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

Immunosuppressive drugs are needed to prevent the rejection of transplanted organs by the immune system. Immunosuppressive antimetabolites act by inte rrupting cell metabolism. Their mechanism of action can be studied in vitro by measuring the inhibition of biochemical activities which is reflected b y changes in the nucleotide content. In our experiments, human peripheral b lood mononuclear cells (PBMC) isolated from healthy volunteers were used. A fter PBMC stimulation with phytohaemagglutinin (PHA) to mimic activation oc curring at a rejection crisis, cells were exposed to varying concentrations of different immunosuppressants (i.e., mycophenolic acid, cyclosporin A an d prednisolone) for 68 h at 37 degreesC. Changes in nucleotide content were observed by determining the concentrations of 15 nucleotides using a newly developed HPLC method. T The results obtained for mycophenolic acid (MPA; final concentrations in a range between 0.1 and 5 mu mol/l), cyclosporin A (CsA; final concentrations between 100 ng/ml and 1 mug/ml) and prednisolone (final concentrations bet ween 0.5 and 10 mu mol/l) are given as percentage changes in nucleotide con tent versus controls and are expressed as mean confidence interval. The pos sibility of synergistic effects was investigated by incubating the cells wi th mixtures of all three immunosuppressive drugs varying the amount of myco phenolic acid. In addition, we have shown the effects of MPA/guanosine co-i ncubation on the intracellular nucleotide levels. Stimulation of peripheral blood mononuclear cells with phytohaemagglutinin led to a significant increase of pyrimidine and purine nucleotides versus c ontrol values (100%). Pyrimidine (CTP, U-DP, UTP) and purine nucleotides (G DP, GTP, ADP, ATP) were elevated up to 153 +/- and 142 +/- 17%, respectivel y. Under co-incubation of cells with MPA, the GTP level decreased in a dose -related manner to 56 +/-3% of control at a NIPA final concentration of 5 m u mol/l. Concomitantly, an increase of UTP values to 203 +/- 18% versus con trol was observed under co-incubation with 1 mu mol/l MPA. Co-incubation of mononuclear cells with guanosine (50 mu mol/l) compensated for the effects of MPA on intracellular GTP levels. Combination of NIPA, CsA and prednisol one did not alter intracellular nucleotide profiles of PBMC compared to tho se under M-PA incubation alone. The depletion of the guanine nucleotide poo l and concomitant increase of uridine nucleotides under the influence of th e immunosuppressive drug mycophenolic acid is caused by its inhibitory effe cts on the key enzyme of de novo purine biosynthesis, inosine 5'-monophosph ate dehydrogenase (IMPDH). (C) 2001 Elsevier Science B.V. All rights reserv ed.