Tacrolimus Research Articles

Tacrolimus Pharmacokinetics is Associated with Gut Microbiota Diversity in Kidney Transplant Patients: Results from a Pilot Cross-Sectional Study.

Clinical use of tacrolimus (TAC), an essential immunosuppressant following transplantation, is complexified by its high pharmacokinetic (PK) variability. The gut microbiota gains growing interest but limited investigations have evaluated its contribution to TAC PKs. Here, we explore the associations between the gut microbiota composition and TAC PKs. In this pilot cross-sectional study (Clinicaltrial.gov NCT04360031), we recruited 93 CYP3A5 non-expressers stabilized kidney transplant recipients. Gut microbiota composition was characterized by 16S rRNA gene sequencing, TAC PK parameters were computed, and additional demographic and medical covariates were collected. Associations between PK parameters or diabetic status and the gut microbiota composition, as reflected by α- and β-diversity metrics, were evaluated. Patients with higher TAC area under the curve AUC/(dose/kg) had higher bacterial richness, and TAC PK parameters were associated with specific bacterial taxa (e.g., Bilophila) and amplicon sequence variant (ASV; e.g., ASV 1508 and ASV 1982 (Veillonella/unclassified Sporomusaceae); ASV 664 (unclassified Oscillospiraceae)). Building a multiple linear regression model showed that ASV 1508 (co-abundant with ASV 1982) and ASV 664 explained, respectively, 16.0% and 4.6% of the interindividual variability in TAC AUC/(dose/kg) in CYP3A5 non-expresser patients, when adjusting for hematocrit and age. Anaerostipes relative abundance was decreased in patients with diabetes. Altogether, this pilot study revealed unprecedented links between the gut microbiota composition and diversity and TAC PKs in stable kidney transplant recipients. It supports the relevance of studying the gut microbiota as an important contributor to TAC PK variability. Elucidating the causal relationship will offer new perspectives to predict TAC inter- and intra-PK variability.

Development and Evaluation of Tacrolimus Loaded Nano-Transferosomes for Skin Targeting and Dermatitis Treatment

Tacrolimus (TRL) is used for the treatment of atopic dermatitis (AD) due to its T-cell stimulation effect. However, its significantly poor water solubility, low penetration and cytotoxicity have reduced its topical applications. Herein, tacrolimus loaded nano transfersomes (TRL-NTs) were prepared, followed by their incorporation into chitosan gel to prepare tacrolimus loaded nano transfersomal gel (TRL-NTsG). TEM analysis of the TRL-NTs was performed to check their morphology. DSC, XRD and FTIR analysis of the TRL-NTs were executed after lyophilization. Similarly, rheology, spreadability and deformability of the TRL-NTsG were investigated. In vitro release, ex vivo permeation and in vitro interaction of TRL-NTsG with keratinocytes and fibroblasts as well as their co-cultures were investigated along with their in vitro cell viability analysis. Moreover, in vivo skin deposition, ear thickness, histopathology and IgE level were also determined. Besides, 6 months stability study was also performed. Results demonstrated the uniformly distributed negatively charged nanovesicles with a mean particle size distribution of 163 nm and zeta potential of -27 mV. DSC and XRD exhibited the thermal stability and amorphous form of the drug, respectively. The TRL-NTsG showed excellent deformability, spreadability and rheological behavior. In vitro release studies exhibited an 8-fold better release of TRL from the TRL-NTsG. Similarly, 6-fold better permeation and stability of the TRL-NTsG with keratinocytes and fibroblasts as well as their co-cultures was observed. Furthermore, the ear thickness (0.6 mm) of the TRL-NTsG was found significantly reduced when compared with the untreated (1.7 mm) and TRL conventional gel treated mice (1.3 mm). The H&E staining showed no toxicity of the TRL-NTsG with significantly reduced IgE levels (120 ng/mL). The formulation was found stable for at least 6 months. These results suggested the efficacy of TRL in AD-induced animal models most importantly when incorporated in NTsG.

Efficacy of Switching to Adalimumab for Maintenance of Remission Following Induction Therapy with Tacrolimus in Patients with Ulcerative Colitis.

Tacrolimus (TAC) effectively induces remission in refractory ulcerative colitis (UC). However, TAC therapy usually lasts for 3 months. Although azathioprine (AZA) is often used in maintenance therapy, the relapse rate remains high. Herein, we evaluated the efficacy of adalimumab (ADA) for remission maintenance in patients with UC after induction therapy with TAC. We prospectively enrolled patients with moderate-to-severe UC who achieved clinical remission after 3 months of TAC therapy with endoscopic non-mucosal healing (Cohort A). After TAC discontinuation, the remission maintenance rate up to 1 year after starting ADA therapy was examined. We retrospectively enrolled patients with UC treated with TAC (Cohort B). Among patients in clinical remission after TAC treatment for 3 months, those who received AZA as remission maintenance therapy after TAC discontinuation constituted the AZA group. Patients in Cohort A who received ADA and AZA as remission maintenance therapy after TAC discontinuation constituted the ADA + AZA group. We compared the remission maintenance rates in the AZA and ADA + AZA groups for up to 5 years after TAC discontinuation. In Cohort A, of the 46 patients with UC treated with TAC, 17 were eligible for analysis after receiving ADA as remission maintenance therapy. A notable 88.2% (15/17) were still in remission 1 year after starting ADA. The ADA + AZA group (n = 16) exhibited a significantly higher relapse-free rate than the AZA group (n = 26) (p < 0.05; log-rank test). switching to ADA for remission maintenance in patients with refractory UC who achieved clinical remission with TAC is clinically useful.

Pharmacokinetics of immunosuppressive agents during hemoperfusion in a sheep model

IntroductionHemoadsorption shows promising signals in organ preservation and post lung transplantation. However, its potential impact on the pharmacokinetics of immunosuppressant drugs (ID) is still unknown.MethodsIn this interventional study, CytoSorb® hemoperfusion was tested in healthy sheep (n = 5) against a sham extracorporeal circuit (n = 3). Seven different ID (tacrolimus (TAC), cyclosporin A (CYA), mycophenolate mofetil (MMF), everolimus (EVER), basiliximab (BAS), methylprednisolone (MP) and prednisolone (PRED)) were administered in clinically relevant doses and combinations. Their levels were measured repeatedly in blood samples from the extracorporeal circulation over 6 h following administration. Population pharmacokinetic modeling analysis (NONMEM® 7.5) was performed.ResultsNegligible clearance was observed for PRED and BAS. For all other substances, a saturable adsorption sub-model with linear decrease of the adsorption effect over the adsorbed amount best described the measured concentrations. The maximum absolute adsorbed amounts (95% CI) for TAC, CYA, MMF, EVER, and MP were 0.040 (0.028–0.053), 1.15 (0.39–1.91), 4.17 (2.00–6.35), 0.0163 (0.007–0.026), and 53.4 mg (20.9–85.9), respectively, indicating an adsorption of less than 5% of the daily administered dosages for all investigated substances.DiscussionIn this large animal model, CytoSorb® hemoperfusion appears to have a limited effect on the clearance of tested ID.

Efficacy and safety of immunosuppressive agents for adults with lupus nephritis: a systematic review and network meta-analysis.

Various immunosuppressive regimens have been developed for the treatment of lupus nephritis (LN). This study aimed to compare the efficacy and safety of immunosuppressive regimens in adults with LN. We systematically searched the PubMed, Embase, and Cochrane Central Register of Controlled Trials databases, including conference proceedings, trial registries, and reference lists, from inception until July 10, 2022. The effects of treatment were compared and ranked using the surface under the cumulative ranking curve (SUCRA). The primary endpoint was total remission. The secondary endpoints were complete remission, systemic lupus erythematosus disease activity index (SLEDAI), relapse, all-cause mortality, end-stage renal disease (ESRD), infection, herpes zoster, ovarian failure, myelosuppression, and cancer. Sixty-two trials reported in 172 studies involving 6,936 patients were included in the network meta-analysis. The combination of tacrolimus (TAC), mycophenolate mofetil (MMF), and glucocorticoid (GC) provided the best result for the total remission rate (SUCRA, 86.63%) and SLEDAI (SUCRA, 91.00%), while the combination of voclosporin (VCS) , MMF and GC gave the best improvement in the complete remission rate (SUCRA, 90.71%). The combination of cyclophosphamide (CYC), MMF and GC was associated with the lowest risk of relapse (SUCRA, 85.57%) and cancer (SUCRA, 85.14%), while the combination of obinutuzumab (OTB), MMF and GC was associated with the lowest risk of all-cause mortality (SUCRA, 84.07%). Rituximab (RTX) plus MMF plus GC was associated with the lowest risk of ESRD (SUCRA, 83.11%), while the risk of infection was lowest in patients treated with azathioprine (AZA) plus CYC plus GC (SUCRA, 68.59%). TAC plus GC was associated with the lowest risk of herpes zoster (SUCRA, 87.67%) and ovarian failure (SUCRA, 73.60%). Cyclosporine (CsA) plus GC was associated with the lowest risk of myelosuppression (SUCRA, 79.50%), while AZA plus GC was associated with the highest risk of myelosuppression (SUCRA, 16.25%). This study showed that a combination of TAC, MMF and GC was the best regimen for improving the total remission rate. The optimal regimen for specific outcomes should be highlighted for high-risk patients.

Low dose tacrolimus exposure and early steroid withdrawal with strict body weight control can improve post kidney transplant glucose tolerance in Japanese patients.

The development of diabetes mellitus (DM) after living donor kidney transplantation (KT) is a risk factor for worsening transplant kidney function, cardiac disease, and cerebrovascular disease, which may affect prognosis after KT. At our institution, all patients' glucose tolerance is evaluated perioperatively by oral glucose tolerance tests (OGTTs) at pre-KT, and 3, 6, and 12 month (mo.) after KT. We analyzed the insulinogenic index (ISI) and homeostasis model assessment beta cell (HOMA-β) based on the immunoreactive insulin (IRI) levels to determine how glucose tolerance changed after KT in 214 patients who had not been diagnosed with DM before KT. In addition, we analyzed the body mass index (BMI) which may also influence glucose tolerance after KT. The concentration of tacrolimus (TAC) in blood was also measured as the area under the curve (AUC) to examine its effects at each sampling point. The preoperative-OGTTs showed that DM was newly diagnosed in 22 of 214 patients (10.3%) who had not been given a diagnosis of DM by the pre-KT fasting blood sugar (FBS) tests. The glucose tolerance was improved in 15 of 22 DM patients at 12 mo. after KT. ISI and IRI deteriorated only at 3 mo. after KT but improved over time. There was a trend of an inverse correlation between HOMA-β and TAC-AUC. We also found inverse correlations between IRI and an increase in BMI from 3 to 12 mo. after KT. Early corticosteroid withdrawal or the steroid minimization protocol with tacrolimus to maintain a low level of diabetogenic tacrolimus and BMI decrease after KT used by our hospital individualizes lifestyle interventions for each patient might contribute to an improvement in post-KT glucose tolerance.

FRI093 Severe Hypertriglyceridemia Post Liver Transplant- A Multifactorial Disorder

Abstract Disclosure: A.R. Saand: None. A.R. Drinnon: None. F. Mubeen: None. J. Shakil: None. Introduction: Hypertriglyceridemia (HTG) is a known complication in solid organ transplant patients. In addition to increasing cardiovascular complications and mortality risk, these metabolic abnormalities also increase the risk of allograft atherosclerosis and vasculopathy. Although medications used in post-transplant management have a well-documented effect of worsening lipid profile, multiple factors may be contributing in the development of severe HTG. Case Description: We describe a 68-year-old male with elevated triglycerides (TG) of 3957 mg/dL on hospital admission for pneumomediastinum. He received liver transplant 2 years prior due to alcohol-related liver cirrhosis. He had normal TGs prior to the transplant. Immunosuppressant regimen included tacrolimus (TAC) and methylprednisone. Lipoprotein electrophoresis was normal. Intravenous insulin infusion was initiated with moderate improvement however, upon transition to oral medications: fenofibrate, omega-3 fatty acids, niacin and rosuvastatin, TGs continued to increase. Reinitiating insulin infusion showed no improvement. TAC was switched to cyclosporin with transient decrease in TGs. Upon discharge amylase and lipase levels remained normal despite TG elevation. When readmitted for an unrelated reason, his TGs were elevated at 2218 mg/dl. Plasmapheresis was initiated but was complicated by anaphylaxis with angioedema requiring intubation. After partial plasmapheresis, TGs significantly decreased and soon returned to normal. Post-discharge, the patient was changed back to previous TAC dosage due to cyclosporin related renal injury. His steroid dose was reduced and icosapent was added, resulting in no further recurrence of HTG. Discussion: Following liver transplant, about 15-40% recipients have increased cholesterol levels and about 40% have HTG. Although, immunosuppressant-induced dyslipidemia is seen frequently, additional risk factors have been identified. A retrospective study of 85 patients post liver transplant showed HTG developed early within the first month in about 67% patients and persisted throughout the first year. Some common factors predictive of HTG post liver transplant include pre-liver transplant hepatocellular disease and post-transplant renal dysfunction. A study in pediatric patients post liver transplant observed that changes in lipoprotein and HDL subfraction post-transplant may lead to hypertriglyceridemia. Our case suggests that HTG may be multifactorial given the persistent elevation of TGs despite the change in immunosuppressant regimen. Patient factors as well as genetic characteristics of the donor liver may be potential causes. Recognizing this variation in patient populations may be extremely important due to high risk for pancreatitis and cardiovascular events. Early identification and management may prevent complications. Presentation: Friday, June 16, 2023

The impact of CYP3A4 and CYP3A5 genetic variations on tacrolimus treatment of living-donor Egyptian kidney transplanted patients.

Tacrolimus (TAC) is the mainstay of immunosuppressive regimen for kidney transplantations. Its clinical use is complex due to high inter-individual variations which can be partially attributed to genetic variations at the metabolizing enzymes CYP3A4 and CYP3A5. Two single nucleotide polymorphisms (SNPs), CYP3A4*22 and CYP3A5*3, have been reported as important causes of differences in pharmacokinetics that can affect efficacy and/or toxicity of TAC. Investigating the effect of CYP3A4*22 and CYP3A5*3 SNPs individually and in combination on the TAC concentration in Egyptian renal recipients. Overall, 72 Egyptian kidney transplant recipients were genotyped for CYP3A4*22 G>A and CYP3A5*3 T>C. According to the functional defect associated with CYP3A variants, patients were clustered into: poor (PM) and non-poor metabolizers (Non-PM). The impact on dose adjusted through TAC concentrations (C0) and daily doses at different time points after transplantation was evaluated. Cyp3A4*1/*22 and PM groups require significantly lower dose of TAC (mg/kg) at different time points with significantly higher concentration/dose (C0/D) ratio at day 10 in comparison to Cyp3A4*1/*1 and Non-PM groups respectively. However, CyP3A5*3 heterozygous individuals did not show any significant difference in comparison to CyP3A5*1/*3 individuals. By comparing between PM and Non-PM, the PM group had a significantly lower rate of recipients not reaching target C0 at day 14. This is the first study on Egyptian population to investigate the impact of CYP3A4*22 and CYP3A5*3 SNPs individually and in combination on the TAC concentration. This study and future multicenter studies can contribute to the individualization of TAC dosing in Egyptian patients.

Alterations in the Gut Microbiome in Liver Recipients with Post-Transplant Diabetes Mellitus

Post-transplant diabetes mellitus (PTDM) increases the risk of graft failure and death in liver transplant (LT) recipients. Experimental studies have indicated that enteric dysbiosis mediated by immunosuppressive tacrolimus (TAC) could contribute to glucose disorders, but no data on human recipients with PTDM have been reported. Here, by combining high-throughput shotgun metagenomics sequencing and metabolomics profiling, we characterized the intestinal microbiome (IM) in LT recipient cohort with or without PTDM and deciphered the potential relationship among IM, TAC dosage, and diabetes. By comparing with both non-PTDM and classical type 2 diabetes mellitus (T2DM), we identified microbial signatures of PTDM, which was characterized by the enriched Proteobacteria and decreased Bacteroidetes. Additionally, the altered microbes, as well as the microbial metabolomics, correlated with the dosage of TAC. Specifically, the levels of beneficial microbes associated with PTDM were lowered in recipients with the high TAC trough concentrations (> 5 ng∙mL–1) than those with low ones (< 5 ng∙mL–1), which was accompanied by reduced faecal metabolites involved in the biosynthesis of α-linolenic acid and arachidonic acid-lowering factors of developing T2DM. Moreover, these microbial signatures linked with the extent of glucose disorders in LT recipients. In summary, the faecal microbiome and metabolome differed between PTDM and non-PTDM patients, which were linked with TAC dosage. This study was the first to explore taxonomic alterations and bacterial gene functions to better understand the contribution of the IM to PTDM.

B-329 Comparison Between Trough and AUC Monitoring of MPA in Patients With and Without Organ Transplantation. Influence of the Co-administered Immunosuppressant

Abstract Background Monitoring of mycophenolic acid (MPA) could be performed by measurement of trough concentrations with target therapeutic range of 1.3 to 3.5 mg/L, or by calculation of area under the drug concentration vs time curve (AUC) utilizing 3 or 4 samples within a dosing interval, with a target range of 30 to 60 mg*h/L. AUC is considered more reliable compared to trough levels, and MPA kinetics are dependent on the type of the co-administered calcineurine inhibitor. This study compares trough vs AUC MPA monitoring received alone or in co-administration with Cyclosporine (CsA) or with Tacrolimus (TAC). Methods 77 transplanted patients receiving MPA, 25 of them on CsA, 49 on TAC, and 20 non-transplanted patients on MPA alone, were evaluated more than once, yielding 260 monitoring consults included for assessment: 45 for MPA alone, 108 for MPA combined with CsA, and 117 consults for the MPA combined with TAC. MPA, CsA, and TAC levels were measured by validated LC/MS/MS methods. Validated pharmacokinetic programs for the calculation of MPA AUC were used, adapted to the type of calcineurine inhibitor. Assessment was made by comparison of the distribution and coincidence between trough levels and AUC with respect to the above therapeutic ranges. Results Transplanted patents: when combined with CsA, MPA AUC was in the target range in 61% of cases, under it—in 31%, and in 8%—over it, while trough levels were 52% under, 42.5% within, and 5.5% over the therapeutic range; agreement between trough MPA and MPA AUC was poor—there was coincidence in 37.5% of sub-therapeutic, 63% of therapeutic and 0% of supra-therapeutic ranges, 3 cases were identified with sub-therapeutic trough and supra-therapeutic AUC; when combined with TAC, MPA AUC was in the target range in 63% of cases, under it—in 13%, and in 24%—over it; trough levels being 23% under, 60% within and 17% over the therapeutic range; agreement between trough MPA and MPA AUC was much better, compared to the combination with CsA: there was coincidence in 56% of sub-therapeutic, 84% of therapeutic and 80% of supra-therapeutic ranges. Nevertheless, 1 case was found with sub-therapeutic trough and supra-therapeutic AUC. In non-transplanted patients MPA AUC was in the target range in 47% of cases, under it—in 13%, and in 40%—over it; trough levels being 18% under, 53% within and 29% over the therapeutic range; agreement between trough MPA and MPA AUC showed coincidence in 75% of sub-therapeutic, 75% of therapeutic and 92% of supra-therapeutic ranges. Conclusion This study confirms that MPA AUC is more relevant than trough monitoring for dose individualization. It also shows that MPA trough levels are much more concordant with MPA AUC in non-transplanted patients and in transplanted patients on TAC, while in combination with CsA, discordance remains significant.

A-383 A Novel Lateral Flow Immunoassay for Health Biomarker and Immunosuppressive Drug Monitoring in Fingerstick Blood in a Point-of-Care Setting

Abstract Background Rapid, CLIA-waived quantitative testing can help eliminate delays in receiving care, improve telemedicine, enhance the management of chronic diseases and clinical trials, and reduce healthcare costs. Intelligent Optical Systems has developed an innovative Enhanced Lateral Flow Assay (ELFA) platform and partnered with the University of California, Irvine Nephrology Division and Wake Forest Institute for Regenerative Medicine for the clinical validation of the assay. The ELFA platform quantitatively measures the immunosuppressive drug tacrolimus (TAC), and health biomarkers such as proinflammatory cytokines IL6, IL8, matrix metalloproteinase-3 (MMP3), glial fibrillary acidic protein (GFAP, an indicator of traumatic brain injury), and cystatin C (CysC, an indicator of kidney function) from fingerstick blood at the point of care. The testing panel is continuously expanding. Methods The ELFA platform is an in vitro, quantitative, single plex or multiplexed lateral flow immunoassay that consists of three main components: (i) simple fingerstick whole blood sampling kit; (ii) lateral flow test strip with running buffer and dried fluorescent labeled affinity reagents; and (iii) miniaturized readout device that can be further developed with a controlled user interface, enabling untrained users to easily record results and instantly transmit them to the physician. Depending on the analytes, the ELFA point-of-care platform has undergone bench testing or clinical validation studies, including: (1) Single plex for TAC: Clinical validation using 5 uL of capillary fingerstick whole blood from kidney transplant recipients (KTR, n = 50) compared to standard LC-MS/MS laboratory method. (2) Multiplexed ELFA for TAC, IL6, and MMP3: In vitro assessment of limit of detection (LoD), detection range, specificity, and repeatability (n = 5) with 10 uL spiked whole blood. (3) Single plex ELFA for GFAP, IL8, and CysC: In vitro determination of the LoD, detection range, CV%, and correlation of spiked and estimated analyte levels with 10 uL of spiked whole blood. For all analytes, a 4-parameter logistic calibration curve with n = 3 for each level of analyte was constructed to determine the LoD, detection range, and CV%. Pearson correlation and Bland-Altman analysis were applied to correlate and compare data between spiked and estimated levels, or between ELFA and standard lab test methods. Results Study (1) demonstrated excellent correlation between ELFA and LC-MS/MS, with a correlation coefficient r-value of 0.89, and at 95% limit of agreement, a mean difference of 0.7 ng/mL in the clinically compared samples from 50 KTRs. Study (2) showed an LoD and range of detection of 2 ng/mL to 20 ng/mL, 1 ng/mL to 5 ng/mL, and 10 ng/mL to 200 ng/mL for the multiplexed detection of TAC, IL6, and MMP3, respectively. The repeatability assessment showed a &amp;lt;10%CV for all analytes. Study (3) showed an LoD and range of detection of 1 ng/mL to 10 ng/mL, 0.5 ng/mL to 5 ng/mL, and 0 to 5 mg/L for GFAP, IL8, and CysC, respectively. Conclusion The ELFA platform delivers minimally invasive, convenient sampling in a POC device using capillary fingerstick blood, and provides accurate, rapid measurements of TAC, which demonstrate good repeatability and strong correlation against LC-MS standard reference measurements.

No Difference Between Tacrolimus and Cyclosporine A on Depression Among Kidney Transplantation Recipients

The 2 main types of calcineurin inhibitors (CNI) are tacrolimus (TAC) and cyclosporine A (CsA); both are needed by patients who receive kidney transplants. A common adverse reaction of TAC is depression, which is listed in its instructions. However, depression occurred rarely, according to the instructions manual for CsA. Scales measuring depression were sent to recipients who had taken TAC or CsA to observe whether there was a difference in depression between patients who consumed the 2 drugs. From September 23rd-December 8th 2022, a questionnaire was sent to kidney transplant recipients online to investigate depression by PHQ-9 score. Then, the questionnaires returned were divided into 2 groups: TAC group and CsA group. The difference of basic characteristics was made to equal by means of propensity score matching (PSM). The scores, degrees of depression, and prevalence of major depression between the 2 groups were compared. Of 259 questionnaires returned, 220 questionnaires were valid. Among them, 170 recipients used TAC and 50 recipients used CsA. There were no significant differences in baseline characteristics after PSM. After PSM, there was no statistically significant difference in PHQ-9 (0.8) score, degree of depression (P=.7), or rate of major depression between the 2 groups. There was no significant difference between kidney transplant recipients taking TAC or CsA in PHQ-9 score, degree of depression, or prevalence of major depression.

Dynamic Monitoring of Intracellular Tacrolimus and Mycophenolic Acid Therapy in Renal Transplant Recipients Using Magnetic Bead Extraction Combined with LC-MS/MS.

Tacrolimus (TAC) and mycophenolic acid (MPA) are commonly used immunosuppressive therapies after renal transplant. Our objective was to quantify TAC and MPA concentrations in peripheral blood mononuclear cells (PBMCs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) and to evaluate and validate the performance of the methodology. A prospective follow-up cohort study was conducted to determine whether intracellular concentrations were associated with adverse outcomes in renal transplants. PBMCs were prepared using the Ficoll separation technique and purified with erythrocyte lysis. The cells were counted using Sysmex XN-3100 and then packaged and frozen according to a 50 µL volume containing 1.0 × 106 cells. TAC and MPA were extracted using MagnaBeads and quantified using an LC-MS/MS platform. The chromatography was run on a reversed-phase Waters Acquity UPLC BEH C18 column (1.7 µm, 50 mm × 2.1 mm) for gradient elution separation with a total run time of 4.5 min and a flow rate of 0.3 mL/min. Mobile phases A and B were water and methanol, respectively, each containing 2 mM ammonium acetate and 0.1% formic acid. Renal transplant recipients receiving TAC and MPA in combination were selected for clinical validation and divided into two groups: a stable group and an adverse outcome group. The concentrations were dynamically monitored at 5, 7, 14, and 21 days (D5, D7, D14, and D21) and 1, 2, 3, and 6 months (M1, M2, M3, and M6) after operation. Method performance validation was performed according to Food and Drug Administration guidelines, showing high specificity and sensitivity. The TAC and MPA calibration curves were linear (r2 = 0.9988 and r2 = 0.9990, respectively). Both intra-day and inter-day imprecision and inaccuracy were less than 15%. Matrix effects and recoveries were satisfactory. The TAC and MPA concentrations in 304 "real" PBMC samples from 47 renal transplant recipients were within the calibration curve range (0.12 to 16.40 ng/mL and 0.20 to 4.72 ng/mL, respectively). There was a weak correlation between PBMC-C0TAC and WB-C0TAC (p < 0.05), but no correlation was found for MPA. The level of immunosuppressive intra-patient variation (IPV) was higher in PBMC at 77.47% (55.06, 97.76%) than in WB at 34.61% (21.90, 49.85%). During the dynamic change in C0TAC, PBMC-C0TAC was in a fluctuating state, and no stable period was found. PBMC-C0TAC did not show a significant difference between the stable and adverse outcome group, but the level of the adverse outcome group was generally higher than that of the stable group. Compared with conventional therapeutic drug monitoring, the proposed rapid and sensitive method can provide more clinically reliable information on drug concentration at an active site, which has the potential to be applied to the clinical monitoring of intracellular immunosuppressive concentration in organ transplantation. However, the application of PBMC-C0TAC in adverse outcomes of renal transplant should be studied further.

Computational simulation on the study of Tacrolimus and its improved dermal retention using Poly(Ԑ-caprolactone) nanocapsules

Tacrolimus (TAC) is a drug from natural origin that can be used for topical application to control autoimmune skin diseases such as atopic dermatitis, psoriasis, and vitiligo. Computational simulation based on quantum mechanics theory by solving Schrödinger Equation for n-body problem may allow the theoretical calculation of drug geometry, charge distribution and dipole moment, electronic levels and molecular orbitals, electronic transitions, and vibrational transitions. Additionally, the development of novel nanotechnology-based delivery systems containing TAC can be an approach for reducing the dose applied topically, increasing dermal retention, and reducing the reported side effects due to the controlled release pattern. Firstly, this paper was devoted to obtaining the molecular, electronic, and vibrational data for TAC by using five semi-empirical (SE) methods and one Density Functional Theory (DFT) method in order to expand the knowledge about the drug properties by computational simulation. Then, this study was carried out to prepare TAC-loaded poly(ԑ-caprolactone) nanocapsules by interfacial polymer deposition following solvent displacement and investigate the in vitro drug permeation using the Franz diffusion cell and the photoacoustic spectroscopy. Computational simulations were compared in the three schemes SE/SE, SE/DFT, and DFT/DFT, where the first method represented the procedure used for geometry optimization and the second one was performed to extract electronic and vibrational properties. Computational data showed correspondence with TAC geometry description and electronic properties, with few differences in HOMO – LUMO gap (Δ) and dipole values. The SE/DFT and DFT/DFT methods presented a better drug description for the UV–Vis, Infrared, and Raman spectra with low deviation from experimental values. Franz cell model demonstrated that TAC was more delivered across the Strat-M® membrane from the solution than the drug-loaded poly(ԑ-caprolactone) nanocapsules. Photoacoustic spectroscopy assay revealed that these nanocapsules remained more retained into the Strat-M® membranes, which is desirable for the topical application.

Dosage optimization of tacrolimus based on the glucocorticoid dose and pharmacogenetics in adult patients with systemic lupus erythematosus

BackgroundThe purpose of the study was to develop a genotype-incorporated population pharmacokinetic (PPK) model of tacrolimus (TAC) in adults with systemic lupus erythematosus (SLE) to investigate the factors influencing TAC pharmacokinetics and to develop an individualized dosing regimen based on the model. In addition, a non-genotype-incorporated model was also established to assess its predictive performance compared to the genotype-incorporated model. MethodsA total of 365 trough concentrations from 133 adult SLE patients treated with TAC were collected to develop a genotype-incorporated PPK model and a non-genotype-incorporated PPK model of TAC using a nonlinear mixed-effects model (NONMEM). External validation of the two models was performed using data from an additional 29 patients. Goodness-of-fit diagnostic plots, bootstrap method, and normalized predictive distribution error test were used to validate the predictive performance and stability of the final models. The goodness-of-fit of the two final models was compared using the Akaike information criterion (AIC). The dosing regimen was optimized using Monte Carlo simulations based on the developed optimal model. ResultsThe typical value of the apparent clearance (CL/F) of TAC estimated in the final genotype-incorporated model was 14.3 L h−1 with inter-individual variability of 27.6%. CYP3A5 polymorphism and coadministered medication were significant factors affecting TAC-CL/F. CYP3A5 rs776746 GG genotype carriers had only 77.3% of the TAC-CL/F of AA or AG genotype carriers. Omeprazole reduced TAC-CL/F by 3.7 L h−1 when combined with TAC, while TAC-CL/F increased nonlinearly as glucocorticoid dose increased. Similar findings were demonstrated in the non-genotype-incorporated PPK model. Comparing these two models, the genotype-incorporated PPK model was superior to the non-genotype-incorporated PPK model (AIC = 643.19 vs. 657.425). Monte Carlo simulation based on the genotype-incorporated PPK model indicated that CYP3A5 rs776746 AA or AG genotype carriers required a 1/2–1 fold higher dose of TAC than GG genotype carriers to achieve the target concentration. And as the daily dose of prednisone increases, the dose of TAC required to reach the target concentration increases appropriately. ConclusionsWe developed the first pharmacogenetic-based PPK model of TAC in adult patients with SLE and proposed a dosing regimen based on glucocorticoid dose and CYP3A5 genotype according to the model, which could facilitate individualized dosing for TAC.

Investigation of the effects of urotensin II receptors in LPS-induced inflammatory response in HUVEC cell line through calcineurin/NFATc/IL-2 pathway

PurposeThe effect of urotensin II (U-II), a powerful endogenous vasoconstrictor substance, on the immune system and its mediators is very important.It was herein aimed to demonstrate the possible relationship between the calcineurin/nuclear factor of activated T-cells cytoplasmic 1/interleukin-2 (CaN/NFATc/IL-2) pathway and urotensin receptors (UTRs) in inflammatory response due to lipopolysaccharide (LPS). MethodsAn LPS-induced inflammation model was used on the human umbilical vein endothelial cells (HUVEC) cell line and drugs were applied accordingly, forming the following groups: Control Group, LPS Group, Agonist Group (10−8 ​M U-II), Antagonist Group (10−6 ​M palosuran), Tacrolimus (TAC) Group (10 ​ng/mL FK-506), Agonist ​+ ​TAC Group, and Antagonist ​+ ​TAC Group. Gene expression analyses were performed using real-time polymerase chain reaction (RT-PCR). ResultsIn the analysis of the cell viability at 48 and 72 ​h, there was a decrease in the Agonist Group, while in the Agonist ​+ ​TAC Group, the cell viability increased. In the Antagonist Group, cell viability was maintained when compared to the LPS Group, while in the TAC Group, this effect was reduced. The mRNA expression levels of UTR, CaN, NFATc, IL-2 receptor (IL-2R), IL-6 and nuclear factor kappa B (NF-κB) were higher in the LPS Group than in the Control Group, and even the UTR, CaN, NFATc, IL-2R were higher with agonist administration. This effect of the agonist was shown to be completely mitigated in the presence of the CaN inhibitor. ConclusionU-II and its receptors can perform key functions regarding the endothelial cell damage via the CaN/NFATc/IL-2 pathway.

Predictive Factors for Efficacy of Oral Tacrolimus Induction Therapy in Moderate to Severe Ulcerative Colitis Patients: Large Multicenter Retrospective Cohort Study.

Tacrolimus (TAC), a calcineurin inhibitor, is used for remission induction therapy in patients with moderate to severe ulcerative colitis (UC), with short-term efficacy and related predictive factors shown in previous cohort studies. However, most studies reported data for only a limited number of patients enrolled from a single center. We performed a large multicenter retrospective cohort study to identify factors related to prediction of clinical remission in UC patients treated with oral TAC. The medical records of patients with moderate to severe UC treated with oral TAC as induction therapy at 7 institutions between April 2009 and March 2017 were retrospectively reviewed. A total of 216 patients who received TAC for induction were analyzed, of whom 123 (56.9%) showed clinical remission at week 12. Logistic regression analysis indicated that previous or current use of antitumor necrosis factor (TNF)-α antibodies (odds ratio [OR], 0.259; P = .006), and concomitant treatment with 5-aminosalicylate (5-ASA) at the baseline (OR, 0.268; P = .005) were independent predictive factors correlated with failure of clinical remission, whereas higher levels of C-reactive protein (OR, 1.124; P = .014) predicted achievement of clinical remission. Results of this multicenter study clearly indicate the efficacy of TAC induction therapy for patients with moderate to severe UC. Notably, previous or current use of anti-TNF-α antibodies was associated with poor achievement of clinical remission by week 12.

CircFOXN2 alleviates glucocorticoid- and tacrolimus-induced dyslipidemia by reducing FASN mRNA stability by binding to PTBP1 during liver transplantation.

We aimed to examine impacts and functional mechanism of circular RNA forkhead box N2 (FOXN2) in tacrolimus (TAC)- and dexamethasone (Dex)-induced lipid metabolism disorders. RNA level and protein contents in TAC, Dex, or combined TAC- plus Dex-treated patients and Huh-7 cells were measured utilizing quantitative real-time (qRT)-PCR and western blotting assays measured the formation of lipid droplet. Total cholesterol (TC) and triglyceride (TG) levels were determined using corresponding commercial kits and Oil red O staining. RNA immunoprecipitation and RNA pull-down verified the binding relationship among circFOXN2, polypyrimidine tract binding protein 1 (PTBP1) and fatty acid synthase (FASN). Male C57BL/6 mice were used to establish a dyslipidemia mouse model to validate the discoveries at the cellular level. Dex treatment significantly promoted TAC-mediated increase of TC and TG in serum samples and Huh-7 cells. Moreover, circFOXN2 was reduced but FASN was elevated in TAC-treated Huh-7 cells, and these expression trends were markedly enhanced by Dex cotreatment. Overexpression of circFOXN2 could reverse the accumulation of TC and TG and the upregulation of FASN and sterol regulatory element binding transcription factor 2 (SREBP2) mediated by Dex and TAC cotreatment. Mechanistically, circFOXN2 reduced FASN mRNA stability by recruiting PTBP1. The protective roles of circFOXN2 overexpression on lipid metabolism disorders were weakened by FASN overexpression. In vivo finding also disclosed that circFOXN2 greatly alleviated the dysregulation of lipid metabolism triggered by TAC plus Dex. CircFOXN2 alleviated the dysregulation of lipid metabolism induced by the combination of TAC and Dex by modulating the PTBP1/FASN axis.NEW & NOTEWORTHY Collectively, our experiments revealed for the first time that circFOXN2 alleviated the Dex- and TAC-induced dysregulation of lipid metabolism by regulating the PTBP1/FASN axis. These findings suggested that circFOXN2 and FASN might be candidate targets for the treatment of Dex- and TAC-induced metabolic disorders.

Physiologically based pharmacokinetic model combined with reverse dose method to study the nephrotoxic tolerance dose of tacrolimus.

Nephrotoxicity is the most common side effect that severely limits the clinical application of tacrolimus (TAC), an immunosuppressive agent used in kidney transplant patients. This study aimed to explore the tolerated dose of nephrotoxicity of TAC in individuals with different CYP3A5 genotypes and liver conditions. We established a human whole-body physiological pharmacokinetic (WB-PBPK) model and validated it using data from previous clinical studies. Following the injection of 1mg/kg TAC into the tail veins of male rats, we developed a rat PBPK model utilizing the drug concentration-time curve obtained by LC-MS/MS. Next, we converted the established rat PBPK model into the human kidney PBPK model. To establish renal concentrations, the BMCL5 of the in vitro CCK-8 toxicity response curve (drug concentration range: 2-80mol/L) was extrapolated. To further investigate the acceptable levels of nephrotoxicity for several distinct CYP3A5 genotypes and varied hepatic function populations, oral dosing regimens were extrapolated utilizing in vitro-in vivo extrapolation (IVIVE). The PBPK model indicated the tolerated doses of nephrotoxicity were 0.14-0.185mg/kg (CYP3A5 expressors) and 0.13-0.155mg/kg (CYP3A5 non-expressors) in normal healthy subjects and 0.07-0.09mg/kg (CYP3A5 expressors) and 0.06-0.08mg/kg (CYP3A5 non-expressors) in patients with mild hepatic insufficiency. Further, patients with moderate hepatic insufficiency tolerated doses of 0.045-0.06mg/kg (CYP3A5 expressors) and 0.04-0.05mg/kg (CYP3A5 non-expressors), while in patients with moderate hepatic insufficiency, doses of 0.028-0.04mg/kg (CYP3A5 expressors) and 0.022-0.03mg/kg (CYP3A5 non-expressors) were tolerated. Overall, our study highlights the combined usage of the PBPK model and the IVIVE approach as a valuable tool for predicting toxicity tolerated doses of a drug in a specific group.

Therapeutic Drug Monitoring of Tacrolimus in Tunisian Renal Transplant Patients during the Tuberculosis Infection: A Retrospective, Observational, Single-centre Analysis.

A retrospective, observational, single-center analysis was performed at the Department of Clinical Pharmacology (National Centre of Pharmacovigilance, Tunisia). We analyzed the database of patients who received TAC from 2009 until 2018. We included samples provided from renal transplant patients infected by Mycobacterium tuberculosis after transplantation. Trough blood levels (C0) were determined using an immunoassay analyzer. The Therapeutic Range (TR) of TAC was considered between 5 and 10 ng/mL. Pharmacokinetic parameters were compared between the period of co-administration of TAC/ATT (period A) and the period during which patients received only TAC (period B). Seven renal transplant patients treated by TAC were included. 41 samples were analyzed (16; period A, 25; period B). Only 6 % of C0 values were found within TR during period A, while this rate was 44% during period B. During period A, 88% of TAC C0 was under the lower limit of TR, indicating a high risk of transplant rejection. The mean C0 and C0/D were significantly lower during period A (3.11±1.53 ng/mL vs 7.11 ± 3.37 ng/mL; p = 0.001 and 33.06 ± 24.89 vs 83.14 ± 44.46; p = 0.0006, respectively), without difference in doses between periods. Considering the results of this study, clinicians are suggested to monitor TAC closely in this particular circumstance.