Calcineurin Inhibitors Cyclosporine Research Articles

#1888 Association between intrapatient variability of serum tacrolimus levels with the development of diabetes in post-kidney transplantation patients

Abstract Background and Aims Kidney transplantation is the most cost-effective renal replacement therapy compared to dialysis. Diabetes is main cause of increased cardiovascular risk and death. Post-transplant Diabetes Mellitus (PTDM) it refers to presence of diabetes in an environment after kidney transplantation; According to the ADA (American Diabetes Association) criteria, the diagnosis must be made with a tolerance test with 75 grams of oral glucose (Oral glucose tolerance test-OGTT) or the determination of glycosylated hemoglobin 1Ac (Hb1Ac) in a post-transplant period of stability of the immunosuppression dose and without infections. There are various risk factors for the development of PTDM: hypomagnesemia, glucocorticoids, calcineurin inhibitors (tacrolimus and cyclosporine); hepatitis C virus and cytomegalovirus infections. In a Mexican kidney transplant recipients in Guadalajara, Andrade SG demonstrated that the incidence of PTDM was 10.1% at 1 year post-transplant. In the Spanish population, Porrini E demonstrated that tacrolimus represents a risk factor for the development of PTDM, increasing the incidence of prediabetes by 33% at 12 months post-transplant. Several mechanisms have been implicated in the association of calcineurin inhibitor and PTDM: decreased insulin secretion, glucose intolerance, reduced pancreatic beta cell mass, increased islet apoptosis and decreased insulin gene expression. Studies mention that tacrolimus (FK) is more diabetogenic than cyclosporine (CsA) especially at levels above 20 ng/mL. The DIRECT study (Diabetes Incidence after Renal Transplantation) showed the incidence in 73 kidney transplant patients with CsA (26%) and 96 FK patients (33.6%, p = 0.046). By the other side, Malik RF mentions that FK levels aren´t a risk factor for the development of PTDM. To demonstrate that the high intrapatient variability of tacrolimus/cyclosporine serum levels correlates with the development of diabetes (PTDM) in kidney transplant recipients at the central military hospital in Mexico City. Method Observational, analytical, longitudinal and retrospective study. All kidney transplant recipients from living donors and cadaveric donors from January 2002 to December 2020 were included. FK and CsA were used as maintenance immunosuppression. Serum levels of FK/CsA, Hb1Ac and glucose were measured at months 0, 3, 6, 9 and 12 and intra-patient variability (IPV) of FK/CsA levels was calculated for each patient. The patients were separated into 2 groups, IPV low and IPV high according to the mean the coefficient of variability (CV). The IPV of the calcineurin inhibitor was estimated by calculating the CV in each patient according to the following equation: CV(%)= (SD/mean trough concentration of FK/ CsA)×100. The diagnosis of PTDM followed the criteria of the American Diabetes Association. The Mann Whitney U test was used. Results 638 kidney transplant recipients were included. The mean age of the recipients was 36.78 (±12.81) years. At the time of kidney transplant, the BMI was 22.67 (±3.90) and 25.76 (±3.99) kg/m² BS at 1 year post-transplant for whole cohort. At 1 year post-transplant, 72.3% patients were on FK and 27.7% on CsA. Hb1Ac for whole cohort was 4.81 (±0.71)% and 4.84 (±0.77)% at month 0 and 12 post-kidney transplant, respectively. FK and CsA levels were 12.41 (±5.31) and 234.34 (±87.80) ng/mL and 9.13 (±5.3) and 194.38 (±92.44) ng/mL in the PTDM and non-PTDM groups, respectively (p = 0.029). 17.96% of patients developed PTDM. Mean CV of the entire cohort was 33.84 (±20.41)%. When performing the analysis of the IPV coefficient by subgroups of maintenance immunosuppression used at 1 year post-transplant: the IPV of FK was 43.39 (±18.49)% and 29.99 (±17.56)% in the PTDM and non-PTDM groups respectively, p = 0.000, which shows that patients with a high IPV coefficient of tacrolimus developed more PTDM than those with low variability, student's t-test to compare IPV was 0.03. The IPV of CsA was 38.61 (±18.76)% and 37.51 (±25.67)% in the PTDM and non-PTDM groups respectively, p = 0.819. Conclusion High intrapatient variability of tacrolimus was correlated with the development of PTDM in Mexican kidney transplant recipients.

Reduced Prevalence of Parkinson's Disease in Patients Prescribed Calcineurin Inhibitors.

Preclinical evidence suggests calcineurin inhibitors (CNIs) combat α-synuclein-induced neuronal dysfunction and motor impairments. However, whether CNIs prevent or treat Parkinson's disease (PD) in humans has never been investigated. We seek to ascertain if prescription of CNIs is linked to a decreased prevalence of PD in a varied patient population and to glimpse into the mechanism(s) and target site through which CNIs might decrease PD prevalence. We analyzed electronic health records (EHRs) from patients prescribed the brain penetrant CNI tacrolimus (TAC), the peripherally restricted CNI cyclosporine (CySp), or the non-CNI sirolimus (SIR). For comparison, EHRs from a diverse population from the same network served as a general population-like control. After propensity-score matching, prevalence, odds, and hazards of PD diagnoses among these cohorts were compared. Patients prescribed CNIs have decreased odds of PD diagnosis compared to the general population-like control, while patients prescribed SIR do not. Notably, patients prescribed TAC have a decreased prevalence of PD compared to patients prescribed SIR or CySp. Our results suggest CNIs, especially those acting within the brain, may prevent PD. The reduced prevalence of PD in patients prescribed TAC, compared to patients prescribed SIR, suggests that mechanisms of calcineurin inhibition- other than immunosuppression, which is common to both drugs- are driving the reduction. Therefore, CNIs may provide a promising therapeutic approach for PD.

Post-transplant cyclophosphamide, calcineurin inhibitor, and mycophenolate mofetil compared to anti-thymocyte globulin, calcineurin inhibitor, and methotrexate combinations as graft-versus-host disease prophylaxis post allogeneic stem cell transplantation from sibling and unrelated donors in patients with acute myeloid leukemia: a study on behalf of the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation.

Post-transplant cyclophosphamide plus calcineurin inhibitor (CNI)(tacrolimus or cyclosporine A) plus mycophenolate mofetil (PTCy/TAC or CSA/MMF) and anti-thymocyte globulin plus CNI (tacrolimus or cyclosporine A) plus methotrexate (ATG/TAC or CSA/MTX) are common graft-versus-host disease (GVHD) prophylaxis regimens. We compared the two regimens in patients with acute myeloid leukemia (AML) undergoing allogeneic transplantation from matched siblings or unrelated donors. 402 received PTCy/TAC or CSA/MMF and 5648 received ATG/TAC or CSA/MTX. Patients in the PTCy-based group were younger (48.7 vs. 51.5 years, p = 0.024) and there was a higher frequency of patient cytomegalovirus seropositivity and female donor to male patient combination in this group (77.8% vs. 71.8%, p = 0.009 and 18.4% vs. 14.4%, p = 0.029, respectively). More patients in the PTCy-based group received reduced-intensity conditioning (51.5% vs. 41%, p < 0.0001). No differences were observed in the incidence of acute GVHD grade II-IV and III-IV (21.2% vs. 20.4%, p = 0.92 and 8.1% vs. 6%, p = 0.1) or 2-year total and extensive chronic GVHD (33.7% vs. 30%, p = 0.09 and 10.7% vs. 11.2%, p = 0.81) between the groups. In the multivariate analysis, all transplant outcomes did not differ between the groups. PTCy/CNI/MMF and ATG/CNI/MTX are alternative regimens for GVHD prophylaxis in AML patients.

Assessing Trans-Inhibition of OATP1B1 and OATP1B3 by Calcineurin and/or PPIase Inhibitors and Global Identification of OATP1B1/3-Associated Proteins.

Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are key determinants of drug-drug interactions (DDIs). Various drugs including the calcineurin inhibitor (CNI) cyclosporine A (CsA) exert preincubation-induced trans-inhibitory effects upon OATP1B1 and/or OATP1B3 (abbreviated as OATP1B1/3) by unknown mechanism(s). OATP1B1/3 are phosphoproteins; calcineurin, which dephosphorylates and regulates numerous phosphoproteins, has not previously been investigated in the context of preincubation-induced trans-inhibition of OATP1B1/3. Herein, we compare the trans-inhibitory effects exerted on OATP1B1 and OATP1B3 by CsA, the non-analogous CNI tacrolimus, and the non-CNI CsA analogue SCY-635 in transporter-overexpressing human embryonic kidney (HEK) 293 stable cell lines. Preincubation (10-60 min) with tacrolimus (1-10 µM) rapidly and significantly reduces OATP1B1- and OATP1B3-mediated transport up to 0.18 ± 0.03- and 0.20 ± 0.02-fold compared to the control, respectively. Both CsA and SCY-635 can trans-inhibit OATP1B1, with the inhibitory effects progressively increasing over a 60 min preincubation time. At each equivalent preincubation time, CsA has greater trans-inhibitory effects toward OATP1B1 than SCY-635. Preincubation with SCY-635 for 60 min yielded IC50 of 2.2 ± 1.4 µM against OATP1B1, which is ~18 fold greater than that of CsA (0.12 ± 0.04 µM). Furthermore, a proteomics-based screening for protein interactors was used to examine possible proteins and processes contributing to OATP1B1/3 regulation and preincubation-induced inhibition by CNIs and other drugs. A total of 861 and 357 proteins were identified as specifically associated with OATP1B1 and OATP1B3, respectively, including various protein kinases, ubiquitin-related enzymes, the tacrolimus (FK506)-binding proteins FKBP5 and FKBP8, and several known regulatory targets of calcineurin. The current study reports several novel findings that expand our understanding of impaired OATP1B1/3 function; these include preincubation-induced trans-inhibition of OATP1B1/3 by the CNI tacrolimus, greater preincubation-induced inhibition by CsA compared to its non-CNI analogue SCY-635, and association of OATP1B1/3 with various proteins relevant to established and candidate OATP1B1/3 regulatory processes.

Chronic GVHD: review advances in prevention, novel endpoints, and targeted strategies.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for many malignant and non-malignant hematologic disorders. Chronic graft-versus-host (cGVHD) disease remains a significant hurdle for long-term survival in patients post allo-HCT, and it remains the leading cause of late non-relapse mortality. The risk factors for development of cGVHD include degree of human leukocyte antigen (HLA) disparity, increasing recipient age, use of peripheral blood stem cells as a source, myeloablative conditioning regimens, prior acute GVHD (aGVHD), and female donor to male recipient. Our biological understanding of cGVHD is mostly derived from transplantation mouse models and patient data. There are three distinct phases in the development of cGVHD. Approaches to prevent GVHD include pharmacologic strategies such as calcineurin inhibitors (cyclosporine, tacrolimus) combined with methotrexate or mTOR inhibitors (sirolimus), and IMP dehydrogenase inhibitors (mycophenolate mofetil). Increasingly, posttransplant cyclophosphamide is emerging as a promising strategy for GVCHD prevention especially in a setting of reduced intensity conditioning. Other approaches include serotherapy (ATG, Campath) and graft manipulation strategies. A significant obstacle to evaluating the response of novel GVHD-directed therapies has been standardized response assessments. This has functioned as a barrier to designing and interpreting clinical trials that are structured around the treatment of cGVHD. Novel endpoints including failure-free survival, Graft-versus-host disease-free, relapse-free survival (GRFS), and current GVHD-free, relapse-free survival (CGRFS) may create a clearer picture for post-HCT outcomes. Targeted therapies including Bruton's tyrosine kinase inhibition, JAK1/2 inhibition, and ROCK2 inhibitors have improved cGVHD therapy, especially in the steroid refractory setting. Continued improvement in prophylactic strategies for cGVHD, identification of accurate cGVHD treatment endpoints, and access to novel therapeutic agents are expected to improve cGVHD outcomes.

Cyclosporine and Voclosporin Resistant Immune Effector Cells to Improve Outcomes after Stem Cell Transplantation

Introduction: Relapse of a patient's underlying malignancy, and complications related to viral reactivation are prominent causes of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Immune effector cells (IEC) are increasingly deployed to prevent and treat these complications. These include anti-CD19 CAR-T-cells used for post-HCT B-ALL relapse, as well as viral specific T cells (VSTs) targeting EBV, CMV, and others. However, the efficacy of these IECs early after HCT may be compromised by the inhibitory effects of immunosuppressive agents used to prevent graft-versus host-disease (GVHD). A commonly employed immunosuppressant used for GVHD prophylaxis is the calcineurin inhibitor (CNI) cyclosporine (CsA). A structural analog, voclosporin (VCS), is now FDA approved for lupus nephritis and in trials to prevent rejection after solid organ transplantation. Given its favorable pharmacodynamic and toxicity profile versus CsA and tacrolimus, VCS is poised to become an important option for HCT patients. Both CsA and VCS interact with calcineurin only in the presence of their immunophilin binding partner - cyclophilin A ( PPIA). Tacrolimus and sirolimus interact with a distinct immunophilin, FKBP12. This creates the opportunity to delete a single immunophilin (eg. PPIA) to create IECs resistant to one class of CNI (CsA/VCS) but sensitive to other immunosuppressive agents, an important safety feature. Here we developed CsA/VCS-resistant IECs by CRISPR/Cas9 knockout (KO) of PPIA. These cells exhibited superior proliferation and preserved effector function in the presence of CsA and VCS. Methods: We developed IECs that are simultaneously resistant to both CsA and VCS via CRISPR-mediated KO of PPIA. Proliferation, cytotoxicity, and cytokine production of PPIA KO conventional T cells (Tcon), CMV (pp65)-specific VSTs, and CD19 CAR-T cells were tested against mock-edited (MOCK) cells in functional in vitro assays with escalating doses of CsA and VCS. Results: PPIA KO Tcon demonstrated high editing efficiency (88-92%), high viability, and robust expansion in culture. Indeed, PPIA KO efficiency was maintained over several weeks in culture, suggestive of no survival disadvantage of cells bearing this deletion. Importantly, when PPIA KO cells were used as Responders in a mixed lymphocyte reaction in the presence of CsA, the edited cells demonstrated significant resistance to drug-mediated suppression of CD4+ and CD8+ T cell proliferation across the dose-response curve (p &amp;lt; 0.01; Fig. 1). Similar results were observed with VCS, with PPIA KO cells significantly resistant to VCS-mediated suppression across otherwise highly suppressive doses (p&amp;lt;0.0001; Fig. 1). Having demonstrated that edited Tcon were resistant to CsA and VCS, we next sought to make PPIA KO IECs. CRISPR-edited VSTs and CD19 CAR-T cells showed similar editing efficiencies to Tcon. Consistent with prior results, CMV-specific PPIA KO responder VSTs were significantly resistant to suppression of proliferation by CsA vs MOCK cells at all doses tested (p=0.0004, not shown). Proliferation in the presence of drug was then assessed in KO CAR-T cells using the CD19+ NALM6 line as stimulators. Division of PPIA KO responder CD19 CAR-T cells was almost completely unaffected by CsA and VCS across escalating doses ( Fig. 2). For example, at an effector-to-target (E:T) ratio of 1:0.1 and a CsA dose of 50 ng/mL, there was 40.6 ± 2.3 % suppression of division in MOCK CD8+ CAR-T cells vs. -0.2 ± 0.4 % in PPIA KO CD8+ CAR-T cells (p&amp;lt;0.0001). Similar findings were observed with CD4+ responder CAR-T cells (p&amp;lt;0.0001, not shown). PPIA KO CAR-T cells also demonstrated intact cytotoxic function with no significant differences in % killing between the MOCK and PPIA KO groups at 3 different E:T ratios. For example, at a 1:1 E:T ratio, there was 59 ± 15% killing vs. 55 ± 12% killing in MOCK vs PPIA KO groups, respectively (not shown). Conclusion: KO of PPIA is an effective strategy for engineering resistance to CsA and VCS incellular therapies. Our results demonstrate that PPIA KO conventional T cells, VSTs, and CAR-T cells were markedly resistant to the otherwise suppressive effects on T cell proliferation that are hallmarks of both CsA and VCS. Engineering CNI-resistance into IECs will enhance the ability to deploy these cells early after transplant to prevent or treat both malignant relapse and viral reactivation.

Taurine promotes muscle fiber type transformation through CaN/NFATc1 signaling in porcine myoblasts.

This study investigated the effect of taurine (TAU) on the muscle fiber type transformation in porcine myoblasts and its molecular mechanisms. The findings revealed that TAU augmented the protein expression of slow MyHC and the enzyme activities of oxidative metabolism markers like malate dehydrogenase and succinic dehydrogenase. Conversely, it curtailed the expression of fast MyHC and glycolytic metabolism enzyme activity of lactate dehydrogenase. Moreover, TAU elevated the expression of genes associated with oxidative fiber while diminishing the expression of those linked to glycolytic fibers, suggesting that TAU promoted the muscle fiber type transformation from glycolytic fiber to oxidative fiber. Additionally, TAU notably enhanced the expression of key molecules of calcineurin (CaN)/nuclear factor of activated T cells c1 (NFATc1) signaling and the CaN activity in porcine myoblasts. However, CaN inhibitor cyclosporine A abolished these effects induced by TAU. Our results indicated that TAU regulated the muscle fiber type transformation from glycolytic to oxidative fiber by activation of CaN/NFATc1 signaling.

Hyperglycemia and Hyperlipidemia with Kidney or Liver Transplantation: A Review.

Although solid organ transplantation in persons with diabetes mellitus is often associated with hyperglycemia, the risk of hyperlipidemia in all organ transplant recipients is often underestimated. The diagnosis of diabetes often predates transplantation; however, in a moderate percentage of allograft recipients, perioperative hyperglycemia occurs triggered by antirejection regimens. Post-transplant prescription of glucocorticoids, calcineurin inhibitors and mTOR inhibitors are associated with increased lipid concentrations. The existence of diabetes mellitus prior to or following a liver transplant is associated with shorter times of useful allograft function. A cycle involving Smad, TGF beta, m-TOR and toll-like receptors has been identified in the contribution of rejection and aging of allografts. Glucocorticoids (prednisone) and calcineurin inhibitors (cyclosporine and tacrolimus) induce hyperglycemia associated with insulin resistance. Azathioprine, mycophenolate and prednisone are associated with lipogenesis. mTOR inhibitors (rapamycin) are used to decrease doses of atherogenic agents used for immunosuppression. Post-transplant medication management must balance immune suppression and glucose and lipid control. Concerns regarding rejection often override those relative to systemic and organ vascular aging and survival. This review focuses attention on the underlying mechanism of relationships between glycemia/lipidemia control, transplant rejection and graft aging.

Use of Immunosuppression and the Risk of Subsequent Overall or Cancer Mortality

To determine the incidence of all-cause and cancer mortality (CM) in association with immunosuppression. Retrospective cohort study at ocular inflammatory disease (OID) subspecialty centers. We harvested exposure and covariate data retrospectively from clinic inception (earliest in 1979) through 2010 inclusive. Then we ascertained overall and cancer-specific mortalities by National Death Index linkage. We constructed separate Cox models to evaluate overall and CM for each class of immunosuppressant and for each individual immunosuppressant compared with person-time unexposed to any immunosuppression. Patients with noninfectious OID, excluding those with human immunodeficiency infection or preexisting cancer. Tumor necrosis factor (TNF) inhibitors (mostly infliximab, adalimumab, and etanercept); antimetabolites (methotrexate, mycophenolate mofetil, azathioprine); calcineurin inhibitors (cyclosporine); and alkylating agents (cyclophosphamide) were given when clinically indicated in this noninterventional cohort study. Overall mortality and CM. Over 187 151 person-years (median follow-up 10.0 years), during which 15 938 patients were at risk for mortality, we observed 1970 deaths, 435 due to cancer. Both patients unexposed to immunosuppressants (standardized mortality ratio [SMR]= 0.95, 95% confidence interval [CI], 0.90-1.01) and those exposed to immunosuppressants but free of systemic inflammatory diseases (SIDs) (SMR= 1.04, 95% CI, 0.95-1.14) had similar mortality risk to the US population. Comparing patients exposed to TNF inhibitors, antimetabolites, calcineurin inhibitors, and alkylating agents with patients not exposed to any of these, we found that overall mortality (adjusted hazard ratio [aHR]= 0.88, 0.89, 0.90, 1.11) and CM (aHR= 1.25, 0.89, 0.86, 1.23) were not significantly increased. These results were stable in sensitivity analyses whether excluding or including patients with SID, across 0-, 3-, or 5-year lags and across quartiles of immunosuppressant dose and duration. Our results, in a cohort where the indication for treatment was proven unassociated with mortality risk, found that commonly used immunosuppressants-especially the antimetabolites methotrexate, mycophenolate mofetil, and azathioprine; the TNF inhibitors adalimumab and infliximab, and cyclosporine-were not associated with increased overall and CM over a median cohort follow-up of 10.0 years. These results suggest the safety of these agents with respect to overall and CM for patients treated with immunosuppression for a wide range of inflammatory diseases. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Calcineurin signaling promotes takotsubo syndrome

Takotsubo syndrome (TTS) is an acute heart failure syndrome that mimics the symptoms of acute myocardial infarction and is often preceded by emotional and/or physical stress. There is currently no treatment for TTS. Here we show that injection of 2.5 mg kg−1 of epinephrine (EPI) into mice recapitulates numerous features of human TTS, including increased myocardial damage and mortality in males. Gene set enrichment analysis of myocardial RNA sequencing after EPI injection revealed significant enrichment of calcineurin-dependent pro-inflammatory gene networks, which was more pronounced in male than in female mice, in agreement with observed sex discrepancies in the mouse phenotype. An increase in calcineurin activity was detected in the circulating cells of patients with TTS, suggesting a systemic nature of the syndrome. Preventive and therapeutic treatment of mice injected with EPI using calcineurin inhibitors cyclosporine and tacrolimus improved heart function and reduced myocardial injury. Our findings suggest that calcineurin inhibition could be a potential therapy for TTS.

Long Term Complications of Immunosuppression Post Liver Transplant

Improvement in immunosuppression has led to a remarkable improvement in short-term and long-term outcomes post-liver transplant (LT). However, with improvements in long-term survival, complications related to immunosuppressive drugs, either directly or indirectly, have also increased. The adverse events could be drug-specific, class-specific, or generic. Calcineurin inhibitors (cyclosporine and tacrolimus) are the backbone of the immunosuppression after LT and the main culprit associated with most of the complications, including renal failure, post-transplant diabetes mellitus (PTDM), and metabolic syndrome. Steroids are also implicated in the development of diabetes, osteoporosis, and metabolic syndrome post-LT. The development of infections and de novo malignancies (DNMs) is a generic effectlinked to the overall cumulative immunosuppression. The development of these complications significantly hampers the quality of life and leads to increased morbidity and mortality post-LT. Thus, it is important to minimize the cumulative immunosuppression dosewhile simultaneously preventing allograft rejection. This review provides up-to-date, comprehensive knowledge of the complications of long-term immunosuppression post-LT along with associated risk factors and strategies to minimize the risk of complications.

#2989 SELECTIVE RENAL DISPOSITION OF THE CALCINEURIN INHIBITORS VOCLSOPORIN, CYCLOSPORINE, AND TACROLIMUS

Abstract Background and Aims The calcineurin inhibitors (CNI) cyclosporine (CSA) and tacrolimus (TAC) were revolutionary immunosuppressants when first introduced for solid organ transplantation in the 1980s. Voclosporin (VCS), a novel CNI, recently became the first oral therapy approved in the United States, Great Britain, and Europe for the treatment of active lupus nephritis based on positive results from Phase 2 and 3 clinical trials. Unlike CSA and TAC, VCS has demonstrated a consistent pharmacokinetic and pharmacodynamic profile, eliminating the need for therapeutic drug monitoring. Further, VCS is associated with a more favorable metabolic profile and has not been associated with electrolyte disturbances. Emerging evidence indicates small molecule therapeutics may display differential disposition within organ tissues. This suggests that CNIs may be differentially distributed and retained in the kidney, potentially explaining the difference in their efficacy and safety profiles. To evaluate renal disposition of CSA, TAC, and VCS, we assessed in mice and humans the disposition of each CNI in the kidney relative to its systemic drug exposure. Method Single 30 mg/kg doses of CSA, TAC and VCS were administered intravenously to mice. Following intravenous administration, kidneys were collected at 15 minutes, 30 minutes, 1 hour and 2 hours, flash frozen in liquid nitrogen, and stored at −20 °C until sectioning. Sections of 10 μm kidney tissue were mounted on indium tin oxide coated glass slides. Matrix of 10 mg/mL α-Cyano-4-hydroxycinnamic acid in 85% acetonitrile/13% ethanol + 2% water + 0.1% trifluoroacetic acid was sprayed on the tissue using an HTX tissue sprayer, dried for 10 minutes in the vacuum, and subjected to Matrix-assisted Laser Desorption and Ionization Mass Spectrometry Imaging (MALDI-MSI). The systemic and renal clearance in humans of CSA and TAC were obtained from the literature; pharmacokinetic data on VCS was obtained from data on file. Renal secretion of each drug was compared to its expected passive filtration based on glomerular filtration rate (GFR), fraction unbound in plasma (fu), and respective systemic drug exposure. Results MALDI-MSI demonstrated significantly higher concentrations of drug and more diffuse tissue disposition of CSA in mouse kidney compared to VCS (Figure 1). CSA was retained in all kidney tissues up to 2 hours post-administration. Higher concentrations and more diffuse disposition of TAC was also noted compared to VCS at 15 and 30 minutes; TAC was distinctively retained in the cortex and medulla. VCS had moderate distribution in the cortex and was rapidly excreted with low levels of drug present in the kidney after 1 hour. According to published data, CSA has a measured renal clearance of 1.48 mL/min in healthy human subjects, representing approximately 10% of expected passive filtration of 12.5 mL/min (Table 1). TAC has a renal clearance of 0.014 mL/min representing &amp;lt;2% of expected passive filtration of 1.25 mL/min. VCS has a renal clearance of 7.82 mL/min representing approximately 200% of its expected passive filtration rate of 3.75 mL/min. Conclusion MALDI-MSI revealed differential retention and distribution of CSA, TAC and VCS in mice, consistent with their respective renal clearances in humans. Higher drug exposure and &amp;gt;90% renal reabsorption was observed for both CSA and TAC in this study, whereas the renal handling of VCS suggested a significant component of tubular secretion. The higher rate of secretion and lower overall exposure of kidney tissue to VCS may be associated with an improved safety profile when compared to the more diffuse distribution and greater renal retention of CSA and TAC.

Protective effect of the novel calcineurin inhibitor voclosporin in experimental colitis.

Acute severe steroid-refractory ulcerative colitis remains a medically challenging condition with frequent need of surgery. It can be treated with the calcineurin inhibitor cyclosporine A with the need for therapeutic drug monitoring and significant toxicity. Recently, a novel calcineurin inhibitor, voclosporin, has been approved for the treatment of lupus nephritis with no need for therapeutic drug monitoring and an improved long-term safety profile. However, the therapeutic effect of voclosporin in acute severe steroid-refractory ulcerative colitis is still uncertain. We aimed to assess the therapeutic potential of voclosporin to ameliorate inflammation in an experimental model of colitis. We used the dextran sodium sulfate-induced model of colitis in C57BL/6 J wildtype mice treated with either cyclosporine A, voclosporin or solvent control. We employed endoscopy, histochemistry, immunofluorescence, bead-based multiplex immunoassays and flow cytometry to study the therapeutic effect of calcineurin inhibitors in a preventive setting. Acute colitis was induced by dextran sodium sulfate characterized by weight loss, diarrhea, mucosal erosions and rectal bleeding. Both cyclosporine A and voclosporin strongly ameliorated the course of disease and reduced colitis severity in a similar manner. Voclosporin was identified as biologically effective in a preclinical model of colitis and may be a potential therapeutic option in treating acute severe steroid-refractory ulcerative colitis.

An analysis of the efficacy of graft-versus-host disease prophylaxis with post-transplant cyclophosphamide in children with acute myeloid leukemia following allogeneic hematopoietic stem cell transplantation from HLA-matched and partially-matched unrelated donors

An analysis of the efficacy of graft-versus-host disease prophylaxis with post-transplant cyclophosphamide in children with acute myeloid leukemia following allogeneic hematopoietic stem cell transplantation from HLA-matched and partially-matched unrelated donors

Cyclosporine ophthalmic emulsion (Verkazia) for vernal keratoconjunctivitis.

The FDA has approved Verkazia (Santen), a 0.1% ophthalmic emulsion formulation of the calcineurin inhibitor cyclosporine, for treatment of vernal keratoconjunctivitis (VKC). Verkazia is the first product to be approved in the US for this indication.

Clinical Implications and Risk Factors for Erectile Dysfunction in Kidney Transplant: A Single-Center Assessment.

Erectile dysfunction (ED) affects the great majority of people undergoing dialysis and also the majority of patients undergoing kidney transplantation. In this study, we investigated the degree of erectile dysfunction (ED), as well as its prevalence, contributory variables, and overall impact after renal transplant. Adult male kidney transplant patients were the subject of an observational, non-interventional study that was conducted at a single center. Age, time and type ofdialysis before transplantation, comorbidities, factors associated with cardiovascular risk, data on sexual history, physical examination, and laboratory results were among the clinical data we examined. In addition to gathering clinical and demographic characteristics, the International Index of Erectile Function (IIEF) questionnaire was used to evaluate sexual function. A total of 170 renal transplanted patients between 20 and 70 years old (mean age: 45.40±11.5) were included in this study. All of the patients had immunosuppressive treatment with a calcineurin inhibitor (cyclosporine or tacrolimus) and had a normalglomerular filtration rate (GFR). The prevalence of sexual dysfunction increased with age (42.6% of patients under 40, 47.4% of patients in the 40-60 age group, and 78.9% of patients over 60). Mild, moderate, and severe ED was noted in 33.5%, 20.6%, and 10.6% of cases, respectively, and 51 (30%) patients reported having a normal sexual function.While calcium channel blockers (122 cases) were the most commonly used antihypertensive medication and chronic glomerulosclerosis (55.3%) was the most common cause of chronic kidney disease (CKD) before transplantation, none of these variables appear to have affected the severity of erectile dysfunction. The only medications associated with sexual dysfunction were alpha-blockers and aspirin (75 mg)(p=0.026 and p=0.013, respectively). Although kidney transplantation has positive impacts on the quality of life, erectile dysfunction is a frequent condition among patients with renal transplants, and it has an increased frequency with age. In our study, it has been observed that only a small percentage of the research group had a normal sexual function, although most of the patients were young, and that alpha-blockers and aspirin (75 mg) are associated with erectile dysfunction.

Blood, cellular, and tissular calcineurin inhibitors pharmacokinetic-pharmacodynamic relationship in heart transplant recipients: the INTRACAR study.

After heart transplantation, calcineurin inhibitors (CNI) (cyclosporin A and tacrolimus) are key immunosuppressive drugs to prevent graft rejection. Whole-blood concentration (Cblood)-guided therapeutic drug monitoring (TDM) is systematically performed to improve graft outcomes. However, some patients will still experience graft rejection and/or adverse events despite CNI Cblood within the therapeutic range. Other pharmacokinetic parameters, such as the intra-graft, or intracellular concentration at the CNI site of action could refine their TDM. Nonetheless, these remain to be explored. The objective of the INTRACAR study was to describe the relationship between whole blood, intra-graft, and intracellular CNI concentrations as well as their efficacy in heart transplant recipients (HTR). In a cohort of HTR, protocol endomyocardial biopsies (EMB) were collected to assess rejection by anatomopathological analysis. Part of the EMB was used to measure the intra-graft concentrations of CNI (CEMB). Cblood, and the concentration inside peripheral blood mononuclear cells (CPBMC), a cellular fraction enriched with lymphocytes, were also monitored. Concentrations in the three matrices were compared between patients with and without biopsy-proven acute rejection (BPAR). Thirty-four HTR were included, representing nearly 100 pharmacokinetic (PK) samples for each CNI. Cblood, CEMB and CPBMC correlated for both CNI. BPAR was observed in 74 biopsies (39.6 %) from 26 patients (76.5 %), all except one of low-grade. None of the PK parameters (Cblood, CEMB, CPBMC, CEMB/blood and CPBMC/blood) was associated with BPAR. In this cohort of well-immunosuppressed patients, no association was observed for any of the PK parameters including Cblood, with the occurrence of BPAR. However, a trend was noticed for the CEMB and CEMB/blood of cyclosporin A. Further studies in higher-risk patients may help optimize the use of CEMB and CPBMC for CNI TDM in HTR.

The Role of N-Acetylcysteine Amide (NACA) As Prophylactic Treatment for Acute Graft-Versus-Host Disease

Aim The current study aimed to investigate the role of N-acetylcysteine amide (NACA) as prophylactic treatment for acute graft-versus-host disease (GvHD) and to explore the underlying mechanism of action. Background GvHD remains one of the major complications after allogeneic hematopoietic cell transplantation (allo-HCT). In addition, GvHD is a significant cause of morbidity, mortality and deteriorated life quality in transplanted patients. At the present, several strategies have been suggested as a prophylactic treatment against GvHD. Due to the good anti-proliferative effects on the donor T cells, calcineurin inhibitors (cyclosporine or tacrolimus) in combination with methotrexate (MTX) or mycophenolate mofetil (MMF) are recommended by several transplantation centers. Recently, several studies have reported good results of post-transplant cyclophosphamide treatment because of its ability to induce apoptosis of donor allo-activated T cells and to promote transplant tolerance. However, inevitable off-target effect and dose-related toxicity frequently occur regardless of the treatment selected. Evidence showed that oxidative stress is elevated in the recipients of allo-HCT and is linked to the pathogenesis of GvHD. Methods Using a classic murine GvHD model with complete MHC-mismatched allo-HCT (C57Bl/6 to Balb/c), the effect of NACA on the occurrence and severity of acute GvHD was evaluated. In order to reveal the underling mechanism of action, clinical manifestations, survival, cytokine profiles, ROS accumulation, T cell allo-responses, and target tissue damage in NACA-treated GvHD mice were scrutinized and compared with the same parameters observed in N-acetylcysteine (NAC)-treated GvHD mice. Results We found that NACA treatment significantly prolonged the survival and decreased GvHD severity score, in mice underwent allo-HCT. Moreover, NACA downregulated the pro-inflammatory cytokines, such as IFNγ, TNFα, IL-1β and IL-6, but upregulated the anti-inflammatory IL-4 in the mouse serum. Data also showed that NACA administration significantly reduced ROS accumulation in the spleen on day +7 and day +14 after transplantation. In addition, a modest dividing defect in donor T cells was observed upon NACA administration regardless of the experienced antigen classes, and the remarkably lower percentage of Ki67 expression in donor T cells substantiated their inhibited proliferation. In the meanwhile, NACA restrained Th1 and Th17 differentiation of donor T cells but enhanced the regulatory polarization. Furthermore, the migration capability of donor T cells was suppressed by NACA as well, resulting in a less severe intervention of the GvHD target organs. Importantly, NACA did not alter the engraftment as was observed in the bone marrow and spleen chimera but facilitated the neutrophil engraftment. Opposite to NACA, the effect of NAC on GvHD was not significant. Conclusion The present investigation showed that NACA, rather than NAC, has a prophylactic effect in acute GvHD mouse model.The protective effect is mediated by antioxidation, anti-inflammation and downregulation of T-cell allo-responses without altering the engraftment and its process.NACA treatment has significantly improved survival, lower the severity score and shortened the recovery time. However, it was not able to fully eliminate the occurrence of acute GvHD.It is important to consider the combination of NACA with other clinical-used drugs in order to achieve complete disease remission and minimize treatment-related toxicity.The encouraging present results in combination with the clinical reported results of the use of NACA may open the door for a new prophylactic treatment of GvHD.

An Italian multicentre distributed data research network to study the use, effectiveness, and safety of immunosuppressive drugs in transplant patients: Framework and perspectives of the CESIT project.

The goal of post-transplant immunosuppressive drug therapy is to prevent organ rejection while minimizing drug toxicities. In clinical practice, a multidrug approach is commonly used and involves drugs with different mechanisms of action, including calcineurin inhibitors (CNI) (tacrolimus or cyclosporine), antimetabolite (antimet) (mycophenolate or azathioprine), inhibitors of mechanistic target of rapamycin (mTOR) (sirolimus or everolimus), and/or steroids. Although evidence based on several randomized clinical trials is available, the optimal immunosuppressive therapy has not been established and may vary among organ transplant settings. To improve the knowledge on this topic, a multiregional research network to Compare the Effectiveness and Safety of Immunosuppressive drugs in Transplant patients (CESIT) has been created with the financial support of the Italian Medicines Agency. In this article, we describe the development of this network, the framework that was designed to perform observational studies, and we also give an overview of the preliminary results that we have obtained. A multi-database transplant cohort was enrolled using a common data model based on healthcare claims data of four Italian regions (Lombardy, Veneto, Lazio, and Sardinia). Analytical datasets were created using an open-source tool for distributed analysis. To link the National Transplant Information System to the regional transplant cohorts, a semi-deterministic record linkage procedure was performed. Overall, 6,914 transplant patients from 2009–19 were identified: 4,029 (58.3%) for kidney, 2,219 (32.1%) for liver, 434 (6.3%) for heart, and 215 (3.1%) for lung. As expected, demographic and clinical characteristics showed considerable variability among organ settings. Although the triple therapy in terms of CNI + antimet/mTOR + steroids was widely dispensed for all settings (63.7% for kidney, 33.5% for liver, 53.3% for heart, and 63.7% for lung), differences in the active agents involved were detected. The CESIT network represents a great opportunity to study several aspects related to the use, safety, and effectiveness of post-transplant maintenance immunosuppressive therapy in real practice.

Puerarin ameliorates myocardial remodeling of spontaneously hypertensive rats through inhibiting TRPC6-CaN-NFATc3 pathway

Puerarin (Pue) has been widely used in the treatment of hypertension and cardiovascular diseases, but the basic mechanism of Pue on myocardial remodeling (MR) of hypertension is not clear. The purpose of this study was to investigate the effect and mechanism of Pue on MR and provide the basis for the clinical application. Thirty male spontaneously hypertensive rats (SHR) and six male Wistar Kyoto rats (WKY) aged 3 months were used in this study, SHR rats were randomly divided into 5 groups, Pue (40 or 80 mg/kg/d, ip) and telmisartan (TELMI) (30 mg/kg/d, ig) were administrated for 12 weeks. We used Echocardiography to detect the cardiac function. Morphology and structure of myocardium were observed. H9C2 cells were subjected to 1 μM Ang Ⅱ in vitro, 100 μM Pue, 0.5 μM Calmodulin-dependent calcineurin (CaN) inhibitor Cyclosporin A (CsA) and 1 μM specific transient receptor potential channel 6 (TRPC6) inhibitor SAR7334 were used in H9C2 cells. Long-term administration of Pue could significantly improve cardiac function, improve morphology and structure of myocardium in vivo. Pue could reduce MR related proteins expression (ACTC1, TGF-β1, CTGF, β-MHC and BNP), attenuate ROS, restore MMP and decrease Ca2+-overload in vitro. Further study indicated that Pue could decrease TRPC6 expression and inhibit nuclear factor of activated T cells 3 (NFATc3) nuclear translocation in vitro. These results suggested that puerarin could ameliorate myocardial remodeling through inhibiting TRPC6-CaN-NFATc3 in spontaneously hypertensive rats.