IMPDH Activity Research Articles

Pharmacokinetics and pharmacodynamics of mycophenolic acid in stable renal transplant recipients treated with low doses of mycophenolate mofetil.

Suboptimal doses of mycophenolate mofetil (MMF) are frequently employed in renal transplant (Tx) patients, with drug-related side effects or low weight. The aim of this study was to compare the mycophenolic acid (MPA) pharmacokinetic profile and its pharmacodynamic effect on patients receiving either standard (2 g) or low (1.5 g or 1 g) MMF doses, in order to evaluate the therapeutic efficacy of such low doses in inhibiting IMPDH activity. Twenty-seven stable renal Tx recipients aged 18-65 years, with a post-Tx follow-up of 38.5 +/- 44.8 months (6-166 months), receiving 1 g (n = 10), 0.75 g (n = 7) and 0.5 g (n = 10) MMF twice a day in association with cyclosporine and prednisone, were included. The control group was made up of untreated healthy volunteers (n = 5). Plasma concentrations of MPA were analyzed by reverse-phase HPLC. IMPDH activity was determined in lymphocytes by the measurement of 3H release from [2,8-(3)H] hypoxantine. The mean value of areas under the concentration-time curves (AUC(0-12)) of MPA throughout the 12-h dosing interval in patients treated with 2 g was higher than the corresponding data in patients receiving 1.5 g or 1 g bid, but no statistical differences were observed between the three groups. There was no correlation between MPA-AUC(0-12) values and MMF dose (expressed in g/day or g/kg per day). Predose MPA concentrations correlated only weakly with the respective MPA-AUC(0-12) values (r2 from 0.385 to 0.655), whereas an acceptable correlation was observed between MPA Cmax and MPA-AUC(0-12) (r2 from 0.626 to 0.759) in 2 g, 1.5 g, and 1 g MMF groups. An inverse relationship between MPA concentrations and IMPDH activity was observed. In general, the maximum MPA concentration was achieved from 1 h to 2 h after dosing, and the maximum inhibition of IMPDH was also from 1 h to 2 h after dosing. The evaluation of IMPDH activity demonstrated that there was a significant statistical difference between samples from 0 to 1 h (P = 0.008) and 0 to 2 h (P = 0.04). In conclusion, concentration-time profiles of renal transplant recipients administered 0.75 g and 0.5 g twice a day are slightly lower than those from the 2 g group, but nor significantly. On the other hand, inhibition of IMPDH activity was comparable in the three groups, indicating considerable interindividual pharmacodynamic variability. Pharmacodynamic monitoring of the degree of immunosuppression and its correlation with MPA plasma concentrations will be assessed further in future studies.

PHARMACOKINETICS (PK) AND PHARMACODYNAMICS (PD) OF MYCOPHENOLIC ACID (MPA) IN STABLE RENAL TRANSPLANT RECIPIENTS TREATED WITH SUBOPTIMAL DOSES OF MYCOPHENOLATE MOFETIL (MMF)

794 Suboptimal doses of MMF are frequently employed in renat transplant (Tx) patients with drug-related side effects or low weight. The aim of this study was to compare the MPA PK profile and its PD effect in patients receiving either standard (2g) or low (1.5 or 1g) MMF doses, in order to assess whether low doses of MMF offers therapeutic plasma concentrations of MPA and adequate inhibition of IMPDH activity. Patients and Methods: 23 Tx recipients aged 13 to 55, with a post-Tx follow-up of 38.5±44.8 months (6-166 months) receiving 1g (n=9); 0.75g (n=5) and 0.5g (n=9) of MMF twice a day, in association with cyclosporine and prednisone were included. For MPA exposure, AUC was calculated using pre-dose, 20, 40, 75 minutes and 2, 4, 6, 8 and 12 hours plasma samples. MPA plasma levels were analyzed by HPLC. IMPDH activity was determined at zero, 1, 2 and 4 hours, by the measurement of 3H release from [2,8-3H] hypoxanthine or [2,8-3H] inosine. Results are shown in Tables I and II.Table I: MPA Pharmacokinetics Table II: MPA Pharmacodynamics This results show a high PK and PD interindividual variability, without statistical differences between the three groups. Group 1g showed a more spiked IMPDH inhibition profile, whereas the profile was more flat in groups 2g and 1.5g. In conclusion, therapeutic drug monitoring of MPA could be useful in managing these patients. This research was funded in part by Productos Roche, S.A. (Spain).

IMP-dehydrogenase inhibition in human lymphocytes and lymphoblasts by mycophenolic acid and mycophenolic acid glucuronide

Inosine 5'-monophosphate dehydrogenase (IMP-DH) activities were measured in human lymphocytes (exhibiting type I IMP-DH activity) and human lymphoblasts (exhibiting type II IMP-DH activity) in the presence of various amounts of mycophenolic acid (MPA) (0-20 mumol/L) and MPA glucuronide (MPAG) (0-200 mumol/L). Moreover, the influences of human serum albumin (HSA) and human plasma on the MPA- and MPAG-mediated effects were investigated. In the presence of water, 2.5 mumol/L MPA decreased the IMP-DH activity measured in lymphocytes by 60%, whereas in lymphoblasts a 80% inhibition was detectable. In the presence of > or = 10 mumol/L MPA, lymphocytic as well as lymphoblastic IMP-DH activities were reduced in a similar manner. The concentration of MPAG required for 50% inhibition was for both cell types > 25 mumol/L and < 50 mumol/L, respectively. MPAG (200 mumol/L) reduced lymphocytic as well as lymphoblastic IMP-DH activity by approximately 80%. With 100 g/L HSA or human plasma as diluent, the inhibitory effects of MPA and MPAG were significantly (P < 0.05) diminished, whereas HSA concentrations < or = 25 g/L only slightly influenced the inhibition of IMP-DH activity by MPA and MPAG. In summary, it can be clearly demonstrated that not only MPA but also MPAG contributes to the inhibition of both IMP-DH isoenzymes, which might be relevant for the immunosuppressive properties of mycophenolate mofetil in transplant patients.

IMP dehydrogenase from Pneumocystis carinii as a potential drug target.

Mycophenolic acid, a specific inhibitor of IMP dehydrogenase (IMPDH; EC 1.1.1.205), is a potent inhibitor of Pneumocystis carinii growth in culture, suggesting that IMPDH may be a sensitive target for chemotherapy in this organism. The IMPDH gene was cloned as a first step to characterizing the enzyme and developing selective inhibitors. A 1.3-kb fragment containing a portion of the P. carinii IMPDH gene was amplified by PCR with two degenerate oligonucleotides based on conserved sequences in IMPDH from humans and four different microorganisms. Northern hybridization analysis showed the P. carinii IMPDH mRNA to be approximately 1.6 kb. The entire cDNA encoding P. carinii IMPDH was isolated and cloned. The deduced amino acid sequence of P. carinii IMPDH shared homology with bacterial (31 to 38%), protozoal (48 to 59%), mammalian (60 to 62%), and fungal (62%) IMPDH enzymes. The IMPDH cDNA was expressed by using a T7 expression system in an IMPDH-deficient strain of Escherichia coli (strain S phi 1101). E. coli S phi 1101 cells containing the P. carinii IMPDH gene were able to grow on medium lacking guanine, implying that the protein expressed in vivo was functional. Extracts of these E. coli cells contained IMPDH activity that had an apparent Km for IMP of 21.7 +/- 0.3 microM and an apparent Km for NAD of 314 +/- 84 microM (mean +/- standard error of the mean; n = 3), and the activity was inhibited by mycophenolic acid (50% inhibitory concentration, 24 microM; n = 2).

Regulation of the signal transduction program by drugs

The purpose of this paper was to clarify critical aspects of the behavior of signal transduction activity in normal and cancer cells. 1. 1. Signal transduction activity in the conversion of phosphatidylinositol through PI and PIP kinases and PLC to IP 3 is regulated at multiple sites. In liver, hepatomas and human carcinomas PIP kinase is the rate limiting enzyme and PLC activity is present in great excess. 2. 2. The steady-state signal transduction activity as measured by the three enzyme activities and IP 3 concentration was markedly up-regulated in rat hepatomas of different growth rates. The steady-state specific activities of the three signal transduction enzymes were elevated in ovarian carcinomas as compared to normal ovary. Increased enzyme activities were also observed in human breast carcinoma cells as compared to normal human breast parenchymal cells. In breast, ovarian and rat hepatoma cells as they go through lag, log and plateau phases, IP 3 concentration in the early lag phase increased 4.5- to 20-fold and PI and PIP kinase activities peaked in mid-log phase. These events returned to baseline levels in the plateau phase. PLC activity did not change. 3. 3. The bone marrow PI and PIP kinase activities in 3-day starvation were decreased to 13% and IP 3 concentration was reduced to 24%; at 1-day refeeding they returned to normal. PLC activity changed little. These alterations are in line with the rapid t 1 2 degradation rates (12 min) of PI and PIP kinases observed in studies with cycloheximide. By contrast, PLC has a long half-life. 4. 4. The molecular action of tiazofurin entails inhibition of IMP DH activity, decrease in GTP and IP 3 concentrations, reduction of ras and myc oncogene expression, and signal transduction enzyme activities. These events are followed by induced differentiation and apoptosis. There are also decreases in enzyme activities which have rapid turnover, including TdR kinase, dTMP synthase, and GPRT. In vitro studies indicated that these events are abrogated by addition of guanine which restores GTP concentration. Therefore, most or all these events were brought about by the reduced GTP concentration in the tiazofurin target cells. 5. 5. Quercetin and genistein are able to inhibit PI and PIP kinase activities and reduce IP 3 concentration in vivo and in tissue culture systems. These flavonoids are also inhibitors of cell proliferation and clonogenic ability in rat hepatoma 3924A and in human OVCAR-5 and MDA-MB-435 cells. Quercetin down-regulated the expression of c- myc and Ki- ras oncogenes and led to induced differentiation and apoptosis in K562 cells. Genistein reduced IP 3 concentration in vivo and in the tissue culture system. Genistein is anti-proliferative and has cytototoxicity in human carcinoma cells. All three drugs, tiazofurin, quercetin and genistein, act, in part at least, through depression of cellular IP 3 concentration although the mechanisms may not be identical. 6. 6. Quercetin and genistein, which attack different targets and different phases of the cell cycle, proved to be synergistic in OVCAR-5 cells. The impact of tiazofurin, genistein and quercetin is of interest because the drugs crucially inhibit the display of the neoplastic program of cells and lead to induced differentiation and apoptosis.

Demonstration of induction of erythrocyte inosine monophosphate dehydrogenase activity in Ribavirin-treated patients using a high performance liquid chromatography linked method

The activity of inosine monophosphate dehydrogenase (IMPDH: EC 1.2.1.14) was measured in erythrocyte lysates using a non-radiolabelled method linked to reversed-phase liquid chromatography (RPLC). The mean activity in erythrocytes from healthy controls using this sensitive method was extremely low (mean 85 pmol/h per mg protein, range 4–183). The elevated erythrocyte IMPDH activity reported previously in hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency was confirmed (mean 234 pmol/h per mg protein). Erythrocyte IMPDH activity of patients with other disorders of purine metabolism, or with leukaemias and lymphomas, showed no marked difference from controls, except in one instance — an immunodeficient child with purine nucleoside phosphorylase (PNP) deficiency, treated with Ribavirin, where a 30-fold increase in activity was found (2670 pmol/h per mg protein). Investigation of erythrocyte IMPDH in other immunodeficient children with normal PNP activity demonstrated that this grossly elevated erythrocyte activity was attributable to induction of IMPDH by Ribavirin therapy.

Effects of human T lymphocyte activation on inosine monophosphate dehydrogenase expression.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the first step in the formation of guanine ribonucleotides from inosine monophosphate and the activity of this enzyme appears to be essential for cell proliferation. Inhibitors of IMPDH have been demonstrated to be effective immunosuppressive agents and to inhibit T cell activation in vitro. IMPDH activity results from the expression of two different genes (types I and II) that encode protein subunits of identical size with 84% amino acid identity. To determine the relative contribution of the expression of these two genes to T cell activation, we have examined the effects of T cell stimulation on IMPDH activity, mRNA levels, and protein. The stimulation of isolated peripheral blood CD28+ T cells with PMA and ionomycin causes a 15-fold increase in IMPDH activity over a 72-h period. This is associated with a 10-fold increase in type II mRNA levels at 48 h. Type I mRNA is expressed at very low levels in resting T cells, but increases 10-fold by 24 h after stimulation. The type I cDNA probe also detects a second larger mRNA species of 4.0 kb that is not detectable in a variety of normal tissues or in a panel of leukemic cell lines. RNase protection assays using RNA probes corresponding to the entire coding region of the type I enzyme reveal a single protected fragment, demonstrating that the 4.0-kb message is the result of alternate splicing in the 5' or 3' untranslated regions or the use of an alternative polyadenylation site. Western blot analysis demonstrates a concomitant increase in total IMPDH protein on T cell activation, although posttranslational modifications do not allow the distinction between type I and type II on isoelectric focusing gels. We conclude that the induction of both type I and type II IMPDH contribute significantly to the T cell proliferative response. Both enzymes therefore should be considered important targets for immunosuppressive therapy.

Inhibition by tiazofurin of inosine 5′-phosphate dehydrogenase (IMP DH) activity in extracts of ovarian carcinomas

Cancer cells have an increased ability to synthesize GTP (guanosine triphosphate) because of increased activity of IMP DH (inosine 5′-phosphate dehydrogenase, EC 1.1.1.205). Because IMP DH activity is rate limiting for de novo biosynthesis of GTP, this enzyme was suggested as a sensitive target for chemotherapy. Tiazofurin (2-β- d-ribofuranosylthiazole-4-carboxamide) is converted in the cells into the active metabolite, TAD, (thiazole-4-carboxamide adenine dinucleotide) which potently inhibits IMP DH activity. By adding TAD to tissue extracts one can determine the extent of inhibition of IMP DH. We applied the IMP DH assay method to extracts of normal ovaries ( N = 11) and epithelial ovarian carcinomas ( N = 10). The IMP DH activity (mean ± SE) in ovarian carcinoma was 21.1 ± 5.8 which was markedly higher than that observed in normal ovaries (2.9 ± 0.7 nmol/hr/mg protein) ( P < 0.05%). The inhibition by TAD of IMP DH activity in ovarian carcinomas ( N = 4) was 81%. the results indicate that IMP DH activity is elevated sevenfold in ovarian carcinomas as compared to normal ovary and can be inhibited by exposure to tiazofurin (TAD). Similar high IMP DH activity and inhibition of the activity by TAD were observed in patients with chronic granulocytic leukemia in blast crisis among whom 70 to 80% remissions were reported. Since there is increased IMP DH activity in human ovarian carcinomas and in OVCAR-5 cells and tiazofurin and TAD inhibit IMP DH activity of these cells and the proliferation of human ovarian carcinoma xenografts in the mouse, tiazofurin may merit serious consideration for a Phase II trial for patients with recurrent/refractory epithelial ovarian carcinoma.

Tiazofurin Down-Regulates Expression of c-Ki-ras Oncogene in a Leukemic Patient

The increased activity in cancer cells of inosine 5'-monophosphate dehydrogenase (IMP DH, EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, was suggested as a sensitive target for chemotherapy. Tiazofurin (NSC 286193), through its conversion to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), is a strong inhibitor of IMP DH. In our clinical trial, tiazofurin caused return to the chronic phase in patients with chronic granulocytic leukemia in blast crisis (Tricot, G.; Jayaram, H.N.; Weber, G.; Hoffman, R. Tiazofurin: Biological effects and clinical uses. Int. J. Cell Cloning 8:161-170; 1990). In K562 human leukemic cells, tiazofurin down-regulated the expression of c-Ki-ras and c-myc oncogenes, which was followed by induced differentiation. We now report down-regulation by tiazofurin of the c-Ki-ras oncogene in a patient with chronic granulocytic leukemia in blast crisis. A single tiazofurin infusion (2,200 mg/m2) on days one and two decreased IMP dehydrogenase activity (the apparent t1/2 was 30 min), GTP concentration (the apparent t1/2 was 6 hr), and expression of ras (the apparent t1/2 was 8 hr) and c-myc (the apparent t1/2 was 38.5 hr) oncogenes in the leukemic cells. No further tiazofurin was given, because on days three and four the chemotherapeutic impact became evident in a tumor-lysis syndrome and the blast cells were cleared from the periphery by day five. The decrease in IMP DH activity, GTP concentration, and expression of c-Ki-ras oncogene were early markers of the successful chemotherapeutic impact of tiazofurin in a patient with chronic granulocytic leukemia in blast crisis.

Cell differentiation and altered IMP dehydrogenase expression induced in human T-lymphoblastoid leukemia cells by mycophenolic acid and tiazofurin

The IMP dehydrogenase inhibitors mycophenolic acid (MPA) and tiazofurin (TZ) induce a time- and dose-dependent inhibition of cell growth, as well as differentiation in T-lymphoid CEM-2 leukemia cells. The differentiated cells have acquired a suppressor/cytotoxic T-lymphocyte phenotype characterized by reactivity with maturation-specific monoclonal antibodies. Coadministration of guanosine and hypoxanthine reduces the growth inhibition and diminishes the induction of differentiation by either MPA or TZ. No such reduction was observed for differentiation induced by phorbol 12-myristate 13-acetate (PMA), another inducer of a suppressor/cytotoxic phenotype in CEM-2 cells. During the first 2 days of treatment with MPA or TZ, a pattern of stable IMPDH mRNA levels and increased amounts of cellular enzyme was observed, perhaps, because of compensation for the inhibitor-mediated decrease in cellular IMPDH activity or a MPA- or TZ-mediated decrease in proteolysis of IMPDH. PMA treatment decreased the levels of IMPDH mRNA, protein, and activity. In addition, treatment of CEM-2 cells with either IMPDH inhibitors or PMA caused different alterations of the ribonucleotide pools. The lack of a consistent pattern of IMPDH expression in CEM-2 cells treated with IMPDH inhibitors or PMA indicates that no general association exists between the induction of cell differentiation and the expression of IMPDH. Nevertheless, our results indicating that IMPDH inhibitors can induce differentiation in CEM-2 cells suggest that this treatment may provide a useful approach to circumvent the differentiation block in some tumor cells.

Tiazofurin: Biological effects and clinical uses

Inosine 5'-phosphate dehydrogenase (IMPDH) activity is increased in all cancer cells. It is the rate-limiting enzyme of guanosine triphosphate (GTP) biosynthesis, and therefore, a sensitive target of chemotherapy. Tiazofurin selectively blocks IMPDH activity. Tiazofurin was found to have an antiproliferative effect on tumor cells in vitro and in the murine system. Based on these findings, Phase I trials were started elsewhere in patients with solid tumors, but were discontinued because of toxicity. In leukemic patients, we were able to demonstrate a good correlation between biochemical parameters (i.e., decline in IMPDH activity and GTP concentrations in blast cells) and clinical response. The most consistent responses to therapy were seen in patients with myeloid blast crisis of chronic myeloid leukemia. Severe toxicity was seen in the earlier patients in the study. However, better patient selection, limitation of treatment duration and earlier recognition and treatment of complications have now made it possible to administer tiazofurin without undue toxicity.