Surface plasmon resonance biosensor combined with lentiviral particle stabilization strategy for rapid and specific screening of P-Glycoprotein ligands.

A novel surface plasmon resonance-based P-gp ligand screening system (SPR-PLSS) combined with lentiviral particle (LVP) stabilization strategy was constructed to screen out potential P-gp inhibitors from natural products. Firstly, we constructed LVPs with high and low expression levels of P-gp. The LVPs can ensure the natural conformation of P-gp based on the principle that LVPs germinated from packaging cells will contain cell membrane fragments and P-gp they carry. Then the LVPs with high P-gp expression for active channel and LVPs with low P-gp expression for reference channel were immobilized on CM5 chip respectively. The affinity detection was thus carried out with the signal reduction on the two channels. The P-gp inhibitors, Valspodar (Val) and cyclosporin (CsA), as positive compounds, were detected to characterize the chip's activity, and the KD of Val and CsA were 14.09 µM and 16.41 µM, respectively. Forty compounds from natural product library were screened using the SPR CM5 chip, and magnolol (Mag), honokiol (Hon), and resveratrol (Res) were screened out as potential P-gp ligands, showing a significant response signal. This work presented a novel P-gp ligand screening system based on LVP-immobilized biosensor to rapidly screen out P-gp ligands from natural product library. Compared with traditional cell experiments which the screening time may take up to several days, our method only takes several hours. Furthermore, this study has also provided solid evidences to support that some complicated membrane proteins would apply to the lentivirus-based SPR screening system.

Liquid chromatography coupled to different atmospheric pressure ionization sources-quadrupole-time-of-flight mass spectrometry and post-column addition of metal salt solutions as a powerful tool for the metabolic profiling of Fusarium oxysporum.

Fusarium oxysporum L11 is a non-pathogenic soil-borne fungal strain that yielded an extract that showed antifungal activity against phytopathogens. In this study, reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to different atmospheric pressure ionization sources-quadrupole-time-of-flight mass spectrometry (API-QTOF-MS) was applied for the comprehensive profiling of the metabolites from the extract. The employed sources were electrospray (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). Post-column addition of metal solutions of Ca, Cu and Zn(II) was also tested using ESI. A total of 137 compounds were identified or tentatively identified by matching their accurate mass signals, suggested molecular formulae and MS/MS analysis with previously reported data. Some compounds were isolated and identified by NMR. The extract was rich in cyclic peptides like cyclosporins, diketopiperazines and sansalvamides, most of which were new, and are reported here for the first time. The use of post-column addition of metals resulted in a useful strategy for the discrimination of compound classes since specific adducts were observed for the different compound families. This technique also allowed the screening for compounds with metal binding properties. Thus, the applied methodology is a useful choice for the metabolic profiling of extracts and also for the selection of metabolites with potential biological activities related to interactions with metal ions.

Differentiation of cyclosporin A from isocyclosporin A by liquid chromatography/electrospray ionization mass spectrometry with post-column addition of divalent metal salt.

RATIONALE: Cyclosporin A (CsA) rearranges to its isomer isocyclosporin A (isoCsA) upon acid hydrolysis and also during ionization in the ion source of the mass spectrometer. It has been reported that both compounds could not be differentiated by tandem mass spectrometry (MS/MS) using atmospheric pressure ionization (API) sources and ambiguously differentiated by using other sources. In order to analyze these compounds which are common fungal metabolites, it is relevant to develop a simple method for their differentiation. METHODS: CsA and isoCsA were analyzed by liquid chromatography/mass spectrometry (LC/MS) with post-column addition of metal ion solutions in a quadrupole time-of-flight instrument equipped with an electrospray ionization (ESI) source. RESULTS: Mass spectra of CsA obtained upon post-column addition of solutions of Ca(II), Cu(II) and Zn(II) showed complexes between cyclosporin and the metal, including [2CsA + Me](2+) and [CsA-H + Me](+). These complexes were not observed in the spectra of isoCsA. The same results were observed at different metal concentrations. CONCLUSIONS: Differentiation via metal complexation in positive ion mode LC/ESI-MS was performed to simultaneously distinguish CsA and its isomer isoCsA.

Comparison of five cyclosporin immunoassays with HPLC.

High performance liquid chromatography (HPLC) is the reference method for cyclosporin (CyA) measurements but therapeutic monitoring of the drug is frequently made using the more practical immunoassays. Cross-reactivity with CyA metabolites may compromise the specificity of immunoassays, particularly in liver graft recipients where metabolites may accumulate. The aim of this study was to compare with HPLC the performance of two recently introduced CyA immunoassays (the AxSYM fluorescent polarisation immunoassay (FPIA) and non-extraction CEDIA assay). The comparison was extended to the well-established TDx monoclonal FPIA (TDx mono) and the enzyme multiplied (EMIT)-specific assays and to the polyclonal FPIA (TDx poly), in which metabolite cross-reactivity is extensive. Assays were performed on 106 blood samples (taken 6 days to 118 months post-liver transplant) and results were compared by non-parametric regression analysis and difference plots. AxSYM and CEDIA showed both constant and proportional bias against HPLC (unlike EMIT) but the mean difference from HPLC was least for AxSYM (2.7 microg/l vs. 11.7, 9.4 and 54 microg/l for CEDIA, EMIT and TDx mono, respectively. (TDx poly - HPLC) values were proportional to all immunoassay results, with slopes of 0.33, 0.38 and 0.45 for EMIT, AxSYM and CEDIA, respectively. Our data suggest close agreement between AxSYM, CEDIA and EMIT results.

Efeito imunossupressor da ciclosporina intra-muscular administrada em diferentes períodos pós-operatórios em um modelo de transplante penetrante de córnea em ratos / Immunosuppressive effect of intramuscular cyclosporine used at different post-operative period on a penetrating keratoplasty model in the rat

Obetivo: Avaliar o efeito imunossupressor da ciclosporina intramuscular (I.M.), administrada por tempo limitado em diferentes períodos do pós-operatório, no transplante penetrante de córnea em um modelo experimental em rato, por meio de avaliaçäo clínica e anátoma-patológica do enxerto corneano. Método: Foram utilizados ratos isogênicos Fischer cmo doadores e Lewis como receptopres, em um modelo ortotópico de transplante de córnea. A administraçäo de ciclosporina I.M. 10mg/kg/ dia foi iniciada em diferentes períodos nos grupos estudados: no pós-operatório. A ciclosporina quando iniciada foi administrada até o 30 dia pós-operatório. UM grupo controle näo recebeu a ciclosporina. I.M. Os enxertos corneanos foram avaliadas clínica e histologicamente. Resultado: Rejeiçäo foi observada nas primeiras três semanas dp pós-operatório em 100 (por cento) dos casos no grupo contole (n=5) que näo recebeu a ciclosporina. Os ratos tratados com ciclosporina (n=15) apresentaram rejeiçäo em apenas um caso, que teve curta evoluçäo e poucos sinais clínicos. Os estudos histológicos confirmaram as avaliaçöes clínicas. O grupo controle apresentou infiltrado no enxerto corneal com predomínio de linfócitos sobre neutrófilos com amsi neovasos, com mais fibrose e com infiltrado inflamatório mais intenso do que os grupos tratados com ciclosporina. Conclusäo: Os dados obtidos indicam que a ciclosporina I.M., pode ter efeito benéfico sobre o controle da rejeiçäo do transplante de córnea, mesmo na sua fase ativa

Avaliaçäo da hipersensibilidade ao dinitroclorobenzeno em ratos tratados ou näo com ciclosporina / Evaluation of hipersensibility to dinitrochlorobenzeno in rats treated or not with cyclosporin-A

Avaliou-se nesse trabalho a resposta imunológica de ratos Wistar, através da hipersensibilidade tipo IV, celular ou retardada, provocada pelo DNCB em cinco grupos de 10 ratos. Para a imunizaçäo, aplicou-se sobre a pele dos animais 50 µl de DNCB, e a reaçäo foi desencadeada com concentraçöes crescentes de 10, 20, 30, 50 e 100 mg/ml na superfície da orelha esquerda e, após 24 horas, foi feita uma relaçäo de peso das orelhas esquerda/direita (controle). Todos os animais desenvolveram reaçöes de hipersensibilidade tipo IV num mesmo padräo clínico. O mesmo teste foi aplicado em cinco grupos de 5 ratos que receberam doses de 5, 7, 10, 30 e 50 mg/kg/dia de ciclosporina. Todos os animais tratados näo responderam ao teste, deduzindo-se que eles estavam imunossuprimidos pela ciclosporina

Measurement of AM19 and other cyclosporine metabolites in the blood of liver transplant patients with stable liver function.

Chronic renal impairment represents on of the major side effects of cyclosporine (CsA) immunotherapy for organ transplantation. The clinical relevance of selective measurement of CsA metabolites to correlating nephrotoxic activities remains inconclusive. A relatively simple and reliable method was developed to measure AM19 and four other CsA metabolites in whole blood by sequential solid-phase extraction and reversed phase high-pressure liquid chromatography (HPLC). The procedures were modified from a well-established method developed to quantitate CsA, the most important difference being in the elution step. The use of acetonitrile/methanol instead of ethylacetate/isopropanol in the elution procedure enhanced the absolute recoveries of metabolites. The washing steps were also slightly modified to recover AM19 quantitatively and specifically. Isocratic chromatographic conditions allowed very good separation of AM19, AM1, AM9, AM1c, AM4N, CsA, and the internal standard, cyclosporin C (CsC). Analytical recoveries for CsA and five of its metabolites ranged from 82 to 92%. No interfering substance from the matrix was found. The detection limit was 10 micrograms/l. The objectives of this study were to measure trough concentration of AM19 in whole blood, expressed as percentage of total metabolites plus parent CsA, and to determine if the blood level of AM19 could be used to predict subsequent changes in serum creatinine level in liver transplant patients. Twenty-three patients, who underwent liver transplantation between January and June 1993 were studied. Trough concentration of CsA in whole blood, serum creatinine and liver enzymes were constantly monitored. AM19 level was determined 5-8 months after transplantation. Preliminary results suggest an inverse relationship between AM19 and serum creatine levels.(ABSTRACT TRUNCATED AT 250 WORDS)

BF3-catalysed methanolysis combined with fast atom bombardment tandem mass spectrometry as a new tool for the preparation and analysis of linear secocyclosporins.

BF3-catalysed methanolysis is presented for cyclic peptide cleavage using cyclosporins as model compounds. The reaction conditions for BF3-catalysed methanolysis of cyclosporins were optimized to favour the formation of linear undecapeptides. The resulting secocyclosporins were analysed by fast atom bombardment tandem mass spectrometry. Only one dominant and two side mechanisms of the ring opening are operating so that the complete sequence determination of all prepared oligopeptides was achieved.

Localization of cyclophilin A and cyclophilin C mRNA in murine kidney using RT-PCR.

Cyclosporin A (CsA), which is widely used as an immunosuppressant, has a nephrotoxic side effect. The mechanism of this nephrotoxicity is not well understood; however, recent studies suggest that cyclophilin (cyp) is responsible for mediating the immunosuppressive action of CsA through the interaction with the Ca(2+)- and calmodulin-dependent phosphatase, calcineurin. While cyp A mRNA is expressed ubiquitously, cyp C mRNA has been shown to be topically expressed, including in the kidney. We examined: (1) distribution of cyp A and cyp C mRNA in microdissected murine nephron segments, using a combination of reverse transcription and polymerase chain reaction (RT-PCR) techniques, and (2) the effect of CsA administration on cyp C mRNA expression in proximal convoluted tubule. Among the nephron segments examined, large signals for cyp C PCR product were detected in proximal convoluted tubule and proximal straight tubule. Our data showed that the distribution of cyp C mRNA was uneven, and it mainly existed in segments that are relatively sensitive to CsA toxicity. In contrast, cyp A mRNA was found to be distributed almost equally along the nephron segments examined. By CsA administration, the signal for cyp C mRNA PCR product was increased. These results suggest that cyp C may play some role in the renal tubular disorder observed in CsA nephrotoxicity.

Thermospray high-performance liquid chromatography/mass spectrometric determination of cyclosporins.

The cyclic undecapeptides cyclosporin (Csp) A, CspD and dihydro CspC (HCspC) were analyzed by high-performance liquid chromatography/thermospray-mass spectrometry (HPLC/TSP-MS) on line with a UV detector. Positive ion partial (1000-1300 u) mass spectra of these compounds could be obtained with 1-2 pmol injected on column. Mass spectra were characterized by signals corresponding to the [M+H] ions as well as fragment ions derived from the loss of 112 (CspA and CspD) or 44, 114 and 114 + 44 (HCspC) mass units from the parent ion. The same qualitative profile was observed for negative-ion acquisition where the ions were formed by proton abstraction. The application of the technique to the characterization of CspA and its major hydroxylated and dealkylated metabolites in human blood samples is presented.

Micro-quantification of cyclosporine and its metabolites and determination of their spectral absorptivities.

We report a micromethod for the analysis of cyclosporine (CsA), based on quantification of its constituent amino acids. The amino acids were released by gas-phase hydrolysis, derivatized with fluorenylmethyl chloroformate, and separated and analyzed in a reversed-phase HPLC system. The imprecision (CV) of the amino acid analysis was less than 4%, and several determinations of the amount of standard CsA were within 1% of the weighed material. The detection limits (signal-to-noise ratio = 2) were 500 fmol for ultraviolet detection and 100 fmol for fluorescence detection. We also used this method to determine the ultraviolet absorptivities of CsA and five metabolites at 210, 214, and 230 nm. The molar absorptivity of most metabolites was about 10% higher than that of CsA, although the metabolite that was oxidized to a carboxyl group on the terminal carbon of N-methyl-butenyl-methyl-threonine (AM1A) had a molar absorptivity about 40% higher than that of CsA.

Isolation of an immunosuppressive metabolite of FK506 generated by human microsome preparations.

Metabolism of FK506, a 23 member macrolide under clinical investigation as immunosuppressant after transplantation, was studied using human liver microsomes. Two fractions isolated by semi-preparative HPLC were identified by negative fast atom bombardment mass spectrometry as FK506 metabolites with mass peaks at m/z = 790 indicating demethylation of the mother compound. The immunosuppressive activity of one metabolite was evaluated in a ConA-stimulated peripheral rat lymphocyte assay. FK506 had an IC50 of 0.186 nmol/L and the metabolite tested of 1.89 nmol/L.

Synthesis of a fluorescent derivative of cyclosporin A for high-performance liquid chromatography analysis.

A direct assay method for use in studies of cyclosporin binding must be highly sensitive and selective since it must be capable of measuring the concentrations encountered in the protein-free matrix. The failure of current HPLC methods to achieve the sensitivity required for binding studies may be attributed to the use of UV detection, which relies on the relatively weak end-absorption of cyclosporin A. A method involving fluorescence derivatization was sought with the aim of increasing HPLC assay sensitivity. A method is described for producing a fluorescent derivative of cyclosporin A, a compound which has no functional groups which are easily derivatized. However, intramolecular rearrangement of cyclosporin A to form its structural isomer, isocyclosporin A, exposes a secondary amine which can be reacted with dansyl chloride to produce a fluorescent derivative. This two-step derivatization procedure was used as the basis of an HPLC fluorescence assay. Although this assay is not sufficiently sensitive to measure concentrations encountered in the protein-free matrix during plasma binding studies, the method does point to the possible development of a more sensitive assay using a derivatizing reagent other than dansyl chloride.

Micro method for liquid chromatographic determination of cyclosporin A in whole blood with use of a rapid extraction procedure.

In this precise, reversed-phase liquid chromatographic procedure, Cyclosporin A (Cy A) is extracted from 300 microL of whole blood with cyclosporin D as internal standard by liquid-liquid extraction. A 20-microL aliquot of the extract, injected onto a Nucleosil octyl analytical column heated at 72 degrees C, is eluted with a mixture of acetonitrile and 0.01 M phosphate buffer, pH 5.5 at a flow rate of 1 mL/min. Detection is set at 210 nm. The chromatography is complete within 10 min. The detection limit is 25 micrograms/L. Between-run CVs range from 2.3 to 6.2% and recovery is 92.1 +/- 6.3%. The major advantages of our extraction procedure are the rapid clean-up method and the small volume required. This procedure is especially suitable for cyclosporine determination in pediatric transplantations.

Chronic ciclosporin nephrotoxicity: a rabbit model.

Ciclosporin (CsA) was administered intravenously for 30 days to New Zealand white rabbits at the following doses: group A, 2.5 mg/kg/day; group B, 5.0 mg/kg/day; group C, 10 mg/kg/day; group D, saline control; group E, vehicle (cremophor-EL) control. Creatinine clearance was significantly reduced (p less than 0.05) at time of sacrifice in group C as compared to controls. There were no apparent differences among the five groups with respect to body weight gain or blood pressure. Morphologically, there were marked changes in the cytoarchitecture of the kidneys from all groups of animals treated with CsA. At the light microscopic level, in contrast to controls, there was the presence of leukocyte infiltration, tubular atrophy, interstitial fibrosis, and arteriolopathy. At the ultrastructural level, numerous vacuoles, lysosomal-like structures, and loss of cellular integrity were seen in the proximal and distal tubules, as well as interstitial fibrosis in the form of numerous collagen fibers. No changes in the glomeruli were seen in any of the CsA-treated groups. These findings are consistent with chronic CsA nephrotoxicity similar to that seen in man.

Absorption and disposition of colloidal drug delivery systems. I. High-performance liquid chromatographic (HPLC) analysis of a cyclosporin emulsion.

The amount of cyclosporin A in an oil-in-water emulsion drug delivery system was determined by HPLC. The direct extraction and analysis of an intact emulsion were compared to the analysis of a cracked emulsion and an olive oil solution of the drug. The intra- and interday variability for the intact emulsion was less than 10% from 35 to 150 micrograms/ml, with recovery of 94%. Comparison of the assay results obtained with the emulsion and the olive oil solution gave a highly correlated regression line with a small intercept and a slope close to unity. Thus, the direct extraction and HPLC analysis of drugs in emulsions may be a viable approach to evaluate drug content.

Açäo da ciclosporina sobre transplantes dentais homógenos: estudo histológico em ratos / Cyclosporin's action upon allogenic dental transplants: histological study in rats

No trabalho avaliamos a açäo da ciclosporina sobre transplantes dentários homógenos em ratos. Foram empregados 36 ratos albinos divididos em dois grupos experimentais. Os animais tiveram o incisivo superior direito extraído e transplantado para um alvéolo homógeno. No Grupo I, controle, os animais receberam uma dose diária de 0,4 ml de soluçäo fisiológica via intra-peritoneal, iniciada 48 horas antes do transplante e mantida durante o período experimental. No Grupo II, tratado, os animais receberam uma dose diária de soluçäo de ciclosporina, na dosagem de 10mg/kg de peso corporal, iniciada 48 horas antes do transplante e mantida durante o período experimental. Os animais foram sacrificados em número de 6 aos 10, 30 e 60 dias pós operatórios. As peças foram processadas e analisadas histologicamente. Os resultados permitem concluir que: 1) a ciclosporina foi efetiva na diminuiçäo do infiltrado inflamatório ao nível do conjuntivo da mucosa gengival, do ligamento periodontal e fundo alveolar; 2) houve marcante reduçäo na ocorrência de reabsorçöes cemento dentinárias e anquilose alvéolo dental; 3) houve deposiçäo cementária sobre superfícies radiculares näo reabsorvidas; 4) näo observamos calcificaçöes semelhantes a tecido ósseo ao nível dos terços médio e apical da polpa dental; 5) näo se desenvolveram estruturas semelhantes a germes dentais

[Cyclosporin levels in semen of renal transplant patients].

Cyclosporin (CsA) has powerful immunosuppressive properties, and its recent application to organ transplantation has resulted in markedly improved graft survival. However, CsA has certain adverse side effects, the most notable being nephrotoxicity and hepatotoxicity. Among other untoward effects, little is known about the effects of CsA on the reproductive organs. Since good graft survival is now expected with CsA, the long term effect of the drug on the gonads should be monitored carefully, particularly for young recipients. Prior to investigation of the effect of CsA on the testis, the level of CsA in semen of the adult renal transplant patients were measured. Twelve samples of semen from eight recipients, mean age 35 (SD 9), were collected by masturbation in the morning just before taking CsA after more than 5 day abstinence, and it was frozen at -80 degrees C. They had been taking immunosuppressants that were a combination of either CsA and prednisolone, or CsA, azathioprine and prednisolone for 5 to 63 months. The dose of CsA was 3.7 mg/kg to 5.0 mg/kg. The range of the level of serum creatinine was 1.1 mg/dl to 2.9 mg/dl. CsA in whole blood was measured by high performance liquid chromatography (HPLC), and CsA in semen was also measured by HPLC after extracting CsA with diethylether. The level of CsA in semen was 27 ng/ml to 165 ng/ml and that in whole blood was 51 ng/ml to 133 ng/ml. The relation between both levels was linear (r = 0.68, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical analysis of effects of cyclosporin A on gingival epithelium.

Cyclosporin A (CSA)-induced gingival overgrowth was immunohistochemically compared with that phenytoin-induced and nonspecific inflammatory gingiva, and CSA concentration was determined for dental plaque. Leu-6+ epithelial dendric cells (EDC) were found to significantly decrease in number in CSA-induced gingival overgrowth, while the ratio of HLA-DR+ EDC to Leu-6+ EDC did not change significantly. The expression of class II major histocompatibility complex antigens, such as HLA-DR, -DP and -DQ on keratinocytes did not change by CSA-treatment. Leu-4+ mononuclear cells in CSA-induced gingival overgrowth were located primarily in the connective tissue far outside the epithelium. CSA concentration was much higher in dental plaque than in blood and other tissues. Immune response thus appears to be suppressed in the epithelial layer of CSA-induced gingival overgrowth through decrease in Leu-6+ HLA-DR+ EDC and T cell infiltration, both due to CSA in dental plaque. DNA polymerase alpha was detected in much fewer basal keratinocytes of CSA- and phenytoin-induced gingival overgrowth. Epithelial hyperplasia may thus be not due to increased keratinocyte proliferation, but rather to enhanced keratinocyte life span.