IMPDH Activity Research Articles

Pharmacokinetics and pharmacodynamics profiles of enteric-coated mycophenolate sodium in female patients with difficult-to-treat lupus nephritis.

Relapsed or resistant lupus nephritis (LN) is considered a difficult‐to‐treat type of LN, and enteric‐coated mycophenolate sodium (EC‐MPS) has been used in this condition. Therapeutic drug monitoring using the area under the plasma mycophenolic acid concentration from 0 to 12 h postdose (MPA‐AUC0–12h) ≥45 μg.h/ml is a useful approach to achieve the highest efficiency. This study assessed EC‐MPS’s pharmacokinetic (PK) and pharmacodynamic (PD) profiles and investigated an optimal level of the single time point of plasma MPA concentration. Nineteen biopsy‐proven patients with class III/IV LN received 1440 mg/day of EC‐MPS for 24 weeks. PK (maximum plasma MPA concentration [C max], time to C max, and MPA‐AUC0–12h) and PD (activity of inosine‐5′‐monophosphate dehydrogenase [IMPDH]) parameters were measured at weeks 2, 8, 16, and 24. We found that IMPDH activity decreased from baseline by 31–42% within 2–4 h after dosing, coinciding with the increased plasma MPA concentration. MPA‐AUC0–12h ≥45 μg.h/ml was best predicted by a single time point MPA concentration at C0.5, C2, C3, C4, and C8 (r 2 = 0.516, 0.514, 0.540, 0.611, and 0.719, respectively), independent of dose, albumin, urine protein/creatinine ratio, and urinalysis. The MPA‐C0.5 cutoff of 2.03 g/ml yielded the highest overall sensitivity of 85% and specificity of 88.2% in predicting MPA‐AUC0–12h ≥45 μg.h/ml. A single timepoint of plasma MPA‐C0.5 ≥2.03 μg/ml may help guide EC‐MPS adjustment to achieve adequate drug exposure. Further study of EC‐MPS used to validate this cutoff is warranted.

Synthesis and Biological Activity of Some [(5-oxazolyl)-phenyl]-thiourea Derivatives

Aims:Synthesis and biological evaluation of some [(5-oxazolyl)-phenyl]-thiourea derivatives as potential antiviral agents.Background:(5-Oxazolyl)-phenyl derivatives were derived from the design of mycophenolic acid structurally related analogues. The (5-oxazolyl)-phenyl fragment is an excellent composition for many novel structure compounds having good pharmaceutical properties, such as immunosuppressive, antiviral and anticancer. In the present study, we present combinations of thiourea group and (5-oxazolyl)-phenyl fragment. The antiviral activity, cytotoxicity and IMPDH activity of the title compounds were evaluated in vitro bioassay.Objective:[(5-Oxazolyl)-phenyl]-thiourea derivatives containing different substituted benzene rings were synthesized by introducing thiourea linker. All the synthesized derivatives were screened for their in vitro antiviral evaluation and inosine monophosphate dehydrogenase activity.Methods:A series of [(5-oxazolyl)-phenyl]-thiourea derivatives were synthesized by the reaction of thiocarbonyldiimidazole with amines. This was an effective method for introducing the thiourea group in the (5-oxazolyl)-phenyl structure. All of the synthesized derivatives were screened for their in vitro antiviral activity against influenza A virus, coxsackievirus B3, herpes simplex virus type 1 and inosine monophosphate dehydrogenase activity.Result:The results of the screening revealed that compounds 4i, 4j, 4k, 7m, 7n and 7o showed comparable activity towards IMPDH as compared to the control drug. Compounds 4k, 4l, 7m and 7n exhibited potent activity towards both RNA virus influenza A virus, coxsackievirus B3 and DNA virus HSV-1 at low micromolar concentrations. The activities of most compounds directly linked to the substituted benzene ring by the thiourea group were superior to those of the compounds which had the methylene linkage.Conclusion:The in vitro biological assays indicated that most of target molecules having combinations of thiourea group and (5-oxazolyl)-phenyl fragment exhibited antiviral activity and IMPDH activity compared the control drugs.

Post-translational regulation of retinal IMPDH1 in vivo to adjust GTP synthesis to illumination conditions.

We report the in vivo regulation of Inosine-5´-monophosphate dehydrogenase 1 (IMPDH1) in the retina. IMPDH1 catalyzes the rate-limiting step in the de novo synthesis of guanine nucleotides, impacting the cellular pools of GMP, GDP and GTP. Guanine nucleotide homeostasis is central to photoreceptor cells, where cGMP is the signal transducing molecule in the light response. Mutations in IMPDH1 lead to inherited blindness. We unveil a light-dependent phosphorylation of retinal IMPDH1 at Thr159/Ser160 in the Bateman domain that desensitizes the enzyme to allosteric inhibition by GDP/GTP. When exposed to bright light, living mice increase the rate of GTP and ATP synthesis in their retinas; concomitant with IMPDH1 aggregate formation at the outer segment layer. Inhibiting IMPDH activity in living mice delays rod mass recovery. We unveil a novel mechanism of regulation of IMPDH1 in vivo, important for understanding GTP homeostasis in the retina and the pathogenesis of adRP10 IMPDH1 mutations.

IMP/GTP balance modulates cytoophidium assembly and IMPDH activity

BackgroundInosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo GTP biosynthesis, plays an important role in cell metabolism and proliferation. It has been demonstrated that IMPDH can aggregate into a macrostructure, termed the cytoophidium, in mammalian cells under a variety of conditions. However, the regulation and function of the cytoophidium are still elusive.ResultsIn this study, we report that spontaneous filamentation of IMPDH is correlated with rapid cell proliferation. Intracellular IMP accumulation promoted cytoophidium assembly, whereas elevated GTP level triggered disassociation of aggregates. By using IMPDH2 CBS domain mutant cell models, which are unable to form the cytoophidium, we have determined that the cytoophidium is of the utmost importance for maintaining the GTP pool and normal cell proliferation in the condition that higher IMPDH activity is required.ConclusionsTogether, our results suggest a novel mechanism whereby cytoophidium assembly upregulates IMPDH activity and mediates guanine nucleotide homeostasis.

The pharmacokinetics and pharmacodynamics of mycophenolate mofetil in younger and elderly renal transplant recipients.

Elderly transplant recipients have a lower incidence of acute rejection, and a higher risk to die from infectious complications. A potential cause may be differences in the pharmacokinetics (PK) or pharmacodynamics (PD) of the immunosuppressive drugs they are taking. This study was designed to comprehensively evaluate the influence of age on the PK and PD of mycophenolic acid (MPA). In this study the PK and PD of MPA was studied in 26 elderly and 54 younger renal transplant recipients treated with mycophenolate mofetil and tacrolimus. Patients were sampled repetitively, both before and during the first 6 months after kidney transplantation. Age-related variability in MPA PK, baseline IMPDH activity, as well as MPA-induced IMPDH inhibition were studied. The IMPDH activity pre-transplantation did not differ between elderly and younger patients. Neither IMPDH activity pre-transplantation nor maximum IMPDH inhibition was significantly correlated with the patients' age. The area under the MPA plasma concentration-time curve (AUC0-12h ) and the area under the effect (IMPDH activity)-time curve (AEC0-12h ) from 0 to 12h were also not significantly different between the two groups. We found no significant differences in EC50 and Emax between elderly and younger patients. Age did not significantly affect the PK or PD of MPA. It is unlikely that the lower incidence of acute rejection in elderly patients, or the higher risk to die from a severe infection in elderly patients is due to different handling of MPA in the elderly.

Mycophenolic Acid-Induced Developmental Defects in Zebrafish Embryos.

With the increasing use of mycophenolic acid (MPA) in solid organ transplantation, some clinical studies indicate that it is also a human teratogen. However, it is unknown by which mechanism MPA acts as a teratogen. Mycophenolic acid was a selective blocker of de novo purine synthesis, and its immunosuppressive effect is mediated by the inhibition of inosine monophosphate dehydrogenase, which could be a target for MPA-induced toxicity as well. The aim of our study was to examine the direct influence of MPA exposure on zebrafish (Danio rerio) embryos. Morphological defects including tail curvature and severe pericardial edema in zebrafish embryos caused by MPA (3.7-11.1 µmol/L) were found in a dose-dependent manner. The teratogenic index (25% lethal concentration value (LC25)/no observed adverse effect level ratio) was 16, which indicated MPA as a teratogen. Quantitative polymerase chain reaction analysis revealed that the expression level of impdh1b and impdh2 was significantly reduced by MPA treatment at 8 µmol/L (equals to LC25 level). All the toxic effects could be partially reversed by the addition of 33.3 µmol/L guanosine. Our results indicated that MPA impairs the development of zebrafish embryos via inhibition of impdh activity, which subsequently caused a guanosine nucleotide depletion in vivo.

Drug target molecules to guide immunosuppression

The individual and interindividual variability of response to immunosuppressants combined with the prevailing concept of lifelong immunosuppression following any organ transplantation motivates the search for methods to further individualize such therapy. Traditional therapeutic drug monitoring, adapting dose according to concentrations in blood, targets the pharmacokinetic variability. It has been increasingly recognized, however, that there is also a considerable variability in the response to a given concentration. Attempts to overcome this variability in response include the efforts to identify relevant targets and methods for pharmacodynamic monitoring. For several of the currently used immunosuppressants there is experimental data suggesting markers that are relevant as indicators for individual monitoring of the effects of these drugs. There are also some clinical data to support these approaches; however what is generally missing, are studies that in a prospective manner demonstrates the benefits and effects on outcome. The monitoring of antithymocyte globulin by lymphocyte subset counts is actually the only well established example of pharmacodynamic monitoring. For drugs such as MPA and mTOR inhibitors, there are candidates such as IMPDH activity expression and p70SK6 phosphorylation status, respectively. The monitoring of CNIs using assays for NFAT RGE, either alone or combined with concentration measurements, is already well documented. Even here, some further investigations relating to the categories of organ transplant, combination of immunosuppressants etc. will be requested. Although some further standardization of the assay is warranted and there is a need for specific recommendations of target levels and how to adjust dose, the NFAT RGE approach to pharmacodynamic monitoring of CNIs may be close to implementation in clinical routine.

Interferon and ribavirin combination treatment synergistically inhibit HCV internal ribosome entry site mediated translation at the level of polyribosome formation.

PurposeAlthough chronic hepatitis C virus (HCV) infection has been treated with the combination of interferon alpha (IFN-α) and ribavirin (RBV) for over a decade, the mechanism of antiviral synergy is not well understood. We aimed to determine the synergistic antiviral mechanisms of IFN-α and RBV combination treatment using HCV cell culture.MethodsThe antiviral efficacy of IFN-α, RBV alone and in combination was quantitatively measured using HCV infected and replicon cell culture. Direct antiviral activity of these two drugs at the level of HCV internal ribosome entry site (IRES) mediated translation in Huh-7 cell culture was investigated. The synergistic antiviral effect of IFN-α and RBV combination treatment was verified using both the CalcuSyn Software and MacSynergy Software.ResultsRBV combination with IFN-α efficiently inhibits HCV replication cell culture. Our results demonstrate that IFN-α, interferon lambda (IFN-λ) and RBV each inhibit the expression of HCV IRES-GFP and that they have a minimal effect on the expression of GFP in which the translation is not IRES dependent. The combination treatments of RBV along with IFN-α or IFN-λ were highly synergistic with combination indexes <1. We show that IFN-α treatment induce levels of PKR and eIF2α phosphorylation that prevented ribosome loading of the HCV IRES-GFP mRNA. Silencing of PKR expression in Huh-7 cells prevented the inhibitory effect of IFN-α on HCV IRES-GFP expression. RBV also blocked polyribosome loading of HCV-IRES mRNA through the inhibition of cellular IMPDH activity, and induced PKR and eIF2α phosphorylation. Knockdown of PKR or IMPDH prevented RBV induced HCV IRES-GFP translation.ConclusionsWe demonstrated both IFN-α and RBV inhibit HCV IRES through prevention of polyribosome formation. The combination of IFN-α and RBV treatment synergistically inhibits HCV IRES translation via using two different mechanisms involving PKR activation and depletion of intracellular guanosine pool through inhibition of IMPDH.

Target Enzyme Activity as a Biomarker for Immunosuppression

Pharmacokinetic drug monitoring has been used for many years to relate immunosuppressant dose to drug exposure in vivo. However, this conventional therapeutic drug monitoring of blood immunosuppressant levels may not necessarily predict the pharmacologic effects on immune cells. The direct determination of target enzyme activity (eg, calcineurin activity, inosine-5'-monophospahte dehydrogenase [IMPDH] activity, p70S6 kinase) may help to better assess the individual response to the immunosuppressant. However, its use is limited by the difficulties of the assay systems, which did not allow yet the prospective assessment of these enzymes in larger patient cohorts with the establishment of validated pharmacodynamic drug monitoring. The most progress regarding a robust and reproducible test system has been achieved with the determination of IMPDH activity as a specific pharmacodynamic parameter of mycophenolic acid activity. This recently validated and standardized assay allows the investigation of IMPDH activity in larger clinical studies. Although the determination of target enzyme activity, eg, by the determination of IMPDH activity, holds promise for a more individualized therapy in transplant medicine, more studies are needed to prospectively validate this approach.

Global Regulation of Nucleotide Biosynthetic Genes by c-Myc

BackgroundThe c-Myc transcription factor is a master regulator and integrates cell proliferation, cell growth and metabolism through activating thousands of target genes. Our identification of direct c-Myc target genes by chromatin immunoprecipitation (ChIP) coupled with pair-end ditag sequencing analysis (ChIP-PET) revealed that nucleotide metabolic genes are enriched among c-Myc targets, but the role of Myc in regulating nucleotide metabolic genes has not been comprehensively delineated.Methodology/Principal FindingsHere, we report that the majority of genes in human purine and pyrimidine biosynthesis pathway were induced and directly bound by c-Myc in the P493-6 human Burkitt's lymphoma model cell line. The majority of these genes were also responsive to the ligand-activated Myc-estrogen receptor fusion protein, Myc-ER, in a Myc null rat fibroblast cell line, HO.15 MYC-ER. Furthermore, these targets are also responsive to Myc activation in transgenic mouse livers in vivo. To determine the functional significance of c-Myc regulation of nucleotide metabolism, we sought to determine the effect of loss of function of direct Myc targets inosine monophosphate dehydrogenases (IMPDH1 and IMPDH2) on c-Myc-induced cell growth and proliferation. In this regard, we used a specific IMPDH inhibitor mycophenolic acid (MPA) and found that MPA dramatically inhibits c-Myc-induced P493-6 cell proliferation through S-phase arrest and apoptosis.Conclusions/SignificanceTaken together, these results demonstrate the direct induction of nucleotide metabolic genes by c-Myc in multiple systems. Our finding of an S-phase arrest in cells with diminished IMPDH activity suggests that nucleotide pool balance is essential for c-Myc's orchestration of DNA replication, such that uncoupling of these two processes create DNA replication stress and apoptosis.

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.

Monitoring of inosine monophosphate dehydrogenase activity in mononuclear cells of children with acute lymphoblastic leukemia: Enzymological and clinical aspects

Inosine 5'-monophosphate dehydrogenase (IMPDH; EC1.1.1.205) catalyzes the rate-limiting step in guanine nucleotide biosynthesis, and may play an important role in treatment of patients with antipurines. We used an HPLC method to measure the IMPDH activity in peripheral blood and bone marrow mononuclear cells (MNC). IMPDH activities were determined in children who were diagnosed with and treated for acute lymphoblastic leukemia (ALL), and in a group of control children. The median IMPDH activity for control children was 350 pmol/10(6) pMNC/hr (range 97-896; n = 47). No gender or age differences were observed. IMPDH activity at diagnosis of ALL was correlated with the percentage of peripheral blood lymphoblasts (r = 0.474; P < 0.001; n = 71). The median IMPDH activity at diagnosis was 410 pmol/10(6) pMNC/hr (range 40-2009; n = 76), significantly higher than for controls (P = 0.012). IMPDH activity significantly decreased after induction treatment, and during treatment with methotrexate (MTX) infusions (median 174 pmol/10(6) pMNC/hr; range 52-516; n = 21). The activity remained low during maintenance treatment with 6-mercaptopurine (6MP) and MTX, at a significantly lower level than for controls (P < 0.004). One year after cessation of treatment IMPDH activity returned to normal values. The decrease of IMPDH activity at remission of ALL seems to be at least partly due to the eradication of lymphoblasts with the type 2 isoform of the enzyme.

3-Hydrogenkwadaphnin targets inosine 5′-monophosphate dehydrogenase and triggers post-G1 arrest apoptosis in human leukemia cell lines

3-Hydrogenkwadaphnin (3-HK) is a recently characterized daphnane-type compound isolated from Dendrostellera lessertii with high anti-tumor activity in animal models. Herein, we report on time- and dose-dependent effects of this compound on growth, differentiation, IMPDH inhibition, cell cycle and apoptosis of a panel of human leukemia cell lines (HL-60, K562 and Molt4). The drug decreased the growth of leukemia cells in less than 24 h of treatment. However, longer exposure times and/or higher concentrations were required to promote cell apoptosis. Cell cycle analysis revealed the accumulation of cells in their G1 phase as early as 12 h after drug exposure but sub-G1 population was recorded after 24 h. Occurrence of apoptosis was constantly accompanied by morphological (staining with DNA-binding dyes) and biochemical (DNA fragments) variations among drug-treated cells. Despite these observations, non-activated normal human PBL were insensitive to the drug action. In addition, treatment of PHA-activated PBL, K562, Molt4 and HL-60 cells with a single dose of the drug for 24 h led to the inhibition of IMPDH activity by almost 37, 38, 44 and 50%, respectively. In contrast, no difference in IMPDH activities were seen between normal PBL and the drug treated PBL cells. Restoration of the depleted GTP concentration by exogenous addition of guanosine (25–50 μM) reversed the drug effects on cell growth, DNA fragmentation and apoptosis. Furthermore, the drug effects were potentiated by exogenous addition of hypoxanthine to the drug-treated cells. Reduction of the drug potency on the non-proliferative (retinoic acid treated) HL-60 cells by almost 40%, compared to the proliferative cells, clearly shows type II IMPDH as one of the main targets of the drug. These results suggest that 3-HK may be a powerful candidate for treatment of leukemia.

Modulation of IMPDH2, survivin, topoisomerase I and vimentin increases sensitivity to methotrexate in HT29 human colon cancer cells

We determined differentially expressed genes in HT29 human colon cancer cells, both after short treatment with methotrexate (MTX) and after the resistance to MTX had been established. Screening was performed using Atlas Human Cancer 1.2K cDNA arrays. The analysis was carried out using Atlas image 2.01 and genespring 6.1 software. Among the differentially expressed genes we chose for further validation were inosine monophosphate dehydrogenase type II (IMPDH2), inosine monophosphate cyclohydrolase and survivin as up-regulated genes, and topoisomerase I (TOP1) and vimentin as down-regulated genes. Changes in mRNA levels were validated by quantitative RT-PCR. Additionally, functional analyses were performed inhibiting the products of the selected genes or altering their expression to test if these genes could serve as targets to modify MTX cytotoxicity. Inhibition of IMPDH or TOP1 activity, antisense treatment against survivin, or overexpression of vimentin, sensitized resistant HT29 cells to MTX. Therefore, these proteins could constitute targets to develop modulators in MTX chemotherapy.

PHARMACOKINETIC AND PHARMACODYNAMIC COMPARISON OF MYCOPHENOLATE MOFETIL (MMF) AND ENTERIC-COATED MYCOPHENOLATE SODIUM (EC-MPS) IN MAINTENANCE RENAL TRANSPLANT PATIENTS WITH TACROLIMUS AS BASIC IMMUNOSUPPRESSION

P736 Aims: The determination of IMPDH activity is a pharmacodynamic (PD) parameter of MPA activity. Enteric-coated mycophenolate sodium (EC-MPS) has been developed to avoid the release of mycophenolic acid (MPA) into the stomach and allow direct delivery of the active agent via small intestinal absorption. This is the first study to compare pharmacokinetics (PK) and the PD response of both drugs under the maintenance immunosuppression with tacrolimus. Methods: In this open-label study, 16 renal transplant patients on MMF and Tacrolimus with or without corticosteroids were converted to EC-MPS, containing an equimolar amount of MPA. Full 12h PK- and PD-profiles were obtained in each patient on MMF following the 2-week open-label run-in period and with EC-MPS 14 days after conversion. For each profile, IMPDH activity, MPAG and MPA concentrations (HPLC) were determined in parallel. IMPDH activity in peripheral mononuclear cells was measured using a non-radioactive procedure. Results: Equimolar doses of both formulations resulted in similar (p=n.s.) Cmax (EC-MPS: 13,57±6,4 vs. MMF: 15,37±8,2ng/ml), and MPA exposure (AUC; EC-MPS: 36,40±15,9 vs. MMF: 33,5±9,6 μg∗h/ml). The MPAG exposure was significantly higher during the treatment with EC-MPS (AUC; EC-MPS: 652,5±407 vs. MMF: 551,1±377 μg∗h/ml). EC-MPS resulted in a significant (p<0.003) later Tmax (30 vs. 90 min). The lowest daytime IMPDH activity was comparable (EC-MPS: 5,94±3,4 vs. MMF: 4,16±2,0 nmol/h/mg, n.s.), as well as the inhibition (EC-MPS: 57±22 vs. MMF: 62±57%). The daytime average IMPDH activity as a measure of the overall PD response was also similar (EC-MPS: 9,9±2,5 vs. MMF: 9,3±2,2nmol/h/mg). Conclusions: Treatment with equimolar doses of MMF and EC-MPS in a tacrolimus based regimen results in an identical drug exposure and similar PD-response, despite minor differences in PK.

Targeted disruption of the inosine 5'-monophosphate dehydrogenase type I gene in mice.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the critical, rate-limiting enzyme in the de novo biosynthesis pathway for guanine nucleotides. Two separate isoenzymes, designated IMPDH types I and II, contribute to IMPDH activity. An additional pathway salvages guanine through the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) to supply the cell with guanine nucleotides. In order to better understand the relative contributions of IMPDH types I and II and HPRT to normal biological function, a mouse deficient in IMPDH type I was generated by standard gene-targeting techniques and bred to mice deficient in HPRT or heterozygous for IMPDH type II. T-cell activation in response to anti-CD3 plus anti-CD28 antibodies was significantly impaired in both single- and double-knockout mice, whereas a more general inhibition of proliferation in response to other T- and B-cell mitogens was observed only in mice deficient in both enzymes. In addition, IMPDH type I(-/-) HPRT(-/0) splenocytes showed reduced interleukin-4 production and impaired cytolytic activity after antibody activation, indicating an important role for guanine salvage in supplementing the de novo synthesis of guanine nucleotides. We conclude that both IMPDH and HPRT activities contribute to normal T-lymphocyte activation and function.

Tmolt4 leukemic type II isoform of IMP dehydrogenase as a target for 1,2,4-triazolidine-3,5-diones, 1-(1-(3-methylphenyl)ethylidineamino)-4,4-diethyl-3,5-azetidinediones, 3,5-isoxazolidinediones, and 4,4-disubstituted-3,5-pyrazolidinediones.

The 1,2,4-triazolidine-3,5-diones, 1-(1-(3-methylphenyl)ethylidineamino)-4,4-diethyl-3,5-azetidinediones, and 4,4-disubstituted-3,5-pyrazolidinediones proved to be potent competitive inhibitors of human Tmolt4 leukemia Type II IMP dehydrogenase [IMPDH] activity, an enzyme isoform which is induced in highly proliferating cells. On the other hand, the 3,5-isoxazolidinediones were shown to be uncompetitive inhibitors of Type II IMPDH activity. The correlation between inhibition of Type II IMPDH activity with the agents' ability to suppress DNA and purine syntheses in these Tmolt4 leukemia cell was positive. Type I IMPDH (i.e., the isoform that is present in normal cells) was not inhibited by these compounds suggesting that these agents would be less toxic to normal cells and have selective inhibition towards proliferating cells.

Inosine-5'-monophosphate dehydrogenase is required for mitogenic competence of transformed pancreatic beta cells.

The relation of inosine-5'-monophosphate dehydrogenase (IMPDH; the rate-limiting enzyme in GTP synthesis) to mitogenesis was studied by enzymatic assay, immunoblots, and RT-PCR in several dissimilar transformed pancreatic ss-cell lines, using intact cells. Both of the two isoforms of IMPDH (constitutive type 1 and inducible type 2) were identified using RT-PCR in transformed beta cells or in intact islets. IMPDH 2 messenger RNA (mRNA) and IMPDH protein were both regulated reciprocally by changes in levels of their end-products. Flux through IMPDH was greatest in rapidly growing cells, due mostly to increased uptake of precursor. Glucose (but not 3-0-methylglucose, L-glucose, or fructose) further augmented substrate uptake and also increased IMPDH enzymatic activity after either 4 or 21 h of stimulation. Serum or ketoisocaproate also increased IMPDH activity (but not uptake). Two selective IMPDH inhibitors (mycophenolic acid and mizoribine) reduced IMPDH activity in all cell lines, and, with virtually identical concentration-response curves, inhibited DNA synthesis (assessed as bromodeoxyuridine incorporation) in response to glucose, serum, or ketoisocaproate. Inhibition of DNA synthesis was reversible, completely prevented by repletion of cellular guanine (but not adenine) nucleotides, and could not be attributed to toxic effects. Despite the fact that modulation of IMPDH expression by guanine nucleotides was readily detectable, glucose and/or serum failed to alter IMPDH mRNA or protein, indicating that their effects on IMPDH activity were largely at the enzyme level. Precursors of guanine nucleotides failed, by themselves, to induce mitogenesis. Thus, adequate IMPDH activity (and thereby, availability of GTP) is a critical requirement for beta-cell proliferation. Although it is unlikely that further increases in GTP can, by themselves, initiate DNA synthesis, such increments may be needed to sustain mitogenesis.

Mechanism of action of the antitumor agents 6-benzoyl-3,3-disubstituted-1,5-diazabicyclo[3.1.0]hexane-2,4-diones: potent inhibitors of human type II inosine 5'-monophosphate dehydrogenase.

The observation that expression of the IMPDH gene is tightly linked with cellular proliferation and transformation has led to an interest in developing inhibitors that deplete intracellular guanine nucleotide pools. IMPDH exists as 2 isoforms, one of which is induced in tumor cells, type II and thus has led to new interest in this target for the design of isoform-selective anticancer chemotherapeutic agents. Several classes of IMPDH inhibitor are now in use or under development; however, only the 1,5-diazabicyclo[3.1.0]hexane-2,4-diones show selectivity for the type II isoform. In the current study, we further evaluated chemical modification of this class to determine the necessary components for selective type II IMPDH inhibition. The 6-benzoyl-3,3-disubstituted-1,5-diazabicyclo[3.1.0]hexane-2,4-diones were effective cytotoxic agents in human leukemias, lymphomas, breast, glioma and HeLa-S3 suspended uterine carcinoma screens with ED(50) values 0.3 to 12 microM. The agents acted as antimetabolites by inhibiting de novo purine biosynthesis at the key regulatory enzyme IMPDH, resulting in suppression of DNA synthesis and dGTP pool levels within 60 min. Furthermore, the derivatives were specific for the type II isoform as opposed to type I, acting in a competitive manner with K(i) values of 5.1 to 63 microM. Addition of the 6-benzoyl moiety to the bicyclic parent ring structure afforded the most potent agent in the novel class of 1,5-diazabicyclo[3.1.0]hexane-2,4-diones that selectively inhibits type II IMPDH activity.

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

Abstract 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 [2,8-3H] hypoxantine. The mean value of areas under the concentration-time curves (AUC0-12) 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-AUC0-12 values and MMF dose (expressed in g/day or g/kg per day). Predose MPA concentrations correlated only weakly with the respective MPA-AUC0-12 values (r2 from 0.385 to 0.655), whereas an acceptable correlation was observed between MPA Cmax and MPA-AUC0-12 (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.