Influence Of Mycophenolic Acid Research Articles

Manipulation of Cellular Redox State was Essential for Triggering Erythroid Differentiation of K562 Cells by Mycophenolic Acid

Reactive Oxygen Species (ROS) play important roles in a variety of normal cellular functions and various pathological events. Uncontrolled ROS production in various types of cancer cells has been globally reported and considered as a hallmark for new drug developments. Accordingly, in this study, we planned to investigate the probable roles of oxidants on erythroid differentiation of K562 cells under the influence of Mycophenolic acid (MPA). Based on our results, MPA attenuated the intracellular SOD activity to provide a suitable redox environment for hemoglobinization of the exposed cells, while the cellular content of glutathione and glutathione peroxidase were augmented at the initial times of MPA-induced erythroid differentiation. These variations were accompanied by the significant reduction in the cellular ROS content in the first 8 h of MPA exposure followed by an enormous increase in ROS content by the 24 h of exposure. Furthermore, while the cells were undergoing erythroid differentiation by MPA, it became evident that p27kip1, apart from its role in cell cycle arrest, was functioning as a sensor of the intracellular oxidants. Overall, it might be concluded that MPA-guided erythroid differentiation was strongly associated with manipulation of ROS content of the chronic myelogenous leukemia cells.

In Vitro Influence of Mycophenolic Acid on Selected Parameters of Stimulated Peripheral Canine Lymphocytes.

Mycophenolic acid (MPA) is an active metabolite of mycophenolate mofetil, a new immunosuppressive drug effective in the treatment of canine autoimmune diseases. The impact of MPA on immunity is ambiguous and its influence on the canine immune system is unknown. The aim of the study was to determine markers of changes in stimulated peripheral canine lymphocytes after treatment with MPA in vitro. Twenty nine healthy dogs were studied. Phenotypic and functional analysis of lymphocytes was performed on peripheral blood mononuclear cells cultured with mitogens and different MPA concentrations– 1 μM (10−3 mol/m3), 10 μM or 100 μM. Apoptotic cells were detected by Annexin V and 7-aminoactinomycin D (7-AAD). The expression of antigens (CD3, CD4, CD8, CD21, CD25, forkhead box P3 [FoxP3] and proliferating cell nuclear antigen [PCNA]) was assessed with monoclonal antibodies. The proliferation indices were analyzed in carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled cells. All analyses were performed using flow cytometry. The influence of MPA on apoptosis was dependent on the mechanism of cell activation and MPA concentration. MPA caused a decrease in the expression of lymphocyte surface antigens, CD3, CD8 and CD25. Its impact on the expression of CD4 and CD21 was negligible. Its negative influence on the expression of FoxP3 was dependent on cell stimulation. MPA inhibited lymphocyte proliferation. In conclusion, MPA inhibited the activity of stimulated canine lymphocytes by blocking lymphocyte activation and proliferation. The influence of MPA on the development of immune tolerance–expansion of Treg cells and lymphocyte apoptosis–was ambiguous and was dependent on the mechanism of cellular activation. The concentration that MPA reaches in the blood may lead to inhibition of the functions of the canine immune system. The applied panel of markers can be used for evaluation of the effects of immunosuppressive compounds in the dog.

Mycophenolic acid affects basic functions of human keratinocytes in the IMPDH-dependent manner

Clinical studies suggest that the immunosuppressant MPA is associated with impaired wound healing. It is believed that the main cause of impairment is the inhibition of inflammatory response. However, it is unknown whether MPA may directly affect epidermal cells. The aim of our study was to examine the direct influence of mycophenolic acid, the selective blocker of de novo purine synthesis, on human epidermal keratinocyte morphology, proliferation, motile activity, and differentiation in in vitro culture. The number of keratinocytes cultured in the presence of MPA was counted and cell motility was measured by a time-lapse computer-aided method. Cell morphology was determined by flow and image cytometry methods. Real-time RT-PCR analysis was employed to investigate the expression of markers of differentiation. We showed that MPA induces irreversible inhibition of cell proliferation, causes cell enlargement and impairs cell locomotion in a time-dependent manner. The level of expression of differentiation markers was significantly reduced by MPA treatment. All these effects were reversed by the addition of guanine. Our results indicated that MPA impairs basic functions of human skin keratinocytes via intracellular guanosine nucleotide depletion, which may be directly reflected in wound healing problems in patients treated with this immunosuppressant.

Mycophenolic acid inhibits albumin-induced MCP-1 expression in renal tubular epithelial cells through the p38 MAPK pathway

Proteinuria is a well-established exacerbating factor in chronic kidney disease. Although the mechanisms of albumin-induced tubulointerstitial damage have been extensively studied, the influence of mycophenolic acid (MPA) on tubular epithelial cells has not been sufficiently elucidated. MPA, the active metabolite of mycophenolate mofetil, is a potent, non-competitive, and reversible inhibitor of inosine-5'-monophosphate dehydrogenase, the rate-limiting enzyme for de novo purine synthesis. Monocyte chemoattractant protein 1 (MCP-1) is a 76-amino-acid chemokine thought to be the major chemotactic factor for monocytes. MCP-1 is found in macrophage-rich areas of atherosclerotic lesions. However, the mechanisms regulating MCP-1 expression by MPA in renal tubular epithelial cells were still unclear. In this study, the inhibitory effect of MPA on MCP-1 expression by albumin-induced renal tubular epithelial cells was investigated, and the roles of p38 mitogen-activated protein kinase (p38 MAPK) pathway were explored. MPA attenuated albumin-induced expression of MCP-1 mRNA and protein. The experiment suggested that MPA actively inhibited protein of MCP-1. The inhibitory effect of MPA on MCP-1 expression was mediated by the sequential attenuation of p38 MAPK expression. These inhibitory effects were partially inhibited by SB203580, a specific inhibitor of p38 MAPK. Taken together, these results suggest that the negative modulation of MCP-1 by MPA is partly dependent on p38 MAPK pathway.

IMP dehydrogenase basal activity in MOLT‐4 human leukaemia cells is altered by mycophenolic acid and 6‐thioguanosine

Objective. Depletion of guanine and deoxyguanine nucleotides by inhibition of inosine 5'‐monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) or introduction of 6‐thioguanine nucleotide antimetabolites are two principles of retarding cell proliferation by interference with the cellular purine nucleotide pool. IMPDH activity may be a promising pharmacodynamic biomarker during immunosuppressive and anticancer pharmacotherapy. The aim of the study was to investigate the impact of mycophenolic acid (MPA) and 6‐thioguanosine (tGuO) on IMPDH basal activity. Material and methods. We studied the IMPDH basal activity (i.e. the enzyme activity following inhibitor exposure, but measured in absence of the inhibitor) in response to increasing concentrations of the IMPDH inhibitor MPA and the antimetabolite tGuO in MOLT‐4 human leukaemia cells. In parallel, IMPDH gene expression and cellular purine nucleotide concentrations were examined. Results. A biphasic concentration‐dependent influence of MPA on the IMPDH basal activity was observed. At concentrations⩽IC50, MPA increased the IMPDH basal activity. The increase was associated with elevated expression of IMPDH2. Despite increased expression, the basal enzyme activity decreased following exposure to high MPA concentrations. The IMPDH2 expression increased modestly in response to tGuO exposure. However, the IMPDH basal activity decreased when the cells were exposed to a proliferation‐blocking tGuO concentration. Conclusions. These findings demonstrate that IMPDH basal activity is influenced by MPA and tGuO, and suggest that reduced IMPDH basal activity is related to the proliferation‐blocking effects of these agents.

Influence of mycophenolic acid and tacrolimus on homocysteine metabolism

The effect of mycophenolate mofetil (MMF) on homocysteine (Hcy) metabolism is unknown. This in vitro study examined whether mycophenolic acid or tacrolimus influences the formation of Hcy as determined by measuring the total Hcy (tHcy) concentrations in supernatants of human renal proximal tubule epithelial cells. Cells were incubated with and without vitamins (folate, vitamin B6 and B12) in the presence of low or high methionine concentrations at different mycophenolic acid (0, or 5, or 20 microg/mL) or tacrolimus (0, or 10, or 25 ng/mL) concentrations for 24, 48 or 72 hours. The concentration of tHcy in culture supernatants was measured by a fluorescence polarization immunoassay. The effect of MMF on tHcy plasma levels was also examined in 454 kidney graft recipients. Comparisons of tHcy levels in culture supernatants over time by four way ANOVA showed that methionine concentration (P < 0.00001), time (P < 0.00001), vitamins (P = 0.002728), and mycophenolic acid concentration (P = 0.000095) were all significant predictors of tHcy concentrations. This was due to significantly lower tHcy levels with using mycophenolic acid at a high concentration versus control at the 48- and 72-hour time points. By contrast, tacrolimus showed no effect in vitro. Among the kidney graft recipients, male patients on MMF therapy showed lower plasma tHcy concentrations as compared to those on azathioprine (P = 0.03). Our study suggests a tHcy lowering effect of MMF in male transplant recipients, which improves the cardiovascular disease risk profile, whereas tacrolimus showed no effect.

Short-term treatment with mycophenolic acid increases bile flow in continuously perfused and cold-preserved rat livers and does not affect hepatic ischemia-reperfusion injury.

Mycophenolate mofetil (MMF) is a new immunosuppressive agent which has been used successfully after kidney and heart transplantation. Experience with MMF after liver transplantation is still limited. In particular, there is no information about influence on ischemia-reperfusion injury (IRI). Therefore, the aim of this investigation was to assess the effects of mycophenolic acid (MPA), the pharmacologically active metabolite of MMF, in the cold-preserved or normal rat liver. Livers of male Sprague-Dawley rats were subjected to cold ischemia in University of Wisconsin (UW) solution (24 h, 4 degrees C) and reperfused for 2 h in the absence or presence of MPA (100 microg/ml, n=5-6 each). Another group received MPA pretreatment for 20 min prior to ischemia ( n=7). In further experiments, livers were perfused with a bile salt-free Krebs-Henseleit buffer in a continuous fashion (controls, n=5). MPA was infused from 20-40 min after starting perfusion in therapeutic concentrations (5 microg/ml, 10 microg/ml, 40 microg/ml, and 100 microg/ml; n=3-6 each). There was no significant influence of MPA on portal pressure nor on postischemic efflux rates of LDH. MPA pretreatment resulted in a significant improvement of bile flow during reperfusion (0.32+/-0.05 microl/min x g liver) compared with controls (0.17+/-0.04 microl/min x g liver, mean+/-SEM). In contrast, postischemic bile flow was not influenced by continuous administration of MPA during the reperfusion period only (0.18+/-0.07 microl/min x g liver). In continuously perfused livers, MPA increased bile salt-independent bile flow (1.00+/-0.06 microl/min x g liver) in a dose-dependent manner, reaching half-maximal effects around 5 microg/ml (1.66+/-0.15 microl/min x g liver) and maximal effects at 40 microg/ml (2.61+/-0.28 microl/min x g liver). In conclusion, neither preischemic nor postischemic administration of MPA influences IRI to hepatocytes significantly after hypothermic liver preservation in UW solution. In contrast to other immunosuppressive agents, MPA exhibits strong choleretic effects, which are related to a stimulation of bile salt-independent bile formation.

Influence of mycophenolic acid on inosine 5′-monophosphate dehydrogenase activity in human peripheral blood mononuclear cells

Background: Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyses the oxidation of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP). Thus, this enzyme plays an important role in the rate-limiting step of the de novo guanine nucleotide biosynthesis, making it a potent target for immunosuppressive drugs. Mycophenolic acid (MPA) is the most potent and specific inhibitor of IMPDH. Method: IMPDH activity is determined via evaluation of XMP formation and the inhibitory influence of MPA in human peripheral blood mononuclear cells (PBMCs) is assessed by means of high-performance liquid chromatography (HPLC). For this objective, we have optimised a method based on solvent-generated ion exchange chromatography by cautiously varying mobile phase parameters. Results: The optimised method renders it possible to separate 18 analytes in 54 min in a single isocratic experiment and to measure the IMPDH activity in the lysate of human PBMCs in dependence on incubation time, substrate, co-substrate and inhibitor concentrations. In this way, we have determined the Michaelis–Menten constants K M and V max for IMP and β-NAD + and the inhibitor constant K i for MPA. Conclusions: The chromatographic method presented in this report allows a rapid, reliable and reproducible quantification of IMPDH activity in PBMCs and therefore represents an attractive tool for the pharmacodynamic monitoring of the effects of MPA in patients under immunosuppressive therapy.

Influence of Mycophenolic Acid and FK–506 on Human Platelet Activation in vitro

Background/Aim: FK–506 (FK) and mycophenolic acid (MPA) are immunosuppressive agents used in kidney transplant recipients. Their effect on posttransplant thromboembolic complications is either controversial (FK) or has not been described (MPA). Thromboembolic events are among the consequences of platelet hyperaggregability which can be identified by measuring platelet aggregation. The aim of this study was to evaluate the in vitro effects of MPA and FK upon platelet activation in healthy subjects. Methods: Platelet–rich plasma from healthy volunteers (n = 18) was incubated with FK (70 ng/ml), FK vehicle, and MPA (30 μg/ml) before platelet aggregation was induced by the platelet agonists adenosine diphosphate (2 and 5 μM) and collagen 0.5 and 1.0 μg/ml). Aggregation was measured by recording the optical density. Results: MPA resulted in a significant decrease in the platelet response to collagen (1.0 μg/ml) in platelet–rich plasma, whereas FK significantly increased platelet aggregation in response to collagen (0.5 μg/ml). The vehicle of FK had no influence on platelet aggregation with either agonist. Conclusions: The decreased platelet–activating response following preincubation with MPA may favor its use in kidney transplant recipients to reduce thromboembolic events. The FK–induced enhancement of platelet aggregation shown in vitro may lead to thromboembolic complications in transplant recipients.