Evaluating Single-Nucleotide Polymorphisms in Inflammasome Proteins and Serum Levels of IL-18 and IL-1ß in Kidney Interstitial Damage in Anti-Neutrophilic Cytoplasmic Antibody-Associated Vasculitis.

The inflammasome regulates the innate inflammatory response and is involved in autoimmune diseases. In this study, we explored the levels of IL-18 and IL-1ß in serum and urine and the influence of various single-nucleotide polymorphisms (SNPs) on kidney lesions at diagnosis in patients with ANCA-associated vasculitis (AAV) and their clinical outcomes. Ninety-two patients with renal AAV were recruited, and blood and urine were collected at diagnosis. Serum and urine cytokine levels were measured by ELISA. DNA was extracted and genotyped using TaqMan assays for SNPs in several inflammasome genes. Lower serum IL-18 (p = 0.049) and the IL-18 rs187238 G-carrier genotype (p = 0.042) were associated with severe fibrosis. The IL-18 rs1946518 TT genotype was associated with an increased risk of relapse (p = 0.05), whereas GG was related to better renal outcomes (p = 0.031). The rs187238 GG genotype was identified as a risk factor for mortality within the first year after AAV diagnosis, independent of the requirement for dialysis or lung involvement (p = 0.013). We suggest that decreased cytokine levels could be a surrogate marker of scarring and chronicity of the renal lesions, together with the rs187238 GG genotype. If our results are validated, the rs1946518 TT genotype predicts the risk of relapse and renal outcomes during follow-up.

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 Role of Inflammasomes in Glomerulonephritis.

The inflammasome is an immune multiprotein complex that activates pro-caspase 1 in response to inflammation-inducing stimuli and it leads to IL-1ß and IL-18 proinflammatory cytokine production. NLRP1 and NLRP3 inflammasomes are the best characterized and they have been related to several autoimmune diseases. It is well known that the kidney expresses inflammasome genes, which can influence the development of some glomerulonephritis, such as lupus nephritis, ANCA glomerulonephritis, IgA nephropathy and anti-GBM nephropathy. Polymorphisms of these genes have also been described to play a role in autoimmune and kidney diseases. In this review, we describe the main characteristics, activation mechanisms, regulation and functions of the different inflammasomes. Moreover, we discuss the latest findings about the role of the inflammasome in several glomerulonephritis from three different points of view: in vitro, animal and human studies.

TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) Is Upregulated in Lymphocytes Stimulated with Concanavalin A.

The glycolytic modulator TP53-Inducible Glycolysis and Apoptosis Regulator (TIGAR) is overexpressed in several types of cancer and has a role in metabolic rewiring during tumor development. However, little is known about the role of this enzyme in proliferative tissues under physiological conditions. In the current work, we analysed the role of TIGAR in primary human lymphocytes stimulated with the mitotic agent Concanavalin A (ConA). We found that TIGAR expression was induced in stimulated lymphocytes through the PI3K/AKT pathway, since Akti-1/2 and LY294002 inhibitors prevented the upregulation of TIGAR in response to ConA. In addition, suppression of TIGAR expression by siRNA decreased the levels of the proliferative marker PCNA and increased cellular ROS levels. In this model, TIGAR was found to support the activity of glucose 6-phosphate dehydrogenase (G6PDH), the first enzyme of the pentose phosphate pathway (PPP), since the inhibition of TIGAR reduced G6PDH activity and increased autophagy. In conclusion, we demonstrate here that TIGAR is upregulated in stimulated human lymphocytes through the PI3K/AKT signaling pathway, which contributes to the redirection of the carbon flux to the PPP.

Integrated miRNA/mRNA Counter-Expression Analysis Highlights Oxidative Stress-Related Genes CCR7 and FOXO1 as Blood Markers of Coronary Arterial Disease.

Our interest in the mechanisms of atherosclerosis progression (ATHp) has led to the recent identification of 13 miRNAs and 1285 mRNAs whose expression was altered during ATHp. Here, we deepen the functional relationship among these 13 miRNAs and genes associated to oxidative stress, a crucial step in the onset and progression of vascular disease. We first compiled a list of genes associated to the response to oxidative stress (Oxstress genes) by performing a reverse Gene Ontology analysis (rGO, from the GO terms to the genes) with the GO terms GO0006979, GO1902882, GO1902883 and GO1902884, which included a total of 417 unique Oxstress genes. Next, we identified 108 putative targets of the 13 miRNAs among these unique Oxstress genes, which were validated by an integrated miRNA/mRNA counter-expression analysis with the 1285 mRNAs that yielded 14 genes, Map2k1, Mapk1, Mapk9, Dapk1, Atp2a2, Gata4, Fos, Egfr, Foxo1, Ccr7, Vkorc1l1, Rnf7, Kcnh3, and Mgat3. GO enrichment analysis and a protein-protein-interaction network analysis (PPI) identified most of the validated Oxstress transcripts as components of signaling pathways, highlighting a role for MAP signaling in ATHp. Lastly, expression of these Oxstress transcripts was measured in PBMCs from patients suffering severe coronary artery disease, a serious consequence of ATHp. This allowed the identification of FOXO1 and CCR7 as blood markers downregulated in CAD. These results are discussed in the context of the interaction of the Oxstress transcripts with the ATHp-associated miRNAs.

PI3K-Akt signaling controls PFKFB3 expression during human T-lymphocyte activation.

Lymphocyte activation is associated with rapid increase of both the glycolytic activator fructose 2,6-bisphosphate (Fru-2,6-P2) and the enzyme responsible for its synthesis, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). PFKFB3 gene, which encodes for the most abundant PFK-2 isoenzyme in proliferating tissues, has been found overexpressed during cell activation in several models, including immune cells. However, there is limited knowledge on the pathways underlying PFKFB3 regulation in human T-lymphocytes, and the role of this gene in human immune response. The aim of this work is to elucidate the molecular mechanisms of PFKFB3 induction during human T-lymphocyte activation by mitotic agents. The results obtained showed PFKFB3 induction during human T-lymphocyte activation by mitogens such as phytohemagglutinin (PHA). PFKFB3 increase occurred concomitantly with GLUT-1, HK-II, and PCNA upregulation, showing that mitotic agents induce a metabolic reprograming process that is required for T-cell proliferation. PI3K-Akt pathway inhibitors, Akti-1/2 and LY294002, reduced PFKFB3 gene induction by PHA, as well as Fru-2,6-P2 and lactate production. Moreover, both inhibitors blocked activation and proliferation in response to PHA, showing the importance of PI3K/Akt signaling pathway in the antigen response of T-lymphocytes. These results provide a link between metabolism and T-cell antigen receptor signaling in human lymphocyte biology that can help to better understand the importance of modulating both pathways to target complex diseases involving the activation of the immune system.

ANRIL as a genetic marker for cardiovascular events in renal transplant patients - an observational follow-up cohort study.

Cardiovascular disease is the leading cause of morbidity and mortality in kidney transplant recipients. Several single-nucleotide polymorphisms (SNPs) in the ANRIL gene pathway have been associated with cardiovascular events (CE). The main objective was to ascertain whether ANRIL (rs10757278) and CARD8 (rs2043211) SNPs could mediate susceptibility to CE. This was an observational follow-up cohort study of renal transplant recipients at Bellvitge University Hospital (Barcelona) from 2000 to 2014. A total of 505 recipients were followed up until achievement of a CE. Patients who did not achieve the endpoint were followed up until graft loss, lost to follow-up or death. Survival analysis was used to ascertain association between genetic markers, clinical data, and outcome. Fifty-three patients suffered a CE after renal transplantation. Results showed a significant association between ANRIL SNP and CE. Homozygous GG for the risk allele showed higher risk for CE than A carriers for the protective allele [HR = 2.93(1.69-5.11), P < 0.0001]. This effect was maintained when it was analyzed in combination with CARD8, suggesting that CARD8 SNP could play a role in the ANRIL mechanism. However, our study does not clarify the molecular mechanism for the CARD8 SNP regulation by ANRIL. ANRIL SNP may predispose to the development of CE after successful kidney transplantation.

Posttransplant peripheral blood donor-specific interferon-γ enzyme-linked immune spot assay differentiates risk of subclinical rejection and de novo donor-specific alloantibodies in kidney transplant recipients.

Noninvasive diagnosis of kidney allograft inflammation in transplant recipients with stable graft function (subclinical rejection) could permit more effective therapy and prevent later development of de novo anti-donor HLA antibodies and/or graft dysfunction. Here we tested whether quantifying posttransplant donor-specific alloreactive T-cells by IFN-γ ELISPOT assay noninvasively detects subclinical T-cell mediated rejection and/or predicts development of anti-donor HLA antibodies. Using an initial cross-sectional cohort of 60 kidney transplant patients with six-month surveillance biopsies, we found that negative donor-specific IFN-γ ELISPOT assays accurately ruled out the presence of subclinical T-cell mediated rejection. These results were validated using a distinct prospective cohort of 101 patients where donor-specific IFN-γ ELISPOT results at both three- and six-months posttransplant significantly differentiated patients with subclinical T-cell mediated rejection at six months, independent of other clinical variables (odds ratio 0.072, 95% confidence interval 0.008-0.653). The posttransplant donor-specific IFN-γ ELISPOT results independently associated with subsequent development of significant anti-donor HLA antibodies (0.085, 0.008-0.862) and with significantly worse two-year function (estimated glomerular filtration rate) compared to patients with a negative test. Thus, posttransplant immune monitoring by donor-specific IFN-γ ELISPOT can assess risk for developing subclinical T-cell mediated rejection and anti-donor HLA antibodies, potentially limiting the need for surveillance biopsies. Our study provides a guide for individualizing immunosuppression to improve posttransplant outcomes.

The combination of CYP3A4*22 and CYP3A5*3 single-nucleotide polymorphisms determines tacrolimus dose requirement after kidney transplantation.

INTRODUCTION: Tacrolimus (Tac) has a narrow therapeutic window and shows large between-patient pharmacokinetic variability. As a result, over-immunosuppression and under-immunosuppression are frequently encountered in daily clinical practice. Unraveling the impact of genetic polymorphisms on Tac pharmacokinetics may help to refine therapy. In this study, the associations of single-nucleotide polymorphisms (SNPs) in drug-metabolizing enzymes (CYP3A) with Tac pharmacokinetics were investigated in renal transplant recipients. PARTICIPANTS AND METHODS: In a cohort of 272 kidney transplant recipients, associations between functional genetic variants (CYP3A4*22 and CYP3A5*3) and dose-adjusted predose Tac concentrations (C0) and daily doses of Tac at days 5-7 and 15 and 1, 3, 6 and 12 months after renal transplantation were evaluated. Patients were genotyped and clustered according to both CYP3A4*22 and CYP3A5*3 allelic status: poor (PM) (CYP3A4*22 carriers with CYP3A5*3/*3), intermediate (IM) (CYP3A4*1/*1 with CYP3A5*3/*3 or CYP3A4*22 carriers with CYP3A5*1 carriers) and extensive CYP3A-metabolizers (EM) (CYP3A4*1/*1 and CYP3A5*1 carriers). RESULTS: EM had an 88% lower dose-adjusted C0 compared with IM. PM had a 26% higher dose-adjusted C0 compared with IM. The percentage of patients with supratherapeutic Tac exposure (C0>15 ng/ml) was significantly higher in PM (43.5%) compared with EM (0%) at days 5-7 after transplantation (P=0.01). About 30% of EM had subtherapeutic exposure (C0<5 ng/ml) at days 5-7 after transplantation (P=0.001). CONCLUSION: The combined CYP3A4 and CYP3A5 genotype of renal transplant recipients has a major influence on the Tac dose required to reach the target exposure.

CD154-CD40 T-cell co-stimulation pathway is a key mechanism in kidney ischemia-reperfusion injury.

Ischemia-reperfusion occurs in a great many clinical settings and contributes to organ failure or dysfunction. CD154-CD40 signaling in leukocyte-endothelial cell interactions or T-cell activation facilitates tissue inflammation and injury. Here we tested a siRNA anti-CD40 in rodent warm and cold ischemia models to check the therapeutic efficacy and anti-inflammatory outcome of in vivo gene silencing. In the warm ischemia model different doses were used, resulting in clear renal function improvement and a structural renoprotective effect. Renal ischemia activated the CD40 gene and protein expression, which was inhibited by intravenous siRNA administration. CD40 gene silencing improved renal inflammatory status, as seen by the reduction of CD68 and CD3 T-cell infiltrates, attenuated pro-inflammatory, and enhanced anti-inflammatory mediators. Furthermore, siRNA administration decreased a spleen pro-inflammatory monocyte subset and reduced TNFα secretion by splenic T cells. In the cold ischemia model with syngeneic and allogeneic renal transplantation, the most effective dose induced similar functional and structural renoprotective effects. Our data show the efficacy of our siRNA in modulating both the local and the systemic inflammatory milieu after an ischemic insult. Thus, CD40 silencing could emerge as a novel therapeutic strategy in solid organ transplantation.

Development of a population PK model of tacrolimus for adaptive dosage control in stable kidney transplant patients.

BACKGROUND: Tacrolimus pharmacokinetics (PK) presents a high variability that hampers its therapeutic use. The aims of this study are to: (1) develop a population pharmacokinetic (PPK) model for tacrolimus and to identify the factors that contribute to the variability of tacrolimus PK in renal transplant patients; and (2) to establish a new Bayesian estimator that can easily and routinely be applied in the hospital. A new PPK model may allow efficacy to be optimized, improve dose regimens, minimize side effects, and decrease the cost of extensive area under the curve (AUC) monitoring. METHODS: PPK analysis of the full PK profiles of 16 patients on 5 occasions was performed with NONMEM 7.2. Biochemical variables (hematocrit, hemoglobin, aspartate aminotransferase, and others) were analyzed. RESULTS: A 2-open-compartment model with interoccasion variability best described the PK of tacrolimus. Three transit compartments provided the best description of the absorption process. The hematocrit, aspartate aminotransferase, and alanine aminotransferase were not significant in the covariate analysis. External validation with 91 patients proved the good predictability of the model with a bias and precision of 0.37 mcg/L (CI 95%, -0.11 to 1.20 mcg/L) and 0.38 mcg/L (CI 95%, 0.02 to 1.21 mcg/L), respectively. A limited sampling strategy using 1 sampling point at predose (trough concentrations) showed a good performance in AUC0-12h estimation with a correlation between AUCfull and AUCLSS, bias and imprecision of r = 0.75, 6.78% (range, -16.26% to 30.06%) and 1.42% (IC 95%, 0.14%-3.61%), respectively. CONCLUSIONS: The PPK model developed provides reliable prior information for Bayesian adaptive control of dosage regimens of tacrolimus to achieve the desired AUC goals in stable renal transplant patients.

Pharmacokinetic modeling of enterohepatic circulation of mycophenolic acid in renal transplant recipients.

Several factors contribute to mycophenolic acid (MPA) between-patient variability. Here we characterize the metabolic pathways of MPA and quantify the effect of combining genetic polymorphism of multidrug-resistant-associated protein-2, demographics, biochemical covariates, co-medication (cyclosporine (CsA) vs. macrolides), and renal function on MPA, 7-O-MPA-glucuronide (MPAG), and acyl-glucuronide (AcMPAG) disposition, in renal transplant recipients, after mycophenolate mofetil. Complete pharmacokinetic profiles from 56 patients (five occasions) were analyzed. Enterohepatic circulation was modeled by transport of MPAG to the absorption site. This transport significantly decreased with increasing CsA trough concentrations (CtroughCsA). MPAG and AcMPAG plasma clearances significantly decreased with renal function. No significant influence of multidrug-resistant-associated protein-2 C24T single-nucleotide polymorphism was found. The model adequately predicted the increase in MPAG/AcMPAG exposures in CsA and macrolide patients with decreased renal function. This resulted in higher MPA exposures in macrolide patients versus CsA patients, and increased MPA exposures with renal function from 25 to 10 ml/min, in macrolide patients, owing to enhanced MPAG enterohepatic circulation. Lower-percentage enterohepatic circulation occurred with higher CtroughCsA and renal function values. The lack of MPA protein-binding modeling did not permit evaluation of the impact of renal function and CtroughCsA on MPA exposures in CsA patients. Thus, dose tailoring of covariates is recommended for target MPA exposure.

Optimal sparse sampling for estimating ganciclovir/valganciclovir AUC in solid organ transplant patients using NONMEN.

BACKGROUND: Ganciclovir and valganciclovir (GCV/VGCV) are used for the treatment and prophylaxis of cytomegalovirus in solid organ transplant (SOT) patients. An area under the time-concentration curve of 40-50 µg × h/mL is related to efficacy. Therapeutic drug monitoring could prevent suboptimal drug exposure and adverse events, but obtaining full concentration profiles is not feasible. Sampling optimization by developing a reliable and clinically applicable limited sampling strategy (LSS) may simplify dose adjustment. METHODS: An LSS was developed using an original pharmacokinetic (PK) data set of 40 full profiles from 20 adult SOT patients. The LSS was developed based on population and Bayesian prediction approaches. Population PK parameters from a previous model were used for simulation or as priors (NONMEM version 7.2). Median percentage of prediction error and median of absolute percentage prediction error were calculated for plasma clearance (CL) and central compartment distribution volume (V(2)). Bias and precisions were compared using 1-way analysis of variance (SPSSv19.0). RESULTS: Sampling windows were designed according to the PK profile previously observed with the entire set of data. The 4 windows selected were distributed from 0.5 to 1.5 hours, 2 to 3 hours, 4 to 5 hours, and 6 to 8 hours. Predose and concentrations beyond 8 hours were not considered in any case because simulated negative concentrations occurred in both cases. Predicted exposure using 3 sampling times (0.5-1.5, 4-5, and 6-8 hours) showed the best predictive performance, by either the population or Bayesian approaches. Bias and imprecision for CL and V(2) were 0 and 0.60%, and -0.78% and 0.78%, respectively. CONCLUSIONS: GCV/VCG area under the time-concentration curve in SOT patients could be predicted with acceptable accuracy for clinical management and dose individualization using LSS. The estimator of GCV/VGC, using 3 concentrations measured at 0.5-1.5, 4-5, and 6-8 hours after drug intake, could be used for dose adjustment.

Immunosuppressive drug combinations after kidney transplantation and post-transplant diabetes: A systematic review and meta-analysis.

Post-transplant diabetes mellitus (PTDM) is a frequent complication after kidney transplantation (KT). This systematic review investigated the effect of different immunosuppressive regimens on the risk of PTDM. We performed a systematic literature search in MEDLINE and CENTRAL for randomized controlled trials (RCTs) that included KT recipients with any immunosuppression and reported PTDM outcomes up to 1 October 2023. The analysis included 125 RCTs. We found no differences in PTDM risk within induction therapies. In de novo KT, there was an increased risk of developing PTDM with tacrolimus versus cyclosporin (RR 1.71, 95%CI [1.38-2.11]). No differences were observed between tacrolimus+mammalian target of rapamycin inhibitor (mTORi) and tacrolimus+MMF/MPA, but there was a tendency towards a higher risk of PTDM in the cyclosporin+mTORi group (RR 1.42, 95%CI [0.99-2.04]). Conversion from cyclosporin to an mTORi increased PTDM risk (RR 1.89, 95%CI [1.18-3.03]). De novo belatacept compared with a calcineurin inhibitor resulted in 50% lower risk of PTDM (RR 0.50, 95%CI [0.32-0.79]). Steroid avoidance resulted in 31% lower PTDM risk (RR 0.69, 95%CI [0.57-0.83]), whereas steroid withdrawal resulted in no differences. Immunosuppression should be decided on an individual basis, carefully weighing the risk of future PTDM and rejection.

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.

Do drug transporter (ABCB1) SNPs and P-glycoprotein function influence cyclosporine and macrolides exposure in renal transplant patients? Results of the pharmacogenomic substudy within the symphony study.

The function of the efflux pump P-glycoprotein (Pgp) and ABCB1 single nucleotide polymorphisms (SNPs) should be considered as important tools to deepen knowledge of drug nephrotoxicity and disposition mechanisms. The aim of this study is to investigate the association of C3435T, G2677T, C1236T, and T129C ABCB1 SNPs with Pgp activity and exposure to different immunosuppressive drugs in renal transplant patients. Patients included in the Symphony Pharmacogenomic substudy were genotyped for ABCB1 SNPs. According to the design, patients were randomized into four immunosuppressive regimens: low and standard dose of cyclosporine (n = 30), tacrolimus (n = 13), and sirolimus (n = 23) concomitantly with mycophenolate and steroids. Pgp activity was evaluated in PBMC using the Rhodamine 123 efflux assay. TT carrier patients on C3435T, G2677T, and C1236T SNPs (Pgp-low pumpers) showed lower Pgp activity than noncarriers. Pgp-high pumpers treated with cyclosporine showed lower values of Pgp function than macrolides. There was a negative correlation between cyclosporine AUC and Pgp activity at 3 months. Results did not show any correlation between tacrolimus and sirolimus AUC and Pgp activity at 3 months. We found an important role of the ABCB1 SNPs Pgp function in CD3(+) peripheral blood lymphocytes from renal transplant recipients. Pgp activity was influenced by cyclosporine but not macrolides exposure.

Measurement of apixaban concentrations in different human biological fluids by UHPLC-MS/MS. Clinical pharmacokinetic application in a subject with chronic kidney disease and nonvalvular atrial fibrillation on haemodialysis.

BACKGROUND: Apixaban's technical sheet does not recommend its use in clinical practice for patients with chronic kidney disease undergoing haemodialysis. However, recent studies indicate that apixaban could be a safe oral anticoagulant in these kinds of patients who do not present valvular atrial fibrillation. We developed and validated ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) procedures for measuring apixaban concentrations in plasma, dialysate liquid, and urine. MATERIAL AND METHODS: Simple protein precipitation was implemented to prepare samples. Chromatographic separations were achieved on an Acquity®-UPLC®-BEHTM (2.1x100 mm id, 1.7 µm) reverse-phase C18 column using a water/acetonitrile non-linear gradient containing 0.1 % formic acid at a 0.4 mL/min flow rate. Apixaban and its internal standard (apixaban-d3) were detected by electrospray ionisation mass spectrometry in positive and multiple reaction monitoring modes, using transitions of 460.3 â†’ 199.0/443.2 and 463.3 â†’ 202.0, respectively. RESULTS: No significant interferences and carry-overs were observed. Precisions, absolute relative biases, normalised-matrix factors, and normalised recoveries were ≤ 12.2%, ≤8.0%, 94.3-105.1%, and 93.9-105.4%, respectively. Linearity was observed between 5 and 500 µg/L for plasma/dialysate liquid and 5-1000 µg/L for urine. CONCLUSIONS: The validated UHPLC-MS/MS procedures could help support a pharmacokinetic study in non-valvular atrial fibrillation subjects with chronic kidney disease undergoing haemodialysis and apixaban-based anticoagulant therapy.

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.

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.

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.