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INFLAMMATION AND IMMUNOPHARMACOLOGY
Tsukuba Research Laboratories, Eisai Co., Ltd., Tsukuba Science City, Ibaraki, Japan (T.Y., A.I., K.S., N.T., M.N.); RIKEN Research Center for Allergy and Immunology, Suehiro-cho, Tsurumi, Yokohama, Kanagawa, Japan (K.-i.S.); Precursory Research for Embryonic Science and Technology, Japan Science and Technology, Kawaguchi, Saitama, Japan (K.-i.S.); and Eisai Research Institute, Eisai Co., Ltd., Wilmington, Massachusetts (S.K.)
Received August 5, 2004; accepted December 16, 2004.
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Abstract |
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-dependent manner. In particular, synthetic retinoid agonists with selectivity to RAR- have been reported to have a remarkable disease-ameliorating effect in some immune disease models via their potent immunosuppressive activities; however, there has been no report in which the effect of RAR--selective agonists in the nephritic models was examined. In this report, we investigated the effect of a newly synthesized RAR--selective retinoid agonist, E6060 [4-{5-[7-fluoro-4-(trifluoromethyl)benzo[b]furan-2-yl]-1H-2-pyrrolyl}benzoic acid], on the disease progression in a murine lupus nephritis model. Female (NZBxNZW)F1 mice were prophylactically treated with E6060 from 5 months of age, and their nephritic (proteinuria, blood urea nitrogen) and immunological parameters (serum anti-DNA autoantibodies and total serum immunoglobulins) were monitored with age up to 10 months old. E6060 at 0.03 and 0.1 mg/kg (once daily, p.o.) significantly improved survival rate and prevented the development of proteinuria in (NZBxNZW)F1 mice. Anti-DNA autoantibodies and total serum IgG were also significantly reduced in the E6060-treated mice. Among IgG isotypes, IgG2a was substantially reduced by E6060 treatment, indicating reduced T helper 1 responses in E6060-treated mice. In accordance with this possibility, elevation of serum interleukin-12 (p40) in old female (NZBxNZW)F1 mice was significantly inhibited by E6060 treatment. Our data suggest that the RAR--selective retinoid E6060 is a promising candidate of new remedy for lupus nephritis in systemic lupus erythematosus patients.
). RAR and RXR each have three subtypes, , 
, and 
. Retinoids act by modulating the transcription of target genes through interaction with these RAR and RXR subtypes, thereby interfering with cellular regulation, growth, and differentiation processes.
Retinoids, such as 4-hydroxyretinamide, all-trans-retinoic acid (ATRA), and 13-cis-retinoic acid, were reported to be effective in some immunological disease models, such as rat adjuvant arthritis (Brinckerhoff et al., 1983) and experimental allergic encephalomyelitis (Racke et al., 1995), which are believed to be caused through inappropriate cellular immune responses. Recently, Am80, a ligand with an affinity specific for RAR-, was reported to inhibit collagen-induced arthritis concomitantly with a prominent reduction in anti-collagen IgG titers (Kuwabara et al., 1996; Nagai et al., 1999). This report was the first to show that a retinoid could reduce antibody production and thereby prevent rat collagen-induced arthritis (Kuwabara et al., 1996). We have also synthesized and reported RAR- subtype-selective retinoid agonists (Kikuchi et al., 2000, Seino et al., 2000; Yoshimura et al., 2000). Among them, E6060 is one of the most potent RAR--selective ligands thus far reported, and it exerts a potent immunosuppressive effect in murine models (Yoshimura et al., 2000).
Lupus nephritis is a devastating disease state associated with 40 to 50% of systemic lupus erythematosus (SLE) patients. Even nowadays, renal dysfunction due to lupus nephritis results in the death of a significant proportion of SLE patients. There are few effective therapies for this disease, and most of them provoke severe toxicity. To date, some synthetic retinoids have been prescribed for SLE patients with refractory dermal complications (Shornick et al., 1991). These retinoids remarkably ameliorated the dermal disorders of the patients without apparent toxicity, but their effect on the autoimmune parameters was not mentioned in those reports. The (NZBxNZW)F1 mouse (B/WF1) is a well known model for lupus nephritis in SLE patients. This mouse resembles numerous autoimmune features of SLE patients, such as abnormal autoantibody formation and deposition of immune complexes (consisting of autoantibodies and components of complement) in the nephrons (Dixon et al., 1971), vasculitis, lymphadenopathy, and splenomegaly. Kinoshita et al. (2003) recently reported that administration of ATRA remarkably inhibited autoimmune nephritis in this mouse lupus model through its profound immunomodulating activities. In addition, Lehrke et al. (2002) and Liebler et al. (2004) showed disease amelioration by various retinoids in anti-Thy-1.1-induced rat nephritis, a model of mesangioproliferative glomerulonephritis. Their studies demonstrated that RAR- and RXR-selective retinoids suppressed the glomerular injury through inhibition of mesangial cell proliferation, glomerular infiltration of monocytes/macrophages, and accumulation of extracellular matrix components. By referring to those findings, we investigated whether our RAR--selective ligand would inhibit the pathology of murine nephritis models. We prophylactically treated female B/WF1 with E6060 and found its remarkable disease-ameliorating effect on various autoimmune parameters in this model.
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Materials and Methods |
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Drug Treatment. E6060 was synthesized at Eisai Tsukuba Research Laboratories. At 5 month of age, female B/WF1 mice were divided into five groups, each consisting of 12 animals. E6060 (0.01, 0.03, and 0.1 mg/kg) was suspended in 0.5% methylcellulose solution and orally administered once a day, 5 times a week, from 5 to 10 months of age. Blood and urine samples were collected every other week, from 6 to 10 months of age, and subjected to assay. At the end of the study, B/WF1 and NZW were sacrificed for histological evaluation of the kidneys.
Antidouble-Stranded (ds) and Single-Stranded (ss) DNA IgG ELISA. We determined anti-ds and ssDNA by means of ELISA according to the previous report (Early et al., 1996), with slight modifications. Plates were precoated with 10 µg/ml poly-L-lysine (Sigma-Aldrich, St. Louis, MO) and then coated with 50 µlof5 µg/ml poly(dA-dT)/poly(dA-dT) dsDNA (Sigma-Aldrich) or 10 µg/ml of heat-denatured calf thymus DNA (ssDNA, Sigma-Aldrich). Wells were blocked and then incubated with serum samples or standard serum for 2 h. The plates were washed, and 50 µl of biotinylated goat anti-mouse IgG (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK) was added to each well. Anti-DNA antibody was detected with 50 µl of peroxidase (POD)-labeled streptavidin (Amersham Biosciences UK, Ltd.) and developed with 50 µl of substrate (400 µg/ml o-phenylenediamine). The amount of either anti-ssDNA or dsDNA IgG in each sample was expressed as units per milliliter, with reference to the standard serum containing 1000 U/ml of each class of anti-DNA.
IL-12 ELISA. Unlabeled or biotin-labeled anti-mouse IL-12 (p40/p70) was purchased from BD Biosciences PharMingen (San Diego, CA). According to the manufacturer's description, both mAbs react with the p40 subunit of IL-12. Mouse IL-12 heterodimer was obtained from R&D Systems (Minneapolis, MN). Assays were performed according to the procedure previously described (Gately, 1991).
Immunoglobulin Isotype ELISA. We measured serum immunoglobulins with specific ELISA systems. We used unlabeled or POD-conjugated goat IgG fraction of anti-mouse IgG Fc, IgG1 (1), IgG2a (2a), or IgM (µ) [all purchased from Valeant Pharmaceuticals (Costa Mesa, CA)] for the first and second antibodies, respectively. The procedure was almost the same as mentioned above, except for the use of POD-labeled second antibodies instead of the biotin-labeled ones. Each immunoglobulin concentration was calculated with reference to the isotype standard [IgG, IgG1, IgG2a, and IgM; all purchased from Zymed Laboratories, South San Francisco, CA].
Blood Urea Nitrogen (BUN). Serum BUN concentration was determined with BUN B test (Wako Pure Chemicals, Osaka, Japan) according to the manufacturer's instructions.
Proteinuria. We determined urinary protein content with Bio-Rad Protein Assay Dye Reagent (Bio-Rad, Hercules, CA). Urinary protein was scored according to the following criteria: 0, <30 mg/dl; ++, 
30 mg/dl; 2+, 100 mg/dl; 3+, 300 mg/dl; and 4+, 1000 mg/dl. Incidence of proteinuria represents the number of mice whose urinary protein score reached 1+ or more.
Renal Histopathology. The left kidney from each mouse was removed at the end of the study (at 10 months of age), fixed in phosphate-buffered 10% formalin, and embedded in paraffin. Five-micrometer sections were stained with hematoxylin-eosin or periodic acid-Schiff. An observer blinded to the source of each section scored all glomeruli and classified the lesion as reported elsewhere (Raij et al., 1984; Uehara et al., 1991). The scoring system was as follows: 0, no lesion; 1+, 
25%; 2+, 50%; 3+, 75%; and 4+, 100% of glomeruli were involved. Injury score for an animal was calculated according to the following formula: glomerular injury score = 
[(degree of lesion) x (percentage of the glomeruli with the same degree of lesion)].
Immunohistochemistry. To detect the deposition of IgG and the third component of complement (C3) in glomeruli, right kidneys were frozen at –80°C until use. Five-micrometer cryostat sections were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG and C3 (Valeant Pharmaceuticals) diluted at 1:500 for 2 h at room temperature under dark conditions. After rinsing with phosphate-buffered saline thoroughly, fluorescein-positive deposits were observed with a fluorescent microscopy (Microphot-FXA; Nikon, Tokyo, Japan) equipped with a camera (FX-35DX; Nikon). For negative control, nonimmune fluorescein isothiocyanate-conjugated goat IgG (Valeant Pharmaceuticals) was used.
Statistics. Unless otherwise mentioned, data are expressed as mean ± S.E.M. Parameters for drug-treated groups were compared with those for control B/WF1 by means of analytical methods indicated in the footnotes under each figure or table. The dose-responsiveness of the effect of E6060 was evaluated using Jonckheere's or Tarone's test. A probability value of 5% (two-sided) was considered statistically significant in all cases. All statistical analysis was conducted using the SAS 6.12 software package (SAS Institute Japan, Tokyo, Japan).
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), the binding affinity values (Ki) of E6060 to RAR-, RAR-, and RAR- were 0.2, 51, and 143 nM, respectively. In accordance with this, its EC50 values in RAR--, RAR--, and RAR--mediated reporter transactivation assays were 0.2, 7, and 40 nM, respectively. E6060 did not induce reporter transactivation through any of the RXR homodimers (RXR-, RXR-, and RXR-) at concentrations up to 3000 nM. In parallel experiments, ATRA, an authentic endogenous retinoid, had almost the same affinity toward each RAR subtype in our binding and transactivation assays. Thus, E6060 is a selective RAR- agonist, without any affinity for RXRs. Survival Rate of B/WF1. Female B/WF1 mice were orally treated with either E6060 (0.01, 0.03, and 0.1 mg/kg) from 5 to 10 months of age, and their survival was monitored during that period. The survival rate among control B/WF1 started to decrease from around 7 months of age. Only 4 of 12 control B/WF1 were alive at 10 months of age. E6060 improved their survival in a dose-dependent manner, and its effect at 0.03 and 0.1 mg/kg was statistically significant (Fig. 1a; p < 0.05 by log-rank test followed by Bonferroni adjustment). In a separate experiment, the plasma concentration of E6060 was determined in normal mice, and it reached maximum at 9 ng/ml (23 nM) and 42 ng/ml (108 nM) at 0.5 h after oral dosing at 0.03 and 0.1 mg/kg, respectively. Those in vivo plasma concentrations are well beyond its EC50 values for activating RARs. E6060-treated animals looked healthy and gained body weight normally unless they developed nephritis. As indicated in Fig. 1b, we didn't observe any body weight loss or inhibition of body weight gain in any E6060-treated groups.
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In another experiment in which B/WF1 mice were therapeutically treated with E6060 at 0.1 mg/kg for 6 months, blood samples were collected from the animals that survived to perform both blood chemical and hematological analysis. Even after the 6-month treatment, E6060 did not show any toxicity, as shown by the evaluation on hepatic markers (i.e., serum aspartate aminotransferase and alanine aminotransferase), or anemia, which is known to be a common complication after usual retinoid treatment (data not shown). Teratogenicity is another severe toxicity with retinoid treatment. Although it is a preliminary finding, like common retinoids, E6060 has been found to be a potent teratogen even at its pharmacological dose (i.e., 0.1 mg/kg) in our preliminary reproductive safety tests. This issue will be investigated more thoroughly in future safety evaluations.
Proteinuria and BUN. The number of mice with proteinuria was also monitored at various assay points and is shown in Fig. 2a. The incidence of proteinuria represents the number of mice whose urinary protein score reached 1+ or more (over 30 mg/dl protein concentration). Proteinuria-positive B/WF1 was observed in the control group already at 6.5 months of age, and all control mice had proteinuria by 10 months of age. E6060 prevented proteinuria development in a dose-dependent manner, and its effect was statistically significant at 0.03 and 0.1 mg/kg. In addition, E6060 prevented the elevation of BUN in B/WF1, and the effect was statistically significant from 0.03 mg/kg. Mean BUN in the control and E6060 0.01-mg/kg groups declined at the latest stage of the experiment. This is due to the fact that many animals in both groups with high BUN level died during that period, and they were excluded from the calculation of mean BUN thereafter.
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Renal Histopathology of B/WF1. As shown in Fig. 3a, renal sections from control B/WF1 revealed remarkable proliferation and periodic acid-Schiff-positive deposits in the mesangial region, massive mononuclear cell infiltrates in interstitium, vasculopathy, and destruction of capillaries. Their glomeruli were injured much more severely than in age- and sex-matched NZW, which served as the non-nephritic control. Due to these histopathological changes, the mean glomerular injury score in control B/WF1 (244.8 ± 27.3) was significantly higher than that of age-matched female NZW (125.4 ± 15.1). E6060 improved the glomerular injury in a dose-dependent manner, and the glomeruli from the animals treated with E6060 at 0.1 mg/kg looked almost normal (Fig. 3a). E6060 at the doses of 0.01, 0.03, and 0.1 mg/kg decreased the scores to 223.9 ± 28.2, 162.4 ± 15.9, and 133.9 ± 12.8, respectively, and the effect of 0.1 mg/kg E6060 was statistically significant (Fig. 3b). In addition, E6060 markedly reduced IgG and complement deposition in the glomeruli compared with vehicle-treated B/WF1 (Fig. 3a).
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Anti-DNA IgG Antibody Production. Serum anti-dsDNA IgG in B/WF1 controls gradually increased just after initiation of the study, and remained elevated until the end of the study. E6060 inhibited the anti-dsDNA IgG production, and its effect was statistically significant at 0.03 and 0.1 mg/kg. At 0.1 mg/kg, it almost completely inhibited the elevation of this parameter throughout the study period (Fig. 4a). In addition to anti-dsDNA, E6060 dose dependently inhibited the production of anti-ssDNA, and its effect was statistically significant at 0.1 mg/kg (Fig. 4b).
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Immunoglobulin Production. In control B/WF1, serum IgG was remarkably elevated within the 3-month period, from 5 to 8 months of age, whereas serum IgM was not. Among IgG subtypes, IgG2a was preferentially elevated compared with IgG1 in our B/WF1 controls. E6060 prevented the elevation of serum IgG in a dose-dependent manner, and its effect was statistically significant at 0.03 and 0.1 mg/kg (Table 1). It also significantly inhibited serum IgG2a elevation in B/WF1 at the same doses. Conversely, E6060 did not reduce, but rather significantly increased, serum IgG1 at 0.1 mg/kg. It was reported that all-trans-retinoic acid also suppressed IG2a production but augmented IgG1 production via a reduction of interferon- (IFN-) production by CD4+ T cells (Carman and Hayes, 1991). Together, it suggests that E6060 exerts its effect by affecting murine antibody production in a manner similar to all-trans-retinoic acid. E6060 did not significantly alter serum IgM of B/WF1 throughout the study. According to these data, selective inhibition of IgG2a production by E6060 mainly contributed to its significant inhibition of total serum IgG elevation in this strain. Since murine IgG2a production and IgG1 production are known to be selectively induced by T helper 1 (Th1) and Th2 cytokines, respectively, we next investigated the effect of E6060 on the serum cytokine levels in B/WF1.
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Serum IL-12. Serum IL-12 (p40) in B/WF1 controls at 10 months of age was significantly higher than that in age-matched female NZW (B/WF1, 863.9 ± 101.2 pg/ml versus NZW, 113.9 ± 23.2 pg/ml; p < 0.05 by Mann-Whitney test). E6060 prevented the elevation of serum IL-12 (p40) in B/WF1 in a dose-dependent manner, and its effect was statistically significant at 0.03 and 0.1 mg/kg (Fig. 5). We could not detect any elevation of other cytokines, i.e., IL-2, IL-4, and interferon-, in our B/WF1 mice (data not shown).
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selective synthetic retinoid, Am80, was reported to inhibit type II collagen-induced arthritis with a prominent reduction in anti-collagen IgG titer (Kuwabara et al., 1996; Nagai et al., 1999). Similarly, our RAR--selective retinoid substantially inhibited not only anti-DNA antibody production (Fig. 4a), but also total IgG and IgG2a elevation in aged female B/WF1 (Table 1). In addition, our recent data indicated that E6060 significantly inhibited the antibody production against T-dependent antigen at 0.1 mg/kg p.o. (data not shown). Hence, prominent inhibition of antibody production may be a common feature among RAR--selective retinoids.
Plasma concentration of E6060 after oral dosing even at 0.03 mg/kg (the minimum effective dose) was enough beyond its EC50 values to cause transcriptional activation via each RAR subtype. Thus, E6060 may have activated all RAR subtypes in B/WF1, and it is not clear through which RAR subtype(s) E6060 exerted its effect in this study, even though it has higher affinity for RAR- than the other RAR subtypes. Nevertheless, it has been reported that retinoid inhibits murine B lymphocyte proliferation in an RAR--dependent manner (Apfel et al., 1992). In addition, our preliminary data indicated that ATRA and other retinoids remarkably inhibit the proliferation of alloantigen-stimulated murine T lymphocytes, and their effect is completely reversed by RAR--selective antagonist (Kikuchi et al., 2001). Taking these findings into consideration, it seems possible to speculate that E6060 exerted its immunosuppressive effects in this model, at least partly, in an RAR--dependent manner.
Among the various autoantibodies, we measured anti-ss and dsDNA IgG because these antibodies, especially the latter, are reliable markers of disease progression in SLE patients (Hahn, 1998). To date, various cytokines have been proposed to be involved in this type of autoantibody production in B/WF1 (Druet et al., 1996). Among them, monoclonal antibody (mAb) against Th2 cytokines, i.e., IL-4 (Nakajima et al., 1997), IL-6 (Finck et al., 1994), and IL-10 (Ishida et al., 1994), suppressed autoantibody production and improved the lupus nephritis in those mice. Additionally, treatment of the mice with recombinant IL-10 enhanced the autoantibody production and exacerbated the renal injury (Ishida et al., 1994). In addition to the Th2-type cytokines, Th1-type cytokines have also been shown to be involved in these processes. Anti-IL-12 mAb treatment substantially reduced anti-dsDNA IgG production, especially of IgG2a and IgG2b subclasses, in aged B/WF1, although it hardly prevented auto-immune nephritis in these mice (Nakajima et al., 1997). In addition, Appleby et al. (1993) reported that IL-12 was an exacerbating factor in murine experimental nephritis models. Anti-IL-12 mAb ameliorated antiglomerular basement membrane-induced renal lesions and antiglomerular basement membrane IgG2a production (Kitching et al., 1999). In agreement with that study, we observed that E6060 selectively reduced the serum level of IgG2a and IL-12, which were both elevated in aged B/WF1. Marked inhibition of IL-12 production by our retinoid is consistent with the recent report showing that retinoids inhibit IL-12 production from activated macrophages through functional interactions between their receptors (RXR and RAR) and nuclear factor-
B, a crucial transcription factor for IL-12 gene expression (Na et al., 1999). Taking these findings together, it is conceivable that the inhibition of IL-12 production by our retinoid contributed to substantial inhibition of autoantibody and serum IgG2a production in female B/WF1. Takano et al. (1999) reported that elevated serum IL-12 in SLE patients, apparently associated with the degree of their organ involvement. In accordance with that finding, E6060 remarkably reduced the renal injury score in aged B/WF1 (Fig. 3). We consider that IL-12 may play an important role not only in elevated antibody production, but also in developing renal involvement in B/WF1.
In addition to IL-12, IFN- is regarded as an important cytokine in this model. IFN- markedly up-regulates IgG2a production in mice (Finkelman et al., 1990). Anti-IFN- mAb or the recombinant soluble IFN- receptor inhibits both autoantibody production and lupus nephritis in B/WF1 (Jacob et al., 1987; Ozmen et al., 1995). In IFN- receptor knockout MRL/lpr mice with lupus nephritis, both autoantibody production and renal injury were remarkably abrogated (Haas et al., 1997). Retinoid has been reported to be an intrinsic factor necessary to inhibit IFN- production (Abb and Deinhardt, 1987; Carman and Hayes, 1991). Retinoid-mediated suppression of human IFN- production is exerted at the transcriptional level (Cippitelli, 1996). More recently, Kinoshita reported a remarkable disease-ameliorating effect of ATRA in B/WF1 mice (Kinoshita et al., 2003). In this report, ATRA preferentially inhibited the production of anti-DNA IgG2a, a subclass thought to be predominantly nephritogenic in B/WF1 (Park et al., 1983), presumably through its predominant inhibition of IFN- production by splenic CD4+ T cells. In this context, we speculate that E6060 also suppressed IFN- production in B/WF1, and this effect contributed to the substantial improvement of the autoimmune disease. The fact that E6060 inhibited IL-12 production in our B/WF1 mice likely supports this notion, since IL-12 is a potent inducer of IFN- production (Hsieh et al., 1993). However, very few mice in this study had a detectable level of serum IFN- when they were sacrificed at 10 months of age, so we could not assess the effect of E6060 on this parameter. Instead, our preliminary observation reveals that E6060-treated B/WF1 had fewer IFN--producing CD4+ T lymphocytes in their spleens than control mice (data not shown). Another preliminary study also shows that E6060 inhibited the up-regulation of antigen-induced ex vivo IFN- production and spleen T cell IFN- mRNA expression in dinitrophenylated keyhole limpet hemocyanin-immunized BALB/c mice (data not shown). Hence, inhibition of IFN- production by E6060 may also contribute, at least in part, to its remarkable disease-modifying effect in B/WF1. This possibility should be investigated more directly in future experiments.
Recently, retinoids with various receptor subtype selectivity were reported to ameliorate the renal injury in an anti-Thy1.1 nephritis model, a model of mesangioproliferative glomerulonephritis (Lehrke et al., 2002; Liebler et al., 2004). According to those reports and others (Wagner et al., 2000; Schaier et al., 2001), retinoids prevent renal damage through inhibition of mesangial cell proliferation, reducing extracellular matrix accumulation, lowering the number of infiltrating mononuclear cells, and reducing inducible nitric-oxide synthase expression by mesangial cells. It is suggested that these actions are mediated, at least in part, through activation of RARs locally expressed in glomerular tissues (Liebler et al., 2004). Like other retinoids, E6060 might have those direct actions on local renal tissues, although we have no direct evidence on this point; thus, this possibility remains to be clarified. To investigate this possibility more directly, we have started to analyze the effect of the drug on Masugi's rat nephritis model, in which renal disease is induced by passive immunization of nephritogenic antibody, suggesting that this nephritis does not depend on antibody formation in the host animals.
Lupus nephritis is a life-threatening disease. Although the survival rate of SLE patients has improved significantly over time, even in the 1990s 10 to 20% of patients lost their lives every 5 years, and the biggest cause of death was renal dysfunction due to lupus nephritis. In addition, there are few approved treatments for lupus nephritis at this time, and more effective drugs are highly desirable. E6060 is one of the most potent compounds that have been tested in female B/WF1 (Corna et al., 1997). Moreover, E6060 did not induce any apparent toxicity up to 0.1 mg/kg, a dose at which it completely prevented the disease progression. However, our preliminary studies indicate that it is a potent teratogen at this dose. Considering that SLE is most common in women at childbearing age, its teratogenic effects should be thoroughly investigated in additional preclinical studies. Accordingly, clinical trial of E6060 in SLE patients should be considered by carefully referring those safety data and might be rationalized only if potential benefits from the treatment seem to outweigh its teratogenic risk and appropriate contraceptive measures for the patients could be successfully introduced.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: RAR, retinoic acid receptor; RXR, retinoid X receptor; ATRA, all-trans-retinoic acid; E6060, 4-{5-[7-fluoro-4-(trifluoromethyl)-benzo[b]furan-2-yl]-1H-2-pyrrolyl}benzoic acid; Am80, 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2,3-naphthyl) carbamoyl]benzoic acid; SLE, systemic lupus erythematosus; NZB, New Zealand Black; NZW, New Zealand White; B/WF1, (NZBxNZW)F1; ELISA, enzyme-linked immunosorbent assay; ds, double-stranded; ss, single-stranded; POD, peroxidase; IL, interleukin; BUN, blood urea nitrogen; IFN-, interferon-; mAb, monoclonal antibody; Th, T helper; ANOVA, analysis of variance.
Address correspondence to: Dr. Toshihiko Yamauchi, Tsukuba Research Laboratories, Eisai Co., Ltd., Tsukuba Science City, Ibaraki, 300-2635, Japan. E-mail: t-yamauchi{at}hhc.eisai.co.jp
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), 0.01 mg/kg E6060 (
), 0.03 mg/kg E6060 (
), or 0.1 mg/kg E6060 (
) from 5 to 10 months of age. a, survival rate of B/WF1 mice. Significant difference from vehicle controls; *, p < 0.05 compared by log-rank test followed by Bonferroni adjustment. b, mean body weight in each treatment group indicated in a time-dependent manner. No statistically significant differences were observed between the control and any E6060-treatment groups at any time points.
, p < 0.05 by Mann-Whitney test.
