Plasma Cyclosporine Research Articles

Abbott's fluorescence polarization immunoassay for cyclosporine and metabolites compared with the Sandoz "Sandimmune" RIA.

A new procedure for measuring cyclosporine in plasma has been introduced by Abbott Laboratories, involving their TDx instrumentation and fluorescence polarization immunoassay. Radioimmunoassay (RIA) and high-performance liquid chromatography are currently the conventional methods for measuring cyclosporine in plasma and whole blood. In an effort to find a method that will decrease the radioactive hazard, the reagent and supply cost, and the labor requirements associated with RIA procedures, we used specimens from transplantation patients to compare the Abbott assay with the Sandoz Sandimmune assay. We believe that the Abbott assay offers some advantages over the Sandimmune RIA procedure, providing a reliable but simpler and less hazardous technology.

Effect of erythromycin on blood cyclosporine concentrations in kidney transplant patients

Patient 1. A 10-year-old boy with renal failure caused by dysgenesis of a congenital single kidney underwent cadaveric renal transplantation in 1984. Immunosuppression was achieved with prednisone and cyclosporine. Plasma concentrations of cyclosporine over the period of 3 years ranged between 70 and 130 ng/ml. Serum creatinine levels over the last months were 4.2 to 4.4 mg/dI (370 to 390 #tool/L). Thirty-six months after transplantation, interstitial pneumonia developed and the patient was given erythromycin in a dose of 40 mg/kg for 10 days. Over 14 days the plasma cyclosporine levels rose from 73 to 373 ng/ml and returned to a level of less than 30 ng/ml within 10 days after erythromycin was discontinued. At the same time, serum creatinine values rose from 4.4 mg/dl (520 t.~mol/L), returning to 4.2 mg/dl after the erythromycin was discontinued. Patient 2. A 10-year-old girl with renal failure secondary to Wegener granulomatosis underwent cadaveric renal transplantation in 1986. Immunosuppression was achieved with prednisone and cyclosporine. After the renal transplant, her serum creatinine levels ranged from 0.9 to 1.2 mg/dl (80 to 106 ~mol/L) and the plasma cyclosporine concentration from 30 to 45 ng/ml. Four months after transplantation, pneumonia developed and erythromycin, 50 mg/kg daily, and co-trimoxazole, 40/8 mg/kg daily, were prescribed. Five days later, facial edema and hypertension

The influence of haematocrit on blood cyclosporin measurements in vivo.

The influence of haematocrit on blood cyclosporin measurements has been studied in 276 paired blood and plasma samples from 21 renal transplant patients. A highly significant correlation was found between blood and plasma cyclosporin concentrations, r = 0.8744, but the correlation between blood or plasma cyclosporin and haematocrit was not significant. The ratio of blood/plasma cyclosporin did not significantly increase with increasing haematocrit. It was concluded that in vivo the influence of haematocrit on the measurement of blood cyclosporin concentrations was negligible.

Cyclosporine absorption in apancreatic dogs

The plasma concentration time course of orally administered cyclosporine was studied in apancreatic dogs with established islet autografts and compared with that in normal control dogs. After oral administration of cyclosporine (20 mg/kg), blood samples were collected at 0, 1, 2, 3, 4, 6, 8, and 24 hours, and the plasma cyclosporine concentrations were measured by radioimmunoassay. The plasma level of cyclosporine increased promptly in both groups after dosing. Peak plasma concentrations ranged from 435 to 1,542 ng/ml and were attained at between 2 and 6 hours in the apancreatic dogs, and concentrations from 602 to 4,414 ng/ml were attained at between 1 and 6 hours in the control dogs. Even though there was substantial variation among animals, the plasma concentration curve of the apancreatic group was quite comparable with that of the control group. The Student's test for unpaired data failed to show any significant differences over the time course. Area under the concentration-time curves, maximum concentration, and times of peak concentration were calculated. These data demonstrated the capacity of apancreatic dogs with islet autografts to absorb cyclosporine when they are stable, nutritionally normal, and have full endocrine reconstitution.

Substitution of 125I-labeled cyclosporine in the radioimmunoassay of plasma cyclosporine.

Substitution of 125I-labeled cyclosporine in the radioimmunoassay of plasma cyclosporine.

Cyclosporine plasma levels six hours after oral administration. A useful tool for monitoring therapy.

Clinical evolution and cyclosporine (CsA) monitoring of 65 transplanted patients (55 kidneys, and 10 kidneys and pancreases) treated with CsA were analyzed retrospectively (45 patients) and prospectively (34 patients). Our results showed the following: (1) nephrotoxicity is not uncommon even with low trough plasma levels of CsA; (2) the T6 value of a CsA pharmacokinetic plasma curve (6 hr after oral drug administration) is a valid expression of a full pharmacokinetic study; (3) when T6 was used prospectively as a monitoring tool and dose adjustments made disregarding concomitant serum creatinine levels, the latter decreased when CsA dose adjustments were made to correct toxic (greater than 350 ng/ml) or subtherapeutic (less than 100 ng/ml) T6, P less than 0.01. At present, serum creatinine for all our patients is 180.2 +/- 8 mumol/L, and no patient has needed to be switched to conventional treatment. The validity of trough plasma levels in patients under CsA oral administration once or twice a day seems questionable, and T6 proved to be more useful. Thus nephrotoxicity and CsA undertreatment may be avoided. This new monitoring tool (T6) will allow the utilization of lower doses of CsA and thus contribute to improved long-term graft function.

Is There a Therapeutic Range for Monitoring Plasma Cyclosporin in Renal Allograft Recipients?

Sixty-two consecutive adult cadaveric renal allograft recipients have been monitored by high-performance liquid chromatography estimations of trough plasma cyclosporin (CsA) concentrations within 30 days of transplantation. The CsA estimations during graft rejection (mean + 1 SD 218.6 + 126.4 ng/ml) were not significantly different from those obtained during stable renal function (232.3 + 172.8 ng/ml), whereas the estimations during episodes of nephrotoxicity (476.0 + 267.3 ng/ml) were significantly greater (P less than 0.001). During stable renal function 73.7% of estimations were within the therapeutic range of 100-350 ng/ml. However, 37.9% of estimations during nephrotoxicity and 76.7% of estimations during rejection episodes were also within this range. The therapeutic window for trough plasma CsA estimations is not clearly defined. It seems that rejection episodes may occur at apparently adequate CsA concentrations, and although most nephrotoxic episodes are associated with elevated concentrations, acute nephrotoxicity may occur at apparently therapeutic values. However, in this study, some patients at risk of nephrotoxicity within 30 days of transplantation were identified as early as 7 days post-transplantation by the simple determination of the mean trough CsA concentration for days 1-7.

Blood protein binding of cyclosporine in transplant patients.

The objective of this study was to compare the binding of cyclosporine to blood proteins between four healthy subjects and five liver and eight renal transplant patients. Fresh heparinized blood was obtained, to which sufficient quantities of tritium-labelled cyclosporine and unlabelled cyclosporine were added to blood samples or red blood cell (RBC) suspensions. Concentrations of cyclosporine in whole blood, plasma, RBC suspension, and phosphate buffer were estimated by liquid scintigraphy. The blood:plasma ratio of cyclosporine in transplant patients was significantly lower (P less than .05) than that in healthy volunteers. The RBC:buffer ratio, a measure of affinity of RBCs for cyclosporine, was highest in those with liver transplants and lowest in those with kidney transplants. The unbound fraction of cyclosporine in plasma was less in transplant patients than in healthy volunteers. The results of this study indicate that there are differences in blood protein binding of cyclosporine between transplant patients that may contribute to the differences in the pharmacokinetics and pharmacodynamics of this drug.

Renal Interstitial Fibrosis and Vascular Changes

Histologic examinations of kidney biopsy specimens from six patients with relapsing polychondritis (n = 1), Behçet's syndrome (n = 3), and chronic uveitis (n = 2) were performed after four to 36 months of treatment with cyclosporine. Five of the patients had a variable degree of focal interstitial fibrosis and tubular atrophy, with and without minimal interstitial inflammation. Arteriolar hyalinization was noted in four and glomerular sclerosis in two patients. These renal lesions could not be attributed to underlying autoimmune disease or previous drug therapy but were similar to those recently reported in kidney and heart recipients receiving long-term cyclosporine. The initial cyclosporine doses were 15 mg/kg body weight in one patient and 10 mg/kg in the others. The maintenance cyclosporine doses ranged from 2.5 to 7.5 mg/kg with appropriate trough cyclosporine plasma levels (60 to 130 ng/mL). A rough correspondence between the extent of the histologic renal changes and the cumulative cyclosporine was seen, whereas serum creatinine increase or the development of hypertension during treatment did not predict the degree of interstitial fibrosis or the presence of arteriolar changes. Neither did a rapid fall in the serum creatinine level after withdrawal of cyclosporine exclude focal irreversible renal lesions. Since the histopathologic changes found in the kidneys are potentially progressive, we believe that, until more is known, long-term cyclosporine treatment should be reserved for situations where more established immunosuppression has failed to control an autoimmune process threatening the function of vital organs.

Cyclosporine: A review of drug monitoring problems and presentation of a simple, accurate liquid chromatographic procedure that solves these problems

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.

The Plasma Time Course of Orally-Administered Cyclosporine in the Dog

The plasma concentration time course of orally-administered cyclosporine has been studied in five adult male mongrel dogs. Cyclosporine concentrations were measured by high performance liquid chromatography with ultraviolet absorbance detection. Cyclosporine plasma concentrations were below assay sensitivity within 24 hours after administration. Peak plasma concentrations ranging from 411.8 to 1735.0 ng/ml were attained between post-administration times of 1 and 2.5 hours. Areas under plasma concentration time course curves (AUC = 4.63 +/- 5.60 micrograms/ml X hr, mean +/- SD) suggest that systemic availability is highly variable between animals. Present and previous findings discourage the oral route of cyclosporine administration for canine transplant research and suggest more extensive pharmacokinetic evaluation of cyclosporine following oral administration in humans.

Small intestinal transplantation

The purpose of this study was to determine whether small intestinal transplantation could be considered as an alternative in the treatment of patients suffering from the short-bowel syndrome. The site of absorption of oral cyclosporine A was determined as were the changes that follow small intestinal transplantation. The interactions between the lipophilic cyclosporine A molecule and fat emulsion solutions used for total parenteral nutrition were investigated. Finally, a technique for harvesting the entire small bowel in man was developed. The absorption of oral cyclosporine A in normal dogs, and in bowel-resected, autotransplanted, and allotransplanted dogs was determined. Cyclosporine A levels were monitored in all animals. This demonstrated that cyclosporine A is absorbed through the small bowel and carried through the lymphatics; that absorption is decreased to 40 percent of normal after autotransplantation or allotransplantation without rejection. Rejection further hampers cyclosporine A absorption. Administration of olive oil alone enhances absorption of cyclosporine A. We also administered cyclosporine A IV to five dogs, with and without a concomitant infusion of fat emulsion solution (Intralipid). No changes in plasma cyclosporine A levels, in the clearance of cyclosporine A, or in the in vivo distribution of cyclosporine A were noted. Finally, dissections in six cadavers and in four brain-dead organ donors were performed, and a reproducible technique for harvesting the small bowel in man was established. In selected patients with the short-bowel syndrome, small intestinal transplant may be considered as an alternative therapy to home total parenteral nutrition.

Solid-phase extraction and liquid chromatography for improved assay of cyclosporine in whole blood or plasma.

In this simple, precise, accurate, and specific isocratic liquid chromatographic procedure for determining cyclosporine, the cyclosporine is extracted from 1 mL of whole blood or from plasma, with 500 micrograms of cyclosporin D added per liter as internal standard, by elution from a Bond-ElutTM C18 extraction column with 300 microL of a mixture of ethanol and tetrahydrofuran. A 100-microL aliquot of the eluate, injected onto a cyano-phase analytical column, is eluted with a mixture of acetonitrile and pH 7.0 phosphate buffer at a flow rate of 1.0 mL/min and at 50 degrees C. Detection is at 210 nm. The chromatography is complete in less than 14.0 min. The method can measure less than 10.0 micrograms/L. Analytical recovery of cyclosporine added to whole blood ranged from 99 to 109% for concentrations up to 2000 micrograms/L. Between-run CVs ranged from 6.4 to 6.6%. None of numerous drugs and steroids tested interfered. Results by radioimmunoassay exceeded by 20 to 350% those measured by the present method.

Effect of hematocrit on cyclosporine (cyclosporin A) in whole blood and plasma of renal-transplant patients.

Cyclosporine concentrations in whole blood and plasma were determined in 25 samples from renal-transplant patients. The portion of cyclosporine carried in plasma varied widely, from 9 to 58%. Radioimmunoassay and liquid chromatography both revealed a statistically significant inverse correlation between hematocrit and the plasma fraction of cyclosporine in the circulating blood. Regression analysis indicates that a 10% increase in hematocrit would decrease the portion of cyclosporine in plasma by 12 to 14%. Studies with cyclosporine-supplemented samples further demonstrated an effect of hematocrit on the drug concentration in plasma without a corresponding effect on the concentration in whole blood. The distribution of cyclosporine between plasma and erythrocytes was unaffected by plasma cholesterol and triglycerides for these transplant patients.

Effects of sample preparation on concentrations of cyclosporin A measured in plasma.

Because cyclosporin A rapidly changes its distribution in blood with changes in temperature, sample preparation affects results for it as measured in plasma. If whole blood is stored at either 4 degrees C or room temperature, results for cyclosporin A in the plasma are lower than in whole blood stored at 37 degrees C and centrifuged at this temperature. Re-equilibration of the former to 37 degrees C before cells are removed increases the analytical recovery of cyclosporin A in plasma; the optimal equilibration interval is 30 min. Use of such re-equilibration, followed by immediate centrifugation at room temperature, increases values obtained for cyclosporin in plasma by 60 to 65% over those determined after non-temperature-standardized collection procedures, but does not significantly improve the correlation between values for plasma and whole blood. Hematocrit and concentrations of cyclosporin A in plasma are inversely related. Correction for hematocrit improves the correlation between results for plasma and whole blood.

On the Measurement of Cyclosporine in Plasma

Departments of Clinical Pharmacology and Haematology St. Vincent's Hospital Darlinghurst, Australia

Semi-automated high-performance liquid chromatographic method for the determination of cyclosporine in plasma and blood using column switching

A sensitive, semi-automated high-performance liquid chromatographic method utilizing column switching is described for the determination of cyclosporine in plasma and blood. This method involves a short and improved manual protein precipitation of the sample followed by an automated clean-up of the supernatant. After automatic loading of the clean supernatant onto an LC-8 column for initial separation, the segment containing cyclosporine is loaded (automatically) onto an LC-18 column for final separation and quantitation. Cyclosporine is detected by its ultraviolet absorption at 202 nm. The rate of analysis was four samples per h running 24 h per day (ca. 100 samples per day). The method is sensitive enough to measure with confidence cyclosporine concentrations of 8 μg/l in plasma and 20 μg/l in blood with a linear response up to 2500 μg/l using only 0.5 ml of sample. No internal standard is required. The method was applied continuously (24 h per day) to approximately 1000 samples without deterioration in method parameters.