SDZ RAD 40-O-(2-hydroxyethyl)rapamycin is a macrolide immunosuppressant that is currently under clinical investigation after organ transplantation. The elucidation of its metabolic pathway is ...essential to improve the understanding of its therapeutic potentials and safety. In this article we describe investigations on the structural identification of some major metabolites of the drug produced by human liver microsomes in vitro. The principles described may be generally applicable for the structural elucidation of complex compound mixtures in biological matrices. Under the conditions of electron impact ionization, SDZ RAD undergoes extensive fragmentation and no information sufficient for structural elucidation is obtained. Therefore, mass spectrometry based on soft electrospray ionization (ESI) in conjunction with collision-induced fragmentation was the method of choice. High-performance liquid chromatography coupled to an ESI mass spectrometer resulted in separation and identification of 16-O-demethyl-SDZ RAD, the ring-opened form of SDZ RAD, and its dehydrate. Additionally, we characterized several demethylated and hydroxylated metabolites.
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)
Helicobacter pylori infection has been considered as a risk factor for gastric carcinoma. Strong evidence exists that reactive oxygen species (ROS) play an important role in carcinogenesis, and in ...vivo investigations have shown increased synthesis of ROS in the gastric mucosa of H.pylori-infected patients. In the present study the direct effects of H.pylori on ROS and DNA synthesis, induction of apoptosis and DNA repair were investigated in the gastric epithelial cell lines AGS and HM02. Incubation of gastric cells with H.pylori extract induced the synthesis of ROS, diminished the levels of reduced glutathione (GSH), induced DNA fragmentation and increased DNA synthesis in gastric cells. Poly(ADP-ribose) formation was increased in gastric cells exposed to H.pylori extract. FACS analysis of gastric cells exposed to H.pylori extract did not reveal any change in the percentage of cells in the G2/M phase of the cell cycle. The radical scavengers MnTBAP (a cell permeable superoxide dismutase mimic), ebselen (a GSH peroxidase mimic) and high doses of catalase completely blocked H.pylori extract-induced elevation in DNA synthesis. Our results indicate that H.pylori extract directly induces the synthesis of ROS in gastric epithelial cells and causes DNA damage.
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.
1Six male and six female stable renal allograft recipients under cyclosporine immunosuppression and without concomitant therapy with drugs known either to induce or inhibit CYP3A enzymes were ...included in the study and received 180 mg day−1 diltiazem for 1 week in a two‐period cross‐over fashion. Cyclosporine (352±56 mg day−1) was given in two daily oral doses. The daily doses were not changed during the study. Blood samples were collected for 12 h after receiving cyclosporine alone and after receiving diltiazem in addition for 1 week. Cyclosporine and nine of its metabolites were quantified using h.p.l.c.
2Co‐administration of diltiazem caused a 1.6 fold increase of the AUC(0,12 h) of cyclosporine and a 1.7 fold increase of the AUC(0, 12 h) of its metabolites. Analysis of the metabolite patterns showed an over‐proportional increase of the AUC(0, 12 h) of the cyclized metabolites AM1c (2.6 fold) and AM1c9 (2.2 fold). The AUC(0, 12 h) values of cyclosporine and the hydroxylated metabolites increased less than two fold.
3Differences of the AUC(0, 12 h) values of cyclosporine with and without diltiazem were significantly higher in female than in male patients (P<0.02). The differences in the AUC(0, 12 h) values of the metabolites, especially AM1c, tended to be higher in female patients as well.
4It is concluded that coadministration of diltiazem not only increases the blood concentration of cyclosporine but also those of its metabolites, leads to a shift of the metabolite pattern towards cyclized metabolites, and that the pharmacokinetic changes under diltiazem administration are more prominent in female than in male patients.
Helicobacter pylori shows a rather high variability of several biochemical markers including lipopolysaccharide structures. This study aimed to determine whether Helicobacter pylori has a potential ...for phenotypic variability and to describe its effects on bacterial pathogenesis. From colonies of three clinical strains of Helicobacter pylori with rough (R) colony morphology, spontaneous phenotypic variants with smooth (S) colony morphology were isolated that occurred with a frequency of 10(-2) to 10(-3), irrespective of growth conditions. R-variant bacteria produced exclusively low-molecular-mass lipopolysaccharide. They exhibited increased lysis in the presence of plain air. In contrast, the S variants produced low- and high-molecular-mass lipopolysaccharide and did not exhibit increased lysis in the presence of plain air. Cocultivation of bacterial cells with AGS stomach cancer cells revealed that R-variant bacteria but not S-variant bacteria effected an inhibition of high molecular-weight glycoprotein biosynthesis and secretion by the host cells. Skirrow supplement added as selective agent to liquid and/or solid media was tolerated to a similar extent among R- and S-variant bacteria, while all variants proved sensitive to metronidazole, amoxicillin and clarithromycin except for the R and S isolates of strain Hp57, which showed resistance to the latter compound. It was concluded that R- and S-variants of Helicobacter pylori may have distinct roles in pathogenesis; nevertheless, these bacteria may be isolated by traditional methods and eradicated by conventional anti-infective therapy.
1. Blood and urine concentrations of the macrolide immunosuppressant FK506 and its metabolites were measured in seven orthotopic liver transplant patients after the first oral dose of FK506 (0.04 +/‐ ...0.02 mg kg‐1) used as primary immunosuppressant. A specific h.p.l.c.‐MS assay was used, allowing the measurement of parent drug and eight metabolites. Results were compared with those obtained using a microparticle enzyme immunoassay (MEIA). 2. Blood drug concentrations were described by an open two compartment model with first‐order absorption giving the following mean data: tmax: 1.9 (h), Cmax: 17.4 (microgram l‐1), AUC: 328.1 (microgram l‐1 h), t1/2,1: 0.74 (h). The terminal elimination half‐life was estimated at about 26 h using the h.p.l.c.‐MS assay. 3. The metabolites found in blood were demethyl‐ FK506 and demethyl‐hydroxy‐FK506, while in urine FK506 and eight of its metabolites were detected.
An analytic technique using liquid chromatography (LC) coupled with electrospray-mass spectrometry (ESI-MS) has been developed for the simultaneous determination of the new immunosuppressant SDZ RAD ...(40-O-2-hydroxy)ethylrapamycin) and cyclosporine (Cs), including their metabolites in blood. With the time-sparing, automated on-line extraction technique, the recovery of SDZ RAD averaged 95% and that of Cs, 94%. The calibration lines were linear from 0.5 to 100 microg/L (r2 = 0.99) for SDZ RAD and from 10 to 1,000 microg/L (r2 = 0.99) for Cs. The method has been tested on blood samples from renal transplant recipients taken between 1 and 5 hours after oral SDZ RAD and Cs administration. In blood, we found the following metabolites: Hydroxy-SDZ RAD, dihydroxy-SDZ RAD, demethyl-SDZ RAD, and the ring-opened form of SDZ RAD. The main metabolite of SDZ RAD in blood was hydroxy-SDZ RAD. This novel LC/ESI-MS technique provided an excellent method for simultaneous quantitative monitoring of SDZ RAD and Cs, including their relevant groups of metabolites in patients treated simultaneously with these immunosuppressants.
1
The mechanism of the gastric antisecretory action of SCH 28080 has been studied utilizing two different in vitro test systems, isolated and enriched parietal cells from the guinea‐pig and ...guinea‐pig gastric membranes purified and enriched with K+/H+‐ATPase.
2
In guinea‐pig isolated and enriched parietal cells SCH 28080 inhibited the acid response to histamine and high K+ concentrations with IC50 values not significantly different from each other.
3
SCH 28080 inhibited the purified K+/H+‐ATPase measured in the presence of 5 mM KCl with an IC50 value of 1.3 μM. Kinetic studies indicated a competitive inhibition of ATPase by SCH 28080 with respect to K+. Studies on Na+/K+‐ATPase showed that this enzyme was only slightly depressed by SCH 28080.
4
It is concluded that SCH 28080 acts with high selectivity on the parietal cell K+/H+‐ATPase, establishing its antisecretory effect by a competitive interaction with the high affinity K+‐site of the gastric ATPase.
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