CsA Dose Research Articles

Response to biological drugs and JAK inhibitors following cyclosporine in patients with atopic dermatitis.

The therapeutic arsenal for atopic dermatitis (AD) has increased in recent years. The use of biologics or Janus kinase inhibitors (JAKi) is advocated following failure or contraindication to cyclosporine (CSA), however, it is not known whether treatment with CSA can impact the response to biologics or JAKi. The aim of this study was to evaluate the effect of previous treatment with CSA on response to biologics or JAKi in patients with AD. This was a retrospective observational study including patients with AD treated for 16 weeks with a biologic or JAKi, who had previously received cyclosporine for at least four weeks. Thirty patients with AD, with a mean age of 25.07±9.91 years, of whom 18 (60%) were women, were included. The mean duration of CSA treatment was 43.39±31.32 weeks. After 16 weeks of biologic or JAKi treatment, 17 (56.7%) patients achieved EASI75. These patients had a higher cumulative dose of CSA (3,6815 vs.76,993.33 mg; p=0.022) and a longer treatment duration (24.5 vs.57.4 weeks; p=0.003). Additionally, a negative correlation was observed between cumulative dose of CSA and EASI or SCORAD at 16 weeks. Previous cumulative dose and longer duration of CSA treatment does not appear to have a negative impact on response to biologics and JAKi in patients with AD.

Protective effects of cyclosporine and its analog NIM-811 in a murine model of hepatic ischemia-reperfusion injury

Background and aimThe liver is susceptible to ischemia-reperfusion injury (IRI) during hepatic surgery, when the vessels are compressed to control bleeding, or liver transplantation, when there is an obligate period of ischemia. The hallmark of IRI comprises mitochondrial dysfunction, which generates reactive oxygen species, and cell death through necrosis or apoptosis. Cyclosporine (CsA), which is a well-known immunosuppressive agent that inhibits calcineurin, has the additional effect of inhibiting the mitochondrial permeability transition pore (mPTP), thereby, preventing mitochondrial swelling and injury. NIM-811, which is the nonimmunosuppressive analog of CsA, has a similar effect on the mPTP. In this study, we tested the effect of both agents on mitigating warm hepatic IRI in a murine model. Materials and methodsBefore ischemic insult, the mice were administered with intraperitoneal normal saline (control); CsA at 2.5, 10, or 25 mg/kg; or NIM-811 at 10 mg/kg. Thereafter, the mice were subjected to partial warm hepatic ischemia by selective pedicle clamping for 60 min, followed by 6 h of recovery after reperfusion. Serum alanine transaminase (ALT) was measured, and the liver tissue was examined histologically for the presence of apoptosis and the levels of inflammatory cytokines. ResultsCompared with the control mice, the mice treated with 10 and 25 mg/kg of CsA and NIM-811 had significantly lower ALT levels (P < 0.001, 0.007, and 0.031, respectively). Moreover, the liver tissue showed reduced histological injury scores after treatment with CsA at 2.5, 10, and 25 mg/kg and NIM-811 (P = 0.041, <0.001, 0.003, and 0.043, respectively) and significant decrease in apoptosis after treatment with CsA at all doses (P = 0.012, 0.007, and <0.001, respectively). Levels of the pro-inflammatory cytokines, particularly interleukin (IL)-1β, IL-2, IL-4, IL-10, and keratinocyte chemoattractant/human growth-regulated oncogene significantly decreased in the mice treated with the highest dose of CsA (25 mg/kg) than those in the control mice. ConclusionsPremedication with CsA or NIM-811 mitigated hepatic IRI in mice, as evidenced by the decreased ALT and reduced injury on histology. These results have potential implications on mitigating IRI during liver transplantation and resection.

Effects of Triazole Antifungal Agents on the Plasma Concentration and Dosage of Cyclosporin in Patients with Aplastic Anaemia

Objectives. This study aimed to investigate the effects of different triazole antifungal agents on the blood concentration and dosage of cyclosporine (CsA) in patients with aplastic anaemia (AA). Methods. This retrospective study enrolled AA patients who received CsA and triazole antifungal agents simultaneously between January 2018 and December 2022. The ratio of CsA blood concentration (ng/mL) to dosage (mg/day) (C/D) co-administration with and without azoles was compared. The effects of different triazole antifungal agents on blood concentrations and dosages of CsA were analysed. Results. The mean C/D ratio of CsA increased 1.97 times when co-administered with posaconazole (POS), while the mean C/D ratio of CsA increased 1.76 times when co-administered with fluconazole (FCZ). Compared with CsA monotherapy, there was a significant difference in CsA concentrations among patients with azoles (P&lt;0.05). The mean dose of CsA decreased was 0.26 (−0.25—1.05) mg/kg/day and 0.18 (−0.50—0.69) mg/kg/day when co-administered with POS and FCZ, respectively. There is a wide interindividual variability in the magnitude of drug interaction between azoles and CsA. Conclusions. Although azoles increased CsA concentration, a wide individual variability was found in the patients with CsA C/D ratio. Therefore, the CsA dose should be adjusted by closely monitoring the blood levels of CsA co-administered with triazole antifungal agents. In addition, we observed that POS had a greater effect on the blood concentration of CsA than FCZ. When adjusting the dose of CsA in clinical practice, the blood concentration of CsA and the type of co-administered triazole antifungal agents should be considered.

Effects of early vibration and resistant strategies on bond performances between high-strength concrete and rebar

The local bond performances between rebar and high-strength concrete (HSC) exposed to early vibration were experimentally investigated in this study. In addition, three vibration-resistant strategies, including additions of special rapid-hardening cement(calcium sulphoaluminate cement, CSA), shrinkage compensation admixture (expansion agent, EA), and toughening metallic fibers (steel fiber), were verified through penetration resistance, compressive, and central pull-out tests, respectively. Finally, two-way ANOVA tests were conducted to understand the significance of each strategy from a statistical perspective. The results demonstrated that early disturbances compromise bond performances by weakening chemical adherence and mechanical interlocking effects, which considerably degrade bond strength and stiffness. The presence of EA delayed the setting and hydration of concrete and prolonged vibration-sensitive duration. However, the later expansive hydrate products effectively strengthen the bond performances and compensate for early-stage vibration-induced damage. In comparison, the synergetic effects between CSA and Portland markedly enhance the hydration reactivity of blends at early stages, which can achieve threshold maturity and vibration resistance within hours. However, excessive CSA (10%) severely compromises the construction time of mixtures and amplifies the cracking potentials of HSC under the constraints of steel bars. The synergetic effects of steel fibers and aggregates formed disordered networks to enhance the vibration resistance and rigid skeletons for bridging cracks of HSC. The two-way ANOVA results indicate that EA significantly improves the mechanical performances of HSC, followed by steel fibers. The additions of expansive agents and steel fibers are highly recommended in engineering with vibration scenarios. In contrast, the dosages of CSA (or similar early-strength agents) should be carefully controlled unless with a robust shrinkage compensation strategy.

Optimized Rabbit Antithymocyte Globulin Protocol Plus Sequential Cyclosporine and Levamisole Regimen for Newly Diagnosed Severe Aplastic Anemia: A Single-Arm, Single-Centre, Prospective, Phase 2 Trial

Background The inferior effect of rATG is due to the early fatal blow caused by the more potent immunosuppression. In order to retain its sustainable immunosuppression while weakening the early fatal blow. We explored a modifying schedule of administration of rATG by prolonging the course of rATG administration based on the equality of total dose, and then reported the better response and survival in severe aplastic anemia (SAA). And previously, we reported the encouraging effectiveness of cyclosporine alternately combined with levamisole (CSA and LMS regimen) in moderate AA, refractory or relapsed SAA, and SAA. We aim to establish whether the optimized rATG protocol plus sequential CSA and LMS regimen could improve SAA outcomes. Methods We conducted a single-arm, prospective, phase 2 trial at The Institute of Hematology &amp; Blood Diseases Hospital, Chinese Academy of Medical Science &amp; Peking Union Medical College (CAMS &amp; PUMC). Patients aged 6 years or older with confirmed, newly diagnosed SAA were eligible. Patients received optimized rATG administration (1.97 mg/kg/d for 9 days) based on the equality of total does with standard protocol (3.55 mg/kg/d for 5 days). And then CSA and LMS were alternatively administrated every other day from day 14. The initial dose of CSA was 3mg/kg/day in adults and 5mg/kg/day in children (6-18 years old). Oral LMS was alternatively administrated at a dose of 150 mg per day in adults and 2.5 mg/kg per day in children (&amp;lt; 40 kg) in three divided doses. The primary endpoint was overall response rate at 6 months. The trial is registered at www.clinicaltrials.gov as NCT02203396. Results: Between Aug 1, 2014 and Sep 30, 2017, 40 patients with newly diagnosed SAA were treated (median age 24 years [range 7-57 years]; 19 [47.5%] male; 18 [45%] VSAA; 1[2.5%] post-hepatitis). All of these patients were transfusion dependent. Demographic data and clinical characteristics of these patients are outlined in Table 1. The 3-month overall response rates (ORRs) was 40.0%, including 2.5% CR and 37.5% PR. The ORRs increased to 60% (15% CR and 45% PR) at 6 months and 72.5% (42.5% CR and 30% PR) at 12 months. With a median follow-up of 77 months (range 0.1-97 months), estimated 8-year OS rate and event-free survival rate were 84.9% (95% CI, 69.35-92.9%) and 66.5 (95%CI, 49.1-79.1%), respectively. No patient discontinued therapy because of treatment-related toxicity. There were two deaths-one due to infection and another due to cerebral hemorrhage. In this cohort, 4/40 patients (5.7%) evolved to clonal disorders. Of the 4 patients, 1 patients progressed to overt PNH, 2 MDS and 1 acute myeloid leukemia (AML). Cytogenetic abnormalities involved chromosome 7 occurred in one cases with AML. A total of 2 (7.1%) patients relapsed at 33 and 79 months after initial response. Both of the 2 relapsed patients acquired response again with the administration of CSA. Conclusions: Front-line sequential optimized rATG protocol with CSA and LMS regimen resulted in encouraging long-term survival. Future randomized studies should evaluate the optimized IST regimen in the treatment of patients with SAA.

Early Initiation of Oral Therapy with Cyclosporine and Eltrombopag for Treatment Naïve Severe Aplastic Anemia: Interim Results of a Phase II Trial

Introduction: Severe aplastic anemia (SAA), a life-threatening bone marrow failure disease, is treated with allogeneic bone marrow transplantation or immunosuppression (IST). Pancytopenia meeting “Camitta” criteria and an empty bone marrow (&amp;lt;30% cellularity) establish the diagnosis of SAA. However, neither diagnosis nor therapeutic interventions are straightforward, resulting in delays of therapy. SAA must be differentiated from hematologic malignancy, and constitutional forms should be excluded by functional or genetic testing. Administration of IST usually occurs at a tertiary care center, and in younger patients' evaluation for BMT is first required. Ongoing immune mediated destruction of stem cells further depletes marrow reserve and increases regenerative stress with such delays. In this phase II study, we tested the feasibility and safety of early initiation of cyclosporine (CSA) to block T cells and eltrombopag (EPAG) to stimulate hematopoiesis followed by standard of care therapies to expedite treatment (NCT04304820). Methods: Twenty-six patients with treatment-naive SAA were enrolled since 2020. Patients were 3 years or older and median age was 35 years. Exclusion criteria included a diagnosis or high clinical suspicion for inherited disease or evidence of myeloid neoplasm. To ensure rapid initiation of therapy, enrollment was not dependent on pending specialized tests (telomere length, chromosomal breakage, cytogenetics, or genomics). Lower dose CSA (2mg/kg) and full dose EPAG were initiated after confirming eligibility. Upon completion of evaluation and confirmation of acquired SAA diagnosis, all subjects received standard IST (horse ATG, CSA, and EPAG; Figure 1A). Primary feasibility endpoint assessed a composite measure of misdiagnosis, non-compliance, and treatment-related serious adverse events (TRSAE) during oral therapy. Secondary endpoints were efficacy at 6 months, relapse, clonal evolution, and overall survival. Results were compared to a historical cohort of 139 patients treated with hATG, CSA, and EPAG initiated the same day (NCT01623167). Results: All patients completed the protocol-defined treatment period, with median time on oral therapy of 17 days (range 0 - 86 days). All subjects ultimately were confirmed to have acquired SAA (no misdiagnoses), all tolerated oral treatment well without any TRSAE, and patients were compliant with local and study-site follow-up. Thus, there were zero events for the primary feasibility endpoint. A single SAE during oral treatment occurred in subject #1, enrolled at the height of the COVID pandemic and was severely ill with persistently positive COVID-19 PCR and recurrent ileitis requiring hospitalization care; his time to standard IST was the longest (86 days). There was significant increase in absolute neutrophil count (ANC; median 0.315x10 3/mL vs 0.570, p=0.041) in a brief interval of oral therapy prior to receiving hATG. There was also a trend towards improvement in absolute reticulocyte count (29x10 3/mL vs. 35.8) and a decrease in absolute lymphocyte count (1.5x10 3/mL vs. 1.3; Figure 1B). Compared to historical cohort, less patients (19% vs 52%) had very SAA (ANC &amp;lt;200 K/uL); age, sex, race, and pre-treatment blood counts were similar in two cohorts. Hematologic response occurred in 68% at 6 months with 45% complete responses, rates similar to the historical cohort (80% and 40%, respectively). Time to platelet and red blood cell transfusion independence was 43 and 51 days, respectively. Only age (18-40 years) was predictive for 6 month response. With median follow-up of 396 days, the cumulative incidence of relapse at 2 years was 17%, lower than in the historical cohort (27%). Clonal evolution to secondary myeloid malignancy was not increased compared to historical cohort; one subject had myelodysplastic syndrome with excess blasts 1 with deletion 20q, and two had transient trisomy 8 without dysplasia. Overall survival was 96.2%; no deaths occurred during study treatment period of oral medications only. Conclusion: Early initiation of CSA and EPAG is a feasible and safe approach to rapidly treat patients with SAA as a bridge to standard therapies and it may lead to improved blood counts, in turn, decreasing early infectious complications. Hematologic response was comparable to our historic cohort. Rates of long-term complications need to be confirmed in a larger cohort with ongoing study enrollment.

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.

Clinical Features and Outcomes in Large Granular Lymphocyte Leukemia-Associated Pure Red Cell Aplasia with STAT3 Mutation

Background Large granular lymphocyte leukemia (LGLL) is a rare disease frequently complicated with pure red cell aplasia (PRCA) [1, 2]. STAT3 mutations have been describled in 30-40% of LGLL patients [3]. The aim of this work was to evaluate whether STAT3 mutations might be associated with specific clinical features and outcomes in LGLL-associated PRCA. Methods and results From January 2016 to July 2023, 81 patients with LGLL-associated PRCA were enrolled in the China Eastern Cooperation Group for Anemia (CECGA) database (ChiCTR2100043485). As an initial step in the STAT3 mutational analysis, we screened all patients with Sanger sequencing or Next-generation sequencing (NGS). The initial dose of CsA was 3-5mg/kg/day, and the serum concentration was adjusted to be 150-200ng/mL based on adverse reactions. After 12 months of maintenance, the dosage was slowly reduced. Cyclophosphamide combined with prednisone (CP) regimen was used as a salvage therapy for patients who failed to CsA. The initial dose of cyclophosphamide and prednisone were 100mg/day and 0.5-1mg/kg/day, respectively. Among the 81 cases, 26% of them were positive for STAT3 mutations. The clinical characteristics of patients with or without STAT3 mutation were summarized in Table 1. Patients with STAT3 mutations had a higher reticulocyte percentage (0.88% vs 0.28%, P=0.039) and red cell distribution width-coefficient of variation (18.8% vs 15.8%, P=0.008) than patients without STAT3 mutations. Y640F mutation were found in 9 of 21 cases. Subgroup analysis showed that patients with Y640F mutation were associated with younger age (44 vs 65 years old, P=0.007) and higer lymphocyte percentage in peripheral blood (63.7% vs 34.4%, P=0.033). Deep sequencing of rearranged T-cell receptor Vβ complementarity-determining region 3 by NGS was conducted in 61 patients. The predominant V-gene of LGLL clonotypes belonged to the TRBV06 family gene was detected in 12 (25%) patients. The expression of TRBV06 family gene was a litter lower in patients with STAT3 mutation than non-mutant groups (8% vs 31%, P=0.189). The complete response rate (CRR) [31% (5/16) vs 33% (19/58), P=0.909] and overall response rate (ORR) [56% (9/16) vs 50% (29/58), P=0.658] of cyclosporine (CsA) treatment were similar in patients with STAT3 mutations or not. In STAT3 mutant group, the CRR [54% (7/13) vs 31% (5/16), P=0.274] and ORR [85% (11/13) vs 56% (9/16), P=0.130] of CP regimen tended to be better than CsA . 6 patients (67%) relapsed among the 9 patients who responded to CsA in STAT3 mutant group. In patients without STAT3 mutation, 19 of 29 (66%) CsA-responders relapsed. There was no siginificant difference in the relapse-free survival between the two group (Figure 1). Discussion/Conclusions In summary, STAT3 mutation was frequently recognized in LGLL-associated PRCA, and the hotspot site was Y640F. Patients with STAT3 mutations responded to CsA as well as those without the mutation. CP regimen could be used as a salvage therapy for patients who failed to CsA. Reference 1. Balasubramanian SK, Sadaps M, Thota S, et al. Rational management approach to pure red cell aplasia. Haematologica. 2018, 103(2): 221-230. 2. Wu X, Cheng L, Liu X, et al. Clinical characteristics and outcomes of 100 adult patients with pure red cell aplasia. Ann Hematol. 2022, 101(7):1493-1498. 3. Barilà G, Teramo A, Calabretto G, et al. Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients. Leukemia. 2020, 34: 1116-1124.

A non-toxic recombinant Clostridium septicum α toxin induces protective immunity in mice and rabbits

Clostridium septicum alpha toxin (CSA) plays significant roles in ruminant's braxy. Genetically engineered CSA has been shown to function as a potential vaccine candidate in the prevention of the disease caused by Clostridium septicum. In the present study, we synthesized a non-toxic recombinant, rCSAm4/TMD by introducing four amino acid substitutions (C86L/N296A/H301A/W342A) and 11-amino-acid deletion (residues 212 to 222). Compared to recombinant CSA, rCSAm4/TMD showed no cytotoxicity to MDCK cells and was not fatal to mice. Moreover, rCSAm4/TMD could protect immunized mice against 5 × mouse LD100 (100% lethal dose) of crude CSA without obvious pathological change. Most importantly, rabbits immunized with rCSAm4/TMD produced high titers of neutralizing antibodies which protected the rabbits against crude CSA challenge. These data suggest that genetically detoxified rCSAm4/TMD is a potential subunit vaccine candidate against braxy.

#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.

Četrdeset godina ciklosporina u kliničkoj praksi

Cyclosporine (CsA) was discovered in the lab of Sandoz in Switzerland in 1972. while searching for an antifungal drug. However, it quickly became an irreplaceable immunosuppressive drug for renal and other solid organ transplantation. It has been found, in the initial experiments, that CsA inhibits both in vitro cell-mediated lysis and lymphocyte sensitization by allogeneic target cells. Clinical trials have demonstrated better one-year graft survival after cadaveric renal transplants when receiving CsA instead of azathioprine. Although improvement has been observed in the rates of one-year renal graft survival and acute rejection, but long-term graft survival rate did not improve. This can be attributed to the nephrotoxic effects of the CsA. This issue is a consequence of hemodynamic effects on renal blood flow and glomerular filtration, effect on renal tubular function and blood vessels. Along with nephrotoxicity, CsA also causes other adverse effects such as hypertension, gingival hyperplasia, hyperkalemia, hypomagnesemia, hyperlipidemia, neurotoxicity, and in some cases thrombotic microangiopathies. However, in recent years CsA nephrotoxicity has been looked at from a different angle, where it has been linked to high CsA doses that used to be administered. Following its use in solid organ transplantation, CsA has been found to have an important role in treating systemic connective tissue diseases, as well as its consequences, primary glomerulonephritis, inflammatory bowel disease, and psoriasis. CsA effectiveness in treating above mentioned diseases is still greater than its side effects, which makes it a base of treatment options for numerous diseases.

Cyclosporine-use-in-post-haematopoietic-stem-cell-transplant-factors-affecting-the-initial-cyclosporine-concentration-and-its-association-with-acute-graft-versus-host-disease

Background: Cyclosporine (CSA) is required as a prophylaxis of graft-versus-host disease (GVHD) following allogeneic haematopoietic stem cell transplant (HSCT). However, subtherapeutic CSA concentration will increase the incidence of acute GVHD which is one of the major concerns. Objective: This study aims to identify the incidence of patients who achieved therapeutic initial CSA level with a standard intravenous CSA dose of 1.5 mg/kg BD, the occurrence of acute GVHD and factors associated with subtherapeutic CSA at the initial concentration in post-HSCT patients. Method: A retrospective single-centred study was conducted which involved 69 patients who underwent allogeneic HSCT between January 2020 and December 2020 in Hospital Ampang. The factors assessed were patients’ demographics, concurrent medications, liver and renal functions. Mann-Whitney test, Kruskal Wallis test and Spearman correlation test were used to identify the factors associated with sub-therapeutic CSA initial concentration. Result: 17.4% had therapeutic initial CSA level (200-400 ng/mL) and among 69 patients, 37.7% of them developed acute GVHD post-transplantation. Besides, only ethnicity and serum creatinine significantly affected the initial CSA levels. There was no significant association between the initial CSA level and the occurrence of acute GVHD. Conclusion: With the standard intravenous CSA dose of 1.5 mg/kg BD, only 17.4% were able to achieve a therapeutic initial CSA level due to the drug pharmacokinetic variability in different individuals. Hence, this study served as a baseline study for the future prospective clinical study to develop a population pharmacokinetic model in optimising the intravenous CSA dose to achieve the desired therapeutic range and improve the transplant outcomes.

Phase I Trial of Brentuximab Vedotin Plus Cyclosporine in Relapsed/Refractory Hodgkin Lymphoma

Introduction Treatment of relapsed/refractory (R/R) Hodgkin lymphoma (HL) following failure of brentuximab vedotin (BV) and PD-1 blockade is a major unmet need. Resistance to BV is associated with multidrug resistance gene 1 (MDR1) overexpression. There are pre-clinical data showing that MDR1 inhibitors such as cyclosporine (CsA) or verapamil (VRP) are synergistic with BV in BV-resistant HL cell lines. Preliminary results of a phase I study evaluating the safety and efficacy of combination of BV with CsA and VRP in R/R HL were previously reported. Here we report the final results of the completed study. Methods This is a single center, open-label phase I investigator-initiated study. Patients (pts) with HL whose disease had relapsed or was refractory to ≥1 prior lines of therapy were enrolled. The dose finding portion followed a 3+3 design with 4 planned dose levels (DL). BV was given at 1.2 mg/kg (DL1) or 1.8 mg/kg (DL2-4) intravenously every 3 weeks. Planned dose of CsA was 5 mg/kg (DL1-2) or 7.5 mg/kg (DL3-4) orally twice daily on days 1-5. Planned dose of VRP was 120 mg orally four times daily on days 1-5 (DL4). Each cycle was 3 weeks. Once the maximum tolerated dose (MTD) was determined, BV-refractory pts were enrolled in an expansion cohort at the MTD. Primary objective was to determine the MTD and safety of the combination. Secondary objectives were overall response rate (ORR), complete response (CR) rate, response duration (DOR), overall survival (OS), and progression-free survival (PFS). Results Twenty-nine pts were enrolled onto the study, 14 in the dose-finding portion and 15 in the dose expansion BV-refractory cohort. With respect to baseline characteristics, 38% of patients were female, 69% had advanced stage disease, 45% had extranodal disease, and 24% had B symptoms. Median age was 36 (20-69) and pts had a median of 5 (3-12) prior lines of therapy. All had prior BV (only 2 BV-sensitive) and 27 had prior PD-1 blockade. Four pts were treated on DL1, 22 on DL2, and 3 on DL3. MTD was DL2. DLTs observed were: DL1 10-day CsA (n=1) grade (Gr) 3 hyperbilirubinemia, abdominal pain, and hypertension, DL2 (n=1) Gr3 abdominal pain and Gr3-4 neutropenia, DL3 (n=2) Gr3 bone pain/constipation/Gr4 lymphopenia (n=1) and Gr3 hyperglycemia (n=1). Median duration of treatment was 3 (1-16) cycles. Most frequent adverse events (AEs) were nausea (90%), hypertension (90%), anemia (86%), fatigue (76%), neutropenia (76%), and leukopenia (76%). All pts had Gr3+ AEs with most frequent being neutropenia (62%). Reasons for treatment discontinuation included death (n=4), disease progression (n=10), toxicity (n=2), patient refusal (n=8), loss to follow-up (n=2), MD decision (n=1), proceeding to HCT (n=1), and study completion (n=1). Treatment-related death occurred in 3 pts (pneumonitis at DL1, respiratory failure and hypotension at DL2). The ORR/CR was 62%/24%. The median DOR and PFS was 5 months and median OS was not reached. Discussion The combination of BV and CsA was effective in BV-refractory R/R HL but was also associated with toxicity, including treatment-related deaths that led to early termination of the clinical trial. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 10121: Hypoimmunogenic Human Induced Pluripotent Stem Cells Derived Cardiomyocytes for Cardiac Regeneration

Introduction: Post-transplant rejection resulting from human leukocyte antigen (HLA) mismatch is a challenge in allogeneic cell therapy. Human amniotic fluid stem cell-derived iPSCs (hAFSC-iPSCs) are proved to have immune privilege. Hypothesis: hAFSC-iPSC derived cardiomyocytes (hAFSC-iPSC-CMs) are less immunogenic. Methods and Results: hAFSC-iPSCs and hAFSC-iPSC-CMs expressed classical HLA-Ia (HLA-A, -B and -C) and non-classical HLA-Ib but did not have HLA-II expression, indicating that these cells may not be highly immunogenic. We did T Cell Assays to confirm low immunogenicity of hAFSC-iPSC-CMs. We did ischemia-reperfusion surgery of left anterior descending coronary artery to create large transmural myocardial infarction in rats. At post-infarct 4 days, we injected human pluripotent stem cell-derived cardiomyocytes (1.0х10 7 cells/kg), including human embryonic stem cell-derived cardiomyocytes (hESC-CMs), hiPSC-CMs and hAFSC-iPSC-CMs, into the infarcted myocardium. Using very low dose of cyclosporine A (CsA, 2.5 mg/kg/day), only hAFSC-iPSC-CMs transplantation could improve post-infarct cardiac function (left ventricular ejection fraction improvement in hAFSC-iPSC-CMs, hESC-CMs and hiPSC-CMs: 13.1 ± 1.8%, 0.6 ± 1.7% and -1.9 ± 1.6%, respectively). Compared with hESC-CMs and hiPSC-CMs, hAFSC-iPSC-CM transplantation remuscularized the infarcted myocardium with infarct size reduction. Using very low dose CsA, these engrafted hAFSC-iPSC-CMs resulted in a 3-month survival in vivo. hAFSC-iPSC-CMs expressing non-classical HLA-Ib evaded detection and attack by natural killer (NK) cells and cytotoxic T cells because these hAFSC-iPSC-CMs activated the protein tyrosine phosphatase SHP-1/SHP-2 signalling pathway of NK cells and cytotoxic T cells which downregulated signal transducer and activator of transcription 1 (STAT-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression. Conclusions: We demonstrated the immune privilege of hAFSC-iPSC-CM for cardiac regeneration. HLA-Ib-expressing hiPSCs with reduced HLA-II expression are less immunogenic and may serve as platforms for regenerative medicine.

Association between the cumulative dose of cyclosporine and liver enzyme abnormalities in dermatology patients managed with a low‐dose regimen

Cyclosporine (CsA) is an immunosuppressive agent that specifically inhibits T cell-related immune responses. There is little evidence regarding the association between low-dose CsA administration and abnormal hepatic function in dermatology patients. This study aimed to examine the association between the cumulative dose of CsA and liver enzyme abnormalities obtained from peripheral blood tests in patients with skin diseases. A retrospective single-center study of 697 patients who were prescribed CsA for skin disease in the outpatient dermatology clinic between 2015 and 2019 were performed. Multiple logistic regression with confounder adjustment was performed to assess the association between the cumulative dose of CsA and liver enzyme abnormalities. Compared to patients with the lowest cumulative dose of CsA (˂7.0 g), patients with the highest cumulative dose of CsA (≥30.6 g) were significantly associated with an increased likelihood of developing liver enzyme abnormalities (odds ratio [OR]=1.96; 95% confidence interval [CI]=1.02-3.79). In the stratified analysis, patients with the highest cumulative dose of CsA (≥30.6 g) were significantly associated with a 1.5-or higher alanine aminotransferase elevation from baseline (OR=2.26, CI=1.08-4.73). Patients prescribed long-term, low-dose CsA up to a high cumulative dose (≥30.6 g) may be associated with an increased risk of developing liver enzyme abnormalities. However, these liver enzyme elevations were not severe in magnitude and were reversible.

Drug-Drug Interactions With Cyclosporine in the Anti-Hepatitis C Viral PrOD Combination Regimen of Paritaprevir/Ritonavir-Ombitasvir and Dasabuvir in Organ Transplant Recipients With Severe Hepatic Fibrosis or Cirrhosis.

The clinical guidelines suggest that the dosing of cyclosporine (CsA), during combination therapy with paritaprevir/ritonavir-ombitasvir and dasabuvir (PrOD), would be only one-fifth of the pre-PrOD total daily dose to be administered once daily. However, this dosing may not be applicable to all patients depending on their clinical condition. This study focuses on the pharmacokinetic dynamics of PrOD with CsA in Asian organ transplant recipients with severe liver fibrosis or cirrhosis who undergo concurrent treatment with PrOD treatment and CsA. The efficacy and safety of PrOD treatment was also evaluated. Data from 7 patients obtained between January 2017 and September 2017 were retrospectively analyzed. Determinations of the blood concentrations of CsA were made, whether used as a single treatment or in combination therapy with PrOD. The combination regimen compared with CsA administered alone resulted in a 4.53-fold and 5.52-fold increase in the area under the concentration-time curve from time 0-12 hours (AUC0-12 h) of CsA on days 1 and 15, respectively. In addition, the maximal concentration, time to maximum concentration, and terminal phase elimination half-life (t1/2) of CsA were increased during the combined treatment of PrOD and CsA. The authors proposed reducing the CsA dosage during PrOD treatment to one-seventh of that of the pre-PrOD treatment of the total daily dose to maintain target CsA levels. All patients achieved sustained virologic responses at week 12. There were no episodes of serious adverse events or graft rejections observed. Although the combination with PrOD significantly affects the pharmacokinetics of CsA, it is effective and safe with regular monitoring of the CsA blood concentrations and appropriate CsA dose adjustment.

Massive eosinophilia despite severe aplastic anemia.

A 47-year-old nonsmoking man developed very severe aplastic anemia (AA) (hemoglobin 56 g/L, neutrophils 110/μl, eosinophils 0/μl, platelets 4000/μl) with fatty marrow (Figure 1A: hematoxylin–eosin stain, ×4 objective). He had a history of bronchial asthma for 3 years and had been treated with inhaled corticosteroid in combination with long-acting β2-agonist (budesonide/formoterol fumarate). In addition, he had developed eosinophilic pneumonia requiring systemic administration of prednisolone (<30 mg/day) twice in the last 1.5 years. His family history was not significant for any serious diseases. Treatment with antithymocyte globulin, eltrombopag olamine, and cyclosporine (CsA) (500 mg/day) eliminated the need for blood transfusions and the CsA dose was gradually reduced. While pancytopenia continued (hemoglobin <97 g/L, neutrophils <1400/μl, platelets <30,000/μl), eosinophilia (>500/μl) appeared 17 months after the start of treatment. This increased to 4550/μl in 4 months after discontinuation of CsA treatment at 33 months. In addition, he presented with a rash with purpura on the legs, for which prednisolone (<20 mg/day) was not effective. Bone marrow examination showed mild hypocellular marrow with eosinophil expansion (43.8%) (Figure 1B: hematoxylin–eosin stain, ×4 objective; Figure 1C: hematoxylin–eosin stain, ×40 objective; Figure 1D: direct Fast Scarlet stain, ×40 objective). Increased blasts and significant dysplasia were not observed. G-banding and fluorescence in situ hybridization for the FIP1L1–PDGFRA re-arrangement detected no chromosomal aberrations. Moreover, skin biopsy revealed eosinophilic vasculitis and panniculitis, compatible with eosinophilic granulomatosis with polyangiitis (Figure 1E: hematoxylin–eosin stain, ×20 objective; Figure 1F: direct Fast Scarlet stain, ×40 objective). After re-administration of CsA (150 mg/day), eosinophilia and skin lesions disappeared in 5 weeks. This case indicates that massive reactive eosinophilia can occur even in AA patients with eosinophil-associated diseases, especially during dose reduction, or after the discontinuation of CsA. None. The authors declare that there is no conflict of interest. Aya Takahashi and Tsutomu Shinohara designed the research, drafted the manuscript, and created the figure. Aya Takahashi performed the skin biopsy. Yoshihito Iwahara, Aya Takahashi, Hisanori Machida, Keishi Naruse, Eiji Takeuchi, and Tsutomu Shinohara analyzed the data. Keishi Naruse contributed to pathological assessment. All authors revised the manuscript critically and approved the final manuscript.

Population pharmacokinetics of ciclosporin in allogeneic hematopoietic stem cell transplant recipients: C-reactive protein as a novel covariate for clearance.

Ciclosporin (CsA), a potent immunosuppressive agent used to prevent graft-versus-host disease in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients, is characterized by large inter-individual variability and a narrow therapeutic range. The aim of this study was to develop a population pharmacokinetic model for CsA in Chinese allo-HSCT recipients and to identify covariates influencing CsA pharmacokinetics. A total of 758 retrospective drug monitoring data points were collected after intravenous infusion or oral administration of CsA from 59 patients. Population pharmacokinetic analysis was performed using nonlinear mixed effects modelling expressed by differential equations. Monte Carlo simulation was applied to optimize dosage regimens. The final model was validated using bootstrap and normalized prediction distribution errors. The results showed that the daily CsA dose, haematocrit, total bile acid, C-reactive protein (CRP) and co-administration of triazole antifungal agent were identified as significant covariates for clearance (CL) of CsA. The typical value of CL was 19.8L/h with an inter-individual variability of 13.1%. The volume of distribution was 1340L. Bioavailability was 67.2% with an inter-individual variability of 8.5%. Dosing simulation based on the developed model indicated that patients with high CRP concentration required a higher daily dose to attain the therapeutic trough concentration. The influence of CRP ultimately on the therapy outcome of CsA is not clear, which needs further study. CRP concentration was identified as a novel marker associated with CsA pharmacokinetics, which should be considered when determining the appropriate dosage of CsA in allo-HSCT recipients.

Factors Affecting Time-Varying Clearance of Cyclosporine in Adult Renal Transplant Recipients: A Population Pharmacokinetic Perspective.

The pharmacokinetic (PK) properties of cyclosporine (CsA) in renal transplant recipients are patient- and time-dependent. Knowledge of this time-related variability is necessary to maintain or achieve CsA target exposure. Here, we aimed to identify factors explaining variabilities in CsA PK properties and characterize time-varying clearance (CL/F) by performing a comprehensive analysis of CsA PK factors using population PK (popPK) modeling of long-term follow-up data from our institution. In total, 3674 whole-blood CsA concentrations from 183 patients who underwent initial renal transplantation were analyzed using nonlinear mixed-effects modeling. The effects of potential covariates were selected according to a previous study and well-accepted theoretical mechanisms. Model-informed individualized therapeutic regimens were also evaluated. A two-compartment model adequately described the data and the estimated mean CsA CL/F was 32.6Lh-1 (relative standard error: 5%). Allometrically scaled body size, hematocrit (HCT) level, CGC haplotype carrier status, and postoperative time may contribute to CsA PK variability. The CsA bioavailability in patients receiving a prednisolone dose (PD) of 80mg was 20.6% lower than that in patients receiving 20mg. A significant decrease (52.6%) in CL/F was observed as the HCT increased from 10.5% to 60.5%. The CL/F of the non-CGC haplotype carrier was 14.4% lower than that of the CGC haplotype carrier at 3months post operation. By monitoring body size, HCT, PD, and CGC haplotype, changes in CsA CL/F over time could be predicted. Such information could be used to optimize CsA therapy. CsA dose adjustments should be considered in different postoperative periods.

Ciclosporin A in bilateral auto-immune chronic posterior uveitis associated with macular oedema: a Long-term Observational Safety and Efficacy Study.

A non-interventional, longitudinal, retrospective follow-up study to assess CsA-induced nephrotoxicity (IN) and its reversibility after withdrawal in patients exhibiting a bilateral chronic posterior uveitis (CPU) associated with cystoid macular oedema (CMO) in at least one eye. Data from medical records between 1986 and 2013. Primary outcome was the renal tolerance during and after CsA treatment assessed by plasma creatinine concentration and glomerular filtration rate (GFR) estimated by Chronic Kidney Disease Epidemiology (CKD-Epi) formula. Secondary outcomes were CsA through concentration, occurrence of cancers and ophthalmologic efficacy assessed by three parameters including CMO, vitreous inflammation, and best-corrected visual acuity BVCA changes. One hundred forty-three patients were followed for renal tolerance. Underlying diseases were Birdshot retinochoroiditis (n = 67), Behçet disease (n = 9), probable sarcoidosis (n = 23), sympathetic ophthalmia (n = 3), idiopathic (n = 41). After CsA discontinuation in 115 patients (mean treatment duration of 5.9 ± 3.8 years) mean plasma creatinine concentration was 82.2 ± 14.2 µmol/L versus 82.1 ± 14.1 µmol/L at baseline, mean GFR was 79.4 ± 13.9 mL/min versus 82.5 ± 14.3 mL/min at baseline, with no significant difference (respectively p = 0.91 and p = 0.09). Blood pressure did not significantly change during follow-up. CMO was completely resorbed in at least one eye, in 70.8% patients (n = 72) at 6 months, in 71.4% patients (n = 49) at 10 years and in 54.2% patients (n = 24) at 20 years. BCVA did not statistically change over time. Early and long-term monitoring of renal tolerance and dual adjustment of CsA doses in inflammatory stages of CPU were associated with reversible CsA IN. CsA could be effective in the treatment of CMO in CPU patients.