Simultaneous determination of sivelestat and its metabolite XW-IMP-A in human plasma using HPLC-MS/MS / 药学学报

A simple and rapid method was developed based on high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to determine sivelestat and its metabolite XW-IMP-A in human plasma. After a simple protein precipitation, the samples and internal standards were analyzed on a C18 column by a gradient elution program. The mobile phase consisted of 30% acetonitrile in methanol and 5 mmol · L(-1) ammonium acetate at a flow rate of 0.7 mL · min(-1). The mass spectrometric data was collected in multiple reaction monitoring mode (MRM) in the negative electrospray ionization. The standard curves were linear in the range of 10.0-15,000 ng · mL(-1) for sivelestat, and 2.50-1000 ng · mL(-1) for XW-IMP-A. The low limits of quantitation were identified at 10.0 and 2.50 ng · mL for sivelestat and XW-IMP-A, respectively. The intra- and inter-day precision were within 11.3% and 13.1% for sivelestat and XW-IMP-A, and accuracy was 0.3% and 0.6% for sivelestat and XW-IMP-A, within the acceptable limits across all concentrations. The method was successfully validated in the pharmacokinetic study of sivelestat in healthy Chinese volunteers.

The basic functions of inosine 5'-monophosphate dehydrogenase and its application in drug discovery / 药学学报

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a key enzyme of de novo GMP biosynthesis. The expression and activity of IMPDH can be affected by diseases and physiological process. It is the drug target for anticancer, antiviral, antimicrobial and immunosuppressive therapeutics. Not only catalytic action but the other biological functions of IMPDH also play an important role in diseases. The basic functions, mechanism of catalysis, classification of inhibitors, biological functions and the latest advances to IMPDH will be illustrated in this review. It is expected to be helpful to the discovery of new inhibitors and biological functions of IMPDH.

Decision Factors on Mycophenolic Acid Dose after Renal Transplantation / 대한이식학회지

BACKGROUND: Triple immunosuppressant therapy including anti-metabolites is the representative immunosuppressive therapy after renal transplantation. This study is to evaluate the factors that influence Mycophenolate sodium (MPS, Myfortic, Novartis, Basel, Switzerland) dosage patterns in renal transplantation patients who take MPS as an inosine monophosphate dehydrogenase (IMPDH) among antimetabolites. METHODS: From May 2007 to April 2008, 16 clinical departments of 14 transplantation centers in Korea retrospectively performed a survey on 650 renal transplantation recipients taking MPS. This survey collected personal information, clinical factors related to transplantation and immunosuppressive therapy. RESULTS: The mean age of the patients was 43.0+/-12.0 (7~75) and the study included 364 males (56.0%) and 286 females (44.0%). The average follow up period after renal transplantation was 49.5+/-53.4 (1~307) months. There were 366 (56.3%) living related cases, 145 (22.3%) living non-related cases and 139 (21.4%) deceased donor cases. Cyclosporine was the most common calcineurin inhibitor (CNI) used in combination therapy with MPS (476 cases, 73.2%) followed by tacrolimus (169 cases, 26.0%). The mean daily dose of MPS was 909.7+/-336.3 (180~1,620)mg and the mean daily dose per kg was 15.3+/-5.9 (2.65~32.73)mg/kg. The daily dose showed significant positive correlation with patient body weight but the daily dose per kg showed negative correlation. The daily dose of MPS was significantly higher in the combination therapy with cyclosporine than that with tacrolimus. The daily dose and the dose per kg decreased with increment of recipient age and post-transplant period. CONCLUSIONS: Our study concluded that MPS dosages correlated with the combined type of CNI, post-transplant period and age.

Mycophenolic Acid Induced Apoptosis in Human Jurkat Cells viathe Generation of Reactive Oxygen Species

PURPOSE: Mycophenolic acid (MPA) is the active agent of mycophenolate mofetil (MMF), which is an immunosuppressive drug. MPA is a selective inhibitor of inosine monophosphate dehydrogenase. The aim of this study was for demonstrate that mycophenolic acid induces apoptosis in human Jurkat cells via the generation of reactive oxygen species (ROS). METHODS: The cells were cultured in the presence or absence of MPA. Flow cytometric analysis was performed after propidium iodide staining. Western blotting for caspase 3, Bcl-2 and Bax proteins was also performed. RESULTS: MPA decreased the viability of Jurkat cells in a dose- and time-dependent manner. The MPA induced apoptotic cell death displayed nuclear fragmentation and sub G0/G1 phase arrest in the Jurkat cells. The expression of caspase-3 proteases in the MPA treated-Jurkat cells increased in a time-dependent manner. Treatment with MPA resulted in increased ROS generation in the Jurkat cells. There was a decreased expression of Bcl-2 and an increased expression of Bax protein in the MPA treated Jurkat cells. CONCLUSION: This result suggests that MPA-induced cytotoxicity is associated with a direct increase of both ROS generation and the expression of Bax protein.

Emergencia de la resistencia antibiótica debida a las ß-lactamasas de espectro extendido (blee): detección, impacto clínico y epidemiología / Emergence of antimicrobial resistance to extended-spectrum ß-lactamases (ESBL): detection, clinic impact and epidemiology

RESUMEN La rápida emergencia de la resistencia antimicrobiana debida a las BLEE tiene un impacto significativo en la salud pública. En los últimos 24 años ha suscitado un gran interés el conocimiento acerca de las BLEE, esta explosión de publicaciones abarca a todos los continentes y más de 30 países, actualmente es motivo de preocupación y se considera un problema de salud pública. Las BLEE son enzimas que producen los gram negativos y confieren resistencia a las penicilinas, a todas las cefalosporinas y al aztreonam, pero no a los carbapenems ni a las cefamicinas y la mayoría son inhibidas por el acido clavulanico. En general las BLEE son derivadas de TEM-1, TEM-2 y SHV-1, difieren entre si de sus progenitoras por unos escasos aminoácidos por lo que su filogenia es cercana. Son comúnmente encontradas en E.coli, Klebsiella sp, y P.mirabilis, no obstante, existen otras BLEE que difieren filogenéticamente de TEM y SHV, como las CTX-M, las carbapenemasas tipo OXA y las metalo-β-lactamasas VIM e IMP, típicamente encontradas en especies de P. aeruginosa, Serratia sp and Enterobacter sp. La producción de BLEE en los patógenos de importancia clínica es un problema serio en los pacientes hospitalizados debido a las implicaciones clínicas, terapéuticas y económicas. Las técnicas para la detección de las BLEE van de lo simple con aspectos fenotipicos hasta las pruebas complejas moleculares de geno-detección específica. El objetivo de esta revisión es discutir el impacto clínico y epidemiológico de las BLEE más prevalentes así como las técnicas para su detección y su seguimiento nosocomial.

ABSTRACT The rapid emerge of antimicrobial resistance dueto ESBL has a significant impact in public health. In the last 24 years, the study of extendedspectrumβ-lactamases (ESBL) has created great interest. This has been documented by publications from all continents and more than 30 countries, and the extent of this problem is a public health concern. ESBLs produced by Gram negative bacilli are enzymes that confer resistance to penicillins, cepaholosporins and aztreonam, but not to carbapenems or cephamycins, and are usually inhibited by clavulanic acid. Most of the ESBLs are derived from TEM-1, TEM-2 and SHV-1, and differ from their progenitors by only a few amino-acids. Thus, their phylogeny is close. ESBLs are usually found in E. coli, Klebsiellasp, and Proteus mirabilis. However, there are some ESBL phylogenetic branches that differ from TEM and SHV, such as CTX-M, OXA carbapenemases, VIM and IMP metalo-β-lactamases, typically found in P. aeruginosa, Serratia sp and Enterobacter sp . ESBL production by different clinical pathogens imply an important clinical problem in nosocomial patients due to medical, therapeutic and economical impact. ESBL detection techniques include simple tests as well as complex detection system involving molecular genotyping. This review discusses the most prevalent ESBLs and their epidemiological and clinical impact. Also, it presents tools and strategies for ESBL detection and molecular tracking at the nosocomial level.

Effects of Inosine Monophosphate Dehydrogenase Inhibition on Platelet-derived Growth Factor- Induced Fibronectin Secretion and Cellular Reactive Oxygen Species in Mouse Mesangial Cells / 대한이식학회지

PURPOSE: Mesangial cell extracellular matrix (ECM) synthesis plays an important role in various renal diseases. Mycophenolic acid (MPA), which is an inhibitor of inosine monophosphate dehydrogenase (IMPDH), inhibits mesangial cell proliferation and ECM synthesis. However, the exact mechanism of MPA has not been clearly elucidated in mesangial cells. To examine the relative importance of IMPDH on the inhibitory action of MPA, we compared the effects of MPA or IMPDH2 siRNA on platelet-derived growth factor (PDGF)-induced fibronectin secretion and cellular reactive oxygen species (ROS) in mouse mesangial cells (MMC). METHODS: MMC were stimulated with PDGF 10 ng/ml with or without MPA 0.1~10micrometer, IMPDH2 siRNA 10~50 nM, or N-acetylcystein (NAC). IMPDH2 siRNA was transiently transfected by lipofectamine for 24 hours. MPA 0.1~10micrometer, ribavirin 10~100micrometer, and NAC 5 mM were administered 1 hour before the stimulation. Cell viability was measured by methylthiazoletetrazolium (MTT) assay, fibronectin secretion by Western blot analysis, and dichlorofluorescein (DCF)-sensitive cellular ROS by flow cytometry. RESULTS: PDGF 10 ng/ml effectively increased fibronectin secretion and cellular ROS in MMC. MPA and NAC at concentration without affecting basal level of fibronectin and cellular ROS ameliorated PDGF-induced fibronectin secretion and cellular ROS. However, IMPDH2 siRNA only partially reduced PDGF- induced fibronectin secretion and cellular ROS in MMC. CONCLUSION: These results suggest that MPA may inhibit PDGF-induced fibronectin secretion partly through IMPDH2 or cellular ROS in MMC, and there may be other mechanisms on the inhibitory action of MPA in mesenchymal cells.

Mizoribine Inhibits Production of Pro-inflammatory Cytokines and PGE2 in Macrophages

BACKGROUND: Mizoribine (MZR) is an imidazole nucleoside isolated from Eupenicillium brefeldianum. MZR is currently in clinical use for patients who have undergone renal transplantation. Therapeutic efficacy of MZR has also been demonstrated in rheumatoid arthritis and lupus nephritis. MZR has been shown to inhibit the proliferation of lymphocytes by interfering with inosine monophosphate dehydrogenase. Since the exact mechanism by which MZR benefits rheumatoid arthritis (RA) is not clear, we investigated the ability of MZR to direct its immunosuppressive influences on other antigen presenting cells, such as macrophages. METHODS: Mouse macrophage RAW264.7 cells were stimulated with lipopolysaccharide in the presence of MZR. To elucidate the mechanism of the therapeutic efficacy in chronic inflammatory diseases, we examined the effects of MZR on the production of pro-inflammatory cytokines, nitric oxide (NO) and prostaglandin E2 (PGE2) in macrophages. RESULTS: MZR dose-dependently decreased the production of nitric oxide and pro- inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukins 1beta (IL-beta) and IL-6 PGE2. Examination of gene expression levels showed that the anti-inflammatory effect correlated with the down-regulation of inducible nitiric oxide synthase expression, cycloxygenase-2 expression and TNF-alpha gene expression. CONCLUSION: In this work, we resulted whether MZR (1.25~10 microgram/ml) inhibited macrophage activation by inhibiting secretion of pro-inflammatory cytokines, NO and PGE2. These findings provide an explanation for the therapeutic efficacy of MZR in chronic inflammation- associated diseases.

The 3' terminal sequence of the inosine monophosphate dehydrogenase gene encodes an active domain in the yeast Schizosaccharomyces pombe

The gua1 gene encoding inosine monophosphate dehydrogenase (IMPDH), which catalyses the first step in de novo biosynthesis of guanosine monophosphate (GMP), was cloned in the yeast Schizosaccharomyces pombe by functional complementation of a gua1ura4-D18 mutant strain from a S. pombe DNA genomic library. Complementation analysis revealed a 1.2 kb fragment which segregation analysis confirmed did not code for a suppressor gene. Only 446 nucleotides of the gua1 gene encoding the IMPDH C-terminal residues were found within this 1.2 kb sequence (GenBank, AJ293460). The comparison of this wild-type fragment with the same fragment from the gua1ura4-D18 mutant revealed that there was a point mutation at position 1261 (guanine -> adenine) from the 5' end, corresponding to the amino acid residue 421 (glycine -> serine) of the enzyme. Dot and Northern analyses showed that the gua1 gene was expressed in transformants as well as in the wild-type and the gua1ura4-D18 mutant, but enzyme activity was only detected in wild-type and transformant cells. It seems likely that a 446 bp fragment from the 3' end of the gua1 gene abolished the point mutation in the mutant strain, suggesting that this fragment participates in the sequences encoding the active domain of IMPDH in S. pombe.

Mizoribine-mediated Apoptosis Signaling in Jurkat T Cells

PURPOSE: Mizoribine (MZR), an inhibitor of Inosine monophosphate (IMP) dehydrogenase which depletes cellular GTP, is clinically used as an immunosuppressive drug. This study was designed to evaluate the mechanism by which MZR exerts the cytotoxic effect on Jurkat T cells. METHODS: Jurkat T cell is a human T lymphocytic cell line. It was obtained from the Korean Type Culture Collection. Cell viability was measured by the MTT assay and flow cytometry. Caspase activity assay, Western blotting, 2-D PAGE, and mitochondrial membrane potential were detected using biochemical analysis. Morphologic finding was observed by Hoechst staining. RESULTS: The data demonstrated that the treatment of MZR decreased cell viability in a dose- and time-dependent manner. MZR-induced cell death was confirmed as apoptosis, which was characterized by chromatin condensation and H2AX phosphorylation. MZR increased the catalytic activity of caspase-3 protease, -8 protease and -9 proteases. The activation of caspase-3 protease was further confirmed by the degradation of polymerase (PARP), a substrate of caspase-3 protease by MZR in Jurkat T cells. Furthermore, MZR induced the changes of the mitochondrial transmembrane potential (MTP) and the cytosolic release of cytochrome c from the mitochondria. In addition, MZR induced the decrease of Bcl-X(L) expression whereas the increase of Bcl-X(S), Bak and Bim expression. Guanosine markedly inhibited cell viability and apoptosis through consistent suppression of the activity of caspase-8 protease, an upstream caspase among the caspase family, H2AX phosphorylation and PARP cleavage in MZR-treated cells. Also, I have screened the expression profile of proteins in the Jurkat T cells by using two-dimensional (2-D) gel electrophoresis. Among 300 spots resolved in the 2-D gels, the comparison of the control versus apoptotic cells revealed that the signal intensity of 10 spots was decreased and 5 spots was increased. CONCLUSION: The results suggest that MZR functions as an inhibitor of IMP dehydrogenase in apoptosis of Jurkat T cells via activation of intrinsic caspase cascades as well as mitochondrial dysfunction.

Mizoribine-mediated Apoptosis Signaling in Jurkat T Cells

PURPOSE: Mizoribine (MZR), an inhibitor of Inosine monophosphate (IMP) dehydrogenase which depletes cellular GTP, is clinically used as an immunosuppressive drug. This study was designed to evaluate the mechanism by which MZR exerts the cytotoxic effect on Jurkat T cells. METHODS: Jurkat T cell is a human T lymphocytic cell line. It was obtained from the Korean Type Culture Collection. Cell viability was measured by the MTT assay and flow cytometry. Caspase activity assay, Western blotting, 2-D PAGE, and mitochondrial membrane potential were detected using biochemical analysis. Morphologic finding was observed by Hoechst staining. RESULTS: The data demonstrated that the treatment of MZR decreased cell viability in a dose- and time-dependent manner. MZR-induced cell death was confirmed as apoptosis, which was characterized by chromatin condensation and H2AX phosphorylation. MZR increased the catalytic activity of caspase-3 protease, -8 protease and -9 proteases. The activation of caspase-3 protease was further confirmed by the degradation of polymerase (PARP), a substrate of caspase-3 protease by MZR in Jurkat T cells. Furthermore, MZR induced the changes of the mitochondrial transmembrane potential (MTP) and the cytosolic release of cytochrome c from the mitochondria. In addition, MZR induced the decrease of Bcl-X(L) expression whereas the increase of Bcl-X(S), Bak and Bim expression. Guanosine markedly inhibited cell viability and apoptosis through consistent suppression of the activity of caspase-8 protease, an upstream caspase among the caspase family, H2AX phosphorylation and PARP cleavage in MZR-treated cells. Also, I have screened the expression profile of proteins in the Jurkat T cells by using two-dimensional (2-D) gel electrophoresis. Among 300 spots resolved in the 2-D gels, the comparison of the control versus apoptotic cells revealed that the signal intensity of 10 spots was decreased and 5 spots was increased. CONCLUSION: The results suggest that MZR functions as an inhibitor of IMP dehydrogenase in apoptosis of Jurkat T cells via activation of intrinsic caspase cascades as well as mitochondrial dysfunction.

Mycophenolic Acid Induced Apoptotic Signal Transduction in Molt-4 T-cells

PURPOSE: Mycophenolic acid (MPA), a selective inhibitor of inosine monophosphate dehydrogenase (IMPDH), is the active metabolite of the immunosuppressive drug, mycophenolate mofetil (MMF). MMF is used to prevent an immune- mediate rejection response following organ transplantation via the inhibition of the IMPDH and GTP biosynthesis pathway. This study was designed to elucidate the mechanism by which MPA exerts its cytotoxic effect on human T lymphocytic and monocytic cell lines. METHODS: MOLT-4 and U937 cell lines were treated with MPA. Cell viability, expression of Bcl2 family proteins and Fas/Fas-L, effects of antioxidants and intracellular Ca2+ regulating agents and apoptosis were measured using a variety of microscopic and biochemical techniques. RESULTS: MPA induced the death of U937 and MOLT-4 cells in dose and time dependent manners, which was revealed an apoptosis with a characteristic ladder pattern of DNA fragmentation. In addition, BAPTA/AM, an intracellular Ca2+ chelator protected MOLT-4 cells from MPA treated apoptosis, although it did not have an additive with thapsigargin, and increases cytosolic Ca2+ stores. However, antioxidants including reduced glutathione (GSH) and N-acetyl-L-cysteine (NAC) did not inhibit the apoptosis of cells by MPA. Furthermore, guanosine suppressed MPA induced apoptosis of MOLT-4 lymphocytes, although adenosine did not. MPA also increased the catalytic activity of caspase family cysteine proteases including caspase-8, 9 and 3 proteases in MOLT-4 cells. Sequential activation indicated that the cleavage of caspase-8 and 9 precedes those of caspase-3. CONCLUSION: The results suggest that MPA induces the apoptotic death of MOLT-4 lymphocytes via the activations of caspase family proteases and the depletion of GTP.

Effects of Mycophenolate Mofetil on Acute Rejection of Allografted Kidney / 대한이식학회지

Mycophenolate mofetil (MMF) is a novel new immunosuppressant which suppress proliferation of T and B lymphocytes by inhibiting inosine monophosphate dehydrogenase. Even though the results we can get now are still preliminary but the positive result such as low incidence of acute rejection episode, is very attractive to clinical therapist. It also has certain effect on rescue therapy of steroid resistant acute rejection. There is little proven report of long-term follow up of this drug but the improvement of early graft survival suggest better long-term result. From March 1997, we used MMF as one of the primary immunosuppressant with cyclosporine and steroid in 47 renal allograft recipient (MMF group) and the early result of this protocol is compared with control group using conventional two drug regimen (cyclosporine and steroid)in 96 recipients. The 2 g of MMF were given daily from 2nd post-transplant day. The acute rejection episode within 3 and 6 months are 12.1 and 12.8% in MMF group and these are statistically significant difference with the results of control group (36.5% and 38.5% respectively, P<0.005). The response rate of acute rejection to steroid pulse therapy was 66.7% in MMF group but has no statistical difference with that of conventional group (84.1%). Steroid resistant severe acute rejection that needed to use OKT3 was developed in 1 case (2.1%) in MMF group but 7 (7.3%) in control group. Among the complication, post-transplant infection occurred in 6 cases (12.8%) of MMF group but in 8 cases (8.3%) in control group. Diarrhea that needed medication developed in 8 cases of MMF group (17.0%), but only one of them is necessary to change his immunosuppressive regimen. Leukopenia also developed in 1 case of each group. In summary, the incidence of acute rejection episode and steroid resistant rejection that is necessary to use OKT3 is significantly decreased in MMF group but the response rate to steroid pulse therapy and complications of both groups showed no statistical difference.

Interactions of praseodymium and neodymium with nucleosides and nucleotides: absorption difference and comparative absorption spectral study.

The interactions of praseodymium(III) and neodymium(III) with nucleosides and nucleotides have been studied in different stoichiometry in water and water-DMF mixtures by employing absorption difference and comparative absorption spectrophotometry. The 4f-4f bands were analysed by linear curve analysis followed by gaussian curve analysis, and various spectral parameters were computed, using partial and multiple regression method. The magnitude of changes in both energy interaction and intensity were used to explore the degree of outer and inner sphere coordination, incidence of covalency and the extent of metal 4f-orbital involvement in chemical bonding. Crystalline complexes of the type [Ln(nucleotide)2(H2O)2]- (where nucleotide--GMP or IMP) were characterized by IR, 1H NMR, 31P NMR data. These studies indicated that the binding of the nucleotide is through phosphate oxygen in a bidentate manner and the complexes undergo substantial ionisation in aqueous medium, thereby supporting the observed weak 4f-4f bands and lower values for nephelauxetic effect (1-beta), bonding (b) and covalency (delta) parameters derived from coulombic and spin orbit interaction parameters.