Background: Endotoxemia, characterized by elevated plasma lipopolysaccharide (LPS), is associated with gut microbiota dysbiosis and increased intestinal permeability. In transplant patients, immunosuppressive drugs may contribute to or exacerbate endotoxemia. LPS can modulate hepatic metabolic enzymes and drug transporters, potentially altering the pharmacokinetics of immunosuppressants.

Aims: Understanding these interactions is crucial for optimizing therapy and improving transplant outcomes.

Methods: We analyzed plasma samples from 48 transplant recipients at one-year post-transplantation, sourced from the CRBIOLIM biobank (Limoges University Hospital) through the PIGREC (heart), STIMMUGREP (lung), and BIOMARGIN (kidney) studies in organ transplant recipients. In addition, samples from 24 healthy volunteers were obtained with informed consent through the Clinical Investigation Center (Limoges University Hospital). Patients included were on a standard immunosuppressive regimen with mycophenolate mofetil (MMF), known to disrupt gut microbiota and promote endotoxemia. Plasma LPS levels were quantified using ELISA and compared between groups (Mann-Whitney test). Patient area under the concentration-time curve (AUC) of MMF, tacrolimus, and cyclosporine was extracted from databases, and correlations with LPS levels were assessed using Spearman’s rank correlation coefficient.

Results: Transplant recipients had significantly higher plasma LPS concentrations than healthy volunteers (99 ± 22 pg/mL vs. 257 ± 32 pg/mL; mean ± SEM; p < 0.01), confirming endotoxemia. Among immunosuppressants, only cyclosporine AUC correlated with LPS levels (Spearman’s ρ=0.818, p=0.002).

Conclusion: The strong correlation between cyclosporine exposure and LPS plasma levels suggests that endotoxemia may influence cyclosporine metabolism by modulating hepatic enzymes and transporters. Further research is needed to elucidate the mechanisms.

Keywords: endotoxemia, lipopolysaccharide, cyclosporine A

Background: The metabolism of azathioprine, an immunosuppressive drug used in inflammatory bowel disease treatment, is significantly influenced by the gut microbiota. This study aims to identify novel gut microbiota biomarkers for the therapeutic drug monitoring of azathioprine.

Methods: The research combines animal experiments and clinical studies. Germ-free rats were employed to establish a model evaluating the impact of gut microbiota on azathioprine pharmacokinetics. Simultaneously, stable Crohn's disease patients receiving azathioprine for over three months were enrolled to explore the correlations between gut microbiota structure and active metabolite levels (6-thioguanosine nucleotides, 6-TGN). Both 16S RNA sequencing and metagenomic sequencing were utilized to identify potential gut microbiota biomarkers for azathioprine's therapeutic drug monitoring.

Results: Variations in gut microbiota composition correlated with differences in 6-TGN levels. Prevotella emerged as indicative bacteria influencing both exposure (AUC, 3148.8±515.4 hour∙ng∙mL-1 in control group vs. 2078.0±290.5 hour∙ng∙mL-1 in germ-free group) and steady-state concentrations of 6-TGN (6-TGN_L: 151.7±29.7, 6-TGN_M: 290.7±58.2, 6-TGN_H: 529.7±130.4; unit: pmol/8×108RBC). Metagenomic analysis revealed significant enrichments of key metabolic enzymes (guanine-methylation-sensitive endonuclease, hypoxanthine phosphoribosyltransferase, and inosine monophosphate dehydrogenase) in the genera Clostridium and Roseburia, potentially contributing to 6-TGN level variations. Known butyrate-producers, including Clostridium and Roseburia, demonstrated a positive correlation with 6-TGN levels (correlation coefficient: 0.483, P＜0.05), suggesting azathioprine alleviates intestinal inflammation through an intrinsic mechanism involving these genera.

Conclusion: Prevotella, Roseburia, and Clostridium constitute novel biomarkers for azathioprine therapeutic drug monitoring, potentially offering implications for personalized treatment strategies.

Keywords: Azathioprine, Gut microbiota, Biomarker, 6-TGN

Background: Tacrolimus, essential for allogeneic stem cell transplant patients, requires therapeutic drug monitoring (TDM) due to high variability and a narrow therapeutic window. Finger-prick dried blood spots (DBS) could replace venous blood draws, enabling home monitoring and improving patients’ quality of life.

Aims: This study aimed at evaluating the clinical applicability of capillary finger-prick sampling for tacrolimus TDM and creatinine determination in the hospital (part 1), and in a homesampling context (part 2), using DBS and Capitainer® qDBS devices.

Methods: Patients collected qDBS samples at home at trough levels over three days. LC-MS/MS analysis methods were used. Analytical and intra-patient variation based on home-sampled duplicates were evaluated as well as therapy adherence. For each homesampling participant, tacrolimus levels were compared to their individual target ranges. Patients’ experience was assessed via questionnaires.

Results: Assisted sampling confirmed the clinical applicability of capillary (q)DBS for tacrolimus TDM and creatinine determination, with qDBS showing better clinical agreement (98% of tacrolimus and 93% of creatinine results within 20% of reference values). Homesampling using qDBS is feasible, with the CV below 8%. In stable-dose patients, within-subject variation for tacrolimus ranged from 4.1% to 24.7%, suggesting strong adherence. Homesampling improved tacrolimus trough level resolution and enabled closer monitoring after dose changes, with positive patient feedback.

Conclusions: Capillary qDBS show great potential as a complement to venous blood draws for tacrolimus TDM and creatinine determination. Homesampling is promising for this patient population, addressing the need for individualized TDM.

Key Words: Therapeutic Drug Monitoring, Tacrolimus, Creatinine, Dried Blood Microsampling

Introduction: Cytochromes P450 (CYP) regulate exposure to medicines and hence medicine efficacy and safety. Understanding variability in CYP abundance and activity caused by physiological and pathological factors is useful for safe and effective dosing of medicines. Liver-derived extracellular vesicles (EVs) offer a novel strategy to characterise CYP abundance and predict variability in exposure to medicines.
Aim: Investigate liver EVs as a strategy to account for variability in CYP enzyme activity associated with physiological and pathological conditions.
Methods: The abundance of 8 CYP enzymes in liver EVs and tissue homogenates prepared from eleven human livers were quantified by LCMS. Linear regression assessed EV-tissue protein correlations. CYP3A4 abundance was quantified in liver-derived EVs isolated from plasma of pregnant (n=9) and non-pregnant (n=3) females, patients with metabolic associated fatty liver disease (MAFLD; n=14) and controls (n=14).
Results: Strong correlations were observed in paired liver EVs and homogenates for all CYP (r = 0.654 to 0.962) supporting EVs as a robust surrogate of hepatic enzyme expression. CYP3A4 expression in liver EVs was significantly elevated in third-trimester pregnancy (3.2-fold, P = 0.003) compared to non-pregnant controls. CYP3A4 abundance was 30 to 70% lower in MAFLD patients, consistent with reported reduced metabolism in liver disease. The reduction in CYP3A4 abundance in MAFLD patients was associated with the severity of disease.
Conclusion: These data support liver EVs as a non-invasive liquid biopsy to characterize variability in medicine exposure cased by physiological and pathological changes. EV protein abundance may complement genotype to enable a comprehensive ADME genomic profile.

Introduction: Tacrolimus (Tac) is a key immunosuppressant in renal transplantation, often combined with anti-thymocyte globulin (ATG) for induction therapy. However, ATG may be obviated in low-risk patients. Tac inhibits nuclear factor of activated T cells (NFAT), reducing gene expression (IL-2, TNF-α, GM-CSF, IFN-γ). Study examines the in-vitro and in-vivo pharmacodynamic (PD) effects of Tac using NFAT residual gene expression (NFAT-RGE) in renal-transplant recipients with/without ATG.

Methods: Sixty patients (≥18 years), planned for de-novo renal-transplant. Blood samples were collected pre-transplant (10ml) and on post-transplantation day 7 (POD 7) (2ml at Tac-C0 and C1.5). Samples were spiked with varying concentrations of Tac (0, 2.5, 5, 10, or 20 ng/ml) and ex-vivo immune activation was done with RPMI 1640 medium, phorbol myristate acetate (PMA), and ionomycin. Total RNA was extracted, cDNA was synthesized. Real-time PCR was performed for gene expression quantification. Fold change was calculated using ∆Ct and ∆∆Ct. Tac concentration was estimated in whole blood samples using LC-MS/MS system with TAC-13C-d2 as the internal standard.

Results: Recipients (n=60) had an average NFAT-RGE of 53.7±21.8% (2.5ng/ml), 36.6±18.7% (5ng/ml), 24.8±16.2% 10ng/ml) and 13.5±11.6% (20ng/ml) in-vitro. In comparison, the POD7 NFAT-RGE was 46.9±22.3% at C0 (11.15±2.7ng/ml) and 19.5±13.1% at C1.5 (16.6±4.3ng/ml). The NFAT-RGE in ATG-induced patients (n=34) at POD7 was 25.13±30.83% compared to 37.68±34.36% in the non-ATG (n=26) at C0. The corresponding values were 11.92±13.11% and 14.29±15.76% for C1.5.

Conclusions: NFAT-RGE could be used as potential biomarker in individualizing immunosuppression in non-ATG triple drug-induced patients.

Keywords: Renal transplantation, Pharmacodynamic biomarkers, tacrolimus, residual gene expression, NFAT

Background: Mycophenolic Acid (MPA), an ester prodrug of Mycophenolate Mofetil i.e. is an immunosuppressive agent, used with tacrolimus to prevent organ rejection in transplant patients. Traditionally, Therapeutic Drug Monitoring (TDM) relies on plasma measurements, but alternative matrices like saliva offers a non-invasive approach for assessing drug exposure. This study investigates the feasibility of estimating MPA from saliva and explores its potential as a surrogate for plasma measurements in renal transplant recipients.

Aims: The study aims to evaluate the feasibility of measuring MPA in saliva, assessing salivary and plasma MPA exposure through area under curve (AUC) and finding their concentration correlation for further clinical outcomes.

Methods: Saliva and plasma samples were collected from 10 adult renal transplant patients with stable allograft ≥4 weeks. Salivary MPA (sMPA) was quantified using High Performance Liquid Chromatography-Fluorescence Detector (HPLC-FLD) and plasma MPA by established HPLC-UV method. The correlation between salivary and plasma MPA concentrations and mean AUC0-12h was calculated.

Results: The mean plasma MPA AUC0-12h was 88.21 ± 35.03 µg*h/mL, while the mean sMPA AUC0-12h was 195.82 ± 126.81 ng*h/mL. In our study, the correlation between plasma and sMPA concentrations was 0.903±0.133,concluding sMPA concentrations may reflects its plasma concentrations.

Conclusions: Out of the 10 patients, 7 exhibited a strong correlation between plasma and sMPA concentrations (>0.900). These findings suggest that saliva may serve as a potential alternative matrix for TDM of MPA, which could aid in non-invasive assessment and optimization for further clinical outcomes.

Keywords: Mycophenolate, Saliva, Therapeutic Drug Monitoring, Area under the Curve, HPLC-FLD

Background: Tacrolimus is one of few immunosuppressants used in pregnancy. Therapeutic drug monitoring often prompts dose increases due to decreasing trough blood concentrations (BC0) throughout pregnancy. Given high erythrocyte-binding, decreasing BC0 may reflect changes in haematocrit, rather than clearance.

Aims: To investigate relationships between BC0, trough plasma concentrations (PC0) and pregnancy outcomes.

Methods: BC0 and PC0 were measured by LC-MS/MS. Biochemistry and outcomes were obtained from clinical records. Relationships between BC0 and PC0 were investigated by linear mixed effect models adjusting for repeated measures and confounders. Effects of BC0 and PC0 on neonatal birth weight, gestational age and maternal plasma creatinine were investigated by linear regression or mixed effects models.

Results: In 9 participants (6 kidney transplant, 3 lupus nephritis) median BC0 and PC0 were 5.4ng/mL (1.6-23.1ng/mL, n=97) and 0.88ng/mL (0.28-4.27ng/mL, n=88). BC0 and PC0 were moderately correlated (R2=0.51; P<0.0001). The PC0/BC0 ratio increased during pregnancy (P<0.01) and with higher albumin (P<0.05). Maternal creatinine increased during third trimester (P<0.001) and with higher dose (P<0.05). 6/8 neonates were <37 weeks gestation, 5/6 weighed <2.5kg. Weight was inversely associated with BC0 (P<0.0001, Padj<0.0004). For every 100ng/L increase in BC0, weight decreased by 60g (95%CI: -90, -30).

Conclusions: BC0 were highly variable in pregnancy and often subtherapeutic. However, routinely increasing dose may be unwarranted given the only moderate relationship between BC0 and PC0, and our preliminary observation of a relationship between high dose, BC0 and poor outcomes (decreased maternal renal function and neonatal weight), which warrants further investigation.

Key words: tacrolimus, pregnancy, immunosuppression