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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., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background and Objectives: An open-label, multicenter, randomized phase II trial was conducted from July 1, 2005 to March 29, 2011 to compare two protocols for treating children with frequently relapsing nephrotic syndrome using microemulsified cyclosporine., Design, Setting, Participants, & Measurements: Ninety-three children with frequently relapsing nephrotic syndrome were randomly assigned to group A (n=46) or group B (n=47). In both groups, the 2-hour postdose cyclosporine level was monitored. For group A, the cyclosporine target was set to 600-700 ng/ml for the first 6 months and 450-550 ng/ml for the next 18 months; for group B, it was set to 450-550 ng/ml for the first 6 months and 300-400 ng/ml for the next 18 months. The primary end point was the sustained remission rate. At the end of the study, if there was no difference in safety profile between the two groups and the sustained remission rate in group A was superior to group B with a decision threshold of 8%, then the regimen for group A would be determined the better treatment., Results: Eight children from an ineligible institution, where cyclosporine levels were not measured, were excluded from all analyses. At 24 months, the sustained remission rate was nonsignificantly higher in group A (n=43) than group B (n=42; 64.4% versus 50.0%; hazard ratio, 0.57; 95% confidence interval, 0.29 to 1.11; P=0.09), and the progression-free survival rate was significantly higher (88.1% versus 68.4%; hazard ratio, 0.33; 95% confidence interval, 0.12 to 0.94; P=0.03). The relapse rate was significantly lower in group A than group B (0.41 versus 0.95 times/person-year; hazard ratio, 0.43; 95% confidence interval, 0.19 to 0.84; P=0.02). The rate and severity of adverse events were similar in both treatment groups., Conclusion: The sustained remission rate was not significantly different between the two treatment groups, but the regimen with the higher 2-hour postdose cyclosporine level target improved progression-free survival and reduced the relapse rate.

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.

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.

The cyclosporine analog Valspodar (PSC 833, Novartis Pharma) is a strong inhibitor of the mdr1 gene product p-glycoprotein (pgp). A phase I/II study was conducted in order to evaluate if addition of Valspodar to treatment with daunorubicin and cytarabine, given to patients with primary refractory or relapsed acute myeloid leukemia, could increase the complete remission rate.Fifty-three patients were treated in cohorts of three to six patients. Twelve patients reached a complete remission in bone marrow, five of whom also normalized their peripheral blood values. Three patients experienced treatment-related deaths from pneumonia, liver failure and cerebral hemorrhage, respectively. It is concluded that Valspodar 10 mg/kg per 24 h in combination with daunorubicin 45 mg/m(2) for 3 days and cytarabine 1 g/m(2) twice daily for 4 days is tolerable in this heavily pre-treated group of patients. Due to the moderate treatment results, the phase II part of the study was ended prematurely. The modulation of only pgp did not give an obvious improvement of the treatment results in this group of patients.

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.

The pharmacokinetics of valspodar (PSC 833), a selective second-generation P-glycoprotein modulator, was evaluated as part of a Phase I study to modulate paclitaxel therapy in 15 patients with refractory malignancies. Valspodar was given intravenously at 1.42 mg/kg/h for 2 hours, followed by a 27-hour continuous infusion at 0.42 mg/kg/h. Serial blood samples were obtained after intravenous infusion of valspodar and paclitaxel. Valspodar disposition was best described by a linear two-compartment model. The median (range) valspodar clearance was 0.40 ml/min/kg (0.07-1.40 ml/min/kg). The 20-fold interpatient variability in valspodar clearance was not correlated with age, body weight, orgender but might be associated with coadministered medications that were metabolized via cytochrome P450 3A-mediated elimination. Valspodar whole-blood concentrations were maintained above the target threshold of 1000 ng/ml for a median of 32 hours. The pharmacokinetic model generated from this study allows for application in future studies to optimize the use of valspodar.

Background: The aim of the study was to investigate the behaviour of plasma levels of endothelin-1 (ET-1), an endothelial peptide with vasoconstrictive and proliferative actions, in patients with cardiac transplantation and in chronic treatment with cyclosporine A, some of whom became hypertensive after cardiac transplantation., Methods: We studied: 1) 18 consecutive patients (15 M, 3F; mean age 53 +/- 7 yrs) who underwent cardiac transplantation about six months ago at least (range 6-108 months); 2) 15 patients with essential arterial hypertension (10 M, 5 F; mean age 42 +/- 15 yrs) without organ damage; 3) 21 normal subjects (15 M, 6 F; mean age 31 +/- 12 yrs). Plasma levels of ET-1 (RIA), haemodynamic and functional renal parameters were determined in all groups and plasma levels of cyclosporine were measured in patients with cardiac transplantation., Results: ET-1 was higher in patients with cardiac transplantation than in the other two groups (p < 0.05); instead there was no difference between patients with essential arterial hypertension and controls (p>0.05). A statistical difference was found between circulating ET-1 in hypertensive transplanted patients. In heart transplanted patients a positive and significative correlation was found between plasma levels of ET-1 and systolic (r=0.525; p<0.037) blood pressure.

Aims: The aim of the study was to investigate the pharmacokinetics and metabolism of the new immunosuppressant SDZ RAD during concomitant therapy with cyclosporin in stable renal transplant patients. Furthermore, we studied the influence of SDZ RAD on the pharmacokinetics of cyclosporin at steady state levels., Methods: SDZ RAD was administered orally in different doses (0.25-15 mg day-1) to seven patients, who were on standard cyclosporin-based immunosuppression. The blood concentrations of both drugs including their main groups of metabolites were measured simultaneously by LC/electrospray-mass spectrometry., Results: The mean area under the blood concentration-time curve to 12 h (AUC(0,12 h)) was 4244 +/- 1311 microg l-1 h for cyclosporin before SDZ RAD treatment and 4683 +/- 1174 microg l-1 h (P = 0.106) on the day of SDZ RAD treatment (95% CI for difference -126, 1003). On both study days Cmax, and tmax of cyclosporin were not significantly different. The metabolite pattern of cyclosporin did not change. The pharmacokinetic data of SDZ RAD dose-normalized to 1 mg SDZ RAD were as follows: AUC(0,24 h): 35.4 +/- 13.1 microg l-1 h, Cmax: 7.9 +/- 2.7 microg l-1 and tmax: 1.5 +/- 0.9 h. The metabolites of SDZ RAD found in blood were hydroxy-SDZ RAD, dihydroxy-SDZ RAD, demethyl-SDZ RAD, and a ring-opened form of SDZ RAD., Conclusions: A single dose of SDZ RAD did not influence significantly the pharmacokinetics of cyclosporin. The most important metabolite of SDZ RAD was the hydroxy-SDZ RAD, its AUC(0,24 h) being nearly half that of the parent compound SDZ RAD.

We established a rapid and sensitive ex vivo bioassay to detect the multidrug resistance (MDR)-inhibitory activity of SDZ PSC 833 ([3'-keto-Bmt1]-[Val2]-cyclosporin (PSC 833)) in two RPMI 8226 human myeloma sublines (parent 8226 and doxorubicin-resistant subline Dox6) in 75% human serum. In vitro sensitivity of the tumor to doxorubicin was determined by 3-h drug exposure growth inhibition assay (MTT assay). PSC 833 in serum restored the IC50 of doxorubicin in the P-glycoprotein (P-gp)-positive resistant subline to the same level as in the sensitive cells at 1 microg/ml, which has been shown to be an achievable concentration in clinical trials. In addition, the cytotoxic effect of doxorubicin was enhanced by PSC 833 in the sera of the patient in whom the blood level was 705.7 ng/ml. However, 10 microg/ml PSC 833 in serum does not cause a complete recovery in the IC90 of doxorubicin in the resistant sublines. This MDR-inhibitory activity was supported by the finding that PSC 833 in serum does not increase accumulation of rhodamine 123 in doxorubicin-resistant cells in an in vitro functional assay. The present study provides evidence that PSC 833 in human serum is effective to modulate P-gp-mediated MDR but insufficient for the reversal of MDR from the clinicopharmacological point of view.

Introduction: This trial was designed to determine the maximum tolerated dose of intravenous daunorubicin (DNR) in combination with valspodar and to test the feasibility of P-glycoprotein modulation using valspodar in elderly patients with previously untreated acute myelogenous leukemia receiving standard induction chemotherapy., Methods: Patients > or =60 years of age with previously untreated AML received valspodar (10 mg/kg/24 h by continuous intravenous infusion [CIV] on days 1-4 with a 2-mg/kg loading dose on day 1) in conjunction with two cycles of induction chemotherapy consisting of cytarabine (200 mg/m(2) CIV on days 1-7), and DNR (35 mg/m(2) [cohort 1] or 45 mg/m(2) [cohort 2] on days 1-3, intravenous bolus). Patients were assessed for dose-limiting toxicities (DLT), response rate, event-free and overall survival, and pharmacokinetics of valspodar and DNR., Results: Valspodar was well tolerated at the lower DNR dose level (ie, 35 mg/m(2)) resulting in a 21% rate of DLT and only three toxic deaths. Treatment-related mortality was unacceptably high at the 45 mg/m(2) DNR dose level. The complete response rate was 49% overall and similar in both cohorts. The median overall survival of patients was 333 days in cohort 1 compared to 98 days in cohort 2. At baseline, 70% of assessable patients were P-glycoprotein positive., Conclusion: Substantial inhibition of P-glycoprotein activity can be achieved in this patient population at clinically tolerable doses of valspodar and DNR. The maximum tolerated dose of DNR was established as 35 mg/m(2). This regimen is being further evaluated in phase III trials.

We present a case of sympathetic ophthalmia. A 41-year-old man suffered a penetrating injury to his right eye. Six weeks after the injury he complained of photophobia and redness in his left eye. Visual acuity without correction was: right eye (RE), no light perception; and left eye (LE), 1.0. Ocular examination of the left eye revealed a shallow anterior chamber and mildly elevated intraocular pressure (25 mmHg). An initial diagnosis of narrow angle glaucoma was made and antiglaucomatous agents were prescribed. One month after diagnosis the vision in his left eye suddenly dropped to 0.04. Ocular examination showed annular serous retinal detachment and scattered yellow-white lesions (Dalen-Fuchs Nodules). Under the diagnosis of sympathetic ophthalmia, high dose intravenous corticosteroid (methylprednisolone, 200 mg daily) and subtenon dexamethasone (4 mg/0.8 cc) were used. Ten days after beginning treatment the vision improved to 0.1 but the retinal detachment was not sealed completely. Oral prednisolone (100 mg daily) and cyclosporine (125 mg bid) therapy replaced the intravenous corticosteroids. The serum level of Cyclosporine was 118 ng/ml. After 3 months the vision improved to 0.6 and the retinal detachment subsided. Renal function and hematocrit status were monitored closely and no abnormal conditions were noted.

The expression of high levels of P-glycoprotein (Pgp) in circulating mononuclear cells allowed us to use an ex vivo assay as a surrogate measure of Pgp antagonism. Efflux of rhodamine from CD56(+) cells was measured before the start of PSC 833 and at varying times thereafter. Patients receiving PSC 833 had decreased rhodamine efflux from their circulating CD56(+) cells. Time course studies showed that following a single oral dose of PSC 833, decreased rhodamine efflux was found in some patients within 15 minutes of treatment. Maximal inhibition was observed at times ranging from 45 minutes to 60 minutes. A dose-response relationship was shown between the concentration of PSC 833 in the blood and the inhibition of rhodamine efflux, with an apparent plateau of the inhibition of rhodamine efflux at approximately 1,000 ng/mL. The Ki, defined as the concentration required for half-maximal inhibition of Pgp-mediated rhodamine efflux, was determined to be in the range of 29 to 181 ng/mL; although results in two patients were distinctly different, with Ki values of 914 and 916 ng/mL. MRK-16 staining was similar among all patients. We conclude that measurement of rhodamine efflux from CD56(+) cells provides a surrogate assay with the potential for monitoring Pgp antagonism in clinical trials.

We have developed a rapid, sensitive and selective method for the determination of the cyclosporin analog PSC 833 in human and mouse plasma using cyclosporin A as internal standard. The assay uses liquid-liquid extraction with diethyl ether for sample clean-up followed by reversed-phase high-performance liquid chromatography with UV detection at 210 nm. Good peak shapes were obtained using a NovaPak Phenyl column operating at 72 degrees C. Good selectivity from endogenous compounds was achieved using a mobile phase composed of methanol-acetonitrile-water (34:34:32). The retention times of cyclosporin A and PSC 833 were approximately 7.8 and 11.7 min, respectively, with two major endogenous peaks at 9.2 and 16.7 min. Selective decreasing of the retention times of cyclosporin A and PSC 833 relative to these interferences occurring upon aging of the column was balanced by increasing the percentage of methanol relative to acetonitrile. No other late eluting peaks were present, resulting in a total analysis time of 20 min per sample. The assay performance in human plasma was good. The absolute recovery of PSC 833 after the sample clean-up step was 48+/-6%. The lower limit of quantitation was 0.05 microM using 500 microl of sample. Within the linear dynamic range of the assay (0.10-5.0 microM) the accuracy was close to 100% and within-day and between-day variation less than 7%. Because of the limited availability of blank mouse plasma, the concentration in samples from mice were determined using calibration curves constructed in human plasma. The lower limit of quantitation in mouse was 0.25 microM using 200 microl of sample. Overall, the performance of the assay in mouse plasma was somewhat less than in human plasma but accuracy and precision were within the ranges that are considered acceptable for bio-analytical assays.

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.

When converting maintenance renal allograft recipients from Sandimmune (cyclosporin A [CsA]) to Neoral (CsA-microemulsion [CsA-ME]), a dose conversion ratio of 1:1 may not be optimal, in part because of the variability in absorption of the CsA formulation of cyclosporine. After conversion using a 1:1 dose ratio, an individualized approach to the management of dosing was applied. In this article, close monitoring, which began at the time of conversion, and rapid response to potentially meaningful changes in cyclosporine trough levels early in the postconversion course were used to maintain patients' cyclosporine troughs at preconversion levels. The results of cyclosporine dose changes after converting stable, maintenance renal transplant patients from CsA (once daily and twice daily) to CsA-ME (twice daily) during 52 wk of follow-up are reported. Most patients (87.2%) required CsA-ME dose reduction to maintain preconversion trough levels, and 64% of the patients attained their CsA-ME maintenance dose by study week 4. Logistic regression analysis identified one significant predictor concerning the week 52 CsA-ME dose: patients converted from CsA doses > or = 4.0 mg/kg per d were more likely to require dose reduction (P < 0.0001). Although firm guidelines for dose modification after conversion from CsA to CsA-ME cannot be provided because of the individual nature of cyclosporine absorption, an individualized approach to patient management is recommended. Patients with higher CsA doses before conversion are particularly likely to require dose reduction early in the postconversion course. With CsA-ME, good absorbers of cyclosporine remain good absorbers, or become better absorbers, whereas poor absorbers become good absorbers.

Cyclosporin A (CyA) is intensively metabolized by the hepatic cytochrome p450 III monooxygenase A system in the human liver, the most important metabolites being M1, M17, and M21. Because CyA and its metabolites have nephrotoxic, hepatotoxic, and neurotoxic side effects, CyA dosage must be calculated to avoid the risk of organ rejection through underdosage and toxic organ damage through overdosage or accumulation of metabolites. In this study, we determined the whole-blood concentrations of cyclosporin and metabolite M17 by high-pressure liquid chromatography (HPLC) and by monoclonal specific and polyclonal nonspecific fluorescence polarization immunoassay (Abbott) in patients after immunosuppressive treatment. Patients with different resorption and metabolization rates showed high individual variations. CyA concentrations in patients with good liver function and low concentrations of CyA metabolites showed a good correlation between the HPLC and the FPIA (TDx-monoclonal assay) methods in ranges between 25 and 180 ng/mL. TDx-monoclonal was not always as precise as HPLC. In cases of metabolic disorders, we found false high CyA concentrations assayed with the immunologic method, caused by a crossreaction of the elevated metabolite concentration. We found that HPLC rendered more information about the extent of immunosuppressive activity and the metabolization rate and showed a good correlation with the concentration of metabolite M17 and total metabolites measured with the Abbott CyA polyclonal kit.

Purpose: To contribute to a better understanding of the physiological role of P-glycoprotein (P-gp) in the adrenal gland, we initiated our studies in rabbits. The aim of our study was to explore the effect of the selective multidrug resistance (MDR) modulator PSC 833 (valspodar) on serum cortisol in rabbits., Methods: Baseline and corticotropin-stimulated serum cortisol levels were measured before and after valspodar treatment in adult male rabbits. Seven rabbits were treated with 50 mg/kg per dose and seven, with 75 mg/kg per dose of valspodar subcutaneously. Serum cortisol levels were determined by radioimmunoassay adjusted for expected values., Results: Serum cortisol levels (baseline as well as corticotropin-stimulated) increased after both valspodar treatment regimens. The increase was dose-dependent and was higher for the baseline than for the corticotropin-stimulated values. Serum valspodar levels exceeding 1000 ng/ml were achieved in all except one animal in each group. We hypothesize that the increased serum cortisol levels were due to increased adrenocorticotropic hormone (ACTH) secretion after valspodar treatment, but, unfortunately, we could not measure ACTH properly in rabbits by means of the commercially available kits., Conclusions: Our study indicates that P-gp is not involved in steroid hormone secretion in the adrenal gland. This is evident from observations that serum cortisol levels were found to have increased rather than decreased in rabbits treated with a P-gp blocker and that the treated animals appeared healthy and normal. Since P-gp was found to play an important role in protection against xenobiotics in some other organs, further studies to explore the protective role of P-gp in the adrenal gland are warranted.

Objective: To assess the effects of pregnancy on cyclosporine levels in six renal allograft patients., Methods: Maternal demographic, laboratory, clinical, and perinatal outcome data were recorded in six pregnant women with previous renal allografts receiving cyclosporine immunosuppression. The cyclosporine and serum creatinine levels were measured before pregnancy, during each trimester, and postpartum., Results: The mean (standard deviation [SD]) maternal age was 29.1 (3.8) years. Parity ranged from 0 to 3. Mean serum creatinine levels tended to be lower during pregnancy than before or after, as did the mean cyclosporine levels. After adjusting for dose, five of six patients had declines in cyclosporine level during pregnancy. The mean (SD) gestational age at delivery was 37.5 (2.8) weeks with a mean (SD) birth weight of 2837 (538) g., Conclusion: Pregnancy in patients with renal allografts can lead to a substantial decline in cyclosporine levels.

Cyclosporin A (CyA) and its metabolites seem to have nephro-, hepato- and neurotoxic side effects. Immunosuppressive therapy is a narrow path between the risk of rejection by underimmunosuppression and toxic organ damage by overdosage. Thus CyA dosage must be calculated to avoid the risks of organ rejection through underdosage and toxic organ damage through overdosage or accumulation of metabolites. In routine monitoring of CyA therapy, it can be important to measure not only the parent drug but also the metabolites. We describe a rapid and isocratic high-performance liquid chromatographic method for measurement of CyA and its metabolites M1, M17 and M21 in whole blood. CyA was detected by ultraviolet absorption at 212 nm with a CN analytical column maintained at 50 degrees C and recycling of hexane-isopropanol as mobile phase for improved long-term column stability and efficiency. The minimum detectable concentration of CyA and the three metabolites was 10 ng/ml blood. Our modified HPLC method for the determination of CyA and its metabolites is a simple (isocratic), rapid (the retention times were 7.1 min for CYD, internal standard, 8.9 min for CyA, 11.0 min for M21, 12.9 min for M17 and 16.3 min for M1) and economical method suitable for measuring the concentration of the major metabolite, M17, and for routine monitoring of CyA-treated patients.

P-glycoprotein (Pgp) is a 170-kDa membrane transporter that mediates drug efflux and is an effector of multidrug resistance. SDZ PSC 833 (PSC), a nonimmunosuppressive cyclosporine that potently modulates Pgp, is currently under clinical evaluation in patients with cancer. We have developed a reversed-phase HPLC assay for determining PSC blood concentrations that utilizes a step gradient with linear segments to resolve PSC into two distinct peaks (likely to be keto and enol isomers). To clinically validate the assay, PSC concentrations were obtained by HPLC from nine patients receiving oral doses of 5 mg/kg every 6 h. Values ranged from 0.91 to 5.4 mg/L during the dosing period, comparable with concentrations of PSC that modulate Pgp in vitro. In addition, we investigated the immunoreactivity of the Abbott TDx cyclosporin A (CsA) monoclonal whole-blood assay for PSC. The TDx CsA assay cross-reacts approximately 17% with PSC as determined by adding known amounts of PSC to whole blood. When PSC concentrations obtained by the TDx CsA assay were divided by 0.17, we found agreement between the TDx CsA assay and the HPLC PSC assay for samples from nine patients.

Objective: The aim of this study was to examine the relationship between erythrocyte-to-plasma distribution ratio of cyclosporin (CsA-EP) and lymphocyte proliferation as an indicator of immunosuppressive activity in renal transplant patients., Methods: A total of 113 whole blood samples obtained from 6 inpatients with renal transplantation were analysed. CsA concentrations in blood and plasma at trough were measured by fluorescence polarization immunoassay using monoclonal antibody, lymphocyte proliferation in response to phytohaemagglutinin was evaluated by the fluorimetric derivatization method using ethidium bromide and the stimulation index (SI) was calculated., Results: There was no correlation between CsA dose and trough levels (vs blood CsA, r2 = 0.052; vs plasma CsA, r2 = 0.054, n = 113). A significant negative correlation between the SI and the CsA-EP was found in individual or all samples (r2 = 0.224, p < 0.0001, n = 113), whereas CsA trough levels in blood or plasma had no correlation with the SI., Conclusion: Although the degree of contribution of CsA-EP to the SI was 22%, the CsA-EP is a more useful predictor of changes in immunosuppressive response than CsA concentration in blood or plasma. The adoption of the CsA-EP as a monitoring index could be helpful in assessing the appropriateness of CsA immunosuppressive therapy.

This study was conducted to determine the effect of clarithromycin (CAM) on the bioavailability of cyclosporin (CYA) in rats, and to compare its effect with that of erythromycin (EM). The area under the blood CYA concentration-time curve (AUCi.v.) values after intravenous administration of CYA (2 mg/kg) in combination with CAM or EM (100 mg/kg, p.o.) were significantly increased compared with those of CYA alone, suggesting that there was metabolic inhibition of CYA in the liver by CAM or EM. The time to reach the peak concentration after oral administration of CYA (10 mg/kg) tended to be longer with increasing doses of both CAM and EM (10 and 100 mg/kg, p.o.). Each AUCp.o. value for the CAM or EM coadministration group, except the EM (100 mg/kg) coadministration group (about 77% increase), was comparable to that for the CYA alone group. Both CAM and EM (10 and 100 mg/kg, p.o.) were shown to delay gastric emptying in a dose-dependent manner. The gastric emptying in the group treated with CAM (100 mg/kg) was significantly lower than that with EM (100 mg/kg). It is suggested that CAM as well as EM might affect the oral bioavailability of CYA by inhibiting its metabolism and simultaneously by changing the gastrointestinal motility in rats. Thus, caution is recommended when administering CYA concomitantly with CAM to humans.

Cyclosporine, a cyclic undecapeptide, is currently the major immunosuppressant used after liver transplantation. Since it is unclear whether or not cyclosporine metabolites play a part in toxicity, high concentrations of metabolites should be avoided. The quantification of cyclosporine metabolites requires immunoassays using nonspecific antibodies cross-reacting with metabolites or high-performance liquid chromatography (HPLC) analysis. Since no guidelines are available to date concerning when such additional analysis is required, it was the aim of this study to define biochemical parameters that parallel cyclosporine elimination and indicate whether or not cyclosporine elimination is impaired, requiring quantification of cyclosporine metabolites. One hundred and thirty adult liver graft recipients were included in a prospective study during their first hospital stay. Cyclosporine and 11 metabolites were quantified in blood every second day using radioimmunoassay and HPLC. When the cyclosporine metabolite patterns in trough blood samples of patients with impaired liver function were compared with those of patients with good liver function, concentrations of metabolites AM19 and AM1A were found to be elevated. Serum concentrations of conjugated and total bilirubin were significantly correlated with blood trough concentrations of AM19 and AM1A, while there was no correlation with cyclosporine or its first-generation metabolites. Distribution statistics showed that liver graft patients with impaired cyclosporine elimination had total bilirubin concentrations in serum > 60 mumol/l L. No correlation was found between bile acids and the concentrations of metabolites AM19 and AM1A, suggesting that the ion-coupled transport system is not quantitatively involved in cyclosporine excretion and that bilirubin and cyclosporine metabolites are eliminated by the same transport system through the biliary membrane. It is concluded that bilirubin and cyclosporine metabolite concentrations are strictly parallel and that the total bilirubin concentration in serum may be used as an indicator of impaired cyclosporine elimination.

Simultaneous kidney-pancreas transplant (SPK) recipients have a higher rate of acute organ rejection than do recipients of a kidney-alone transplant. The etiology of this increased number of rejection episodes is unknown. Cyclosporine (CsA) monitoring in SPK recipients is an important tool in the care of these patients. Many centers use the monoclonal fluorescence polarization immunoassay (mFPIA) to monitor CsA levels in this group of organ recipients. The reported specificity of the mFPIA for the CsA parent compound has varied in several studies but has not been systematically studied in recipients of an SPK transplant. Twelve recipients of an SPK transplant from 1 month to 3 months after surgery had serial simultaneous whole blood high-performance liquid chromatography (HPLC) and mFPIA CsA concentrations evaluated. A control group of 15 nondiabetic kidney-alone recipients underwent a similar evaluation. The average mFPIA/HPLC ratio in recipients of an SPK was 1.71 +/- 0.05 (r = 0.94), whereas it was 1.56 +/- 0.03 (r = 0.98) in the kidney-alone group (p > 0.01). Interpatient variability (range, 1.41 to 2.13) was very high in the SPK group, whereas intrapatient variability was lower. Seven patients had AUC profiles. The average AUC mFPIA/HPLC ratio was not significantly different from each patient's respective trough ratios. Our study suggests that the mFPIA overestimates CsA parent compound to a greater extent in SPK recipients than in kidney-alone recipients. In addition, the large interpatient variability in the specificity of the mFPIA makes assessing individual patient's levels problematic.

Chronic renal impairment represents on of the major side effects of cyclosporine (CsA) immunotherapy for organ transplantation. The clinical relevance of selective measurement of CsA metabolites to correlating nephrotoxic activities remains inconclusive. A relatively simple and reliable method was developed to measure AM19 and four other CsA metabolites in whole blood by sequential solid-phase extraction and reversed phase high-pressure liquid chromatography (HPLC). The procedures were modified from a well-established method developed to quantitate CsA, the most important difference being in the elution step. The use of acetonitrile/methanol instead of ethylacetate/isopropanol in the elution procedure enhanced the absolute recoveries of metabolites. The washing steps were also slightly modified to recover AM19 quantitatively and specifically. Isocratic chromatographic conditions allowed very good separation of AM19, AM1, AM9, AM1c, AM4N, CsA, and the internal standard, cyclosporin C (CsC). Analytical recoveries for CsA and five of its metabolites ranged from 82 to 92%. No interfering substance from the matrix was found. The detection limit was 10 micrograms/l. The objectives of this study were to measure trough concentration of AM19 in whole blood, expressed as percentage of total metabolites plus parent CsA, and to determine if the blood level of AM19 could be used to predict subsequent changes in serum creatinine level in liver transplant patients. Twenty-three patients, who underwent liver transplantation between January and June 1993 were studied. Trough concentration of CsA in whole blood, serum creatinine and liver enzymes were constantly monitored. AM19 level was determined 5-8 months after transplantation. Preliminary results suggest an inverse relationship between AM19 and serum creatine levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Gel chromatography combined with specific and non-specific cyclosporin radioimmunoassays was adopted for quantitative analysis of cyclosporin and metabolites in free and protein-bound forms in blood compartments of kidney transplant patients. The analytical method was proved to be useful for the purpose, although plasma protein-bound forms of neither cyclosporin nor metabolites could be quantitated in the system. The present study also provided, by gel chromatographic analysis, additional examples to prove that concentrations of cyclosporin metabolites in blood compartments may not be deduced or inferred simply from those of cyclosporin.

Previous studies in vitro and in rodent transplantation models have suggested that an analogue of cyclosporin, SDZ-IMM-125, has immunosuppressive properties at least equivalent to that of cyclosporin. As the bioavailability of the drug was considered to be greater than that of cyclosporin, it was hoped that lower doses could be used with the avoidance of nephrotoxicity. Renal allografts were undertaken between beagle dogs from two partially inbred breeding colonies. After transplantation, SDZ-IMM-125 was given orally at a dosage of 5, 7.5, 10 or 20 mg/kg/day, and graft survival compared to that in dogs given cyclosporin 10 mg/kg or in untreated animals. The results showed that SDZ-IMM-125 is immunosuppressive in dogs and prolongs graft survival up to 50 days at a dosage of 20 mg/kg/day. However, at this dose histological changes suggestive of liver toxicity were observed in one dog, and mild anaemia was produced,but there was no evidence of nephrotoxicity. Absorption profiles suggested that the drug is rapidly absorbed and metabolized, and that a more frequent daily dosage may be appropriate. Overall, there appeared to be no significant advantage for the analogue SDZ-IMM-125 over cyclosporin. The transplant model was associated with a high spontaneous renovascular thrombosis rate, particularly after cyclosporin administration, which was prevented by the daily administration of aspirin.

the time course of intravenously administered cyclosporine (1 mg kg-1) and its metabolite AM1, AM9, and AM1c were examined in the blood, liver, and spleen of naive Lewis rats. Cyclosporine concentration versus time data for all three tissues were qualitatively similar, following a biexponential model C = Ae-gamma 1t + Be-gamma 2t with maximum cyclosporine concentrations reached at 1 h. Whole-blood cyclosporine clearance, terminal half-life, mean residence time, steady state volume of distribution, and hepatic extraction ratio (calculated from blood data) were similar to previously reported results. Cyclosporine in the liver showed the largest area under the concentration-time curve, mean residence time, and disposition and terminal half-lives. Spleen cyclosporine mean residence time and and terminal half-life were not significantly different from blood parameters. Metabolites AM1, AM9, and AM1c showed almost parallel time courses in all three tissues. The hydroxylated derivative AM9 was the major metabolite found in all tissues, with twofold greater levels in the liver compared to the blood and spleen. Slightly less AM1 was found in the liver relative to blood and spleen, where it was present in equal amounts. AM1c levels in the liver were not different from those in the spleen and were greater than observed for blood. The results obtained above were reflected in preliminary studies using liver transplanted rats treated with multiple doses of cyclosporine. Both blood and liver biopsy levels of CyA, AM1, and AM9 post-transplant showed twofold to fourfold decreases from day 3 ( samples taken 4 h post-CyA-dose) and concentrations were not significantly different from similarly sampled naive controls. More importantly, the metabolite/CyA ratios did not vary significantly between liver and blood in the two groups. For naive rats, and liver transplanted animals not undergoing rejection, changes in blood cyclosporine levels seem to predict variations in tissue concentrations.

A cyclosporine derivative, dihydrocyclosporine D, was used for the evaluation of milk fat globule membrane (MFGM) as an emulsifier of lipophilic cyclopeptides. As compared with olive oil formulation, MFGM emulsion significantly enhanced the blood and lymphatic fluid concentrations of the cyclosporine derivative after intraduodenal dosing in rats. Thus, it was suggested that MFGM can be used as an intestinal absorption enhancer of cyclosporines.

The immunosuppressant, SDZ IMM 125 (IMM), is a derivative of cyclosporin A (CyA). The disposition kinetics of IMM in plasma, blood cells, and various tissues of the rat was characterized by a physiologically based pharmacokinetic (PBPK) model; the model was then applied to predict the disposition kinetics in dog and human. Accumulation of IMM in blood cell is high (equilibrium blood cell/plasma ratio = 8), although the kinetics of drug transference between plasma and blood cell is moderately slow, taking approximately 10 min to reach equilibrium, implying a membrane-limited distribution into blood cells. A local PBPK model, assuming blood-flow limited distribution and tissue/blood partition coefficient (KP) data, failed to adequately describe the observed kinetics of distribution, which were slower than predicted. A membrane transport limitation is therefore needed to model dynamic tissue distribution data. Moreover, a slowly interacting intracellular pool was also necessary to adequately describe the kinetics of distribution in some organs. Three elimination pathways (metabolism, biliary secretion, and glomerular filtration) of IMM were assessed at steady state in vivo and characterized independently by the corresponding clearance terms. A whole-body PBPK model was developed according to these findings, which described closely the IMM concentration-time profiles in arterial blood as well as 14 organs/tissues of the rat after intravenous administration. The model was then scaled up to larger mammals by modifying physiological parameters, tissue distribution and elimination clearances; in vivo enzymatic activity was considered in the scale-up of metabolic clearance. The simulations agreed well with the experimental measurements in dog and human, despite the large interspecies difference in the metabolic clearance, which does not follow the usual allometric relationship. In addition, the nonlinear increase in maximum blood concentration and AUC with increasing dose, observed in healthy volunteers after intravenous administration, was accommodated quantitatively by incorporating the known saturation of specific binding of IMM to blood cells. Overall, the PBPK model provides a promising tool to quantitatively link preclinical and clinical data.

OG 37-325 (nva-cyclosporine, cyclosporine G) is structurally similar to cyclosporine A (CsA). We hypothesized that OG 37-325 may, therefore, be metabolized by P450 3A, an enzyme recently shown to metabolize CsA. To test this hypothesis, we employed the erythromycin breath test (ERMBT) to measure P450 3A activity on multiple occasions in twenty OG 37-325-treated renal transplant recipients. When stable dosing was achieved, there was a measured 6-fold variation in the ratio of 12-hr whole-blood parent compound trough concentration (ng/ml, HPLC) to daily OG 37-325 dose (mg/kg) ([OG 37-325]/dose). In support of our hypothesis, there was an inverse correlation between the ERMBT result and the [OG 37-325]/dose ratio (r = -0.71, P < 0.001, n = 20); that is, patients with higher P450 3A activity generally required higher OG 37-325 doses to attain target blood levels. We also found that intrapatient variation in the [OG 37-325]/dose ratio observed over the course of the study correlated with changes in the ERMBT results (r = -0.67, P = 0.002). Inter- and intrapatient differences in [OG 37-325]/dose ratio were not predicted by patient age, serum cholesterol, blood hematocrit, or traditional liver chemistries. We conclude that P450 3A is generally rate limiting in the elimination of OG 37-325 in adult renal transplant recipients. Therefore, most drug interactions observed with CsA should also be expected with OG 37-325. We also conclude that intrapatient changes in OG 37-325 dosing requirements largely result from changes in P450 3A activity. The ERMBT may therefore provide useful information concerning patient compliance and may also serve as a useful guide to OG 37-325 dosing adjustments.

The steady-state concentrations of cyclosporin G (OG37-325) (CsG) and six of its metabolites (GM1, GM9, GM4N, GM1c, GM1c9, GM19) were measured throughout the 12-h dosing interval in six renal transplant recipients receiving CsG as prophylaxis against acute cellular rejection. The mean 12-h whole-blood trough concentrations (micrograms/L) were CsG, 131 +/- 26; GM1, 79 +/- 55; GM9, 110 +/- 114; GM4N, 28 +/- 18; GM1c, 31 +/- 18; GM1c9, 216 +/- 145; and GM19, 303 +/- 217. The relative concentration of the primary metabolites (GM1, GM9, GM4N) remained stable with respect to CsG throughout the dosing interval, whereas that of the secondary metabolites increased. The secondary metabolites GM19 and GM1c9 exhibited extensive between-patient variation. We investigated the effect of these metabolites on commercially available monoclonal antibody-based fluorescence polarization immunoassays (FPIA) and RIAs adapted for measurement of CsG. The 12-h whole-blood trough concentrations measured by FPIA and RIA exceed those measured by HPLC by 19% and 36%, respectively. These measured biases corresponded closely with the calculated biases (FPIA 19%, RIA 28%) based on the known cross-reactivities of CsG metabolites and their concentrations. These results suggest that cross-reactivity with metabolites account for a large part of the bias observed in immunoassays of CsG.

The immunosuppressive efficacy of CsA and its derivative SDZ IMM-125 was compared in highly mismatched mongrel dogs in receipt of renal allografts. At an equal dose of 15 mg/kg/day and in the same drug vehicle, SDZ IMM-125 was not superior to CsA in prolonging allograft survival. Whole blood levels of SDZ IMM-125 were lower than those achieved for CsA. No specific drug-related side effects were noticed in this model.

Cyclosporin G (CsG) is less nephrotoxic than cyclosporin A (CsA) and is undergoing clinical trials for use as an immunosuppressive agent after renal transplantation. Three assays for whole-blood CsA-HPLC, RIA (INCSTAR, Cyclo-Trac SP), and FPIA (Abbott TDx)--were adapted for use with CsG and were assessed for analytical suitability and to determine which assay was capable of deriving CsG values rapidly after transplantation. The assays were acceptable in terms of sensitivity, linearity, analytical recovery, and precision. When considering blood samples (n = 107) from renal transplant recipients receiving a low dose of CsG (5 mg/kg per day) and a high dose (10 mg/kg per day), we obtained the following correlation data: RIA = 0.974HPLC + 27.89 (r = 0.9798, Sy/x = 39.24); FPIA = 0.964HPLC + 33.59 (r = 0.9819, Sy/x = 36.66); and FPIA = 0.977RIA + 9.50 (r = 0.9894, Sy/x = 28.12). The FPIA of CsG is recommended as the most rapid method, although it is the most expensive. HPLC, RIA, and FPIA were capable of accurately deriving projected CsG concentrations at various stages of the clinical trial when the low- and high-dose regimes were tapered over a period of 16 weeks.

Immunoassays of cyclosporin A (CsA) have been routinely used to measure CsG. We investigated the cross-reactivities of CsG and its metabolites, as well as the proportion CsG constitutes in relation to total drug measured, for six CsG metabolites (GM1, GM9, GM4N, GM1c, GM1c9, GM19) in the following CsA assays: Sandimmune selective RIA (SS), Sandimmune nonselective RIA (NS), Cyclotrac SP-RIA (CT), fluorescence polarization immunoassay (FPIA), and enzyme immunoassay (EMIT). The cross-reactivity of CsG in these assays was as follows: SS, FPIA, CT, approximately 100%; NS, approximately 40%; EMIT, < 2%. The cross-reactivities of CsG metabolites were investigated in all assays except EMIT and varied among metabolites and assays. The most significant variance was found with the NS assay, where most of the metabolites exhibited cross-reactivities of > 40%. In contrast, in the SS, FPIA, and CT assays, cross-reactivities of < 5% were observed for most of the metabolites. The ranking of cross-reactivities of CsG metabolites in the assays is SS = CT < FPIA < NS. The degree of cross-reactivity did not change significantly when the SS, CT, and FPIA assays were calibrated with CsG instead of CsA--whether parent CsG was present or not. The data suggest that the SS, CT, and FPIA methods would be suitable for the routine monitoring of CsG.

Two commercial monoclonal immunoassays for monitoring cyclosporin A were used to measure whole-blood concentrations of the immunosuppressant cyclosporin G (CsG) in renal transplant patients. We performed a three-way comparison of these two immunoassays and HPLC. Although the two immunoassays agreed favorably, both the monoclonal fluorescence polarization immunoassay and the monoclonal RIA yielded higher CsG results for patients' specimens than did the liquid-chromatographic assay. The experimental data indicate that the observed differences are most likely due to the cross-reactivity of CsG metabolites in the immunoassays.

Chronic tubulointerstitial nephropathy during long-term cyclosporine A (CsA) use has led to a search for equally effective but safer analogues. In this study we evaluated one of these analogues, cyclosporine G (CsG), in a rat model of chronic cyclosporine nephrotoxicity. CsG has immunosuppressive effects equivalent to CsA when dosed on a weight basis. Pair-fed Sprague-Dawley rats kept on a low-salt rice diet were given CsA 15 mg/kg, CsG 15 mg/kg, CsG 25 mg/kg, or vehicle subcutaneously. After 21 days, CsA animals had a lower glomerular filtration rate, measured by inulin clearance (0.16 +/- 0.04 ml/min/100 g) and higher serum creatinine (0.94 +/- 0.06 mg/dl) than CsG 15 mg/kg (GFR: 0.41 +/- 0.10 ml/min/100 g and serum creatinine: 0.68 +/- 0.09 mg/dl), CsG 25 mg/kg (GFR: 0.39 +/- 0.16 ml/min/100 g) or control rats (GFR: 0.62 +/- 0.06 ml/min/100 g; serum creatinine: 0.56 +/- 0.03 mg/dl), respectively (P < 0.05). The CsA group had considerable cortical and medullary injury (interstitial fibrosis and tubular atrophy), whereas both groups of CsG animals had more limited changes. Despite the same or larger doses of CsG on a weight basis, cyclosporine blood levels were significantly lower in CsG than CsA rats. We conclude that CsG, an analogue of cyclosporine with immunosuppressive activity equivalent to that of CsA, produced less nephrotoxicity in a model of chronic renal injury in rats, using both functional and structural parameters.

In a series of 256 recipients of paediatric liver transplants, from 1984 to 1990, four patients presented with sudden onset seizures not explained by conventional work-up. None had a family or personal history of seizures. Infectious causes were excluded. There were no glucose or electrolyte disturbances. Seizures were not induced by systemic or intracranial hypertension. One child out of four had transient white matter and cortex focal lesions on computed tomography of the brain. One to 10 days before seizures all four children presented with supratherapeutic concentrations of serum cyclosporin that were determined by a non-specific method that measured the parent compound plus its metabolites. The supratherapeutic concentrations were not found with the specific method measuring cyclosporin alone. It is concluded that these seizures may correspond to a toxic effect of cyclosporin, probably due to one or several metabolites, as suggested by the discrepancy between specific and non-specific methods of determination.

We report here on the distribution of cyclosporin G (CsG), an analog of cyclosporin A, in whole blood. CsG has a temperature-dependent distribution between erythrocytes (RBCs) and plasma. After 30 min at 37 degrees C, the plasma/whole blood and plasma/RBC ratios were relatively constant (0.7-0.8) up to CsG at 1000 micrograms/L. At 5000 micrograms/L, this ratio increased to 1.0-1.1 for plasma/whole blood and 1.7 for plasma/RBC. The primary CsG metabolites GM1 and GM9 were sequestered within RBCs to a greater extent than was the parent drug. In whole blood, approximately 2% of CsG was bound to granulocytes, 6% to lymphocytes, and 50-55% to RBC, and 35-40% was found in the plasma fraction. The free fraction of the drug as determined by ultracentrifugation was 5-6% and 13-17% of total drug at 37 and 4 degrees C, respectively. In plasma the drug was primarily associated with high-density lipoprotein (50-60%) and to a lesser degree with low-density (20-30%) and very-low-density (10%) lipoproteins.

Three modifications of the Chem-1 determination of serum creatinine were tested. Two different algorithms developed for compensating the interference of bilirubin were compared with the current one by analysing bilirubin-enriched albumin solutions and 82 icteric serum samples. A number of known other interfering substances were also tested. The modification involving parallel bichromatic measurement plus bilirubin correction appeared to give the best performance. However, despite the improvements, the determination is not yet completely satisfactory.

A sensitive high-performance liquid chromatographic method for the analysis of the immunosuppressant cyclosporin G (OG 37-325, Nva2-cyclosporine, CsG) in whole blood has been developed. Sample preparation, employing cyclosporin A (CsA) as internal standard, involves organic extraction with methyl t-butyl ether under sequential acidic and basic conditions. Chromatography is performed using a 2 mm inside diameter x 25 cm column packed with 5 microns octyl (C8) material. An isocratic mobile phase comprised of acetonitrile:methanol:water, at a flow rate of 0.4 ml/min, is utilized. Separation is monitored at 230 nm. Data are also presented that demonstrate the use of CsG as an alternative internal standard to cyclosporin D for liquid chromatographic determinations of CsA.

Anaemia is common in children following cardiac transplantation. In a series of 5 children with anaemia beyond the immediate post-operative period one had a hypochromic, microcytic anaemia which corrected with oral iron. The other four had normochromic, normocytic anaemias unresponsive to iron or folate supplementation and associated with inappropriately low levels of erythropoietin. Subcutaneous administration of low dose human recombinant erythropoietin to these four patients resulted in correction of their anaemia. Our findings suggest that erythropoietin deficiency is an important cause of anaemia in transplant recipients and should be sought in cases of anaemia refractory to conventional haematinic therapy. In cases of proven erythropoietin deficiency, treatment with erythropoietin is effective, acceptable to patients and preferable to repeated blood transfusion.