Metabolism and excretion study of DW116, a new fluoroquinolone, in rats

Metabolite identification and urinary and biliary excretion of the new fluoroquinolone antibacterial agent DW116 [1-(5-fluoro-2-pyridyl)-6-fluoro-7-(4-methyl-1-piperazinyl)-1,4-dihydro-4-oxoquinoline-3-carboxylic acid, hydrochloride] after oral administration have been studied in Sprague-Dawley rats. The excretion kinetics were monoexponential. Most of the drug was eliminated via the hepatic and renal routes. Mean renal clearance of DW116 was 73.4 ml/hr/kg and mean biliary clearance was 83.8 ml/hr/kg. The major metabolite excreted in the bile was identified as the glucuronide ester of the parent drug using base-hydrolysis of the conjugate metabolite followed by co-HPLC with standard compound,¹⁹F-NMR and LC-MS methods. The glucuronide conjugate was also found in urine. The mean urinary recoveries of free and total (free plus glucuronide ester) DW116 were 28.6±2.7% and 36.4±1.8% of the administered dose and the corresponding biliary recoveries were 14.4±5.5% and 37.0±7.6%, respectively.     

Induction of apoptosis in HepG2 human hepatocellular carcinoma cells by a novel derivative of ursodeoxycholic acid (UDCA)

The effects of ursodeoxycholic acid (UDCA) and its novel derivative, named as HS-1030, on the proliferation of HepG2, human hepatocellular carcinoma cells were investigated. Whereas UDCA had no significant effect in a concentration range we have tested, HS-1030 inhibited the proliferation of HepG2 cells in a concentration dependent manner. Surprisingly, HS-1030 had no effect on the proliferation of Human Chang liver cell which is a normal liver cell line. We also found that proliferation-inhibitory effect of HS-1030 was due to the induction of apoptosis of HepG2 cells, which was confirmed by observing the internucleosomal DNA fragmentation and morphological changes (i.e. cell shrinkage, nuclear condensation and the formation of apoptotic bodies). These results suggest that HS-1030 may be a good candidate as a drug for the treatment of liver cancer.     

Inhibitory effects of plant extracts on adjuvant-induced arthritis

Twenty seven plant extracts were selected on the basis of ancient literature search for rheumatoid arthritis or similar syndrome. Methanol extract of each plant was prepared and administered orally to rats everyday at a dose of 200 mg/kg/day. Experimental arthritis was induced by subplantar injection of heat-killedMycobacterium butyricum to right hind paw of rats. This treatment provoked swelling of the treated paw in two phases, acute primary swelling and secondary arthritic swelling. An inhibition of secondary swelling was considered to be antiarthritic activity. Several plant methanol extracts such asAkebia quinata (caulis),Ephedra sinica (herba) andSophorae subprostrata (radix) were found to show significant inhibitory activity against secondary swelling at the dose tested. Our results strongly suggested an antiarthritic potential of these plant extracts.     

Evaluation of a microplate assay specific for heavy metal toxicity

A rapid, quantitative microbial assay, which is specific for heavy metal toxicity, has been developed. The assay (MetPLATE) is in a 96-well microtitration plate format and is suitable for determining toxicity characteristics such as median inhibitory concentrations. The sensitivity of MetPLATE to heavy metals [Cu, Zn, Cd, Pb, Hg, Cr(III)] was generally higher than Microtox and was of the same order as or better than Daphnia and fish bioassay. MetPLATE was insensitive to organic compounds at concentrations higher than those found in the environment. Six out of 10 industrial wastewaters or process waters surveyed were toxic. Heavy metal analysis of these waters confirmed the presence of heavy metals in the toxic samples. MetPLATE can be run concurrently with other assays for general toxicity to help determine the nature of chemicals causing toxicity.     

Beta-amyloid precursor protein is detectable on monocytes and is increased in Alzheimer's disease

Using the anti-beta-amyloid precursor protein (betaAPP) monoclonal antibodies 4G8, 6E10 and 22C11 and flow cytometry, we report that human circulating peripheral blood monocytes display surface immunoreactivity for betaAPP. In contrast, circulating lymphocytes do not possess cell surface betaAPP immunoreactivity, despite similar levels of betaAPP expression. Immunoblotting analysis showed that monocytes, but not lymphocytes, possess an 82 kDa C-terminal betaAPP fragment consistent with a processed transmembrane species. Monocyte surface betaAPP was upregulated approximately threefold by activation with lipopolysaccharide and interferon-gamma, activation did not produce detectable betaAPP on the cell surface of lymphocytes. Surface betaAPP immunoreactivity was reduced in a normal aged population compared to normal young controls (Young = 81.07 +/- 13.67 mean fluorescence units, Aged = 36.74 +/- 3.81, p < 0.01), but was significantly increased in AD subjects compared to age-matched healthy controls (AD = 60.31 +/- 7.42, p < 0.05). Our data suggest that a proportion of peripheral A beta may be derived from monocyte/macrophages, and that defects in brain cell processing of betaAPP in AD may be shared by this readily accessible peripheral cell.     

Processing of the beta-amyloid precursor protein in ex vivo human brain cells

We studied distribution and processing of the Alzheimer's beta-amyloid precursor protein (betaAPP) in immediately ex vivo human brain cells obtained during neurosurgical procedures. Immunoblotting and flow cytometry studies revealed that brain cells supported betaAPP as a transmembrane holoprotein. Brain cells in short-term suspension culture were competent to process betaAPP into Abeta as shown by [35S]methionine pulse-chase studies. Brain cell Abeta was immunoprecipitated as SDS-stable dimers and higher-order multimers. Cleavage of cell surface betaAPP with trypsin prior to metabolic labeling reduced cellular Abeta by approximately 50%. We conclude that plasmalemmal betaAPP in human brain cells is a source of cellular Abeta, presumably via endosomal-lysosomal processing.     

cAMP potentiates beta-amyloid-induced nitric oxide release from microglia

The beta-amyloid peptide (Abeta) has been known to activate microglia and to induce release of nitric oxide (NO). In this study, we examined the effect of cAMP on Abeta-induced microglial activation using cultured rat brain microglia. Dibutyryl-cAMP (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX) significantly potentiated Abeta(25-35)- or Abeta(1-42)-induced NO release in a dose-dependent manner. The increase in NO release was due to the increased expression of inducible nitric oxide synthase (iNOS). However, forskolin, an adenylate cyclase activator, weakly increased NO release at 10-50 microM but caused a decrease at 100 microM. These results suggest that increase in intracellular cAMP could potentiate microglial activation induced by Abeta.     

Effect of intravesical nitric oxide therapy on cyclophosphamide-induced cystitis

PURPOSE: This study was conducted to examine effects of nitric oxide (NO) donors on bladder hyperactivity induced by cyclophosphamide (CYP)-induced cystitis. MATERIALS AND METHODS: Female Sprague-Dawley rats received a single intraperitoneal injection of CYP (100 mg./kg.), and then their micturition pattern including mean micturition volume and the number of micturitions during 24 hours was recorded in a metabolic cage before and after CYP treatment. Forty-eight hours after CYP injection, bladder function under urethane anesthesia was evaluated by cystometry with continuous saline infusion (0.04 ml. per minute) or under isovolumetric conditions (0.8 ml. bladder volume). NO donors, S-nitroso-N-acetyl-penicillamine (SNAP, 2 mM) or sodium nitroprusside (SNP, 1 mM), and an NO synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME, 20 mM) were administered intravesically. Direct action of SNAP on bladder afferent neurons was also tested in a patch-clamp recording study. RESULTS: The number of micturitions significantly increased during the first 24 hours after CYP injection (19.0 +/- 0.88 versus 92.1 +/- 16.3 micturitions/24 hours, mean +/- SE, n = 25) (p <0.001). There was no significant difference in total micturition volume before (12.3 +/- 1.0 ml./24 hours) and after CYP treatment (15.6 +/- 1.5 ml./24 hours). During continuous infusion cystometry, intercontraction interval (ICI) was smaller in CYP-injected rats than in control rats. In CYP-injected animals, NO donors increased the ICI, but did not change the amplitude of bladder contractions. Continuous intravesical infusion of the NOS inhibitor did not alter the cystometric parameters. During cystometry under isovolumetric conditions, contraction frequency was decreased after NO donor administration. NO donors did not influence bladder activity in control rats. In patch clamp recordings, when SNAP (500 microM) was directly applied to dissociated afferent neurons innervating the urinary bladder, high-voltage-activated Ca2+ channel currents were suppressed by approximately 30%. CONCLUSIONS: Intravesical NO donors can suppress CYP-induced bladder hyperactivity. We hypothesize that the effect of NO donors is not due to smooth muscle relaxation, but rather due to an inhibitory effect on bladder afferent pathways that was manifested by an increase in intercontraction interval without changes in contraction amplitude. NO donors may be considered as a possible treatment of CYP-induced and other types of bladder inflammation.     

Apoptosis induced by inhibition of contact with extracellular matrix in mouse collecting duct cells

Cell-matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation and aspects of cell growth control. Detachment from the matrix epithelial cells induces programmed cell death, and this cell detachment induced apoptosis has been referred to as 'anoikis'. This study was undertaken to determine whether apoptosis is induced by inhibition of contact with extracellular matrix (ECM) in collecting duct cells and to investigate the signaling mechanisms of the process. Upon detachment from ECM, mouse inner medullary collecting duct cells (mIMCD-3) and mouse outer cortical collecting duct cells (M-1), which were derived from an SV40 transgenic mouse, entered into programmed cell death. Forced suspension of mIMCD-3 or M-1 cells did not affect the expression of Bcl-2-related proteins and did not activate c-Jun NH(2)-terminal kinase. Detachment of cells from ECM activated p38 mitogen-activated protein kinase (p38), but its inhibition with SB203580 did not protect cells from anoikis. Detachment of cells from matrix inhibited NF-kappaB activity, and the inhibition of NF-kappaB activity by overexpression of nonphosphorylatable I-kappaB increased detachment-induced apoptotic cell death in M-1 cells. Forced suspension of M-1 cells still activated p53 activity. Caspase-8 was activated during anoikis, but the time course of its activation was in accordance with DNA fragmentation. These results indicate that detachment from ECM induces apoptosis in the kidney collecting duct cells. Changes in expression levels of Bcl-2-related proteins or activation of JNK/p38 kinase are not critical for anoikis. Decrease in NF-kappaB activity and activation of p53 induced by inhibition of interaction with ECM play roles in anoikis in SV-40-transformed collecting duct cells. Caspase-8 is activated during detachment-induced apoptosis, the mechanisms of which are independent of activation of cell death receptors. Copyright Copyright 1999 S. Karger AG, Basel