Determination of the effects of mycophenolic acid on the nucleotide pool of human peripheral blood mononuclear cells in vitro by high-performance liquid chromatography

Abstract

Immunosuppressive drugs are needed to prevent the rejection of transplanted organs by the immune system. Immunosuppressive antimetabolites act by interrupting cell metabolism. Their mechanism of action can be studied in vitro by measuring the inhibition of biochemical activities which is reflected by changes in the nucleotide content. In our experiments, human peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers were used. After PBMC stimulation with phytohaemagglutinin (PHA) to mimic activation occurring at a rejection crisis, cells were exposed to varying concentrations of different immunosuppressants (i.e., mycophenolic acid, cyclosporin A and prednisolone) for 68 h at 37°C. Changes in nucleotide content were observed by determining the concentrations of 15 nucleotides using a newly developed HPLC method. The results obtained for mycophenolic acid (MPA; final concentrations in a range between 0.1 and 5 μmol/l), cyclosporin A (CsA; final concentrations between 100 ng/ml and 1 μg/ml) and prednisolone (final concentrations between 0.5 and 10 μmol/l) are given as percentage changes in nucleotide content versus controls and are expressed as mean±confidence interval. The possibility of synergistic effects was investigated by incubating the cells with mixtures of all three immunosuppressive drugs varying the amount of mycophenolic acid. In addition, we have shown the effects of MPA/guanosine co-incubation on the intracellular nucleotide levels. Stimulation of peripheral blood mononuclear cells with phytohaemagglutinin led to a significant increase of pyrimidine and purine nucleotides versus control values (100%). Pyrimidine (CTP, UDP, UTP) and purine nucleotides (GDP, GTP, ADP, ATP) were elevated up to 153±14% and 142±17%, respectively. Under co-incubation of cells with MPA, the GTP level decreased in a dose-related manner to 56±3% of control at a MPA final concentration of 5 μmol/l. Concomitantly, an increase of UTP values to 203±18% versus control was observed under co-incubation with 1 μmol/l MPA. Co-incubation of mononuclear cells with guanosine (50 μmol/l) compensated for the effects of MPA on intracellular GTP levels. Combination of MPA, CsA and prednisolone did not alter intracellular nucleotide profiles of PBMC compared to those under MPA incubation alone. The depletion of the guanine nucleotide pool and concomitant increase of uridine nucleotides under the influence of the immunosuppressive drug mycophenolic acid is caused by its inhibitory effects on the key enzyme of de novo purine biosynthesis, inosine 5′-monophosphate dehydrogenase (IMPDH).