The temporal profile of calcineurin inhibition by cyclosporine in vivo

BACKGROUND: Cyclosporine (CsA) acts by inhibiting the phosphatase calcineurin (CN), but the time course and extent of inhibition in vivo are unknown. We examined the effect of single oral CsA doses on CN activity in humans and mice in vivo. METHODS: In humans, blood CsA levels were determined and CN activity was measured in whole blood and in blood leukocytes of patients up to 12 hr after CsA dosing (just before the second dose). Samples were collected from patients receiving a first single dose (2.5 mg/kg), and up to 14 days later after repeated dosing. In mice, after CsA dosing (12.5-200 mg/kg) by oral gavage, CsA levels in blood and tissue (spleen, kidney) were determined and CN activity was measured in spleen and kidney. RESULTS: In humans, peak CsA levels of 800-2285 microg/L at 1-2 hr produced 70-96% CN inhibition. Inhibition correlated closely with the rise and fall of CsA levels with no observable lag at the times sampled. Repeated doses showed similar CN inhibition to first dose, with no significant adaptation. In mice, CsA peaked at 1 hr in blood, spleen, and kidney, with higher concentrations in spleen and kidney than in blood. CN inhibition closely followed CsA concentrations/doses, and was greater in kidney than spleen. CONCLUSION: Thus CsA induces partial CN inhibition that varies directly with the blood and tissue levels, and may be greater in some tissues due to higher drug accumulation. The high CsA concentrations and CN inhibition in kidney may be relevant to nephrotoxicity.     

Modulation of experimental cyclosporine nephrotoxicity by inhibition of thromboxane synthesis

The clinical usefulness of Cyclosporine is limited by its intrinsic nephrotoxicity. A potential mechanism of CsA-mediated renal injury may involve an alteration in the prostaglandin-thromboxane (PG-TX) cascade. In our studies, pharmacological manipulation of the PG-TX system in normal and nephrotoxic animals was conducted using a specific thromboxane synthetase inhibitor U63,557A, and the cyclooxygenase inhibitor indomethacin. Administration of CsA 50 mg/kg/day for 7 days to Sprague Dawley rats resulted in a 99% increase in urinary thromboxane B2 excretion compared with controls (48.2 +/- 3.1 vs. 24.2 +/- 2.6 ng/24 hr, P less than 0.001), while plasma levels remained unchanged. Glomerular and tubular function was significantly reduced at this time, with a 48% decrease in creatinine clearance (CCr), and a 25% reduction in the fractional excretion of sodium (FeNa) (P less than 0.001). Histological injury included cortical tubular vacuolization and necrosis. Administration of indomethacin 8 mg/kg/day to both normal and CsA-treated rats resulted in a significant reduction in prostanoid excretion. Indomethacin alone had no adverse effect on glomerular function; however, when coadministered with CsA an exaggerated decrease in renal function was observed. CCr in this group fell by a further 27% compared with the CsA-50 group, while FeNa decreased by 76% (P less than 0.001). Histologic injury intensified, with an increase in vacuolization and necrosis. In contrast, coadministration of U63,557A with CsA prevented the rise in urinary TXB2 excretion, improved CCr by 20% (P less than 0.05), and restored FeNa to control levels. The severity of CsA-induced vacuolization was significantly diminished. Selective inhibition of thromboxane production may therefore be valuable in mitigating the clinical nephrotoxicity of CsA.     

Low-dose cyclosporine with mycophenolate mofetil induces similar calcineurin activity and cytokine inhibition as does standard-dose cyclosporine in stable renal allografts

One strategy to minimize nephrotoxicity in maintenance immunosuppression in renal transplantation is reduction of cyclosporine (CsA) with addition of mycophenolate mofetil (MMF). This approach seems safe, but concern exists about whether it yields adequate immunosuppression in the long term. Thus, we investigated the pharmacodynamic response to CsA in stable renal allografts treated with standard CsA (n = 17, CsA-C0h > or = 125 ng/mL) and low CsA plus MMF (n = 18 CsA-C0h <100 ng/mL). Patients treated with MMF without CsA (n = 13) and healthy subjects (n = 7) were used as controls. We observed that inhibition of calcineurin (CN) activity in peripheral blood mononuclear cells (PBMC), as well as interleukin (IL)-2 and interferon (IFN)-gamma production were similar in Standard-CsA and Low-CsA+MMF groups. Moreover, addition of MMF to a low CsA dose regime improved the correlation between CsA-C2h and both CN activity and IL-2 production. Thus, our results suggest that MMF could be synergistic with the pharmacodynamic effect of low CsA in maintenance immunosuppression.     

Differential inhibition of the T cell activation pathway by dexamethasone and cyclosporine

We examined the inhibitory capacity of dexamethasone (DEX) and cyclosporine (CsA) on T cell activation using various accessory cell (AC)-dependent and AC-independent stimuli. We found that CsA strongly inhibited T cell activation in each of the assays used: allogeneic T cell stimulation, phorbol myristate acetate plus concanavalin A, PMA plus anti-CD3 monoclonal antibody (2C11), or PMA plus ionomycin (IONO) T cell activation. DEX was a potent inhibitor of allogeneic stimulation and of the PMA+Con A- or PMA + 2C11-induced T cell stimulation. PMA + IONO stimulation, however, was not affected by DEX. When inhibition occurred, both drugs suppressed [3H]TdR incorporation, IL-2 production, and IL-2 mRNA accumulation, indicating that the sites of interference of these drugs in the T cell activation pathway are located proximal to IL-2 mRNA accumulation. However, the difference in the effects of DEX and CsA in PMA + IONO stimulation suggests that DEX and CsA differentially affect T cell activation.     

Organ-specific response to inhibition of mitochondrial metabolism by cyclosporine in the rat

To evaluate organ-specific metabolic changes after in vivo cyclosporine (CyA) treatment, male Wistar rats were treated with 10 mg/kg per day CyA orally for 6 days. Blood, kidney, liver, and heart tissues were extracted and analyzed by magnetic resonance spectroscopy (MRS). CyA decreased the energy balance [adenosine triphosphate (ATP)/adenosine diphosphate (ADP)] in all organs (kidney [control]: 50%, liver: 64%, and heart: 62%, all P<0.01) due to decreased activity of the mitochondrial Krebs cycle and oxidative phosphorylation. As a compensatory effect, anaerobic glycolysis (lactate) was increased. This was reflected in the low glucose level in the kidney and heart, but not in the liver where a significant decrease in glycogen was seen. Only in the kidney was mitochondrial inhibition accompanied by decreased polyunsaturated fatty acid (PUFA) concentrations and elevated lipid peroxidation. The metabolic marker for nephrotoxicity, trimethylamine-N-oxide (TMAO), was elevated. While CyA decreased mitochondrial homeostasis in all organ systems, cellular adaptation was different and most efficient in the liver.The results of this study were presented in part at the 2002 Winter Symposium of the American Society of Transplant Surgeons, Miami, 25–27 January 2002     

Cimetidine reduces cyclosporine inhibition of interleukin-2 production

Cimetidine has been shown to up-regulate proliferative and cytotoxic immune responses, which are mediated in part by an increase in interleukin-2 (IL-2) production. Cyclosporine achieves its immunosuppressive effect mainly through inhibition of IL-2 production. A recent clinical report of renal allograft recipients with elevated serum creatinine levels while on a histamine type-2 receptor antagonist raised concern whether the cimetidine increase in IL-2 production was counterbalancing the cyclosporine inhibition of IL-2 and thereby increasing alloreactivity to the transplant. We therefore measured IL-2 production of mitogen-stimulated murine spleen cells with or without cimetidine and cyclosporine and in combination of these two drugs. Cimetidine (10(-4) and 10(-5) M) completely reversed the cyclosporine (0.1 ng/ml and 1 ng/ml)-induced 25 and 41% inhibitions of IL-2, respectively. Cyclosporine (10 ng/ml) reduced IL-2 by 64% and cimetidine partially reversed this inhibition to 48%. All cimetidine groups which reduced the cyclosporine effect on IL-2 were statistically significant (P less than 0.05). These data raise concern about the safety of giving histamine type-2 receptor antagonists to allograft recipients without increasing alloreactivity due to a relative increase in IL-2.     

Influence of HLA phenotypes on the inhibition of in vitro alloreactivity by cyclosporine

Interindividual variations in the immunosuppressive effect of Cyclosporine have been observed in clinical organ transplantation. Searching for an in vitro correlate we investigated a possible relation between inhibition of alloresponsiveness by CsA and the HLA phenotypes of the responder or stimulator in mixed lymphocyte reactions. Peripheral blood mononuclear cells from 28 healthy volunteers were used as responder or stimulator cells (gamma-irradiated) and the inhibitory effect of graded amounts of CsA was determined in 130 criss-cross combinations. Sensitivity of alloresponsiveness to the drug was expressed as the dose causing 50% inhibition (ED50) and was read from the inhibition curves generated after four-parameter logistic curve fitting. ED50 ranged from 0.35 ng/ml to 33.4 ng/ml and correlated only weakly with the magnitude of the response (r = 0.12). In MLC with HLA DR4-positive responder cells, ED50 was significantly lower (Pc = 0.0035, Kruskal Wallis) when compared with MLC with responder cells of other DR haplotypes. For HLA DR5-positive responder cells ED50 was significantly higher (Pc = 0.042) when compared with DR5-negative responder cells. No significant correlation between ED50 and any particular haplotype of the stimulator cells could be observed. Sensitivity to CSA did not differ in MLC with 1 or 2 mismatches in the HLA-DR locus. In summary, we found that sensitivity of in vitro alloreactivity was different for particular HLA DR phenotypes, which may have important implications for the immunosuppressive therapy of transplanted patients with cyclosporine.     

Inhibition of leukocyte chemotaxis by immunosuppressive agents. Specific inhibition of lymphocyte chemotaxis by cyclosporine

Since chemotaxis is an important mechanism by which leukocytes are recruited to allografts during rejection, the effect of immunosuppressant agents on in vitro chemotactic responses of neutrophils, monocytes, and lymphocytes was studied. Cyclosporine caused profound inhibition of lymphocyte chemotaxis to three different chemotactic factors at pharmacologic levels but had no effect on neutrophil or monocyte chemotaxis. Methylprednisolone and azathioprine both inhibited chemotactic responses of neutrophils and monocytes as well as lymphocytes. These data suggest that immunosuppressant agents are potent inhibitors of leukocyte chemotaxis and that drugs differ as to which leukocyte cell type they inhibit. The selective action of cyclosporine might explain why this drug is ineffective in reversing established rejection episodes in which monocytes and neutrophils are also involved. Inhibition of leukocyte chemotaxis may be an important mode of action of immunosuppressant drugs, and drug regimes might be adjusted to cause selective inhibition of particular cell types.     

Immunophilins may limit calcineurin inhibition by cyclosporine and tacrolimus at high drug concentrations

BACKGROUND: The immunosuppressive drugs cyclosporine (CsA) and tacrolimus (FK506 or FK) are qualitatively similar but differ in molar potency. Both drugs sterically inhibit the phosphatase activity of calcineurin (CN) but differ in molar potency. In our study we explored whether differential inhibition of CN explained the differences in molar potency of FK versus CsA. METHODS: We compared their effects on NFATC2 dephosphorylation using Western analysis, interferon-gamma production using ELISA, and CN phosphatase activity using the CN assay in human peripheral blood leukocytes (PBL) and mouse spleen cell suspension. RESULTS: The FK concentration inhibiting 50% (IC50) of all three activities was approximately 0.2 microg/ml in human PBL, versus 5-20 microg/ml for CsA. Although inhibition of interferon-gamma secretion and NFATC2 dephosphorylation was complete, inhibition of CN phosphatase activity was incomplete with both drugs at saturation, particularly with FK. Inhibition of CN phosphatase activity was incomplete whether FK treatment was in vivo in mouse or in vitro in various human and mouse tissues, especially brain. Exogenous FKBP12 or CyPA increased CN phosphatase inhibition, suggesting that incomplete inhibition of CN phosphatase activity reflected limiting amounts of active immunophilin. CONCLUSIONS: These data contradict the prevailing assumption that immunophilins are abundant and not limiting for inhibition of CN by CsA or FK. Further, the observation that FK and CsA completely inhibit immune function without completely inhibiting CN suggests that the inhibition of immune function is not mediated by general CN inhibition but by inhibition of a subset of CN which is critical for lymphocyte activation.     

Age-associated differences in the inhibition of mitochondrial permeability transition pore opening by cyclosporine A

Background: Inhibiting mitochondrial permeability transition pore (mPTP) opening is a key protection of the myocardium from ischemia/reperfusion (I/R) injury. Here, we investigated age‐associated differences in the ability of cyclosporine A (CsA) to protect the heart and to modulate mPTP opening during I/R injury in vivo and its opening induced by reactive oxygen species (ROS) in vitro. Methods: Fischer 344 male rats were assigned from their respective age groups, young or old groups, to (1) I/R or (2) I/R+CsA. All animals were subjected to 30 min of ischemia following 120 min of reperfusion to determine myocardial infarct size in vivo. To measure mPTP opening in vivo, left ventricular tissues were collected 10 min after reperfusion and nicotinamide adenine dinucleotide (NAD⁺) levels were measured. In parallel experiments, rat ventricular myocytes were prepared from young and old hearts, loaded with tetramethylrhodamine ethylester and then subjected to oxidative stress in the presence or absence of CsA, and the mPTP opening time was measured using laser scanning confocal microscopy. Results: CsA reduced myocardial infarct size in young I/R rats. Whereas CsA failed to significantly affect myocardial infarct size in old I/R rats, NAD⁺ levels were better preserved in young CsA‐treated rats, but this relative improvement was not observed in old rats. CsA also significantly prolonged the time necessary to induce mPTP opening in young cardiomyocytes, but not in cardiomyocytes isolated from the old rats. Conclusions: mPTP regulation is dysfunctional in the aged myocardium and this could account for loss of cardioprotection with aging.     

Enhancement of immunosuppression by substitution of fish oil for olive oil as a vehicle for cyclosporine

As previously reported, acute cyclosporine-induced nephrotoxicity is characterized by a decline in glomerular filtration rate and a selective intrarenal production of the vasoconstrictor thromboxane (TxA2), but not vasodilator prostaglandin E2 (PGE2), or prostacyclin (PGI2), cyclooxygenase metabolites. Fish oils (FO), that are rich in n-3 polyunsaturated fatty acids have a high affinity for cyclooxygenase but serve as poor substrate inhibit TxA2 synthesis. We have shown that when FO replaces olive oil (OO) as the vehicle for CsA, CsA-induced nephrotoxicity and increased TxA2 synthesis are obviated in rodent models. In this study, we demonstrate that the FO vehicle for CsA does not compromise CsA's immunosuppressive properties as deduced from studies of a delayed-type hypersensitivity (DTH) model in BALB/c mice and in a rat heart transplant model. In fact, concurrent FO administration with CsA actually enhances immunosuppression. A dose of CsA incapable of blunting DTH when injected in OO was suppressive when given in FO. Administration of as little as 0.05 ml of FO vehicle potentiated the suppressive action of CsA. In addition, nonconcurrent dietary supplementation of FO in animals receiving CsA caused an increase in the immunosuppressive action of CsA in DTH. FO alone reduced DTH as compared with OO, but was far less effective than CsA plus FO. Furthermore, doses of CsA (5 mg/kg/day or 1.5 mg/kg/day), which are subtherapeutic when administered with OO, prolonged engraftment of Lewis recipients of Lewis x Brown-Norway F1 hearts when CsA was solubilized with FO. These studies indicate that concurrent administration of CsA and FO potentiates the activity of CsA and thus increases its therapeutic index. Thus, CsA plus FO is potentially a safe, potent antirejection therapy worthy of clinical testing, especially insofar as FO prevents CsA-induced acute nephrotoxicity in the rodent.     

Comparison of the temporal profile of calcineurin inhibition by cyclosporine and tacrolimus in renal transplant patients

Calcineurin phosphatase (CaN) activity has been the focus of several recent studies on renal transplant patients as the calcineurin inhibitors tacrolimus (FK) and cyclosporine (CsA) are still the cornerstone in the immunosuppressive treatment. The aim of this study was to compare the temporal inhibition profiles of CaN using CsA or FK in two groups of renal transplant patients. Nineteen tacrolimus-treated and 10 cyclosporine-treated renal transplant patients had blood samples drawn before and at 1, 2, 3, 4, and 6 hours after ingestion of drug. CaN activity was measured as the release of 32P from a previously phosphorylated peptide and radioactivity quantitated by liquid scintillation counting. Results were expressed as units CaN. Blood concentrations of tacrolimus were determined with an IMx method and of CsA with an EMIT assay. FK-treated patients showed maximal inhibition of CaN activity at 1 to 3 hours, returning to the predose level 4 hours after drug intake. CsA-treated patients showed a gradual decrease in CaN activity with a nadir after 3 hours, failing to return to predose levels during the observation period. Both groups showed a significant rise in drug blood concentrations. To conclude, we have demonstrated that two widely used immunosuppressants, CsA and FK, show different CaN inhibitory profiles in renal transplant patients.     

Late graft dysfunction after prolonged cold ischemia of the donor kidney: inhibition by cyclosporine.

BACKGROUND: The present study was devised to elucidate the influence of prolonged cold ischemia on the development of chronic transplant dysfunction (CTD) in kidney isografts (Brown Norway-->Brown Norway; BN-->BN) and in kidney allografts (BN-->Wistar Agouti/ Rij [WAG]) under temporary cyclosporine (CsA) therapy. METHODS: To induce ischemic injury, BN donor kidneys were preserved for 24 hr in 4 degrees C University of Wisconsin solution before transplantation. Renal function (proteinuria), histomorphology according to the BANFF criteria for CTD, and infiltrating cells were assessed. Grafts were examined both early at days 2, 3, 6, and 10, and late at week 26 (allografts) or at week 52 (isografts). RESULTS: Nonischemic isografts preserved a normal function and morphology. Ischemic isografts developed a progressive proteinuria over time and demonstrated significantly more glomerulopathy with macrophage (Me) infiltration and intimal hyperplasia than nonischemic controls at week 52. During the initial 10 days, there was an increased infiltration of MHC class II+ cells, predominantly CD4+ cells and Mphi, coinciding with up-regulated intercellular adhesion molecule-1 expression. CsA treatment in ischemic isografts inhibited infiltration of MHC II+ cells in the early stage, which was accompanied by significantly less renal damage at week 52 compared with untreated controls (proteinuria: 59+/-8 vs. 134+/-19 mg/24 hr; BANFF score: 2.8+/-0.4 vs. 4.3+/-1.0). Under CsA therapy, 24-hr cold ischemia of the allograft affected neither the onset or progress of proteinuria, nor the histomorphology (BANFF score: 7.8+/-2.4 vs. 7.3+/-1.9). In both ischemic and nonischemic allografts, intercellular adhesion molecule-1 expression and mononuclear cell infiltration (CD4, CD8, Mphi was abundantly present during the first 10 days and function deteriorated rapidly. CONCLUSIONS: Prolonged cold ischemia plays a role in the induction of CTD, but its deleterious effect can be successfully inhibited by CsA. Therefore, the alloantigeneic stimulus is the overriding component in the multifactorial pathogenesis of CTD.     

Growth factor receptors as targets for lung cancer therapy

Dysregulated signal transduction of growth factor receptors contributes to the process of malignant transformation by promoting cell proliferation, motility, and invasion through extracellular matrix as well as angiogenesis. Epidermal growth factor receptors (EGFR), and to a lesser extent HER2/neu, is overexpressed in the majority of nonsmall cell lung cancer (NSCLC) compared with normal tissue, making them ideal targets for the development of novel therapeutics for this disease. Multiple clinical trials have demonstrated that antireceptor strategies employing antagonistic monoclonal antibodies or low molecular weight tyrosine kinase inhibitors against EGFR are well tolerated and occasionally result in objective clinical responses in patients with advanced NSCLC. This report provides an overview of the molecular basis and the preclinical evidence supporting clinical development of anti-EGFR therapy as well as results of phase I-III clinical trials of these compounds in treating patients with solid tumors including NSCLC.     

绿茶多酚改善环孢素A抑制血管舒张的作用

目的 观察绿茶多酚(GTP)对环孢素A(CsA)抑制血管舒张作用的改善,并探讨其机制.方法 将30只SD大鼠随机平均分为3组:CsA组、对照组和CsA+GTP组.建模5周后,检测体质量、肾功能:血尿素氮(BUN)、肌酐(Cre);并取胸主动脉环,观察乙酰胆碱(Ach)诱发的血管舒张反应、左旋硝基精氨酸甲酯(L-NAME)和吲哚美辛预处理对舒张反应的影响、去内皮细胞的血管舒张反应.并检测血管组织一氧化氮(NO)水平.结果 实验5周后,CsA组大鼠体质量(253.2±8.1)g低于对照组(292.1±9.5)g;CsA+GTP组体质量(287.9±9.7)g高于CsA组;CsA组BUN、Cre含量高于对照组;CsA+GTP组BUN、Cre低于CsA组;差异均有统计学意义(P<0.05).CsA组大鼠Ach引起的动脉环的最大舒张度为(42.5±4.3)%,低于对照组的(81.2±7.6)%和CsA+GTP组的(70.1±6.5)%,差异有统计学意义(P<0.05).用L-NAME预处理后,CsA组和CsA+GTP组动脉环的舒张幅度分别为(40.3±3.7)%和(45.8±4.2)%,均低于对照组的(79.4±6.8)%;用吲哚美辛预处理后对照组和CsA+GTP组的舒张反应高于CsA组;差异均有统计学意义(P<0.05).去内皮细胞的各组血管中,血管舒张反应被明显抑制,各组血管的舒张百分比差异无统计学意义(P>0.05).CsA组大鼠血管组织中NO含量显著低于对照组和CsA+GTP组,差异有统计学意义(P<0.05).结论 CsA可导致血管组织NO水平下降,引起NO介导的内皮依赖性血管舒张功能异常.应用绿茶多酚后,则升高其血管组织中NO水平,改善内皮依赖性的血管舒张功能.     

绿茶多酚改善环孢素A抑制血管舒张的作用

Objective To observe the alleviation effect of green tea polyphenols (GTP) on cyclosporine A (CsA)-induced inhibition of vasodilation, and to study the underlying mechanism. Methods Thirty SD rats were randomly divided into three groups: CsA group, control group and CsA + GTP group. Five weeks after the treatment, the body weight, kidney function (BUN, Cr) of the rats were measured. Then thoracic aorta rings were mounted on a bath system, and the Ach-induced vasodilation, the effect of L-NAME and indomethacin pretreatment on the vasodilation and the denuded vasodilation were evaluated. The contents of nitric oxide (NO) in the vascular tissues were measured. Results Five weeks later, the body weight of the rats in CsA group (253.2 ±8.1) g was lighter than that in control group (292.1 ±9.5) g (P < 0.05);the body weight in CsA + GTP group (287.9 ± 9.7) g was heavier than that in CsA group ( P < 0.05);The levels of BUN and Cr in CsA group were higher than in control group (P <0.05);The levels of BUN and Cr in CsA + GTP group were lower than in CsA group (P <0.05). The maximal response for Achinduced vasodilation in CsA group was (42.5 ±4.3)% , significantly lower than (81.2 ±7. 6)% in control group and (70.1 ± 6.5) % in CsA + GTP group (both/P < 0.05). After pretreatment with L-NAME, the vasodilation in CsA group and CsA + GTP group was (40.3 ±3.7)% and (45.8 ±4.2)% respectively, lower than in control group (79. 4 ± 6. 8)% ;After pretreatment with indomethacin, the vasodilation in control group and CsA + GTP group was higher than in CsA group (both P < 0.05). The vasodilation in the denuded groups was inhibited, but there was no significant difference between groups (P>0.05). In CsA group, the content of NO in vascular tissues was lower than that in control group and CsA + GTP group (both P < 0.05). Conclusion CsA might decrease the NO content in vascular tissues and induce abnormal endothelium-dependent vasodilation which is mediated by NO. The administration of GTP could increase the NO content and alleviate the CsA-induced inhibition of the endothelium-dependent vasodilation.