CsA Blood Research Articles

B-267 Validation of the Newly Developed Fully Automated Chemiluminescent Enzyme Immunoassay (CLEIA) Reagent for Measurement of Cyclosporine in the Blood

Abstract Background Cyclosporine (CsA) is an immunosuppressive drug requiring therapeutic drug monitoring (TDM) for its narrow-ranged medication level. Approximately 60% of CsA in the blood exist as molecular complexes with proteins like cyclophilin in lymphocytes and erythrocytes. Measurement of CsA in the blood by immunoassay or liquid chromatography-tandem mass spectrometry (LC-MS/MS) requires an extraction process for CsA from the whole blood specimen, which is often manual complicated and time-consuming process causing a risk of variation in the measurement values among different operators. In addition, the small (cyclic polypeptide composed of 11 amino acids) and hydrophobic property of CsA, make it difficult to establish antibodies for a sandwich immunoassay. In this study, we have validated a novel fully automated sandwich immunoassay reagent for measurement of blood CsA that used a combination of newly-established CsA antibodies and iTACT® (immunoassay for total antigen including complex via pretreatment) technology. Methods The reagent was developed and validated on a fully automated immunoassay system, LUMIPULSE® L2400 (FUJIREBIO INC). Twenty uL whole blood specimens are sampled after mixed with suction/discharge stirring to prevent that blood cells settle to the bottom of a container on the analyzer, and then treated with a pretreatment solution to obtain free CsA. The treated samples are assayed by a two-step sandwich immunoassay. Limit of Detection (LoD), Limit of Quantitation (LoQ), within-laboratory precision, linearity, no interference, and the correlation of this assay and LC-MS/MS were validated, following the current CLSI guidelines protocols. Data were analyzed using Microsoft Excel statistical tool Analyse-it. Results The LoD and the LoQ were 2.7 ng/mL and 10.8 ng/mL respectively. The within-laboratory precision showed CV 1.6-2.9 %. Linearity was demonstrated over the range 13.1-2381.5 ng/mL. No interference was observed with unconjugated (≤19.7 mg/dL) or conjugated bilirubin (≤20.1 mg/dL), chyle (≤1480 FTU), RF (rheumatoid factor, ≤1000 IU/mL), and HAMA (human anti-mouse antibody, ≤1010 ng/mL). The correlation coefficient and the regression slope of this assay and LC-MS/MS were 1.0 and 1.0, respectively (N=120). Conclusions The performance of our novel CsA assay, which use LUMIPULSE L2400, was satisfactory for routine analysis. A unique fully-automated process, including the pretreatment process of whole blood specimens, for measurement would contribute the low LoQ and the low Within-laboratory CV. In addition, the results are obtained in 35-minute. We expect this novel immunoassay could reduce the burden on operators and turnaround time.

Development and Validation of a U-HPLC-MS/MS Method for the Concurrent Measurement of four Immunosuppressants in Whole Blood.

This study aimed to develop and validate a U-HPLC-MS/MS method for simultaneous determination of four immunosuppressants in human whole blood. The method was based on the injection of 20 µL of calibrators and controls pretreated with the liquid phase extraction method for chromatography separation and mass spectrometry determination. LPE offline was performed by adding 0.1 mol/L ZnSO4 and acetonitrile, while separation of target compounds was achieved within 2.5 minutes by a Zorbax Eclipse XDB-C8 column using ammonium acetate and ACN mixed with formic acid as solvents. The assay offers ng/mL detection limits (from 1.1 to 12.4 ng/mL), accuracy (% deviation from -4.4% to 5.6%), precision (CV less than 15% at all QC levels), and linearity (from 23.4 to 948 ng/mL for CsA, from 2.11 to 45.5 ng/mL for TAC, SIR and EVR). The recovery and matrix results were acceptable, and the carryover was less than 1%. The results of method comparison show that IA-based methods overestimated the concentration of drugs compared with the MS-based method. Comparing our MS-based method with external LC-MS/MS showed that the results were within 2 SDs. We have developed a reliable assay for the analysis of CsA, TAC, SIR and EVR in whole blood using U-HPLC-MS/MS.

Effects of Citrus grandis peels on cyclosporin concentration and immune responses in mice

A previous study indicated that the coadministration of decoction of Citri Grandis Pericarpiurn (CGP), the peels of Citrus grandis (Rutaceae), significantly increased the blood level of cyclosporin (CsA) and resulted in acute intoxication of pigs. In this study, we aimed to measure the effects of CGP on the distribution of CsA in both lymphoid and non-lymphoid tissues as well as on the immune cell function in mice. Our results showed that coadministration of 3 g/kg CGP with CsA (Neoral®, 10 mg/kg) significantly decreased the concentrations of CsA in blood, liver, kidney and spleen by 37 %, 45 %, 46 % and 51 %, respectively, after a two week treatment. The decrease in CsA level correlated positively with the CGP dose. Pharmadynamically, coadministration of CGP restored the phagocytosis activity in blood decreased by CsA. Moreover, the nitric oxide production of activated macrophages and helper T cell type 1 (Th1) cytokines production suppressed by CsA were also reversed by the coadministration of CGP. In conclusion, coadministration of CGP significantly decreased the systemic exposure of CsA and resulted in higher macrophage and Th1 type activities than giving CsA alone in mice. Therefore, the combined use of CGP with CsA requires close monitoring clinically.

Cyclosporine May Inhibit the Effect of Extra-Physiologic Oxygen Shock/Stress on Peripheral Blood Stem Cells

Introduction: Adequate numbers of stem cells with preserved multi-potency and self-renewing capabilities are necessary for successful hematopoietic reconstitution after bone marrow transplantation. Although hematopoietic stem cells (HSC) reside in the bone marrow under a hypoxic microenvironment (1-4% O2), human HSC are collected and processed in ambient air (21% O2) (Spencer et al., Nature 508:269-73). Exposure of murine bone marrow and human cord blood to ambient air for as little as 30 minutes triggered stem cell differentiation from quiescent pluripotent long term stem cells into activated multipotent progenitors (MPPs), a phenomenon called extra-physiologic oxygen shock / stress (EPHOSS)(Mantel et al., Cell 161:1553-65). The effect of EPHOSS on HSCs is mediated through reactive oxygen species (ROS) which open the mitochondrial permeability transition pore (MPTP) and trigger stem cell differentiation. Cyclosporine (CSA) inhibits the MPTP regulator cyclophilin D and prevents MPTP opening (Kroemer et al., Physiol Rev 87:99-163). Currently, mobilized peripheral blood stem cells (PBSCs) are the major source of grafts for hematopoietic cell transplantation. We hypothesized that EPHOSS is detrimental to human PBSCs similar to murine bone marrow and human cord blood stem cells and CSA will protect human PBSCs from the effects of EPHOSS and inhibit their differentiation from pluripotent long term HSC into short term MPPs.Study design and methods. This is a proof-of-concept, prospective, non-interventional study. We obtained G-CSF mobilized PBSCs from healthy related donors under an IRB approved protocol. All donors provided written informed consent. Blood containing HSCs was collected from each donor with minimal exposure to ambient air (<60 seconds). The sample was immediately split and incubated for 60 minutes with and without CSA (50 µg/mL). CSA treated and untreated PBSCs were immunophenotyped by multi-parameter flow cytometry for HSCs (CD34+CD38-CD90+CD45RA-), MPP (CD34+CD38-CD90-CD45RA-) and common lymphoid progenitors (CD34+CD38+CD127+). We used a CD34 ISHAGE-based gating strategy to accurately enumerate HSC and MPP.Results. In four separate experiments, CSA treated PBSCs had a higher median (range) number of HSCs/106 total nucleated cells (TNC) compared to untreated PBSCs ((297 (51 - 512) versus 185 (47 -392), respectively). CSA treated PBSCs also had a higher median (range) HSC:MPP ratio compared to untreated PBSCs ((0.56 (0.40 - 0.70) versus 0.43 (0.30 - 0.50), respectively) suggesting that the differentiation of HSC to MPP upon exposure to air was decreased in the CSA treated PBSCs.Conclusions / future directions. This is the first report describing the effect of EPHOSS in human PBSCs. Our preliminary data suggest that EPHOSS promotes the differentiation of human PBSCs from HSC to MPP and that CSA may inhibit this process. Further confirmatory and mechanistic studies exploring the contribution of MPTP and ROS to EPHOSS are ongoing. The results from this research may potentially change the current practice of collecting PBSCs in ambient air. DisclosuresMcCarthy:Janssen: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Chen:Bellicum Pharmaceuticals: Research Funding.

Simultaneous determination of cyclosporine and tacrolimus in human whole blood by ultra-high performance liquid chromatography tandem mass spectrometry and comparison with a chemiluminescence microparticle immunoassay

Overestimation of immunoassays for cyclosporine (CsA) and tacrolimus (TAC) analysis in human whole blood is a problem. The liquid chromatography tandem mass spectrometry is recommended as a golden method for CsA and TAC analysis. The aim of the study is to develop and validate an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of CsA and TAC in human whole blood and evaluate its agreement with a chemiluminescence microparticle immunoassay (CMIA). The UHPLC-MS/MS method for simultaneous determination of CsA and TAC in human whole blood was developed and validated according to the guidelines. A total of 177 CsA and 220 TAC samples were determined by UHPLC-MS/MS and CMIA, and the agreement of the two methods was evaluated by Bland-Altman plot. The calibration range of UHPLC-MS/MS method was 5 to 2000 ng/mL for CsA and 0.2 to 80 ng/mL for TAC. The inaccuracy and imprecision were −13.33% to 11.80% and <11.74% for CsA and −8.94% to 6.53% and <10.84% for TAC, respectively. Evaluated by Bland-Altman plot, the mean overestimation of CMIA compared to UHPLC-MS/MS was 53.7% for CsA and 48.1% for TAC.

Analytical performance evaluation of the Elecsys® Cyclosporine and Elecsys® Tacrolimus assays on the cobas e411 analyzer

BackgroundCyclosporine (CsA) and tacrolimus (TAC) are immunosuppressant drugs that are often used to treat autoimmune diseases and as transplantation therapy; therefore, their concentrations need to be monitored carefully. We herein evaluated the analytical performance of the Elecsys® Cyclosporine and Elecsys® Tacrolimus assay kits, which have been newly developed to measure CsA and TAC concentrations in the whole blood. MethodsWe used residual whole blood samples from autoimmune disease and transplantation patients who were being treated with CsA or TAC. CsA concentrations were measured using an affinity chrome-mediated immunoassay (ACMIA) and an electrochemiluminescence immunoassay (ECLIA). TAC concentrations were measured using a chemiluminescence immunoassay (CLIA) and ECLIA. We investigated assay precision, linearity, lower limit of quantitation (LOQ), stability of calibration, influence of interference substances and the hematocrit, correlation of ACMIA with ECLIA, and correlation of CLIA with ECLIA. ResultsWithin-assay coefficients of variation were 1.8−3.6% (CsA: 94−1238 ng/mL) and 2.9−3.9% (TAC: 2.1−17.8 ng/mL), whereas day-to-day coefficients of variation ranged between 3.0−4.1% (CsA) and 2.8−3.9% (TAC). The limits of quantitation were defined as the concentration at which the CV was approximately 10%. Each lower LOQ obtained was 16 ng/mL (CsA), and 0.95 ng/mL (TAC). CsA and TAC calibrations were stable for at least 21 days. Neither the presence of conjugated bilirubin, unconjugated bilirubin, chyle, and rheumatoid factor nor the hematocrit affected these assays. A method comparison using a standardized major axis regression analysis of ACMIA and ECLIA was r=0.995, y=0.924x −1.175, n=200 (CsA), while that of CLIA and ECLIA was r=0.994, y=1.080x −0.197, n=200 (TAC). ConclusionsThe analytical performances of the Elecsys® Cyclosporine and Elecsys®Tacrolimus assays were acceptable. Furthermore, CyA and TAC concentrations may be simultaneously measured using a single pretreatment which is of benefit if patients have to undertake conversion between these two drugs. Additionally, it benefits the workflow in the clinical laboratory. Thus, the Elecsys® Cyclosporine and Elecsys® Tacrolimus assays may be suitable for routine therapeutic drug monitoring.

Could salivary cyclosporine dosage replace the whole blood cyclosporine measurements in renal transplant patients?

Background:Cyclosporin (CsA) has been extensively used as the immunosuppressant of choice in renal transplantation. Currently available approaches to assess CsA levels, both in serum and blood, fail to accurately reflect the concentration of the pharmacologically active drug fraction. Free CsA levels in biological fluids (blood or saliva) have been advocated to play an important role. Traditional salivary CsA monitoring tests are based on available archaic salivary techniques that are nonspecific and require large amounts of saliva. The aim of this study was to assess salivary CsA correlation using a novel and more accurate technique and to correlate with CsA levels in blood.Material and Methods: Patients provided blood samples of 2 ml and 2 ml of unstimulated saliva on the same day 2 h after the morning CsA dose (C2). Whole blood levels of CsA were determined using the monoclonal fluorescent polarization immunoassay (FPIA) kit. The FPIA kit was adapted to salivary testing by using a novel extraction method developed and patented under the name of Middle East Research Institute (MERI). Wilcoxon signed rank test compared the differences in blood and salivary CsA. Pearson's correlation coefficient assessed the linear association between blood and salivary CsA concentrations. All analyses were performed using IBM-SPSS version 23 (IBM Corp, Armonk, NY, USA).Results: No significant correlation was observed between blood and salivary CsA levels.Conclusion: Salivary CsA concentrations at C2 cannot adequately replace C2 blood levels as an indicator of CsA bioavailability despite improved performance of monoclonal FPIA and application of the MERI technique. More studies may be warranted to design more reliable and less invasive procedures for therapeutic drug monitoring.

The study of cyclosporin A modified intraocular lens preventing posterior capsular opacification in rabbit eyes

To investigate the safety and efficacy of cyclosporine A sustained release from modified intraocular lens for preventing posterior capsular opacification (PCO) in rabbit eyes. Forty-five New Zealand albino rabbits undergoing phacoemulsification in their right eyes were randomly and equally divided into three groups. Group A had implanted original IOL, group B had implanted PLGA-IOL(IOL coated with polylactide-glycoli acid), and group C had implanted CsA-PLGA-IOL (CsA loaded PLGA-IOL). All the 45 eyes were examined by a slit-lamp microscope. The intraocular pressures were recorded. Anterior chamber flare and aqueous humor cells were graded at different time point after surgery. The concentrations of CsA in the aqueous humor and blood were determined by high performance liquid chromatography. Anterior segment tissue was histologically examined. Wet posterior capsules were weighed. PCO was graded 6 months later. The mean concentrations of CsA in group C at 2 h,1 d,3 d,7 d,14 d,30 d,60 d after operation were (11.47±2.42) mg/L, (10.30±2.15) mg/L, (6.71±1.45) mg/L, (4.81±1.16) mg/L, (6.11±0.84) mg/L, (2.53±0.77) mg/L, (0.86±0.28) mg/L. The concentrations of CsA in blood were undetectable. During the early days after operation, the reactions of the anterior chamber in group A and B were more severe than group C. The initial appearance of PCO in group C was much later than in the other two groups, and the grade of PCO in group C was much lower than the other two groups. The mean weights of wet posterior capsules in group A(312.86±52.91) mg and B(310.64±62.42) mg were much heavier than that of group C(56.93 ± 24.24) mg. Histological observation showed that there was remarkably less accumulation of lens materials on the posterior capsules in group C than in the other two groups. No toxic actions were found in intraocular tissues in group C. Our study suggested that Cyclosporin A modified intraocular lens could effectively and safely prevent the formation and development of posterior capsular opacification in rabbit eyes for a relatively long term.

Accidental Overdose of Oral Cyclosporine in Haematopoietic Stem Cell Transplantation: A Case Report and Literature Review.

A 26-year-old woman developed symptoms of acute toxicity during cyclosporine (CsA) therapy for graft-versus-host disease prophylaxis. The standard regimen included CsA in a dose of 1.5 mg/kg (120 mg) every 12 h, but, as a medication error, she received a high dose of 500 mg of oral CsA. After 2 h, she developed nausea and vomiting and, subsequently, flushing, chest tightness, tremor and vertigo. Laboratory and clinical examinations revealed high blood CsA concentrations (1000 ng/mL after 12 h) with a mild increase in blood pressure. Therefore, the patient was diagnosed with an acute CsA overdose. Before confirmation of the overdose by measurement of drug concentrations, the second dose was administered at its routine time because of uncertainty about the aetiology of the symptoms. The third dose was withheld, and the patient was monitored closely for clinical and laboratory presentations until the time when the abnormalities were relieved. CsA administration was then resumed with the correct prescription. The patient was discharged with successful engraftment and normal biochemical laboratory results after 1 month. Evaluation with the Naranjo assessment score indicated a probable relationship between the patient’s symptoms and overdosage with the suspected drug. Currently, detailed presentations of acute CsA toxicity cases due to overdose are limited in the medical literature. Evaluation of the patient’s medical and laboratory records, with cooperation of all responsible clinical staff, along with a review of the literature, were very helpful in discovery of the toxicity incident. Vigilance of health care providers with regard to medication errors and early detection of toxicity symptoms can decrease CsA-related morbidity and mortality in the future.

Impact of Cyclosporine Levels on the Development of Acute Graft versus Host Disease after Reduced Intensity Conditioning Allogeneic Stem Cell Transplantation

We analyze the impact of cyclosporine (CsA) levels in the development of acute graft-versus-host disease (aGVHD) after reduced intensity conditioning allogeneic hematopoietic transplantation (allo-RIC). We retrospectively evaluated 156 consecutive patients who underwent HLA-identical sibling allo-RIC at our institution. CsA median blood levels in the 1st, 2nd, 3rd and 4th weeks after allo-RIC were 134 (range: 10–444), 219 (54–656), 253 (53–910) and 224 (30–699) ng/mL; 60%, 16%, 11% and 17% of the patients had median CsA blood levels below 150 ng/mL during these weeks. 53 patients developed grade 2–4 aGVHD for a cumulative incidence of 45% (95% CI 34–50%) at a median of 42 days. Low CsA levels on the 3rd week and sex-mismatch were associated with the development of GVHD. Risk factors for 1-year NRM and OS were advanced disease status (HR: 2.2, P = 0.02) and development of grade 2–4 aGVHD (HR: 2.5, P < 0.01), while there was a trend for higher NRM in patients with a low median CsA concentration on the 3rd week (P = 0.06). These results emphasize the relevance of sustaining adequate levels of blood CsA by close monitoring and dose adjustments, particularly when engraftment becomes evident. CsA adequate management will impact on long-term outcomes in the allo-RIC setting.

Assessment of cyclosporine serum concentrations on the incidence of acute graft versus host disease post hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment option for hematological disorders. Cyclosporine (CsA) is one of the major immunosuppressive agents for the prophylaxis against graft versus host disease (GvHD). In this retrospective study, we evaluated the effects of CsA serum levels on the incidence of acute GvHD and transplant outcomes. Retrospective study in 103 adult patients received Hematopoitic Stem Cell Transplantation (HSCT) in the Hematology-Oncology, Bone Marrow Transplantation center at Shariati Hospital in Tehran, Iran. All participants received prophylactic regimen of cyclosporine plus methotrexate. CsA dose titration was done according to patients᾽ serum levels and drug toxicity. Serum levels tested on the twice weekly basis in first 4 weeks after transplantation. Acute GvHD (grades II-IV) developed in 44 patients (43%, 95%CI: 33%-52%). The median time to ANC and PLT recovery was 13 days (range: 9-31 days) and 16 days (range: 0-38 days), respectively. Univariate analysis of risk factors related to aGvHD (grade II-IV) development showed a higher risk of incidence of aGvHD (grades II-IV) for patients having the lowest blood CSA concentration (<200 ng/mL) in the third weeks after transplantation (36% vs. 12%, P = 0.035). The only risk factors related to incidence of aGvHD grades III-IV was also blood CsA concentration at 3(rd) week post-transplant (15% vs. 3%, P = 0.047). The CsA concentration at 3(rd) week was not related to disease free survival and overall survival (P = 0.913 vs. P = 0.81) respectively. Higher CsA serum levels in the third week post HSCT significantly decreased incidence of acute GvHD.

Feasibility of Pre- and Postoperative Gemcitabine-plus-Cisplatin Systemic Chemotherapy for the Treatment of Locally Advanced Urothelial Carcinoma in Kidney Transplant Patients

ObjectiveTo investigate the feasibility of pre- and postoperative gemcitabine-plus-cisplatin (GC) adjuvant chemotherapy for the treatment of locally advanced urothelial carcinoma in kidney transplant patients. MethodsSeven kidney transplant patients diagnosed with locally advanced urothelial carcinoma were treated with a pre- and postoperative GC adjuvant chemotherapy between January 2008 and March 2012. Gemcitabine (800 mg/m2) was administered at as an intravenous infusion on days 1 and 8. A total cisplatin dosage of 100 mg/cycle was administered on 2 days (50 mg/d on days 2 and 3) as an intravenous infusion. A single treatment cycle lasted 21 days. At the beginning of chemotherapy, the cyclosporine (CSA) dosage was reduced by 25 mg/d (on day 1 through day 8) if the blood CSA concentration was well maintained and did not fluctuate significantly. In addition, mycophenolate mofetil was reduced by 500 mg/d, while azathioprine was reduced by 25 mg/d (on day 1 through day 16). One cycle of GC neoadjuvant chemotherapy was given before operation, and several GC cycles were given after operation according to the patients' situation. Retrospective analysis was performed on the clinical data, chemotherapy regimen, chemotherapy efficacy, and side effects of the 7 patients. ResultsThe 7 patients were all treated with 1 course of presurgical chemotherapy. The seven patients completed 24 treatment cycles of chemotherapy in total. The average GC medication period per patient was 3.4 cycles. The postsurgery follow-up was 6 to 36 months (average-22.1); all of the patients survived. There was 1 case of complete remission (14.5%), 2 of partial remission (28.5%), and 4 of stable disease (57%), with one case of T4N1M0 and three cases of T3N0M0. The overall efficacy was 43%. The toxicity and side effects associated with the GC regimen were largely associated with myelosuppression. The other side effects included reversible nephrotoxicity, gastrointestinal tract and skin reactions, as well as phlebitis. Hematologic toxic reactions included reversible leukopenia, thrombocytopenia, and anemia. There was 1 case of degree III anemia and 1 case of degree II; 5 cases of degree III and 1 of degree II leukopenia; and 3 of degree II thrombocytopenia. Gastrointestinal reactions included nausea, vomiting, and constipation. There were 2 cases of degree III and 4 cases of degree II nausea and vomiting as well as 2 cases of degree III and 3 cases of degree II constipation. There were 3 cases of degree I phlebitis (43%) and 2 cases of degree I skin erythema. The nephrotoxicity reactions were all reversible. Both liver function and grafted kidney function were not significantly altered after chemotherapy compared with prior to chemotherapy. None of the patients suffered renal allograft rejection after chemotherapy; none required additional antirejection drug treatments. The original antirejection treatment regimen was restored after the patients completed the chemotherapy treatment cycles. ConclusionWe confirmed the efficacy of applying a GC regimen to treat locally advanced urothelial carcinoma in kidney transplant patients. The side effects were tolerable and reversible with minor impacts on graft function.

The effect of ketoconazole on whole blood and skin ciclosporin concentrations in dogs

Ciclosporin (CSA) is approved for the treatment of canine atopic dermatitis. Ciclosporin is metabolized by liver cytochrome P450 enzymes, a process inhibited by ketoconazole (KTZ). The aims of this study were to determine skin and blood CSA concentrations when CSA was administered alone at 5.0 (Treatment 1) or 2.5 mg/kg (Treatment 2) and when CSA was administered at 2.5 mg/kg concurrently with KTZ at 5 (Treatment 3) or 2.5 mg/kg (Treatment 4). We hypothesized that skin and blood CSA concentrations in Treatment 1 would not differ from those obtained with T3 or T4. In a randomized cross-over study, six healthy research dogs received each of the treatments (Treatment 1, 2, 3 and 4) once daily for 7 days. After the first, fourth and seventh dose for each treatment, a peak and trough skin punch biopsy sample and whole blood sample were collected and analysed with high-performance liquid chromatography-tandem mass spectrometry. Data were analysed using a repeated measures approach with PROC MIXED in SAS. Pairwise comparisons were performed with least squares means and Tukey-Kramer adjustment for multiple comparisons. Mean blood CSA concentrations in Treatment 1 were not different from those in Treatment 2 or 4, but were less than in Treatment 3. Mean skin CSA concentrations in Treatment 1 were greater than in Treatment 2, not different from those in Treatment 4, and less than those in Treatment 3. Administration of CSA and KTZ concurrently at 2.5 mg/kg each may be as effective as CSA alone at 5.0 mg/kg for treatment of canine atopic dermatitis.

Influence of Serum Uric Acid Levels in Response to the Conversion From Mycophenolate Mofetil to Mizoribine in Kidney Transplant Recipients

BackgroundCurrently, hyperuricemia is reported to be one of the most common side effects of mizoribine (MZR). However, previous clinical trials have not specially focused on MZR-related hyperuricemia. MethodsThirty-nine kidney recipients underwent a switch from mycophenolate mofetil (MMF) to MZR due to adverse clinical events. The control group included 39 kidney recipients continuing MMF without obvious adverse clinical events. They all received cyclosporine (CsA), prednisone, and MMF before enrollment. Every control was matched with a study group patient based on each pair undergoing kidney transplantations during the same week. Over the 24-month follow-up, every recipient underwent a physical examination including blood pressure, and laboratory screening of serum uric acid (sUA), creatinine, albumin, and CsA trough concentrations. We also calculated the estimated glomerular filtration rate (eGFR). ResultsThe sUA level of the study group significantly and consistently increased during the first 5 months, and then decreased. By 18 months, sUA concentrations were not significantly different between the 2 groups (P = .068). There were more new hyperuricemic cases in the study group at 24 months. There were no significant differences in blood pressure, eGFR, and CsA levels between the 2 groups. ConclusionAdministration of MZR was associated with a significant, transient increase in sUA. The increased uric acid suggested that MZR interferes with purine metabolism. The decrease should be associated with hypoxanthine-guanine phosphoribosyl transferase (HGPRT) enzyme activity, which improved under long-term MZR treatment. Therefore, recipients who receive MZR should be monitored more frequently for sUA during the first 5 months, followed by standard monitoring after 18 months.

Rosuvastatin Attenuates Inflammation, Apoptosis and Fibrosis in a Rat Model of Cyclosporine-Induced Nephropathy

Background/Aim: Cyclosporine (CsA)-induced kidney injury is characterized by renal dysfunction with inflammatory cell infiltrations, apoptosis and fibrosis. Pleiotropic effects of statins may exert anti-inflammatory, antiapoptotic and antifibrotic actions beyond lipid control. The aim of this study is to investigate whether rosuvastatin (RUS) has anti-inflammatory, antiapoptotic and antifibrotic effects on chronic CsA-induced nephropathy in a rat model. Methods: Male Sprague-Dawley rats fed a low-sodium diet were divided into three treatment groups: control (0.9% saline injection), CsA (15 mg/kg/day by subcutaneous injection), CsA + RUS (10 mg/kg/day by gastric gavage). Renal function, CsA level and lipid levels were measured at the end of 4 weeks. The expression of ED-1, transforming growth factor-β₁ (TGF-β₁) and α-smooth muscle actin (α-SMA) for inflammation and fibrosis were examined by Western blot analysis. The expression levels of apoptosis-associated factors were examined by Western blot analysis. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase-mediated biotin nick end-labeling (TUNEL) method. Results: Kidney function was decreased in CsA-treated rats compared with controls, which was attenuated by RUS. RUS did not affect the lipid level or the blood CsA level. TUNEL staining showed that RUS inhibited CsA-induced tubular apoptosis. RUS decreased CsA-induced increased expression of Bax/Bcl-2 ratio. The expressions of ED-1, α-SMA, TGF-β₁, Smad2/3, Smad4 and p-JNK were increased in CsA-treated rats, which were attenuated by RUS. Tubular atrophy and interstitial fibrosis in CsA-treated rats were attenuated by RUS supplementation. Conclusion: RUS supplementation attenuates proinflammatory and apoptosis-related factors and inhibits the fibrotic pathways including the smad-dependent and smad-independent pathways in a rat model of CsA-induced nephropathy.

Anti‐apoptotic and anti‐oxidant effects of grape seed proanthocyanidin extract in preventing cyclosporine A‐induced nephropathy

Although the pathogenesis of cyclosporine (CsA) nephropathy is not completely understood, it is attributed to oxidative damage and apoptosis. Grape seed proanthocyanidin extract (GSPE) is a molecule with anti-oxidant and anti-apoptotic properties. Our aim was to demonstrate the effects of GSPE in preventing CsA nephropathy. Twenty-four Sprague-Dawley rats were divided into four groups. The control, GSPE, CsA and CsA+GSPE groups were given 1 mL olive oil, 100 mg/kg GSPE, 25 mg/kg CsA and 100 mg/kg GSPE+25 mg/kg CsA, respectively. On day 21, blood samples were taken for blood urea nitrogen (BUN), creatinine and CsA levels, and renal tissue was used for total oxidant system (TOS), total anti-oxidant system (TAS), oxidative stress index (OSI) and malondialdehyde (MDA) measurements. In addition to renal histopathology, apoptosis staining was performed on renal tissue. The BUN, creatinine, TOS, OSI, MDA, histopathological score, and apoptotic index exhibited increases in the CsA group. In the CsA+GSPE group, however, BUN, creatinine, OSI, MDA, renal histopathological score and apoptotic index (AI) decreased and TAS levels increased. In addition, there was no difference between the CsA and CsA+GSPE groups with regard to CsA levels. We demonstrated that GSPE prevents CsA nephropathy and that this effect is achieved by anti-apoptotic and anti-oxidant activity. We also achieved a significant recovery in kidney functions without affecting CsA plasma levels.

P-Glycoprotein-Based Loperamide–Cyclosporine Drug Interaction at the Rat Blood–Brain Barrier: Prediction from In Vitro Studies and Extrapolation to Humans

We have shown that the rat can quantitatively predict the verapamil-cyclopsorine A (CsA) drug-drug interaction (DDI) at the human blood-brain barrier (BBB). In addition, the potency (EC(50)) of CsA to inhibit rat BBB P-gp can be predicted from in vitro studies in MDRI-transfected cells. To assess if these excellent agreements extend to other substrates, we determined the magnitude of P-gp-based DDI at the rat BBB between loperamide (Lop) or its metabolite, N-desmethyl Lop (dLop), and escalating CsA blood concentrations. The percent increase in the brain:blood Lop concentration ratio was described by the Hill equation, E(max) = 2000%, EC(50) = 7.1 μM and γ = 3.7. The potency (EC(50)) of CsA to inhibit P-gp at the rat BBB was independent of the substrate used (verapamil, Lop, or dLop). Like the verapamil-CsA DDI, the potency (EC(50)) of CsA to inhibit rat BBB P-gp could be predicted from studies in MDRI-transfected cells. When (11)C-Lop was coadministered with a 10 mg/kg iv infusion of CsA (1) yielding ~5.6 uM CsA blood concentration to healthy volunteers, the brain distribution of (11)C-radioactivity was increased by 110%. (1) When corrected for diffusible Lop metabolite(s), this translates into an increase in (11)C-Lop brain distribution of 457%. Based on our rat data, we estimated a similar value at 5.6 μM blood CsA concentration, 588% increase in Lop brain distribution. These data support our conclusion that the rat is a promising model to predict P-gp based DDI at the human BBB.

Renal Graft Function and Low-Dose Cyclosporine Affect Mycophenolic Acid Pharmacokinetics in Kidney Transplantation

In kidney transplantation, the pharmacokinetics of mycophenolic acid (MPA), the active compound of mycophenolate mofetil (MMF), is influenced by concomitant immunosuppressive therapy, including cyclosporine (CsA). However, whether in the setting of immunosuppressive therapy minimization CsA still affects MPA pharmacokinetics, particularly in relation to varying degree of renal graft function deterioration, remains ill defined. One hundred thirty-five complete MPA profiles were sequentially collected from 56 kidney transplant recipients given MMF and low-dose CsA as part of their immunosuppressive therapy. MPA pharmacokinetic parameters were correlated with blood CsA area under the curve (AUC0-12) and graft function as measured glomerular filtration rate (GFR). The relative contribution of CsA exposure and GFR to MPA kinetics in relation to other clinical parameters was determined by multivariate analysis. Dose-adjusted MPA AUC0-12 negatively correlated with CsA AUC0-12. MPA exposure significantly increased when CsA AUC0-12 was below 2000 ng hr/mL. Stratification of MPA profiles according to stages of renal dysfunction showed that dose-adjusted MPA AUC0-12 was higher (P<0.001) in patients with severe (stage 4: 70.37±27.93 μg hr/mL/g MMF) than in those with mild (stage 2: 47.46±11.66 μg hr/mL/g MMF) or moderate (stage 3; 47.48±15.22 μg hr/mL/g MMF) renal insufficiency. At multivariate analysis GFR, serum albumin and hemoglobin levels, use of gastroprotective medications, and time posttransplant were identified as independent determinants of MPA AUC0-12. In stable renal transplant recipients given MMF, tapering CsA dose and deterioration of renal graft function contribute to increased MPA exposure. Thus, monitoring plasma MPA pharmacokinetics should be advised, especially in patients on minimized CsA therapy with severe renal insufficiency.

Feasibility study of replacing TDXFLX by AxSYM in determination of whole blood concentration of ciclosporin

Objective:To study the correlation of whole blood concentrations of ciclosporin(CsA) determined by AxSYM and TDXFLX,and evalute the feasibility of replacing TDXFLX with AxSYM.Methods:A total of 113 blood samples from outpatients and inpatients,who had underwent renal transplantation or hematopoietic stem cell transplantation in Changhai Hospital,Second Military Medical University,were determined by AxSYM and TDXFLX monoclonal analyzer simultaneously.Linear regression analyses was performed with TDXFLX results as x-axis and AxSYM results as y-axis to evaluate the correlation.Paired t-test was performed with these data.A P value less than 0.05 was taken as significantly different.Results:There was a good correlation between AxSYM and TDXFLX results.The linear equation was y=1.001 9 x-6.101 7(n=113,r=0.988 3).The results determined by AxSYM were significantly lower than those by TDXFLX by average 4.76%(n=113,P0.05).Conclusion:AxSYM can replace TDXFLX for routine assay of whole blood CsA concentration in our hospital.

Prospective, Randomized Study of the Efficacy of Systemic Cyclosporine in High-Risk Corneal Transplantation

Immunologic rejection remains a major cause of graft failure in high-risk corneal transplantation. This study was conducted to elucidate the efficacy and safety of systemic cyclosporine (CsA) in high-risk corneal transplantation. Prospective, randomized, open-labeled clinical trial with a parallel-group study. Patients underwent high-risk corneal transplantation at the Department of Ophthalmology, Tokyo Dental College, Chiba, Japan. High-risk was defined as corneal neovascularization in more than 1 quadrant or a history of corneal grafting. Patients were assigned to either a systemic CsA group or a control group. Administration of CsA was continued for at least 6 months with blood CsA concentration 2 hours after administration of approximately 800 ng/mL, unless undesirable side effects developed. The main outcome measures were graft clarity, endothelial rejection, and local and systemic complications. Forty patients were enrolled and 39 (18 men, 21 women; mean age, 67.4 ± 11.9 years) were analyzed. In the CsA group, CsA was discontinued within 6 months in 7 patients because of side effects. With a mean follow-up of 42.7 months, endothelial rejection developed in 6 and 2 eyes in the CsA and control groups, respectively. No differences were observed in the rates of graft clarity loss between the 2 groups (P = .16, Kaplan-Meier analysis). No positive effect of systemic CsA administration for suppressing rejection in high-risk corneal transplantation was observed. With a relatively high incidence of systemic side effects, the results suggest that this protocol should not be recommended for corneal transplant recipients, especially those of advanced age.