Search

Introduction: Cyclosporine is a molecule used in ophthalmology for the prevention of corneal graft rejection. The systemic use of this product can lead to serious adverse side effects that can be avoided using the topical formulation of cyclosporine. However, cyclosporine application can induce ocular irritation., Material and Methods: The aim of this study is to evaluate the cytotoxicity of four formulations of 2% cyclosporine eye drops: Sandimmum intravenous solution diluted with NaCl 0.9%, Sandimmun oral solution diluted in castor oil or corn oil after ethanol evaporation, and Sandimmun oral solution diluted in castor oil without previous ethanol evaporation. Two tests--the Draize test and the evaluation of cytotoxicity of adherent alive cells with cold light cytofluorimetry on microplates--were used in this study., Results: These tests demonstrated that the aqueous solution shows more toxicity than the other formulations, and the type of oil and ethanol concentration influence cell viability., Conclusion: These results helped the Pharmacy unit choose the vehicles for a safe cyclosporine eye drop formulation and thus decrease the side effects of cyclosporine eye drop instillation with a decrease in ethanol concentration compared to published formulations.

Infectious crystalline keratopathy is an exceptional but serious complication of penetrating keratoplasty. Streptococcus viridans and a steroid treatment are very often found. The treatment is always lengthy and difficult. The high number of bacteria and the steroid treatment, necessary to prevent rejection, worsen the infection. We report a case of Streptococcus crystalline keratopathy that evolved, despite the treatment, simultaneously toward abscess and acute rejection. Topical cyclosporin treatment was used to maintain an immunosuppression without worsening the infection.

Tacrolimus (FK506) and cyclosporin A (CsA) are two potent immunosuppressants mainly metabolized by hepatic cytochrome P-450 3A (CYP3A) monooxygenase. The aim of this study was to compare the toxic effects of the two drugs on hepatocytes in primary culture as a function of their metabolism and to explore the variations of cytotoxicity when both drugs are associated. The cytotoxicity of FK506 and CsA, as expressed by their IC50 values, was of the same order but with a switch according to whether hepatocytes were induced or uninduced by dexamethasone, CsA being more toxic in its native form and FK506 through its metabolism. Similar results were obtained with the intracellular calcium content. When both drugs were associated at their IC50 values, the expected additive cytotoxic effect was not observed. Moreover, when small quantities of FK506 were added to CsA at its IC50, cell viability improved in the induced cultures. It is hypothesized that the interaction between the two drugs relies on a mechanism involving both competition of FK506 and CsA for CYP3A and of their immunophilin complexes for a common site on the calcineurin-calmodulin complex.

Thalidomide (Thd) has been shown to have interesting immunosuppressive properties and strong action against TNF-alpha. It is used for treating a variety of immune-mediated pathology and inflammatory diseases. The purpose of this work was to evaluate the in vitro and in vivo immunosuppressive effects of Thd and its derivative, N-Hydroxythalidomide (H-Thd), alone and in combination with cyclosporin A (CsA), upon different in vitro lymphocyte activation pathways and in vivo local graft-versus-host-reaction (GvHR). At different concentrations, both Thd and H-Thd alone inhibited the lymphocyte proliferation induced by alloantigen (MLR), mitogens (Con A, PWM) and superantigen (SEB) with an activity of 50-75% that of CsA, however, in some tests, immunosuppressive potency of H-Thd was shown to be higher than that of Thd. In vivo using GvHR, Thd and H-Thd alone proved as active as CsA. The association in vitro and in vivo of each compound with CsA at different low concentrations, produced an additive effect as strong as CsA used alone at high therapeutic concentrations. In summarizing, this study revealed that: (1) despite its weaker potency in vitro than that of CsA, H-Thd presents interesting immunosuppressive properties similar to, and in some cases, better than Thd, and (2) the combination of H-Thd or Thd with CsA at suboptimal concentrations leads to high activity.

FK506 and cyclosporin A (CsA) are two potent immunosupressants with similar toxicity profile. Nephrotoxicity is the main adverse effect of both compounds. The aim of this study is to compare the in vitro nephrotoxic effects on renal epithelial cell line LLC-PK1 by measuring cell viability and energy status as evaluated by concentrations of ATP and ATP metabolites. Cell viability (expressed as IC50 was assessed via thiazolyl blue (MTT) assay after incubation for 4-24 h with FK506 or CsA. ATP and its metabolites were determined by HPLC after 4 and 6 h incubation with FK506 or CsA alone at the respective IC50. Both FK506 and CsA decreased cell viability to similar extents, in a dose- and time-dependent manner. After 4 h incubation, both drugs decreased ATP levels (-25%) and increased uric acid levels. However, the latter percentage increase was twofold higher with CsA (18%) than with FK506 (9%). The energy charge, calculated according to levels of adenine nucleotides, was decreased by 10% in FK506-treated cells and by 27% in CsA-treated cells. At the end of 6-h incubation, FK506-treated cells maintained ATP levels coupled with energy charge at near control levels whereas the levels were 32% lower in CsA treated cells. Compared to the 4 h-incubation, the increase in uric acid was similar for FK506 but was doubled with CsA. The decrease in cell integrity and ATP depletion induced by CsA in LLC-PK1 cells was only transiently observed with FK506. By preserving energy status, FK506 leads to fewer metabolic disturbances than CsA in the renal epithelial cell line LLC-PK1, demonstrating a minor potential nephrotoxicity.

The present study was undertaken to evaluate the effects of platelet activating factor (PAF) antagonists, PMS 536 and PMS 549, on LLC-PK1 toxicity induced by Cyclosporin A (CsA). The LLC-PK1 cell line was used as an in vitro model. CsA cytotoxicity was determined in relation with ATP content. Alkaline phosphatase and N-acetyl-beta-glucosaminidase activities, which are directly correlated with tubular cell damage, were used as markers for renal injury. CsA alone provoked in the LLC-PK1 cell line a marked decrease in cell viability (55%) and membrane integrity (56%), and a significant increase in AP and NAG activities and in oxidized glutathione level. The ATP decrease and the ADP increase, resulting in a decline of the ATP/ADP ratio, is indicative of an anoxic energy charge. Co-treatment with CsA plus PMS 536 or PMS 549 resulted in a minor decrease in cell viability and in significant membrane integrity recovery. Moreover, the ATP depletion and the increase in ATP metabolites, hypoxanthine and uric acid induced by CsA were strongly prevented by PAF antagonists. In contrast, GSSG level remained high as in CsA-treated cells, but GSH level was in the range of controls. Our results suggest that both PAF antagonists attenuate CsA oxidative injury and prevent energy metabolism disturbances probably by maintaining cell integrity. The lipophilicity of both molecules may be responsible for membrane stabilization and may confer the protective effects observed in energy metabolism. The results obtained with PMS 536 and PMS 549 are indicative of interactions between PAF and CsA in renal injury and suggest the therapeutic potential of these PAF-antagonists against CsA-induced nephrotoxicity.

Cyclosporin A (CsA) is in vivo mainly metabolized by hepatic cytochrome P450 IIIA to more than 21 metabolites, the major ones known as: M1, M17 and M21. The aim of this work is to explore the in vitro metabolism of CsA after incubation, in the presence of NADPH, with renal or hepatic microsomes obtained from rabbits pretreated with rifampycin (enzyme inducer) or erythromycin (enzyme inhibitor). The presumed metabolites were separated by semi-preparative high-performance liquid chromatography (HPLC) and identified in each collected fraction by fluorescence polarization immunoassay (FPIA) (HPLC-FPIA) using a non-specific polyclonal antibody. They were also analyzed by HPLC-mass spectrometry (MS) using fast atom bombardment (HPLC-MS-FAB). Five collected fractions gave positive results with FPIA. The major metabolites found were M1, M17 and M21 after identification by HPLC-MS-FAB and comparison with three corresponding standard metabolites. The CsA biotransformation rates were calculated by the amount of unmetabolized CsA and were linear with time. These mean rates (Vm) for 12-min incubation by renal microsomes of rabbits treated with rifampicin or erythromycin or untreated (control) were 0.11, 0.02 and 0.04 nmol/min x mg microsomal protein, respectively. These rates were 15-, 37-, and 30-fold lower than those obtained with hepatic microsomes of rabbits treated identically. As CsA metabolites are less cytotoxic than the parent drug, this weak renal biotransformation of CsA after in vitro incubation should be one of the mechanisms of its in vivo nephrotoxicity.

This study was designed to assess Cyclosporin A (CsA) nephrotoxicity in the rabbit-possibly a more sensitive species than the rat-and to explore the mechanism of this toxicity with special attention to glutathione metabolism disturbances and cytochrome P-450 level in the kidney. CsA given for three days at a daily dose of 50 mg/kg (s.c.) induced nephrotoxicity as assessed by histological abnormalities and by a significant increase in blood urea nitrogen and urinary enzyme activities: N-acetyl-beta-D-glucosaminidase and L-gamma-glutamyl-transferase. This observed renal injury was of the same order as that obtained in the rat. In addition, there was a significant increase in oxidized glutathione content (40%) while reduced glutathione level remained unchanged. Concurrently, there was a significant decrease in renal cortex glutathione reductase (49%) and to a lesser extent in glutathione peroxidase activities (16%) whereas that of glutathione-S-transferase was not modified. A significant increase in renal cortex cytochrome P-450 (3-fold versus controls) was also observed. The mechanism of CsA nephrotoxicity is to be related to a cytochrome P-450 induction. This event could induce the observed impairments in renal glutathione metabolism and Na+K(+)-ATPase activity, via a possible increase in eicosanoid metabolism.

The clinical usefulness of cyclosporin A (CsA) in organ transplantation is limited by its intrinsic nephrotoxicity, the mechanisms of which have not yet been completely elucidated. Various processes could be responsible for the CsA-induced nephrotoxicity including an interaction with glutathione metabolism, a lipoperoxidative process, or an interaction with platelet-activating factor (PAF). Therefore, the aim of this study was to assess the potential role of PAF in CsA-induced nephrotoxicity using a new PAF antagonist, BN50726. CsA alone provoked a significant increase in serum urea, urinary gamma-glutamyl transferase activity and oxidized glutathione level of kidney cortex in the rat. In contrast, when rats were treated with CsA plus BN50726, urinary gamma-glutamyltransferase activity was similar to that of controls and oxidized glutathione was significantly lower than that measured in rats treated with CsA alone. However, serum urea remained increased as it was in the CsA group. Our results suggest that BN50726 is able to prevent the damage of renal membranes probably by an antiperoxidative activity. These data indicate the potential therapeutic capacity of a PAF antagonist, BN50726, in reducing CsA nephrotoxicity and suggest interactions between PAF and CsA in the mechanism of renal injury.

Rabbit renal microsomes were used to investigate the effect on lipid peroxidation (LPO) of cyclosporin A (CsA) and its first generation metabolites M1, M17 and M21, and of two natural CsA analogs: cyclosporins C and D (CsC & CsD respectively), at concentrations of 1 to 10 micrograms/ml. No induction of lipid peroxidation was observed with these substances except CsA at a high level (10 microgram/ml); but at a concentration of CsA that would be reached in vivo, no inductive effect on LPO was noted. A moderate inhibitory effect upon LPO was observed with CsC and M21, but its biological significance is questionable.