Monophosphate Dehydrogenase Activity Research Articles

The combination of exposure to Tacrolimus, mycophenolic acid, Inosine 5'-Monophosphate Dehydrogenase activity and inhibition in the first week define early histological outcomes in renal transplant recipients.

Therapeutic drug monitoring is routine for Tacrolimus, while levels are not routinely monitored for mycophenolic acid (MPA). This study investigated the effect of early post-transplant pharmacokinetics (PK) of MPA and Tacrolimus along with the pharmacodynamics (PD) of MPA on biopsy-proven acute rejection (BPAR) after renal transplantation. A prospective PK/PD study with limited sampling (three blood samples) was conducted in renal transplant recipients on week 1, around Day 6 (n=42) and at the 3rd-month biopsy on Day 90 (n=23). The partial exposures (area under curve [AUC]0-3.5h ) of both MPA and Tacrolimus obtained during the first week were more predictive of rejection (combined clinical and subclinical rejection) by Day 90 than their trough concentrations or Day 90 exposures. Patients with rejection had significantly worse renal function (eGFR) and a comparatively lower exposure to MPA during the first post-transplant week. The lower MPA exposure was also associated with sub-optimal inosine monophosphate dehydrogenase (IMPDH) inhibition in patients with rejection, and the probability of rejection was higher in the presence of an increased pre-transplant IMPDH activity. A composite of parameters, including MPA exposure and IMPDH activity was found to predict acute rejection and may be beneficial along with tacrolimus monitoring early after renal transplantation.

Inosine monophosphate dehydrogenase activity and mycophenolate pharmacokinetics in children with nephrotic syndrome treated with mycophenolate mofetil

Some studies have shown that the area under the concentration-time curve (AUC) of mycophenolic acid (MPA) should be higher for children with nephrotic syndrome (NS) than after renal transplantation. The pharmacodynamic aspect of MPA, the activity of inosine monophosphate dehydrogenase (IMPDH), has not been studied in children with NS. The study included 21 children (4-16 years old) with NS treated with mycophenolate mofetil. MPA and its glucuronide plasma concentrations were determined using validated high-performance liquid chromatography-ultraviolet (HPLC-UV). The separate HPLC-UV method was applied for IMPDH activity determination. The variability was expressed by the coefficient of variation (CV). IMPDH activity and MPA concentration (Ctrough ) before the morning dose amounted to 29.95 μmol s-1 mol-1 adenosine monophosphate (AMP) (range, 6.71-98.60 μmol s-1 mol-1 AMP) and 1.72 μg/mL (range, 0.39-4.34 μg/mL), respectively, whereas the area under the effect-time curve from 0 to 4h and MPA AUC0-4 were 130.36 μmol s-1 mol-1 AMP × h (range, 23.58-306.57 μmol s-1 mol-1 AMP × h) and 24.63 μg h/mL (range, 12.21-67.48 μg h/mL), respectively. IMPDH activity decreased concomitantly with MPA concentration increase, however, the variability of the pharmacodynamic parameters was greater than of the pharmacokinetics. The median degree of maximum IMPDH inhibition was 61%. MPA Ctrough and predicted AUC were lower than in our previous study. Only a few MPA pharmacokinetic parameters correlated with the pharmacodynamics. IMPDH activity did not correlate with the children's age and did not differ between boys and girls. MPA clearance was the highest in younger children (median, 10.54 L/m2 /h) and cholesterol correlated negatively with the children's age (r=-0.659, P=0.003). IMPDH minimum activity and the degree of maximum IMPDH inhibition were similar to those obtained in renal transplant recipients. IMPDH activity does not undergo developmental or gender-specific regulation in children with NS. MPA underexposure might be more frequent in younger children, especially with high cholesterol and triglycerides levels because of high MPA clearance.

Predictors of Serological Response to SARS-CoV-2 Vaccination in Kidney Transplant Patients: Baseline Characteristics, Immunosuppression, and the Role of IMPDH Monitoring.

Immunosuppression increases the risk of severe coronavirus disease 2019 (COVID-19). Morbidity and mortality of this disease in kidney transplant patients are higher than in the general population. As the vaccination response of transplant patients is weak, serological monitoring was performed. In this cohort study, we analyzed the determinants of vaccination response. All patients had no history of COVID-19. With anti-spike IgG monitoring, 148 responders and 415 non-responders were identified. We compared both groups using multivariate analyses of the cohort and a sub-cohort of mycophenolic-acid-treated patients. We investigated the influence of patient characteristics, immunosuppression, and erythrocyte inosine monophosphate dehydrogenase (IMPDH) activity. In responders, the time after transplantation was longer (13.5 vs. 8.5 years), the glomerular filtration rate was higher (56.9 vs. 47.8 mL/min/1.73 m2), and responders were younger (53.0 vs. 57.4 years). Heterologous vaccination was more effective than homologous vaccination. Calcineurin inhibitors plus mycophenolate reduced the seroconversion rate. No seroconversion was observed in belatacept patients. In mycophenolate-treated patients, IMPDH activity was a significantly better predictor of response than mycophenolate dose (AUC 0.84 vs. 0.62, p < 0.001). Immunosuppression strongly affects vaccine response. Modifications to immunosuppression should be considered in order to facilitate this response. Erythrocyte IMPDH activity can be used to guide mycophenolate treatment.

The Application of Inosine 5'-Monophosphate Dehydrogenase Activity Determination in Peripheral Blood Mononuclear Cells for Monitoring Mycophenolate Mofetil Therapy in Children with Nephrotic Syndrome.

In pediatric nephrotic syndrome, recommended mycophenolic acid (MPA) pharmacokinetics are higher than those for transplant recipients. In MPA therapeutic monitoring, inosine-5′-monophosphate dehydrogenase (IMPDH) activity may be useful. We modified the method established for renal transplant recipients and determined IMPDH activity in peripheral blood mononuclear cells (PBMCs) from healthy volunteers and children (4–16 years) with nephrotic syndrome treated with mycophenolate mofetil (MMF). From children, four blood samples were collected, and MPA concentrations were also determined. IMPDH activity was calculated using xanthosine monophosphate (XMP) normalized with adenosine monophosphate (AMP), both determined with the HPLC-UV method. The modified method was accurate, precise, and linear for AMP and XMP within 0.50–50.0 μmoL/L. Mean IMPDH activity in volunteers was 45.97 ± 6.24 µmoL·s−1·moL−1 AMP, whereas for children, the values were variable and amounted to 39.23 ± 27.40 µmoL·s−1·moL−1 AMP and 17.97 ± 15.24 µmoL·s−1·moL−1 AMP before the next MMF dose and 1 h afterward, respectively. The modified method may be applied to IMPDH activity determination in children with nephrotic syndrome treated with MMF. IMPDH activity should be determined after one thawing of PBMCs due to the change in AMP and XMP concentrations after subsequent thawing. For children, the lowest IMPDH activity was observed concomitantly with the highest MPA concentration.

Inosine 5'-Monophosphate Dehydrogenase Activity for the Longitudinal Monitoring of Mycophenolic Acid Treatment in Kidney Allograft Recipients.

Mycophenolic acid (MPA) is a standard immunosuppressant in organ transplantation. A simple monitoring biomarker for MPA treatment has not been established so far. Here, we describe inosine 5'-monophosphate dehydrogenase (IMPDH) monitoring in erythrocytes and its application to kidney allograft recipients. IMPDH activity measurements were performed using a high-performance liquid chromatography assay. Based on 4203 IMPDH measurements from 1021 patients, we retrospectively explored the dynamics early after treatment start. In addition, we analyzed the influence of clinically relevant variables on IMPDH activity in a multivariate model using data from 711 stable patients. Associations between IMPDH activity and clinical events were evaluated in hospitalized patients. We found that IMPDH activity reflects MPA exposure after 8 weeks of constant dosing. In addition to dosage, body mass index, renal function, and coimmunosuppression affected IMPDH activity. Significantly lower IMPDH activities were found in patients with biopsy-proven acute rejection as compared to patients without rejection (median [interquartile range]: 696 [358-1484] versus 1265 [867-1618] pmol xanthosine-5'-monophosphate/h/mg hemoglobin, P < 0.001). The highest IMPDH activities were observed in hospitalized patients with clinically evident MPA toxicity as compared to patients with hospitalization not related to MPA treatment (1548 [1021-2270] versus 1072 [707-1439] pmol xanthosine-5'-monophosphate/h/mg hemoglobin; P < 0.001). Receiver operating characteristic curve analyses underlined the usefulness of IMPDH to predict rejection episodes (area, 0.662; confidence interval, 0.584-0.740; P < 0.001) and MPA-associated adverse events (area, 0.632; confidence interval, 0.581-0.683; P < 0.001), respectively. IMPDH measurement in erythrocytes is a novel and useful strategy for the longitudinal monitoring of MPA treatment.

Pharmacodynamic Monitoring of Mycophenolic Acid Therapy: Improved Liquid Chromatography–Tandem Mass Spectrometry Method for Measuring Inosin-5′-Monophosphate Dehydrogenase Activity

Mycophenolic acid (MPA), a powerful inhibitor of lymphocyte proliferation, is widely used in transplantation medicine and as a glucocorticoid-sparing agent in rheumatic and inflammatory diseases. As inosine-5'-monophosphate dehydrogenase (IMPDH), the target enzyme of MPA, shows high interindividual variability in its basal activity, the assessment of IMPDH activity in addition to pharmacokinetic monitoring has emerged as a strategy to individualize MPA pharmacotherapy. A liquid chromatography-tandem mass spectrometry method was developed to measure IMPDH activity in peripheral blood mononuclear cells from lithium-heparinized blood. Stable isotope-labeled analogs of analytes were used as internal standards for the quantitative analyses of xanthosine-5'-monophosphate (XMP) and adenosine-5'-monophosphate (AMP). IMPDH activity was expressed as enzymatic production of XMP per time normalized to the AMP concentration. Validation and evaluation of the new method were performed by using blood samples from healthy volunteers (n = 10). Linearity was demonstrated over the concentration ranges of 0.25-80 μM for XMP and 4-80 µM for AMP (R > 0.99). Between-day and within-day assay precisions and accuracies were within the acceptance criterion of ±15%. Matrix effects were fully compensated by the coelution of internal standards. Specific and linear XMP production (R > 0.99) and the inhibition of IMPDH activity by MPA at clinically relevant doses were demonstrated. In this study, a liquid chromatography-tandem mass spectrometry method to measure IMPDH activity was established and fully evaluated for matrix and ion suppression effects. The method enabled precise quantification of IMPDH activity for the improvement of pharmacokinetic/pharmacodynamic therapeutic drug monitoring approaches to optimize immunosuppressive treatment with MPA.

Effect of mycophenolic acid on inosine monophosphate dehydrogenase (IMPDH) activity in liver transplant patients

Due to the development of immunosuppressants, the focus in transplanted patients has shifted from short-term to long-term survival as well as a better adjustment of these drugs in order to prevent over- and under-immunosuppression. Mycophenolic acid (MPA) is a noncompetitive inhibitor of inosine monophosphate dehydrogenase (IMPDH) and approved for prophylaxis of acute rejection after kidney, heart, and liver transplantation, where it has become a part of the standard therapy. Targeting inosine monophosphate IMPDH activity as a surrogate pharmacodynamic marker of MPA-induced immunosuppression may allow a more accurate assessment of efficacy and aid in limiting toxicity in liver transplanted patients. Assess IMPDH-inhibition in liver transplant recipients and its impact on biliary/infectious complications, acute cellular rejection (ACR) and liver dependent survival. This observational cohort study comprises 117 liver transplanted patients that were treated with mycophenolate mofetil (MMF) for at least 3 months. Blood samples (BS) were collected and MPA serum level and IMPDH activity were measured before (t(0)), 30minutes (t(30)) and 2h after (t(120)) MMF morning dose administration. Regarding MPA, we assessed the area under the curve (AUC). Patients were prospectively followed up for one year and assessed for infectious and biliary complications, episodes of ACR and liver dependent survival. The MPA levels showed a broad interindividual variability at t(0) (2.0±1.8ng/ml), t(30) (12.7±9.0ng/ml) and t(120) (7.5±4.3ng/ml). Corresponding IMPDH activity was at t(o) (23.2±9.5 nmol/h/mg), at t(30) (16.3±8.8 nmol/h/mg) and t(120) (18.2±8.7 nmol/h/mg). With regard to MPA level we found no correlation with infectious or biliary complications within the follow-up period. Patients with baseline IMPDH(a) below the median had significant more viral infections (6 (10.2%) vs. 17 (29.3%); P=0.009) with especially more cytomegalovirus (CMV) infections (1 (3.4%) vs. 6 (21.4%); P=0.03)). Furthermore, patients with baseline IMPDH(a) above the median developed more often non-anastomotic biliary strictures (8 (13.6%) vs. 1 (1.7%), P=0.03). We found the group reaching the combined clinical endpoint of death and re-transplantation showing significantly lower MPA baseline values (t(0) 0.9±0.7 vs. 2.1±1.8μg/ml Mann-Whitney-U: P=0.02). We calculated a simplified MPA(AUC) with the MPA level at baseline, 30 and 120minutes after MPA administration. Whereas we found no differences with regard to baseline characteristics at entry into the study patients with MPA (AUC) below the median experienced significantly more often the combined clinical endpoint (12.1% (7/58) vs. 0.0% (0/57); P=0.002) and had a reduced actuarial re-transplantation-free survival (1.0 year vs. 0.58 years; Log-rank: P=0.007) during the prospective one-year follow-up period. In univariate and multivariate analysis including gender, age, BMI, ACR, MPA (AUC) and IMPDH(a) only BMI, MPA (AUC) and IMPDH(a) were independently associated with reduced actuarial re-transplantation-free survival. MPA-levels and IMPDH-activity in liver transplanted patients allows individual risk assessment. Patients with higher IMPDH inhibition acquire more often viral infections. Insufficient IMPDH inhibition is associated with development of non-anastomotic bile duct strictures and reduced re-transplantation-free survival.

Analiza DNK oštećenja izazvanog tiazofurinom u humanim ćelijama pune krvi primenom in vitro komet testa

Objective. Inosine 5'-monophosphate dehydrogenase (IMPDH) activity in cancer cells is increased. Tiazofurin selectively inhibits the activity of IMPDH, and it has been granted for the treatment of different cancers and new viral diseases. Its widespread use was limited because exposure to tiazofurin under certain circumstances was found to have a higher frequency of severe non-hematologic toxicity. Therefore, the objective of this study was to examine genotoxic action and inducement of DNA damage of tiazofurin using the comet assay. Methods. The ability of tiazofurin to induce DNA damage was evaluated using single-cell gel electrophoresis (SCGE) technique/comet assay. Human whole blood cells were exposed to three final concentrations of tiazofurin (1µM/mL, 2 µM/mL, and 5 µM/mL) for 30 min in vitro. Results. Our results indicate that tiazofurin produced a significant level of DNA damage on whole blood cells after 30 min of exposure vs. control. All tested concentrations were significantly comet-forming, in a concentration-dependent manner. Conclusion. Our investigation on the tiazofurin-treated cells and their relationship to the formation of DNA damage demonstrated that the genotoxic effect was induced after exposure to tiazofurin under described conditions.

Characterizing the pharmacokinetics and pharmacodynamics of immunosuppressant medicines and patient outcomes in elderly renal transplant patients.

This review examines what is currently known about the pharmacokinetics and pharmacodynamics of commonly prescribed immunosuppressant medicines, tacrolimus, cyclosporine, mycophenolate and prednisolone, in elderly renal transplant recipients, and reported patient outcomes in this cohort. Renal transplantation is increasing rapidly in the elderly, however, currently, long-term patient outcomes are relatively poor compared to younger adults. Some studies have suggested that elderly recipients may have higher dose-adjusted exposure and/or lower clearance of the calcineurin inhibitors tacrolimus and cyclosporine; with one study reporting up to 50% reduction in tacrolimus exposure in the elderly. Elderly transplant recipients do not appear to have higher dosage-adjusted exposure to mycophenolic acid (MPA). The effects of ageing on the pharmacokinetics of prednisolone are unknown. Only one study has examined how aging effects drug target enzymes, reporting no difference in baseline inosine 5'-monophosphate dehydrogenase (IMPDH) activity and MPA-induced IMPDH activity in elderly compared to younger adult renal transplant recipients. In elderly transplant recipients, immunosenescence likely lowers the risk of acute rejection, but increases the risk of drug-related adverse effects. Currently, the three main causes of death in elderly renal transplant recipients are cardiovascular disease, infection and malignancy. One study has showed that renal transplant recipients aged over 65 years are seven times more likely to die with a functioning graft compared with young adults (aged 18-29 years). This suggests that an optimal balance between immunosuppressant medicine efficacy and toxicity is not achieved in elderly recipients, and further studies are needed to foster long-term graft and patient survival. Lower maintenance immunosuppressant targets in elderly recipients may decrease patient susceptibility to drug side effects, however, further studies are required and appropriate targets need to be established.

Optimization and application of an HPLC method for quantification of inosine-5′-monophosphate dehydrogenase activity as a pharmacodynamic biomarker of mycophenolic acid in Chinese renal transplant patients

BackgroundThe immunosuppressive agent mycophenolic acid (MPA) is a non-competitive, reversible inhibitor of inosine-5′-monophosphate dehydrogenase (IMPDH). Thus, IMPDH activity can serve as a potential pharmacodynamic biomarker to optimize dosing of MPA. MethodsPeripheral blood mononuclear cells were isolated from 2 mL blood samples and an in vitro enzymatic reaction was subsequently performed for 120 min. To determine IMPDH activity in Chinese healthy volunteers and renal transplant patients, a high performance liquid chromatography assay was established and validated by subtracting adenosine monophosphate (AMP) from blank samples for eliminating exogenous AMP interference. ResultsThe accuracy of our method ranged between −0.8% and 12.5%, and the precision ranged between 0.7% and 6.3%. The mean value of IMPDH activity across 11 healthy volunteers was 46.60 ± 14.28 μmol/s/mol AMP. A negative relationship between MPA concentration and IMPDH activity was observed in four renal transplant patients treated with MPA 13 days post-transplantation, while the inhibitory rate of IMPDH activity ranged from 24% to 42%. ConclusionA bioanalytical assay for IMPDH quantification was optimized and evaluated. The differences in the pharmacodynamics of MPA between Asians and Caucasians may provide some evidence for dosing differences among ethnicities.

Pharmacokinetic and Pharmacodynamic Markers of Mycophenolic Acid Associated with Effective Prophylaxis for Acute Graft-Versus-Host Disease and Neutrophil Engraftment in Cord Blood Transplant Patients

Mycophenolate mofetil (MMF) is a prodrug of mycophenolic acid (MPA) and is frequently used to prevent acute graft-versus-host disease (aGVHD) in patients receiving hematopoietic stem cell transplantation (HSCT). However, optimal doses of MMF and target MPA concentrations in HSCT patients have not been defined. In this study, relationships between pharmacokinetic or pharmacodynamic markers of MPA and successful aGVHD prevention and neutrophil engraftment were evaluated to inform individualized MPA treatments in HSCT patients. We recruited 35 patients undergoing cord blood transplantation (CBT) who were treated with MMF. Area under the concentration–time curves from 0 to 24 hours (AUC0-24) for free MPA and MPA acyl glucuronide (AcMPAG) at 1 week after the start of MMF treatments were significantly higher in patients with gastrointestinal (GI) aGVHD at stage ≥I than those at stage 0. Patients with faster neutrophil engraftment had higher free MPA AUC0-24 at 1 week after the start of MMF treatments compared with those with slower neutrophil engraftment. Inosine-5′-monophosphate dehydrogenase activity in peripheral blood mononuclear cells and single nucleotide polymorphisms in genes that were previously associated with MPA pharmacokinetics and pharmacodynamics were not an independent predictor for the clinical outcomes. Receiver-operating characteristic model analyses showed that cutoff values of AUC0-24 for successful GI aGVHD prevention were .689 and 15.6 µg⋅hour⋅mL−1 for free MPA and AcMPAG, respectively. In addition, the cut-off value of free MPA AUC0-24 for neutrophil engraftment by day 25 was .405 µg⋅hour⋅mL−1. In conclusion, free MPA AUC0-24 may be a better predictor of the prevention of GI aGVHD and neutrophil engraftment compared with that of total MPA in patients receiving CBT. Hence, monitoring of the free MPA AUC0-24 between .405 and .689 µg⋅hour⋅mL−1 could be considered informative of individualized MPA treatments in CBT patients.

Population pharmacokinetics and pharmacodynamics of mycophenolic acid using the prospective data in patients undergoing hematopoietic stem cell transplantation.

Mycophenolate mofetil (MMF), a prodrug of mycophenolic acid (MPA), is used to suppress GvHD in patients undergoing hematopoietic stem cell transplantation (HCT). The purpose of this study was to construct a population pharmacokinetic and pharmacodynamic model in HCT patients for individualized MPA therapy. Blood samples were obtained from 49 HCT patients after starting MMF therapy. Population pharmacokinetic and pharmacodynamic parameters were obtained using the program NONMEM. MPA was described via a one-compartment model with a first-order elimination, and 30.9% of MPA glucuronide (MPAG) was found in the enterohepatic circulation. Inosine-5'-monophosphate dehydrogenase (IMPDH) activity was modeled as a maximal inhibitory model with a half-maximal inhibitory concentration (IC50) of 3.59 μg/mL against MPA concentrations. Simulations based on the obtained pharmacokinetic and pharmacodynamic parameters revealed that decreased creatinine clearance increases the MPAG concentration followed by an increased MPA concentration; therefore, IMPDH activity decreases. Diarrhea decreases the enterohepatic circulation of MPAG and consequently reduces MPA concentration. The IC50 for MPA exhibited a positive association with C-reactive protein. Dosage adjustment based on plasma MPA concentration is required especially for patients with renal dysfunction and/or diarrhea.

Human monocyte-targeted non-viral siRNA delivery for the suppression of inflammatory macrophages

Mycophenolate mofetil (MMF) is a prodrug of mycophenolic acid (MPA) and is frequently used to prevent acute graft-versus-host disease (aGVHD) in patients receiving hematopoietic stem cell transplantation (HSCT). However, optimal doses of MMF and target MPA concentrations in HSCT patients have not been defined. In this study, relationships between pharmacokinetic or pharmacodynamic markers of MPA and successful aGVHD prevention and neutrophil engraftment were evaluated to inform individualized MPA treatments in HSCT patients. We recruited 35 patients undergoing cord blood transplantation (CBT) who were treated with MMF. Area under the concentration–time curves from 0 to 24 hours (AUC0-24) for free MPA and MPA acyl glucuronide (AcMPAG) at 1 week after the start of MMF treatments were significantly higher in patients with gastrointestinal (GI) aGVHD at stage ≥I than those at stage 0. Patients with faster neutrophil engraftment had higher free MPA AUC0-24 at 1 week after the start of MMF treatments compared with those with slower neutrophil engraftment. Inosine-5′-monophosphate dehydrogenase activity in peripheral blood mononuclear cells and single nucleotide polymorphisms in genes that were previously associated with MPA pharmacokinetics and pharmacodynamics were not an independent predictor for the clinical outcomes. Receiver-operating characteristic model analyses showed that cutoff values of AUC0-24 for successful GI aGVHD prevention were .689 and 15.6 µg⋅hour⋅mL−1 for free MPA and AcMPAG, respectively. In addition, the cut-off value of free MPA AUC0-24 for neutrophil engraftment by day 25 was .405 µg⋅hour⋅mL−1. In conclusion, free MPA AUC0-24 may be a better predictor of the prevention of GI aGVHD and neutrophil engraftment compared with that of total MPA in patients receiving CBT. Hence, monitoring of the free MPA AUC0-24 between .405 and .689 µg⋅hour⋅mL−1 could be considered informative of individualized MPA treatments in CBT patients.

40 - Allogeneic Hematopoietic Stem Cell Transplantation for Patients Aged 60 Years or Older with Myelodysplastic Syndrome in Japan

Mycophenolate mofetil (MMF) is a prodrug of mycophenolic acid (MPA) and is frequently used to prevent acute graft-versus-host disease (aGVHD) in patients receiving hematopoietic stem cell transplantation (HSCT). However, optimal doses of MMF and target MPA concentrations in HSCT patients have not been defined. In this study, relationships between pharmacokinetic or pharmacodynamic markers of MPA and successful aGVHD prevention and neutrophil engraftment were evaluated to inform individualized MPA treatments in HSCT patients. We recruited 35 patients undergoing cord blood transplantation (CBT) who were treated with MMF. Area under the concentration–time curves from 0 to 24 hours (AUC0-24) for free MPA and MPA acyl glucuronide (AcMPAG) at 1 week after the start of MMF treatments were significantly higher in patients with gastrointestinal (GI) aGVHD at stage ≥I than those at stage 0. Patients with faster neutrophil engraftment had higher free MPA AUC0-24 at 1 week after the start of MMF treatments compared with those with slower neutrophil engraftment. Inosine-5′-monophosphate dehydrogenase activity in peripheral blood mononuclear cells and single nucleotide polymorphisms in genes that were previously associated with MPA pharmacokinetics and pharmacodynamics were not an independent predictor for the clinical outcomes. Receiver-operating characteristic model analyses showed that cutoff values of AUC0-24 for successful GI aGVHD prevention were .689 and 15.6 µg⋅hour⋅mL−1 for free MPA and AcMPAG, respectively. In addition, the cut-off value of free MPA AUC0-24 for neutrophil engraftment by day 25 was .405 µg⋅hour⋅mL−1. In conclusion, free MPA AUC0-24 may be a better predictor of the prevention of GI aGVHD and neutrophil engraftment compared with that of total MPA in patients receiving CBT. Hence, monitoring of the free MPA AUC0-24 between .405 and .689 µg⋅hour⋅mL−1 could be considered informative of individualized MPA treatments in CBT patients.

Determination of inosine 5'-monophosphate dehydrogenase activity in red blood cells of thiopurine-treated patients using HPLC.

Thiopurine drugs are commonly used in immune diseases and to a lesser extent, in transplant rejection prophylaxis: however interindividual variability in drug response and in the occurrence of adverse events is observed. Genetic variation in thiopurine S-methyltransferase (TPMT) doesn't completely explain the occurrence of all adverse events and drug response variability. The potential implication of other enzymes involved in thiopurine metabolism, such as ITPA, has been investigated over the last decade but little data is available on inosine 5'-monophosphate dehydrogenase (IMPDH) in patients treated with thiopurine drugs. The authors reported a HPLC method to determine IMPDH activity in the red blood cells (RBCs) of thiopurine-treated patients. IMPDH activity was evaluated by enzymatic conversion of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP). The XMP formed was analyzed on a Luna® NH2 stationary phase, a weak anion exchange phase that exhibits both ionic and hydrophobic properties. XMP was eluted below 15min. Intra-assay and inter-assay precisions were below 9% for RBCs supplemented with 2, 40 and 80μmol/L of XMP. IMPDH activity was measured in adults without thiopurine treatment as well as in adult and paediatric patients treated with thiopurines. A wide interindividual variability in IMPDH activity in RBCs was observed. No difference in IMPDH activity was found between untreated subjects and adult and paediatric patients on thiopurine therapy (median value 11.8, 7.9 and 7.7nmol XPM/g Hb/h respectively). The method described is useful in the determination of IMPDH phenotype from patients on thiopurine therapy and in the investigation of the potential relationship between IMPDH activity in RBCs and the occurrence of adverse events and drug response variability.

Mycophenolic acid exposure and complement fraction C3 influence inosine 5'-monophosphate dehydrogenase activity in systemic lupus erythematosus.

Background Mycophenolate mofetil has recently been reported to be effective against systemic lupus erythematosus. The influence of the pharmacokinetics of mycophenolic acid, the active form of mycophenolate mofetil and the major inactive mycophenolic acid phenolic glucuronide on the activity of the target enzyme inosine 5'-monophosphate dehydrogenase, is expected to be revealed. The aim of this study was to identify the factors associated with inosine 5'-monophosphate dehydrogenase activity in systemic lupus erythematosus patients. Methods Fifty systemic lupus erythematosus patients in remission maintenance phase (29 received mycophenolate mofetil [MMF+] and 21 did not [MMF-]) were enrolled. Median and interquartile range of dose of mycophenolate mofetil were 1500 and 1000-1500 mg/day, respectively. Stepwise multiple linear regression analysis was performed to assess the dependence between inosine 5'-monophosphate dehydrogenase activity and 25 predictor values including predose plasma concentrations of free mycophenolic acid and mycophenolic acid phenolic glucuronide. Results Median and interquartile range of predose total plasma concentrations of mycophenolic acid and mycophenolic acid phenolic glucuronide were 2.73 and 1.43-5.73 and 25.5 and 13.1-54.7 µg/mL, respectively. Predose inosine 5'-monophosphate dehydrogenase activity was significantly higher in MMF+ than MMF- patients (median 38.3 and 20.6 nmoL xanthosine 5'-monophosphate/g haemoglobin/h, P<0.01). The plasma concentration of free mycophenolic acid phenolic glucuronide, complement fraction C3 and body weight were significant predictors accounting for interindividual variability in the inosine 5'-monophosphate dehydrogenase activity (adjusted R2 = 0.52, P < 0.01) in a multivariate analysis. Conclusions Predose inosine 5'-monophosphate dehydrogenase activity was higher in systemic lupus erythematosus patients receiving mycophenolate mofetil therapy. Inosine 5'-monophosphate dehydrogenase activity may be determined by mycophenolic acid exposure and complement fraction C3 in systemic lupus erythematosus patients.

Neural crest requires Impdh2 for development of the enteric nervous system, great vessels, and craniofacial skeleton

Mutations that impair the proliferation of enteric neural crest-derived cells (ENCDC) cause Hirschsprung disease, a potentially lethal birth defect where the enteric nervous system (ENS) is absent from distal bowel. Inosine 5′ monophosphate dehydrogenase (IMPDH) activity is essential for de novo GMP synthesis, and chemical inhibition of IMPDH induces Hirschsprung disease-like pathology in mouse models by reducing ENCDC proliferation. Two IMPDH isoforms are ubiquitously expressed in the embryo, but only IMPDH2 is required for life. To further understand the role of IMPDH2 in ENS and neural crest development, we characterized a conditional Impdh2 mutant mouse. Deletion of Impdh2 in the early neural crest using the Wnt1-Cre transgene produced defects in multiple neural crest derivatives including highly penetrant intestinal aganglionosis, agenesis of the craniofacial skeleton, and cardiac outflow tract and great vessel malformations. Analysis using a Rosa26 reporter mouse suggested that some or all of the remaining ENS in Impdh2 conditional-knockout animals was derived from cells that escaped Wnt1-Cre mediated DNA recombination. These data suggest that IMPDH2 mediated guanine nucleotide synthesis is essential for normal development of the ENS and other neural crest derivatives.

Hypoxanthine-guanine phosphoribosyltransferase and inosine 5′-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa)

Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5′-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5′-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5′-monophosphate (IMP), adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), ATP, guanosine 5′-diphosphate (GDP), guanosine 5′-triphosphate (GTP), and xanthosine 5′-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

Recipient Pretransplant Inosine Monophosphate Dehydrogenase Activity in Nonmyeloablative Hematopoietic Cell Transplantation

Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5′-monophosphate to xanthosine 5′-monophosphate (XMP). We developed a highly sensitive liquid chromatography–mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNCs) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation but not with chronic GVHD, relapse, nonrelapse mortality, or overall mortality. We conclude that quantitation of the recipient's pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient's sensitivity to MMF. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients.

Pharmacokinetic and Pharmacodynamic Analysis of Inosine Monophosphate Dehydrogenase Activity in Hematopoietic Cell Transplantation Recipients Treated with Mycophenolate Mofetil

A novel approach to personalizing postgrafting immunosuppression in hematopoietic cell transplantation (HCT) recipients is evaluating inosine monophosphate dehydrogenase (IMPDH) activity as a drug-specific biomarker of mycophenolic acid (MPA)-induced immunosuppression. This prospective study evaluated total MPA, unbound MPA, and total MPA glucuronide plasma concentrations and IMPDH activity in peripheral blood mononuclear cells (PMNCs) at 5 time points after the morning dose of oral mycophenolate mofetil (MMF) on day +21 in 56 nonmyeloablative HCT recipients. Substantial interpatient variability in pharmacokinetics and pharmacodynamics was observed and accurately characterized by the population pharmacokinetic-dynamic model. IMPDH activity decreased with increasing MPA plasma concentration, with maximum inhibition coinciding with maximum MPA concentration in most patients. The overall relationship between MPA concentration and IMPDH activity was described by a direct inhibitory maximum effect model with an IC50 of 3.23 mg/L total MPA and 57.3 ng/mL unbound MPA. The day +21 IMPDH area under the effect curve (AUEC) was associated with cytomegalovirus reactivation, nonrelapse mortality, and overall mortality. In conclusion, a pharmacokinetic-dynamic model was developed that relates plasma MPA concentrations with PMNC IMPDH activity after an MMF dose in HCT recipients. Future studies should validate this model and confirm that day +21 IMPDH AUEC is a predictive biomarker.