FR252921, a novel immunosuppressive agent isolated from Pseudomonas fluorescens no. 408813. I. Taxonomy,fermentation, isolation,physico-chemical properties and biological activities of FR252921, FR252922 and FR256523

Novel immunosuppressive agents, FR252921, FR252922 and FR256523 were isolated from the cultured broth of a bacterial strain No. 408813. The strain was identified Pseudomonas fluorescens from morphological and physiological characteristics. FR252921, FR252922 and FR256523, novel compounds containing macrolactone ring, showed immunosuppressive activity against murine splenocyte proliferation stimulated with lipopolysaccharide (LPS) or anti-CD3 mAb in vitro.     

Selective immunosuppression of prodigiosin 25-C and FK506 in the murine immune system.

The immunosuppressive effects of prodigiosin 25-C were studied in comparison with FK506. Both prodigiosin 25-C and FK506 suppressed T cell proliferation in response to concanavalin A (con A) or phytohemagglutinin (PHA) more significantly than that to lipopolysaccharide. However, prodigiosin 25-C inhibited con A-mediated mitogenic response more strongly than PHA-mediated one. FK506 showed no selectivity among those responses. In addition, when higher concentration of con A was used an inhibitory effect of prodigiosin 25-C became more evident whereas that of FK506 became less evident. Furthermore, prodigiosin 25-C affected neither interleukin-2 (IL-2) production nor IL-2 receptor (IL-2R) and transferrin receptor (TF-R) expression in vitro, though FK506 extensively inhibited IL-2 production and significantly suppressed IL-2R and TF-R expression. When comparing the effects of prodigiosin 25-C and FK506 in vivo by injecting antigens of different nature to a mouse, prodigiosin 25-C selectively inhibited cytotoxic T lymphocyte (CTL) activity induced by an allogenic mastocytoma, P815, without affecting production of antibody against a thymus dependent (TD) antigen, sheep red blood cell (SRBC). On the contrary, FK506 significantly inhibited both CTL induction and the antibody production. When Brucella abortus, a thymus independent (TI) antigen, and SRBC were simultaneously challenged to a mouse, neither prodigiosin 25-C nor FK506 affected antibody production against the TI antigen while the effect on the TD antigen were the same as described above. The present results revealed the unique immunosuppressive property of prodigiosin 25-C which was different from that of FK506.     

Study of FK-binding protein: FK506-metabolite complexes by electrospray mass spectrometry: correlation to immunosuppressive activity.

Electrospray ionization mass spectrometry was used to study several non-covalent FK-binding protein (FKBP) immunosuppressant complexes in the gas phase. Relative FKBP binding affinities were determined from the signal ratio for the 7+ charge states of bound and unbound complexes as a function of capillary exit voltage. All complexes displayed a 1:1 binding stoichiometry. The relative gas-phase binding affinities were found to be well correlated with in vitro FKBP binding and in vitro immunosuppression (rapamycin > FK506 > or = 31-demethyl FK506 > 13-demethyl FK506 > Cyclosporin A; CsA). The method demonstrates potential as a simple, rapid, and automatable technique for prediction of the immunosuppressive activity of FKBP:drug complexes.     

Biologicals and Immunologicals Overview: Recent developments in macrolide immunosuppressants

Rapamycin and FK506 are structurally related macrolide antibiotic compounds which exhibit potent immunosuppressive properties in higher eukaryotes. It is now well established that these powerful immunosuppressive macrolides, as well as the structurally unrelated undecapeptide Cyclosporin A, all mediate their immunosuppressive effects via inhibition of the specific signal transduction pathways that induce T-lymphocyte activation. Despite the structural similarity between rapamycin and FK506, and that both FK506 and rapamycin bind to FK506 binding proteins through a common transition state mimic structure, the FK506 binding protein 12-rapamycin complex does not bind to calcineurin. FK506 acts at an early stage of the distinct intracellular signal transduction pathway, inhibiting T-cell receptor-mediated transition from a resting state, Go, to the G₁ phase of the cell cycle (activation). Thus, the FK506-FKBP12 and cyclosporin A-cyclophilin complexes act similarly to each other, inhibiting a family of Ca²⁺-dependent signalling pathways by forming a termolecular complex with calcineurin, a Ca²⁺, calmodulin-dependent protein phosphatase target. In contrast to FK506, rapamycin inhibits cytokine-driven T-lymphocyte proliferation rather than activation of cells induced by antigen-T-cell receptor interaction. Rapamycin selectively interacts with a family of Ca²⁺-dependent and Ca²⁺-independent signalling pathways, and as a consequence induces its effects at a later stage of the intracellular signalling pathway, arresting the transition from the G₁ phase to the S phase of the cell cycle (progression). Recent studies have indicated that rapamycin mediates its immunosuppressive activity by selectively inhibiting a specific protein phosphorylation p70 S6 serine/threonine protein kinase cascade. This overview describes recent developments in both the current understanding of the molecular basis of the mechanism of action by which these macrolide immunosuppressive agents exert their activity and the influence on the development of several new diagnostic screening assays. The activity of an extensive number of novel semi-synthetic and naturally produced macrolide compounds in vitro and in vivo tests are evaluated, as well as a broad spectrum of new therapeutic applications.     

FK506 ameliorates spontaneous locomotor activity in collagen-induced arthritis: implication of distinct effect from suppression of inflammation

FK506 (tacrolimus), an immunosuppressive drug, improves quality of life (QOL) for patients with rheumatoid arthritis (RA). However, the mechanism of FK506 behind the improvement in QOL is still uncharacterized. To explain the improvement of QOL by FK506, we investigated the effect of FK506 on spontaneous locomotor activity in rats with collagen-induced arthritis (CIA). CIA was induced in 7- to 8-week-old female Lewis rats by immunization with bovine type II collagen. After initiation of paw inflammation (paw swelling, histopathological analysis), CIA rats were therapeutically administered FK506 or methotrexate (MTX) from day 15. Therapeutic treatment with FK506 ameliorated spontaneous locomotor activity without suppressing paw inflammation in CIA rats from day 27. FK506 also improved hyperalgesia and grip strength from day 27. Therapeutic treatment with MTX did not improve spontaneous locomotor activity, and simultaneously did not recover hyperalgesia or grip strength in CIA rats. Our results indicate that spontaneous locomotor activity in CIA rats correlates mainly with hyperalgesia and muscle strength, but not paw inflammation, implying that therapeutic treatment with FK506 ameliorates spontaneous locomotor activity via improvement of hyperalgesia and muscle strength in CIA rats.     

P-glycoprotein function in peripheral blood mononuclear cells of myasthenia gravis patients treated with tacrolimus

Tacrolimus hydrate (FK506) reduces the symptoms of myasthenia gravis (MG) due to its immunosuppressive properties. A drug efflux pump P-glycoprotein (P-gp) actively transports FK506 out of target cells, thereby reducing their efficacy. We investigated the influence of FK506 therapy on the P-gp function of peripheral-blood mononuclear cells (PBMCs) in MG patients. Six MG patients treated with FK506 (MG(FK+)), four MG patients treated without FK506 administration (MG(FK-)), and 18 healthy subjects were included in this study. P-gp function was estimated by transporter activity that was inferred from a decrease in fluorescent P-gp substrate Rhodamine 123 (Rh123) and its inhibition by cyclosporine A (CsA). The P-gp efflux function in MG (FK+) patients assessed by the Kolmogorov-Smirnov (KS) statistic D was lower than in the healthy subjects (p=0.0084). However, PBMC sensitivity to FK506 in MG (FK+) patients was significantly higher compared to that of the healthy subjects (p=0.02). There was a significant correlation between the Rh123 efflux activity and PBMC sensitivity to FK506 in vitro (p=0.011). The data raise the possibility that FK506 treatment attenuated P-gp function in the PBMCs of the MG patients.     

Effect of FK-506 and cyclosporine on human T and B lymphoproliferative responses.

The macrolide antibiotic FK-506 causes a strong inhibition of human T and B cell proliferation in vitro in concentrations 100-fold lower than cyclosporine (CsA). However, B cell activation appears to be less sensitive to the immunosuppressive action of FK-506 than T cell responses. There was no indication of a synergistic interaction of the two agents. In contrast, CsA added together with FK could counteract the immunosuppressive activity of the latter drug.     

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.     

他克莫司在932例肾移植患者中的应用

目的 :评价他克莫司 (FK506)用于肾移植患者免疫抑制治疗的效果与安全性。方法 :对肾移植术后应用FK506的932例患者的资料进行回顾性分析。结果 :肾移植术后即应用FK506的547例患者 ,急性排斥反应发生率低 ;由环孢素A (CsA)改用FK506的385例患者 ,大部分排斥反应缓解 ,肝功能改善。FK506的主要不良反应有血糖升高和神经系统毒性。结论 :FK506是一种强效免疫抑制剂 ,还可用于CsA不能逆转排斥反应的挽救治疗。     

他克莫司在932例肾移植患者中的应用评价

目的 :评价他克莫司 (FK506)用于肾移植患者免疫抑制治疗的效果与安全性。方法 :对肾移植术后应用FK506的932例患者的资料进行回顾性分析。结果 :肾移植术后即应用FK506的547例患者 ,急性排斥反应发生率低 ;由环孢素A (CsA)改用FK506的385例患者 ,大部分排斥反应缓解 ,肝功能改善。FK506的主要不良反应有血糖升高和神经系统毒性。结论 :FK506是一种强效免疫抑制剂 ,还可用于CsA不能逆转排斥反应的挽救治疗。     

Protective effect of green tea extract and tea polyphenols against FK506-induced cytotoxicity in renal cells

The nephrotoxicity induced by the immunosuppressive drug FK506 (tacrolimus or fujimycin), limits its usefulness in widespread application, and the underlying mechanism has not been completely understood. The primary targets of FK506 in the kidney are the proximal tubular epithelial cells. In this study, the protection of green tea extract against FK506-induced cell death of LLC-PK1 cells was investigated. FK506 caused a significant decrease in survival of the cells, but the addition of green tea extract reduced this effect in a dose-dependent manner. Treatment of the cells with 50 microM (41.1 microg/ml) FK506 induced a significant increase in annexin V-positive/propidium iodide-negative cells from 2.68 to 14.5%, whereas the addition of 6.25, 12.5, and 25 microg/ml of green tea extract caused a significant protective effect in apoptotic cells from 14.5 to 6.51, 3.20 and 3.02%, respectively. The effect of five different constituent tea polyphenols was also examined. Epigallocatechin-gallate and epigallocatechin significantly reduced FK506-induced cytotoxicity but epicatechin and catechin had no effect on cell viability. Furthermore, changes in cytochrome c release and caspase activation, which characterize apoptosis, were studied. Epigallocatechin-gallate and epigallocatechin suppressed a significant release of cytochrome c and activation of caspase-3 in FK506-treated LLC-PK1 cells.     

Microbial transformation of immunosuppressive compounds. I. Desmethylation of FK506 and immunomycin (FR 900520) by Actinoplanes sp. ATCC 53771.

The immunosuppressants FK506 and FR 900520 were desmethylated by Actinoplanes sp. ATCC 53771 to yield various O-desmethylated products. The products were isolated and purified by solvent extraction and HPLC chromatography, and identified by NMR and MS spectroscopy.     

Targeting FK506 binding proteins to fight malarial and bacterial infections: current advances and future perspectives

There is an urgent need for the design and development of new and selective drugs for the treatment of malaria and bacterial infections as these pathogens are developing resistance to presently available therapies. Malaria is a life threatening disease in many countries and responsible for almost one million deaths annually. In particular, drug-resistant malarial parasites are hindering effective control of malaria and prompting to find novel druggable targets and develop compounds with mechanism of action different from the conventional drugs. In this quest, efforts were made to determine three-dimensional structures of Plasmodium falciparum and Plasmodium vivax FK506 binding proteins which bind the macrolides (FK506 and rapamycin) and also demonstrate peptidylprolyl cis-trans isomerase activity in a similar manner as human FKBP12. Previous studies revealed that the immunosuppressive drug FK506 exhibits potential anti-malarial activity by binding FK506 binding domains (FKBD). This review focuses on three different types of FK506 binding proteins/domains in pathogens, their structural characteristics and biological roles. Binding ability of these proteins with the macrolides has opened new possibilities to develop selective inhibitors for these novel targets to combat the life threatening infections.     

FK506 induces chondrogenic differentiation of clonal mouse embryonic carcinoma cells, ATDC5

FK506 (Tacrolimus) and cyclosporin A exert their immunosuppressive effects via a common mechanism, calcineurin inhibition, after binding to intracellular proteins termed immunophilins: FK506-binding protein (FKBP) and cyclophilin. In this study, FK506 was found to induce chondrogenic differentiation of ATDC5 cells (clonal mouse embryonal carcinoma cells) in a concentration-dependent manner (0.1-1000 ng/ml). Immunohistochemical staining showed that ATDC5 cells induced to differentiate by FK506 produced proteoglycan and type II collagen, main components of the extracellular matrix of cartilage. Rapamycin, an immunosuppressant that binds to FKBP, antagonized the effect of FK506. Cyclosporin A did not induce chondrogenesis at concentrations up to 1000 ng/ml. Taken together, these results suggest that FK506 induces chondrogenic differentiation of ATDC5 cells via a calcineurin-independent mechanism, after binding to FKBP.     

Effects of the immunosuppressants cyclosporin A and FK 506 on exocytosis in the rat exocrine pancreas in vitro.

1. We have examined the effects of the immunosuppressive drugs cyclosporin A (CsA) and FK 506 on exocytosis in two in vitro preparations of the exocrine pancreas-lobules and dispersed acini. 2. In lobules taken from starved rats and stimulated with the secretagogue caerulein, both CsA and FK 506, given shortly before stimulation, caused a dose-dependent inhibition of amylase secretion. In lobules from rats that had been pretreated in vivo with the protease inhibitor FOY-305 to stimulate secretion maximally, both CsA and FK 506 inhibited secretion of newly synthesized proteins, whereas only FK 506 inhibited caerulein-stimulated amylase release. 3. These different effects of the immunosuppressants on amylase release were reflected in their effects on degranulation, as revealed by electron microscopy. Control acinar cells in lobules from FOY-305-treated rats were almost completely degranulated, whereas treatment with FK 506, but not CsA, caused the accumulation of zymogen granules close to the apical plasma membrane. 4. In dispersed acini, stimulated with the cholinomimetic secretagogue bethanechol, both CsA and FK 506 reduced the secretory response, to about 45% of control; IC50 values were 50 nM and 3 nM, respectively. A similar partial inhibition of exocytosis was seen in acini permeabilized with the bacterial toxin streptolysin O and stimulated with 10 microM Ca2+. 5. These results demonstrate that the immunosuppressants cause an inhibition of exocytosis in the exocrine pancreas that is both rapid in onset and potent.(ABSTRACT TRUNCATED AT 250 WORDS)