Nils Tore Vethe

Determination of inosine monophosphate dehydrogenase activity in human CD4+ cells isolated from whole blood during mycophenolic acid therapy.

Inosine 5′-monophosphate dehydrogenase (IMPDH) is an established target in immunosuppression following organ transplantation. In lymphocytes, reversible inhibition of this enzyme by mycophenolic acid (MPA) results in reduced production of guanine and deoxyguanine nucleotides and thereby retarded proliferation of activated cells. In order to examine MPA pharmacodynamics in renal allograft recipients, the authors have developed an assay for the determination of IMPDH activity in CD4+ cells directly isolated from a small blood volume. Paramagnetic beads coated with anti-CD4 antibodies were utilized for the cell isolation. The intracellular MPA concentration was restored by incubating the cells in microfiltrated plasma from the original sample. Inosine 5′-monophosphate (IMP; substrate) and nicotine adenine dinucleotide (NAD; co-factor) were added to cell lysates, and IMPDH activity was quantified as the xanthosine 5′-monophosphate (XMP) production rate (pmol/106 cells/min) determined by liquid chromatography after hydrolytic cleavage to xanthine. The reaction kinetics were saturated with IMP and NAD concentrations of 1.79 μmol/L and 0.38 μmol/L, respectively. The production rate was linear in the interval 0.13 to 8.7 pmol XMP/min. Total interseries CVs based on seven replicates at each MPA concentration 0, 2.2, and 8.6 μg/mL were 25%, 16%, and 13%, respectively. When a single 1 gram mycophenolate mofetil dose was administered to a healthy individual, the measured IMPDH activity was 13% of predose value at the MPA peak concentration. The present assay allows reliable determination of IMPDH activity in CD4+ cells during MPA exposure, reducing the potential influence of sample preparation on the measured enzyme activity to a minimum. The assay may be applied to assess MPA pharmacodynamics during immunosuppressive treatment, maintaining the influence of intracellular MPA on the IMPDH activity.

Expression of IMPDH1 and IMPDH2 after transplantation and initiation of immunosuppression.

Background. Mycophenolic acid (MPA) mediates immunosuppressive effects by inhibiting inosine monophosphate dehydrogenase (IMPDH). Induction of IMPDH activity has been observed in whole blood and erythrocyte samples during immunosuppressive therapy. Information concerning the mechanisms for increased IMPDH activity is limited and the potential implications of induction have been debated. Methods. Whole blood, CD4+ cell, and reticulocyte samples were collected from 30 renal transplant patients pre- and posttransplantation. The expressions of two IMPDH isoforms, type 1 and 2, were analyzed by real-time reverse-transcription polymerase chain reaction and quantified using a housekeeping gene index. The IMPDH activity was determined by ultraviolet high-performance liquid chromatography. Results. Transplantation and the initiation of immunosuppressive therapy was associated with increased IMPDH1 (50–88%, P<0.0005) and decreased IMPDH2 (42–56%, P<0.0005) expression. In CD4+ cells, however, IMPDH2 increased (15%, P=0.009). These changes are probably related to glucocorticoid effects. Two weeks posttransplant, MPA-treated patients displayed elevated IMPDH 1 and 2 in reticulocytes, suggesting enzyme induction in these cells during prolonged MPA therapy. Patients with acute rejection during follow-up demonstrated higher IMPDH2 expression in CD4+ cells pretransplant than nonrejecting patients (median expression 1.26 vs. 0.87 respectively, P=0.017). Conclusions. Knowledge of changes in IMPDH 1 and 2 expression after transplantation and initiation of immunosuppression is important considering the action of MPA on IMPDH and the potential for pharmacodynamic monitoring of MPA by measuring IMPDH activity. The expression of IMPDH2 in CD4+ cells pretransplant may be an indicator of immune activation.

Inosine monophosphate dehydrogenase activity in renal allograft recipients during mycophenolate treatment.

Objective. Mycophenolic acid (MPA) exerts its immunosuppression by inhibiting inosine 5′‐monophosphate dehydrogenase (IMPDH), depleting activated lymphocytes of guanine nucleotides and retarding their proliferation. An optimal strategy for monitoring has not been established for mycophenolate mofetil (MMF) in renal transplantation, and clinical investigations of the pharmacokinetic‐pharmacodynamic relationship are warranted. Material and methods. Mycophenolic acid pharmacokinetics and whole blood cell IMPDH activity were investigated in two separate groups of renal allograft recipients. One group was studied within the 12‐h dose interval, while the second group was examined by pre‐dose samples pre‐transplant and then repeatedly during 8 weeks post‐transplant. Results. An inverse relationship between plasma MPA and IMPDH activity within the dose interval was demonstrated. Minimum IMPDH activity was a median 8 % of values pre‐MMF dose, coinciding with the MPA peak. Six hours post‐dose, IMPDH activity had returned to pre‐dose values. Patients receiving MMF had a 4.5‐fold higher pre‐dose enzyme activity than transplanted patients without MMF. During the 8 weeks post‐transplant, the median MPA trough concentration was fairly stable. Following an initial decrease during the first 4 days post‐transplant, IMPDH activity gradually increased during the 40 days post‐transplant, reaching 5‐fold the pre‐transplant values. Conclusions. Provided that the changes in IMPDH activity in whole blood cells predict the clinical effect, these pharmacokinetic‐pharmacodynamic findings may prove useful in the attempts to identify optimal timing and range for the monitoring of mycophenolate in renal transplantation. The question of whether MPA concentrations or measurements of IMPDH activity per se will be the optimal way of monitoring this immunosuppressant remains open and will only be answered by prospective clinical testing.

Use of generic tacrolimus in elderly renal transplant recipients: precaution is needed.

Background Proper bioequivalence studies comparing original with generic immunosuppressive drugs in patients are limited, especially in the increasing population of elderly renal transplant recipients. We performed an open-label, single-center, prospective, randomized, cross-over study and compared steady-state pharmacokinetics (PK) of a generic tacrolimus (Tacni) formulation with the original (Prograf) in renal transplant recipients older than 60 years. Methods Twenty-eight patients, with a median age of 69 years (range, 60 to 78), were randomized at time of transplantation to receive original or generic tacrolimus, and 25 (21 men, 4 women) provided two evaluable 12-hr PK profiles. The PK investigations were performed in a stable phase approximately 6 and 8 weeks postengraftment. After the first PK investigation, tacrolimus formulations were switched (1:1 dose ratio). Results Generic tacrolimus did not meet the bioequivalence criteria; the area under the curve0–12 ratio of generic-original tacrolimus formulation was 1.17 (90% confidence interval, 1.10–1.23) and the Cmax ratio was 1.49 (90% confidence interval, 1.35–1.65). The generic formulation also showed a shorter time to Cmax (Tmax) (P=0.04). Importantly, the lack of bioequivalence was not reflected in the standard monitoring parameter, trough concentrations (P=0.80). Conclusion Generic tacrolimus (Tacni) was not found to be bioequivalent to the original formulation in elderly renal transplant recipients. The significantly higher systemic exposure of tacrolimus, despite similar trough concentrations, may in the long run increase the risk of adverse effects.

Pharmacodynamics of mycophenolic acid in CD4+ cells: a single-dose study of IMPDH and purine nucleotide responses in healthy individuals.

Mycophenolate mofetil is used in rejection prophylaxis after allograft transplantation. The highly variable pharmacokinetics and pharmacodynamics (PD) of the active moiety mycophenolic acid (MPA) render this drug attractive for therapeutic monitoring. The aim of this study was to characterize the exposure-response relationship for MPA to guide future strategies for individualized therapy based on PD monitoring. A single-dose (100, 250, 500, and 1000 mg mycophenolate mofetil) crossover exposure-response study of MPA PD in CD4+ cells was performed in 5 healthy individuals. The activity of inosine 5′-monophosphate dehydrogenase (IMPDH) at time 0 ranged from 1.2 to 7.2 pmol per 106 cells/min. IMPDH was strongly inhibited by MPA; MPA EC50 (concentration required for 50% inhibition) of 2.3 mg/L was determined by a pooled data analysis. Decreased IMPDH gene expression was associated with the exposure to MPA. There were no immediate reductions of guanine nucleotides. On the contrary, a trend toward increased guanosine triphosphate was observed. IMPDH activity AUC0-12h approached maximum reduction at MPA AUC0-12h 22 mg·h/L (corresponding to the 500 mg dose), whereas plasma concentrations exceeding approximately 6 mg/L did not further increase the IMPDH inhibition. The results suggest that guanine nucleotides in circulating lymphocytes may not serve as immediate response biomarkers to MPA. Strategies for preventing over- or underexposure to MPA may be developed by means of IMPDH activity combined with MPA concentration measurement.

Determination of cyclosporine, tacrolimus, sirolimus and everolimus by liquid chromatography coupled to electrospray ionization and tandem mass spectrometry: Assessment of matrix effects and assay performance

Abstract Objective. The immunosuppressants cyclosporine, tacrolimus, sirolimus and everolimus are used in rejection prophylaxis after transplantation. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has become a widely used methodology for monitoring of the drug levels to ensure therapeutic exposure. The main objective of the study was to evaluate the existence and potential influence of matrix effects on LC-MS/MS measurements of the immunosuppressants in clinical blood samples. Methods. The samples were prepared by protein precipitation and thereafter analysed by reversed-phase chromatography coupled to MS/MS via an electrospray interface. Assay performance including within- and between-series imprecision and deviations from external controls were examined. Elution of overall matrix components and glycerophosphocholines were investigated. The MS/MS signals were monitored in post-column infusion experiments, and post-precipitation addition of compounds provided a basis for quantification of the matrix effects. The influence of matrix effects on assay performance was investigated after dilution of quality controls with blood from multiple individuals. Results. Between-series coefficients of variation were ≤ 5.1, ≤ 6.6, ≤ 11.0 and ≤ 7.4 %, and the mean deviations from external controls were −10.3, −6.7, 15.6 and 4.3% for cyclosporine, tacrolimus, sirolimus and everolimus, respectively. The elution of matrix components including glycerophosphocholines overlapped to some extent with the target compounds, and the average ion suppression ranged from 8.5–21%. However, the drugs and internal standards were correspondingly influenced. Conclusion. The internal standards consistently corrected the between-individual variability of matrix effects. These findings consolidate the reliability of the assay.

Mycophenolate pharmacokinetics and inosine monophosphate dehydrogenase activity in liver transplant recipients with an emphasis on therapeutic drug monitoring

Abstract Background. The pharmacokinetics of the immunosuppressant mycophenolic acid (MPA) demonstrates high inter- and intra-patient variability. Variation in the binding of MPA to albumin has been postulated to be an important factor in this variability, and monitoring of free MPA has been suggested to improve therapeutic drug monitoring (TDM) of MPA. Inosine monophosphate dehydrogenase (IMPDH) is the target enzyme for MPA, therefore the IMPDH activity in lymphocytes can serve as a marker of the MPA-specific response. This study aimed to explore how the albumin concentration influences the free concentration of MPA in liver transplant recipients and to assess whether alteration in the free MPA influences IMPDH activity in CD4 + cells. Methods. Blood samples were taken from 20 liver transplant recipients on two separate occasions (days 3–5 and 16–21). Total and free concentrations of MPA, and IMPDH activity were measured during the first 4 h of each dose interval. Results. Albumin levels correlated with the free fraction of MPA. However, the total MPA and free MPA were equal predictors of the immunosuppressive response as defined by IMPDH activity. Conclusion. Total and free MPA are equally good predictors of the immunosuppressive effect exerted by MPA as defined by IMPDH activity. IMPDH activity measurements represent a promising approach to TDM in patients treated with MPA.

No change in insulin sensitivity in renal transplant recipients converted from standard to once-daily prolonged release tacrolimus

BACKGROUND New-onset diabetes after transplantation may be associated with the use of tacrolimus (Tac) causing impaired insulin release or reduced insulin sensitivity. Such effects have not been studied in renal transplant recipients receiving traditional twice-daily tacrolimus (TacBID) and then compared to the new once-daily prolonged release formulation of tacrolimus (TacOD). METHODS We performed a prospective crossover study of 20 stable non-diabetic renal transplant recipients. All patients underwent one hyperglycaemic clamp on TacBID (3.8 ± 2.2 mg/day) and a new clamp 4-6 weeks after a 1:1 mg/day switch to TacOD (4.0 ± 2.8 mg/day). RESULTS Tac trough concentrations decreased from 6.6 ± 2.9 to 5.4 ± 1.4 μg/mL (P = 0.037) and Tac max from 21.3 ± 8.4 to 15.2 ± 3.5 μg/L (P = 0.001). Tac AUC(0-24) was reduced from 265 ± 112 to 218 ± 47 μg × h/L (P = 0.12). The hyperglycaemic clamp did not detect any change in insulin sensitivity index after conversion [0.26 ± 0.21 versus 0.26 ± 0.25 μmol/min/kg/(pmol/L insulin), P = 0.99] nor any change in first (334 ± 274 versus 353 ± 248 μIU × min/mL, P = 0.41) or second phase insulin secretion (224 ± 155 versus 263 ± 210 μIU × min/mL/mmol glucose, P = 0.60) on TacBID versus TacOD. CONCLUSIONS Conversion from standard TacBID to TacOD on a 1:1 mg basis is safe. In spite of a reduced Tac exposure, there was no change in insulin release or sensitivity in renal transplant recipients.

Rimonabant affects cyclosporine A, but not tacrolimus pharmacokinetics in renal transplant recipients.

Background. Obesity is a common problem following renal transplantation. Rimonabant, a cannabinoid-1 receptor blocker, offers a new approach for reducing obesity. Methods. The potential pharmacokinetic interaction between rimonabant and cyclosporine A (CsA, n=10) and tacrolimus (Tac, n=8) was assessed in stable renal transplant recipients 6.2 (0.9–21.7) years posttransplant. A 12-hour pharmacokinetic profile was obtained before and after two months of concomitant treatment with 20 mg rimonabant each morning. Results. Rimonabant treatment induced a moderate, but significant increase in CsA AUC0–12 (19.8±16.1 %, P=0.005). Cmax and C2 values tended to increase whereas C0 remained unaffected. Tac pharmacokinetics was not significantly affected by rimonabant treatment. Eleven of 18 patients experienced adverse events. Two patients reported depressions and one reported severe nightmares. Conclusions. The effect on CsA pharmacokinetics is probably of marginal clinical relevance since trough concentrations were unaltered, but CsA concentrations should probably be more closely monitored if rimonabant treatment is initiated, preferably by C2 monitoring.

Mycophenolate pharmacokinetics and pharmacodynamics in belatacept treated renal allograft recipients – a pilot study

BackgroundMycophenolic acid (MPA) is widely used as part of immunosuppressive regimens following allograft transplantation. The large pharmacokinetic (PK) and pharmacodynamic (PD) variability and narrow therapeutic range of MPA provide a potential for therapeutic drug monitoring. The objective of this pilot study was to investigate the MPA PK and PD relation in combination with belatacept (2nd generation CTLA4-Ig) or cyclosporine (CsA).MethodsSeven renal allograft recipients were randomized to either belatacept (n = 4) or cyclosporine (n = 3) based immunosuppression. Samples for MPA PK and PD evaluations were collected predose and at 1, 2 and 13 weeks posttransplant. Plasma concentrations of MPA were determined by HPLC-UV. Activity of inosine monophosphate dehydrogenase (IMPDH) and the expressions of two IMPDH isoforms were measured in CD4+ cells by HPLC-UV and real-time reverse-transcription PCR, respectively. Subsets of T cells were characterized by flow cytometry.ResultsThe MPA exposure tended to be higher among belatacept patients than in CsA patients at week 1 (P = 0.057). Further, MPA concentrations (AUC0–9 h and C0) increased with time in both groups and were higher at week 13 than at week 2 (P = 0.031, n = 6). In contrast to the postdose reductions of IMPDH activity observed early posttransplant, IMPDH activity within both treatment groups was elevated throughout the dosing interval at week 13. Transient postdose increments were also observed for IMPDH1 expression, starting at week 1. Higher MPA exposure was associated with larger elevations of IMPDH1 (r = 0.81, P = 0.023, n = 7 for MPA and IMPDH1 AUC0–9 h at week 1). The maximum IMPDH1 expression was 52 (13–177)% higher at week 13 compared to week 1 (P = 0.031, n = 6). One patient showed lower MPA exposure with time and did neither display elevations of IMPDH activity nor IMPDH1 expression. No difference was observed in T cell subsets between treatment groups.ConclusionThe significant influence of MPA on IMPDH1 expression, possibly mediated through reduced guanine nucleotide levels, could explain the elevations of IMPDH activity within dosing intervals at week 13. The present regulation of IMPDH in CD4+ cells should be considered when interpreting measurements of IMPDH inhibition.