities of CW-270033 against wild-type strains and clinical isolates were compared with those of imipenem and meropenem. CW-270033 was more active than imipenem against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) clinical isolates, but was slightly less active than meropenem. Against the Gram-positive clinical isolates methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), CW-270033 was slightly more active than meropenem, but was less active than imipenem. CW-270033 displayed potent in-vivo activity against E. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. aureus SMITH in the mouse systemic infection model; the efficacy of CW-270033 in this model was 2—7 fold higher than that of meropenem. This activity was comparable to the in-vitro activity of CW-270033. An intravenous injection of CW-270033 showed that the half-life of CW-270033 in serum in mice was about 20 min, which was about two times that of meropenem. CW-270033 was also found to be resistant to hydrolysis by the mouse renal dehydropeptidase I (DHP-I) enzyme.