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We synthesized an 125I-labeled-histamine-cyclosporin C tracer, to obviate the use of tritiated tracer in radioimmunoassay of cyclosporine. With this tracer, the assay results varied linearly with concentration up to at least 800 micrograms/L. The within-assay CV was 6.6% at 39 micrograms/L, 4.2% at 100 micrograms/L, and 7.0% at 300 micrograms/L (n = 15). The between-assay CV was 10.0, 6.4, and 7.8% for the same respective concentrations. Comparison with an assay involving tritiated tracer (x) showed good agreement of results: y = 3.81 + 0.927x (r = 0.975, n = 604). Analytical recovery ranged from 100 to 106%. We also compared another commercially available radioiodinated tracer ("125Iodocyclosporin"; Immunonuclear Corp.). Our tracer appeared to be more specific for cyclosporine, as determined by assaying chromatographic fractions of bile extract from a patient being treated with cyclosporine. Results with use of our tracer compared favorably with those obtained with the tritiated tracer, and our assay has the advantages of gamma counting vs liquid-scintillation counting.

The influence of Cyclosporine A (CyA) on the development of renal injury in in situ immune complex glomerulonephritis (ICGN) in rats was examined. Left kidneys of male Wistar rats were perfused with cationized human IgG followed by an intravenous injection of rabbit anti-human IgG. CyA was given orally in a dosage of either 2.5, 15 or 25 mg/kg body weight per day in two daily injections. The amount of immune complexes deposited in the left kidney was not affected by CyA application as shown by isotopic studies. There were no differences between the control and CyA groups in respect to immunofluorescence, light and electron microscopy. Proteinuria was significantly inhibited with all treatment schedules, even when rats were first treated five days after inducing nephritis. These results show that CyA inhibits proteinuria without affecting glomerular cell accumulation/proliferation in in situ ICGN, and indicates that cellular activation at glomerular level might be prevented by CyA.

Since 1981, at the University of Minnesota, and more recently at Washington University in St. Louis, cyclosporine has been used as the main immunosuppressive agent for heart transplantation. It was initially combined with prednisone and given in a manner similar to that described at Stanford. In late 1983, concern regarding the nephrotoxic side effects of cyclosporine were heightened due to the fact that a potential recipient had chronic renal insufficiency secondary to renal damage suffered during previous heart surgery. In this patient it was decided to use lower doses of cyclosporine and to add azathioprine to maintain adequate immunosuppression. Initially, the same prednisone therapy was employed. This patient had an uncomplicated course following heart transplantation and was discharged with a normal renal function. This experience was the origin of a trial consisting of using cyclosporine, azathioprine, and prednisone as immunotherapy for heart transplantation. This report describes the results of this therapy in 17 patients.

A fully automated high-performance liquid chromatographic column-switching system is presented for the determination of cyclosporin A in whole blood. After blood proteins were precipitated with acetonitrile, the supernatant was automatically loaded on to a cyanopropyl column for initial separation, and then the fraction containing cyclosporin A was loaded on to a trimethylsilica column for final separation and quantitation. Cyclosporin A was detected by ultraviolet absorption at 205 nm. The minimum detectable concentration of cyclosporin A was 5 ng/ml in 100 microliter of blood. The coefficient of variation of the method was 1.755, 1.748 and 0.655% in whole blood when spiked at the 170, 425 and 850 ng/ml levels, respectively. One assay was completed in 15 min.

It is reported on the blood level-oriented oral long-term therapy with cyclosporin-A (Sandimmun) in 20 patients after kidney transplantation. The Cy-A-concentrations were measured in the whole blood by means of a radioimmunoassay (Sandoz) under steady state conditions. The mathematical analysis of the steady state daily minimum concentrations in the whole blood depending upon the dosage rate allows individual calculations of the dosage for the stabilisation of the therapeutic concentration desirable for the individual patient in each case. In the majority of the patients a non-linear relation between the steady state blood concentration and the dosage rate was present. The observation of a defined therapeutic area under the conditions of the ambulatory treatment could be achieved without any problems. The control intervals were 4-6 weeks. The long-term stability of the individual pharmacokinetic parameters can at present not be judged reliably, since the average observation time is still too short. In 6 patients who are controlled on the way demonstrated already for several months no systemic changes in the behaviour of the blood level were to be seen.

18 renal transplant recipients receiving cyclosporin, prednisone, and azathioprine were given ketoconazole, a potent inhibitor of the cytochrome P-450 enzyme system. Within a month ketoconazole-induced blockade of cyclosporin metabolism allowed a significant reduction (451 vs 106 mg/day; 77%) of the mean dose of cyclosporin without altering cyclosporin whole blood trough levels, although maximum blood levels were almost halved. This dose reduction was maintained in patients followed up for up to 13 months. Renal and hepatic function were unchanged after the addition of ketoconazole. This drug interaction has the potential to reduce dramatically expenditure on cyclosporin in transplant recipients.

The new Abbott TDx cyclosporine and metabolites fluorescent polarization immunoassay procedure provides a 20-min sample turn-around time, using 50 microliters of sample for the analysis of cyclosporine in whole blood. A precipitation agent and a lysing agent are utilized as a pretreatment step. The range of the whole blood assay is from 0 to 2,000 ng/ml, with a sensitivity of 50 ng/ml. Precision studies at 3 control levels provided coefficients of variation of 2.1-4.8% for both assays. In order to compare this assay with the currently used Sandoz polyclonal radioimmunoassay (RIA) method. 200 whole blood samples were obtained from 20 renal, cardiac, and hepatic transplant recipients. The mean whole blood cyclosporine concentrations for samples above the sensitivity level were as follows: TDx 754 ng/ml (+/- 31) and RIA 619 ng/ml (+/- 22). Blood TDx levels correlated strongly with RIA levels, with a regression coefficient of r = 0.915. This new assay provides reliable blood cyclosporine concentrations that correlate well with RIA measurements. This assay offers rapid sample turn-around times, making same-day results for outpatient drug monitoring possible.

Cyclosporine (CSA) is biotransformed to many metabolites which may contribute to its immunosuppressive and nephrotoxic activity. We report a rapid and sensitive, automated column-switching high-performance liquid chromatographic (HPLC) method for measuring CSA-M17 in whole blood; the method also separates CSA-M1. CSA metabolite standards were isolated by a preparative-scale HPLC method. Samples were prepared by protein precipitation with acetonitrile followed by dilution with water. CSA-M17 was initially separated on a C8 column; final separation was on a C18 column. The inter-day relative standard deviation at 50 ng/ml was 8% (n = 3). Limit of detection was 20 ng/ml.

Although cyclosporine (CsA) displays high immunosuppressive efficacy due to potent selective inhibition of T cell, but not nonspecific, immune functions, the pleiotropic toxicities of the drug result in a low therapeutic index. Thus for a given individual there is at best only a narrow dosage range producing immunosuppression not beclouded by toxicity. Selection of the appropriate CsA dose to achieve this state is complicated by marked inter- and intraindividual variability in drug pharmacokinetics and pharmacodynamics (1). Even considering renal transplant recipients solely, pharmacokinetic variations in drug absorption, volume of distribution, and metabolism as estimated by clearance rates are so great that strategies based on median population values are not useful for a great proportion of patients. Thus it is necessary to devise a CsA strategy that tailors therapy to compensate for interindividual variations. Implementation of such a strategy not only standardizes drug therapy, but also reveals the clinical impact of interindividual differences in the profile of CsA metabolites and in pharmacodynamic effects of a given quantity of CsA, reflecting both the therapeutic actions on the immune system and toxic effects on target organs. Thus a dosing strategy that achieves uniform drug levels by compensating for pharmacokinetic variation is essential for the eventual dissection of a rational CsA regimen.

In order to evaluate the antihypertensive effectiveness and interaction with ciclosporin A (CS-A) nitrendipine, a dihydropyridine derivative calcium entry blocking agent, was used in 16 (13 men, 3 women) hypertensive renal posttransplant patients followed by the Nephrology Department of Hacettepe University Hospital. The patients did not receive any antihypertensive drug for a 7-day period. They were then given 20 mg/day nitrendipine for 3 weeks. At the end of this period, mean (+/- SE) supine blood pressure fell from 163/108 +/- 3.6/1.87 to 141/87 +/- 3.8/2.2 mm Hg (p less than 0.01), while the heart rate was unchanged. 14 of 16 patients achieved full control of blood pressure levels with 20 mg/day nitrendipine, and only 2 patients needed a higher dosage of 30 mg/day (20 + 10 mg). After 3 weeks of treatment no significant variations in blood chemistry or renal functional parameters were noticed. There was also no difference between blood CS-A levels before and after treatment with nitrendipine (218.06 +/- 33 vs. 222.68 +/- 26 ng/ml, p greater than 0.05). We conclude that short-term therapy with nitrendipine in renal post-transplant patients does not appear to be harmful and longer term studies are needed to fully evaluate safety and efficacy of this drug. Because it influences neither blood chemistry nor renal functional parameters and blood CS-A level, it may be preferable to other calcium channel blocking agents in this group of patients.

The method of measurement of cyclosporine concentrations in renal transplant recipients varies between centers and employs either high-performance liquid chromatography (HPLC) or radioimmunoassay (RIA). The merit of using HPLC for identifying the parent compound versus the RIA technique, which also measures certain cross-reactive metabolites that accumulate during renal impairment, is controversial. As a result of the lack of uniformity among centers, an abundance of complex literature that describes the disposition of this potent immunosuppressive agent, as well as a wide range of guidelines for therapeutic monitoring, has evolved. To examine the influence of assay methodology on the repeated determination of cyclosporine in the immediate postoperative period, a time when renal function is often unstable, eight renal transplant recipients were studied after i.v. and oral administration on up to four separate occasions. Whole-blood samples were analyzed by HPLC and RIA. Intravenous kinetic analysis yielded a mean total body clearance of 0.24 +/- 0.2 L/min (RIA) and 0.31 +/- 0.1 L/min (HPLC) (p greater than 0.05), the mean volume of distribution was 2.17 +/- 0.6 L/kg (RIA) and 2.75 +/- 1.2 L/kg (HPLC) (p greater than 0.05), and a mean half-life was 11.7 +/- 4.4 h (RIA) and 12.8 +/- 3.8 h (HPLC) (p greater than 0.05). The mean bioavailability was 0.36 +/- 0.23 (RIA) and 0.28 +/- 0.15 (HPLC) (p greater than 0.05). Regression of the 12-h cyclosporine (RIA versus HPLC) concentration yielded a line described by the following equation: RIA = 72 + 1.6 (HPLC).(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of continuous immunosuppression on the immune system of 2 neonates born to heart-lung transplant recipients was analyzed. T cells, T cell subpopulations, B cells and cyclosporine levels were measured in maternal and cord blood at the time of delivery. Cyclosporine, both parent compound, and metabolites were found in cord blood but in lower amounts than that in the mother. This discrepancy was more pronounced for parent compound than metabolites. Infants born to immunosuppressed mothers had more T and B cells than the mother and had similar numbers of T and B cells to that found in cord blood from 10 babies born to nonimmunosuppressed mothers. One infant had fewer activated T cells and a higher CD4/CD8 ratio due to increased numbers of CD4 cells than normal. In conclusion, neither of these babies had a lymphocyte profile suggestive of chronic immunosuppression.

Alternative combined immunosuppressive therapy was tested in canine orthotopic bowel transplantation. Despite sporadic long-term survival, cyclosporine is still questionably effective. Triple-drug therapy (cyclosporine, azathioprine, and prednisone) combined with antilymphocyte serum or with a short segment graft was effective in reducing the early postoperative mortality due to acute rejection but did not alter the long-term survival rate. There was no apparent relationship between the serum cyclosporine levels and survival. The long-term survivors (longer than 100 days) maintained relatively low serum trough levels of cyclosporine. These suggest that orthotopic bowel transplantation in the dog, and probably in the human as well, requires improved immunosuppressive regimens.

Cyclosporine is an immunosuppressant used to prevent the rejection of transplanted kidneys, hearts, and livers. Cyclosporine suppresses T-lymphocyte function without causing myelosuppression, and its pharmacokinetics are highly variable. Compared with conventional immunosuppressive drug therapy, both patient and graft survival improved in patients treated with cyclosporine. Patients treated with cyclosporine also had less complicated hospital courses than patients receiving conventional immunosuppressants. The adverse effects from cyclosporine are reversible but include nephrotoxicity, hepatotoxicity, malignancies, hirsutism, and minor neurologic complications. Intravenous cyclosporine doses range from 2-9 mg/kg/d and oral doses range from 10-50 mg/kg/d; the dosage should be individualized based on the clinical status of the patient as well as blood concentrations of the drug. Monitoring cyclosporine blood concentrations is necessary in the postoperative management of transplant patients. Cyclosporine has contributed to the improved success of transplantation and the recognition of transplantation as a new therapeutic option for several diseases.

Ciamexon is a new immunomodulating agent with promising effects in treatment of newly-diagnosed insulin dependent diabetic patients. Ciamexon seems to have fewer adverse toxic properties than Cyclosporin A. Therefore, the effects of both substances on islet cell function were studied. Collagenase-isolated mouse islets were cultured free-floating for 3 or 7 days in medium RPMI 1640 plus 10% fetal calf serum in the presence of Cyclosporin A or Ciamexon (0.1 microgram/ml, 1 microgram/ml). Culture of the islets in the presence of 0.1 microgram/ml Ciamexon neither reduced the number of viable islets nor impaired insulin secretion as was found in Cyclosporin A. These results should be taken into account when treating early Type 1 diabetes or when transplanting islet cells.

The metabolic and immunological effects of cyclosporin given to prevent diabetes in BB rats were examined. Diabetes-prone (BBdp) and normal (BBn) BB rats received either oral cyclosporin (10 mg X kg-1 X day-1 or its vehicle from age 30-150 days. Six of 21 (29%) vehicle-treated rats became glycosuric, with hyperglycemia, weight loss, and unremitting insulin requirements, and showed destruction of islet beta-cells. Five of 24 (21%) cyclosporin-treated rats became glycosuric, but none demonstrated weight loss, all required insulin only intermittently after onset, and all showed persistence of islet beta-cells. Cyclosporin induced hypoinsulinemic glucose intolerance in BBn rats. Cyclosporin inhibited the normal rise with age of peripheral blood lymphocyte cell numbers, identified with monoclonal antibodies. OX19+ (pan-T) and W3/25+ helper T-lymphocytes were affected, and there was an increase in the large W3/13+ OX19- population characteristic of BBdp rats; in addition, this subset appeared in BBn rats. Cyclosporin also caused the appearance and/or increase in both BBdp and BBn rats of W3/25+ OX19- and OX8+ OX19- subsets. Suppressor/cytotoxic (OX8+) T-lymphocytes and Ia+ cells were less affected. The incidence of hyperglycemia and glycosuria was therefore unaltered by cyclosporin, although the diabetic syndrome was milder. BBn rats receiving cyclosporin showed glucose intolerance, suggesting that in BBdp rats, the net effects of immunosuppression on beta-cell destruction may have been counterbalanced by the direct effect on the same cells. The attenuation of diabetes in BBdp rats occurred through further immunosuppression rather than by correction of its preexisting immunodeficiency.

H2-receptor antagonists, such as cimetidine (C), ranitidine (R) and famotidine (F) seem to be effective in the prevention and treatment of stress ulcer in transplant recipients receiving cyclosporin A (CyA). The aim of this study was to detect the possible synergistic nephro- and hepato-toxicity of these drugs, assaying the serum creatinine (SC), ALT, AST levels, and the histological features of 45 young male Sprague-Dawley rats, divided into nine groups of five rats each. After 10 days of treatment the results showed: (i) serum CyA levels were increased in the group receiving daily CyA (5 mg/kg) + R(5 mg/kg) (2430 +/- 403 ng/ml; p less than 0.05 vs. controls) and in the group receiving daily CyA (5 mg/kg) +/- C (10 mg/kg) (2440 +/- 265 ng/ml; p less than 0.01 vs. controls); (ii) ALT and AST levels were increased in this latter group (ALT 223 +/- 133 UL, AST 114.67 +/- 39 UL; p less than 0.01 vs. controls); (iii) SC levels were normal; and (iv) steatosis of the liver was observed in these two groups. These findings suggest that C and R, but not F, may inhibit the hepatic cytochromes P-450 which are involved in the oxidative metabolism of the drugs. Furthermore, the high serum CyA levels seem to play a major role in the appearance of biochemical and histological damage to the liver.

In view of the reported increase in thromboembolic episodes following cyclosporine A (CyA) therapy, the effect of this drug on platelet aggregation and thromboxane A2 release was investigated. The addition of CyA, at therapeutic concentrations to platelet rich plasma from normal subjects in vitro was found to increase aggregation in response to adrenaline, collagen and ADP. Ingestion of CyA by healthy volunteers was also associated with enhanced platelet aggregation. The CyA-mediated enhancement of aggregation was further enhanced by the addition in vitro of therapeutic concentrations of heparin. Platelets from renal allograft recipients treated with CyA also showed hyperaggregability and increased thromboxane A2 release, which were most marked at "peak" plasma CyA concentration and less so at "trough" concentrations. Platelet hyperaggregability in renal allograft patients on long-term CyA therapy tended to revert towards normal following the replacement of CyA with azathioprine. Hypertensive patients with renal allografts on nifedipine therapy had normal platelet function and thromboxane release in spite of CyA therapy. These observations suggest that CyA-mediated platelet activation may contribute to the pathogenesis of the thromboembolic phenomena associated with the use of this drug. The increased release of thromboxane A2 (a vasoconstrictor) may also play a role in mediating CyA-related nephrotoxicity.

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.

We have analyzed and compared the cyclosporine concentrations in whole blood specimens from heart and kidney transplant patients, using three different methods: high-performance liquid chromatography (HPLC), polyclonal radioimmunoassay, and monoclonal radioimmunoassay. HPLC results for heart transplant patients correlated well with the monoclonal (specific antibody) results but did not correlate with the polyclonal (nonspecific antibody) results. All three methods correlated well for renal transplant patients; however, polyclonal results were consistently higher than either monoclonal or HPLC results. We conclude that the results from the monoclonal (specific antibody) RIA are equivalent to HPLC results for both kidney and heart transplant patients.

The ability of a new cyclosporine (Cs) derivative, (Nva2)-Cs (CsG), to suppress rejection of lung and heart allografts in rats was determined and compared with that of CsA. Left lungs were transplanted orthotopically; hearts were transplanted heterotopically into the abdomen. (Nva2)-Cs was used in three experimental protocols: (1) single or three (Nva2)-Cs injections given to lung-transplanted rats, (2) daily oral (Nva2)-Cs treatment at different doses compared with similar CsA treatments in heart allografted rats, and (3) An 11-day (Nva2)-Cs treatment starting at increasing intervals after transplantation of hearts. (Nva2)-Cs was found to be immunosuppressive, and effective even when the treatment started as late as four days after transplantation. However, (Nva2)-Cs was less effective than CsA in suppressing rejection of lung and heart allografts at low doses. Because (Nva2)-Cs is possibly not nephrotoxic, it might be a useful drug if used in higher doses than CsA or in combination with other immunosuppressive agents.

Cs pharmacokinetic profiles using HPLC have aided in predicting necessary dosage alterations for specific groups of transplant patients. Additional information has been gained by HPLC profiles in nontransplant subjects who are healthy or have a stable disease state. The clinician now knows that liver disease not only impairs Cs elimination but may also have a pronounced effect upon drug absorption. While the cardiac failure patient may have reversible inhibition of Cs clearance, other factors may affect the distribution of the drug to lower dosage requirements. Impaired renal function is not an impediment to Cs elimination, but malabsorption similar to that observed in liver and bone marrow transplant patients may still occasionally complicate therapy. Pharmacokinetic information on Cs must be integrated into the complex care plan of a transplant patient to optimally utilize and monitor this pharmacologic agent.

The initiation of a liver transplant program in a pediatric hospital prompted the development of a high-performance liquid chromatography method for monitoring cyclosporine, which requires minimal amounts of blood and produces fast, reliable results. A protein-free filtrate is prepared by mixing 250 microliters of whole blood with 750 microliters of organic solvent (acetonitrile and methanol in the ratio of 9:1) containing the internal standard cyclosporine D. After centrifugation the supernatant is subjected to a quick clean-up using two types of disposable cartridges (C18 and silica Bond Elut), and the eluate is dried, reconstituted in equal parts of acetonitrile and water, and subjected to a final heptane wash to remove late eluting peaks. Chromatography is performed at 75 degrees C using a supelcosil C18 column (15 cm X 4.6 mm) with 3 microns packing and a mobile phase of 77/23 of acetonitrile/phosphate buffer at pH 2.5. Flow rate is 0.6 ml/min, providing a chromatography time of 10 min. Absorbance is measured at 214 nm at a sensitivity setting of 0.01 absorbance units full scale. Recovery for cyclosporine A is between 92 and 104%, and the lower level of sensitivity is 15 micrograms/L. Between-day precision provided coefficient of variation values less than 5% for a control serum of 150 micrograms/L. The method is linear to 3,000 micrograms/L. No interfering substances have been found. The method has proved to be consistent, reliable, and simple enough to be performed by all staff members. Column life for a system used daily is at least 3 months.