tacrolimus and thiazolyl-blue

Cyclosporine and tacrolimus are calcineurin inhibitors that are used to prevent acute rejection in renal transplant recipients. The lymphocyte immunosuppressant sensitivity test (LIST) can predict the pharmacological efficacy of these immunosuppressive agents for renal transplant recipients. There is a correlation between cyclosporine and tacrolimus pharmacological efficacy as evaluated by LIST by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay procedure prior to renal transplantation. However, the LIST can also evaluate patients before and after the transplantation.. The present study examined the relationship between cyclosporine and tacrolimus pharmacological efficacy by LIST using the MTT assay in 16 renal transplant recipients at 1, 3 and 12 months after transplantation, as well as before the operation.. The relationship of cyclosporine and tacrolimus pharmacological efficacy gave a significant Kendall and Spearman's coefficient correlation in these transplant recipients by the LIST using the MTT assay procedure immediately prior to renal transplantation (rk = 0.711, rs = 0.877, p < 0.01). Furthermore, correlations between the cyclosporine and tacrolimus IC50 values were also observed with a significant Kendall and Spearman's coefficient correlation at 1 and 12 months after transplantation (rk1month = 0.65, rs1month = 0.829, p < 0.01, and k12month = 0.433, rs12month = 0.603, p < 0.01, respectively). However, no statistically significant relationship was observed between the pharmacological efficacies of the calcineurin inhibitors at 3 months after transplantation (rk3month = 0.117, rs3month = 0.1, p > 0.05).. Both cyclosporine and tacrolimus exhibit pharmacological efficacy by the inhibition of calcineurin. However, the correlation between cyclosporine and tacrolimus pharmacological efficacies may be altered, due to immunosuppressive therapy or clinical events at 3 months after renal transplantation.

Topics: Cyclosporine; Humans; Immunosuppressive Agents; Kidney Transplantation; Lymphocytes; Tacrolimus; Tetrazolium Salts; Thiazoles

Immunosuppression considerably affects hepatitis C virus (HCV) recurrence and the outcome of antiviral treatment after liver transplantation. Recent findings have suggested that the calcineurin inhibitor tacrolimus (Tac), unlike cyclosporine A (CsA), interferes with the antiviral activity of interferon-alpha (IFN-alpha) in vitro. The aim of this study was to more extensively investigate the effects of calcineurin inhibitors on IFN-alpha signaling and antiviral activity in subgenomic and infectious HCV models. Treatment with Tac and CsA did not affect Huh7 cell proliferation at doses of 10 to 500 ng/mL; however, it completely inhibited T cell proliferation. In contrast to previous reports, Tac had no effect on IFN-alpha-stimulated reporter gene expression, even at the dose of 5 microg/mL. Furthermore, in Huh7 subgenomic HCV replicon cells, treatment with Tac had no significant effect on the suppression of viral replication by IFN-alpha. In the infectious HCV model, treatment with IFN-alpha effectively inhibited both viral RNA replication and de novo production of virus particles, and neither was attenuated at any concentration of Tac. CsA had no significant effect on IFN-alpha-stimulated reporter gene expression; however, as shown previously, a combination of CsA (at 500 ng/mL and higher) and IFN-alpha resulted in enhanced inhibition of viral replication in both the subgenomic and infectious HCV models. In conclusion, our study shows no evidence that Tac or CsA interferes with IFN-alpha-mediated inhibition of HCV replication and virion production in vitro. Therefore, no further mechanistic arguments have been found to break the clinical controversy about the choice of calcineurin inhibitors during posttransplantation antiviral therapy.

Topics: Antiviral Agents; Calcineurin Inhibitors; Cell Proliferation; Cell Survival; Gene Expression Regulation; Hepacivirus; Hepatitis C; Humans; Immunosuppressive Agents; Interferon-alpha; T-Lymphocytes; Tacrolimus; Tetrazolium Salts; Thiazoles

Tacrolimus is an agent used in clinical immunosuppressive drug therapies. A wide spectrum of adverse effects has been reported in association with this immunosuppressor, including neurotoxic effect. The upper limit of therapeutic blood concentrations of tacrolimus has been described as 30 ng/ml in immunosuppressed patients. We investigated the effect of this therapeutic dose of tacrolimus on the expression and activity of the multidrug resistance protein 1 (MDR1 or Pgp, P-glycoprotein) and ATP-binding cassette transporters A5 (ABCA5) in human brain microvascular endothelial cells (HBMEC), derived from Blood-Brain Barrier (BBB) endothelium, these being the most predominantly expressed transcripts in these cells. The expression and activity of MDR1 transporter decreased with 30 ng/ml tacrolimus. The cell viability was not changed with the therapeutic dose used. By contrast, ABCA5 transcripts, of unknown role as yet, increased their expression at this concentration. We propose that the secondary cytotoxic effects of this immunosuppressor on CSN, besides the functional blockade related to multidrug resistance proteins, such as MDR1, and probably ABCA5, could be linked to variations in the expression levels of these proteins at the BBB.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Blood-Brain Barrier; Blotting, Western; Caspase 3; Cells, Cultured; Endothelial Cells; Fluoresceins; Humans; Immunosuppressive Agents; Reverse Transcriptase Polymerase Chain Reaction; Tacrolimus; Tetrazolium Salts; Thiazoles

The dose of calcineurin inhibitors in renal transplantation has been adjusted, based on the therapeutic drug monitoring data. However, the data do not always correlate with clinical drug efficacy. In vitro response of peripheral-blood mononuclear cells to immunosuppressive drugs is reported to correlate with the recipient-response to therapeutic efficacy of the drug. We report, here, usefulness of a lymphocyte immunosuppressant sensitivity test for the estimation of individual drug sensitivity in renal transplant recipients. The LIST we have developed includes MTT assay procedures without the use of radioisotope-labeled compounds, which is convenient for general hospital use. Utilizing this procedure, we compared the pharmacological efficacy between cyclosporine A and tacrolimus in 41 renal transplant recipients.

Topics: Adult; Calcineurin Inhibitors; Cyclosporine; Female; Graft Rejection; Humans; Immunoassay; Immunosuppressive Agents; Kidney Transplantation; Lymphocyte Activation; Lymphocytes; Male; Middle Aged; Tacrolimus; Tetrazolium Salts; Thiazoles

Neuron-astrocyte interactions are critical for signalling, energy metabolism, extracellular ion and glutamate homeostasis, volume regulation and neuroprotection in the CNS. Glutamate uptake by astrocytes may prevent excitotoxic glutamate elevation and determine neuronal survival. However, an excess of glutamate can cause the death of astrocytes. FK506, an inhibitor of calcineurin, and an immunosuppressive drug, is neuroprotective in animal models of neurologic diseases, including focal and global ischaemia. In the present work, we demonstrate that a single injection of FK506 60 min after a transient middle cerebral artery occlusion (MCAo) significantly decreases the number of terminal deoxynucleotidyl transferase nick-end labelling (TUNEL)-positive cells in the ischaemic cortex and striatum. Using 3-D confocal microscopy we found that, 24 h after MCAo, many TUNEL-positive cells in the ischaemic striatum and cortex are astrocytes. Furthermore, we demonstrate that exposure of cultured cortical astrocytes to 50-100 mM Glu for 24 h induces apoptotic alterations in nuclear morphology, DNA fragmentation, dissipation of mitochondrial transmembrane potential (DeltaPsi) and caspase activation. FK506 (1 muM) efficiently inhibits Glu-induced apoptosis of cultured astrocytes, DNA fragmentation and changes in mitochondrial DeltaPsi. Our findings suggest that modulation of glutamate-induced astrocyte death early after reperfusion may be a novel mechanism of FK506-mediated neuroprotection in ischaemia.

Topics: Animals; Apoptosis; Astrocytes; Blotting, Western; Caspases; Cell Size; Cell Survival; Cells, Cultured; Cerebral Cortex; DNA Fragmentation; Enzyme Activation; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Glial Fibrillary Acidic Protein; Glutamic Acid; In Situ Nick-End Labeling; Indoles; Infarction, Middle Cerebral Artery; Male; Membrane Potentials; Mitochondria; Neuroprotective Agents; Rats; Reperfusion Injury; Tacrolimus; Tetrazolium Salts; Thiazoles

The aim of the present study was to test the hypothesis that exposure of astrocytes depleted of glutathione (GSH) to simulated ischemia conditions in vitro and treated with immunosuppressant immunophilin ligands (cyclosporin A (CsA) and FK506) can increase intracellular GSH levels and that such mechanism may be responsible, at least in part, for their protective effects. In addition, we also compared the antioxidant properties of these immunosuppressants with N-acetylcysteine (NAC), a precursor of GSH synthesis. GSH depletion was induced by 24 h pretreatment with L-buthionine sulfoximine (BSO). Cultures of rat astrocytes were exposed to CsA (1-50 microM) and FK506 (1-1000 nM) and NAC (100 or 200 microM). We examined the effects of these compounds on apoptosis, cell viability, reactive oxygen species production and GSH content. Our study demonstrated that toxicity of simulated ischemia conditions were enhanced when intracellular GSH was depleted, and immunosuppressants (especially 100 nM FK506 and 10 microM CsA) effectively prevented ischemia toxicity in GSH depleted astrocytes. In addition, we have shown that interfering with the generation of GSH and attenuation, the rise of oxidative stress level by NAC may be a powerful tool for prevention of ischemia-induced glial cell damage.

Topics: Acetylcysteine; Animals; Apoptosis; Astrocytes; Benzimidazoles; Brain Ischemia; Cell Separation; Cells, Cultured; Cyclosporine; Fluorescent Dyes; Free Radical Scavengers; Glutathione; Immunophilins; Immunosuppressive Agents; Ligands; Mitochondria; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Tacrolimus; Tetrazolium Salts; Thiazoles

The mechanism of the neuroprotective effect of FK506 in relation to nitric oxide (NO) production has not been clarified in vivo. We have investigated the effect of FK506 on ischemia-induced NO production in association with the pathogenesis of delayed neuronal death (DND) in rats.. In vivo microdialysis was performed in the hippocampus of male Sprague-Dawley rats (250-350 g). Dialysate samples were collected every 3 min. In the ischemia group (n=16), global ischemia was induced for 21 min and reperfusion was achieved. In the FK506 treatment group (n=25), FK506 (1 mg/kg, i.v.) was administered 21 min prior to the onset of global ischemia. Sham operations were done (n=15). The levels of NO(2)(-) in the dialysate samples were determined by the Griess reaction. The animals were decapitated 7 days after ischemia. Coronal brain sections were stained with hematoxylin and eosin.. In the ischemia group, the NO(2)(-) level significantly increased during ischemia. In the FK506 treatment group, there was no significant change in the NO(2)(-) level during ischemia. In histological examinations, FK506 treatment showed a neuroprotective effect against DND.. The effect of FK506 inhibiting NO production contributes to the neuro-protective effect of FK506 on DND in the hippocampus.

Topics: Analysis of Variance; Animals; Brain Ischemia; Cell Count; Cell Death; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Hippocampus; Humans; L-Lactate Dehydrogenase; Male; Microdialysis; Neuroblastoma; Neurons; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Rats; Rats, Sprague-Dawley; Reperfusion; Staining and Labeling; Tacrolimus; Tetrazolium Salts; Thiazoles

Changes in mitochondrial integrity, reactive oxygen species release and Ca2+ handling are proposed to be involved in the pathogenesis of many neurological disorders including methylmalonic acidaemia and Huntington's disease, which exhibit partial mitochondrial respiratory inhibition. In this report, we studied the mechanisms by which the respiratory chain complex II inhibitors malonate, methylmalonate and 3-nitropropionate affect rat brain mitochondrial function and neuronal survival. All three compounds, at concentrations which inhibit respiration by 50%, induced mitochondrial inner membrane permeabilization when in the presence of micromolar Ca2+ concentrations. ADP, cyclosporin A and catalase prevented or delayed this effect, indicating it is mediated by reactive oxygen species and mitochondrial permeability transition (PT). PT induced by malonate was also present in mitochondria isolated from liver and kidney, but required more significant respiratory inhibition. In brain, PT promoted by complex II inhibition was stimulated by increasing Ca2+ cycling and absent when mitochondria were pre-loaded with Ca2+ or when Ca2+ uptake was prevented. In addition to isolated mitochondria, we determined the effect of methylmalonate on cultured PC12 cells and freshly prepared rat brain slices. Methylmalonate promoted cell death in striatal slices and PC12 cells, in a manner attenuated by cyclosporin A and bongkrekate, and unrelated to impairment of energy metabolism. We propose that under conditions in which mitochondrial complex II is partially inhibited in the CNS, neuronal cell death involves the induction of PT.

Topics: Animals; Antimycin A; Bongkrekic Acid; Brain; Calcimycin; Calcium; Catalase; Cell Survival; Cyclosporins; Dose-Response Relationship, Drug; Drug Interactions; Electron Transport Complex II; Enzyme Inhibitors; Female; In Vitro Techniques; Ionophores; Malonates; Membrane Potentials; Methylmalonic Acid; Mitochondria; NADP; Neurons; Nitro Compounds; Oxygen Consumption; PC12 Cells; Permeability; Propionates; Rats; Rotenone; Tacrolimus; Tetrazolium Salts; Thiazoles; Uncoupling Agents

FK506, cyclosporin A (CsA), and its structural analog cyclosporin G (CsG) are immunosuppressant drugs mainly metabolized by hepatic cytochrome P-450 3A (CYP 3A) oxygenase. FK506 metabolites exhibit greater toxicity than the parent drug, while CsA metabolites are far less toxic than CsA itself. The aim of our study was to compare the toxicity of CsG with CsA and FK506 as a function of CYP 3A induction. Hepatocytes from Wistar rats with or without dexamethasone (DEX) induction (200 mg/kg per day, p.o for 4 days) were used in primary culture. The DEX-inductive effect on CYP 3A was assessed by SDS-PAGE. After 6 h incubation with CsG, CsA or FK506 (5 to 200 microM), cell viability (expressed as IC50), intracellular calcium content and apoptosis were evaluated. Concerning cytotoxicity, IC50 values for CsG, CsA and FK506 were 75, 50 and 180 microM respectively in non-induced cultures, and 150, 120 and 25 microM in induced cultures. For intracellular calcium content, a dose-dependent increase was observed in all cultures. However this increase is more important for CsG and CsA in non-induced cultures (150%) compared to induced cultures (110%) at 150 microM. Conversely for FK506, this increase is greater in induced cultures (150%) than in non-induced cultures (127%). Estimation of the percentage of apoptotic cells shows similar variations. Our results show that the toxicity of the three drugs in rat hepatocytes is dependent on CYP 3A induction: increased for FK506, decreased for CsA and CsG. Moreover, with regard to the three tests used, the toxic effects of CsG are close to those of CsA, indicating that CsG metabolites are also less toxic than the parent drug.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Calcium; Cell Survival; Cells, Cultured; Coloring Agents; Cyclosporine; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Flow Cytometry; Immunosuppressive Agents; Intracellular Fluid; Liver; Male; Oxidoreductases, N-Demethylating; Rats; Rats, Wistar; Tacrolimus; Tetrazolium Salts; Thiazoles

FK506 and cyclosporin A (CsA) are two potent immunosupressants with similar toxicity profile. Nephrotoxicity is the main adverse effect of both compounds. The aim of this study is to compare the in vitro nephrotoxic effects on renal epithelial cell line LLC-PK1 by measuring cell viability and energy status as evaluated by concentrations of ATP and ATP metabolites. Cell viability (expressed as IC50 was assessed via thiazolyl blue (MTT) assay after incubation for 4-24 h with FK506 or CsA. ATP and its metabolites were determined by HPLC after 4 and 6 h incubation with FK506 or CsA alone at the respective IC50. Both FK506 and CsA decreased cell viability to similar extents, in a dose- and time-dependent manner. After 4 h incubation, both drugs decreased ATP levels (-25%) and increased uric acid levels. However, the latter percentage increase was twofold higher with CsA (18%) than with FK506 (9%). The energy charge, calculated according to levels of adenine nucleotides, was decreased by 10% in FK506-treated cells and by 27% in CsA-treated cells. At the end of 6-h incubation, FK506-treated cells maintained ATP levels coupled with energy charge at near control levels whereas the levels were 32% lower in CsA treated cells. Compared to the 4 h-incubation, the increase in uric acid was similar for FK506 but was doubled with CsA. The decrease in cell integrity and ATP depletion induced by CsA in LLC-PK1 cells was only transiently observed with FK506. By preserving energy status, FK506 leads to fewer metabolic disturbances than CsA in the renal epithelial cell line LLC-PK1, demonstrating a minor potential nephrotoxicity.

Topics: Adenosine Triphosphate; Animals; Cell Survival; Cyclosporine; Energy Metabolism; Immunosuppressive Agents; Kidney; LLC-PK1 Cells; Nucleotides; Swine; Tacrolimus; Tetrazolium Salts; Thiazoles; Time Factors

FK506, CsA, and rapamycin are potent inhibitors of T lymphocyte activation; relatively little is known of their effects on cells of the monocyte/macrophage lineage. Studies were undertaken to determine the effects of these drugs on the proliferative response of bone marrow-derived mononuclear phagocytes (BMMP) to CSFs. Rapamycin inhibited the proliferation of BMMP cultured in the presence of 10% L cell-conditioned medium, used as a source of macrophage CSF. The inhibition by rapamycin was dose dependent and apparent at concentrations of 0.1 nM or greater. In a similar fashion, rapamycin inhibited the proliferation of BMMP stimulated by the recombinant forms of murine IL-3 and murine granulocyte-macrophage CSF, and human macrophage CSF. In contrast, neither FK506 nor CsA at concentrations as high as 1000 nM diminished the proliferation of BMMP cultured under identical conditions. FK506, but not CsA, blocked the inhibitory effects of rapamycin on the response of BMMP to CSFs. In summary, these data indicate that rapamycin inhibits the proliferation of BMMP in response to CSFs. These results imply that patients receiving rapamycin, but not FK506 or CsA, may have an impaired ability to generate a functional mononuclear phagocyte population.

Topics: Animals; Bone Marrow Cells; Cell Count; Cell Division; Colony-Stimulating Factors; Concanavalin A; Culture Media, Conditioned; Cyclosporine; Female; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Phagocytes; Polyenes; Sirolimus; Spleen; T-Lymphocytes; Tacrolimus; Tetrazolium Salts; Thiazoles; Thymidine; Tritium