Yoshitaka Ohkubo

Tacrolimus suppressed the production of cytokines involved in atopic dermatitis by direct stimulation of human PBMC system. (Comparison with steroids).

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.

FK506 potently inhibits T cell activation induced TNF-α and IL-1β production in vitro by human peripheral blood mononuclear cells

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)‐α and interleukin (IL)‐1β, with a view to assessing this immunosuppressive agent as a potential anti‐rheumatic drug. We employed an in vitro model which produces TNF‐α and IL‐1β 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‐α and IL‐1β production at concentrations less than 1 ng ml−1. Flow cytometric analysis of intracellular TNF‐α and IL‐1β 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‐α and IL‐1β 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.

Inflammatory responses in cuff-induced atherosclerosis in rabbits

Cuff-treatment of the rabbit carotid artery produced a diffuse intimal thickening which resembled early lesions of atherosclerosis. A limited amount of focal endothelial damage occurred first (0.5 h), leukocytes infiltrated the subendothelium and extensive endothelial denudation occurred at 24 h. At 3 days, the regenerating endothelium covered the denuded area, and the media was edematous. At 7 days proliferation of intimal cells became visible. Maximum intimal thickening occurred at 3 weeks. Daily injection of dexamethasone (0.01-10 mg/kg i.m.) and ticlopidine (1-100 mg/kg i.m.) dose-dependently attenuated the intimal thickening. Indomethacin had little effect. Inflammatory exudate from zymosan-activated air pouch induced chemotaxis of rat smooth muscle cells (SMC) in vitro. Similar chemotactic activity was observed with leukotriene B4 (LTB4) but not with the other lipoxygenase products tested. The exudate contained reasonable amounts of LTB4, which would account for its chemotactic activity. Dexamethasone inhibited the chemotaxis by the exudate and proliferation of SMC. These results are discussed in relation to the mechanism of atherogenesis. It is concluded that leukocytes play a major role in cuff-induced intimal thickening, and that their products cause endothelial denudation and SMC chemotaxis. Involvement of platelet aggregation in atherogenesis is also suggested.

Characterization of a novel aldose reductase inhibitor, FR74366, and its effects on diabetic cataract and neuropathy in the rat☆

FR74366 (FK366) ([3-(4-bromo-2-fluorobenzyl)-7-chloro-2,4-dioxo-1,2,3,4- tetrahydroquinazolin-1-yl] acetic acid) is a chemically novel aldose reductase (AR) inhibitor. It exhibited a highly potent, reversible, and mixed type inhibition of partially purified AR from the rat sciatic nerve (IC50 = 3.6 nmol/L) and rat lens (IC50 = 4.4 nmol/L). FR74366 inhibited sorbitol accumulation in the isolated human erythrocyte (IC50 = 1.6 mumol/L), rat lens (IC50 = 39 mumol/L), and rat sciatic nerve (IC50 = 17 mumol/L) incubated with high glucose concentrations. The oral administration of FR74366 to streptozotocin (STZ)-induced diabetic rats for 2 weeks decreased sorbitol levels (ED50 = 3.7 mg/kg for sciatic nerve, 23 mg/kg for lens, 52 mg/kg for retina, and 62 mg/kg for renal cortex). Administration of FR74366 to diabetic rats for 17 weeks delayed cataract formation and admixture of 0.028% FR74366 in the diet completely inhibited the cataract formation. Moreover, the recovery of reduced motor nerve conduction velocity by FR74366 in diabetic rats was demonstrated in prevention and reversal experiments. This recovery effect correlated well with reduction of accumulated sorbitol and fructose levels and normalization of decreased myoinositol levels. The duration and tissue specificity of inhibitory effects of FR74366 on sorbitol accumulation also correlated well with the levels of FR74366 in various tissues of diabetic rats. These data indicate that both decreases in tissue sorbitol levels and improvement of functional defects reflect FR74366 levels in tissue rather than plasma in diabetic rats. These results, taken together, suggest that FR74366, which is currently undergoing clinical trials in Japan and the United States, will be a useful therapeutic agent for diabetic complications.

Differential effects of physostigmine and pilocarpine on the spatial memory deficits produced by two septo-hippocampal deafferentations in rats

Rats that had received two kinds of septo-hippocampal deafferentations, medial septum (MS) lesion and fimbria-fornix (FF) transection, were assayed for brain cholineacetyltransferase (ChAT) activity and spatial memory in an 8-arm radial maze task. Both lesions produced profound and long-lasting spatial memory impairments, which were characterized by a reduction in the numbers of correct arm choices and first correct choices, a reduction in the percent of correct choices and an increase in the number of errors. The degree of memory impairment was severer in FF- than in MS-lesioned rats, and paralleled that of decreases in ChAT activity in the hippocampus. MS lesion reduced ChAT activity in the hippocampus by approximately 45%, while FF lesion almost completely depleted the activity. An intraperitoneal injection of physostigmine (0.0032-0.32 mg/kg), an acetylcholinesterase (AChE) inhibitor, significantly ameliorated the spatial memory deficit induced by MS lesion, but hardly affected that by FF lesion. In contrast, intraperitoneal doses (0.032-3.2 mg/kg) of pilocarpine, a muscarinic agonist, showed a significant improvement of both types of memory deficit with bell shaped dose-response curves. The drug was more potent in the FF- than in the MS-lesioned rats. These results suggest that the septo-hippocampal cholinergic system plays a crucial role in the maintenance of spatial memory, and that the degree of septo-hippocampal deafferentation affects the efficacy of cholinergic drugs.

Prevention of progressive joint destruction in collagen‐induced arthritis in rats by a novel matrix metalloproteinase inhibitor, FR255031

1 FR255031 (2‐[(7S)‐7‐[5‐(4‐ethylphenyl)‐2‐thienyl]‐1,1‐dioxido‐4‐(2‐pyridinylcarbonyl)hexahydro‐1,4‐thiazepin‐7‐yl]‐N‐hydroxyacetamide) is a novel synthetic matrix metalloproteinase (MMP) inhibitor that inhibits human collagenases (MMP‐1, MMP‐8 and MMP‐13), gelatinases (MMP‐2 and MMP‐9) and membrane type 1 MMP (MT1‐MMP/MMP‐14). FR255031 also inhibits rat collagenase and gelatinase. We studied the effect of FR255031 and Trocade, an inhibitor of collagenase and MMP‐14, on a rat collagen‐induced arthritis (CIA) model. 2 Rat CIA was induced by intradermal injection of type II collagen (IIC) and oral administration of FR255031 or Trocade was performed for 28 days. Body weight loss, hind paw swelling, elevation of serum anti‐IIC antibody, and histological and radiographic scores were evaluated. 3 FR255031 markedly inhibited cartilage degradation in a dose‐dependent manner in the CIA model, but Trocade failed to prevent the degradation. 4 FR255031 at a dose of 100 mg kg−1 also had statistically significant effects on bone destruction and pannus formation and on the recovery of body weight loss on day 28. 5 These results indicate that FR255031 is effective for rat CIA, especially on joint cartilage destruction. These data suggest that as well as collagenases or MT‐MMP, gelatinases are also involved in joint destruction in arthritis.

Effects of FK506 and other immunosuppressive anti-rheumatic agents on T cell activation mediated IL-6 and IgM production in vitro

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.

Role of cyclooxygenase-2, but not cyclooxygenase-1, on type II collagen-induced arthritis in DBA/1J mice

The purpose of this paper is to explore the contribution of isoforms of cyclooxygenase (COX) to chronic inflammation in DBA/1J mice with type II collagen-induced arthritis (CIA). To address this question pharmacologically, we tested the effects of selective inhibitors of COX-1 and COX-2 on paw edema and the formation of arachidonic acid metabolites in the inflamed paws immunized with type II collagen (CII). Oral administration of FR140423 (3-(difluoromethyl)-1-(4-methoxyphenyl)-5-[4-(methylsulfinyl)phenyl]pyrazole), a selective inhibitor of COX-2, showed a dose-dependent anti-inflammatory effect in mouse CIA with ED(50) value of 0.20mg/kg. Indomethacin, a non-selective inhibitor of COX, also inhibited paw edema in this arthritic model. In contrast, the selective COX-1 inhibitors, FR122047 (1-[(4,5-bis(4-methoxyphenyl)-2-thiazoyl)carbonyl]-4-methylpiperazine hydrochloride) and SC-560 (5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole), had no effect in mouse CIA model. The increase of prostaglandin (PG) E(2) and thromboxane (TX) B(2) in the mouse inflamed paws was associated with the development of paw edema induced by CII. FR140423 dose dependently inhibited the levels of PGE(2) and TXB(2) in the CIA mouse paws with ED(50) values of 0.20 and 0.12 mg/kg, respectively, similar to indomethacin. In contrast, FR122047 and SC-560 had no effect. These results suggest that COX-2, but not COX-1, contributes to the edema and the formation of PGE(2) and TXB(2) in mouse CIA model.

Effects of FK506 (tacrolimus hydrate) on chronic oxazolone-induced dermatitis in rats

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.

Topical Application of FK506 (Tacrolimus) Ointment Inhibits Mite Antigen-Induced Dermatitis by Local Action in NC/Nga Mice

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)-γ were detected, even though the IL-4+/IFN-γ– 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-γ 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.