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The European InnoMed-PredTox project was a collaborative effort between 15 pharmaceutical companies, 2 small and mid-sized enterprises, and 3 universities with the goal of delivering deeper insights into the molecular mechanisms of kidney and liver toxicity and to identify mechanism-linked diagnostic or prognostic safety biomarker candidates by combining conventional toxicological parameters with "omics" data. Mechanistic toxicity studies with 16 different compounds, 2 dose levels, and 3 time points were performed in male Crl: WI(Han) rats. Three of the 16 investigated compounds, BI-3 (FP007SE), Gentamicin (FP009SF), and IMM125 (FP013NO), induced kidney proximal tubule damage (PTD). In addition to histopathology and clinical chemistry, transcriptomics microarray and proteomics 2D-DIGE analysis were performed. Data from the three PTD studies were combined for a cross-study and cross-omics meta-analysis of the target organ. The mechanistic interpretation of kidney PTD-associated deregulated transcripts revealed, in addition to previously described kidney damage transcript biomarkers such as KIM-1, CLU and TIMP-1, a number of additional deregulated pathways congruent with histopathology observations on a single animal basis, including a specific effect on the complement system. The identification of new, more specific biomarker candidates for PTD was most successful when transcriptomics data were used. Combining transcriptomics data with proteomics data added extra value., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Cyclophilins are target molecules for cyclosporin A (CsA), an immunosuppressive antimicrobial drug. We have previously reported the in vitro anti-Trypanosoma cruzi activity of H-7-94 and F-7-62 non-immunosuppressive CsA analogues. In this work, we continue the study of the parasiticidal effect of H-7-94 and F-7-62 CsA analogues in vitro and in vivo and we analyse 3 new CsA derivatives: MeIle-4-CsA (NIM 811), MeVal-4-CsA (MeVal-4) and D-MeAla-3-EtVal-4-CsA, (EtVal-4). The most efficient anti-T. cruzi effect was observed with H-7-94, F-7-62 and MeVal-4 CsA analogues evidenced as inhibition of epimastigote proliferation, trypomastigote penetration, intracellular amastigote development and in vivo T. cruzi infection. This trypanocidal activity could be due to inhibition of the peptidyl prolyl cis-trans isomerase activity on the T. cruzi recombinant cyclophilins tested. Furthermore, CsA and F-7-62 derivative inhibited the efflux of rhodamine 123 from T. cruzi epimastigotes, suggesting an interference with a P-glycoprotein activity. Moreover, H-7-94 and F-7-62 CsA analogues were not toxic as shown by cell viability and by aminopyrine-N-demethylase activity on mammalian cells. Our results show that H-7-94, F-7-62 and MeVal-4 CsA analogues expressed the highest inhibiting effects on T. cruzi, being promissory parasiticidal drugs worthy of further studies.

Background: PSC 833 is a second-generation P-glycoprotein (Pgp) antagonist developed to reverse multidrug resistance (MDR). The authors conducted a Phase I study of orally administered PSC 833 in combination with vinblastine administered as a 5-day continuous infusion., Methods: Seventy-nine patients with advanced malignant disease were enrolled in the trial and treated with escalating doses of PSC 833. Pharmacokinetic interactions between PSC 833 and vinblastine were anticipated. Accordingly, when dose limiting toxicities were observed, the dose of vinblastine was reduced as PSC 833 was escalated. Three schedules and two formulations of PSC 833 were used in the study., Results: The maximum tolerated doses of PSC 833 were 12.5 mg/kg orally every 12 hours for 8 days for the liquid formulation in combination with 0.9 mg/m(2) per day vinblastine as a continuous intravenous infusion (CIV) for 5 days; and 4 mg/kg orally every 6 hours for 8 days for the microemulsion formulation in combination with 0.6 mg/m(2) per day vinblastine CIV for 5 days. The principal toxicities for PSC 833 were ataxia and paresthesias and for the combination, constipation, fever. and neutropenia. Increased oral bioavailability and increased peak and trough concentrations were observed with the microemulsion formulation. Significant interpatient variability in pharmacokinetic parameters was observed. Ten patients studied at the MTD for PSC 833 (4 mg/kg orally every 6 hours for 8 days) had inhibition of rhodamine efflux from CD56 positive peripheral lymphocytes as a surrogate for Pgp antagonism. Among 43 evaluable patients with clear cell carcinoma of the kidney, 3 patients had complete responses, and 1 patient had a partial response., Conclusions: PSC 833 in combination with vinblastine can be administered safely to patients provided the vinblastine dose is adjusted for pharmacokinetic interactions. The high interpatient variability is a significant confounding factor. Surrogate studies with CD56 positive cells suggest that Pgp inhibition in the clinical setting is achievable. Improved methods for predicting pharmacokinetic interactions should improve future studies., (Copyright 2001 American Cancer Society.)

Administration of antiasthmatic drugs in the form of inhalation particles may alter the cytokine network in the airways, independently of their pharmacological actions. Changes induced by drugs not well tolerated may potentially contribute to the immunopathology of the disease, a strongly undesirable effect. In this study, cell viability assays and characterization of the cellular profile of cytokines and chemokines were performed in order to investigate the response of human bronchoalveolar macrophages and bronchial epithelial cells in culture to inhalation particles of the cyclosporine derivative IMM 125. Interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and IL-8 were assayed by enzyme-linked immunosorbent assay (ELISA) in the supernatants of bronchoalveolar macrophages, and RANTES, granulocyte--macrophage colony-stimulating factor (GM-CSF), and IL-8 in those of bronchial epithelial cells. Cells were studied both under basal and stimulated conditions (lipopolysaccharide and TNFalpha were used for activating macrophages and epithelial cells, respectively). The immunosuppressant FK 506 and the glucocorticoid Budesonide served as comparison. IMM 125 did not affect cell viability (except at high concentrations and long time periods). Moreover, IMM 125 did not induce an increase in the secretion of any of the cytokines and chemokines measured with respect to nontreated cells, except for a slight increase in IL-8, an effect that was also observed for FK 506, Budesonide, and inert latex particles, and was therefore regarded as nonspecific. Furthermore, IMM 125 significantly decreased the secretion of TNFalpha, IL-1beta by macrophages, and GM-CSF by epithelial cells, suggesting an antiinflammatory potential. In conclusion, the present in vitro results point to a good tolerance of human airways to IMM 125 inhalation particles.

Background: One adverse side-effect of the immunosuppressive drug cyclosporin A (CsA) is a decrease in glomerular filtration rate (GFR). This effect might be the result of increased glomerular contractions. The present study compared the contractile effects of CsA, cyclosporin G (CsG) and the novel cyclosporin derivative IMM 125 in isolated rat glomeruli and primary cultures of rat mesangial cells., Methods: Interactive image analysis was used to measure glomerular and mesangial cell contraction., Results: CsA, CsG, and IMM 125 at concentrations of 0, 10(-9), 10(-8), 10(-7), 10(-6) and 10(-5) M caused a time-dependent and a concentration-dependent contraction of isolated glomeruli and mesangial cells 30 min after incubation. In glomeruli, CsA was more potent than CsG and IMM 125. In mesangial cells, IMM 125 also exhibited the lowest contractile activity, while CsA and CsG were almost equally myoreactive. The absolute degree of the glomerular contraction was proportional to the number of contracting mesangial cells in one glomeruli. The number of responding cells after incubation with IMM 125 and CsG were lower compared to CsA, which might explain the different response with CsG and CsA in both models., Conclusions: Since the concentrations used in these experiments were close to that reached in rat serum after treatment with CsA, the present results suggest that the contractile effects of IMM 125 and CsG in isolated glomeruli were clearly smaller compared to CsA, which might reflect the cyclosporins induced GFR changes in vivo.

The novel immunosuppressive drug O-hydroxyethyl-D(Ser)8-cyclosporine (SDZ IMM 125) and cyclosporine A (CyA) were compared in different in vitro models with respect to hepatocellular side effects. SDZ IMM 125 was less lipophilic than CyA and also decreased liposomal membrane anisotropy less. Furthermore, SDZ IMM 125 increased Na+ and Ca++ permeability across the liposomal membranes significantly more than CyA. The uptake of CyA and SDZ IMM 125 into freshly isolated rat hepatocytes was neither saturable, Na+ dependent or temperature sensitive, nor could it be inhibited vice versa, indicating passive diffusion. The diffusion coefficient of CyA was about two times higher than that of SDZ IMM 125, reflecting its higher lipophilicity. In primary hepatocyte monolayers the cellular concentrations of CyA were about two times higher than that of SDZ IMM 125. As an indicator of cholestasis the saturable uptake of cholyltaurine into isolated cells was found to be apparently competitively inhibited to the same extent by both compounds. In isolated perfused rat livers SDZ IMM 125 caused a significantly greater decrease in bile flow than did CyA. Release of lactate dehydrogenase from hepatocyte primary cultures and from isolated perfused livers were determined as parameter of cell damage. In both systems the cytotoxicity of SDZ IMM 125 was significantly higher than that of CyA. The data suggest that SDZ IMM 125 causes greater cholestatic and cytotoxic effects than CyA at equimolar cellular exposure.

Conventional methods that are used to overcome multidrug resistance (MDR) often involve the coadministration of chemosensitizers and anticancer drugs. The cyclosporin analogue SDZ PSC 833 [(3'-keto-Bmt1)-(Val2)-cyclosporin] (PSC 833) has been shown to possess powerful chemosensitization properties in vitro, in addition to being intrinsically nontoxic. However, coadministration of PSC 833 with anticancer drugs, such as daunorubicin, doxorubicin (DOX), and Taxol, have resulted in the exacerbation of anticancer drug toxicity, which is due to altered anticancer drug pharmacokinetics. Here, we hypothesized that optimization of the anticancer drug delivery, using liposomal carriers, may, by avoiding these adverse interactions, offer a significant advantage over nonencapsulated drugs. Toxicity studies were conducted in normal BDF1 mice, with i.v. DOX (free or liposome encapsulated) administration and p.o. PSC 833 in single and multiple dosage regimens over a 15-day study period. p.o. administration of PSC 833, at a dose of 100 mg/kg, reduced the maximum tolerated dose (MTD) of i.v administered free drug by 2.5-3-fold, in single- and multiple-dose regimens. In contrast, PSC 833 administration resulted in only a 20% reduction of the MTD for DOX encapsulated in 100-nm 1,2 distearoyl-sn-glycero-3-phosphocholine/cholesterol liposomes (55:45 molar lipid ratio) in a single-dose regimen and had no effect on the liposomal DOX MTD for the day 1, 5, and 9 treatment schedule. Modest modulation of P-glycoprotein-mediated MDR was observed in the murine P388/ADR solid tumor model when PSC 833 was administered with free DOX at the MTD. In contrast, liposomal DOX combined with PSC 833 resulted in tumor growth inhibition that was comparable to that observed for drug-sensitive P388/WT tumors. This efficacy of P388/ADR tumors treatment was dependent on PSC 833 because treatment with liposomal DOX alone provided significantly less antitumor activity. Pharmacokinetic and tissue distribution data demonstrated that DOX encapsulated in 1,2 distearoyl-sn-glycero-3-phosphocholine/cholesterol liposomes exhibited comparable plasma elimination and tissue distribution properties in the presence and absence of PSC 833, whereas free DOX displayed reduced plasma elimination rates and altered tissue distribution in the presence of PSC 833. These results provide evidence that PSC 833 can induce P-glycoprotein modulation and chemosensitize MDR tumors in the absence of altered DOX pharmacokinetics when liposomal carriers are used. This suggests that the improved tumor selectivity of anticancer drugs that are administered in liposomal formulations may avoid the complications that are associated with free drug-MDR-reversing agent combinations and enhance the therapy of multidrug-resistant tumors.

In the present study it was investigated whether and by which mechanisms the co-administration of interleukin-3 (IL-3) and the P-glycoprotein blocker PSC 833 can augment mitoxantrone (MX) and daunorubicin (DAU) cytotoxicity in two human growth factor dependent P-glycoprotein (P-gp) positive myeloid leukemic cell lines, Mo-7 and GF-D8. Cytotoxicity was determined in MTT assay. Increased cytotoxicity occurred in Mo-7 cells preincubated with 24h IL-3 followed by 1 h MX (cell survival: 85% +/- 6 vs 68% +/- 2, at 0.05 microM MX, P < 0.005) or DAU (79% +/- 8 vs 62% +/- 9 at 0.8 microg/ml DAU, P < 0.05). Similar results were obtained for the GF-D8 cell line. In this cell line, at 0.5 microM MX the cell survival decreased from 84% +/- 13 to 61% +/- 19 (P < 0.05) and at 5.0 microg/ml DAU from 102% +/- 8 to 69% +/- 5, (P < 0.002). The IL-3 administration did not affect the P-gp and bcl-2 protein expression, cellular MX concentration or MX efflux but coincided with an increased percentage of cells in S-phase and topoisomerase II (topo II)-alpha mRNA and topo II activity especially in the Mo-7 cell line. PSC 833 enhanced DAU cytotoxicity in both cell lines. The administration of IL-3 plus PSC 833 in the Mo-7 cell line resulted in an additive effect on DAU cytotoxicity. At 0.8 microg/ml DAU and 2 microg/ml PSC 833, the percentage surviving cells decreased from 62% +/- 9 in the absence of IL-3 to 37% +/- 3 in the presence of IL-3 (P < 0.01). The additive effect of combined treatment was most pronounced in GF-D8 cells which also had the highest P-gp expression. In contrast, PSC 833 did not modulate the MX effects, irrespective of the presence of IL-3. In summary, the results demonstrate that the combined administration of IL-3 and PSC 833 can enhance the cytotoxic effects of DAU but not MX in these P-gp positive cell lines whereas the effects of MX could be modulated by factors which influence topo II activity.

This study compares the toxic effects of native cyclosporia A (CyA) with those of targeted CyA that is conjugated with the anti-rat-thymocyte antibody of rabbit origin via the N-(2-hydroxypropyl)methacrylamide (HPMA) carrier bearing digestible, reactive oligopeptide side chains. Ten toxic doses of native CyA (50 mg/kg i.p.) given to young adult rats in the course of 14 d produced a severe renal lesion-diffuse microvacuolization of the proximal tubules in the deep cortex, and hypergranulation of juxtaglomerular regions. Severe atrophy of the thymic medulla was documented by morphometry. In the cortex the epithelial reticular (but not deep interdigitating) cells showed ultrastructural signs of severe degeneration and lysis. The immature CD4+8+ double-positive cortical lymphocytes were preserved whereas the single-positive medullary thymocytes were greatly depleted; there was also a restriction of MHC class II antigen expression in the medulla. The number of medullary B cells was increased. The cytokeratin net was focally shrunken in the cortex and almost negative in the medulla, with loss of Hassall's corpuscles. After ten corresponding doses of antibody-targeted conjugated CyA no damage to the renal tubules and arterioles appeared and the antiGBM or immune-complex deposition was absent. The thymus had a normal medulla with numerous mature thymocytes and the cortical epithelial reticulum remained well preserved. Thus, the main toxic effects of CyA could be eliminated by targeting. The T-cell-targeted drug was tested for preserved immunosuppressive properties and non-toxic character of HPMA copolymer carrier.

ASSESSMENT OF RENAL FUNCTION: There are a number of methods of evaluating renal function, including measurements of glomerular filtration, renal plasma flow, tubular function, micro- and macroalbuminuria and urinary sediment. Of these, microalbuminuria, glomerular filtration and renal plasma flow are the most appropriate. RENAL EFFECTS OF CALCIUM ANTAGONISTS: Calcium antagonists have important effects on renal function, including a reduction in renal vasoconstriction, increased renal blood flow and, in some circumstances, reduced protein excretion. In particular, these agents can reverse the mild renal vasoconstriction that is seen in the offspring of hypertensive patients. The renal effects of calcium antagonists have been studied in animal models, where radioimaging techniques have shown a biphasic haemodynamic response. RENAL PROTECTION WITH CALCIUM ANTAGONISTS: Two important beneficial effects of calcium antagonists are prevention of acute renal failure and protection against cyclosporin nephrotoxicity. Calcium antagonists have thus been used therapeutically in renal transplant patients and in patients with acute renal failure secondary to the effects of nephrotoxic agents.

Joint, muscle, and peripheral nerve allotransplantation was done with short-term cyclosporine immunosuppression. To investigate the effectiveness of this regimen, the allografts were examined after withdrawal of cyclosporine. Using inbred rats, vascularized orthotopic allotransplantation of the knee joint, rectus femoris muscle, and great saphenous nerve was done across a major histocompatibility complex barrier. Cyclosporine was administered for 4 to 6 weeks postoperatively, and the grafts were observed until Week 12. Long-term administration of cyclosporine and nonvascularized transplantation were used as controls. Although rejection of the allografts could be delayed for 2 to 3 weeks after the withdrawal of cyclosporine, all transplanted joints, muscles, and nerves eventually were rejected completely and immunotolerance could not be induced. The joint allografts at first achieved bony union, but eventually were destroyed because of pathologic fractures. In the group treated with long-term immunosuppression, the allografts showed no rejection and functional improvement was obtained. However, rats given a high dose (10 mg/kg per day) of cyclosporine died from adverse effects of the drug by Week 12. In the nonvascularized treatment group, the results were poor in every patient, and the need for graft vascularization for skeletal tissue allotransplantation was confirmed.

In the mouse, the P-glycoprotein-directed chemosensitizer SDZ PSC 833 could both restore a therapeutic window for doxorubicin against multidrug-resistant tumors, by inhibiting P-glycoprotein function, and increase the anti-cancer drug efficacy against drug-sensitive tumors, by increasing doxorubicin bioavailability. Since the success of such combined chemotherapy treatments might have been limited by the myelotoxicity of doxorubicin and the P-glycoprotein expression on some blood cells, their lymphotoxicity and myelotoxicity was studied on normal B6D2F1 mice, and whenever possible, the persistence of blood cell alterations was also searched for in scid recipients of lymphohaematopoietic grafts from the donor mice. Analyzed parameters were blood, lymphoid and myeloid cell numbers, proliferative responses to T- and B-cell mitogens, and serum immunoglobulin levels. Cell alterations caused by doxorubicin alone were potentiated by SDZ PSC 833, but did not persist in scid recipients. Chemotherapy regimens combining SDZ PSC 833 and doxorubicin, and known for their therapeutic benefit for multidrug-resistant tumor-bearing mice, only caused a rather mild toxicity for the lympho-myeloid system of normal mice.

Cyclosporine A (CsA) nephrotoxicity has been suggested to be aggravated in the presence of ischemia, as occurs after renal transplantation. Cyclosporine G (CsG) may be less nephrotoxic than CsA. This study evaluated in the rat (1) the effect of CsA and CsG on blood flow and the function of the kidney subjected to 60 min of warm ischemia and (2) the protective effect of the calcium antagonist verapamil (VP). After left nephrectomy, ischemia was induced in the right kidney by the clamping of the kidney pedicle for 60 min, which resulted in a significant increase in serum creatinine (SCr) to 2.30 +/- 0.25 mg/dL by Day 1 with 25% mortality by Day 7. The administration of CsA or CsG (20 mg/kg i.v. daily for 7 days) after 60 min of renal ischemia significantly increased SCr and mortality compared with ischemia alone. In another set of experiments, 60 min of warm ischemia was applied to the right kidney and RBF was measured in both kidneys with a laser Doppler flowmeter. Blood flow in the ischemic kidney returned to the preischemic level by 15 min after the removal of the vascular clamp in the control animals. In contrast, in animals treated with CsA, a significant decrease in RBF was seen in both kidneys; however, blood flow in the ischemic kidney was significantly lower than that in the nonischemic kidney. CsG also decreased RBF in both kidneys, although in the left (nonischemic) kidney, RBF remained significantly higher with CsG than with CsA.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Cyclosporin G (CsG) is an analogue of cyclosporin A (CsA) with strong immunosuppressive activity. We compared these two drugs in a rat model in which salt depletion promotes irreversible renal interstitial fibrosis with renal dysfunction in animals given CsA for three weeks. When both drugs were given in the same dosage on a weight basis (15 mg/kg/day, subcutaneously), CsA blood levels were higher than CsG (3305 vs. 1824 ng/ml, P < 0.001). This could be explained by a higher CsG clearance (6.4 vs. 4.3 ml/min/kg in CsA, P < 0.0001) resulting in smaller CsG area under the curve. There was also lower renal and hepatic CsG tissue concentrations. CsA induced a dramatic decrease in GFR, 0.14 in CsA versus 0.67 ml/min/100 g in control, P < 0.001, and increased urinary excretion of N-acetyl beta-D-glucosaminidase (NAG), 21 in CsA versus 13 IU/gCr in control rats, P < 0.001. CsG-treated and control rats had similar GFR and urinary NAG. When CsA dosage was decreased to 7.5 mg/kg blood levels were similar to those found with CsG 15 mg/kg. CsA at this dose caused a reduced GFR (0.29 ml/min/100 g) and an increased urinary NAG (20 IU/gCr) (P < 0.01 vs. control for both). Both dosages of CsA induced considerable cortical and medullary injury (interstitial fibrosis and tubular atrophy), more severe than the histological damage found in CsG-treated rats. Neither drug promoted significant changes in liver function or histology.(ABSTRACT TRUNCATED AT 250 WORDS)

The potential nephrotoxicity of cyclosporine A (CsA), its three main metabolites: M1, M17 and M21, and its two analogues: cyclosporines C and D (CsC, CsD) was evaluated in vitro in suspensions of freshly isolated rabbit renal proximal tubular cells. This assessment involved the measure of enzyme release in the incubation media and the determination of Na+/K(+)-ATPase activity and glutathione content directly in the tubular cells. In vitro nephrotoxicity results of the six compounds tested could be respectively schematized as: CsA > CsD > CsC > M21 > M17, M1 It would be interesting to promote the study of promising CsC because of its low nephrotoxicity and its high immunosuppressive potency as previously reported.

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.

We studied the effect of ciclosporin (Cs) on rats which underwent a 90% hepatectomy. Rats were divided into two groups: group 1 (without Cs pretreatment) and group 2 (with Cs pretreatment). Animals were given a 4-day treatment of Cs (10 mg/kg/day) prior to hepatectomy. The 1-week survival, serum biochemistry and parameters for hepatocyte proliferation (indices for mitosis and 5-bromo-2-deoxyuridine uptake) were serially investigated. Although Cs pretherapy significantly upregulated liver cell proliferation in group 2, there was no improvement in the survival rate of the immunosuppressed animals (group 2) compared to the controls (group 1). The implications of Cs pretherapy are discussed in the setting of extensive hepatectomy.

Renal side-effects are frequently observed after parenteral administration of pentamidine. In this study in a rat model, the nephrotoxicity was assessed by measuring urinary loss of tubular cells, malate dehydrogenase activity and creatinine clearance. In addition, we studied the influence of other nephrotoxins such as tobramycin, amphotericin B and cyclosporin on the pentamidine-associated nephrotoxicity and proved the possibilities of reducing this toxicity by coadministration with other drugs. The tubular toxicity of pentamidine (1, 10 or 20 mg/kg daily) is dose-related and reversible. The toxicity can be reduced by coadministration of fosfomycin (1 x 500 or 2 x 250 mg/kg daily) and D-glucaro-1,5-lactam (2 x 5 mg/kg daily) and enhanced by tobramycin (2 x 2.5 mg/kg daily), amphotericin B (1 mg/kg daily) and cyclosporin (10 mg/kg daily). Furthermore, an increase in the creatinine clearance in pentamidine-treated rats can be obtained with both verapamil (2 x 1.5 mg/kg daily) and enalapril (5 mg/kg daily).

The immunosuppressant cyclosporine and the long-acting somatostatin analog SMS 201-995 (octreotide acetate) may have to be given simultaneously in diseases such as pancreatic transplantation. The aim of the study was to evaluate the effects of SMS 201-995 on the pharmacokinetics of cyclosporine, and to assess whether the addition of SMS 201-995 altered some of the cellular immune and toxic (renal, hepatic, glucose tolerance) effects of cyclosporine. Male Wistar rats were treated with cyclosporine 10 mg/kg/day, SMS 201-995 100 micrograms/kg b.i.d., a combination of the two drugs, or the vehicle alone. The addition of SMS 201-995 delayed the peak plasma levels of cyclosporine without affecting the through levels, and did not alter the effects of cyclosporine on the mononuclear cell subsets. This combination caused significant increases in plasma creatinine and hepatic enzymes, suggesting renal and hepatic toxicity, and severe glucose intolerance. If present in humans, the appearance of severe glucose intolerance with combined administration of SMS 201-995 and cyclosporine for pancreatic transplantation could be misinterpreted as rejection and lead to inappropriate interventions.

The effects of 40 days of treatment with Cyclosporine A (CSA) on plasma and urine free amino acids were investigated in sham-operated (C) and partially nephrectomized (Pnx) female Fischer 344 rats. High Dose CSA (30 mg/kg/day ip) was associated with reduced weight gain, increased plasma urea nitrogen, and hypoproteinemia in C and Pnx animals. These animals also demonstrated increased plasma levels of alanine, markedly reduced levels of tryptophan, and an increase in urinary excretion of methylhistidines. C but not Pnx animals also showed a significant increase in plasma serine and a decrease in plasma taurine. CSA treatment of group C resulted in a progressive aminoaciduria involving substrates of the neutral and acidic renal amino acid transport systems; however, the renal excretion of taurine and beta-alanine by these animals was markedly reduced as compared to vehicle treated controls. High dose CSA exacerbated aminoaciduria in Pnx animals, but in this group, the excretion of beta amino acids was also increased. Our findings demonstrate that chronic CSA toxicity in rodents with normal renal function is characterized by increased muscle protein catabolism, significant reductions in plasma tryptophan, and an apparent decrease in whole body taurine pools. With the exception of the taurine abnormalities. CSA treatment had similar effects on Pnx animals; however, in this group, CSA-induced pathological changes were superimposed on the changes due to renal insufficiency per se. CSA toxicity as identified by the parameters investigated in this study was no more severe in Pnx animals with moderate chronic renal insufficiency than in controls with intact renal function.

Several approaches have been attempted to manage renal allograft dysfunction in cyclosporine-prednisone (CsA-Pred)-treated patients. Conversion to conventional therapy and perioperative triple drug have been associated with high rates of acute rejection episodes, infections, or neoplasms. We report our experience in delayed addition of azathioprine (1-2 mg/kg/day) to CsA/Pred protocol in three groups of patients. Group I (n = 9) had chronic renal function deterioration due to chronic rejection; group II (n = 10) had repeated or severe acute rejection episodes despite adequate CsA levels; and group III (n = 8) had CsA toxicity despite drug tapering. In group I, serum creatinine (SCr) had risen from 2.2 +/- 0.9 to 2.9 +/- 0.7 mg/dl over the 6 months prior to Aza addition (P less than 0.05), renal function declining at a rate of -0.14 +/- 0.12 Cr-1/year. In the 6-month post-Aza, renal function improved at a rate of 0.06 +/- 0.06 Cr-1/year and during the entire follow-up at a rate of 0.04 +/- 0.12 Cr-1/year (P less than 0.05) with stable CsA levels (288 +/- 167 vs. 251 +/- 172 ng/dl, NS). In group II response was worse, though the rate of declining renal function prior to Aza (-0.10 +/- 0.10 Cr-1/year) was almost stopped after Aza. In group III there was very good response to Aza addition, as 7 out of 8 patients improved graft function (baseline SCr 2.5 +/- 0.7 mg/dl vs. 1.9 +/- 0.6 mg/dl at last follow-up, P less than 0.05), with significantly decreased CsA levels (480 +/- 97 vs. 268 +/- 120, P less than 0.05). One patient from group II died from pneumonia, and 6 patients (1 from group I and 5 from group II) lost their grafts. Fifteen patients improved graft function, and 9 worsened after addition of Aza. The bad-responders had significantly higher SCr at baseline compared with the good-responders (3.8 +/- 1.8 vs. 2.7 +/- 0.6 mg/dl, P less than 0.01). Amelioration of chronic graft dysfunction can be achieved by delayed addition of Aza to CsA-Pred in patients with chronic rejection or CsA toxicity. This is accompanied by low rate of acute rejection, good patient and graft survival, and low rate of infections. A worse outcome can be seen in patients with high-baseline SCr levels, suggesting the need for addition of Aza in the initial chronic graft dysfunction.

Sprague-Dawley rats were treated for 14 days with rapamycin (RAP; 1.5 mg/kg/day i.p.), cyclosporine (15 mg/kg/day by gavage), both drugs in combination, or appropriate drug vehicles. Hematological parameters and biochemical indices of renal and hepatic function were determined throughout the experimental period, at the end of which the rats were killed and tissues examined histologically. There was a significant reduction in weight gain in RAP- but not CsA-treated animals, while rats given both drugs showed a reduction in body weight over the 14-day experimental period. There were no significant alterations in absolute or differential white blood cell counts or in T or B cell numbers, except in the drug combination group, in which an absolute lymphopenia was detected on day 14. Small but significant increases in urinary flow rate (UFR) were found with either drug alone, and there was a marked (4-fold) increase in UFR in response to drug combination. Both RAP and CsA caused a small elevation in serum creatinine concentrations, but only with CsA was there a significant elevation in urinary enzyme activity and reduction in 51Cr. EDTA clearance. The drug combination exacerbated renal impairment, the extent of which was greater than the additive effect of either drug alone. Hyperbilirubinemia of similar magnitude was observed in rats receiving either CsA alone or in combination with RAP. In contrast to its effect on renal function, however, the CsA+RAP combination was without additional effect on liver function compared with the minor changes seen with either drug alone. Plasma and urinary glucose levels were elevated in all drug treatment groups and especially in animals given both drugs. RAP administration did not significantly affect whole-blood CsA concentrations, although the possibility of a pharmacokinetic interaction cannot be totally excluded. Histological studies revealed striking thymic medullary atrophy in all drug-treated animals. In addition, all rats given RAP showed focal myocardial necrosis of overall mild-moderate severity. Kidneys of RAP-treated rats appeared normal, whereas mild, focal, acute tubular necrosis was evident in all CsA-treated animals. Pancreases of all drug-treated animals were normal.

Cyclosporine is used widely to prevent transplant rejection. Unfortunately, this drug causes both renal glomerular and tubular damage. To develop a stable reproducible noncatabolic animal model of cyclosporine nephrotoxicity, rats were fed a special liquid diet and daily administered 7.5 mg/kg of cyclosporine intramuscularly. Twenty-one days after this regimen was started, the body weight of cyclosporine-treated rats had increased 15%, a value only 38% less than the increase in pair-fed controls. After 21 days, glomerular filtration rate fell 34% in cyclosporine-treated rats (p less than 0.001) compared with the pair-fed controls. Simultaneously, citrate and N-acetylglucosaminidase excretion increased significantly. When isolated proximal tubules obtained after 21 days from the cyclosporine and pair-fed rats were incubated by using 1.0 mmol/L glutamine and 1.0 mmol/L citrate substrates, ammonia production was reduced 40% and glucose production reduced 20% (p less than 0.001) in the cyclosporine-treated rats. Discontinuing cyclosporine for 7 days partially reversed all these changes. The glomerular filtration rate in cyclosporine-treated rats now was only 15% below that in controls (p less than 0.05), N-acetylglucosaminidase excretion was the same in both groups, and urinary citrate excretion in the cyclosporine-treated rats fell from 122% to 62% above that in controls. In vitro differences in tubular ammonia and glucose production also narrowed. In summary, these experiments describe a stable reproducible noncatabolic and at least partially reversible rat model for studying cyclosporine nephrotoxicity.

Cyclosporin-A (CsA) has greatly influenced the outcome of organ transplantation and has also been effective in the treatment of many autoimmune diseases. Unfortunately, it has deleterious effects on bone remodelling, causing a high turnover bone loss, with bone resorption exceeding bone formation. Salmon calcitonin (SCtn) has been shown to inhibit bone resorption in high turnover states such as Paget's disease and postmenopausal osteoporosis. In an attempt to attenuate the high turnover bone remodelling caused by CsA alone, we studied the bone mineral effects of CsA in combination with SCtn in male Sprague-Dawley rats. Group A (n = 20) received vehicle as control, group B (n = 20) received CsA (15 mg/kg BW) by daily gavage and SCtn vehicle sc, group C (n = 20) received SCtn (1.3 IU/kg BW) daily sc and CsA vehicle, and group D (n = 20) received a combination of CsA and Ctn daily, as described above. Rats were bled weekly for determination of circulating biochemical bone parameters. Eight rats from each group were killed on day 14 (short term), and the remaining rats were killed on day 28 (long term). Tibiae were removed for bone histomorphometry after death, which revealed a reduction of trabecular bone volume and an increase in osteoclast number induced by CsA alone. These changes were significantly attenuated by the combination of CsA and SCtn to resemble the histomorphometry of the control group. The inhibition of osteoclast number by SCtn is the most plausible mechanism by which the combination therapy attenuates the high turnover bone loss induced by CsA alone.

The effect of the selective A1-adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX, 0.1 mg kg-1 i.v.) administered twice daily to rats has been assessed on the development of renal dysfunction induced by four daily injections of cyclosporin (60 mg kg-1 i.p.). The series of cyclosporin injections resulted in a polyuria accompanied by a 64-70% increase in plasma urea and creatinine concentrations and a 50% reduction in inulin clearance. However, cyclosporin administration resulted in no change in p-aminohippurate clearance nor was there any evidence of tubular necrosis or vascular damage. CPX treatment did not improve any index of renal function perturbed by cyclosporin. The findings provide evidence that adenosine does not play a role in the pathophysiology of cyclosporin nephrotoxicity.

Male Sprague-Dawley rats were given oral cyclosporine A or control vehicle, and renal protein synthesis and renal ATP levels were examined. Acute oral cyclosporine A at 5, 10, 25 and 50 mg/kg/day for 6 days reduced [3H]L-leucine incorporation by isolated renal microsomes to 76.2, 56.8, 44.3 and 29.5% of control incorporations, respectively. No significant changes in renal ATP levels were detected by NMR spectroscopy after acute oral cyclosporine A administration at the doses indicated. However, during chronic exposure to cyclosporine A at doses of 5 and 25 mg/kg/day for 30, 60 and 90 days, there was a recovery of renal microsomal protein synthesis by day 30 at 5 mg/kg/day, and by day 45 at 25 mg/kg/day. NMR spectroscopy of the kidneys of these rats demonstrated decreases in renal ATP level by day 60 in animals given cyclosporine A at 25 mg/kg/day. Cyclosporine A administration produced a renal acidosis and up to a 40% decrease in renal ATP level by day 90 in rats fed cyclosporine A at 25 mg/kg. No apparent histologic abnormalities were observed in the ATP-deficient renal tissue by NMR imaging. Reductions in renal ATP level suggest that the recovery of renal microsomal protein synthesis is aberrant in the continued presence of cyclosporine A, or that mitochondria are direct sites of cyclosporine A toxicity.

Much is known about the renin-angiotensin system during cyclosporine treatment: plasma renin or prorenin activity seems to be raised in man and animals; hyperplasia of the juxtaglomerular apparatus has been reported in man and animals; renin concentration is known to be increased in animal kidneys; renin-angiotensin-like alterations in vascular contractility have been identified in animal vessels. Since the major functional side effects of cyclosporine are systemic vasoconstriction and renal vasoconstriction, the renin-angiotensin system could be involved. Also, an infrequent complication in man, vasculopathy, develops only in the afferent arteriole, where renin production is most abundant. Hence, this information is now reviewed and incorporated into a single pathophysiological concept, with the implication that renin activation plays a central role in the pathogenesis of cyclosporine-induced functional and structural lesions, merits further attention.

The authors assessed the ocular toxicity and pharmacokinetics of subconjunctivally and intravenously administered cyclosporine in New Zealand white rabbits. Fifteen rabbits received a subconjunctival injection of 5 (five animals), 10 (five animals) or 25 (five animals) mg of cyclosporine in 0.1 mL (intravenous solution of Sandimmune [50 mg/mL]); 5 mg was found to be the maximum tolerable dose. This dose was given as a bolus to 36 other rabbits either subconjunctivally (18 animals) or intravenously (18 animals). In both groups the cyclosporine concentrations in the ocular compartments, blood and urine were measured by means of high-pressure liquid chromatography at 0.5, 1, 2, 4, 8 and 12 hours, three animals being assessed at each interval. Subconjunctival administration resulted in peak cyclosporine concentrations of 718 ng/mL in the aqueous humour and 1078 ng/mL in the vitreous humour, compared with no detectable levels in the aqueous humour and a peak concentration of 292 ng/mL in the vitreous following intravenous administration. The peak blood cyclosporine levels were 10 times lower after subconjunctival injection than after intravenous injection. The results indicate that subconjunctival administration is superior to intravenous administration in enhancing the ocular absorption of cyclosporine while minimizing systemic exposure in the rabbit.

The ability to prepare purified islet Beta-cell aggregates was used to examine the survival of this cell type after allotransplantation in diabetic BB rats. The aggregates were intraportally implanted in numbers that were previously found to correct a streptozotocin-induced diabetic state in syngeneic or allogeneic Brown Norway recipients. When the grafts were prepared from RT1u/l donors, which shared the MHC-class I antigen with the BB recipients (RT1u/u), their implant sites became diffusely infiltrated by inflammatory cells and their metabolic function was completely lost within 5 weeks. MHC-class I incompatible islet Beta-cell allografts (RT1n/n) exhibited a longer survival, in particular when combined with other islet endocrine cells and/or when covered by a 5-week cyclosporin treatment. In the latter combination, 10 of 12 BB rat recipients remained normoglycaemic over the 10-week observation period, their liver implants presenting a comparable insulin reserve and similarly discrete mononuclear cell infiltration as streptozotocin-diabetic Brown Norway rats receiving this treatment. However, administration of cyclosporin to diabetic BB rats was associated with a morbidity that was not observed in drug-treated streptozotocin-diabetic Brown Norway animals or in untreated diabetic BB rats. It is concluded that MHC-incompatible islet Beta cells can induce a long-term normalization in diabetic BB rats provided that they are implanted under conditions which allow allograft acceptance. The standardized preparation of purified islet Beta-cell grafts can help assessing the conditions for successful transplantations in diabetes with an autoimmune origin.

Functional and morphologic techniques were used to study the renal changes induced by a long-term exposure of normal rats to cyclosporine A (CsA), as well as their potential reversibility. CsA treatment for 3 months resulted in a significant (P less than 0.01) reduction of glomerular filtration rate (GFR) as compared with vehicle-treated animals. Histological examination of the kidneys showed mild glomerular damage characterized by ischemic lesions, increased mesangial matrix, and intracapillary hypercellularity in the CsA-treated group, but not in the vehicle-treated group. Proximal tubular abnormalities and limited areas of interstitial fibrosis were also present in the CsA group. A complete reconstruction of glomerular corpuscle was used to evaluate the consequence of CsA-induced renal ischemia on capillary tuft volume. The results showed that in rats administered CsA for 3 months glomerular volume distribution was shifted toward small glomeruli. Prolongation of CsA administration for 5 months did not result in a further decrease in GFR, and was associated with the appearance of a subset of glomeruli that became larger than normal. We have also investigated whether, once established, CsA-induced renal injury is reversible. In rats that were administered CsA for 3 months and then treatment discontinued for 2 months, GFR returned to pretreatment values and partial reversibility of morphologic changes was observed. Morphometric analysis showed that 2 months after withdrawal of CsA, glomerular volume distribution was almost comparable to that observed in vehicle-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The acute cardiotoxicity of cyclosporine was investigated in isolated cardiomyocytes from adult rats. In a first study, myocytes were incubated with CsA ranging from 1 to 10 micrograms/ml and paced by electrical-field stimulation. After 30 min of stimulation the number of surviving rod-shaped myocytes was significantly reduced at 2.5 micrograms/ml (77.9%) and 5 micrograms/ml CsA (64.2%) as compared with the drug vehicle methanol (88.8%, P less than 0.05) with a further decrease at 10 micrograms/ml CsA (30.1% vs. 81.2%, P less than 0.005). In a second study, with the use of digital image processing of fura-2 fluorescence, the mean intracellular free calcium concentration, integrated over 1 sec, of single myocytes in the presence of 5 micrograms/ml CsA, the solvent methanol, or pure Krebs Ringer Hepes buffer was measured. Starting 2 Hz field stimulation increased the intracellular free calcium concentration from 100.1 to 177.9 nM in buffer and from 145.7 to 200.6 nM calcium with methanol. In contrast, there was a 3-fold increase of the intracellular free calcium concentration with 5 micrograms/ml CsA from 128.8 to 376.1 nM calcium. The intracellular free calcium during electrical stimulation was significantly higher with CsA than with the solvent (376.1 nM vs. 200.6 nM, P less than 0.001). In a further study, myocytes were incubated with calcium ranging from 0.5 to 8 mM calcium in the presence of 5 micrograms/ml CsA or the solvent methanol and electrically stimulated. Here, with increasing extracellular calcium the number of rod-shaped myocytes decreased significantly with CsA as compared with the solvent (P less than 0.02). The data suggest that CsA exerts a dose-dependent toxic effect on isolated rat cardiomyocytes that depends on the extracellular calcium concentration. There is direct evidence that CsA increases the intracellular free calcium concentration in rat cardiomyocytes.

Cyclosporine was administered (11 mg/kg/day) to pregnant mice to study the effects of passively transferred CsA on the developing immune system. Placental transfer of CsA was shown by the detection of fetal-tissue levels ranging from 400 to 1500 ng CsA/g tissue. Treatment clearly altered the developing immune system. Thymuses from the day-18 embryos exposed to CsA were partially depleted of CD4+CD8- single positive cells. Eleven of 50 offspring born to CsA-treated mothers developed significant levels of IgG autoantibodies to gastric antigens. In addition, two animals that received CsA in utero developed an extensive mononuclear cell infiltrate in the gastric mucosa resembling autoimmune gastritis. These results raise the possibility that the administration of CsA during pregnancy may result in potential long-term effects on the developing immune system. Offspring of such pregnancies may suffer an increased incidence or severity of autoimmune diseases.

Chronic cyclosporine nephrotoxicity is a poorly understood drug side-effect characterized by renal cortical interstitial scarring. To evaluate procollagen mRNA levels as an early factor in the development of this form of renal fibrosis, we measured renal procollagen alpha 1 (I), alpha 1 (III), alpha 1 (IV) and beta-actin mRNA levels in rats treated with cyclosporine (CsA) or the olive oil vehicle (OO) for one or four weeks. Renal morphology was similar without atrophy or fibrosis in one week CsA and OO and four week OO rats. Four week CsA rats had focal cortical interstitial fibrosis and tubular atrophy. Cortical procollagen alpha 1 (I) mRNA levels were increased in CsA versus OO rats at one week (P less than 0.02) and four weeks (P less than 0.02). One week medullary procollagen alpha 1 (I) and all other one week medullary, and one and four week cortical procollagen and beta-actin mRNA levels were no different in CsA versus OO rats. The early increase in renal cortical procollagen alpha 1 (I) mRNA levels precedes renal morphologic abnormalities, and may represent an important step in the pathogenesis of cyclosporine-induced renal cortical fibrosis.

The effect of cyclosporine on the immunopathogenesis of viral myocarditis was studied using a murine viral myocarditis model. Mice in the treated group had a higher mortality rate compared with those of the infected control group before day 15 (47/67 vs. 14/31, p less than 0.05). On day 7, treated mice showed higher titers of anti-heart autoantibody than the control group (12 +/- 7 vs 4 +/- 2, p less than 0.05), but no significant difference was seen on day 14 (28 +/- 15 vs. 39 +/- 34). Histologic lesions, lymphocyte subsets in the peripheral blood and heart in situ, the neutralizing antibody, and virus concentrations in the heart showed no significant differences between these groups. This study suggests that with the use of cyclosporine the production of anti-heart autoantibody was enhanced in the early stages of viral myocarditis in mice, and was associated with higher mortality rate.

The characteristics of cyclosporin A transport have been studied in cultured kidney epithelial cell line LLC-PK1. The uptake of cyclosporin A by LLC-PK1 cells was time-dependent and reached a steady state at about 30 min. The initial uptake was saturable and was inhibited by cyclosporin A analogs, cyclosporin C and D and by verapamil, but not by metabolic inhibitors such as 2,4-dinitrophenol and rotenone. Cyclosporin A uptake as well as efflux was strongly dependent on temperature. The Arrhenius plot for cyclosporin A uptake was biphasic, whereas the Arrhenius plot for sulfanilamide uptake, which is transported by a simple diffusion, was linear. These results indicate that a specific mechanism is concerned in the transport of cyclosporin A in LLC-PK1, cells.

The cardiotoxic effect of cyclosporine in the inbred rat was investigated in the isolated perfused heart model. Diastolic and systolic function was studied in 5 groups of (Lewis x Brown Norway) F1 rats treated with different doses of cyclosporine for different time intervals and in a group which received the vehiculum cremophor. Of the cyclosporine-treated rats, one group was studied four weeks after drug discontinuation to assess reversibility of the effect. To control for the nephrotoxic side effects of cyclosporine, a group of animals that had undergone 1.5-1.75 renal ablation was included. Left ventricular pressures were measured with a balloon catheter; stepwise increases in preload were obtained by small increments in the balloon volume. Over the range of left ventricular volumes studied, no differences were found in diastolic pressure between the groups. Peak systolic pressures were significantly lower in hearts obtained from cyclosporine treated rats which had received 15 or 7.5 mg/kg/24 h subcutaneously for three weeks (P less than 0.05). Our results indicate that cyclosporine treatment resulted in a reversible decrease in myocardial contractile force apparently unrelated to decreases in renal function or to changes in mean arterial pressure.

An important step in minimizing the number of animal experiments in medical research is the study of in vitro model systems. We propose the use of "shock protein" formation, which is a cellular response to cell-damaging stress as an assay to monitor cardiotoxicity. Isolated and cultured cardiac myocytes were prepared by a trypsin digestion method from 18-day-old fetal mice. These cells respond to typical substances inducing "shock protein" formation in other cellular systems as well as to known cardiotoxins with the de novo synthesis of "shock proteins." Pharmaceuticals relevant in transplant medicine were tested for possible cardiotoxic effects: Cyclosporine A evokes "shock protein" formation at subtherapeutic concentrations. Azathioprine and methyl-prednisolone exert the same effect but at concentration ranges highly above the therapeutic level. The ability to induce "shock protein" synthesis obviously seems to be restricted to toxic drugs. The data presented demonstrate that the proposed in vitro model system for cardiotoxicity is animal saving and sensitive.

In some circumstances cyclosporine (CyA) induces autoimmune phenomena, and a lethal form of syngeneic graft-vs-host disease (SGVHD) can be induced in rats by the administration of CyA. Although several rat strains develop this disease, in this study, we report that of six mouse strains tested, only the DBA/2 strain developed SGVHD. A comparison of the effect of CyA on thymocytic and peripheral T-cell populations revealed that CyA-induced alterations were similar in the two species, although more marked in rats. Notably, CyA-treated syngeneic bone marrow chimeras (SBMC) had transiently increased numbers of peripheral CD4+ CD8+ T-cells, expressing a marker normally limited to thymocytes. Examination of T-cell receptor (TCR) V beta expression in CyA-treated mice revealed a normal pattern of clonal deletion in all strains (including disease-prone DBA/2 mice), suggesting that CyA may induce autoimmunity without blocking intrathymic clonal deletion.

In this report we have approached two questions relating to the mechanism of action of cyclosporin A (CsA). First, we address whether the major cytosolic protein for CsA, cyclophilin, is directly involved in mediating the immunosuppressive activity of this drug, and, in particular, whether inhibition of this protein's peptidyl-prolyl cis-trans isomerase (PPIase) activity results in inhibition of murine T cell activation. Second, we ask whether the nephrotoxicity observed with CsA is related to inhibition of PPIase-dependent pathways in cells other than lymphocytes. Using a series of 61 cyclosporin analogues, we generally found a good correlation between cyclophilin binding and immunosuppressive activity for the majority of analogues analyzed. However, a number of compounds of distinct structural classes were found that could interact with cyclophilin but were much less immunosuppressive than expected. The inability of these analogues to inhibit lymphocyte activation could not be explained by their failure to enter the cell and bind to cyclophilin under the conditions used in the cellular assays. Surprisingly, a nonimmunosuppressive analogue, MeAla-6, which bound well to cyclophilin and was active as a PPIase inhibitor, did not induce renal pathology in vivo. Furthermore, another analogue, MeBm2t, which was immunosuppressive in vitro, possessed little or no activity as a PPIase inhibitor. These findings pose serious questions concerning a direct role of cyclosporin in mediating CsA's immunosuppressive and nephrotoxic activities. In addition, they raise doubts about whether PPIase has a direct function in lymphocyte signal transduction.

A patient with Felty's syndrome was treated with cyclosporine, initially 10 mg/kg/day and then 4 mg/kg/day. The neutrophil count increased by 6 weeks and was normal at 3 months. Over the subsequent 27 months the neutrophil count was closely related to the cyclosporine dosage and there was no evidence of cyclosporine toxicity. This case indicates that cyclosporine may have a role in the treatment of Felty's syndrome.

Alterations in renal prostaglandin production have recently been postulated to modulate the decrease in renal blood flow associated with cyclosporine nephrotoxicity. In particular, increases in renal production of the potent vasoconstrictor thromboxane A2 have been implicated in the pathogenesis of this disorder. The present study was undertaken to explore the relationship between alterations in urinary thromboxane B2 excretion (UTxB2V) and CsA nephrotoxicity in two rat models. Male Sprague-Dawley (SD) rats were treated for 14 days with CsA 50 mg/kg/day (n = 8) or olive oil (C) (n = 9) by gavage. Creatinine clearance (Ccr), urine flow (V), and urinary excretion rates of sodium, N-acetyl-beta-D-glucosaminidase (NAG), glucose, and TxB2 were determined before and after treatment. A similar study was conducted using Fischer rats (CsA: n = 10, C: n = 13). In Fischer rats, CsA caused a 35% decrease in Ccr (P = 0.01), a 33% decrease in sodium excretion (P = 0.02), and a greater than 2-fold increase in NAG excretion (P = 0.03), while V, glucose excretion, and UTxB2V did not change. Although similar changes in sodium and NAG excretion were seen after CsA administration in SD rats, Ccr was not affected. Additional findings in SD rats included a 3-fold increase in V (P less than 0.01), a 24-fold increase in glucose excretion (P = 0.03), and a 5-fold increase in UTxB2V (P = 0.04). Thus, Fischer rats developed CsA nephrotoxicity in the absence of increased UTxB2V. In contrast, SD rats failed to develop nephrotoxicity despite a marked increase in UTxB2V. We conclude that changes in renal TxA2 production are unrelated to the development of CsA nephrotoxicity.