Olga Millán

Effect of mycophenolate mofetil on immune response and plasma and lymphatic tissue viral load during and after interruption of highly active antiretroviral therapy for patients with chronic HIV infection a randomized pilot study

Summary: The main goal of this study was to assess the role of mycophenolate mofetil (MMF) during interruption of highly active antiretroviral therapy (HAART). Seventeen patients with early-stage chronic HIV type 1 infection were treated with HAART for 12 months. They were then randomized (day 0) to receive MMF (HAART–MMF group, n = 9) or to continue the regimen (HAART group, n = 6) for 120 additional days. At day 120 in the HAART–MMF group, HAART was discontinued, and MMF administration was continued. The primary end point of the study was the number of individuals maintaining a plasma viral load (VL) set point of <200 copies/mL after at least 6 months off HAART. At day 120, all patients in both groups had undetectable plasma VLs. After 6 months off HAART, 5 of 9 patients in the HAART–MMF group versus 1 of 6 patients in the HAART group maintained a plasma VL of <200 copies/mL (P = 0.28). According to the ability of their plasma to inhibit cellular proliferation, patients were reclassified and divided into an inhibition group (n = 6) and a no inhibition group (n = 9). The doubling time of VL rebound was significantly higher in the inhibition group (mean ± SE, 10.22 ± 1.3) than in the no inhibition group (mean ± SE, 4.6 ± 1.6; P = 0.03). Moreover, 5 of 6 patients in the inhibition group maintained a plasma VL of <200 copies/mL versus 1 of 9 patients in the no inhibition group (P = 0.005) after 6 months off HAART. We found that combining MMF and HAART delayed VL rebound and improved control of viral replication without HAART but only when inhibition of lymphocyte proliferation was achieved.

Sequential determination of pharmacokinetics and pharmacodynamics of mycophenolic acid in liver transplant patients treated with mycophenolate mofetil.

Background. In liver transplantation, mycophenolate mofetil (MMF) is habitually administered using fixed doses. We assessed whether mycophenolic acid (MPA) monitoring could be advisable in liver transplant patients. Methods. In 15 liver transplant patients receiving tacrolimus, daclizumab and MMF (1 g bid, orally), we determined the 12-hour plasma MPA pharmacokinetic profile after one dose of MMF at days 6, 10, and 16, and months 3 and 6. The inhibitory capacity of serum MPA on proliferation of CEM cells, a cell line insensitive to other immunosuppressants, was also determined. Results. A large interindividual variability in MPA profiles was observed at any time. Regardless of a gradual increase in individual MPA AUC and C0 over time following transplantation, a substantial proportion of patients had these parameters below the ranges recommended in other organ transplantations throughout the study. When MPA AUC and C0 were within the recommended ranges, CEM proliferation was inhibited by almost all serum samples, but when these pharmacokinetic parameters were below the recommended ranges, CEM proliferation was very variable and, therefore, unpredictable. No relationship between MPA pharmacokinetics and the efficacy of MMF could be established (only one patient developed rejection), probably due to the concomitant administration of tacrolimus and daclizumab. Gastrointestinal symptoms were the only adverse events with a significant relationship with MPA levels. Conclusions. During the first postoperative months, exposure to MPA is low in a considerable proportion of liver transplant patients receiving MMF at a fixed dose of 1 g bid. MPA monitoring appears necessary in these patients.

Intracellular IFN-γ and IL-2 expression monitoring as surrogate markers of the risk of acute rejection and personal drug response in de novo liver transplant recipients

Biomarker monitoring is needed in transplantation to reflect individual response to immunosuppressive drugs and graft outcome. We evaluated intracellular expression and soluble production of interferon-(IFN)-γ and interleukin-(IL)-2 as predictive biomarkers of acute rejection (AR) and personal drug response. Pharmacokinetic-pharmacodynamic profiles were determined in 47 de novo liver recipients treated with tacrolimus, mycophenolate mofetil and prednisone. Of the 47 patients, AR occurred in nine. There were no differences in drug concentrations between rejectors and non-rejectors. A pre-transplantation cut-off value of 55.80% for %CD8(+)-IFN-γ(+) identified patients at high risk of AR with a sensitivity of 75% and a specificity of 82%. In the first week post-transplantation, patients with a % inhibition for soluble IFN-γ, %CD8(+)-IFN-γ(+) and %CD8(+)-IL2(+) lower than 40% developed AR, showing low susceptibility to immunosuppressive drugs. Therefore, effector-T-cell response monitoring may help physicians to identify personal response to treatment and patients at high risk of AR.

Biomarkers of immunoregulatory status in stable liver transplant recipients undergoing weaning of immunosuppressive therapy

Biomarkers that reflect immune response recovery and predict clinical events during withdrawal or minimization of immunosuppressive therapy have not been evaluated. This study aimed to evaluate whether immune response recovers after withdrawal of long-term immunosuppressive treatment in stable liver transplant patients and to determine whether specific biomarkers reflect immune response reactivity and predict rejection. Pharmacokinetic-pharmacodynamic profiles were determined in 24 patients and 80 healthy donors before immunosuppressive treatment reduction began, at 50%, and at complete withdrawal. In patients who rejected, effector-T-cell response mediated by soluble IFN-γ, %CD4(+)IFN-γ and %CD8(+)IL-2/IFN-γ were significantly increased, while TGF-β1 production and the TGF-β1/IFN-γ ratio were significantly decreased. In patients with rejection, soluble IFN-γ and %CD8(+)IL-2 were significantly higher before immunosuppressive treatment was reduced. Further studies are required, but this battery of biomarkers performed in whole blood could be a useful tool to monitor immunosuppressive treatment minimization or withdrawal protocols and identify patients at increased risk of rejection.

Is the intracellular ATP concentration of CD4+ T-Cells a predictive biomarker of immune status in stable transplant recipients?

Background. Intracellular ATP (iATP) production in CD4+-T cells has been proposed as a biomarker for routine personalized monitoring in transplant recipients. The aim of our study was to measure this biomarker in adult renal and liver transplant recipients receiving moderate immunosuppressive treatment (IST) during the maintenance period and to evaluate whether this biomarker can effectively predict clinical status (stability, rejection, or infection) and drug effect. Methods. iATP concentration and trough blood levels of IST were evaluated in 84 adult transplant recipients (50 renal and 34 liver) during the maintenance period. One hundred and fifty healthy donors were also recruited as a control group. In addition, 22 of 34 liver transplant recipients were included in a clinical study in which IST was withdrawn gradually until total elimination. Results. Most of the patients evaluated had iATP levels in the moderate immune response zone during a posttransplantation maintenance period with no relevant clinical events. iATP levels were significantly lower in transplant patients with infection (n=3) than in those free of infection. In the IST withdrawal study, 10 of 22 liver transplant recipients have achieved complete withdrawal and 12 receive 50% of IST. So far, there have been eight rejections once IST began to be withdrawn. None of these patients had iATP in the strong immune response zone (risk of rejection). Conclusions. iATP seems to be more effective in identifying overimmunosuppressed patients with a high risk of adverse events (IST-safety) than in identifying those with a high risk of rejection (IST-efficacy) and should be used in combination with other biomarkers of immunosuppressive effect to improve the efficacy of IST.

Should IFN-γ, IL-17 and IL-2 be considered predictive biomarkers of acute rejection in liver and kidney transplant? Results of a multicentric study.

Acute rejection (AR) remains a major challenge in organ transplantation, and there is a need for predictive biomarkers. In the present multicenter study, we prospectively examined a series of biomarkers in liver and kidney recipients. Intracellular expression of IFN-γ, IL-17 and IL-2 and IL-17 soluble production were evaluated both pre-transplantation and post-transplantation (1st and 2nd week, 1st, 2nd and 3rd month). 142 transplant patients (63 liver/79 kidney) were included in the study. Twenty-eight recipients (14 liver/14 kidney) developed AR. Pre- and post-transplantation intracellular expression of %IFN-γ(+) in CD4(+)CD69(+) and in CD8(+)CD69(+) and soluble IL17 identified liver and kidney transplant patients at high risk of AR. Pre-transplantation, %IL-2(+) in CD8(+)CD69(+) also identified kidney patients at high risk. We constructed pre- and post-transplantation risk prediction models, based on a composite panel of biomarkers, which could provide the basis for future studies and will be a useful tool for the selection and adjustment of immunosuppressive treatments.

Pharmacokinetic and pharmacodynamic correlations of cyclosporine therapy in stable renal transplant patients: evaluation of long-term target C2

We investigated the relationship between the pharmacokinetics and pharmacodynamics of cyclosporine in 15 stable renal transplant patients in order to define an effective and safe therapeutic range. The area under the curve of the first 4 h (AUC0−4), trough (C0) and 2 h (C2) levels showed median values of 1655 ng×h/ml, 114 ng/ml and 384 ng/ml, respectively. C2 showed a strong correlation with AUC0–4 (r=0.942, p=0.0005). C0 correlated poorly with C2 and AUC0–4 (r=0.596, p=0.019 and r=0.538, p=0.031, respectively). Calcineurine activity (CNa) was 6.74% at 0 h and 3.90% at 2 h, representing significant reductions (82% and 89.6%, respectively; p<0.0005) compared with normal healthy controls (median basal value 37.4%). IL-2 production was 349 pg/ml at 0 h and 276.35 pg/ml at 2 h; both results were significantly lower (reductions of 44.5% and 56.1%, respectively; p=0.04 and 0.005) than the controls of 629.1 pg/ml. IFN-γ at 2 h post-dose (8.16 UI/ml) was significantly lower (72.1% reduction, p=0.005) than in controls (29.2 UI/ml). There was a good correlation between CNa and IFN-γ production, particularly at 2 h post-dose (r=0.537, p=0.007), and a fair correlation between CNa and IL-2 concentration (p=0.030, r=0.426). C2 showed an inverse significant correlation with CNa (Spearmans p=0.000, r=−0.753), IL-2 (p=0.000, r=−0.725) and IFN-γ (p=0.000, r=−0.701) production. In treated patients, the Emax inhibitory sigmoidal model showed that a C2 of 279 ng/ml was needed to achieve a 50% inhibition (EC50) of IL-2 and INF-γ production. The results demonstrated a significant inhibition of calcineurin activity and IL-2 and IFN-γ production in patients receiving cyclosporine monotherapy compared to healthy controls. A median C2 value of 384 ng/ml was associated with a good degree of inhibition of CNa and IL-2 and IFN-γ synthesis, and the lack of rejection episodes and relevant toxicity.

T-cell function monitoring in stable renal transplant patients treated with sirolimus monotherapy.

AbstractBackground: Sirolimus is an agent with lymphocyte-specific features similar to those of calcineurin inhibitors but with a different mechanism of action and safety profile. To optimize the use of sirolimus-based immunosuppression, further investigation of appropriate pharmacokinetic (sirolimus exposure) and pharmacodynamic (sirolimus T-cell immunomodulator effect) monitoring is required to determine personalized target concentrations. Aim: The main objective of the study was to evaluate the feasibility and reproducibility of combined pharmacokinetic and pharmacodynamic monitoring and to apply biomarkers of immunosuppression in stable kidney transplant recipients receiving sirolimus monotherapy. Methods: Fourteen renal transplant patients treated with sirolimus monotherapy (median 2 years) were included in this study. Pharmacokinetic and pharmacodynamic parameters were evaluated in each patient three times: at inclusion in the study (day 1), then again at 3 and 6 months. Results: The median sirolimus concentration was 11.5 ng/mL. CD4+ T-cell adenosine triphosphate (ATP) concentrations (150 ng/mL) and interleukin (IL)-10 production (50.9 ng/mL) were significantly lower in treated patients than in healthy controls (n = 95) [301 ng/mL; 278 ng/mL, respectively]. Median inhibition of T-cell proliferation was 60% (31–96%) in treated patients. Interferon-γ and transforming growth factor-β production was found to be similar to those in the healthy controls. Our results suggest an association between low ATP and IL-10 concentrations and the presence of infection. Conclusions: The sequential measurement of these biomarkers in stable renal transplant recipients treated with monotherapy could be useful to evaluate the biological effect of sirolimus in each patient and to establish personalized therapy taking into account the individual response to the drug.

Impact of donor and recipient CYP3A5 and ABCB1 genetic polymorphisms on tacrolimus dosage requirements and rejection in Caucasian Spanish liver transplant patients

Studies of liver transplant (LT) patients, mainly in Asians, have evaluated the influence of the CYP3A5*1 allele and P‐glycoprotein gene ABCB1 on tacrolimus pharmacokinetics or biopsy‐proven acute rejection (BPAR) incidence, with no conclusive results. To investigate these issues, 98 Caucasian Spanish LT patients with tacrolimus, mycophenolate mofetil and steroids and 88 cadaveric donors were genotyped for the SNPs CYP3A5 6986G>A, ABCB1 1236C>T, ABCB1 2677G>A/T and ABCB1 3435C>T;. On day 7 post‐LT, patients with a native CYP3A5*1 allele had significantly lower tacrolimus trough concentrations C0 (P = .03) and dose‐adjusted concentrations C0/D (P = .02) than CYP3A5 *3/*3 homozygotes. Three months post‐LT, patients carrying a liver with CYP3A5*1 had significantly lower C0/D (P = .03) and took significantly higher tacrolimus doses (P = .03) than the corresponding *3/*3 homozygotes. ABCB1 SNPs showed no significant association with tacrolimus variables. The 3‐month incidence of BPAR was 10.2%, with no statistically significant differences related to CYP3A5 (14.3% in expresser vs. 9.5% in non‐expresser) or ABCB1 genotype of either patient or donor. We conclude that in Caucasian Spanish LT patients, a native or graft‐borne CYP3A5*1 allele tends to lower tacrolimus concentrations and increase dosage needs, but has no significant impact on the incidence of BPAR.

Pharmacokinetics and pharmacodynamics of low dose mycophenolate mofetil in HIV-infected patients treated with abacavir, efavirenz and nelfinavir.

BackgroundThe use of mycophenolate mofetil in combination with highly active antiretroviral therapy (HAART) has been proposed in order to inhibit HIV replication. Due to the low doses involved, pharmacokinetic-pharmacodynamic monitoring is recommended.ObjectiveThe aim of this study was to characterise the pharmacokinetic and pharmacodynamic monitoring of low doses of mycophenolate mofetil (0.25g twice daily) in HIV-infected patients treated with HAART and after programmed discontinuation of HAART, in order to assess whether low doses of this immunosuppressive agent provide a biological effect.MethodsMycophenolic acid (MPA) plasma levels (assessed by high-performance liquid chromatography) and the capacity of patients’ sera to inhibit cell line proliferation (assessed by 3H-thymidine uptake) were measured post-dose at 0, 20, 40 minutes and 1, 2, 4, 6, 8, 10 and 12 hours in nine HIV-infected patients treated with a combination of abacavir, nelfinavir and efavirenz (HAART) and mycophenolate mofetil 0.25g twice daily at days 7, 28, 120 and 150 (30 days without HAART) after the treatment initiation. A control group of eight patients was treated with HAART alone.ResultsIn the 35 post-dose curves analysed, no differences were found in MPA levels between days 7, 28, 120 and 150: area under the plasma concentration-time curve — mean value 15.3 mg · h/L, range 10.4−24.4 mg · h/L; minimum plasma concentration — mean value 0.60 mg/L, range 0.20–4.67 mg/L; maximum plasma concentration mean value 2.60 mg/L, range 0.94–7.98 mg/L. Pretreatment patients’ sera did not inhibit proliferation. Post-treatment patients’ sera inhibited proliferation to <40% in 25 of 35 curves at 0 hours (six of nine patients), in 34 of 35 curves at 1 hour, in 32 of 35 curves at 2 hours, in 22 of 35 curves at 4 hours, and in 8 of 35 curves at 12 hours. The MPA level versus proliferation inhibition had a concentration that produces 50% of the maximum drug effect (EC50) of 0.33 mg/L. Viral load at day 150 was >200 copies/mL in all control patients and in three of nine patients receiving mycophenolate mofetil. These three patients were the only ones repeatedly unable to inhibit pre-dose proliferation to <40%.ConclusionsMycophenolate mofetil pharmacokinetic profiles in HIV patients under HAART are not significantly different from those found in transplant patients. Sera from the majority of patients receiving low doses of mycophenolate mofetil inhibited lymphocyte proliferation during most of the inter-dose interval, despite low MPA plasma levels. For some patients, higher doses may be necessary: the capacity of sera to inhibit proliferation may help to identify these patients.