Mycophenolic Acid Research Articles

A validated UPLC-MS/MS method for the quantification of immunosuppressive drugs in peripheral blood mononuclear cells using liquid-liquid extraction with low temperature purification without complex pretreatment steps

Immunosuppressive drugs (ISDs) are given to avoid the allograft rejection after transplantation. The concentrations of ISDs should be closely monitored owing to their wide inter-individual variability in its pharmacokinetics and narrow therapeutic window. Currently, the whole blood concentration measurement is the major approach of therapeutic drug monitoring of clinical ISDs in organ transplantation. Its correlation with the efficacy of ISDs remains elusive. While the acute rejection after transplantation may occur even when whole-blood ISDs concentrations are within the target range. Since the site of action of ISDs are within the lymphocyte, direct measurement of drug exposure in target cells may more accurately reflect the clinical efficacy of ISDs. Although several methods have been developed for the peripheral blood mononuclear cells (PBMCs) extraction and drug concentration measurement, the complex pre-processing has limited the study of the relationship between intracellular ISDs concentrations and the occurrence of rejection. In this study, the extraction of ISDs in PBMCs was carried out by the liquid-liquid extraction with low temperature purification, without centrifugation. The lower limit of quantitation were 0.2 ng/mL for cyclosporine A, tacrolimus and sirolimus, 1.0 ng/mL for mycophenolic acid, and the within-run and between-run coefficient of variations were both less than 12.4 %. The calibration curves of mycophenolic acid had a linear range (ng/mL): 1.0–128.0 (r2 = 0.9992). The calibration curves of other three ISDs had a linear range (ng/mL): 0.2–20.48 (r2 > 0.9956). A total of 157 clinical samples were analyzed by the UPLC-MS/MS for ISDs concentration in blood or plasma ([ISD]blood or plasma) and the concentration within PBMCs ([ISD]PBMC). Although there was strong association between [ISD]PBMC and [ISD]blood or plasma, the large discrepancies between concentration within [ISD]blood or plasma and [ISD]PBMC were observed in a small proportion of clinical samples. The developed method with short analysis time and little amounts of blood sample can be successfully applied to therapeutic drug monitoring of ISDs in PBMCs for analysis of large numbers of clinical samples and is helpful to explore the clinical value of ISDs concentration in PBMCs.

Clinical validation of two volumetric absorptive microsampling devices to support home-based therapeutic drug monitoring of immunosuppression.

Dried blood volumetric absorptive microsamples (VAMS) may facilitate home-based sampling to enhance therapeutic drug monitoring after transplantation. This study aimed to clinically validate a liquid chromatography-tandem mass spectrometry assay using 2 VAMS devices with different sampling locations (Tasso-M20 for the upper arm and Mitra for the finger). Patient preferences were also evaluated. Clinical validation was performed for tacrolimus and mycophenolic acid by comparison of paired VAMS and venipuncture samples using Passing-Bablok regression and Bland-Altman analysis. Conversion of mycophenolic acid VAMS to serum concentrations was evaluated using haematocrit-dependent formulas and fixed correction factors defined a priori. Patients' perspectives, including useability, acceptability and feasibility, were also investigated using established questionnaires. Paired samples (n =50) were collected from 25 kidney transplant recipients. Differences for tacrolimus whole-blood concentration were within ±20% for 86 and 88% of samples from the upper arm and fingerstick, respectively. Using correction factors of 1.3 for the upper-arm and 1.47 for finger-prick samples, 84 and 76% of the paired samples, respectively, were within ±20% for mycophenolic acid serum concentration. Patient experience surveys demonstrated limited pain and acceptable useability of the upper-arm device. Tacrolimus and mycophenolic acid can be measured using 2 common VAMS devices with similar analytical performance. Patients are supportive of home-based monitoring with a preference for the Tasso-M20 device.

Mycotoxins in vegetating corn plants from experimental mono-sowing

The aim of present study was to reveal the mycotoxin contamination of vegetative maize (Zea mays L.) plants during the periods of leaf formation and panicle emergence. Early maturing hybrids of the varieties Krasnodarsky 194 MV, Ladozhsky 175 MV and Competence®, resistant to fungal diseases, were grown in the spring-summer period of 2023 on the experimental field of the Russian State Agrarian University – Moscow Agricultural Academy (Moscow) with sod-podzolic soil and the application of NPK fertilizers 16:16:16. For mycotoxicological analysis, aerial parts of plants were collected weekly from the phase of formation of the 3rd leaf (18 days after sowing. Samples of seedlings, leaves and stems (total number – 172), after drying, were ground in a laboratory mill and extracted with a mixture of acetonitrile and water in a volumetric ratio of 84:16 with a consumption of 10 ml per 1 g of sample. Mycotoxin content was determined in extracts after 10-fold dilution with phosphate-salt buffer solution pH 7.5 by indirect competitive enzyme immunoassay. T-2 toxin, deoxynivalenol, diacetoxiscirpenol, roridin A, sterigmatocystin and PR toxin were absent in the samples. Corn seedlings and leaves contained cyclopiazonic acid (CPA), emodin (EMO), mycophenolic acid (MPA), alternariol (AOL), ergot alkaloids (EA), aflatoxin B1 (AB1) and single samples – zearalenone (ZEN), ochratoxin A (OA) and citrinin (CIT) in concentrations from 16 up to 35 μg/kg. Fumonisins of group B are found only in early seedlings. The permanent contaminant of the stems was MPA, whereas EA and CIT were absent. In the seedlings and leaves of all hybrids, the detection of AOL and EA remained stable during the change of development phases, as did the average concentrations of CPA (about 100 μg/kg), AOL (from 17 to 27 μg/kg), EMO (35–58 μg/kg), MPA (28–41 μg/kg), EA (6–18 μg/kg) and AB1 (2 μg/kg). In the stems of plants in phases 7–9 of leaf and sweeping, variation in cases of detection of CPA, EMO, AOL, AB1 and OA was noted by varieties.

Limited Sampling Strategies Fail to Accurately Predict Mycophenolic Acid Area Under the Curve in Kidney Transplant Recipients and the Impact of Enterohepatic Recirculation.

Therapeutic drug monitoring for mycophenolic acid (MPA) is challenging due to difficulties in measuring the area under the curve (AUC). Limited sampling strategies (LSSs) have been developed for MPA therapeutic drug monitoring but come with risk of unacceptable performance. The authors hypothesized that the poor predictive performance of LSSs were due to the variability in MPA enterohepatic recirculation (EHR). This study is the first to evaluate LSSs models performance in the context of EHR. Adult kidney transplant recipients (n = 84) receiving oral mycophenolate mofetil underwent intensive MPA pharmacokinetic sampling. MPA AUC0-12hr and EHR were determined. Published MPA LSSs in kidney transplant recipients receiving tacrolimus were evaluated for their predictive performance in estimating AUC0-12hr in our full cohort and separately in individuals with high and low EHR. None of the evaluated LSS models (n = 12) showed good precision or accuracy in predicting MPA AUC0-12hr in the full cohort. In the high EHR group, models with late timepoints had better accuracy but low precision, except for 1 model with late timepoints at 6 and 10 hours postdose, which had marginally acceptable precision. For all models, the good guess of predicted AUC0-12hr (±15% of observed AUC0-12hr) was highly variable (range, full cohort = 19%-61.9%; high EHR = 4.5%-65.9%; low EHR = 27.5%-62.5%). The predictive performance of the LSS models varied according to EHR status. Timepoints ≥5 hours postdose in LSS models are essential to capture EHR. Models and strategies that incorporate EHR during development are required to accurately ascertain MPA exposure.

Immunosuppressive drugs and cancer risk

The use of immunosuppressive medications is an area of interest for organ transplantation. These medications have been repurposed for the treatment of cancer in recent years. In the context of cancer therapy, the use of immunosuppressive medications such as cyclosporine, tacrolimus, sirolimus, mycophenolate acid mofetil (MMF), mycophenolate, and azathioprine is discussed. Treatment for cancer frequently includes methods designed to use the immune system to find and destroy cancer cells. Cancer cells can, however, avoid immune surveillance and eradication because the tumor microenvironment frequently has immune-suppressive components. Drugs that suppress the immune system, which were initially designed to avoid organ rejection, have demonstrated potential for obstructing these inhibitory pathways and boosting antitumour immune responses. Tacrolimus and cyclosporine, two immunosuppressive medications frequently used in transplantation, have potential anticancer effects. The immune system and stifling tumor-promoting inflammatory responses, can prevent tumor growth, angiogenesis, and metastasis. Another immunosuppressive drug, sirolimus, has shown anticancer effects by reducing angiogenesis and tumor cell growth while promoting immune-mediated tumors. Mycophenolate medicines, such as MMF and mycophenolate, have strong immunosuppressive properties. By inhibiting the enzymes responsible for purine production, these enzymes aim to inhibit lymphocyte proliferation. By altering immune cell function and reducing tumor growth, these medications have demonstrated promise in the treatment of cancer. Azathioprine, a medication that suppresses the immune system and is frequently used to treat autoimmune illnesses, has also been investigated as a possible cancer treatment. Although immunosuppressive medications have potential in the treatment of cancer, their usage needs to be carefully addressed due to potential side effects and the delicate balance between immune suppression and antitumour action. The immune system plays a crucial role in identifying and eliminating abnormal cells, including those associated with cancer.

Development of an HPLC method using relative molar sensitivity for the measurement of blood concentrations of nine pharmaceutical compounds

We developed a reliable high-performance liquid chromatographic analysis method using a relative molar sensitivity (RMS) technique that does not require an authentic, identical reference analyte material to quantify blood serum carbamazepine, phenytoin, voriconazole, lamotrigine, meropenem, mycophenolic acid, linezolid, vancomycin, and caffeine levels for routine blood concentration measurements. Carbamazepine and caffeine were also used as non-analyte reference materials to calculate the RMS of each analyte. The RMS was calculated from the ratio of the slope of the calibration equation (analyte/non-analyte reference material), then used to quantify analytes in control serum samples spiked with carbamazepine, phenytoin, voriconazole, meropenem, mycophenolic acid, linezolid or vancomycin. In addition, the concentrations of these six drugs in control serum samples determined by the proposed RMS method agreed well with that obtained using a conventional method. The proposed RMS method is a promising tool for the clinical determination of nine drugs, given the accuracy, precision, and efficiency of quantifying these analytes.

Influence of UDP-Glucuronosyltransferase Polymorphisms on Mycophenolic Acid Metabolism in Renal Transplant Patients

This study aims to evaluate the effects of the UDP-glucuronosyltransferases (UGTs) polymorphisms on mycophenolic acid (MPA) metabolites in renal transplant patients. Total 11 single nucleotide polymorphisms (SNPs) of UGT1A1, UGT1A7, UGT1A8, UGT1A9, UGT1A10 and UGT2B7 were genotyped in 79 renal transplant patients. The associations of SNPs and clinical factors with dose-adjusted MPA area under the plasma concentration-time curve (AUC/D), the dose-adjusted concentration (C0/D) of MPAG and the dose-adjusted plasma concentration (C0/D) of AcMPAG were analyzed. In the univariate analysis, UGT1A1 rs4148323, age and anion gap were associated with MPA AUC/D. MPA AUC/D in patients with the GA genotype of UGT1A1 rs4148323 was higher than that in patients with the GG genotype. UGT1A1 rs4148323, UGT1A9 rs2741049 and clinical factors, including age, serum total bilirubin, adenosine deaminase, anion gap, urea, creatinine, were associated with MPAG C0/D. In addition, UGT2B7 rs7438135, UGT2B7 rs7439366, and UGT2B7 rs7662029 were associated with AcMPAG C0/D. Multiple linear regression analysis showed that UGT1A9 rs2741049 and indirect bilirubin were negatively correlated with MPAG C0/D (P=0.001; P=0.039). UGT2B7 rs7662029 was positively correlated with AcMPAG C0/D (P=0.008). In conclusion, the study shows a significant influence of UGT1A9 rs2741049 and UGT2B7 rs7662029 polymorphisms on the metabolism of MPA in vivo.

Acute Lupus Pneumonitis with Cytomegalovirus Co-infection in Patients with Lupus Nephritis

Introduction: Most patients with systemic lupus erythematosus (SLE) show signs of pulmonary involvement. The clinical manifestations of Lupus Pneumonitis are the similar to those of acute interstitial pneumonia. The use of cyclophosphamide in lupus nephritis (LN) is associated with high CMV titers. Ganciclovir is the main choice of therapy for CMV pneumonia infection. Case presentation: A 19-year-old female with previous history of lupus nephritis presented with worsening dyspnea, productive cough with yellowish sputum and hemoptysis. Following physical, laboratory and radiological examinations, the patient was diagnosed with acute lupus pneumonitis, with a differential diagnosis of pneumonia infection. During treatment in the intensive care unit (ICU), she was put on ventilator and received routine hemodialysis due to pulmonary edema. She was given intravenous antibiotics before the culture results came out, but there was no clinical improvement. Once the culture results returned negative, the antibiotics were discontinued and IV pulse methylprednisolone was started. There was significant clinical, radiological, and laboratory improvements. After discharge, the patient experienced hemoptysis again due to CMV pneumonia infection and was given ganciclovir therapy with satisfactory results. Conclusion: In patients with advanced LN and pulmonary involvement, distinguishing between infection and SLE flares may be challenging, which can cause dilemma in diagnosis and treatment decisions. Adequate oxygenation with ventilator, hemodialysis, and administration of ganciclovir and mycophenolic acid provides significant improvements in patient care.

Pharmacodynamic Effect of mTOR Inhibition-based Immunosuppressive Therapy on T- and B-cell Subsets After Renal Transplantation.

The mammalian target of rapamycin inhibitor (mTORi) therapy after kidney transplantation is solely monitored pharmacokinetically, not necessarily reflecting PI3K-Akt-mTOR pathway blockade efficacy leading to potential under-or overimmunosuppression. In this cross-sectional study, phosphoflow cytometry was used to determine the efficacy of mTOR inhibition in peripheral T- and B-lymphocyte subsets by assessing p70S6 kinase (p70S6K) phosphorylation in renal transplant recipients upon treatment with a combination of either mTORi and calcineurin inhibitors (n = 18), or mTORi with mycophenolic acid (n = 9). Nine dialysis patients with end-stage renal disease and 17 healthy age-matched volunteers served as controls. mTORi treatment reduced p70S6K phosphorylation in CD4+, CD8+ T, and CD19+ B cells compared with healthy controls (HCs). Subpopulation analysis of CD4+ T cells and CD19+ B cells revealed a significant reduction of p70S6K phosphorylation in CD4+CD45RA-CD25- Th cells (P < 0.05), CD24hiCD38hi transitional B cells (P < 0.001), CD24+CD38- memory B cells (P < 0.001), and CD24intCD38int-naive B cells (P < 0.05) upon mTORi treatment, whereas CD4+CD45RA-CD25++CD127- regulatory T cells and CD24-CD38hi plasmablasts were not affected. Compared with mTORi + mycophenolic acid therapy, mTORi + calcineurin inhibitor treatment exhibited an even stronger inhibition of p70S6K phosphorylation in CD4+CD45RA-CD25- Th cells and CD8+ T cells. However, trough levels of mTORi did not correlate with p70S6K phosphorylation. mTORi selectively inhibited p70S6K phosphorylation in select lymphocyte subtypes. Assessing p70S6K phosphorylation by phosphoflow cytometry may serve as an approach to understand cell subset specific effects of mTORi providing detailed pharmacodynamic information for individualizing immunosuppression.

Therapeutic drug monitoring of mycophenolic acid and azole antifungals on two distinct LC-MS/MS instruments

BackgroundAs an active metabolite of a commonly prescribed immunosuppressant, mycophenolic acid (MPA) levels are often monitored to prevent organ rejection following a transplant. Triazoles are often prescribed for treatment of invasive fungal infections in immunocompromised patients. Due to the variability in individual pharmacokinetics and drug-drug interactions, therapeutic drug monitoring is recommended for triazole antifungals. A multiplex LC-MS/MS assay has been developed that can quantify both MPA and triazole drugs in serum. MethodsA sample preparation procedure was established to spike in internal standard compounds and precipitate proteins. Reversed-phase chromatographic separation was performed on a C18 column with an analysis time of five minutes per sample. The mass spectrometer was operated in multiple reaction monitoring mode. The method was validated on two HPLC systems interfaced with either a Triple Quad 6500 or an API 4000 instrument. ResultsThe multiplex assay was linear over a wide dynamic range with analyte measurable ranges of 0.4–48 μg/mL for MPA, 0.1–12 μg/mL for posaconazole, and 0.2–24 μg/mL for voriconazole, itraconazole, hydroxyitraconazole, and isavuconazole. The between-day and intraday imprecisions were less than 10 %. Limits of detection were below 0.04 ug/mL with limits of quantitation below 0.2 μg/mL. Method comparison studies against the current in-house method met acceptance criteria. The instrument comparison study demonstrated a strong correlation between data collected from the two systems. ConclusionA robust multiplex LC-MS/MS assay was developed and validated for monitoring MPA and triazoles drug levels in a clinical laboratory. The assay performance on two distinct instruments was acceptable and comparable.

Vibrational study of mycophenolic acid and its quantification with electrochemically assisted surface-enhanced Raman spectroscopy

Mycophenolic acid (MPA) is an immunosuppressant widely used in transplantations, for which therapeutic drug monitoring (TDM) has proven beneficial. There is furthermore a need to develop affordable, rapid, and benchtop analytical methods to perform TDM of MPA in a point-of-need setting. In this paper, we report for the first time the Raman fingerprint region and surface-enhanced Raman spectroscopy (SERS) vibrations of MPA through density functional theory calculations. The output of these simulations was compared with experimental data from electrochemically assisted SERS, i.e. application of a well-defined potential or sequence of potentials in SERS assays. The systematic electrochemically assisted SERS experimental study was carried out to understand the adsorption behaviour of MPA on gold nanopillars (AuNPs) when different potentials were applied. We found that MPA interacts with the AuNPs through its benzofuran heterocycle and carboxylic acid group. Applying different potentials on the SERS substrate resulted in band shifts, which we hypothesise to originate from molecular reorientation on the metal surface. Moreover, an electrochemically assisted SERS assay was developed and carried out on a portable Raman spectrometer. We show that MPA detection can be performed in less than 5 min in a clinically relevant concentration range (1 – 50 µM), with improved repeatability compared to a SERS assay without any applied potential (overall RSD decrease from 34.6 % to 18.5 %).

Gut microbiota biotransformation of drug glucuronides leading to gastrointestinal toxicity: Therapeutic potential of bacterial β-glucuronidase inhibition in mycophenolate-induced enteropathy

AimsDrug-induced enteropathy is often associated with the therapeutic use of certain glucuronidated drugs. One such drug is mycophenolic acid (MPA), a well-established immunosuppressant of which gastrointestinal adverse effects are a major concern. The role of bacterial β-glucuronidase (β-G) from the gut microbiota in MPA-induced enteropathy has recently been discovered. Bacterial β-G hydrolyzes MPAG, the glucuronide metabolite of MPA excreted in the bile, leading to the digestive accumulation of MPA that would favor in turn these adverse events. We therefore hypothesized that taming bacterial β-G activity might reduce MPA digestive exposure and prevent its toxicity. Main methodsBy using a multiscale approach, we evaluated the effect of increasing concentrations of MPA on intestinal epithelial cells (Caco-2 cell line) viability, proliferation, and migration. Then, we investigated the inhibitory properties of amoxapine, a previously described bacterial β-G inhibitor, by using molecular dynamics simulations, and evaluated its efficiency in blocking MPAG hydrolysis in an Escherichia coli-based β-G activity assay. The pharmacological effect of amoxapine was evaluated in a mouse model. Key findingsWe observed that MPA impairs intestinal epithelial cell homeostasis. Amoxapine efficiently blocks the hydrolysis of MPAG to MPA and significantly reduces digestive exposure to MPA in mice. As a result, administration of amoxapine in MPA-treated mice significantly attenuated gastrointestinal lesions. SignificanceCollectively, these results suggest that the digestive accumulation of MPA is involved in the pathophysiology of MPA-gastrointestinal adverse effects. This study provides a proof-of-concept of the therapeutic potential of bacterial β-G inhibitors in glucuronidated drug-induced enteropathy.

Mycophenolate Metabolite Trough Concentrations Are Not Well Correlated With Dosing or Adverse Outcomes in Pediatric Heart Transplant Recipients.

Although mycophenolate metabolite trough concentrations in serum are routinely obtained for pediatric orthotopic heart transplant (OHT) recipients, limited data support this practice. We sought to investigate the relationship of mycophenolic acid (MPA) and MPA glucuronide (MPAG) serum concentrations to dosing and adverse outcomes among pediatric OHT patients. This retrospective study included OHT recipients ages 0 to 21 years who received mycophenolate mofetil (MMF) with MPA and MPAG serum trough concentration monitoring. The primary outcome was the relationship between MPA and MPAG serum concentrations and dosing. Secondary outcomes included the relationship of adverse outcomes to either MPA and MPAG concentrations or dosing. A total of 98 patients with 1287 MPA and MPAG trough serum concentrations (each) were included. The median initial MMF dose was 40.3 mg/kg/day (IQR, 35.12-51.83) and 1164.4 mg/m2/day (IQR, 1080.77-1206.86). There was no correlation between either MPA or MPAG serum concentrations and mg/kg dosing, or mg/m2 dosing. When comparing the adverse effect of bone marrow suppression with no adverse effect, the median MPA serum trough concentration was 2 (IQR, 1.1-3.2) versus 1.6 (IQR, 0.8-2.5), p = 0.003. When comparing the adverse effect of infection with no adverse effect, median MPA serum trough concentration was 0.9 (IQR, 0.49-1.7) versus 1.6 (IQR, 0.8-2.5), p < 0.001. The clinical utility of this finding is of uncertain benefit. There was no association between MPAG serum concentrations and any adverse outcome (p = 0.053). We did not identify a correlation between mycophenolate serum trough concentrations and either adverse outcomes or dosing. Based on these results, we discourage routine monitoring of mycophenolate trough concentrations.

Gastrointestinal Cytomegalovirus Infection in a Transplantation Recipient With a Negative Serum Viral Load: Case Report

Kidney transplant recipients are vulnerable to infections, especially cytomegalovirus (CMV) disease. It is recommended that clinicians plan their prophylaxis and therapeutic regimens based on viral load testing. CMV viral load monitoring testing provides useful information for identifying virologic response and possible antiviral resistance. Due to the paucity of medical literature on guiding viral therapy in cases of CMV tissue disease with nondetectable serum viral load, we intend to provide physicians with evidence on how to guide medical therapy in these cases. A 49-year-old Hispanic male recipient of a kidney transplant from a cadaver donor presented to the emergency department with anorexia, asthenia, diarrhea, weight loss, and supraclavicular and mediastinal adenomegalies at 2 months post-transplantation. Both patients were serum IgG- and IgM-positive for CMV, which classified them as intermediate risk for developing CMV disease or tissue-invasive disease (donor-positive/recipient-positive [D+/R+]). The patient was induced with basiliximab and methylprednisolone and received maintenance therapy with tacrolimus, mycophenolic acid, and prednisone. Real-time polymerase chain reaction analyses were performed due to suspicion for BK virus, B19 parvovirus, Epstein-Barr virus, and CMV, with an undetectable viral load for all. A biopsy specimen taken from the gastrointestinal tract confirmed CMV infection, which was corroborated through immunocytochemistry. Histopathologic testing is a possible option for patients with CMV tissue disease symptoms but no detectable serum viral load. Clinical observation is fundamental when viral monitoring is difficult.

IMPDH Inhibition Decreases TERT Expression and Synergizes the Cytotoxic Effect of Chemotherapeutic Agents in Glioblastoma Cells.

IMP dehydrogenase (IMPDH) inhibition has emerged as a new target therapy for glioblastoma multiforme (GBM), which remains one of the most refractory tumors to date. TCGA analyses revealed distinct expression profiles of IMPDH isoenzymes in various subtypes of GBM and low-grade glioma (LGG). To dissect the mechanism(s) underlying the anti-tumor effect of IMPDH inhibition in adult GBM, we investigated how mycophenolic acid (MPA, an IMPDH inhibitor) treatment affected key oncogenic drivers in glioblastoma cells. Our results showed that MPA decreased the expression of telomerase reverse transcriptase (TERT) in both U87 and U251 cells, and the expression of O6-methylguanine-DNA methyltransferase (MGMT) in U251 cells. In support, MPA treatment reduced the amount of telomere repeats in U87 and U251 cells. TERT downregulation by MPA was associated with a significant decrease in c-Myc (a TERT transcription activator) in U87 but not U251 cells, and a dose-dependent increase in p53 and CCCTC-binding factor (CTCF) (TERT repressors) in both U87 and U251 cells. In U251 cells, MPA displayed strong cytotoxic synergy with BCNU and moderate synergy with irinotecan, oxaliplatin, paclitaxel, or temozolomide (TMZ). In U87 cells, MPA displayed strong cytotoxic synergy with all except TMZ, acting primarily through the apoptotic pathway. Our work expands the mechanistic potential of IMPDH inhibition to TERT/telomere regulation and reveals a synthetic lethality between MPA and anti-GBM drugs.

#1327 mTOR inhibitors in kidney transplant recipients: long-term efficacy and discontinuation causes

Abstract Background and Aims mTOR inhibitors use in kidney transplantation showed conflicting results, but their use as add-on treatment to calcineurin inhibitors (CNI) is still considered a valuable option. High frequency of drug discontinuation hinders their use in clinical practice. We present a real-life study on long term efficacy of mTOR inhibitors use vs mycophenolic acid (MPA) and the causes of drug discontinuation. Method This is an observational retrospective monocentric study. We analyzed data from 773 patients that underwent kidney transplantation at Hospital Clinic between 2013 and 2019. Induction treatment was based either on basiliximab or a lymphocyte-depleting agent together with methylprednisolone. Maintenance treatment was based either on MPA or mTORi with a CNI. Baseline differences were accounted for with the Inverse Propensity of Treatment Weighting (IPTW). Outcomes explored were death with a functioning graft, graft loss or biopsy-proven rejection. Predictors of outcome were explored through logistic regression or Cox-regression as appropriate. Results 335/773 (43.3%) of the patients received MPA as maintenance treatment, whereas 438/773 (56.6%) received an mTOR inhibitor. Average follow-up was 3.36 years (SD ± 2.04 years). No outcome resulted statistically significant at the logistic regression weighted with IPTW in the subgroup mTORi vs MPA: odds ratio was 1.16 (0.75-1.63, p = 0.57) for death, 0.87 (0.54-1.41, p = 0.59) for graft loss and 1.15 (0.79-1.67, p = 0.45) for biopsy-proven acute rejection. The per-protocol analysis confirmed these results as not statistically significant. Strongest predictors of death resulted having a deceased donor of cardiac death (HR 2.17 (1.06-4.40, p = 0.03), having a delayed graft function (HR 1.9 (1.26-2.87, p = 0.002) and being readmitted within three months from the surgery (HR 1.76 (1.21-2.55, p = 0.003). Diabetic nephropathy as cause of kidney disease was significant as well (HR 2.01 (1.24-3.26, p = 0.004). Proportion of patients with a CMV infection were significantly lower in the mTORi than MPA subgroups (33.5% vs 41.5%, p = 0.02). Proportion of patients developing neoplasias were not significantly different (10% vs 11.3%, p = 0.55). A total of 307/773 (39.7%) of patients changed their immunosuppressive treatment at a median time of 110 days (26.5-375.5). In the MPA subgroup 77/335 (22.9%) changed treatment, whereas in the mTORi subgroup 230/438 (52.5%). The causes of change due to drug-related events were the most represented and related to mTOR treatment: lymphocele in 36/773 (4.6%), complications of the surgical wound in 31/773 (4.1%) and edemas in 23/773 (2.9%) were the most common. Of note, unspecific physician preference was cause of discontinuation in a total of 66/773 (8.5%) patients. Conclusion Our data from a tertiary-center, real-life experience show that mTOR inhibitors are a solid alternative to MPA based protocols, with effectiveness proven at the multivariate and weighted analysis. mTOR treatment has a high rate of discontinuation, with most of the causes represented by post-intervention complications; interestingly, physician preference represent a frequent cause. Further experience and research is needed to optimize mTORi treatment, in order to maintain a therapeutic option for transplanted patients.

#2047 Trough-level guided dosing is reliable for mycophenolate mofetil but not for enteric-coated mycophenolic sodium

Abstract Background and Aims While trough level (TL) guided dosing is usus for calcineurin inhibitors, mycophenolic acid (MPA) was marketed as a one-size-fits-all drug. TL has been reported to be a poor surrogate marker of MPA exposure by reason of complex pharmacokinetics. Measurement of area under the curve (AUC) remains the gold-standard for MPA dosing. Method We measured MPA TL and AUC in 299 KTRs at the University Hospital of Zurich. KTRs with agreement between TL and AUC target range of 40-60 mg/Lxh were compared to KTRs with missing agreement between TL and AUC for differences regarding baseline characteristics and co-medication. Results For mycophenolate mofetil (MMF, n = 218) a high correlation between TL and AUC was observed, p &amp;lt; 0.0001, r = 0.85, while correlation was only low for enteric-coated mycophenolate sodium (EC-MPS, n = 81), p = 0.002, r = 0.34, Fig. 1. However, a high correlation between MPA level 4 hours after EC-MPS intake (C4L) and AUC was found, p &amp;lt; 0.0001, r = 0.94. ROC analysis defined an MMF-TL range of &amp;gt;2.05 mg/l (sensitivity 68%, specificity 89%) and &amp;lt;3.05 mg/l (sensitivity 70%, specificity 81%) correlating with an AUC of 40-60 mg/Lxh. Lack of proton pump inhibitors (PPI) use was significantly associated with incorrect classification (n = 71/218). For EC-MPS C4L a range of &amp;gt;1.25 mg/l (sensitivity 98, specificity 100) and &amp;lt;4.4 mg/l (sensitivity 93, specificity 91) was defined for an AUC of 40-60 mg/Lxh. Here, non-tacrolimus-based immunosuppression was significantly associated with incorrect classification (n = 7/81). Conclusion MMF dosing according to TL highly correlates with AUC especially in KTRs treated with PPI. In contrast, EC-MPS dosing according to C4L is preferred especially in context of tacrolimus-based immunosuppression.

#1639 Nephrotic syndrome with anti-nephrin antibodies—case report of minimal change glomerulonephritis

Abstract Background and Aims Minimal change disease (MCD) in adults is one of the causes of nephrotic syndrome. Recently, anti-nephrin antibodies localized in glomerular podocytes have been described in a certain subset of patients with MCD, supporting the autoimmune etiology of the disease. Method A 79-year-old man with arterial hypertension, hypothirosis and mild dementia presented with new-onset nephrotic syndrome. Peripheral and periocular edema, hypoalbuminemia (serum albumin 17 g/l), proteinuria 15 g/day and hypercholesterolemia (serum cholesterol 6.7 mmol/l) were noted. Blood pressure was 133/85 mmHg. Creatinine was 165 mcmol/l on admission and increased to 361 mcmol/l in the following days. Immunologic tests were negative, including antibodies against phospholipase A2-receptor (anti-PLA2R) and thrombospondin-7A (anti-THSD7A). Anti-HBc was positive, HBV DNA was below 15 IU/ml. Liver tests and abdominal ultrasound were normal, with no signs of liver disease. Results Renal biopsy revealed 18 glomeruli with global sclerosis of one glomerulus (1/18) and 5% interstitial fibrosis. Tubular damage was noted, which could be attributed to severe proteinuria. Immunofluorescence showed discrete IgG podocyte deposits with a uniform kappa/lambda distribution and a high probability of idiopathic minimal change disease due to anti-nephrin antibodies. Electron microscopy showed diffuse effacement of the podocyte foot processes without deposits. The serum taken after the biopsy and before the start of immunosuppressive therapy was positive for anti-nephrin antibodies. Due to a possible pharmacokinetic interaction with tenofovir, which the patient was receiving because of his anti-HBc positivity, we decided not to use cyclosporine. In addition to standard therapy with angiotensin convertase inhibitors, calcium channel blockers and furosemide, the patient was treated with low-dose oral glucocorticoids and mycophenolic acid. In addition, he received rituximab. After a few weeks, the oral glucocorticoids and mycophenolic acid were discontinued. The nephrotic syndrome regressed completely and renal function improved. Conclusion Anti-nephrin antibodies have contributed to a subgroup of patients with MCD, they can be found in a renal biopsy and are also detected in the serum of these patients. Targeted anti-B-cell therapy should be introduced to improve the prognosis of patients.

#1505 Late-onset lupus nephritis: clinical-epidemiological, histological, prognostic and therapeutical implications: a single center experience

Abstract Background and Aims Systemic lupus erythematosus (SLE), a chronic autoimmune disease, and lupus nephritis (LN), are typically diagnosed in women in fertile age. Late-onset SLE is defined as a disease onset over 50 years. Our main objective was to analyze the clinical, histological, prognostic and therapeutical features related to LN in our late-onset SLE population. Method A single center and retrospective study was performed comparing clinical-demographic, histological, prognostic and therapeutical data of 45 LN biopsied patients from 1994 to 2023. We divided the study population according to the age of onset of LN into classic-onset (&amp;lt; 45 years) and late-onset (&amp;gt; 45 years). Results Late-onset LN patients showed a higher association with Sjögren's and antiphospholipid syndrome, more cardiovascular comorbidities at presentation, poorer renal function at diagnosis and higher SDI score. On histology they showed more non-proliferative LN classes (II followed by V). Late-onset LN responders needed more time to achieve response but had fewer relapses. Non responsive patients had more incidence of acute kidney injury (AKI) and more glomerulosclerosis and interstitial fibrosis-tubular atrophy (IFTA) at presentation, as well as more need for renal replacement therapy (RRT) at diagnosis and at follow-up. The principal independent variables associated with relapse were the younger age at SLE diagnosis and an initial partial renal response (PRR). Patients of both groups who experienced relapse received higher doses of intravenous cyclophosphamide and less mycophenolic acid (MFA) analogues. Patients who received induction with MFA analogues were more likely to achieve complete renal response (CRR) or non-renal response (NRR). Conclusion late-onset LN is often accompanied by poor renal function at presentation. It seems imperative to establish a tailored approach in this fragile population in order to avoid unnecessary immunosuppression.

#2751 CD14++CD16+ monocytes and CD8+ T cells are independently associated with annual change in eGFR and proteinuria in kidney transplant recipients

Abstract Background and Aims Immune cellular responses are implicated in all clinical aspects of kidney transplantation. There is little data regarding the additional value of regular monitoring of immune cells subsets expression in kidney transplant recipients (KTRs). The aim of our prospective study was a longitudinal follow-up analysis of monocytes subpopulations, natural killer (NK) cells and lymphocytes subsets including regulatory T cells (Tregs) in the circulation of KTRs and potential clinical correlations. Method 48 stable KTRs were initially enrolled. All KTRs were under immunosuppressive regimen with corticosteroids, mycophenolate mofetil or mycophenolate acid and calcineurin inhibitors (CNIs). Exclusion criteria were history or development during follow-up of acute rejection, cardiovascular disease, malignancy and active or chronic infections. Patients were followed for 12 months. The peripheral blood immune cell subsets CD14++CD16-, CD14++CD16+ and CD14+CD16++ absolute values and percentages out of total monocytes and NK cells (CD3+CD16+56+), CD3-CD19+ B lymphocytes, CD3+ CD4+ T cells, CD3+CD8+ T cells and Tregs (CD4+CD25+ FoxP3+) absolute values and percentages out of total lymphocytes were measured by flow cytometry at baseline (T0) and after 12 months (T1). Clinical and laboratory parameters were recorded at T0 and T1. Delta (Δ) eGFR and Δ spot urine protein to creatinine ratio (ΔUPCR) were defined as their respective differences between T1 and T0. Likewise, Delta (Δ) of immune cells subtypes or laboratory indices was defined as their respective difference between T1 and T0. Results 35 KTRs (mean age 53 ± 9.28 years, 71% males, mean transplant vintage 96 ± 66 months, 63% on Tacrolimus and 37% on Cyclosporine) were included in the final analysis. Significant differences were observed between T0 and T1 in monocytes number (653 ± 244 and 538 ± 197/μL respectively, p = 0.001), monocytes percentage (7.6 ± 2.9 and 6.6 ± 2.2% respectively, p = 0.006) as well as in the number of classical CD14++CD16- monocytes (534 ± 225 and 452 ± 185/μL respectively, p = 0.04). The rest immune cells subsets did not show any significant differences between T0 and T1. Mean eGFR declined from 58 ± 17 at T0 to 53 ± 18 ml/min/1.73 m2 at T1 (p = 0.004). No significant changes were observed between T0 and T1 in median UPCR [0.16 (IRQ, 0.09-0.56) at T0 and 0.16 (IQR, 0.10-0.70) gr protein/gr creatine at T1, p = 0.489], in median CRP [4.0 (IQR, 3-7) at T0 and 5 (IQR, 2.5-7.5) mg/L) at T1, p = 0.919], in mean ESR (20 ± 14 at T0 and 22 ± 19 mm/hour at T1, p = 0.381) or other parameters, including CNIs blood levels. ΔeGFR was correlated with the T0 percentage of monocytes (rho = 0.359, p = 0.037), the T0 number and T0 percentage of CD14++CD16+ monocytes (rho= 0.502, p = 0.003 and rho = 0.438, p = 0.008 respectively). On the other hand, a borderline inverse correlation was observed between ΔeGFR and ΔCD14++CD16+ monocytes number (rho = −0.339, p = 0.05). Additional correlates of ΔeGFR included serum albumin at T0 (rho = 0.395, p = 0.021) and Δuric acid (rho = 0.567, p = 0.000). At stepwise linear regression analysis, CD14++CD16+ monocytes (β = 0.338, p = 0.04) and Δuric acid (β = 0.477, p = 0.006) remained independent significant correlates of ΔeGFR. ΔUPCR was significantly correlated with the percentage of B-lymphocytes (rho = 0.385, p = 0.027) and CD4+ T-cells (rho = 0.352, p = 0.044) at T0, and inversely correlated with the T0 percentage of T-lymphocytes (rho = −0.402, p = 0.02) and CD8+ T cells (rho = −0.603, p = 0.000) as well as with Δhemoglobin (rho = −0.385, p = 0.027). At stepwise linear regression analysis, only the CD8+ T cells percentage at T0 remained independently correlated to ΔUPCR (β = –0.379, p = 0.03). Conclusion The results of our study indicate that intermediate CD14++CD16+ monocytes are associated with annual eGFR change whereas CD8+ T cells negatively correlate with the annual trend of proteinuria change in KTRs. Further research is needed to clarify underlying pathophysiological mechanisms for these findings. Finally, future studies including larger cohorts and with longer follow-up are required to specify the potential utility of immune subpopulations monitoring as potential prognostic biomarkers in KTRs.