Tacrolimus versus cyclosporin A: a comparative study on rat renal allograft survival.

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.

Further metabolism of FK506 (tacrolimus). Identification and biological activities of the metabolites oxidized at multiple sites of FK506.

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.

Interaction of tacrolimus(FK506) and its metabolites with FKBP and calcineurin.

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.

Isolation, identification, and biological activities of oxidative metabolites of FK506, a potent immunosuppressive macrolide lactone.

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.

Synthesis and antiinflammatory and analgesic properties of 2-amino-1H-benzimidazole and 1,2-dihydro-2-iminocycloheptimidazole derivatives.

2-Amino-1H-benzimidazoles (3) and 1,2-dihydro-2-iminocycloheptimidazoles (4) were synthesized and evaluated for antiinflammatory and analgesic activities. The compounds in the series 3 were synthesized via phenylthioureas (6) or 2-chloro-1H-benzimidazole (12). Most of 4 were synthesized by two methods. One was the reaction of carbodiimides (14) with 2-amino-2,4,6-cycloheptatrien-1-one (method A). The other was the reaction of guanidines (15) with 2-chloro-2,4,6-cycloheptatrien-1-one (method B). Some of the compounds 3 and 4 exhibited potent antiinflammatory and analgesic activities when compared to timegadine (1) or tiaramide hydrochloride (HCl) (17). It was of interest that 1-(2-benzothiazolyl)-2-cyclohexylimino-1,2-dihydrocycloheptimid azole (4e) showed superior analgesic activity to timegadine or tiaramide HCl (ED50 = 1.7 mg/kg p.o. in the acetic acid-induced writhing test, ED30 = 14.0 mg/kg p.o. in Randall-Selitto method) in spite of no effect on prostaglandin E2 synthesis.

A possible mechanism of fever induced by Nocardia rubra cell wall skeleton (N-CWS) in experimental animals.

The characteristics of fever elicited by the cell wall skeleton of Nocardia rubra (N-CWS) and by lipopolysaccharide (LPS) were compared in rabbits, and the possible involvement of the antigenicity of N-CWS was investigated in guinea pigs. In rabbits, fever of more than 0.5 degree C developed after an intravenous (i.v.) injection of 10 micrograms/kg or more of N-CWS, and was monophasic with 30-100 micrograms/kg but biphasic with the highest dose of 300 micrograms/kg. LPS elicited fever with similar characteristics at doses of 0.01-0.1 microgram/kg. With both compounds, the fever was inhibited by indomethacin. Tolerance to N-CWS and LPS appeared after dosing with 30 or 0.1 micrograms/kg, respectively for 10 d. In guinea pigs sensitized with N-CWS, challenge with 1 or 10 micrograms/kg of N-CWS 10 d later, which did not induce fever in the nonsensitized animals, caused fever of more than 0.5 degree C, and delayed-type hypersensitivity (DTH) appeared. N-CWS also elicited fever in nonsensitized guinea pigs bearing N-CWS-sensitized lymphocytes or anti-N-CWS antibody; the fever was higher in the guinea pigs sensitized with the lymphocytes than in those with the anti-N-CWS antibody. In brief, single injections of N-CWS and of LPS elicited fever with similar characteristics, although the potency of N-CWS was weaker. With N-CWS, the fever is proposed to be triggered by the antigenicity of the compound itself, because doses as low as 1 or 10 micrograms/kg elicited fever along with immunological response in N-CWS-sensitized animals, but not in nonsensitized ones.

Effects of FR50948, a new orally active antiallergic agent, in experimental allergic models.

The antiallergic activity of sodium 10-(2,3-dimethyl pentanamido)-4-oxo-4H-pyrimido [1,2-C] quinazoline-3-carboxylate-hydrate (FR50948) was studied and compared with the activities of sodium cromoglycate (SCG) and lodoxamide. FR50948 had inhibitory effects on type I and type III allergic reactions, but not on type II and IV allergic reactions. FR50948 also had weak inhibitory effects on inflammation (carrageenin paw edema and adjuvant arthritis) and SRS release from rat neutrophils, but no antagonistic effects to histamine and serotonin. The inhibitory effect of FR50948 on IgE-mediated type I allergic reactions was essentially the same as those of SCG and lodoxamide, because FR50948 inhibited the histamine release from rat peritoneal mast cells and had cross tachyphylaxis with SCG in the rat PCA test. However, FR50948, like lodoxamide, had a stronger activity than SCG and was effective by the oral route, unlike SCG which was effective only by the parenteral route. Furthermore, the inhibitory effects of FR50948 on type III reactions and inflammatory reactions were much more potent than those of SCG and equal to those of lodoxamide, and the effect on IgG-mediated PCA was stronger than that of either reference drug. These results suggest that FR50948 will be beneficial in clinical use.

Type II collagen-induced murine arthritis. I. Induction and perpetuation of arthritis require synergy between humoral and cell-mediated immunity.

Immunization of DBA/1 mice with type II collagen resulted in typical and progressive arthritis, which is associated with the production of high titer of anti-collagen antibody and the induction of cell-mediated immunity as exemplified by delayed type hypersensitivity response as well as lymphokine production. In contrast, administration of heat-denatured collagen into DBA/1 mice failed to induce the arthritis. These mice produced only marginal antibody, whereas they developed comparable cell-mediated immunity to that induced by immunization with native collagen, and therefore the inoculation of heat-denatured collagen provided the regimen capable of inducing preferentially cell-mediated immunity without the generation of high level of antibody. Inasmuch as administration of antibody induced only marginal and transient joint swelling not associated with typical histologic lesion, the synergistic effect of humoral and cell-mediated immunities was investigated using antibody preparation and the regimen to induce selectively cell-mediated immunity. The results demonstrate that administration of antibody into DBA/1 mice pre-sensitized with heat-denatured collagen resulted in potent and progressive arthritis. Such synergy was further confirmed by the induction of arthritis in T cell-depleted DBA/1 mice that had been adoptively transferred with antibody and lymphoid cells from heat-denatured collagen-sensitized mice. Moreover, it was revealed that the nature of cells capable of transferring cell-mediated immunity was of Thy-1+ and L3T4+ Lyt-2-. These results indicate that anti-collagen antibody and L3T4+ T cell-mediated cellular immunity are crucially required for the perpetuated development of type II collagen-induced arthritis.

[Host mediated anti-tumor effect of Nocardia rubra cell wall skeleton on syngeneic tumor in mice].

The mode of action of Nocardia rubra cell wall skeleton (N-CWS) on Meth A fibrosarcoma (Meth A) was studied in BALB/c mice. N-CWS suppressed or regressed the intradermal growth of syngeneic Meth A cells in normal BALB/c and athymic BALB/c mice. The intradermally and subcutaneously infiltrated cells harvested from injection sites of N-CWS in normal mice showed in vitro cytotoxic activity against Meth A cells. Pretreatment of normal BALB/c mice with immunosuppressing agents such as hydrocortisone, carrageenan, or silica particles significantly reduced the anti-tumor effect of N-CWS. The growth of Meth A cells, rechallenged into BALB/c mice in which Meth A cells had once been suppressed or regressed by N-CWS treatment, was also inhibited, but not in similarly treated athymic nude mice. This resistant mechanism was shown to be dependent out cellular components but not on humoral components by the Winn Assay. The present results suggest that N-CWS exerts its anti-tumor activity by mediation of the immune system of the host and that the main effector cells in the early stage of tumor rejection are macrophages; T cells may also be involved in the later stage.

Antitumour activity of purified arabinogalactan-peptidoglycan complex of the cell wall skeleton of Rhodococcus lentifragmentus.

Antitumour activity of arabinogalactan peptidoglycan (AP) complex (peptidoglycan and arabinogalactan liberated by an acid or alkaline treatment from Rhodococcus lentifragmentus AN-115 cell wall skeleton) was examined in mice and compared with that of the cell wall skeleton. The growth of syngeneic fibrosarcoma Meth A cells after implantation in BALB/c mice was significantly suppressed by AP complex, and also regressed after intratumoral injection of AP complex on days 1, 4 and 7 after tumour implantation. Although the activity of peptidoglycan was less than that of AP complex, peptidoglycan also showed both tumour-suppressive and regressive activities. Arabinogalactan did not show antitumour activity. It is interesting that peptidoglycan has an important role in the effect against tumours.

Effect of auranofin on autoimmune disease in a mouse model.

The effect of an oral gold preparation, auranofin, on the autoimmune disease mouse MRL/l was examined. Oral administration of auranofin on consecutive days from 6 weeks of age reduced anti-DNA antibody production, IgM rheumatoid factor production, hypergammaglobulinemia, polyclonal B cell activation and renal disease, but did not prevent massive lymphadenopathy or restore the low level of either IL-2 production or mitogen response associated with 1pr gene. In contrast to the effect on autoantibody production, little suppressive activity on the immune response to exogenous antigen SRBC was observed. These results indicate that autoimmune disease in MRL/l mice can be prevented without abrogation of T cell abnormalities and that autoimmune-selective suppression can be induced by chemical compound(s) like auranofin.

[The anti-inflammatory effect of auranofin].

The anti-inflammatory effects of auranofin were studied and compared with those of indomethacin, gold sodium thiomalate (GST) and D-penicillamine. Auranofin was active as indomethacin in inhibiting carrageenan induced paw edema in rats, but was less potent than indomethacin in inhibiting UV-induced erythema in guinea pigs. Auranofin inhibited Arthus type paw edema and reverse PCA reaction in rats, on which indomethacin was ineffective. The inhibitory activity of auranofin on adjuvant arthritis was weaker than that of indomethacin. In in vitro experiments, auranofin did not show any suppression of cyclooxygenase activity, but was capable of suppression of lysosomal enzyme release and chemotaxis of neutrophils and macrophages. In addition to these anti-inflammatory activities, auranofin had almost equal anti-analgesic and anti-pyretic activity to that of indomethacin. The above results indicated that the anti-inflammatory profiles of auranofin and indomethacin differ, so we can expect new therapeutic activities of auranofin. GST had similar anti-inflammatory and anti-analgesic profiles to those of auranofin; however, the activities were less potent than auranofin and devoid of anti-pyretic activity. D-penicillamine did not show any anti-inflammatory, anti-analgesic or anti-pyretic activity.

Potentiation of tumoricidal properties of murine macrophages by Nocardia rubra cell wall skeleton (N-CWS).

Intraperitoneal injection of squalene-treated cell wall skeleton of Nocardia rubra (N-CWS) caused increase in number of peritoneal exudate cells (PEC). Adherent macrophages obtained from N-CWS-treated PEC suppressed growth of methylcholanthrene-induced fibrosarcoma (Meth-A), when injected intradermally with the tumor cells into BALB/c mice. The macrophages showed strong cytotoxicity against Meth-A cells in vitro. When treated with 10 micrograms/ml of N-CWS in vitro, proteose peptone-induced macrophages acquired tumoricidal property but resident macrophages showed no cytotoxicity after the treatment. In the supernatant of spleen cells cultured for 72 hours in the presence of N-CWS (10 micrograms/ml), the presence of (a) factor(s) with macrophage activating effect was observed. This factor, shown to be identical to macrophage activating factor (MAF) in molecular weight, showed synergy with N-CWS in potentiating macrophage cytotoxicity against tumor cells.