Immunophilin ligands decrease release of pro-inflammatory cytokines (IL-1beta, TNF-alpha and IL-2 in rat astrocyte cultures exposed to simulated ischemia in vitro

The aim of present study was to evaluate the effects of immunophilin ligands (cyclosporin A, FK506 and rapamycin) on the simulated ischemia-induced release of pro-inflammatory cytokines (IL-1beta, TNF-alpha and IL-2) in rat primary astrocyte cell cultures. Astrocytes were exposed to cyclosporin A (CsA) (0.25, 0.5, 1, 10, 20 and 50 microM), FK506 (1, 10, 100, 1000 nM) and rapamycin (10, 100, 500 and 1000 nM). In vitro simulated ischemia significantly increased secretion of IL-1beta, TNF-alpha and IL-2 by astrocyte cultures deprived of microglia (by shaking and incubating with L-leucine methyl ester). CsA (at concentrations of 10-50 microM), FK506 (at all used concentrations) and rapamycin (in dose-dependent manner) significantly attenuated IL-1beta release after 24 h exposure to ischemic conditions. Immunophilin ligands at all used concentrations significantly decreased TNF-alpha levels in culture media after 24 h exposure to ischemia. Moreover, significant decrease in IL-2 secretion at 0.25, 0.5, 1 and 50 microM CsA and FK506 at concentrations of 100 and 1000 nM were observed. The results suggest that immunophilin ligands may regulate glial activity during ischemia by affecting the release of pro-inflammatory cytokines.     

Cardioprotective effects of the calcineurin inhibitor FK506 and the PAF receptor antagonist and free radical scavenger, EGb 761, in isolated ischemic/reperfused rat hearts

Effects of the calcineurin inhibitor FK506, the platelet-activating factor (PAF) antagonist, and free radical scavenger Ginkgo biloba extract, EGb 761, and their combination on reperfusion-induced ventricular fibrillation (VF), ventricular tachycardia (VT), and recovery of cardiac function were studied after 30 min of global ischemia followed by 2 h of reperfusion in isolated rat hearts. In the first series of studies, rats received a daily (oral) dose of 0, 1, 5, 10, 20, or 40 mg/kg/day FK506 for 10 days. FK506 dose-dependently reduced the incidence of reperfusion-induced total (irreversible plus reversible) VF from a value of 92% for untreated animals to 92% (NS), 83% (NS), 67% (NS), 33% (p<0.05), and 25% (p<0.05), for doses of 1-40 mg/kg/day, respectively, with effects on incidence of VT showing the same pattern. FK506, between 20 and 40 mg/kg/day, also resulted in significant recovery of postischemic cardiac function. In the second series of studies, rats were treated with EGb 761 alone or in combination with FK506. Whereas no significant reduction in arrhythmias or improvement in cardiac function resulted from a single intervention of EGb 761 at 25 mg/kg/day, combined treatment of rats with 25 mg/kg/day of EGb 761 and 1 or 5 mg/kg/day of FK506 resulted in a reduction in total and irreversible VF of 92% and 92% to 42% (p<0.05) and 33% (p<0.05), 25% (p<0.05) and 8% (p<0.05), respectively, versus untreated control animals, paralleled by similar effects on the incidence of VT and accompanied by significant improvements in postischemic cardiac function. Our results demonstrate a novel cardioprotective characteristic of FK506 and suggest that combination therapy by using FK506 plus EGb 761 synergistically improves postischemic cardiac function, while reducing the incidence of reperfusion-induced VF and VT, which may expand the clinical utility of FK506 and allow therapy with FK506 at lower doses than are currently useful.     

Assessment of antacid characteristics of drugs containing a combination of aluminium and magnesium salts using the "artificial stomach" model

Antacid characteristics of three drugs containing aluminium and magnesium salts (combination of clay with aluminium and magnesium hydroxides, aluminium and magnesium hydroxide mixture and hydrotalcite) have been studied in a dynamic situation simulated by the "artificial stomach" model, simultaneously taking into account both gastric fluxes, a constant secretory flux and variable emptying fluxes. Therapeutic doses of the drugs were added 1. to 100 ml of 0.1 N HCl, without or with 1% or 5% meat extract, and 2. 100 ml of pooled human gastric juice (96 mmol/l, pH 1.1). In addition, antacid activity of 0.5 g aluminium and magnesium hydroxides, taken alone or in combination, were evaluated when added to 100 ml of 0.1 N HCl. In aqueous HCl solution or in human gastric juice, the three antacid drugs exhibited 1. a neutralising activity characterised by pH-rise and 2. a buffering capacity close to pH 3.8. In addition, hydrotalcite exhibited also buffering capacity at pH 1.2. The antacid-induced capacity, expressed as H+ mmol, to recover initial pH were very similar, indicating that antacid physiochemical properties are similar in HCl solution or in gastric juice. H+ consumption depended upon emptying fluxes. The same antacid characteristics were observed when antacids were mixed with 1% meat extract while 5% meat extract resulted in a modification of antacid characteristics. Therefore the antacid capacities of respective mixtures were of smaller magnitude (50-60%) than the sum of the activities of antacids plus meat extracts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability and sorption of FK 506 in 5% dextrose injection and 0.9% sodium chloride injection in glass, polyvinyl chloride, and polyolefin containers.

The effects of the diluent, the storage container, light, and infusion through various types of tubing on the stability and sorption of FK 506 were studied. Solutions of FK 506 in 0.9% sodium chloride injection or 5% dextrose injection were stored at room temperature (24 +/- 2 degrees C) in glass i.v. bottles, polyvinyl chloride (PVC) minibags, and polyolefin containers. FK 506 solution in 0.9% sodium chloride injection was stored in plastic syringes at room temperature and either exposed to normal room light or stored in the dark. FK 506 solution in 5% dextrose injection was placed in plastic syringes and infused through PVC anesthesia extension tubing, PVC i.v. administration set tubing, and fat emulsion tubing over a two-hour period. The infused samples and samples collected from the containers and syringes at intervals up to 48 hours were analyzed for FK 506 concentration by high-performance liquid chromatography. FK 506 concentrations remained greater than 90% of initial concentration for admixtures in 5% dextrose injection stored in glass bottles for 48 hours and for admixtures in 5% dextrose injection or 0.9% sodium chloride injection stored in polyolefin containers for 48 hours. No change in concentration was measured for admixtures in 0.9% sodium chloride injection stored in plastic syringes, and exposure to light did not affect the stability of FK 506 solution. No substantial change in concentration occurred in FK 506 solution in 5% dextrose injection infused through PVC anesthesia extension tubing, PVC i.v. administration set tubing, or fat emulsion tubing. FK 506 admixtures prepared with 5% dextrose injection or 0.9% sodium chloride injection should be stored in polyolefin containers. If polyolefin containers are not available, solutions should be prepared with 5% dextrose injection and stored in glass bottles.     

FK506 ameliorates renal injury in early experimental diabetic rats induced by streptozotocin

Calcineurin (CaN) plays an important role in glomerular hypertrophy and extracellular matrix accumulation in early diabetic nephropathy. Cyclosporine (CSA), a CaN inhibitor, has been shown to reduce renal injury in streptozotocin-induced diabetic rats. We examined whether FK506, which immunosuppressive action was 10–100 times of CSA, inhibits progression of diabetic nephropathy in experimental diabetic rats. Diabetes was induced with streptozotocin in rats, and FK506 (0.5 or 1.0 mg/kg) was orally administered once a day for 4 weeks. Increased relative kidney weight was significantly reduced by FK506 treatment with 1.0 mg/kg (p < 0.05), and elevated 24 hour urinary albumin excretion rate was markedly attenuated by FK506 treatment with 0.5 and 1.0 mg/kg (p < 0.05, 0.01). Elevated glomerular volume was significantly attenuated by FK506 treatment with 0.5 and 1.0 mg/kg (p < 0.05), and increased indices for tubulointerstitial injury were only ameliorated by FK506 treatment with 1.0 mg/kg (p < 0.01). Western blot analysis noted that the expression of CaN protein was increased 2.4 fold in the kidney from diabetic rats, and FK506 treatment with 0.5 and 1.0 mg/kg could reduce increased expression of CaN protein by 38.0% and 73.2%. The expression of 1α (IV) collagen, p65, p-p65, OPN, α-SMA and TGF-β1 protein in kidney was significantly increased in diabetic rats and reduced by FK506 treatment (p < 0.05, 0.01). Our results show that FK506 could ameliorate renal injury in early experimental diabetic rats, which mechanism may be at least partly correlated with suppression on increased CaN in renal tissue in diabetic rats.     

Effects of water-soluble tacrolimus-PEG conjugate on insulin-dependent diabetes mellitus and systemic lupus erythematosus

The effects of a water-soluble tacrolimus-PEG conjugate (KI-102) on insulin-dependent diabetes mellitus and systemic lupus erythematosus were investigated. KI-102 was stable at pH 4.0-4.5 and 4°C. The area under the concentration-time curve, the time of maximum concentration, and the maximum concentration were 43.4 ng·h/mL, 0.85 h, and 8.1 ng/mL, respectively, similar to those of FK506. Mice that administered KI-102 at 4.32 mg/kg had the plasma glucose concentrations that decreased to 7.5 mmol/L after 170 days, similar to that of mice administered FK506 at 0.6 mg/kg. There were no incidences of diabetes when KI-102 was administered at 86.4 mg/kg after 24 weeks. The group that administered 43.2 mg/kg had decreases in the concentrations of β-hydroxybutyrate (60%), triglyceride (24%), and cholesterol (30%). KI-102 administered at 180 mg/kg reduced serum anti-dsDNA antibody activity by 64% compared with a control. Urinary albumin concentration in the same group decreased 81% compared with the control. These results indicate that KI-102 may be practically applicable as prodrug of FK506.     

Implication of nitric oxide synthase activity in the genesis of water immersion stress-induced gastric lesions in rats: the protective effects of FK506

Background: Nitric oxide (NO) is a potent cytoprotective substance of gastric mucosa. FK506, an immunosuppressive drug, shows anti-gastric ulcer effects equivalent to famotidine, an H₂ blocker, in rats. This study was designed to evaluate the cytoprotective mechanism of FK506 on gastric mucosa in relation to the changes in NO synthase activity. Methods: Gastric lesions were induced in rats by water immersion stress. Changes in NO synthase activity during water immersion stress treatment, and effects of FK506 on NO synthase activity were determined enzymatically. Gastric mucosal interleukin (IL)-1β and IL-2 were measured by immunoradiometric assay. Gastric mucosal blood flow was measured by hydrogen gas clearance technique. Results: FK506 mitigated gastric lesions developed by water immersion stress. Stress-induced lesions were exacerbated by N^G-monomethyl-L -arginine (L -NMMA), a specific inhibitor of NO synthase, while sodium nitroprusside, a NO donor, mitigated the lesions. Water immersion stress increased NO synthase activity in the early phase (0.5 h after stress treatment) and decreased it in the late phase (6 h after). Decrease in NO synthase activity in the late phase was significantly mitigated by FK506, though it did not affect changes in NO synthase activity in the early phase. Water immersion stress increased gastric mucosal IL-1β and IL-2 contents 6 h after stress treatment, and these increases were prevented by FK506. FK506 itself did not affect gastric mucosal blood flow. L -NMMA treatment significantly decreased gastric mucosal blood flow. In contrast, gastric mucosal blood flow was significantly increased by sodium nitroprusside. Conclusions: Increase in NO synthase activity might contribute to cytoprotection, and a decrease in activity might be a harmful factor for the gastric mucosa. Preservation of NO synthase activity by FK506 might be involved in FK506's protective effects on the gastric mucosa.     

Pharmacokinetic/pharmacodynamic analysis of tacrolimus-induced QT prolongation in guinea pigs

Recently, several reports of clinical cases of QT prolongation and torsades de pointes, associated with the use of tacrolimus (FK506), have come to light. We have previously demonstrated FK506-induced QT prolongation in guinea pigs [Minematsu T., et al., Life Sci., 65, PL197-PL202 (1999)]. We now examined the relationship between QTc prolongation and the pharmacokinetics of FK506 in guinea pigs, in order to evaluate the arrhythmogenicity of FK506 when compared with that of quinidine sulfate (QND). Thus, dose-response relationships for FK506 (0.01 or 0.1 mg/h/kg) or QND (30 mg/h/kg) were investigated during and after intravenous infusion and also following intravenous bolus administration of FK506 (0.2 mg/kg). The dose-response relationship between plasma drug concentration and QTc prolongation for FK506 and QND were subsequently analyzed using an effect compartment model. The pharmacodynamic parameters thus obtained were as follows: kE0 2.72 x 10(-4) (min-1), Emax 27.1 (ms), EC50 0.376 (ng/ml) for FK506; and kE0 0.148 (min-1), K 8.41 (ms.ml/microgram) for QND. The anti-clockwise hysteresis observed for FK506-induced QT prolongation was successfully analyzed by the present pharmacokinetic/pharmacodynamic model, which may provide a rational basis for developing a clinical dosing regimen to avoid possible QT prolongation induced by FK506.     

The effects of FK506 on the development and expression of morphine tolerance and dependence in mice

FK506 is an immunophilin-binding ligand that inhibits calcineurin and decreases nitric oxide (NO) production in the nervous tissues. We examined the effects in mice of systemic treatment with FK506 on the induction and expression of morphine (s.c.) tolerance and dependence and compared them with the effects of the non-specific NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), and specific inducible NO synthase inhibitor, aminoguanidine. FK506 (0.5-10 mg/kg, s.c.) exerted inhibitory effects on both development and expression of tolerance to morphine-induced antinociception. FK506 also significantly decreased the expression of morphine dependence, as assessed by naloxone-precipitated (2 mg/kg, i.p.) withdrawal syndrome, but a similar effect was not found for the development of morphine dependence. A similar pattern of effects was observed with L-NAME (3-20 mg/kg, i.p.), while aminoguanidine (50-100 mg/kg, i.p.) did not alter tolerance or dependence. Examining the possible interaction between their inhibitory effects on tolerance and dependence, we combined the subeffective doses of FK506 (0.5 or 1 mg/kg) with L-NAME (3 mg/kg) or aminoguanidine (100 mg/kg). The combination of FK506 with L-NAME, but not with aminoguanidine, significantly decreased the development and expression of tolerance and expression of dependence. These data show the effectiveness of FK506 on morphine tolerance and dependence and suggest an additive effect between FK506 and the inhibition of constitutive NO synthesis in this regard.     

The immunosuppressant drug FK506 prevents Fas-induced apoptosis in human hepatocytes

FK506 is a potent immunosuppressive drug used for the prevention of graft rejection in organ transplantation. Experimental and clinical studies have shown correlations between apoptosis and graft rejection, and apoptosis also plays a role in cell death after ischemia-reperfusion injury in the rat liver. Fas-mediated apoptosis is very likely involved in allograft rejection and experimental evidence has shown a decrease of FasR expression in mouse hepatocytes produced by the drugs. On the basis of these findings we have investigated the protective effect of FK506 in comparison with cyclosporine A (CsA) on Fas-induced apoptosis, by analysing the activation of downstream effector caspases in human hepatocytes. Apoptosis was induced by treatment with agonistic antibodies against FasR, which resulted in a significant activation of caspase-3 after 12 h. Prevention of the downstream activation of the caspase cascade and apoptosis was observed when hepatocytes were pre-treated for 3 h with immunosuppressant drugs. A significant reduction (ca. 30-40%) of caspase-3 activation by 5 microM FK506 and CsA was observed. Along with less activation of caspase-3 a decrease of apoptotic DNA fragmentation was found. In addition, FK506 significantly reduced not only caspase-8 but also caspase-9 activation, to a similar extent as CsA, thus suggesting a protective effect at the mitochondrial level of this drug, as has already been reported for CsA. These effects of FK506 help to explain its strong anti-rejection properties and suggest promising benefits of pharmacological preconditioning on ischemia-reperfusion injury following liver transplantation.     

Rapamycin, but not FK506 and GPI-1046, increases neurite outgrowth in PC12 cells by inhibiting cell cycle progression

Immunophilin ligands such as rapamycin, FK506 and GPI-1046 have been reported to increase neurite outgrowth in vitro and to have neuroprotective activity in vitro and in vivo. In this study, however, FK506 and GPI-1046 (0.1-1000 nM) had little effect on neurite outgrowth in PC12 cells in either the presence or absence of nerve growth factor. In contrast, rapamycin markedly increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=10 nM). Unlike FK506 and GPI-1046, rapamycin is an inhibitor of cell cycle progression. Other cell cycle inhibitors such as ciclopirox and flavopiridol also increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=250 nM and 100 nM, respectively). The neuroprotective effects of FK506, rapamycin and GPI-1046 were also tested in a rodent model of permanent focal cerebral ischemia. FK506 and rapamycin decreased infarct volume by 40% and 37%, respectively, whereas GPI-1046 was ineffective. These data do not support the previous suggestion that FK506 and GPI-1046 increase neurite outgrowth of PC12 cells in vitro. Rapamycin increases neurite outgrowth of PC12 cells, an effect that can be ascribed to its ability to inhibit cell cycle progression. The neuroprotective effect of FK506 and rapamycin against cerebral ischemia is probably not due to differentiation of neuronal precursors or stimulation of neuronal regeneration.     

Modulation of energy status and cytotoxicity induced by FK506 and cyclosporin A in a renal epithelial cell line

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. Received: 8 January 1997 / Accepted: 6 March 1997     

The effects of FK506, a specific calcineurin inhibitor, on methamphetamine-induced behavioral change and its sensitization in rats

RATIONALE: FK506 inhibits calcineurin activity, resulting in the inhibition of calcium-dependent intracellular processes. Recent studies have suggested that intracellular calcium is likely to be involved in methamphetamine (MAP)-induced locomotor activity and stereotyped behavior, and in the development of sensitization to MAP. OBJECTIVES: We investigated the effects of FK506 on MAP-induced behavioral changes and the development of sensitization in rats. METHODS: In experiment 1, animals were administered IP 2 mg/kg FK506 or vehicle followed 10 min later by MAP (1, 2, 4 and 8 mg/kg, IP). Another set of animals were administered FK506 (0.1, 2, 5 and 10 mg/kg) followed by 2 mg/kg MAP. Locomotor activity and stereotyped behavior were assessed. In experiment 2, animals received repeated IP injections of 2 mg/kg MAP pretreated with 2 mg/kg FK506 or vehicle for 5 consecutive days. One week later, rats were challenged with 1 mg/kg MAP. RESULTS: Pretreatment with 2 mg/kg FK506 caused a rightward shift of the inverted U-shaped response curve of the locomotor activity induced by 1-8 mg/kg MAP. The same pretreatment significantly attenuated augmentation of the MAP-induced stereotyped behavior. FK506 at doses of 0.1-10 mg/kg dose-dependently inhibited the behavioral response induced by 2 mg/kg MAP. Coadministration of 2 mg/kg FK506 with 2 mg/kg MAP for 5 consecutive days resulted in significant suppression of the behavioral response to challenge with 1 mg/kg MAP. CONCLUSIONS: These results suggest that calcineurin plays an important role in MAP-induced behavioral changes and sensitization, especially the latter.     

Effect of tacrolimus (FK506) on ischemia-induced brain damage and memory dysfunction in rats

The behavioral and neurohistological protective effects of tacrolimus (FK506) were examined in rats subjected to 15-min global forebrain ischemia. Learning and memory performance were evaluated in an aversive, non-food-motivated, eight-arm radial maze. In one experiment, naive rats were rendered ischemic, and 15 days later they were tested for acquisition of a spatial task (postoperative training). In a complementary experiment, rats were trained for 8 days and then subjected to ischemia (preoperative training); 15 days later (on Day 24 of testing) they were retested for retention of cognition. FK506 (1.0 mg/kg) was given intravenously at the beginning of reperfusion, followed by doses applied intraperitoneally 6, 24, 48 and 72 h postischemia. Behavioral performance was expressed by latency to find the goal box, and number of errors. Ischemia did not affect acquisition performance. In contrast, retention of cognition was markedly impaired by ischemia, particularly working memory (P<.05-.001). This ischemia-induced, retrograde amnesia was significantly reduced by FK506 compared to vehicle alone on Day 24, as measured by latency and working memory errors (P<.025). A neuroprotective effect of FK506 was also seen on working memory, when postischemic performance was compared to that prior to ischemia (P>.05, Day 24 vs. Day 8, paired samples), in contrast to the significant, retrograde amnesia found in the ischemic, vehicle-treated group (P<.01). FK506 also significantly reduced the extent of hippocampal CA1 cell loss; however, this effect did not correlate with behavior. The present results suggest that the histological, neuroprotective effect of FK506 may be accompanied by a reduction in cognitive impairment, as assessed in a novel, non-food-motivated, eight-arm radial maze after transient, global, cerebral ischemia in rats.     

The effects of flurbiprofen, aspirin, cimetidine, and antacids on the gastric and duodenal mucosa of normal volunteers. An endoscopic and photographic study

This 7-day, single blind, randomized endoscopic tolerance study compared daily doses of 100, 150 and 200 mg flurbiprofen with 2600 mg aspirin. After seven days the flurbiprofen 100 and 150 mg groups had significantly less gastric irritation than the flurbiprofen 200 mg and aspirin groups. Flurbiprofen showed a linear dose-response relationship with respect to gastric injury and serum drug levels. Four subjects each on aspirin and flurbiprofen with the most severe injuries continued on their medications plus cimetidine and antacids for four more weeks. Both drug groups showed clinical improvement in the gastroduodenal area. In conclusion, flurbiprofen and aspirin therapy can be tolerated in the presence of gastroduodenal irritation by concomitantly administering cimetidine and antacids.     

一种新型海洋多糖纳米材料的制备及载药特性研究

目的 以天然海洋多糖聚甘露糖醛酸(PM)为原料,制备一种生物相容、生物可降解纳米材料,以难溶性药物他克莫司（FK506）为模型药物制备载药胶束,并对其体外释药特性进行考察。方法 以油胺为疏水端,与PM通过酰胺反应制备纳米材料,利用FT-IR、1H-NMR对其结构进行表征。通过超声乳化法制备FK506胶束,利用原子力显微镜与马尔文激光粒度仪分析载药胶束的形态、粒径及zeta-电位。利用高效液相色谱法测定FK506胶束的载药量和包封率。采用体外透析法分析FK506胶束的体外释放特性。结果 油胺与PM等摩尔比投料反应的枝接率为56.8%,FK506胶束外形为球状且分散均匀,粒径大小在 100~150 nm,zeta-电位为?50 mV,多分散系数＜0.2,载药量和包封率分别为17.6±0.21% 和97.7±1.19%。体外释药分为2个阶段,前12 h释放较快,释放药物的64.5±5.8%,后一阶段药物释放较慢,至48 h时可释放药物89.2±6.9%。结论 成功制备生物可降解的PM纳米材料,以其为载体制得的FK506胶束可以提高FK506的溶解性,在体外具有明显的缓释特征,PM纳米材料具有良好的应用前景。     

FK506 Microparticles Mitigate Experimental Colitis with Minor Renal Calcineurin Suppression

No HeadingPurpose. FK506 microparticles providing selective colonic drug delivery were tested for their efficiency in a local treatment to the inflamed gut tissue in inflammatory bowel disease (IBD). Because FK506 proved its distinct mitigating potential in the treatment of IBD, risking, however, severe adverse effects, a more selective delivery to the site of inflammation may further improve efficiency and tolerability.Methods. A model colitis was induced to male Wistar rats by trinitrobenzenesulfonic acid. FK506 was entrapped into microspheres (MS) prepared with the pH-sensitive polymer Eudragit P-4135F in order to allow drug delivery to the colon. Clinical activity score, colon/body weight index, and myeloperoxidase activity were determined to assess the inflammation, and adverse effects of FK506 resulting from its systemic absorption were quantified as well.Results. The clinical activity score and myeloperoxidase activity decreased after the administration of all FK506-containing formulations. The MS formulations proved to be as efficient in mitigating the experimental colitis as the subcutaneous drug solution (myeloperoxidase activity, MS: 9.64 ± 6.6 U/mg tissue; subcutaneous: 7.48 ± 6.96 U/mg) and to be superior to drug solution given by oral route (oral: 12.66 ± 5.46 U/mg; untreated colitis control: 21.88 ± 4.12 U/mg). The FK506 subcutaneous group exhibited increased levels of adverse effects, whereas the FK506-MS group proved its potential to retain the drug from systemic absorption as evidenced by reduced nephrotoxicity.Conclusions. The development of this selective delivery system for FK506 should be given particular consideration in the treatment of IBD, as it allows therapy that profits from FK506s high immune suppressive effect with a simultaneously reduced nephrotoxicity.     

Induction of choleresis by immunosuppressant FK506 through stimulation of insulin-like growth factor-I production in the liver of rats

FK506 (Tacrolimus) is an effective immunosuppressant currently used worldwide in organ transplantation. Based on our recent findings that insulin-like growth factor-I (IGF-I) is important for the stimulation of choleresis in vivo, in this study we investigated the effect of FK506 on bile flow and the plasma and hepatic levels of IGF-I in rats. Intravenous treatment of rats with FK506 resulted in a significant increase in bile flow, whereas cyclosporin A induced a significant decrease. A significant increase in plasma levels of IGF-I was observed in rats 30 min after a single intravenous administration of FK506. Oral treatment of rats with FK506 for 1 week also resulted in an increase in both plasma and hepatic levels of IGF-I. Overall, this study showed that FK506 treatment increased bile flow and also induced an increase in the plasma and hepatic levels of IGF-I in rats, suggesting that a stimulation of hepatic IGF-I production by FK506 may contribute to its choleretic profile.     

Proteasomal degradation of IRS-2, but not IRS-1 by calcineurin inhibition: attenuation of insulin-like growth factor-I-induced GSK-3beta and ERK pathways in adrenal chromaffin cells

The ability of calcineurin to regulate IRS-1 and IRS-2 levels has not been examined in any given cells, although calcineurin inhibition by therapeutic immunosuppressants produced cytoprotective and cytotoxic effects (e.g., new-onset of diabetes mellitus, seizure). Chronic (>or=3h) treatment of cultured bovine adrenal chromaffin cells with cyclosporin A or FK506 decreased IRS-2 protein level by approximately 50% (IC(50)=200 or 10nM), without changing IRS-2 mRNA level, and insulin receptor, insulin-like growth factor-I (IGF-I) receptor, IRS-1, PI3K/PDK-1/Akt/GSK-3beta and ERK1/ERK2 protein levels. When the cells were washed to remove the test drug, the decreased IRS-2 level restored to the control level. Cyclosporin A or FK506 treatment inhibited calcineurin activity (IC(50)=500 or 40 nM, in vitro assay). Rapamycin, an FK506-binding protein ligand unable to inhibit calcineurin, failed to decrease IRS-2, but reversed FK506-induced decreases of calcineurin activity and IRS-2 level. Pulse-label followed by polyacrylamide gel electrophoresis revealed that cyclosporin A or FK506 accelerated IRS-2 degradation rate (t(1/2)) from >24 to approximately 4.2h, without altering IRS-2 synthesis. IRS-2 reduction by cyclosporin A or FK506 was prevented by lactacystin (proteasome inhibitor), but not by calpeptin (calpain inhibitor) or leupeptin (lysosome inhibitor). Cyclosporin A or FK506 increased serine-phosphorylation and ubiquitination of IRS-2. Cell surface (125)I-IGF-I binding capacity was not changed in cyclosporin A- or FK506-treated cells; however, IGF-I-induced phosphorylations of GSK-3beta and ERK1/ERK2 were attenuated by approximately 50%, which were prevented by rapamycin or lactacystin. Thus, calcineurin inhibition decreased IRS-2 level via proteasomal IRS-2 degradation, attenuating IGF-I-induced GSK-3beta and ERK pathways.     

Protective effect of FK506 (tacrolimus) in pentylenetetrazol-induced kindling in mice

The repeated administration of otherwise subconvulsant dose of pentylenetetrazol (PTZ) is known to produce chemical kindling in animals. In our study, chronic administration of subconvulsant dose of PTZ (40 mg/kg) produced chemical kindling in mice. Pretreatment with L-arginine (50-100 mg/kg ip) potentiated the PTZ-induced kindling, whereas N(omega)-nitro-L-arginine methyl ester (L-NAME) (10-20 mg/kg ip) showed a protective effect. FK506, a potent neuroprotective agent, dose dependently (0.5-1 mg/kg po) decreased the kindling score. When given in combination, L-NAME potentiated the protective effect of lower dose of FK506 (0.5 mg/kg) on PTZ-induced kindling. L-Arginine (50-100 mg/kg) reversed the protective effect of FK506 (1 mg/kg) and L-NAME (20 mg/kg). Biochemical studies showed the potential role of free radical toxicity in the kindled mice, as there was an increased lipid peroxidation as indicated by elevated malondialdehyde (MDA) and nitrite levels and decrease in GSH and superoxide dismutase (SOD) levels. FK506 pretreatment significantly reversed the elevated MDA and nitrite levels, GSH and SOD depletion induced by PTZ treatment. In conclusion, the results of the present study suggest the possible neuroprotective action of FK506 against PTZ-induced kindling.