4SC-101, a novel small molecule dihydroorotate dehydrogenase inhibitor, suppresses systemic lupus erythematosus in MRL-(Fas)lpr mice

Abstract

Immunosuppressive treatments of systemic lupus (SLE) remain associated with significant toxicities; hence, compounds with better toxicity profiles are needed. Dihydroorotate dehydrogenase (DHODH) inhibition with leflunomide has proven to be effective in autoimmune diseases including SLE, but leflunomide can cause a variety of side effects. We hypothesized that 4SC-101, a novel DHODH inhibitor with a more favorable toxicity profile, would be as effective as high-dose cyclophosphamide (CYC) in controlling experimental SLE of female MRL(Fas)lpr mice. Daily oral gavage of 30, 100, and 300 mg/kg 4SC-101 from 12 to 22 weeks of age was compared with either vehicle or CYC treatment (30 mg/kg/week, i.p.) in terms of efficacy and toxicity. Three hundred milligrams per kilogram 4SC-101 was as effective as CYC in depleting spleen autoreactive T cells, B cells, and plasma cells as well as the respective DNA and RNA serum autoantibodies. This was associated with a comparable amelioration of the renal, dermal, and pulmonary SLE manifestations of MRL(Fas)lpr mice. However, even the highest dose of 4SC-101 had no effect on bone marrow neutrophil counts, which were significantly reduced in CYC-treated mice. Together, the novel DHODH inhibitor 4SC-101 is as effective as high dose CYC in controlling SLE without causing myelosuppression. Hence, DHODH inhibition with 4SC-101 might be suitable to treat active SLE with fewer side effects than CYC.

Trial registration: ClinicalTrials.gov NCT00820365 NCT01010581.

Figures

Figure 1

Structure and biochemistry of 4SC-101. 4SC-101, 2-(3-fluoro-3′-methoxybiphenyl-4-carbamoyl)-cyclopent-1-enecarboxylic acid, is a novel inhibitor of human DHODH discovered by molecular design based on the X-ray structure of human DHODH. Different from leflunomide, this compound has virtually no effect on kinases and has therefore a much cleaner pharmacological profile.

Figure 2

Pharmacokinetics of 4SC-101 in mice. Twelve-week-old female MRLlpr/lpr mice received a single dose of either 30, 100, or 300 mg/kg 4SC-101 by oral gavage, and plasma samples were obtained at different intervals as indicated. The graph illustrates plasma 4SC-101 levels over time as geometric means of three data points per dose and sampling time.

Figure 3

4SC-101 reduces systemic autoimmunity in MRLlpr/lpr mice. A: Mesenteric lymph node weight was determined at 22 weeks after ten weeks of vehicle, 4SC-101, or CYC treatment. Data are means ± SEM. *P < 0.05; ***P < 0.001 versus vehicle group. B and C: Spleen cell suspensions were prepared for flow cytometry by using specific antibodies that identify T cell (B) and B cell (C) subsets. Data are expressed as mean percentages of all splenocytes ± SEM. *P < 0.05; **P < 0.01 versus vehicle group. D and E: Serum samples were obtained at 22 weeks of age from mice of all groups, and total serum IgG and IgG isotype levels (D) and anti dsDNA IgG levels (E) were determined by enzyme-linked immunosorbent assay. Data are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle group.

Figure 4

4SC-101 improves renal histopathology in MRLlpr/lpr mice. A: Renal sections of 22-week-old MRLlpr/lpr mice were stained with periodic acid Schiff and antibodies for IgG and CD3 (T cells) as indicated. Images are representative for seven to 12 mice in each group (original magnification, ×100 [PAS], ×400 [IgG], ×100 [CD3]). B: Morphometry was performed on IgG and PAS stained sections to quantify glomerular IgG deposits (score ranging from 0 to 3), the lupus nephritis disease activity index (score ranging from 0 to 24), and the lupus nephritis chronicity index (score ranging from 0 to 12). C: CD3+ T cells and Mac2+ interstitial cells were counted in 15 high power fields from renal sections of MRLlpr/lpr mice from all groups. Mac2+ cells were also counted in 15 glomeruli from mice of all groups of MRLlpr/lpr mice from all groups. Data represent means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle control group; ND, not detected.

Figure 5

4SC-101 improves GFR and proteinuria in MRLlpr/lpr mice. At 22 weeks of age, GFR (A) and urinary albumin/creatinine ratios (B) were determined in MRLlpr/lpr mice from all groups as described in Materials and Methods. Note the dose-dependent increase in GFR and decline in albuminuria in 4SC-101-treated MRLlpr/lpr mice. Data represent means ± SEM. *P < 0.05 versus vehicle control group.

Figure 6

4SC-101 and cutaneous lupus in MRLlpr/lpr mice. MRLlpr/lpr mice of all groups were regularly checked for skin manifestations, which typically occur in the facial or neck area. A: Representative images of hematoxylin and eosin stained skin biopsies from selected groups are shown as indicated (original magnification, ×100). A semiquantitative assessment of skin pathology allowed to calculate a composite skin injury score (B), which was derived from adding the scores of seven different criteria (C) as indicated. CYC-treated mice had no signs of skin disease. Data represent means ± SEM. *P < 0.05 versus vehicle control group, **P < 0.01 versus placebo control.

Figure 7

4SC-101 improves autoimmune lung disease in MRLlpr/lpr mice. A: Lung sections of 22-week-old MRLlpr/lpr mice were stained with periodic acid Schiff. Images are representative for 7 to 12 mice in each group (original magnification, ×100). B: Morphometry was used to quantify the peribronchiolar and perivascular inflammation by using a score ranging from 0 to 3. Data represent mean scores ± SEM. **P < 0.01; ***P < 0.001 versus vehicle control group.

Figure 8

4SC-101 and bone marrow cells in MRLlpr/lpr mice. Flow cytometry was used to quantify neutrophils and monocytes in bone marrows of 22-week-old female MRLlpr/lpr mice from all treatment groups. Monocytes were identified as 7/4bri Ly6G−. Neutrophils were identified as 7/4+Ly6G+. Data are shown as mean percentages ± SEM from at least five to six mice in each group. **P < 0.01, ***P < 0.001 versus vehicle control group.