C2 Blood Levels Research Articles

Association Between CYP3A4 and CYP3A5 Genotypes and Cyclosporine's Blood Levels and Doses among Jordanian Kidney Transplanted Patients.

Cyclosporine is used as an immunosuppressive agent in kidney transplantation. It has a narrow therapeutic window. Cyclosporine is predominantly metabolized by CYP3A4 and CYP3A5. The most common Single Nucleotide Polymorphisms (SNPs) affecting cyclosporine metabolism (CYP3A4*1B, CYP3A4*1G, CYP3A4*22 and CYP3A5*3) were investigated among Jordanian kidney transplanted patients to find out the genotypes and allele frequencies of these SNPs. Additionally, this study investigated whether genotypes of CYP3A4 and CYP3A5 affect C2 blood levels, dosing of cyclosporine and the prevalence of acute rejection. Blood samples of 109 adult patients taking cyclosporine as their primary immunosuppressant for kidney transplantation were collected from the Prince Hamzah Hospital, Amman, Jordan. Patients' first C2 blood levels and their first two given doses were collected. Patients were genotyped for the four SNPs using Polymerase Chain Reaction- restriction Fragment Length Polymorphism (PCR-RFLP) assay method. Allele frequencies among Jordanian patients for CYP3A4*1B, CYP3A4*1G, CYP3A4*22 and CYP3A5*3 were 0.037, 0.399, 0.037 and 0.271, respectively. There was a significant association between CYP3A4*22 and mean difference in the second and first given doses (P=0.034). There was a big difference between CYP3A4*22 and the mean of the first C2 blood levels (P=0.063). There was a strong association between CYP3A4*22 and the mean difference between the second and first given doses. There was a trend of significant difference between the mean of the first C2 blood levels among heterozygous CYP3A4*22 patients. Pharmacogenomics may hold promise in assisting the prediction of the best cyclosporine dose and C2 blood level among Jordanian kidney transplant patients.

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.

Change of Cyclosporine Absorption over the Time After Kidney Transplantation

BackgroundAlthough the immunosuppressant cyclosporine (CsA) is widely used after kidney transplantation over the long term, there is still no firm consensus on the best way to monitor of CsA blood levels.ObjectivesCyclosporine (CsA) assay is critical for the management of renal transplant recipients due to inter– and intra–patient variation in CsA absorption and metabolism. Patients and Methods: In a retrospective cross sectional study, blood levels of CsA (through and 2 hours post dose) measured at least 5 times during 3 years post transplantation, in 7702 kidney transplant recipients from different transplant center of Tehran, IR Iran between 2008 and 2012. Cyclosporine absorption (CA) calculated C2/C0 ratio.ResultsCA had a significant correlation with allograft function (P = 0.000, r =.0.285), this correlation was stronger than its relationship with C0 and C2 blood levels (P = 0.000 and P = 0.000 as well as r = 0.033 and r = 0.090, respectively). In univariate analysis during different times after transplantation, C0 and C2 blood levels significantly decreased over three years follow up (P = 0.000), (P = 0.000); While, CA reversely increases over the time (P = 0.000). In linear regression model overall CA levels had correlation with lower age of recipient (P = 0.02), hypokalemia (P = 0.001), higher level of creatinine (P = 0.02) and triglyceride (P = 0.001).ConclusionsThe present study shows that CsA absorption changes trough the post-transplant time and appears to increases over time in long–term period after kidney transplantation.

Everolimus and Minimization of Cyclosporine in Renal Transplantation: 24-Month Follow-Up of the EVEREST Study

The association of cyclosporine A (CsA) with everolimus can exert synergistic effects on the alloimmune response. CsA inhibits the synthesis of interleukin-2 while everolimus inhibits the response to interleukin-2. Moreover, the interaction with CsA increases the bioavailability of everolimus (1). These data suggest that it is possible to reduce the doses of both drugs when given in combination. Previously, we reported the 1-year results of a randomized trial in 285 de novo renal transplants, 278 from deceased donors (2). Patients of group A received low-concentration CsA (C2 maintenance levels 350–500 ng/mL) plus everolimus (C0 levels 3–8 ng/mL), while patients of group B received very low-concentration CsA (C2 maintenance levels 150–300 ng/mL) and everolimus at higher target levels (C0 8–12 ng/mL). One-year graft survival rate was better in group B (98% vs. 90%; P=0.007). The mean serum creatinine levels at 1 year were, respectively, 1.51±0.55 mg/dL and 1.55±0.62 mg/dL. After the 1-year study was completed, 215 patients (110 in group A and 115 in group B) accepted to enter the extension study and to continue the study treatment up to 2 years. At 24 months, the mean everolimus dosages were 1.45±0.6 mg/day in group A (blood levels 6.17±2.1 ng/mL) and 2.24±1.1 mg/day in group B (blood levels 8.15±3.5 ng/mL), and the dosages of CsA were 1.62±0.5 mg/kg/day (C2 levels 407.6±159 ng/mL) and 1.38±0.7 mg/kg/day (C2 blood levels 330.7±159 ng/mL), respectively; the prednisone dosages were 5.14±1.28 mg/day in group A and 4.91±1.10 mg/day in group B. Four deaths (two in group A and two in group B) occurred within 6 months since transplantation; another patient of group A died after the first year because of myocardial infarction. Fifteen grafts were lost in group A and six in group B (P=0.043). In group A, only one graft was lost after the first year because of recurrent focal glomerular sclerosis. In group B, three failures occurred in the second year; two because of chronic rejection and one because of infection. The 24-month graft survival, including death as a cause of graft failure, was 87.4% in group A and 94.4% in group B (P=0.048). Acute rejection after the first year occurred in just one patient of group B. The cumulative probability of biopsy-proven rejection at 2 years was 15.0% in group A and 15.7% in group B. The mean levels of serum creatinine at 24 months were 1.45±0.5 mg/dL in group A and 1.52±0.7 mg/dL in group B (P=not significant [NS]). Mean glomerular filtration rate according to Nankivell et al. (3) was 65.8±19.7 mL/min in group A versus 61.8±22.0 mL/min in group B. The slopes of serum creatinine and glomerular filtration rate between 6 months and 2 years were practically flat (data not shown). At 24 months, systolic blood pressure was 135.6±16.1 mm Hg in group A and 135.8±18.8 mm Hg in group B; diastolic blood pressure was 81.2±9.8 mm Hg in group A versus 81.7±10.4 mm Hg in group B. Most patients were on antihypertensive medication. Mean hemoglobin levels were 13.1±1.6 g/dL in group A and 13.0±1.7 g/dL in group B. Plasma cholesterol levels were 228.0±41 mg/dL in group A and 235.1±54.5 mg/dL in group B (P=NS). Triglycerides levels were 193.3±84.9 mg/dL versus 224.4±120.6 mg/dL (P=NS). 3-hydroxy-3-methyl-glutaryl-Co (HMG-CoA) reductase inhibitors were administered in 83% of patients in both groups. Proteinuria more than 0.2 g/day occurred in 13 patients of group A (9.1%) and in 20 of group B (14.1%). The overall incidence of infections requiring hospitalization was similar in the two groups (10.5% in group A and 14.5% in group B). Diabetes was diagnosed in 7 (4.9%) patients in group A and in 13 patients (9.2%) in group B (P=NS). One patient (0.7%) in group A and three patients in group B (2.1%) developed nonmelanoma skin cancer. Twelve patients (four in group A and eight in group B) discontinued treatment because of adverse events after the first year (Table 1).TABLE 1: Adverse events leading to discontinuation of treatment between the first and the second yearFrom these results, it is possible to conclude that minimal doses of CsA in association with everolimus may offer excellent graft survival and stable renal function at 2 years. Claudio Ponticelli1 Maurizio Salvadori2 Maria Piera Scolari3 Franco Citterio4 Paolo Rigotti5 Antonella Veneziano6 Marta Bartezaghi6 on behalf of EVEREST Study 1Division of Nephrology Humanitas Scientific Institute Rozzano (Milano), Italy 2Division of Nephrology Careggi Hospital Firenze, Italy 3Renal Transplant Unit S. Orsola Malpighi University Hospital Bologna, Italy 4Department of Surgery Catholic University Hospital Roma, Italy 5Surgery Division Policlinic University Hospital Padova, Italy 6Medical Department Novartis Italy Origgio (Varese), Italy

Automated monitoring of C2 and C0 blood levels of mycophenolic acid and cyclosporine on the Abbott Architect c8000

Objectives: Evaluation of the performance of the EMIT 2000 Cyclosporin assay using 2 sets of assay calibrators and the EMIT 2000 mycophenolic acid assay to measure C0 and C2 concentrations on the Abbott Architect c8000 analyzer. Design and methods: Imprecision studies were performed. Cyclosporin concentration was assayed by EMIT on the c8000, by ACMIA on the Dimension and by LC-MS/MS while mycophenolic acid was analyzed by EMIT on c8000 and on Dimension and by HPLC. Results: Agreement between cyclosporin and mycophenolic acid concentrations assayed on the c8000 and on the Dimension was very good. Method comparison between the c8000 and LC-MS/MS resulted in a relative bias of 15.7% for C0 and 11.5% for C2 concentrations. Relative bias of the mycophenolic acid concentrations assayed on the c8000 and the HPLC was 37.7%. Conclusions: When reported properly to the clinician mycophenolic acid and cyclosporine blood levels can be monitored using the EMIT assays on the c8000 consolidating standard routine workflow and reducing reagent costs significantly.

Cyclosporin-A in the Treatment of Nephrotic Syndrome: The Importance of Monitoring C0 (Trough) and C2 (Two Hours After its Administration) Blood Levels

Cyclosporin-A (CsA) is often used in the treatment of nephrotic syndrome. The effectiveness of CsA and the value of C2 blood levels in the treatment of nephrotic syndrome, due to various glomerular diseases, were studied. Forty-two nephrotic patients (M/F 21/21), with well-preserved renal function (creatinine clearance 87+/-20 ml/min) were included in the study. The original diagnoses were minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN), IgA nephropathy (IgAN), and lupus nephritis (LN). All patients were treated with prednisolone and CsA for 24 months. Cyclosporin-A C0 and C2 blood levels were determined at regular intervals. Remission of the nephrotic syndrome was observed in all patients with MCD, IgAN and LN, in 75% with FSGS and in 83% with MN. Relapses were observed in some patients with MCD (25%) and MN (36%). The C0 levels were 93+/-15 ng/ml and the corresponding C2 levels were 498+/-110 ng/ml. However, significantly lower (340+/-83 ng/ml) or higher (680+/-127 ng/ml) to the average C2 levels were found in 6 patients (14%). No relation of C0 and C2 levels with the remission and relapse rate of the nephrotic syndrome and with renal function impairment was observed. Small doses of CsA with prednisolone are effective in the treatment of nephrotic syndrome. Although an individual variation of C2 was observed for the same target C0 levels, no relation of C2 levels was found with the remission or relapse rate of the nephrotic syndrome.

Comparison of different cyclosporine immunoassays to monitor C0 and C2 blood levels from kidney transplant recipients: Not simply overestimation

Background Immunoassays used for the measurement of cyclosporine (CsA) usually show cross-reactivity for CsA metabolites, usually resulting in unacceptable bias. Methods To assess the performance of different immunoassays, CsA concentrations were analyzed in 132 samples using ACMIA, EMIT-VIVA, CEDIA-PLUS, and HPLC. Samples were collected from kidney transplant patients monitored with the traditional blood CsA trough level ( C 0, n=73) and the new sampling at 2-h post CsA dosing ( C 2, n=59). Results Overall, the correlations between HPLC and other methods were good ( r values ranging from 0.85 to 0.97). The use of C 2 concentrations to monitor CsA exposure were associated with an overall better performance of all the immunoassays as compared with C 0 values. However, none of the immunoassays agreed with the guidelines proposed in the Lake Louis Consensus Conference. Of note, the CEDIA-PLUS was the only that provided a linear relationship with HPLC for both sampling times. A false positive case associated with ACMIA was also documented in blood samples from a patient withdrawn from CsA for 1 month. Conclusion These data suggest that the performance of some of the most used immunoassays is not satisfactory, eventually leading to incorrect therapeutic decision guided by erroneous CsA monitoring.

Clinical outcomes and C2 cyclosporin monitoring in maintenance renal transplant recipients: 1 year follow-up study

Cyclosporin A (CsA) concentration monitoring with 2 h post-dosing levels (C2) correlates with the incidence of rejection and graft outcome in de novo renal transplant patients. The advantages of this policy beyond the first 12 months remain a matter of debate. The purpose of the present work was to evaluate the C2 target ranges on CsA monitoring after the first year in stable kidney transplant patients. We studied 142 patients, 94 on CsA-steroids and 48 on triple therapy (CsA-azathioprin-steroids), transplanted for 104+/-42 months and with a serum creatinine of 1.53+/-0.52 mg/dl. C2 and C0 measurements were performed at baseline and at least twice more during the year of follow-up. The mean annual C2 blood levels in double therapy patients showed C2 in 23 (24.5%) of <600 ng/ml; in 53 (56.4%) of between 600 and 850 ng/ml; and in 18 patients (19.1%) of >850 ng/ml. In the triple therapy group, C2 in 12 (25%) was <500 ng/ml, in 24 (50%) between 500 and 700 ng/ml and in 12 patients (25%) >700 ng/ml. In both groups, higher C2 levels were associated with a better absorption of the drug measured by the ratio C2/C0 and C2/dose. There were no differences in incidence of infections, need for hospitalization and the presence of hypertension, hyperuricaemia, hypercholesterolaemia or diabetes between patients with low and high C2 blood levels. However, serum creatinine was higher in triple therapy patients with lower C0 levels (P = 0.004). In 135 patients (90 on double and 45 on triple therapy), renal function remained stable during follow-up and 120 of them (89%) had C2 values under the recommended ranges. C2 monitoring in maintenance patients enabled us to identify overexposure to CsA. Target levels of C2 should be adjusted according to the immunosuppressive regime. C2 levels between 600 and 800 ng/ml in double therapy patients and between 500 and 700 ng/ml in triple therapy patients are sufficient to give an adequate immunosuppression. The superiority of C2 with respect to C0 levels could not be demonstrated.

A COMPARISON OF C0 AND C2 MONITORING IN RENAL TRANSPLANT PATIENTS

A78 Aims: To compare the effects of Cyclosporine (CyA) C0 and C2 level monitoring on acute rejection (AR) and infection rates, toxicity and graft survival in patients with renal transplant (tx). Methods: Prospectively evaluated 10 patients with C2 monitoring (Group C2) were compared to a group of 10 patients with C0 monitoring (Group C0). In C2 Group, C0 levels were also recorded but drug doses were adjusted according to C2 levels. CyA doses, C0 and C2 blood levels, AR and infection rates, drug toxicity and relation of these to drug levels, creatinine levels, GFR, blood pressures (BP), serum glucose, lipids, liver function tests, and Hct levels were analysed in 5 time periods (≤1 month, >1-≤3 month, >3- ≤6 months, >6-≤12 months, >12-≤18 months post-tx). Results: Two groups were similar in mean age, sex, donor type and antilymphocyte treatment. Delayed graft function was seen in 1 patient in Group C0. 2 patients in GroupC0 and 1 patient in Goup C2 returned to hemodialysis during the study. All patients had at least 6 months’ follow-up, 14 patients completed the study. CyA beginning dose was 8 mg/kg/day in all patients. Adjusted drug doses of patients in Group C2 were higher than the doses in Group C0 in all time periods; the differences were significant in the first and last time periods (6.34 mg/kg and 4.47 mg/kg in first period and 3.1 mg/kg and 1.7 mg/kg in last period, p<0.05). C0 levels were similar in the two groups in all time periods. Mean creatinine levels were lower in Group C2 throughout the study; the differences between the groups were significant in the first and last time periods (1.12 mg/dl and 1.80 mg/dl in first and 1.05 mg/dl and 1.74 mg/dl in last time period, p<0.05). GFR was higher in Group C2, the differences were significant in the first and 4th periods (p<0.05). Transient nephrotoxicity was seen in 1 patient in each group. Transient hepatotoxicity was seen in 1 patient in Group C2. Mean systolic BP and mean diastolic BP were lower in Goup C2 in 1st and 4th periods, respectively (p<0.05). Infection rate was significantly higher in Group C0 in the 3rd period. No significant relation was found between C0 or C2 levels and infection rates in any time period. AR was seen in 3 patients in Group C0 and 2 patients in Group 2. No significant differences in the other parameters analysed were found between the groups. Conclusions: C2 monitoring results in better graft function with similar adverse effects when compared to C0 monitoring in renal transplant patients.

Total cholesterol correlates with cyclosporine C2 levels in kidney transplant recipients under maintenance immunosuppression

The aim of this study was to assess the relationship between cyclosporine (CyA) trough level (C0) and 2-hour postdose (C2) and total cholesterol (TC) in kidney transplant (KT) recipients on Neoral maintenance immunosuppression. In KT recipients who had more than 5 years of follow-up, stable graft function, and stable Neoral dose, we measured C2 and C0 blood levels, serum creatinine, mean total cholesterol (TC) over the last 5 years, prednisone dose, use of beta-blockers and thiazides. Correlations between C0 and C2 levels and TC were performed with the Pearson coefficient. Receiver operating characteristics (ROCs) were used to define the threshold with greater accuracy for significant variables at the correlation test. Statistical tests were performed with SPSS 9.5 The C2 correlated with TC (0.31; P = .008) whereas C0 did not. The C2 level was an independent predictor for TC after adjusting for recipient age, gender, dose of prednisone, creatinine clearance, and use of beta-blockers and thiazides (B coefficient = 1.124 E–3; P = .009). A threshold C2 value of 700 μg/L yielded to a TC level of 5.2 mmol/L. This is the first study to report a correlation between C2 levels and TC. Although C2 explained a small fraction of TC variability, it is an independent predictor of TC in KT recipients on Neoral maintenance immunosuppression. A long-term C2 value under 700 μg correlates with better control of hypercholesterolemia.

Cyclosporine absorption profiling and therapeutic drug monitoring using c2 blood levels in stable renal allograft recipients

Cyclosporine absorption profiling and therapeutic drug monitoring using c2 blood levels in stable renal allograft recipients

NEORAL DOSE MONITORING WITH 2 HOUR POST-DOSE LEVELS IN HEART TRANSPLANT PATIENTS RECEIVING ANTITHYMOCYTE GLOBULIN INDUCTION: A PILOT STUDY

624 We have previously reported that cyclosporine (CsA) 2-hour post-dose levels (C2) correlate better with the area-under-the-curve than trough levels (C0) in stable heart transplant (HTx) pts (more than one year after HTx) treated with Neoral. We also found that Neoral dose adjustment with C2 blood levels of 300-600 ng/ml (EMIT) resulted in 30% dose reduction and initial lower serum creatinine (Scr) compared to C0, without evidence of acute rejection. Purpose: to determine the efficacy and side-effect profile of Neoral dose monitoring with CsA C2 levels during the first year after HTx. Methods: 7 consecutive males (53±9 yr.) who underwent HTx between 4/1996 and 8/1996 were followed for one year. Five pts had a primary diagnosis of ischemic and 2 had dilated cardiomyopathy. Initial immunosuppession consisted of a sequential combination of antithymocyte globulin (5 days), azathioprine 1.5 mg/kg/day, methylprednisolone 500 mg intra-operatively, then prednisone(0.3 mg/kg/day at 1 month; 0.07 mg/kg/day at 12 months), and Neoral started at 2 mg/kg/day on day 2-3 after HTx. Neoral dose was monitored with C2 (range 300-600 ng/ml), irrespective of C0. Acute rejection was assessed by routine endomyocardial biopsies (EMB), the left ventricle ejection fraction (LVEF) was assessed by echocardiography. Coronary angiography was performed at one year (graft atherosclerosis was defined as any stenosis). Results: 3 pts (43%) presented with a reversible(methylpednisolone 500 mg on 3 consecutive days) acute rejection episode 17-42 days after HTx (grade 2, 3A and 3A, respectively). The incidence of acute rejection did not differ from the one observed in 20 pts (50%) who underwent HTx between 5/1994 and 1/1996 (same immunosuppression including Sandimmune). The LVEF was 63±11% within the first month and 60±0% one year after HTx. One pt (14%) presented some evidence of graft atherosclerosis. There was no mortality during follow-up. Neoral dose, Scr and CsA levels are shown below. TableConclusion: in HTx pts receiving antithymocyte globulin induction, Neoral dose monitoring with C2 resulted in a low dose without increasing the incidence of acute rejection. Renal function remained stable throughout the first year after HTx. A prospective randomized study should compare Neoral dose monitoring with C0 vs. different ranges of C2 in order to determine a therapeutic range.