Search PubMed⌕ Search

Biomedical subjects

U Christians

Publications and source records attributed to U Christians.

At least 73 records · Page 4Linked to original sources
PubMed1996

Identification of drugs inhibiting the in vitro metabolism of tacrolimus by human liver microsomes.

1. Tacrolimus, an immunosuppressive macrolide, is metabolized by enzymes of the cytochrome P450 3A subfamily. In this study, 34 drugs were tested for their interactions with tacrolimus metabolism by human liver microsomes. 2. Fifteen drugs which inhibit the in vitro metabolism of tacrolimus were identified: bromocriptine, corticosterone, dexamethasone, ergotamine, erythromycin, ethinyloestradiol, josamycin, ketoconazole, miconazole, midazolam, nifedipine, omeprazole, tamoxifen, troleandomycin and verapamil.

Bromocriptine↗
PubMed1996

Metabolism of the macrolide immunosuppressant, tacrolimus, by the pig gut mucosa in the Ussing chamber.

1. The macrolide tacrolimus (FK506), used as an immunosuppressant, is a cytochrome P450 (CYP) 3A substrate in the liver. The metabolism of tacrolimus and the transport of its metabolites in the pig gut was studied in the Ussing chamber. Tacrolimus and its metabolites were quantified by h.p.l.c./mass spectrometry. 2. In the Ussing chamber, demethyl, didemethyl, hydroxy and hydroxy-demethyl tacrolimus were generated. Their formation was concentration- and time-dependent. The metabolite pattern was not different from that after incubation of tacrolimus with human small intestinal microsomes. 3. The metabolite formation was highest in the duodenum and declined in the order duodenum > jejunum > ileum > colon > stomach. 4. Since tacrolimus metabolism was inhibited by the specific CYP3A inhibitors, troleandomycin and ketoconazole, we concluded that these enzymes are involved in intestinal metabolism of tacrolimus. 5. Tacrolimus metabolites re-entered the mucosa chamber (> 90%) and passed through the small intestinal preparation into the serosa chamber. 6. It is concluded that tacrolimus is metabolized in the intestine, that the metabolites are able to re-enter the gut lumen and also enter into the portal vein and that small intestinal metabolism and transport is at least in part responsible for the low oral bioavailability of tacrolimus.

Animals↗
PubMed1996

Drug interactions and interindividual variability of ciclosporin metabolism in the small intestine.

The undecapeptide ciclosporin is used as immunosuppressant after organ transplantation and for therapy of immune diseases. Low and variable bioavailability of ciclosporin has been attributed to its metabolism in the small intestine. The aim of the present study was to investigate drug interactions and interindividual variability of ciclosporin metabolism in the small intestine. Ciclosporin metabolism was studied in vitro using microsomes isolated from the small intestine of humans and pigs. The metabolites generated were quantified by HPLC and identified by mass spectrometry. Using specific antibodies and inhibitors, we showed that, as in the liver, cytochrome P450 3A (CYP 3A) enzymes are responsible for ciclosporin metabolism in the human small intestine. Of the 28 xenobiotics included in the study, 16 drugs, all well-known CYP 3A inhibitors, inhibited ciclosporin metabolism in the small intestine. In the small intestine of different patients, the rate of metabolism varied by a factor of 10. Ciclosporin was metabolized faster by small intestine microsomes from female (n = 4) than from male (n = 10) patients (p < 0.009).

Adult↗
PubMed1996

Sensitive and specific quantification of sirolimus (rapamycin) and its metabolites in blood of kidney graft recipients by HPLC/electrospray-mass spectrometry.

Sirolimus (rapamycin) has a macrolide structure and is under clinical investigation as an immunosuppressant after organ transplantation. An HPLC/mass spectrometry assay to quantify sirolimus in blood was developed. 28-O-Acetyl sirolimus was used as internal standard. Blood samples were extracted with C18 columns. The extracts were injected into an HPLC system and isocratically eluted with methanol/1% formic acid (90/10 by vol) from a 150 X 4 mm C18 analytical column. The HPLC system was connected to a triple-stage quadrupole mass spectrometer with an electrospray interface and positive ions were detected. The limit of quantification in 1 mL of blood was 0.25 microgram/L and the calibration curve in blood was linear up to 250 microgram/L. The recovery from blood was 88 +/- 26% and interassay variation at 1 microgram/L was 19% and at 15 microgram/L 9.3%. Hydroxy, dihydroxy, demethyl, and didemethyl sirolimus as well as sirolimus were detected in blood of kidney graft patients.

Acetic Anhydrides↗
PubMed1996

Tacrolimus (FK506) metabolite patterns in blood from liver and kidney transplant patients.

The metabolite patterns of tacrolimus in blood were evaluated in 41 kidney and liver graft recipients. Trough concentrations of tacrolimus and its metabolites were measured by HPLC-mass spectrometry and microparticle enzyme immunoassay in parallel. A statistically significant correlation between results of both assays was observed for kidney and liver transplant patients (r = 0.77, P <0.001 and r = 0.71, P <0.001, respectively). The main metabolites in blood were demethyl, demethylhydroxy, didemethyl, didemethylhydroxy, and hydroxy tacrolimus. These metabolites added up to 42% (range 0-145%) of the tacrolimus concentration in liver transplant patients and to 44.8% (range 16-152%) in kidney transplant patients. During episodes of impaired liver function, concentrations of tacrolimus and its metabolites were increased compared with normal liver function, indicating accumulation of metabolites, in particular second-generation metabolites such as didemethyl and didemethylhydroxy tacrolimus. Stepwise regression analysis including tacrolimus, its metabolites, and liver function parameters suggested a model including serum activities of gamma-glutamyltransferase, alkaline phosphatase, and alanine aminotransferase as predictors for increased concentrations of demethyl tacrolimus, didemethyl tacrolimus, and the parent drug.

Adolescent↗
PubMed1996

Cyclosporin-A and its metabolites in the anterior chamber after topical and systemic application as determined with high-performance liquid chromatography-electrospray mass spectrometry.

Penetration of cyclosporin A (CSA) into the anterior chamber through the intact cornea after topical application is difficult due to its hydrophobic structure. Following systemic application the anterior-chamber levels of CSA are reported to be higher. CSA metabolites are more hydrophilic than CSA. Only high-performance liquid chromatography-electrospray mass spectrometry allows exact quantification of the CSA level and the identification of all CSA metabolites. We studied the anterior-chamber levels of CSA and different CSA metabolites after topical and systemic application. CSA and CSA-metabolite anterior-chamber levels were measured in 49 patients after topical application of CSA 2% eye drops preceding routine cataract surgery with 2 different application schemes and in 7 patients receiving systemic CSA after high-risk penetrating keratoplasty. After topical application the average CSA level measured in the anterior chamber was 81 ng/ml. The CSA-metabolite levels were much higher, reaching an average of 378 ng/ml. After systemic therapy the anterior-chamber levels of CSA and of the metabolites were much more balanced at 256 and 317 ng/ml, respectively. CSA penetrates into the anterior chamber after topical eye-drop application, but these levels are much lower than those measured after systemic CSA therapy. After topical application the CSA metabolites might play an important role; they are found in the anterior chamber in much higher concentrations than is CSA, and the metabolite pattern differs from that seen after systemic therapy. The relevance of these findings to the immunosuppressive activity of the CSA metabolites, however, remains unclear.

Administration, Topical↗
PubMed1996

Characterization of glucuronidated phase II metabolites of the immunosuppressant cyclosporine in urine of transplant patients using time-of-flight secondary-ion mass spectrometry.

The immunosuppressant, cyclosporine, is metabolized in the liver and small intestine to > 30 metabolites. Metabolism and immunosuppressive and toxic potentials of the metabolites are still unclarified. Therefore, search and determination of new metabolites remain an important part of cyclosporine research. In this study, cyclosporine metabolites were determined in 42 urine samples of transplant patients using time-of-flight secondary-ion MS. Besides the known metabolites of phase I and phase II, other groups of new phase II metabolites were detected, and most of them were identified as glucuronidated phase I metabolites. All metabolites were found in the urine of heart, kidney, and bone marrow graft patients, with frequencies in the range of 74% and 12%. The most intensive group of these metabolites was also detected in a HPLC fraction, together with the known glucuronidated AM1c. The concentration of this new metabolic group could be estimated to < or = 5/ml. In conclusion, this work demonstrated that time-of-flight secondary-ion MS is a powerful tool in pharmacological investigations. Furthermore this study showed that phase II metabolism is an important metabolic pathway of cyclosporine in transplant patients.

Animals↗
PubMed1996

Structural identification of three metabolites and a degradation product of the macrolide immunosuppressant sirolimus (rapamycin) by electrospray-MS/MS after incubation with human liver microsomes.

Sirolimus is a macrolide immunosuppressant that is metabolized by cytochrome P450 3A enzymes to several demethylated and/or hydroxylated metabolites, the exact structures of which have not yet been identified. In addition, sirolimus undergoes degradation in organic solvents and biological fluids. The fragmentation pattern of sirolimus after collision activated dissociation was identified. We used electrospray/MS/MS in combination with collision activated dissociation to elucidate the structures of several sirolimus metabolites and that of a degradation product after incubation of sirolimus with human liver microsomes. The following metabolites were identified: 39-O-demethyl sirolimus, 16-O-demethyl sirolimus, 12-hydroxy sirolimus, as well as the structure of the degradation product 34-hydroxy sirolimus. After incubation with human liver microsomes, 69.7% of the sirolimus derivatives detected were sirolimus, 9.3% 39-O-demethyl sirolimus, 9.3% 34-hydroxy sirolimus, 4.6% 12-hydroxy sirolimus and other hydroxylated metabolites, 2.2% 16-O-demethyl sirolimus, 3% dihydroxylated metabolites (m/z of [M + Na]+ = 968.5), 1.2% trihydroxylated metabolites (m/z of [M + Na]+ = 984.5), and 0.9% tetrahydroxylated metabolites (m/z of [M +Na]+ = 1000.5). Analysis of the fragments of the di-, tri-, and tetrahydroxylated metabolites showed that the hydroxylated sites were located between C(10) and C(27). The intensities of additional fragments was not sufficient to completely identify their structures.

Humans↗
PubMed1995

Alternative cyclosporine metabolic pathways and toxicity.

There are some indications from clinical studies (41,43) for aberrant cyclosporine metabolism resulting in formation of potentially toxic metabolites. When the activity of cytochrome P450 3A enzymes is low, more substrate is available for hypothetical alternative pathways of cyclosporine. There are several reasons for low P450 3A activity in a liver graft such as inter-individual genetic variability (43,49,84), cold ischemia and reperfusion damage, changes of the P450 activity during cholestasis (85) or other liver diseases (86), the influence of cytokines (87) and drug interactions such as inhibition or enzyme induction (88). Furthermore, low concentrations of cytochrome P450 3A influence the cyclosporine blood trough concentrations. The P450 3A concentration as estimated by the erythromycin breath test can be used to calculate the initial cyclosporine dose required to obtain cyclosporine blood trough concentrations in the therapeutic window (89). In vitro such alternative pathways comprising 3-methylcholanthrene-inducible (44,46,47) and/or ethinyl estradiol-inducible cytochrome P450 enzymes (48) could be identified and resulted in production of cyclized cyclosporine metabolites. The exact identification of the P450 enzymes involved requires metabolism of cyclosporine using reconstituted purified enzymes or single P450 enzymes expressed in cell lines. In addition, it remains to be clarified whether cyclosporine itself or its metabolite AM1 is the substrate for cyclization. Because cyclized metabolites have a low affinity to cyclophilin (58,59) they are mainly found in plasma. When more cyclized metabolites are formed primarily the concentration of cyclosporine metabolites in plasma increases. The free fraction of cyclosporine at 37 degrees C was found to be 1%-1.5% (90,91) of the cyclosporine concentration in blood. To date, nothing is known about the free fraction of cyclosporine metabolites. Because distribution characteristics of the cyclized metabolites in blood and urine are different from those of cyclosporine, it can be speculated that the free fraction of the cyclized metabolites is higher than that of cyclosporine. This might be reflected by a higher renal clearance resulting in relatively higher concentrations in urine compared with blood (61; Figure 3). If this is the case, a shift in the metabolite pattern with increased concentrations of cyclized metabolites will lead to an overproportional increase of the free fraction of cyclosporine metabolites. Although it is tempting to assume that cyclization is the alternative pathway explaining cyclosporine toxicity in patients with low concentrations of P450 3A enzymes in the liver (Figure 6), this has not yet been proven and will require not only quantification of P450 3A but of the complete P450 enzyme pattern in the liver in combination with characterization of the cyclosporine metabolite pattern by HPLC with special respect to the cyclized metabolites AM1c and AM1c9. Also, it is still unclear whether or not the cyclized metabolites contribute to cyclosporine toxicity. At least, it is unlikely that they are involved in covalent binding to macromolecules in the liver and kidney (44,71). In a clinical study using an HPLC method which allowed the specific quantification of 16 cyclosporine metabolites it was shown that the blood trough concentrations of the cyclized metabolite AM1c9 is elevated during early nephrotoxicity in liver graft recipients (82) and it was shown in an in vitro model that AM1c9 increases endothelin production and therefore might have a negative effect on renal hemodynamics.(ABSTRACT TRUNCATED)

Animals↗
PubMed1995

An organotypical in vitro model of the liver parenchyma for uptake studies of diagnostic MR receptor agents.

Testing of receptor-specific MR contrast agents targeted to the liver is hampered by a shortage of viable in vitro models with in vivo-like hepatocellular morphology. Coated pits are ultrastructural signs of an active receptor mediated endocytosis in hepatocytes. Expression of coated pits by matrix overlaid hepatocytes was studied by transmission electron microscopy. Binding of a rhodaminated asialoglycoprotein receptor agent (MION-ASF-rh) was assessed by fluorescence microscopy. Fluorescence of cells exposed to MION-ASF-rh with D(+)-galactose reduced fluorescent light emission to a level of 58% of MION-ASF-rh-induced fluorescence. After preincubation with the hepatotoxin CCl4 a dose-dependent decrease in fluorescent light emission resulted. Hepatocytes maintained a homogeneous cell surface expression, with microprojections, coated pits, and vesicles on both sinusoidal surfaces. Matrix overlaid primary hepatocytes constitute a viable, morphologically and functionally differentiated model. This model can be used to study receptor binding, uptake, and blockage of diagnostic magnetopharmaceuticals under controlled conditions.

Animals↗
PubMed1995

Parallel blood concentrations of second-generation cyclosporine metabolites and bilirubin in liver graft recipients.

Cyclosporine, a cyclic undecapeptide, is currently the major immunosuppressant used after liver transplantation. Since it is unclear whether or not cyclosporine metabolites play a part in toxicity, high concentrations of metabolites should be avoided. The quantification of cyclosporine metabolites requires immunoassays using nonspecific antibodies cross-reacting with metabolites or high-performance liquid chromatography (HPLC) analysis. Since no guidelines are available to date concerning when such additional analysis is required, it was the aim of this study to define biochemical parameters that parallel cyclosporine elimination and indicate whether or not cyclosporine elimination is impaired, requiring quantification of cyclosporine metabolites. One hundred and thirty adult liver graft recipients were included in a prospective study during their first hospital stay. Cyclosporine and 11 metabolites were quantified in blood every second day using radioimmunoassay and HPLC. When the cyclosporine metabolite patterns in trough blood samples of patients with impaired liver function were compared with those of patients with good liver function, concentrations of metabolites AM19 and AM1A were found to be elevated. Serum concentrations of conjugated and total bilirubin were significantly correlated with blood trough concentrations of AM19 and AM1A, while there was no correlation with cyclosporine or its first-generation metabolites. Distribution statistics showed that liver graft patients with impaired cyclosporine elimination had total bilirubin concentrations in serum > 60 mumol/l L. No correlation was found between bile acids and the concentrations of metabolites AM19 and AM1A, suggesting that the ion-coupled transport system is not quantitatively involved in cyclosporine excretion and that bilirubin and cyclosporine metabolites are eliminated by the same transport system through the biliary membrane. It is concluded that bilirubin and cyclosporine metabolite concentrations are strictly parallel and that the total bilirubin concentration in serum may be used as an indicator of impaired cyclosporine elimination.

Administration, Oral↗
PubMed1995

Simplified high-performance liquid chromatography-mass spectrometry assay for measurement of tacrolimus and its metabolites and cross-validation with microparticle enzyme immunoassay.

In this study, a modified, specific assay for measurement of tacrolimus and its metabolites in blood and urine from transplant patients using high-performance liquid chromatography (HPLC) linked to mass spectrometry (MS) is described. Samples were prepared for HPLC-MS by modified solid-liquid extraction. The original two-step washing procedure was replaced by a single washing step, and samples were eluted with acetonitrile/water instead of dichloromethane, thus avoiding an evaporation step. Samples were injected automatically every 3 min into the HPLC-MS system. Time-consuming gradient elution was replaced by isocratic elution. This procedure resulted in a lower limit of quantitation of 0.2 microgram/L. The interassay variability was 14.5% for 5 micrograms/L and 15.8% for 25 micrograms/L. The intrassay variability was 11.2% for 5 micrograms/L and 4% for 25 micrograms/L. The recovery for tacrolimus in blood was 90.4% for 1 microgram/L, 78.9% for 10 micrograms/L, and 81.3% for 25 micrograms/L. Measurement of tacrolimus and its metabolites in samples from various transplant patients showed that the main metabolites found in blood and urine are demethyl-tacrolimus, di-demethyl-tacrolimus and demethyl-hydroxy-tacrolimus. Cross validation of the modified HPLC-MS assay with a microparticle enzyme immunoassay showed a significant correlation between the two assays, with r = 0.915.

Acetonitriles↗