Effect of cyclosporin A or tacrolimus on the function of blood–brain barrier cells

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Abstract

Recently, it has been reported that continuous treatment with cyclosporin A or tacrolimus induces encephalopathy in transplant patients. The mechanism of immunosuppressant-induced encephalopathy is unclear. We investigated the cytotoxicity to brain capillary endothelial cells and the effect of these two drugs on P-glycoprotein function using mouse brain capillary endothelial (MBEC4) cells. The transcellular transport of [3H]sucrose was significantly increased and the cellular viability, based on 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and trypan blue exclusion test, was decreased by cyclosporin A (approximately 50% at 5 μM; P<0.005), while tacrolimus showed a much smaller effect. These findings indicate that the toxicity of cyclosporin A was greater than that of tacrolimus. The uptake of [3H]vincristine, a substrate of P-glycoprotein, was increased by these two drugs. The expression of P-glycoprotein in MBEC4 cells was reduced, but there was no effect on mdr1b mRNA levels. The decrease in the expression of P-glycoprotein may be due to the inhibition of the turnover of P-glycoprotein, which involves translation. In conclusion, the direct cytotoxic effect on the brain capillary endothelial cells and the inhibition of P-glycoprotein may be partly involved in the occurrence of immunosuppressant-induced encephalopathy.

Introduction

Cyclosporin A and tacrolimus are immunosuppressants widely used in organ transplantation to prevent fatal graft-vs.-host disease or to suppress rejection symptoms (Powels et al., 1980; Kahan, 1992; Borel et al., 1995). However, the immunosuppressive potency of these two drugs is high, and it is well-known that they induce multiple organ toxicity (Hamilton et al., 1981; Adu et al., 1983; Gupta et al., 1991). Recently, the occurrence of encephalopathies such as seizure, confusion, motor paralysis and cortical blindness in transplant patients using these two drugs was reported (De Groen et al., 1987; Humphreys and Leyden, 1993; Shimizu et al., 1994; Appignani et al., 1996; Hinchey et al., 1996). The mechanism of immunosuppressant-induced encephalopathy is unclear. However, several investigators suggested that increased blood drug levels, due to hypocholesterolemia or concurrently used drugs, enhanced the distribution of drugs to the brain, that nephrotoxicity or hypertension induced by the two drugs caused edema of the white matter, and that virus infection due to the immunosuppressive effect caused virus encephalopathy (Brat et al., 1992; Shimizu et al., 1994; Worthmann et al., 1994; Hinchey et al., 1996). It has also been reported that cyclosporin A inhibits cell growth and induces apoptosis characterized by chromatin condensation, DNA ladder and shrinkage of the cell body in rat C6 glioma cells in a dose-dependent manner (Mosieniak et al., 1997), suggesting that the direct toxicity of the immunosuppressants entering the brain may trigger the encephalopathy.

Cyclosporin A and tacrolimus are known as substrates and inhibitors of P-glycoprotein, a multi-drug efflux pump, (Takeguchi et al., 1993; Tanaka et al., 1996). The transport of cyclosporin A across the blood–brain barrier is restricted by P-glycoprotein, so that the distribution of cyclosporin A in the brain is low despite its high lipophilicity (Tsuji et al., 1993; Shirai et al., 1994). P-glycoprotein is widely expressed in various tissues such as the brush-border membrane of renal proximal tubules, the bile canalicular membrane of hepatocytes and the capillary endothelial cells of the brain (Rothenberg and Ling, 1989; Postan et al., 1991), and P-glycoprotein could be related to the regulation of the pharmacokinetic behavior of drugs including self-defense. P-glycoprotein in brain capillary endothelial cells is localized on the apical surface and serves as an efflux pump for many lipophilic agents transported by passive diffusion across the cell membrane such as antitumor agents, cyclosporin A and tacrolimus in an ATP-dependent manner (Sugawara et al., 1990). In this study, we hypothesized that the permeability of blood–brain barrier to cyclosporin A and tacrolimus is enhanced and that encephalopathy is induced as a result of the enhanced brain distribution of these two drugs as a result of direct injury of the brain capillary endothelial cells and/or the depressed function of P-glycoprotein.

To clarify the mechanism of immunosuppressant-induced encephalopathy, we examined the effect of cyclosporin A or tacrolimus, using the mouse brain endothelial (MBEC4) cells as a model of brain capillary endothelial cells (Tatsuta et al., 1992; Tatsuta et al., 1994), on the function of the blood–brain barrier (cell viability and permeability of test substances by simple diffusion or P-glycoprotein-mediated active efflux transport, and expression of P-glycoprotein and its mRNA level).

Section snippets

Reagents and antibodies

Cyclosporin A and tacrolimus were kindly supplied by Sandoz (Basel, Switzerland) and Fujisawa Pharmaceutical (Osaka, Japan), respectively. [3H]Sucrose (specific activity; 12.3 Ci/mmol) and [3H]vincristine sulphate (specific activity; 10.5 Ci/mmol) were purchased from Amersham International (Buckinghamshire, UK), and [α-32P]dCTP (specific activity; 3000 Ci/mmol) was purchased from NEN Research Products (MA, USA). Polyclonal anti-mdr (Ab-1) was purchased from Oncogene Research Products (MA, USA).

Effect of cyclosporin A or tacrolimus on the transport of [3H]sucrose across MBEC4 cells

Firstly, to study the effect of cyclosporin A or tacrolimus on the permeability of MBEC4 cells, we measured the transcellular transport of [3H]sucrose in MBEC4 cells exposed to cyclosporin or tacrolimus for 24 h. The transcellular transport of [3H]sucrose both from the luminal to the abluminal side and from the abluminal to the luminal side was significantly increased by the exposure of MBEC4 cells to cyclosporin A at a concentration of 5–10 μM, while tacrolimus showed no effect in the range of

Discussion

Recently, it has been reported that cyclosporin A or tacrolimus induce serious neurotoxicity, encephalopathy, and that several factors may participate in the occurrence of encephalopathy (De Groen et al., 1987; Brat et al., 1992; Worthmann et al., 1994; Hinchey et al., 1996). Because the transport of both drugs across the blood–brain barrier is restricted by P-glycoprotein (Tsuji et al., 1993; Shirai et al., 1994), these drugs rarely have side effects on the central nervous system. Cyclosporin

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