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Background: Tacrolimus (TAC), a calcineurin inhibitor, is used for remission induction therapy in patients with moderate to severe ulcerative colitis (UC), with short-term efficacy and related predictive factors shown in previous cohort studies. However, most studies reported data for only a limited number of patients enrolled from a single center. We performed a large multicenter retrospective cohort study to identify factors related to prediction of clinical remission in UC patients treated with oral TAC., Methods: The medical records of patients with moderate to severe UC treated with oral TAC as induction therapy at 7 institutions between April 2009 and March 2017 were retrospectively reviewed., Results: A total of 216 patients who received TAC for induction were analyzed, of whom 123 (56.9%) showed clinical remission at week 12. Logistic regression analysis indicated that previous or current use of antitumor necrosis factor (TNF)-α antibodies (odds ratio [OR], 0.259; P = .006), and concomitant treatment with 5-aminosalicylate (5-ASA) at the baseline (OR, 0.268; P = .005) were independent predictive factors correlated with failure of clinical remission, whereas higher levels of C-reactive protein (OR, 1.124; P = .014) predicted achievement of clinical remission., Conclusions: Results of this multicenter study clearly indicate the efficacy of TAC induction therapy for patients with moderate to severe UC. Notably, previous or current use of anti-TNF-α antibodies was associated with poor achievement of clinical remission by week 12., (© The Author(s) 2023. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Oral tacrolimus therapy is effective for refractory ulcerative colitis (UC), but dose adjustment according to the trough concentrations which varies largely among individuals, is required. This study aimed to identify factors to predict the tacrolimus dose required for achieving the target trough level for remission induction of UC., Methods: Forty-seven consecutive UC patients who were treated with tacrolimus were retrospectively analyzed. Tacrolimus doses were adjusted every 2 or 3 days to achieve trough concentrations of 10-15 ng/mL. The dose required for reaching the target trough level was analyzed based on disease characteristics, course of trough concentrations, and gene polymorphism related to tacrolimus metabolism., Results: Median daily dose of tacrolimus required for achieving the target trough level was 0.19 (0.07-0.42) mg/kg, and patients were divided into high or low dose group (< 0.2 mg/kg or > 0.2 mg/kg). The value of initial trough concentration/starting dose was higher in the low dose group than in the high dose group (1.35 ng/mL/mg vs. 0.78 ng/mL/mg, p < 0.0001). Although presence of CYP3A5 *1 was more frequently observed in the high dose group, initial trough concentration was the only significant factor for determining requirement of high dose of tacrolimus (OR = 28.0, 95% confidence interval 3.20 - 631)., Conclusions: The most practical predictor of the dose required for achieving the target trough concentration was the trough concentration measured 2 or 3 days after starting tacrolimus therapy. Our findings would make tarcolimus administration for UC safer, easier and more effective.

The effect of tacrolimus on epididymal biochemical markers was investigated following single daily subcutaneous doses of 1, 2 and 3 mg kg(-1) day(-1) for 2 weeks to male adult rats. The tacrolimus 2 and 3 mg kg(-1) day(-1) groups showed a significant and dose-dependent decrease in sperm count in the cauda epididymis. Among tissue levels of L-carnitine, alpha-glucosidase and acid phosphatase, only L-carnitine level in the cauda epididymis was significantly reduced in the tacrolimus 3 mg kg(-1)day(-1) group. However, no significant difference was seen in the plasma L-carnitine. It was suggested that lowering of L-carnitine in the cauda epididymis was attributable to the adverse effect on epididymal function to transport and/or concentrate L-carnitine. Since L-carnitine has been reported to have antioxidant potential, antioxidant defense enzymes in the cauda epididymis such as superoxide dismutase (SOD), catalase, glutathione peroxidase and glutathione reductase were evaluated. The results showed no significant differences in activities, confirming that the treatment with tacrolimus did not affect the activities of these antioxidant enzymes. In conclusion, this study indicates that tacrolimus induces a decrease in L-carnitine level in the cauda epididymis, which is probably caused by impairment of epididymal function to transport and/or concentrate L-carnitine from bloodstream, and a decrease in sperm count.

Background: FK506 ointment (tacrolimus ointment, protopic) is a new drug therapeutically effective for patients with atopic dermatitis (AD). However, the mechanism of action of FK506 ointment on AD is not fully understood., Methods: We examined the effect of FK506 ointment on mite antigen-induced dermatitis in NC/Nga mice. Clinical symptoms and ear thickness were recorded, and histopathological studies and in vitro analyses were performed., Results: Topical application of FK506 ointment (0.03-0.3%) suppressed the development of dermatitis. In the lesional skin, both interleukin (IL)-4 and interferon (IFN)-gamma were detected, even though the IL-4+/IFN-gamma- T helper 2 (Th2) population was predominant in the regional lymph nodes (LNs). Topical application of FK506 treatment reduced the elevated level of both IL-4 and IFN-gamma in the skin, but did not decrease the expansion of the Th2 population in the LNs., Conclusions: Topical application of FK506 ointment suppresses dermatitis by inhibiting the activation of inflammatory cells locally, without systemic immune suppression, in this AD model., (Copyright 2004 S. Karger AG, Basel)

Background: It is well-known that FK506 strongly inhibits cytokine production by T cells in vitro. However, less evidence is available from in vivo studies of ocular allergy., Objective: To study the anti-inflammatory effect of FK506 eye drops on late and delayed-type responses in several animal models of ocular allergy., Methods: Rats and guinea-pigs were sensitized with egg albumin (EA) in adjuvant and later challenged by topical EA application to their eyes to examine the late response. Biopsy specimens of conjunctiva were stained with haematoxylin-eosin or stained for T cells and eosinophils. In addition, rats, rabbits and guinea-pigs were sensitized with complete Freund's adjuvant and later challenged by injecting purified protein derivatives for the delayed-type response. Bulbar conjunctival oedema and hyperaemia were graded by score in rabbits, and Evans blue (EB) extravasation was measured in rats and guinea-pigs. FK506 (0.01-1%) and steroid (0.1%) eye drops were instilled in the eyes of animals several times, before and after challenge., Results: FK506 eye drops inhibited T cell and eosinophil infiltration in the late response and EB extravasation in the delayed-type response in rats. Also, they inhibited conjunctival oedema, hyperaemia and ocular mucus in the delayed-type response in rabbits. These effects were similar to those of steroid eye drops (betamethasone sodium phosphate, fluorometholone). FK506 eye drops also inhibited inflammatory cell infiltration, the loss of conjunctival epithelium and decrease of goblet cells in the late response as well as EB extravasation in the delayed-type response in guineapigs, a steroid-resistant species., Conclusion: FK506 eye drops inhibit late and delayed-type responses in animal models of ocular allergy.

Chronic allergic contact dermatitis was induced in rat ear by repeated application of oxazolone. This dermatitis was accompanied by sustained ear swelling and marked epidermal hyperplasia. In the induced ear, there was marked inflammatory cell infiltration into the dermis site and the interferon-gamma amount increased in both protein and mRNA, while the interleukin-4 amount changed minimally. Topical administration of FK506 (tacrolimus hydrate) dramatically suppressed ear swelling and epidermal hyperplasia as well as the increase in interferon-gamma expression. Betamethasone valerate also showed suppressive effects, but 1,25-dihydroxyvitamin D(3) (calcitriol) had no effect. These results suggest that interferon-gamma plays an important role in dermatitis and this model could be a useful pharmacological model for chronic dermatitis featuring epidermal hyperplasia in which interferon-gamma plays a crucial role, such as psoriasis. FK506 demonstrating suppressive effects as potent as those of betamethasone valerate shows potential as a topically usable drug for such skin disorders.

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.

Objective and Design: The anti-arthritic properties of FK506 were compared with methotrexate (MTX) in established adjuvant-induced arthritis (AIA) in rats., Material: Female Lewis rats., Treatment: Arthritic rats were orally administered with FK506 (1-5.6 mg/kg) and MTX (0.1-1 mg/kg) from days 15-24., Methods: Arthritis was induced by injection of Mycobacterium tuberculosis into the right hind footpad on day 0. Efficacy was determined on the basis of paw inflammation measured by paw volume and histological change, hyperalgesia and grip strength. Grip strength measurement was employed as an indication of function of paws in arthritic rats. Peripheral white blood cell (WBC) counts and thymus weights were measured, mainly as indicators of toxic side effects., Results: FK506 suppressed paw inflammation and hyperalgesia without toxic effects on WBC and thymus in established AIA. MTX slightly suppressed paw inflammation and hyperalgesia at the highest dose (1 mg/kg). Toxic effects were observed at lower doses than the effective treatment dose with MTX. Grip strength was found to decrease during development of AIA. FK506, but not MTX, treated rats recovered grip strength loss., Conclusions: The results show that FK506 is more effective and less toxic than MTX in treating established AIA in rats.

Tacrolimus (FK506) ointment showed remarkable efficacy against atopic dermatitis in animal models and clinical trials. The suppressive effect of tacrolimus on the production of the cytokines involved in atopic dermatitis (IL-2, IL-3, IL-4, IL-5, IFN-gamma and GM-CSF) from human peripheral blood mononuclear cells (PBMC) was investigated. We constructed a new cytokine production system in which T cells are activated by direct stimulation in vitro with anti-CD3/CD2 or anti-CD3/CD28 antibody combination. Tacrolimus inhibited the production of these cytokines by both stimulations. In a comparative study with steroids (alclometasone dipropionate and betamethason valerate) in anti-CD3/CD2 system, tacrolimus and both steroids inhibited Th1 cytokines (IL-2, IFN-gamma), Th2 cytokines (IL-4, IL-5) and IL-3, GM-CSF (produced by both Th1 and Th2). The suppressive effect of tacrolimus on cytokine production was stronger than that of alclometasone dipropionate and equal to or stronger than that of betamethason valerate. The effective dose of tacrolimus (IC50, 0.02-0.11 ng/ml) is almost the same as for Th1 and Th2 cytokines, and 1 ng/ml of tacrolimus suppressed all cytokines completely. These results suggest that tacrolimus suppresses the allergic cytokines from T cells, and that tacrolimus ointment is effective against atopic dermatitis through the inhibition of cytokine production.

The objective of this study was to investigate the therapeutic potential of FK506 and other immunosuppressive agents for the treatment of rheumatoid arthritis (RA), focusing on the effects on in vitro IL-6 production and IL-6-mediated immune response. We employed an in vitro model producing IL-6 via T cell activation in human PBMC, based on the hypothesis that T cells play a central role in the pathogenesis of RA. FK506 potently inhibited IL-6 production from PBMC stimulated with anti-CD3 and anti-CD28 monoclonal antibody (anti-CD3/CD28). Cyclosporin A (CsA) also inhibited the anti-CD3/CD28 induced IL-6 production but was about 100 times less potent than FK506. Dexamethasone (DEX) inhibited both anti-CD3/CD28 and LPS induced IL-6 production at almost the same concentration. Methotrexate (MTX) did not affect cytokine production. Anti-CD3/CD28 stimulated PBMC culture supernatants were found to enhance IgM production in SKW6.4 cells. The effects of anti-CD3/CD28 stimulated culture supernatants in the presence of agents on IgM production in SKW6.4 cells were investigated. FK506 and CsA led to suppression of IgM production induced by culture supernatants probably via inhibition of IgM inducible cytokine production from PBMC. DEX profoundly enhanced IgM production, although IL-6 production from PBMC was strongly inhibited by the agent. MTX decreased IgM production although it has no inhibitory effect on IL-6 production. The present study suggests that FK506 is the most effective among the four agents for the suppression of IL-6 production and IL-6-mediated autoantibody production in T cell activation related autoimmune diseases such as RA.

To understand the mechanism of action of FK506 (Tacrolimus) on neutrophil chemotaxis, we examined its effect on human neutrophil chemotaxis and neutrophil chemoattractant production by peripheral blood mononuclear cells. FK506 and cyclosporin A had no direct suppressive effect on neutrophil chemotaxis induced by interleukin-8, leukotriene B(4), complement 5a (C5a), zymosan-activated serum and formyl-Met-Leu-Phe (fMLP). FK506 and cyclosporin A only slightly suppressed the chemotactic activity of platelet-activating factor (PAF). Dexamethasone did not inhibit the chemotactic activity of any chemoattractant. The supernatant of peripheral blood mononuclear cells stimulated with anti-CD3 and CD2 antibodies induced neutrophil chemotaxis. FK506 and cyclosporin A suppressed the chemotactic activity of the supernatant in parallel to the suppression of interleukin-8 production by peripheral blood mononuclear cells. Anti-interleukin-8 antibody completely suppressed the chemotactic activity of the supernatant without drugs. These studies indicate that FK506 may exert a beneficial effect on human inflammatory diseases by suppressing neutrophil chemotaxis secondary to inhibition of chemoattractant (for example, interleukin-8) production by leukocytes.

The aim of this study was to elucidate the in vitro inhibitory potency of FK506 on production of the inflammatory cytokines, tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta, with a view to assessing this immunosuppressive agent as a potential anti-rheumatic drug. We employed an in vitro model which produces TNF-alpha and IL-1beta through T cell activation. Human peripheral blood mononuclear cells (PBMC) were cultured with immobilized anti-CD3/CD28 monoclonal antibody in this model. FK506 inhibited anti-CD3/CD28 induced TNF-alpha and IL-1beta production at concentrations less than 1 ng ml(-1). Flow cytometric analysis of intracellular TNF-alpha and IL-1beta positive cells showed that FK506 potently suppresses inflammatory cytokine production from CD14+ monocytes as well as from T cells. Cyclosporin A (CsA) and dexamethasone (DEX) also inhibited the anti-CD3/CD28 induced cytokine production, but were less potent than FK506. FK506 and CsA, but not DEX, specifically inhibited anti-CD3/CD28 induced inflammatory cytokine production without affecting the lipopolysaccaride (LPS) induced effect. Methotrexate (MTX) was completely inactive for suppressing cytokine production under either condition. Anti-CD3/CD28 stimulated PBMC culture supernatants were found to enhance the expression of adhesion molecules in human vascular endothelial cells. FK506, CsA and DEX led to the suppression of adhesion molecule expression probably by inhibiting cytokine production from PBMC. The inhibitory potency of agents on TNF-alpha and IL-1beta production was compared with cytotoxicity and FK506 was not cytotoxic at concentrations several orders of magnitude greater than those required for cytokine inhibition. These results strongly suggest that FK506 may be most effective to specifically prevent T cell activation mediated inflammatory cytokine production in a clinical setting.

Histamine release and cytokine production by mast cells and basophils are thought to be closely involved in the pathogenesis of allergic diseases. Some reports show that FK506 (tacrolimus hydrate) inhibited histamine release and cytokine production by mast cells and basophils. However, as the effects of FK506 has not been compared with those of clinically used drugs in those reports, the clinical relevancy of FK506 inhibition remained unclear. In this paper, we compared the actions of FK506 with those of steroids or disodium cromoglycate (DSCG) which has been clinically used. FK506 inhibited histamine release by Brown-Norway rat peritoneal mast cells more potently than steroids and especially DSCG. FK506 also inhibited histamine release by a mast rat basophilic leukemia (RBL)-1 cell line and human peripheral blood basophils, whereas steroids failed to inhibit histamine release by human basophils. FK506 as well as steroids inhibited TNF-alpha and IL-4 production by RBL-1 cells. FK506 was therefore more effective than steroids and DSCG in inhibiting histamine release, and it also had the ability of inhibiting cytokine production by mast cells as steroids do. We concluded that FK506 might regulate allergic diseases via these actions, judging from the viewpoint of clinical relevancy.

The effects of FK506 (tacrolimus hydrate) ointment on cutaneous allergic reactions in mice and rats were investigated. FK506 ointment showed significant suppressive effects on delayed allergic reactions in both species, and especially in rats, its inhibitory action was much stronger than that of alclometasone dipropionate, a so-called medium class steroid ointment. On the other hand, FK506 ointment did not inhibit the immediate allergic reaction in rats. FK506 ointment suppressed the delayed allergic reactions in locally passively sensitized mice to the same degree as that in actively sensitized mice. Accordingly, it is speculated that FK506 ointment inhibits the activation of sensitized T lymphocytes (Th1 cells) already accumulated in the dermis.

Although tacrolimus has been studied in a wide variety of experimental animal models, we are the first group to systematically study the effect of tacrolimus on rat renal allograft survival, as primary therapy and as anti-rejection therapy, in comparison with cyclosporin A (CyA). Renal grafts were transplanted from BN to LEW rats. Tacrolimus and CyA were administrated orally from day 0 for 50 days as primary therapy after grafting. Allografts were rejected after a median survival time (MST) of 8 days. Both tacrolimus und CyA significantly prolonged renal allograft survival, in a dose-dependent manner compared with the allograft controls. The most effective dose was 3.2 mg/kg, per day for tacrolimus, and 10 mg/kg per day for CyA. There was no significant difference in renal function between the group treated with the most effective dose of tacrolimus and the CyA-treated group. The percentage of detectable serum IL-2 level was 45% in the allograft control group, but was undetectable in groups treated with the most effective dose of tacrolimus or CyA at days 3 and 6 after grafting. On the other hand, no side effects were noted in recipient rats by daily inspection, body weight change, and histological studies, although minimal tubular vacuolation was encountered in the group treated with CyA 32 mg/kg per day. In addition, the most effective doses of tacrolimus and CyA were studied as anti-rejection therapy. All of the 5 recipients treated with tacrolimus from days 2-14, and 3 of the 5 treated from days 4-16 after grafting, survived for more than 50 days. However, the MST was 19 days for recipients treated with CyA from days 2-14, and 13 days for those treated from days 4-16 after grafting. In summary, tacrolimus as primary therapy induced rat renal allograft survival with renal function and side effects comparable with those of CyA. Interestingly, when both agents were used as anti-rejection therapy, tacrolimus, but not CyA, could significantly overcome ongoing renal allograft rejection in the rat.

The effects of tacrolimus ointment on immediate, late and delayed-type cutaneous allergic reactions and normal skin thickness were investigated in mice and compared with those of steroid ointments. Tacrolimus ointment had no effect on ear edema in the immediate phase of the biphasic reaction and did not inhibit passive cutaneous anaphylaxis, but in the late phase of the biphasic reaction, it inhibited the ear edema. It also showed a clear inhibitory effect on the delayed-type reaction. These evidence suggest that the clinical effect of tacrolimus ointment against atopic dermatitis (AD) may be mainly due to its inhibitory action on late and delayed-type reactions. The steroid ointments inhibited all the reactions mentioned above, and the effects were more potent than those of tacrolimus. Moreover, they also decreased the normal ear thickness, suggesting that the inhibitory effect of the steroid ointments was partially due to skin atrophic action. The same ointments applied to the ears during the induction phase showed an enhancement of delayed-type reaction at the effector phase. Tacrolimus ointment did not show such a rebound effect or skin atrophy. Thus, tacrolimus ointment was expected to be more useful than the steroids for the treatment of AD.

To characterize the metabolic pathway of FK506 (tacrolimus), FK506 or its 31-O-desmethyl metabolite was incubated with liver microsomes prepared from dexamethasone-treated rats in the presence of a NADPH-generating system under aerobic conditions. Besides the four oxidized metabolites already reported, four new metabolites were isolated and identified by HPLC, mass spectrometry, and NMR spectroscopy, and their biological activities were examined. The di-demethylated metabolites at the 15- and 31-, 13- and 31-, and 13- and 15-methoxy groups of FK506, were designated respectively as M-V, M-VI, and M-VII. The fourth, M-VIII, was the metabolite produced after O-demethylation at the 31-methoxy group and formation of a fused 10-membered ring structure through the 19- to 22-carbon of the macrolide ring after oxidation of the 19-methyl group, and of the 36- and 37-vinyl group of FK506. The immunosuppressive activity of the isolated metabolites was estimated in a mouse mixed lymphocyte reaction system and the IC50 values for M-V, M-VI, M-VII, M-VIII, and FK506 were > 1000, 8.78, > 1000, 15.27, and 0.11 ng/ml, respectively. Reactivity of the metabolites with mouse anti-FK506 monoclonal antibody was studied and immunocrossreactivity of M-V was 92.3% of FK506, but no reactivity was observed for M-VI, M-VII, and M-VIII. FK506 thus was metabolized at multiple sites by rat hepatic microsomes and the metabolites formed (M-V) - (M-VIII) exhibited weak or negligible immunosuppressive activity.

Tacrolimus(FK506) is a strong immuno-suppressant and shows its activity through inhibiting IL-2 mRNA transcription by forming pentameric complex with intracellular receptor(FK506 binding protein 12 kDa or FKBP12), Ca2+, calmodulin, and calcineurin. Here, we report the binding activity to FKBP12, the pentameric complex formation and Con-A response inhibiting activities of 7 metabolites. C15-demethylated metabolite(M-3) needed higher quantity to compete in Con-A assay and in pentamer formation assay, although it binds more strongly to FKBP12. The result suggests that the ability to form a pentameric complex is not a two step reaction with the first binding to FKBP12, but a single step reaction by components for the pentamer formation.

To characterize structures and biological activities of FK506 metabolites, FK506 was incubated with liver microsomes prepared from phenobarbital-treated rats in the presence of NADPH generating system under aerobic condition. Oxidative metabolites formed in the reaction medium were isolated and identified. Purified samples were analyzed by HPLC, mass spectrometry, and NMR spectroscopy. M-I, M-II, and M-III were the O-demethylated metabolites at the 13-, 31-, and 15-positions of FK506, respectively, and M-IV was the monohydroxylated metabolite at the 12-position. M-I was the dominant metabolite in this reaction system. M-II and M-III retained the tetrahydropyrane ring in their structures like FK506, but M-I and M-IV had rearranged structures in which the tetrahydropyrane ring was changed to a tetrahydrofuran ring. Measuring the immunosuppressive activity in the mouse mixed lymphocyte reaction system, IC50 values for M-I, M-II, M-III, M-IV, and FK506 were 1.65, 0.23, > 127, 5.52, and 0.15 nM, respectively. Reactivity of the metabolites with mouse anti-FK506 monoclonal antibody was studied and immunocross-reactivity of M-I, M-II, M-III, and M-IV with the antibody were nil, 109.0, 90.5, and 8.8% of FK506, respectively. These results indicate that rat hepatic microsomes oxidatively metabolize FK506 to four metabolites, and some of them exhibit pharmacological activity.