Guiding the starting dose of the once-daily formulation of tacrolimus in "de novo" adult renal transplant patients: a population approach.

Aims: The once-daily extended-release tacrolimus formulation (ER-Tac) has demonstrated similar efficacy and safety to the twice-daily immediate-release formulation (IR-Tac), but few population-based pharmacokinetic models have been developed in de novo kidney transplant patients to optimize doses. Therefore, this study aimed i) at developing a population pharmacokinetic model for ER-Tac in de novo adult kidney transplant patients ii) and identifying genetic factors and time-varying covariates predictive of pharmacokinetic variability to guide tacrolimus dosage during the early post-transplant period. Methods: A total of 1,067 blood tacrolimus concentrations from 138 kidney transplant patients were analyzed. A total of 29 out of 138 patients were intensively sampled for 24 h on the day 5 post-transplantation; meanwhile, for the remaining patients, concentrations were collected on days 5, 10, and 15 after transplantation. Tacrolimus daily doses and genetic and demographic characteristics were retrieved from the medical files. Biochemistry time-varying covariates were obtained on different days over the pharmacokinetic (PK) study. A simultaneous PK analysis of all concentrations was carried out using the non-linear mixed-effects approach with NONMEM 7.5. Results: A two-compartment model with linear elimination and delayed absorption best described the tacrolimus pharmacokinetics. Between-patient variability was associated with oral blood clearance (CL/F) and the central compartment distribution volume (Vc/F). Tacrolimus concentrations standardized to a hematocrit value of 45% significantly improved the model (p < 0.001). This method outperformed the standard covariate modeling of the hematocrit-blood clearance relationship. The effect of the CYP3A5 genotype was statistically (p < 0.001) and clinically significant on CL/F. The CL/F of patients who were CYP3A5*1 carriers was 51% higher than that of CYP3A5*1 non-carriers. Age also influenced CL/F variability (p < 0.001). Specifically, CL/F declined by 0.0562 units per each increased year from the value estimated in patients who were 60 years and younger. Conclusion: The 36% between-patient variability in CL/F was explained by CYP3A5 genotype, age, and hematocrit. Hematocrit standardization to 45% explained the variability of tacrolimus whole-blood concentrations, and this was of utmost importance in order to better interpret whole-blood tacrolimus concentrations during therapeutic drug monitoring. The dose requirements of CYP3A5*/1 carriers in patients aged 60 years or younger would be highest, while CYP3A5*/1 non-carriers older than 60 years would require the lowest doses.

Tools for a personalized tacrolimus dose adjustment in the follow-up of renal transplant recipients. Metabolizing phenotype according to CYP3A genetic polymorphisms versus concentration-dose ratio.

BACKGROUND AND JUSTIFICATION: The strategy of the concentration-dose (C/D) approach and the different profiles of tacrolimus (Tac) according to the cytochrome P450 polymorphisms (CYPs) focus on the metabolism of Tac and are proposed as tools for the follow-up of transplant patients. The objective of this study is to analyse both strategies to confirm whether the stratification of patients according to the pharmacokinetic behaviour of C/D corresponds to the classification according to their CYP3A4/5 cluster metabolizer profile. MATERIALS AND METHODS: 425 kidney transplant patients who received Tac as immunosuppressive treatment have been included. The concentration/dose ratio (C/D) was used to divide patients in terciles and classify them according to their Tac metabolism rate (fast, intermediate, and slow). Based on CYP3A4 and A5 polymorphisms, patients were classified into 3 metabolizer groups: fast (CYP3A5*1 carriers and CYP34A*1/*1), intermediate (CYP3A5*3/3 and CYP3A4*1/*1) and slow (CYP3A5*3/*3 and CYP3A4*22 carriers). RESULTS: When comparing patients included in each metabolizer group according to C/D ratio, 47% (65/139) of the fast metabolizers, 85% (125/146) of the intermediate and only 12% (17/140) of the slow also fitted in the homonym genotype group. Statistically lower Tac concentrations were observed in the fast metabolizers group and higher Tac concentrations in the slow metabolizers when compared with the intermediate group both in C/D ratio and polymorphisms criteria. High metabolizers required approximately 60% more Tac doses than intermediates throughout follow-up, while poor metabolizers required approximately 20% fewer doses than intermediates. Fast metabolizers classified by both criteria presented a higher percentage of times with sub-therapeutic blood Tac concentration values. CONCLUSION: Determination of the metabolizer phenotype according to CYP polymorphisms or the C/D ratio allows patients to be distinguished according to their exposure to Tac. Probably the combination of both classification criteria would be a good tool for managing Tac dosage for transplant patients.

CYP3A5*3 and CYP3A4*22 Cluster Polymorphism Effects on LCP-Tac Tacrolimus Exposure: Population Pharmacokinetic Approach.

The aim of the study is to develop a population pharmacokinetic (PopPK) model and to investigate the influence of CYP3A5/CYP3A4 and ABCB1 single nucleotide polymorphisms (SNPs) on the Tacrolimus PK parameters after LCP-Tac formulation in stable adult renal transplant patients. The model was developed, using NONMEM v7.5, from full PK profiles from a clinical study (n = 30) and trough concentrations (C0) from patient follow-up (n = 68). The PK profile of the LCP-Tac formulation was best described by a two-compartment model with linear elimination, parameterized in elimination (CL/F) and distributional (CLD/F) clearances and central compartment (Vc/F) and peripheral compartment (Vp/F) distribution volumes. A time-lagged first-order absorption process was characterized using transit compartment models. According to the structural part of the base model, the LCP-Tac showed an absorption profile characterized by two transit compartments and a mean transit time of 3.02 h. Inter-individual variability was associated with CL/F, Vc/F, and Vp/F. Adding inter-occasion variability (IOV) on CL/F caused a statistically significant reduction in the model minimum objective function MOFV (p < 0.001). Genetic polymorphism of CYP3A5 and a cluster of CYP3A4/A5 SNPs statistically significantly influenced Tac CL/F. In conclusion, a PopPK model was successfully developed for LCP-Tac formulation in stable renal transplant patients. CYP3A4/A5 SNPs as a combined cluster including three different phenotypes (high, intermediate, and poor metabolizers) was the most powerful covariate to describe part of the inter-individual variability associated with apparent elimination clearance. Considering this covariate in the initial dose estimation and during the therapeutic drug monitoring (TDM) would probably optimize Tac exposure attainments.

A prospective controlled, randomized clinical trial of kidney transplant recipients developed personalized tacrolimus dosing using model-based Bayesian Prediction.

For three decades, tacrolimus (Tac) dose adjustment in clinical practice has been calculated empirically according to the manufacturer's labeling based on a patient's body weight. Here, we developed and validated a Population pharmacokinetic (PPK) model including pharmacogenetics (cluster CYP3A4/CYP3A5), age, and hematocrit. Our study aimed to assess the clinical applicability of this PPK model in the achievement of Tac Co (therapeutic trough Tac concentration) compared to the manufacturer's labelling dosage. A prospective two-arm, randomized, clinical trial was conducted to determine Tac starting and subsequent dose adjustments in 90 kidney transplant recipients. Patients were randomized to a control group with Tac adjustment according to the manufacturer's labeling or the PPK group adjusted to reach target Co (6-10 ng/ml) after the first steady state (primary endpoint) using a Bayesian prediction model (NONMEM). A significantly higher percentage of patients from the PPK group (54.8%) compared with the control group (20.8%) achieved the therapeutic target fulfilling 30% of the established superiority margin defined. Patients receiving PPK showed significantly less intra-patient variability compared to the control group, reached the Tac Co target sooner (5 days vs 10 days), and required significantly fewer Tac dose modifications compared to the control group within 90 days following kidney transplant. No statistically significant differences occurred in clinical outcomes. Thus, PPK-based Tac dosing offers significant superiority for starting Tac prescription over classical labeling-based dosing according to the body weight, which may optimize Tac-based therapy in the first days following transplantation.

The Effect of Intracellular Tacrolimus Exposure on Calcineurin Inhibition in Immediate- and Extended-Release Tacrolimus Formulations.

Despite intensive monitoring of whole blood tacrolimus concentrations, acute rejection after kidney transplantation occurs during tacrolimus therapy. Intracellular tacrolimus concentrations could better reflect exposure at the site of action and its pharmacodynamics (PD). Intracellular pharmacokinetic (PK) profile following different tacrolimus formulations (immediate-release (TAC-IR) and extended-release (TAC-LCP)) remains unclear. Therefore, the aim was to study intracellular tacrolimus PK of TAC-IR and TAC-LCP and its correlation with whole blood (WhB) PK and PD. A post-hoc analysis of a prospective, open-label, crossover investigator-driven clinical trial (NCT02961608) was performed. Intracellular and WhB tacrolimus 24 h time-concentration curves were measured in 23 stable kidney transplant recipients. PD analysis was evaluated measuring calcineurin activity (CNA) and simultaneous intracellular PK/PD modelling analysis was conducted. Higher dose-adjusted pre-dose intracellular concentrations (C0 and C24) and total exposure (AUC0-24) values were found for TAC-LCP than TAC-IR. Lower intracellular peak concentration (Cmax) was found after TAC-LCP. Correlations between C0, C24 and AUC0-24 were observed within both formulations. Intracellular kinetics seems to be limited by WhB disposition, in turn, limited by tacrolimus release/absorption processes from both formulations. The faster intracellular elimination after TAC-IR was translated into a more rapid recovery of CNA. An Emax model relating % inhibition and intracellular concentrations, including both formulations, showed an IC50, a concentration to achieve 50% CNA inhibition, of 43.9 pg/million cells.

Comparison of Three Renal Function Formulas for Ganciclovir/Valganciclovir Dose Individualization in CMV-Infected Solid Organ Transplantation Patients Using a Population Approach.

BACKGROUND AND OBJECTIVE: The gold standard treatment of established cytomegalovirus infection or prevention in solid organ transplantation is the intravenous administration of ganciclovir (GCV) or oral administration of valganciclovir (VGCV), both adjusted to the renal function. In both instances, there is a high interindividual pharmacokinetic variability, mainly owing to the wide range of variation of both the renal function and body weight. Therefore, accurate estimation of the renal function is crucial for GCV/VGCV dose optimization. This study aimed to compare three different formulas for estimating the renal function in solid organ transplantation patients with cytomegalovirus infection, for individualizing antiviral therapy with GCV/VGCV, using a population approach. METHODS: A population pharmacokinetic analysis was performed using NONMEM 7.4. A total of 650 plasma concentrations obtained after intravenous GCV and oral VGCV administrations were analyzed, from intensive and sparse sampling designs. Three different population pharmacokinetic models were built with the renal function given by Cockcroft-Gault, Modification of Diet in Renal Disease, or Chronic Kidney Disease EPIdemiology Collaboration (CKD-EPI) formulas. Pharmacokinetic parameters were allometrically scaled to body weight. RESULTS: The CKD-EPI formula was identified as the best predictor of between-patient variability in GCV clearance. Internal and external validation techniques showed that the CKD-EPI model had better stability and performed better compared with the others. CONCLUSIONS: The model based on the more accurate estimation of the renal function with the CKD-EPI formula and body weight as a size metric most used in the clinical practice can refine initial dose recommendations and contribute to GCV and VGCV dose individualization when required in the prevention or treatment of cytomegalovirus infection in solid organ transplantation patients.

Cardiovascular Risk Following Conversion to Belatacept From a Calcineurin Inhibitor in Kidney Transplant Recipients: A Randomized Clinical Trial.

Rationale & Objective: In kidney transplant recipients (KTRs), a belatacept-based immunosuppressive regimen is associated with beneficial effects on cardiovascular (CV) risk factors compared with calcineurin inhibitor (CNI)-based regimens. Our objective was to compare the calculated CV risk between belatacept and CNI (predominantly tacrolimus) treatments using a validated model developed for KTRs. Study Design: Prospective, randomized, open-label, parallel-group, investigator-initiated, international multicenter trial. Setting & Participants: KTRs aged 18-80 years with a stable graft function (estimated glomerular filtration rate > 20 mL/min/1.73 m2), 3-60 months after transplantation, treated with tacrolimus or cyclosporine A, were eligible for inclusion. Intervention: Continuation with a CNI-based regimen or switch to belatacept for 12 months. Outcomes: Comparison of the change in the estimated 7-year risk of major adverse CV events and all-cause mortality, changes in traditional markers of CV health, as well as measures of arterial stiffness. Results: Among the 105 KTRs randomized, we found no differences between the treatment groups in the predicted risk for major adverse CV events or mortality. Diastolic blood pressure, measured both centrally by using a SphygmoCor device and peripherally, was lower after the belatacept treatment than after the CNI treatment. The mean changes in traditional cardiovascular (CV) risk factors, including kidney transplant function, were otherwise similar in both the treatment groups. The belatacept group had 4 acute rejection episodes; 2 were severe rejections, of which 1 led to graft loss. Limitations: The heterogeneous baseline estimated glomerular filtration rate and time from transplantation to trial enrollment in the participants. A limited study duration of 1 year. Conclusions: We found no effects on the calculated CV risk by switching to the belatacept treatment. Participants in the belatacept group had not only lower central and peripheral diastolic blood pressure but also a higher rejection rate. Funding: The trial has received a financial grant from Bristol-Myers Squibb. Trial Registration: EudraCT no. 2013-001178-20.

Prolonged-Release Once-Daily Formulation of Tacrolimus Versus Standard-of-Care Tacrolimus in de novo Kidney Transplant Patients Across Europe.

Background: Tacrolimus is the calcineurin inhibitor of choice for preventing acute rejection episodes in kidney transplant patients. However, tacrolimus has a narrow therapeutic range that requires regular monitoring of blood concentrations to minimize toxicity. A new once-daily tacrolimus formulation, LCP-tacrolimus (LCPT), has been developed, which uses MeltDose™ drug-delivery technology to control drug release and enhance overall bioavailability. Our study compared dosing of LCPT with current standard-of-care tacrolimus [immediate-release tacrolimus (IR-Tac) or prolonged-release tacrolimus (PR-Tac)] during the 6 months following de novo kidney transplantation. Comparisons of graft function, clinical outcomes, safety, and tolerability for LCPT versus IR-Tac/PR-Tac were also performed. Methods: Standard immunological risk patients with end-stage renal disease who had received a de novo kidney transplant were randomized (1:1) to LCPT (N = 200) or IR-Tac/PR-Tac (N = 201). Results: Least squares (LS) mean tacrolimus total daily dose from Week 3 to Month 6 was significantly lower for LCPT than for IR-Tac/PR-Tac. Although LS mean tacrolimus trough levels were significantly higher for LCPT than IR-Tac/PR-Tac, tacrolimus trough levels remained within the standard reference range for most patients. There were no differences between the groups in treatment failure measures or safety profile. Conclusion: LCPT can achieve similar clinical outcomes to other tacrolimus formulations, with a lower daily dose. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT02432833.

Dual Costimulatory and Coinhibitory Targeting with a Hybrid Fusion Protein as an Immunomodulatory Therapy in Lupus Nephritis Mice Models.

Systemic lupus erythematosus is a complex autoimmune disorder mostly mediated by B-cells in which costimulatory signals are involved. This immune dysregulation can cause tissue damage and inflammation of the kidney, resulting in lupus nephritis and chronic renal failure. Given the previous experience reported with CTLA4-Ig as well as recent understanding of the PD-1 pathway in this setting, our group was encouraged to evaluate, in the NZBWF1 model, a human fusion recombinant protein (Hybri) with two domains: CTLA4, blocking the CD28-CD80 costimulatory pathway, and PD-L2, exacerbating the PD-1-PD-L2 coinhibitory pathway. After achieving good results in this model, we decided to validate the therapeutic effect of Hybri in the more severe MRL/lpr model of lupus nephritis. The intraperitoneal administration of Hybri prevented the progression of proteinuria and anti-dsDNA antibodies to levels like those of cyclophosphamide and reduced the histological score, infiltration of B-cells, T-cells, and macrophages and immune deposition in both lupus-prone models. Additionally, Hybri treatment produced changes in both inflammatory-related circulating cytokines and kidney gene expression. To summarize, both in vivo studies revealed that the Hybri effect on costimulatory-coinhibitory pathways may effectively mitigate lupus nephritis, with potential for use as a maintenance therapy.

Preformed T cell alloimmunity and HLA eplet mismatch to guide immunosuppression minimization with tacrolimus monotherapy in kidney transplantation: Results of the CELLIMIN trial.

Personalizing immunosuppression is a major objective in transplantation. Transplant recipients are heterogeneous regarding their immunological memory and primary alloimmune susceptibility. This biomarker-guided trial investigated whether in low immunological-risk kidney transplants without pretransplant DSA and donor-specific T cells assessed by a standardized IFN-γ ELISPOT, low immunosuppression (LI) with tacrolimus monotherapy would be non-inferior regarding 6-month BPAR than tacrolimus-based standard of care (SOC). Due to low recruitment rates, the trial was terminated when 167 patients were enrolled. ELISPOT negatives (E-) were randomized to LI (n = 48) or SOC (n = 53), E+ received the same SOC. Six- and 12-month BPAR rates were higher among LI than SOC/E- (4/35 [13%] vs. 1/43 [2%], p = .15 and 12/48 [25%] vs. 6/53 [11.3%], p = .073, respectively). E+ patients showed similarly high BPAR rates than LI at 6 and 12 months (12/55 [22%] and 13/66 [20%], respectively). These differences were stronger in per-protocol analyses. Post-hoc analysis revealed that poor class-II eplet matching, especially DQ, discriminated E- patients, notably E-/LI, developing BPAR (4/28 [14%] low risk vs. 8/20 [40%] high risk, p = .043). Eplet mismatch also predicted anti-class-I (p = .05) and anti-DQ (p < .001) de novo DSA. Adverse events were similar, but E-/LI developed fewer viral infections, particularly polyoma-virus-associated nephropathy (p = .021). Preformed T cell alloreactivity and HLA eplet mismatch assessment may refine current baseline immune-risk stratification and guide immunosuppression decision-making in kidney transplantation.

Sustained Inhibition of Calcineurin Activity With a Melt-Dose Once-daily Tacrolimus Formulation in Renal Transplant Recipients.

Tacrolimus (Tac) is the cornerstone calcineurin inhibitor in transplantation. Extended-release Meltdose formulation (Tac-LCP) offers better bioavailability compared with immediate-release formulation (Tac-IR). We postulated that the less fluctuating pharmacokinetic (PK) profile of Tac-LCP might maintain a sustained inhibition of calcineurin activity (CNA) between dose intervals. Higher concentrations (peak plasma concentration (Cmax )) after Tac-IR may not result in a more potent CNA inhibition due to a capacity-limited effect. This study was aimed at evaluating the pharmacodynamic (PD)/PK profiles of Tac-IR compared with Tac-LCP. An open-label, prospective, nonrandomized, investigator-driven study was conducted. Twenty-five kidney transplant recipients receiving Tac-IR were switched to Tac-LCP. Before and 28 days after conversion, intensive CNA-PD and PK sampling were conducted using ultra-high-performance liquid chromatography-tandem accurate mass spectrometry. PD nonlinear mixed effects model was performed in Phoenix-WinNonlin. Statistically significant higher Cmax (P < 0.001) after Tac-IR did not result in lower CNA as compared with after Tac-LCP (P = 0.860). Tac-LCP showed a statistically more maintained CNA inhibition between dose intervals (area under the effect-time curve from 0 to 24 hours (AUE0-24h )) compared with Tac-IR, in which CNA returned to predose levels after 4 hours of drug intake (373.8 vs. 290.5 pmol RII·h/min·mg prot, Tac-LCP vs. Tac-IR; P = 0.039). No correlation was achieved between any PD and PK parameters in any formulations. Moreover, Tac concentration to elicit a 50% of the maximum response (half-maximal inhibitory concentration) was 9.24 ng/mL. The higher Cmax after Tac-IR does not result in an additional CNA inhibition compared with Tac-LCP attributable to a capacity-limited effect. Tac-LCP may represent an improvement of the PD of Tac due to the more sustained CNA inhibition during dose intervals.

Dual and Opposite Costimulatory Targeting with a Novel Human Fusion Recombinant Protein Effectively Prevents Renal Warm Ischemia Reperfusion Injury and Allograft Rejection in Murine Models.

Many studies have shown both the CD28-D80/86 costimulatory pathway and the PD-1-PD-L1/L2 coinhibitory pathway to be important signals in modulating or decreasing the inflammatory profile in ischemia-reperfusion injury (IRI) or in a solid organ transplant setting. The importance of these two opposing pathways and their potential synergistic effect led our group to design a human fusion recombinant protein with CTLA4 and PD-L2 domains named HYBRI. The objective of our study was to determine the HYBRI binding to the postulated ligands of CTLA4 (CD80) and PD-L2 (PD-1) using the Surface Plasmon Resonance technique and to evaluate the in vivo HYBRI effects on two representative kidney inflammatory models-rat renal IRI and allogeneic kidney transplant. The Surface Plasmon Resonance assay demonstrated the avidity and binding of HYBRI to its targets. HYBRI treatment in the models exerted a high functional and morphological improvement. HYBRI produced a significant amelioration of renal function on day one and two after bilateral warm ischemia and on days seven and nine after transplant, clearly prolonging the animal survival in a life-sustaining renal allograft model. In both models, a significant reduction in histological damage and CD3 and CD68 infiltrating cells was observed. HYBRI decreased the circulating inflammatory cytokines and enriched the FoxP3 peripheral circulating, apart from reducing renal inflammation. In conclusion, the dual and opposite costimulatory targeting with that novel protein offers a good microenvironment profile to protect the ischemic process in the kidney and to prevent the kidney rejection, increasing the animal's chances of survival. HYBRI largely prevents the progression of inflammation in these rat models.

Functional immune monitoring of BK Virus and donor-specific T-cell effector immune responses to guide treatment decision-making after kidney transplantation; an illustrative case report and literature review.

Differential diagnosis between Polyoma virus associated-nephropathy (PVAN) and T-cell mediated rejection (TCMR) might be challenging, as respective treatment approaches are totally opposite. Here we report the illustrative case of a kidney transplant recipient with PVAN who developed a persistent acute TCMR after full abrogation of viral infection through immunosuppression modulation. By simultaneous functional immune monitoring of BKV and donor-specific T-cell responses using IFN-γELISPOT assay, we retrospectively demonstrated the predominant effector mechanisms responsible of allograft injury and thus, potential guidance for treatment decision-making. Furthermore, the evidence of an efficient T-cell alloimmunity abrogation accompanied by a sustained anti-viral response after sirolimus addition, promotes the potential benefit of converting patients to an mTOR-based immunosuppression in case of PVAN.

Mycophenolic acid interferes the transcriptional regulation and protein trafficking of maturation surface markers in dendritic cells.

BACKGROUND: The ability of dendritic cells (DCs) to regulate adaptive immunity makes them interesting cells to be used as therapeutic targets modulating alloimmune responses. Mycophenolic acid (MPA) is an immunosuppressor commonly used in transplantation, and its effect on DCs has not been fully investigated. METHODS: Monocyte-derived DCs were obtained from healthy volunteers and cultured for 7 days. Cells were treated with MPA on day 2 and matured by lipopolysaccharide (LPS) stimulation. Functionality of mature DC (mDCs) was evaluated by allogeneic mixed lymphocytes reaction. Surface expression of maturation markers (CD40, CD83, CD86, and ICAM-1) was analyzed in both immature DCs (iDCs) and mDCs by flow cytometry. To assess transcriptional regulation and protein subcellular location, RT-PCR and confocal microscopy were used, respectively. RESULTS: MPA decreased surface expression of all maturation markers in mDCs and significantly abrogated DCs-induced allogeneic T-cell proliferation after MPA pre-treatment. In iDCs, the reduced surface protein expression after MPA paralleled with mRNA downregulation of their genes. In mDCs, the mRNA levels of ICAM-1, CD40 and CD83 were enhanced in MPA-treated mDCs with an increase in the expression of CD83 and ICAM-1 near the Golgi compared to non-treated mDCs. In contrast, mRNA levels of CD86 were diminished after MPA treatment. CONCLUSIONS: The reduced surface markers expression in mDCs exerted by MPA produced a decline in their capacity to activate immune responses. Moreover, the inhibition of guanosine-derived nucleotide biosynthesis by MPA treatment leads to DC maturation interference by two mechanisms depending on the marker, transcriptional downregulation or disrupted intracellular protein trafficking.

Cellular Immunity to Predict the Risk of Cytomegalovirus Infection in Kidney Transplantation: A Prospective, Interventional, Multicenter Clinical Trial.

BACKGROUND: Improving cytomegalovirus (CMV) immune-risk stratification in kidney transplantation is highly needed to establish guided preventive strategies. METHODS: This prospective, interventional, multicenter clinical trial assessed the value of monitoring pretransplant CMV-specific cell-mediated immunity (CMI) using an interferon-γ release assay to predict CMV infection in kidney transplantation. One hundred sixty donor/recipient CMV-seropositive (D+/R+) patients, stratified by their baseline CMV (immediate-early protein 1)-specific CMI risk, were randomized to receive either preemptive or 3-month antiviral prophylaxis. Also, 15-day posttransplant CMI risk stratification and CMI specific to the 65 kDa phosphoprotein (pp65) CMV antigen were investigated. Immunosuppression consisted of basiliximab, tacrolimus, mycophenolate mofetil, and corticosteroids in 80% of patients, whereas 20% received thymoglobulin induction therapy. RESULTS: Patients at high risk for CMV based on pretransplant CMI developed significantly higher CMV infection rates than those deemed to be at low risk with both preemptive (73.3% vs 44.4%; odds ratio [OR], 3.44 [95% confidence interval {CI}, 1.30-9.08]) and prophylaxis (33.3% vs 4.1%; OR, 11.75 [95% CI, 2.31-59.71]) approaches. The predictive capacity for CMV-specific CMI was only found in basiliximab-treated patients for both preemptive and prophylaxis therapy. Fifteen-day CMI risk stratification better predicted CMV infection (81.3% vs 9.1%; OR, 43.33 [95% CI, 7.89-237.96]). CONCLUSIONS: Pretransplant CMV-specific CMI identifies D+/R+ kidney recipients at high risk of developing CMV infection if not receiving T-cell-depleting antibodies. Monitoring CMV-specific CMI soon after transplantation further defines the CMV infection prediction risk. Monitoring CMV-specific CMI may guide decision making regarding the type of CMV preventive strategy in kidney transplantation. CLINICAL TRIALS REGISTRATION: NCT02550639.

Noncanonical immunomodulatory activity of complement regulator C4BP(ß-) limits the development of lupus nephritis.

Lupus nephritis is a chronic autoimmune-inflammatory condition that can lead to end-stage kidney disease. Presently available immunosuppressive treatments for lupus nephritis are suboptimal and can induce significant side effects. Recently, we characterized a novel immunomodulatory activity of the minor isoform of the classical pathway complement inhibitor, C4BP(ß-). We show here that C4BP(ß-) treatment prevented the development of proteinuria and albuminuria, decreased significantly the formation of anti-dsDNA antibodies and, locally, mitigated renal glomerular IgG and C3 deposition and generation of apoptotic cells. There was a consequent histological improvement and increased survival in lupus-prone mice. The therapeutic efficacy of C4BP(ß-) was analogous to that of the broad-acting immunosuppressant cyclophosphamide. Remarkably, a comparative transcriptional profiling analysis revealed that the kidney gene expression signature resulting from C4BP(ß-) treatment turned out to be 10 times smaller than that induced by cyclophosphamide treatment. C4BP(ß-) immunomodulation induced significant downregulation of transcripts relevant to lupus nephritis indicating immunopathogenic cell infiltration, including activated T cells (Lat), B cells (Cd19, Ms4a1, Tnfrsf13c), inflammatory phagocytes (Irf7) and neutrophils (Prtn3, S100a8, S100a9). Furthermore, cytokine profiling and immunohistochemistry confirmed that C4BP(ß-), through systemic and local CXCL13 downregulation, was able to prevent ectopic lymphoid structures neogenesis in aged mice with lupus nephritis. Thus, due to its anti-inflammatory and immunomodulatory activities and high specificity, C4BP(ß-) could be considered for further clinical development in patients with systemic lupus erythematosus.

Validation and evaluation of four sample preparation methods for the quantification of intracellular tacrolimus in peripheral blood mononuclear cells by UHPLC-MS/MS.

Rejection and toxicity occur despite monitoring of tacrolimus blood levels during clinical routine. The intracellular concentration in lymphocytes could be a better reflection of the tacrolimus exposure. Four extraction methods for tacrolimus in peripheral blood mononuclear cells were validated and evaluated with UHPLC-MS/MS. Methods based on protein precipitation (method 1), solid phase extraction (method 2), phospholipids and proteins removal (method 3) and liquid-liquid extraction (method 4) were evaluated on linearity, lower limit of quantification (LLOQ), imprecision and bias. Validation was completed for the methods within these requirements, adding matrix effect and recovery. Linearity was 0.126 (LLOQ)-15 µg/L, 0.504 (LLOQ)-15 µg/L and 0.298 (LLOQ)-15 µg/L with method 1, 2 and 3, respectively. With method 4 non-linearity and a LLOQ higher than 0.504 µg/L were observed. Inter-day imprecision and bias were ≤4.6%, ≤10.9%; ≤6.8%, ≤-11.2%; ≤9.4%, ≤10.3% and ≤44.6%, ≤23.1%, respectively, with methods 1, 2, 3 and 4. Validation was completed for method 1 and 3 adding matrix effect (7.6%; 15.0%) and recovery (8.9%; 10.8%), respectively. The most suitable UHPLC-MS/MS method for quantification of intracellular tacrolimus was protein precipitation due to the best performance characteristics and the least time-consuming rate and complexity.

Measurement of calcineurin activity in peripheral blood mononuclear cells by ultra-high performance liquid chromatography-tandem mass spectrometry. Renal transplant recipients application (pharmacodynamic monitoring).

BACKGROUND: Therapeutic drug monitoring of calcineurin inhibitor, tacrolimus (TAC), is routinely used in post-transplantation. Currently, measurement of calcineurin activity has been proposed as a promising clinical tool to evaluate efficacy and to optimize drug dosing. The main aim of our study was to develop a method to measure phosphatase calcineurin activity (CNA) in peripheral blood mononuclear cells (PBMCs) by ultra-high performance liquid chromatography-tandem mass spectrometry and to validate it following FDA and EMA guidelines. METHODS: This methodology is based on monitoring the Ca2+-dependent dephosphorylation of a phosphopeptide substrate. CNA was evaluated in 5 healthy volunteers and in 5 renal transplant patients receiving twice-daily formulation of TAC before drug intake. Moreover, we studied pharmacodynamic effect of TAC and blood concentrations of TAC in different drug dose intervals (0, 1, 3, 6 and 12 h). RESULTS: Our results showed linearity in the range 0.04-2.00 µM with a lower limit of quantification of 0.04 µM. Coefficients of variation and absolute relative biases were <4.3% and 10.3%, respectively. The mean recovery for peptide was 91.6 ±â€¯4.0%. Matrix effect study displayed ion suppression, and no carry-over and interferences were observed. There were no differences in CNA between healthy and TAC-treated patients. Furthermore, CNA showed maximum inhibition at 1 h after drug intake when TAC reached the highest blood concentration. CONCLUSIONS: This method improves the extraction phase of PBMCs and achieves faster determination compared to other techniques, bringing us closer to be applied in daily laboratory practice.