Liquid chromatography-tandem mass spectrometry method for simultaneous determination of cyclosporine A and its three metabolites AM1, AM9 and AM4N in whole blood and isolated lymphocytes in renal transplant patients.

A LC-MS/MS method was developed and validated for the determination of cyclosporine A (CsA) and its three phase 1 metabolites AM1, AM9, and AM4N in whole blood and lymphocytes isolated on the Histopaque gradient. 200 microL of whole blood was precipitated with 10 mol/L zinc sulfate in acetonitrile/methanol (40:60, v/v) and lymphocytes isolated from 1.5 mL blood were extracted with acetonitrile/methanol (40:60, v/v). The analytes and internal standard cyclosporine D were separated on RP column BEH C18, 2.1 x 50 mm, 1.7 microm using gradient LC-MS/MS analysis in positive electrospray mode. Time of analysis was 5 min. Linearity in blood was 5-2000 microg/L for CsA, AM1, and AM9; 2-500 microg/L for AM4N; and 2-500 microg/L for all substances in lymphocytes. Coefficient of variations was 1.8-9.8% and recovery was 92.0-110.0%. The method was used in early and chronic renal transplant patients for therapeutic drug monitoring of CsA to compare either its share in lymphocytes as target organ or binding to one lymphocyte. The same parameters were calculated for all metabolites tested.

The role of candidate genetic polymorphisms in the interaction between voriconazole and cyclosporine in patients undergoing allogeneic hematopoietic cell transplantation: An explorative study.

PURPOSE: To evaluate polymorphisms in genes of drug metabolizing enzymes and transporters involved in cyclosporine and/or voriconazole disposition among patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT). METHODS: DNA from forty patients was genotyped using the DMETPlus array. The average ratio of cyclosporine concentration/dose (C/D in (ng/mL)/(mg/kg)) per participant's weight was computed using available trough levels and daily doses. RESULTS: The C/D cyclosporine ratio was significantly higher when it was administered with voriconazole as compared to when it was administered alone: median: 116.75 vs. 25.40 (ng/mL)/(mg/kg) with and without voriconazole respectively, (P < 0.001). There was also a significant association between the C/D cyclosporine ratio combined with voriconazole and the ABCB1 2677 G > T > A (rs2032582) genetic polymorphism (P = 0.05). In parallel, ABCB1 variant allele carriers had higher creatinine in combination therapy with a median creatinine (mg/dL) of 0.74 vs. 0.56 for variant allele carriers vs. reference; P = 0.003. Interestingly, CYP2C9, CYP2C19, and CYP3A5 extensive metabolizers tended to be associated with lower cyclosporine C/D ratio when combined with voriconazole, but the results were not statistically significant. CONCLUSION: To the best of our knowledge, this is the first pharmacogenetic study on the interaction between voriconazole and cyclosporine in patients undergoing allo-HCT. Results suggest that the ABCB1 2677 G > T > A genetic polymorphism plays a role in this interaction with cyclosporine related nephrotoxicity. Pre-emptive genotyping for this genetic variant may be warranted for cyclosporine dose optimization. Larger studies are needed to potentially show significant associations with more candidate genes such as CYP3A4/5, CYP2C9, and CYP2C19, among others.

Development of a liquid chromatography-mass spectrometry (LC/MS) assay method for the quantification of PSC 833 (Valspodar) in rat plasma.

A liquid chromatography-mass spectrometry (LC/MS) assay method was developed for the quantification of PSC 833 in rat plasma, using amiodarone as internal standard (IS). Separation was achieved using a C(8) 3.5 microm (2.1 mm x 50 mm) column heated to 60 degrees C with a mobile phase consisting of acetonitrile-ammonium hydroxide 0.2% (90:10 v/v) pumped at a rate of 0.2 mL/min. Detection was accomplished by mass spectrometer using selected ion monitoring (SIM) in positive mode. An excellent linear relationship was present between peak height ratios and rat plasma concentrations of PSC 833 ranging from 10 to 5000 ng/mL (R(2)>0.99). Intra-day and inter-day coefficients of variation (CV%) were less than 15%, and mean error was less than 10% for the concentrations above the limit of quantification. The validated limit of quantification of the assay was 10 ng/mL based on 0.1 mL rat plasma. The method limit of detection, based on an average signal-to-noise (S/N) ratio of 3, was found to be 2.5 ng/mL. The assay was capable of measuring the plasma concentrations of PSC 833 in rats injected with a single dose of 5 mg/kg of the drug. PSC 833 and IS eluted within 4 min, free of interfering peaks. The method was found to be fast, sensitive, and specific for the quantification of PSC 833 in rat plasma.

Acute Graft Rejection and Formation of De Novo Donor-Specific Antibodies Triggered by Low Cyclosporine Levels and Interferon Therapy for Recurrent Hepatitis C Infection After Liver Transplantation: A Case Report.

BACKGROUND: We report a case of acute rejection of a liver graft, together with the occurrence of de novo donor-specific antibodies (DSAs), in a 53-year-old Japanese man who had undergone deceased-donor liver transplantation. METHODS: The graft rejection was triggered by low cyclosporine levels and pegylated interferon treatment for the recurrence of hepatitis C virus (HCV) infection 18 months after transplantation. Although the graft was ABO-compatible, pre-formed DSA B51 was detected; therefore, total plasma exchange was performed and intravenous rituximab (500 mg/body) was administered before transplantation. RESULTS: DSA was absent 6 months after transplantation. HCV recurrence was treated with pegylated interferon-α-2a. Renal function deteriorated with this anti-HCV therapy, with serum cyclosporine levels decreasing to 50 ng/mL. A rapid virologic response was achieved, but liver function deteriorated after 3 months of anti-HCV therapy, with histologic evidence of acute cellular rejection and formation of de novo DSAs. Anti-thymocyte globulin was administered for 5 days, which led to immediate improvement in liver function. However, renal function declined, warranting hemodialysis. The patient recovered 2 months after acute rejection, although de novo DSAs persisted. CONCLUSIONS: Careful immunologic monitoring may be required for patients receiving interferon therapy for HCV infection to maintain sufficient blood levels of immunosuppressive agents and to prevent acute liver graft rejection.

Availability of PSC833, a substrate and inhibitor of P-glycoproteins, in various concentrations of serum.

BACKGROUND: P-glycoproteins are membrane-associated transporters that can render cells resistant to a variety of chemotherapeutic drugs. Reversal agents are (preferably nontoxic) drugs that can inhibit these P-glycoproteins and thereby overcome multidrug resistance. PSC833, a cyclosporin A analog, is a reversal agent that has shown potential in in vitro experiments and in clinical trials. We tested PSC833 to determine whether it is a transported substrate of human and murine P-glycoproteins associated with multidrug resistance (encoded by the human MDR1 gene and its murine homolog, mdr1a) and whether it can completely inhibit these P-glycoproteins under simulated in vivo conditions. METHODS: Monolayers of polarized LLC-PK1 pig kidney cells transfected with complementary DNA containing either MDR1 or mdr1a sequences were used to measure the directional transport of P-glycoprotein substrates under various serum conditions. RESULTS: In contrast to two previous studies, we found that PSC833 is transported by both the MDR1 and the mdr1a P-glycoproteins, albeit at a low rate. PSC833 has a very high affinity for the MDR1 P-glycoprotein, and its Michaelis constant (Km) for transport is 50 nM, fourfold lower than for cyclosporin A. Inhibition of drug transport by PSC833 is approximately eightfold less effective in 100% fetal bovine serum than in tissue culture medium containing 10% serum. The concentration of PSC833 necessary to fully inhibit transport of digoxin and paclitaxel (Taxol) under complete (i.e., 100%) serum conditions is higher than the plasma concentrations achieved in clinical trials. CONCLUSIONS: Although PSC833 binds efficiently to the MDR1 P-glycoprotein and is released only sluggishly, the high concentrations of PSC833 necessary to inhibit this P-glycoprotein under complete serum conditions in our in vitro system suggest that it may be difficult for PSC833 alone to produce total inhibition of P-glycoprotein activity in patients.

Identification of cyclophilin as the erythrocyte ciclosporin-binding protein.

Previous studies on the distribution of circulating ciclosporin have shown that the majority of the drug is associated with erythrocytes. In order to investigate the nature of ciclosporin-erythrocyte binding, binding studies were performed on isolated erythrocytes. At therapeutic concentrations (approx. 0.5 microgram/ml in whole blood) greater than 90% of the erythrocyte associated ciclosporin was found in the cytosol. The cytosolic binding capacity was approximately (2-2.5).10(5) molecules of ciclosporin per cell. A lower affinity binding of the drug to the plasma membrane occurred only at higher ciclosporin concentrations. The ciclosporin-binding species was purified from erythrocyte cytosol using ciclosporin-Affigel affinity chromatography. This revealed a 16 kDa protein, similar in size to the ciclosporin-binding protein, cyclophilin, previously identified in lymphocyte cytosol. Immunochemical analysis using rabbit anti-bovine spleen cyclophilin antisera revealed that the erythrocyte ciclosporin-binding protein was either cyclophilin or a closely related protein. It is concluded that intracellular ciclosporin-binding within erythrocytes is mostly attributable to the presence of a single protein or protein family represented by cyclophilin. The presence of (2-2.5).10(5) copies of this binding protein within each erythrocyte is responsible for the ciclosporin found associated with erythrocytes.

Immunological and metabolic concomitants of cyclosporin prevention of diabetes in BB rats.

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.

Successful long-term survival of pancreatic islet allografts in spontaneous or pancreatectomy-induced diabetes in dogs. Cyclosporine-induced immune unresponsiveness.

Nineteen pancreatectomized beagles and three spontaneously diabetic dogs were recipients of canine islet allografts from one or more unrelated donors. The islets, enriched 30-45-fold for endocrine cells and contained in a packed cell volume of less than 1.5 ml, were engrafted in the livers of recipient animals. Treatment of diabetic recipients with cyclosporine (CsA) was begun 3-5 days before islet transplantation and the initial dosage was adjusted to attain and maintain CsA serum trough levels between 400 and 600 ng/ml. Five dogs with CsA levels less than this (155 +/- 35 SEM ng/ml) at the time of transplantation promptly rejected their grafts, whereas rejection was encountered in only 1 of 17 diabetic animals in which the initial level exceeded 400 ng/ml. CsA was discontinued 30, 60, or 90 days after continuous therapy in 10 animals. Graft failure was observed 2 mo after stopping CsA in 1 animal and 5 mo in the other. Eight other islet allograft recipients have sustained fasting euglycemia for 7 and 8 mo in 2 and for at least 2 mo in the remainder. These results demonstrate that short-term CsA therapy prolongs survival of islet allografts and induces a state of immune unresponsiveness to islet alloantigens in dogs with experimental and spontaneous diabetes. The findings are unique for a nonrodent mammal and thus hold promise that similar results may be achieved for islet allografts of other mammalian species, including humans.

Synthesis-secretion coupling of insulin. Effect of cyclosporin.

This study investigated the effects of cyclosporin (Cs) on insulin secretion and synthesis from the endocrine pancreas. With in vitro perfused pancreases from control and Cs-treated rats (1, 5, 10, or 25 mg.kg-1.day-1 for 2 wk), a dose-response relationship between Cs dose and inhibition of insulin secretion was demonstrated. Examination of the dynamic secretory response to a glucose stimulus (200 mg/dl) over a 3-h perfusion revealed an inhibition of all three secretory phases. Similarly, the ability of the pancreases to synthesize insulin decreased as a function of Cs dose. Reversibility of Cs toxicity on the pancreas was established by 2 wk after cessation of treatment. To evaluate the effect of Cs treatment in vivo, intravenous glucose tolerance tests were performed. Rats treated with 25 mg.kg-1.day-1 Cs for 2 wk had significantly lower k values (slope of log glucose concentration over time) than controls. At 10 mg.kg-1.day-1, although curves that appeared abnormal were observed, k values were not significantly different from those of controls. In summary, this study demonstrates the profound inhibitory effect of Cs on the endocrine pancreas.

Cyclosporin-induced remission of IDDM after early intervention. Association of 1 yr of cyclosporin treatment with enhanced insulin secretion. The Canadian-European Randomized Control Trial Group.

A randomized double-blind placebo-controlled trial was undertaken to determine whether cyclosporin enhances remission of insulin-dependent diabetes mellitus (IDDM) through the 1st yr after diagnosis. Dosage with insulin was minimized with target control of blood glucose levels less than or equal to 7.8 mM (140 mg/dl) before meals. Metabolic control was evaluated by serial determinations of glycosylated hemoglobin levels, and endogenous secretion of insulin was evaluated by determination of the levels of glucagon-stimulated insulin-connecting peptide (CP) in the plasma at 3-mo intervals. A compound definition of remission required a glucagon-stimulated CP level in plasma greater than or equal to 0.6 nM or a non-insulin-receiving state (NIR) in which target control of glycemia was maintained without administration of insulin. A clinical definition of remission required only the NIR state as defined. One hundred eighty-eight patients aged 10-35 yr entered the study within 6 wk of initiation of insulin therapy and within 14 wk of onset of symptoms and were studied for 1 yr. There were no significant differences in metabolic control between the two treatment groups during the study. The anticipated adverse effects of cyclosporin were not more frequent or severe than in other experience with the drug, but histological changes attributable to cyclosporin were present in some kidney biopsies obtained from selected patients after 1 yr. At 1 yr, by the compound definition, 33% of the cyclosporin-group and 21% of the placebo-group patients were in remission, when the corresponding rates for NIR remissions were 24 and 10%.(ABSTRACT TRUNCATED AT 250 WORDS)

The cyclosporin PSC 833 increases survival and delays engraftment of human multidrug-resistant leukemia cells in xenotransplanted NOD-SCID mice.

Circumvention of chemoresistance in cancer may involve several modulator drugs with high affinity for the multidrug transporter P-glycoprotein (Pgp), which is expressed in a number of multi-resistant malignancies. Pgp acts as a membrane efflux pump with broad substrate specificity including antineoplastic drugs and endogenous substances such as certain cytokines and sphingolipids. Therefore, the consequence of Pgp blockade could be far more complex than intracellular drug retention. In the present study exposure of the Pgp inhibitor, PSC 833 (1200 ng/ml), to Pgp expressing KG1a/200 human leukemia cells provoked cell cycle arrest and apoptosis in vitro. This finding was put to test in vivo using a xenotransplant model of KG1a/200 human cells intravenously inoculated into non-obese diabetic severe combined immunodeficient (NOD-SCID) mice. The animals were randomly allocated to receive treatment with PSC 833 (n = 32) or placebo (n = 24). PSC 833 (30 mg/kg) was subcutaneously injected six or 12 times separated by 48-96 h. The overall mean whole blood concentration of PSC 833 was 1191 +/- 60 ng/ml (s.e.m.) at 20 h after administration. Tumor engraftment was significantly reduced in the treatment group (P = 0.037), which also had prolonged survival compared to control animals (P = 0.0016). This is the first study that demonstrates antileukemic effects of a Pgp inhibitor as single agent therapy in vivo, and the present data raise the possibility of alternative exploitation of modulators in cancer chemotherapy.

Short-term changes in renal function, blood pressure, and electrolyte levels in patients receiving cyclosporine for dermatologic disorders.

We compared the changes in renal function, blood pressure (BP), and concentrations of serum potassium, magnesium, and urate (uric acid) in two groups of patients not given transplants. Group 1, comprising 21 psoriatic patients, was treated with 14 mg/kg per day of oral cyclosporine for 4 weeks in a prospective, placebo-controlled study; group 2, comprising 28 patients with diverse cutaneous diseases, was given 6 mg/kg per day of oral cyclosporine for 1 to 3 months in a prospective, open-labeled study. Renal function (determined by serum urea nitrogen [SUN] and creatinine levels and urinalysis), BP, serum electrolyte levels (potassium and magnesium), and urate level were measured weekly for the first 4 weeks in both groups, and then, after 2 and 3 months of therapy, in group 2 only. During the first 4 weeks in group 1 patients, there were significant increases in values of SUN, creatinine, BP, potassium, and urate, and a significant decrease in the serum magnesium value. When data for the two groups were combined, the changes from pretherapy values in each of the above measures (except systolic BP) during the first 4 weeks correlated significantly with cyclosporine trough levels. In group 2, the changes that occurred in the first 4 weeks in the SUN value, SUN/creatinine ratio, and BP were magnified over the subsequent 8 weeks of treatment. In the combined group for the first 4 weeks of therapy, duration of therapy, independent of cyclosporine trough levels, correlated with changes in SUN, creatinine, and urate levels, but not with changes in the potassium or magnesium level or in BP. We conclude that the cyclosporine blood level was a better discriminant than cyclosporine dosage in the analysis of renal dysfunction and hypertension in these patients.

Clinical trials of cyclosporin in IDDM.

The results of uncontrolled trials in immunomodulation of insulin-dependent diabetes mellitus (IDDM) led to randomized controlled trials in Canada and Europe. In the Canadian open study, the rate of clinical remissions (target control of glycemia maintained with less than or equal to 0.15 U.kg-1.day-1 insulin) was unexpectedly high among 81 subjects who had been treated with cyclosporin for at least 3 mo (mean serum trough levels approximately 125 ng/ml by radioimmunoassay). Subjects entered the study within 14 wk of onset of symptoms and received 6 wk of insulin therapy. The clinical remission rate at 1 yr was 46%; of these patients, 84% were not receiving insulin. An effect on beta-cell function was suggested by recovery of plasma glucagon-stimulated C-peptide levels into the normal range in many patients, with maintenance of levels through 1 yr in patients in remission. On the basis of these findings, the French and Canadian-European study groups conducted randomized double-blind controlled trials of cyclosporin, which confirmed the results of the open studies in terms of clinical remission. The Canadian-European study also demonstrated enhancement of beta-cell function by cyclosporin by 3 mo, which was maintained for 1 yr. In the Canadian open study, most patients relapsed within a few weeks after discontinuation of cyclosporin, indicating the need for longer-term immunomodulatory therapy for maintenance of remission. The nature and degree of structural change in kidney biopsies from patients in these studies are under assessment. The results strongly support the hypothesis that autoimmune mechanisms mediate beta-cell damage in many patients with IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporin-A radioimmunoassay: a modified method for whole blood determination.

Cyclosporin-A levels were determined by radioimmunoassay in plasma or whole blood, using split samples collected from patients receiving this agent as the only form of immunosuppression following allogeneic bone marrow transplantation. In the plasma assay the temperature at which centrifugation took place was critical since the mean levels were approximately 30% higher with separation at 37 degrees C in comparison to 20 degrees C or lower. Furthermore, the level in whole blood samples was 2.4 times higher than that from the matching serum. In addition, anticoagulated blood that had been frozen and then thawed was technically more difficult to pipette and resulted in a recovery of only 83% of the cyclosporin when compared with assay using fresh blood. In contrast, consistent measurements were obtained either when whole blood was stored at 4 degrees C and then well mixed and diluted in buffer immediately prior to use or when such buffered samples were frozen and thawed immediately before analysis. The latter modifications render the whole blood assay a practical and reliable means for monitoring cyclosporin-A concentrations and may avoid excessive and the potentially nephrotoxic levels achieved when plasma levels are held in ranges previously considered therapeutic.

Cutaneous papillomatous hyperplasia in cyclosporine-A treated beagles.

All twelve Beagle dogs undergoing long-term therapy (26 weeks) with the immunosuppressive drug cyclosporine-A (30 mg/kg), developed cutaneous papillomatous hyperplasia. By week 7 all dogs developed generalized lesions distributed over the entire body. These occurred as irregular, oval, sessile, unpigmented, firm masses. The incidence and severity of the skin lesions varied among dogs and anatomic site, with no correlation to the blood level of cyclosporine. Microscopic analysis revealed that the epidermis formed short papillary folds on broad fibrovascular stalks and was hyperkeratotic and acanthotic. Mild hyperplasia of hair follicles and sebaceous glands was also evident. A mild diffuse infiltrate of lymphocytes and plasma cells was present in the papillary dermis. No histopathologic changes typical of papillomavirus infection were identified, nor were papillomavirus group-specific antigens or viral DNA detected. Other cutaneous side effects included hyperkeratosis of footpads, increased growth of hair and nails, and hyperkeratinization of the haired skin of the prepuce. All cutaneous lesions regressed spontaneously within 8 weeks following termination of cyclosporine administration. The hyperplastic lesions may have resulted from the action of cyclosporine via the T-lymphocyte system. Conversely a direct action of this drug on epithelial cells may have stimulated proliferation and keratinization.

Trough levels and concentration time curves of cyclosporine in patients undergoing renal transplantation.

We determined the AUC of cyclosporine (24 hours) nonspecifically by RIA and specifically by HPLC after an oral and an intravenous dose of cyclosporine in 58 patients undergoing renal transplantation. The RIA/HPLC concentration ratio of cyclosporine changed continuously during the first 12 hours after administration. The ratio was higher after oral than after intravenous administration and varied from patient to patient. The predictive value of trough levels for the corresponding AUCs was better when trough levels were assessed 24 than 12 hours after administration. Trough levels assessed by RIA poorly predicted AUCs measured specifically by HPLC. Therefore if, in the future, therapeutic cyclosporine monitoring has to be improved, trough levels should be assessed 24 hours after the last dose by means of a specific HPLC method.

Age-dependent cyclosporine: pharmacokinetics in marrow transplant recipients.

We evaluated the effect of age on cyclosporine pharmacokinetics in 69 nonobese patients aged 10 months to 56 years (median 22 years) undergoing allogeneic bone marrow transplantation for treatment of aplastic anemia or hematologic malignancy. Cyclosporine pharmacokinetics were studied during the first 2 posttransplant weeks after an intravenous dose of 2.6 to 3.5 mg/kg. Serum cyclosporine concentrations were measured by HPLC. Cyclosporine concentration-time data were fitted to a two-compartment model with a nonlinear regression program. There was a significant inverse linear correlation between age and both total systemic clearance (CL) (r = 0.42; P less than 0.001) and volume of distribution at steady-state (Vss) (r = 0.33; P less than 0.01). Mean (+/- SE) cyclosporine CL was 82 +/- 21, 45 +/- 5, 38 +/- 9, 44 +/- 8, and 20 +/- 3 ml/min/kg and mean cyclosporine Vss was 34 +/- 11, 28 +/- 10, 15 +/- 4, 14 +/- 5, and 4.7 +/- 0.7 L/kg in patients 0 to 10 (n = 12), 11 to 20 (n = 19), 21 to 30 (n = 12), 31 to 40 (n = 17), and greater than 40 (n = 9) years old, respectively. Patients 0 to 10 years old had a significantly higher cyclosporine CL than those 11 to 40 or greater than 40 years old and also had a significantly larger Vss than those greater than 40 yrs old (P less than 0.05). Age-related differences in CL or Vss were also observed when these parameters were normalized by body surface area.(ABSTRACT TRUNCATED AT 250 WORDS)

The erythromycin breath test as a predictor of cyclosporine blood levels.

The daily dose of cyclosporine required to attain a desired blood level can vary greatly among patients. Because elimination of cyclosporine depends on its metabolism in the liver by an enzyme (cytochrome P-450IIIA) that also demethylates erythromycin, we reasoned that the ability of patients to demethylate a test dose of erythromycin might be useful in estimating their appropriate daily doses of cyclosporine. Accordingly, the [14C-N-methyl] erythromycin breath test was administered to 32 patients before they received 3.0, 5.0, or 7.5 mg/kg/day cyclosporine to treat psoriasis. We found that a simple mathematical equation incorporating just the 14CO2 production, the age of the patient, and the daily dose of cyclosporine accounted for almost 80% (R2 = 0.78) of the interpatient variability in cyclosporine blood levels we observed. Our data indicate that P-450IIIA activity largely accounts for the relationship between dose of cyclosporine and blood levels for an individual patient. We conclude that the erythromycin breath test may be a convenient guide for cyclosporine dosing.

Cyclosporine kinetics in renal transplantation.

The pharmacokinetics of cyclosporine were evaluated in 41 recipients of a cadaveric renal transplant. Cyclosporine was taken by mouth (mean dose 14 mg/kg) on one study day and was intravenously infused over 2 hours (mean dose 4.7 mg/kg) on the next study day. Cyclosporine was extracted from whole blood and analyzed by HPLC. After intravenous infusion, cyclosporine exhibited multicompartmental behavior. The mean (+/- SD) terminal disposition rate constant was 0.065 +/- 0.036 hours-1 and the harmonic mean t 1/2 was 10.7 hours. The harmonic mean total body clearance of cyclosporine was 5.73 ml/min/kg and the mean apparent volume of distribution was 4.5 +/- 3.6 L/kg. The absorption of oral cyclosporine was slow and incomplete. Peak blood cyclosporine concentrations (means = 1,103 ng/ml) were reached between 1 and 8 hours after oral dosing (means = 4 hours). The mean relative bioavailability was 27.6% +/- 20%. Oral bioavailability was less than 10% in 17% of our subjects. The absorption and clearance of cyclosporine were highly variable. We conclude that the variability in the kinetics of cyclosporine makes trough blood level monitoring essential in the management of patients who receive renal transplants.

Antibodies to cyclosporine A (CsA) by a novel route and their use to monitor cyclosporine levels by radioimmunoassay (RIA).

Anti-cyclosporine antibodies were generated in rabbits, using an antigen derived from CsA. CsA was linked to carrier proteins by means of a photoactive cross-linking reagent, 4-benzoylbenzoic acid (BBa), an alpha, beta-unsaturated ketone. In the presence of CsA, photolysis of BBa results in hydrogen abstraction and random insertion into the cyclosporine molecule, generating a population of CsA with carboxyl groups at various positions. Immunization with CsA-BBa-bovine serum albumin gave rise to high affinity antibodies to CsA, with Kd = 9.8 +/- 2.8 x 10(-11) M, as determined by Scatchard analysis. Specificity was determined by competition experiments in a radioimmunoassay (RIA), using a panel of six cyclosporine derivatives, substituted at different positions. The derivatives could be arranged into three groups according to their affinities. One derivative with the lowest affinity had a bulky O-t-butyl-D-serine in place of D-alanine in position 8. Serum CsA levels in 25 transplant patients were measured by RIA, and compared to levels determined with a commercially available polyclonal antibody, which is routinely used clinically. The rabbit antiserum gave values that correlated well with the results using the commercial antibody.