Search

Despite the fact that cyclosporine (CsA) has been used clinically for a number of years, there is still uncertainty about the efficacy of monitoring its blood levels. Few if any studies have documented clear differentiation of rejection, immunosuppression, and toxicity on the basis of CsA concentrations alone. The issues in CsA monitoring include selection of sample matrix, analytical method, dosing interval and the timing of trough measurements, the temporal relationship between measurements and physiological events such as toxicity, the concurrent presence of multiple other immunosuppressive agents, and the lack of "gold standards" for determining rejection, adequate immunosuppression, and toxicity. In contrast to using CsA levels for the differential diagnosis of rejection and toxicity, there is evidence that maintenance of CsA concentrations within a therapeutic window results in a lower prevalence of toxicity and rejection.

Cyclosporine (CsA) is being increasingly used for the treatment of disorders other than transplantation. In contrast to transplant recipients, most of these patients can have CsA therapy discontinued without life-threatening consequences, and the dose of CsA can therefore be restricted in order to limit the incidence and severity of toxicity, including nephrotoxicity. The utility of either CsA blood levels or pharmacokinetic profiling to ensure adequacy of therapy or to prevent incipient as well as overt toxicity has not been confirmed in this group of patients, and prevention of nephrotoxicity usually depends on limiting the dose of CsA and careful assessment of renal function. Frequent measurement of CsA levels beyond the initiation period in patients with autoimmune and other nontransplant diseases cannot be currently justified, and should be reserved for those situations where drug interactions, unexpected toxicity or the possibility of inadequate therapy is likely.

The Canadian Quality Assurance Program was initiated in June 1989, and is a voluntary program which currently encompasses all 32 laboratories involved in the measurement of cyclosporine (CsA) across Canada. Two whole blood samples from control or clinical patients (kidney, liver and heart) containing unknown concentrations of CsA are circulated to each participating laboratory monthly, and analyzed by all techniques employed within that laboratory. Four analytical methods are currently employed: HPLC (n = 4). Sandimmun SP (n = 3), CycloTrac SP (n = 27) and TDx (n = 3). Four laboratories reported survey results in more than one methodology. Results from all participating centers are analyzed monthly. The mean, SD, standard deviation index and range are reported to each laboratory with information coded to preserve confidentiality. Accuracy, precision, recovery, analytical specificity, linearity and blank studies have been performed. This report covers the period from June 1989 to April 1990.

Cyclosporine in whole blood samples from renal and cardiac transplant recipients was measured by high-performance liquid chromatography (HPLC) and by CYCLO-Trac SP specific radioimmunoassay (RIA). The fresh samples assayed by RIA were remeasured in a second laboratory after storage at -20 degrees C for two to six months and good interlaboratory agreement was obtained on the 59 samples assayed (y = 0.926x + 14.8 micrograms/L, r = 0.982). The calibration curve for RIA was not influenced by fresh whole blood, hemolyzed whole blood or serum matrix from normal volunteers. However, comparison of the RIA results from patient samples with those from an HPLC procedure showed that RIA values averaged about double those measured by HPLC. The difference is attributable to differences between the blood matrix of transplant and nontransplant subjects, rather than exclusively to the apparent nonspecific nature of the antibody.

The monitoring of cyclosporine levels in whole blood and serum has become a routine procedure for the post-transplant management of immunosuppression. We have developed fluorescence polarization immunoassays for cyclosporine in whole blood and in serum using a monoclonal antibody. These assays are fast (20 determinations in less than 22 min), sensitive (25 micrograms/L for whole blood samples and 10 micrograms/L for serum samples), and precise (CV less than 7% in both assays). Cross-reactivities with AM1 (Metabolite 17) and AM4N (Metabolite 21) are less than 8.5 and 2.5%, respectively, for both assays.

We developed a sensitive radioimmunoassay (CYCLO-Trac SP) that specifically measures cyclosporine A in serum, plasma and whole blood of transplant patients. The specific monoclonal antibody was from Sandoz and the tracer was an 125I derivative of cyclosporine C. The assay is performed at room temperature for 1 h followed by a 20 min centrifugation. The sensitivities of the assays are 2.6 ng/mL and 8.7 ng/mL for the serum/plasma assay and the whole blood assay, respectively. Within-run and between-run CVs for both types of assays using cyclosporine concentrations of 80 and 58 ng/mL (serum) and 186 and 199 ng/mL (whole blood) were less than 5% and 9%, respectively. Averaged recovery of serum/plasma and whole blood assays at various levels ranged from 93% to 115%. Interferences by bilirubin, triglyceride, cholesterol, hemoglobin, OKT-3, azathioprine, methylprednisolone and 20 other drugs were insignificant. Multicenter proficiency studies showed an excellent correlation between the CYCLO-Trac SP and the specific 3H-Sandimmune assay from Sandoz: whole blood assay (r = 0.998) and serum assay (r = 0.997).

Monitoring blood levels of Cyclosporine (CsA) has been the basis for adjusting individual dosage regimens in the clinic. Radioimmunoassays using polyclonal antisera reacted with CsA and some CsA metabolites, leading to overestimation when compared with high-performance liquid chromatographic measurements of CsA. Monoclonal antibodies (mAbs) have the potential to discriminate between closely related molecules. MAbs with high affinity for CsA have been prepared and their fine-specificity characterized by cross-reactivity studies using a large series of CsA-derivatives. According to the known sites of metabolism on the CsA molecule and to its three-dimensional structure, it was possible to predict which mAb would be suitable for recognizing native Cs specifically.

Adult Sprague-Dawley rats were given cyclosporin A (CyA), frusemide (Fr) or both drugs daily for 14 days. The doses of CyA (50 mg/kg) and Fr (5 mg/kg) were approximately 3-6 times and twice respectively those used in man. Fr on its own produced a diuresis lasting approximately 3 hr. This was characterized by a 10-fold increase in urine flow rate, a 40-fold increase in the rate of sodium excretion, and by 2- and 4-fold increases in urea and creatinine clearance rates, respectively. In addition, there was a doubling in urinary N-acetyl-beta-D-glucosaminidase (NAG) activity. After 4 days of combination treatment with CyA and Fr, the diuretic-induced increases in urine flow rate, sodium excretion and urinary NAG activity were similar to those following frusemide alone. However, urea and creatinine clearances did not increase during the diuresis. Fr itself did not impair renal function, but rats receiving only CyA did show elevations in serum urea and creatinine, with reductions in clearance rates, which progressed with time. There was also an increase in NAG enzymuria. When the two drugs were exhibited together, renal function was more severely impaired. All animals given CyA showed proximal renal tubular cell vacuolation: in half the damage was confined to the straight segment, while the rest showed additional severe convoluted segment change. Renal function was most abnormal in those rats in which both segments were affected. All animals given both drugs showed both straight and convoluted tubular abnormalities and a 2-fold increase in serum CyA levels. CyA-induced disturbances in hepatic function and lymphoid tissue atrophy were unaffected by the addition of Fr, nor did Fr affect the immunosuppressive action of CyA.

We measured cyclosporine in whole blood samples from renal and heart transplant patients by high performance liquid chromatography and by two radioimmunoassays with use of specified monoclonal antibodies. In particular, we evaluated the analytical performance of a new specific radioimmunoassay with an iodinated tracer. The reproducibility of the method is satisfactory (within-run CV 7.1 to 9.5% and between-run CV 7.2 to 10.3%). The limit of detection is 10.3 micrograms/L and the analytical recovery between 99 and 114%. The results obtained with samples from both renal heart transplant patients agree well with those obtained by HPLC and by a specific RIA that uses a tritiated tracer.

The present study was designed to examine inter-relationships between serum cyclosporin (CsA) levels, hepatic drug metabolising enzyme activity and CsA induced nephrotoxicity. CsA (25 mg/kg p.o.) was administered daily to male Sprague-Dawley rats: groups of animals were killed on days 0, 4, 7, 10 and 14 and thereafter at weekly intervals over the 7-week course of the experiment. Nephrotoxicity was evaluated by measuring tubular enzymuria and by light microscopy and serum CsA levels (parent drug plus certain metabolites) were determined by radioimmunoassay. The hepatic microsomal mono-oxygenase enzyme system was monitored by measurement of cytochrome P-450, aminopyrine N-demethylase and NADPH-cytochrome c reductase. Nephrotoxicity appeared within 4 days of starting treatment and continued for 4 weeks. Between weeks 4 and 6 there was a period of complete remission followed by the return of renal damage. Aminopyrine N-demethylase activity fell during the first 4 weeks. During the period of remission, however, N-demethylase activity rose to a point significantly higher than pretreatment values and serum CsA levels fell to their lowest concentration. With relapse, hepatic N-demethylase activity again fell below normal and serum drug levels rose to their pre-remission values. From the third week onward, changes in NADPH-cytochrome c reductase activity paralleled those in N-demethylase activity. The hepatic microsomal concentration of cytochrome P-450 did not, however, change significantly during the 7-week period of CsA treatment. Our results suggest that the spontaneous remission of CsA-induced nephrotoxicity is due to a reduction in circulating drug levels caused by increased hepatic CsA metabolism.

The aim of this study was to determine the effect of altered hepatic drug metabolism on the nephrotoxic and immunosuppressive properties of cyclosporin A (CsA) in the rat. From a consideration of the structures of those CsA metabolites identified so far, it seemed probable that the metabolism of CsA would occur at the hepatic cytochrome P-450 (cyt P-450) enzyme system. CsA (50 mg/kg/24 hr) administered orally for 14 days resulted in significant increases in both serum urea concentration and urinary N-acetyl-beta-D-glucosaminidase activity, accompanied by renal proximal tubular vacuolation. The concomitant administration of either Aroclor 1254 (25 mg/kg/24 hr, i.p.) or phenobarbitone (PB) (40 mg/kg/24 hr, i.p.) but not 3-methylcholanthrene (3-MC) (15 mg/kg/72 hr, i.p.) resulted in abolition of the nephrotoxicity, assessed both biochemically and histologically, whilst the suppressive effect on the humoral response to SRBC was unaltered. Phenobarbitone also significantly decreased serum CsA concentrations. These results suggest that the PB-inducible set of cyt P-450 isoenzymes may be responsible or partly responsible for hepatic CsA metabolism.

Many studies involving large numbers of patients prove the efficacy of cyclosporine to accomplish immunosuppression following hetertopic organ transplant. In long-term follow-up, cyclosporine produces a higher level of nephropathy than does conventional immunosuppression consisting of azathioprine and prednisone. The degree of nephropathy appears to be related to blood concentration and the effect can be minimized by maintaining therapeutic trough blood concentrations. Other significant side effects (central nervous system toxicity and hirsutism) can also be minimized by low blood concentrations. Development of lymphoma secondary to Epstein-Barr virus exposure is unrelated to blood concentration. Two methods are available for therapeutic drug monitoring: radioimmunoassay (RIA) and high pressure liquid chromatography (HPLC). RIA on plasma is a standard, rapid means of obtaining a result, but that result is inaccurate due to metabolite cross-reactivity. The concentration of cyclosporine in plasma is widely variable and unrepresentative of the whole blood concentration. Plasma concentration is dependent upon the temperature of plasma separation. Whole blood analysis avoids this problem. HPLC procedures allow for whole blood analysis but are tedious and time-consuming. We present here a simple, accurate HPLC procedure that is reproducible (CV = 4.9%), sensitive (to 50 ng/mL), and fast (preparation time - 5.7 minutes, chromatography time - 20 minutes). This procedure correlates (r = 0.98) with a reference HPLC procedure and has been used in our clinical laboratory for analysis of more than 4000 specimens without apparent problem. No interferences have been identified.

In a study of three groups of high-risk keratoplasty patients, Group A (n = 19) received topical corticosteroids only; two grafts (10.5%) survived. Group B (n = 20) received both topical and systemic corticosteroids; one graft (5%) survived. There was no significant difference between Group A and Group B (P = .8095). Group C (n = 18) received systemic cyclosporine in addition to topical and systemic corticosteroids; 16 grafts (88.9%) survived, which was a significant improvement compared to results in the other two groups (P = .0001). No patient suffered any permanent side effects related to the use of cyclosporine.