A New Small Molecule For Treating Inflammation and Chorioretinal Neovascularization in Relapsing-Remitting and Chronic Experimental Autoimmune Uveitis

Abstract

Purpose.: We investigated the effect of PP-001, a new small molecule inhibitor of dihydro-orotate dehydrogenase in two experimental rat experimental autoimmune uveitis (EAU) models: a spontaneously relapsing-remitting model and a monophasic/chronic disease model that results in late chorioretinal neovascularization. Both of the diseases are induced by immunization with autoantigen peptides.

Methods.: Prevention was tested using daily oral applications of PP-001 after immunization with the retinal S-antigen peptide PDSAg (for induction of monophasic uveitis and neovascularization) or the interphotoreceptor retinoid-binding protein peptide R14 (for induction of spontaneously relapsing-remitting EAU). Treatment to inhibit relapses and neovascularization was tested using PP-001 daily after the first attack of R14-induced or after onset of PDSAg-induced EAU. Uveitis was graded clinically and histologically. The effect of PP-001 on cytokine secretion and proliferation was evaluated using rat T-cell lines.

Results.: Preventive feeding of PP-001 abrogated both types of EAU. Starting treatment after the resolution of the first attack led to a significant reduction of the number and intensity of relapses in R14-induced EAU. PP-001-treatment initiated after onset or after peak of PDSAg-induced EAU significantly reduced neovascularization (as determined by histology). Proliferation of antigen-specific T-cell lines and secretion of IFN-γ, IL-17, IL-10, IP-10, and VEGF were efficiently suppressed by PP-001.

Conclusions.: We investigated a new dihydroorotate dehydrogenase inhibitor as treatment for primary and recurrent disease in relapsing-remitting and chronic rat models of experimental autoimmune uveitis. The small molecule compound PP-001 suppressed proliferation and cytokine secretion of autoreactive T cells (i.e., IFN-g, IL-17, and VEGF) and chorioretinal neovascularization in chronic EAU.

Introduction

Experimental autoimmune uveitis (EAU) in rats can be induced with retinal autoantigens and peptides. The best characterized autoantigens include R14 peptide from interphotoreceptor retinoid-binding protein (IRBP) and PDSAg, derived from retinal S-antigen. Following immunization of rats with complete Freund's adjuvant (CFA), both peptides induced a panuveitis with infiltration of inflammatory cells in the chamber segment as well as in the posterior segment, resulting in retinal destruction. The two peptides elicited differently regulated diseases¹ and different disease courses. Peptide R14 induced spontaneously relapsing-remitting disease,^2–4 which is highly suitable for testing therapeutic intervention instead of preventive treatment for uveitis. EAU induced with the peptide PDSAg presented with only one clinically observable course of intraocular inflammation without any relapses but results in the formation of chorioretinal neovascularization, visible by histology beginning 4 weeks after immunization (Diedrichs-Möhring M, Wildner G, unpublished data, 2005). Chorioretinal neovascularization is a rare but severe late sequela of autoimmune uveitis⁵ and the main complication in wet AMD. 

Rat EAU is mediated by autoantigen-specific T-cell subtypes Th1 and Th17. The PDSAg peptide induces T cells that produce more IL-17, whereas R14-specific T cells tend to secrete more IFN-γ.³ Investigation of intraocular T cells in PDSAg- and R14-induced EAU have revealed dynamic populations of T cells with coexpression of IFN-γ and IL-17 proteins. During the course of uveitis, intraocular T-cell populations in R14-induced disease show a predominant shift toward IFN-γ production, whereas in PDSAg-induced EAU, the population of IL-17–positive cells increases during the resolution of inflammation.⁴ 

Targeting T-cell activation can be an effective approach for the treatment of some autoimmune diseases. Here we tested effects of PP-001, a third-generation small molecule inhibitor of dihydroorotate dehydrogenase (DHODH), against the induction and progression of experimental rat EAU. 

The enzyme DHODH is needed for de novo synthesis of pyrimidines. A blockade of DHODH can be effective in targeting activated lymphocytes, as these cells increase their pyrimidine pool 8-fold during proliferation and thus depend on de novo synthesis.⁶ Inhibition of DHODH by the use of leflunomide is currently under evaluation in rheumatoid arthritis and multiple sclerosis.⁷ 

To identify new DHODH inhibitors, we used an in silico iterative screening algorithm (4Scan, Ref. 8). The 300 top scoring molecules were chemically synthesized and subjected to medicinal chemistry analysis to identify potential leading compounds. The most promising leading agents were improved for enhanced DHODH inhibition. PP-001, a biphenyl-4-ylcarbomyl thiophene carboxylic acid, was identified as a promising inhibitor of DHODH with a 50% inhibitory concentration (IC₅₀) of <4 nM, which is more than 150-fold more potent than leflunomide (IC₅₀ of 650 nM). In addition, PP-001 also showed improved specificity compared to previous DHODH inhibitors and did not have the side effect of tyrosine kinase inhibition as observed with leflunomide.⁹ 

Preventive treatment of EAU induced with peptide R14 or PDSAg by PP-001 was tested. A dosage of 30 mg/kg of body weight (BW) PP-001 given daily was found to almost completely inhibit EAU induced with PDSAg as well as with R14. Furthermore, in vitro PP-001 treatment suppressed the proliferation of antigen-specific T-cell lines and secretion of IFN-γ, IL-17, IL-10, IP-10, and VEGF in a dose-dependent manner. 

Therapeutic treatment was effective in preventing and reducing the number and intensity of relapses of R14-induced uveitis and was also found to reduce chorioretinal neovascularization in the later, chronic stage of PDSAg-induced disease. Our data show that PP-001 is a potent new drug that is useful for therapeutic treatment of autoimmune uveitis and especially for preventing long-term complications of uveitis. 

Materials and Methods

Animals

Lewis rats (Lew/Orl Rj) were bred in our colony or purchased from Janvier (Le-Genest-Saint-Isle, France) and maintained in sterile isolators. They had unlimited access to rat chow and water. Male and female animals were used for experiments at 6 to 9 weeks old. All experiments were approved by the Review Board of the Regierung von Oberbayern and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Induction of EAU

Uveitogenic peptides PDSAg and R14 were derived from the sequence of bovine S-antigen (PDSAg: amino acids [aa] 341–354, FLGELTSSEVATEV) or human IRBP (R14: aa 1169–1191, PTARSVGAADGSSWEGVGVVPDV). Animals were immunized subcutaneously in both hind legs with a total volume of 200 μL of both the peptides (15 μg peptide) emulsified in CFA, supplemented with Mycobacterium tuberculosis strain H37RA (Becton-Dickinson Co., Heidelberg, Germany) to a final concentration of 2.5 mg/mL. 

Application of PP-001

Animals were fed PBS, vehicle (solution of 80% phosphate buffer, pH 7.4, and 20% Phosal 50PG [phosphatidylcholine 50%; Phospholipid GmbH, Cologne, Germany]), or PP-001 as indicated until termination of experiments. For “prevention” of EAU, rats were fed PP-001 from the day of immunization; for “treatment” (prevention of relapses), R14-immunized animals were fed beginning from resolution of the primary course of EAU (score: ≤0.5, between days 15 and 17 post immunization) until termination of the experiment. To prevent choroidal neovascularization, PDSAg-immunized rats were fed daily with 25 mg/kg BW PP-001 or 10 mg/kg BW cyclosporin A (CyA) starting on day 9 (onset of EAU) or day 15 (peak disease); or with vehicle control (PP-001: 80% phosphate buffer [Unilever, Hamburg, Germany], pH 7.4, plus 20% Phosal 50PG; CyA: 10% corn oil in PBS) starting on day 9. 

Grading of Uveitis

Clinical Grading.

Rats were examined with an ophthalmoscope daily starting on day 7 for clinical signs of uveitis, and were graded as described previously.¹⁰ The clinical uveitis score considers only the inflammation in the anterior part of the eye. Briefly, a score of 0.5 = dilated iris vessels, partial inflammatory cell infiltrates of the iris rim, and a hazy anterior chamber; 1 = complete circular infiltration of the iris rim; 2 = pupil area completely filled with cells and fibrin; 3 = formation of hypopyon; and 4 = anterior chamber completely filled with cells, fibrin, and blood. 

Experimental autoimmune uveitis is shown either as the time course of mean (±standard error [SE]) daily clinical uveitis scores of both eyes of each rat/group or as mean (±SE) maximum clinical score obtained for each eye/group during the entire experiment. A “relapse of EAU” was defined as a clinical score of >0.5 after the first attack of disease and a period of complete absence of all clinical signs of inflammation. 

Mean Area Under the Curve.

Mean (±SE) area under the curve defined the average intensity of uveitis in each eye in a group over a certain period of time and was calculated as the summation of the daily clinical uveitis scores for each eye of each rat in a group/number of eyes. 

Histological Grading.

For histology, eyes from killed animals were embedded in Tissue-Tek O.C.T. (Paesel and Lorey, Frankfurt/Main, Germany) and immediately snap frozen in methyl butane at −80°C. Cryosections of rat eyes were stained with hematoxylin and graded as described previously.¹⁰ Briefly, a score of 0.5 described destruction of less than 25% of the photoreceptor outer segments, whereas the maximum score of 4 represented total destruction of the retinal architecture. Histological grading is shown as the mean (±SE) score of all eyes per group. 

Determination of Neovascularization

Neovascular areas were counted in the same sections as those used for histological grading. Three different sections of each eye were evaluated to determine the mean numbers of neovascularized areas for each eye. Mean (±SE) numbers of neovascularized areas were shown for all eyes per group (see Fig. 5B). 

Peptide-Specific T-Cell Lines

Rats were immunized with 25 μg of the respective peptide as previously described for induction of EAU. After 10 to 12 days, popliteal, inguinal, and para-aortal lymph nodes were collected, and single-cell suspensions were stimulated with the respective antigen peptides at a final concentration of 10 μg/mL RPMI1640 medium supplemented with penicillin and streptomycin, l-glutamine, essential and non-essential amino acids (all from PAA, Coelbe, Germany), 12.5 μM mercaptoethanol, and 1% normal rat serum (first restimulation). After 3 days, cells were expanded in culture medium supplemented with 10% spleen-conditioned medium (from concanavalin A-stimulated rat spleen cells) and 5% fetal calf serum for 4 days. 

For restimulation, 0.5 × 10⁶ to 2 × 10⁶ T cells/mL were incubated with (8-Gy) irradiated rat thymocytes (3× to 10× more thymocytes than T cells) as antigen-presenting cells and 10 μg/mL specific antigen peptide in culture medium as described for the first stimulation. After 2 days of culture, cells were expanded with conditioned medium for 5 days as described above, followed by another cycle of restimulation (third restimulation). For tissue cultures, PP-001 was dissolved with dimethyl sulfoxide (DMSO) and further diluted to the concentrations indicated with RPMI1640 medium. 

XTT Assay

To determine the effect of PP-001 on proliferation and survival of the T cells in vitro, the test substance was added to triplicate microwell cultures during the first, second, and third stimulations with antigen. (For primary restimulation, 2 × 10⁶ lymph node cells per mL were used, and no addition of antigen-presenting cells was necessary; for second and third restimulations, 0.5 × 10⁶ T cells and 2.5 × 10⁶ irradiated thymocytes per milliliter were used and 20 μg of the respective antigen peptide/mL.) 

PP-001 dissolved in DMSO (vehicle) was added to final concentrations of 3, 10, and 30 μM to triplicate microwell cultures of 100 μL (final volume). As vehicle control, DMSO was added according to the concentration of DMSO in the cultures with 30 μM PP-001 (1:1000 dilution). Cells were cultured for 72 hours (first in vitro stimulation) or 48 hours (second/third in vitro stimulations) in the presence of PP-001 or vehicle control before survival and proliferation were determined by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide (XTT) cell proliferation assays (R&D Systems, Wiesbaden, Germany) according to the manufacturer's instructions. Optical density (OD) was measured at 490 nm after 5 hours, and results are shown as mean (±SE) OD. 

Cytokine Assay

For analysis of secreted cytokines and chemokines, using the Milli-Plex Pro Rat 27-plex assay for rat cytokines/chemokines (Merck/Millipore, Schwalbach, Germany), the cells were cultured as described for the XTT assay. Supernatants (25 μL) from triplicate cultures of the XTT assay described above were collected and pooled after 24 and 48 hours and immediately frozen at −70°C until used. Cytokine/chemokine concentrations were measured with the Bio-Plex200 (Bio-Rad, Munchen, Germany). 

Statistics

Statistical analyses were performed using InStat 3 software (GraphPad, La Jolla, CA, USA). Nonparametric ANOVA (Kruskal-Wallis test with Dunn's multiple comparison test) was performed to determine significance levels. 

Results

Prevention of EAU

Starting on the day of immunization with PDSAg or R14, rats were fed daily with 18 or 30 mg/kg BW PP-001; control groups received vehicle or PBS, respectively (Figs. 1A–D). EAU in the group immunized with PDSAg and fed with 18 mg of PP-001/kg was completely suppressed until day 15, when the control groups had already reached their peak disease, and then increased slightly to a moderate disease intensity significantly below that of the control groups (Figs. 1A, 1C). The group treated with the higher dose of 30 mg/mL was almost devoid of any signs of disease, except for one animal with a very mild case of disease (score: 0.5 in both eyes) and very low clinical and histological uveitis scores. The same effect was observed in R14-immunized rats fed 30 mg/kg (Figs. 1B, 1D), with a disease score of 1 in one rat and 0.5 in all other animals. The lower dose (18 mg/kg) had no effect on R14-induced EAU. 

Figure 1

 

Prophylactic oral treatment of EAU in rats with PP-001. Daily oral treatment with PP-001 was initiated on the day of immunization. The clinical courses (mean ± SE daily uveitis scores from both eyes [n = 8–16]) (A, C) and mean ± SE maximal clinical (black columns) and mean ± SE histological scores (gray columns) (B, D) of EAU induced with PDSAg (A, B) and R14 (C, D). Data are combined from two independent experiments. (B, D) “mg/kg” indicates the daily oral dose of PP-001 according to the bodyweight of the rats. Black brackets and asterisks show significant differences for clinical scores, gray brackets and asterisks for histological scores. *P < 0.05; **P < 0.001 shown only for comparison with vehicle controls.

Treatment of EAU to Prevent Relapse

The spontaneously relapsing course of R14-induced uveitis allowed for investigation of PP-001 treatment during the clinical disease course seen in patients. Rats were immunized with R14-CFA, and oral treatment with either 25 mg/kg PP-001, PBS, or vehicle was initiated individually after resolution of clinical signs of EAU (score: <0.5), between days 15 and 17 post immunization (Fig. 2A). Compared to the control groups fed with PBS or vehicle only, the PP-001-treated group showed only 2 relapses in 14 eyes (14%), whereas the PBS-fed group showed 9 and the vehicle-treated group 10 relapses (Table 1). In both control groups, one eye had two relapses. This showed an incidence of 57% for eyes and 71% for rats in the PBS-treated group and 64% for eyes and 85% for rats in the vehicle-treated group. The PP-001-treated group had an incidence of relapses that dropped to 14% (for both, affected eyes and animals). From day 18, all animals received treatment, and the time course (Fig. 2A) and calculation of mean area under the curve (Fig. 2B) showed a significant amelioration of EAU in the PP-001-treated group compared with that in the group treated with vehicle or PBS only (P < 0.05: PP-001 versus PBS group; P < 0.01: PP-001 versus vehicle group). Relapses were usually weaker than the primary course of uveitis, and because relapses are not synchronized (as the primary course of disease), single relapses were not visible in the time course showing the mean daily clinical scores. In the PBS-treated group four of the nine relapses had a score of 2, three relapses showed a score of 1, and the final two relapses had scores between 0.5 and 1. In the vehicle-treated group, we observed three relapses with scores of 2 and seven relapses with scores of 1. In the PP-001-treated group, only two relapses had scores of 2 and 1, respectively. 

Figure 2

 

Therapy to prevent relapses of EAU. (A) Time course shows mean ± SE daily clinical uveitis scores from both eyes (n = 14 eyes/treatment group) of all animals/group. Treatment with PBS, vehicle, or 25 mg/kg PP-001 was initiated individually between days 15 and 17, after the resolution of the first course of EAU with a clinical score of ≤0.5 (hatched area); gray area shows the daily treatment of all animals. Doses of PP-001, vehicle, and PBS were adjusted to the bodyweight of the rats by feeding different volumes. (B) The mean ± SE area under the curve was calculated for all eyes for the primary course of disease (left column) from onset until day 17; for the relapses from days 18 to 31, the mean ± SE area under the curve was calculated for each treatment group.

Table 1

 

Reduction of Relapses of R14-Induced EAU After Treatment With PP-001

Relapse	PBS	Vehicle	PP-001, 25 mg/kg
Total relapses	9	10	2
Rats with relapses, n = 7	5 (71%)	6 (85%)	1 (14%)
Eyes with relapses, n = 14	8 (57%)*	9 (64 %)*	2 (14%)

 Relapses are defined as a clinical score of >0.5 if the score had previously decreased to ≤0.5. Oral treatment was initiated individually in each animal after resolution of the primary course of uveitis (day 15–17 post immunization).

 * One eye had 2 relapses.

In Vitro Stimulation of Rat T Lymphocytes

T-cell lines specific for PDSAg or R14 were restimulated with their respective antigen peptide for up to three cycles. During each antigen stimulation, the effects of PP-001 (at 3, 10, and 30 μM) on cell proliferation and viability and antigen-specific cytokine/chemokine secretion were investigated from triplicate cultures. Figure 3 shows the inhibitory effect of PP-001 on the viability and proliferation of six independent T-cell lines as determined by XTT assay. Dimethyl sulfoxide at a dilution of 1:1000 was applied as vehicle control for the highest concentration of PP-001 (30 μM). Although DMSO had no effect on PDSAg- and R14-specific T cells during antigen-stimulation, PP-001 efficiently suppressed proliferation and viability even at the lowest concentration of 3 μM in both PDSAg- and R14-specific cell lines. In all experiments, the highest suppression was achieved with 30 μM PP-001. 

Figure 3

 

Inhibition of T-cell lines was determined by XTT assay. T cells of various cycles of restimulation (1.–3. restimulation as indicated) were cultivated with the respective peptide only or in combination with various concentrations of PP-001 (diluted from a stock solution in DMSO) as indicated. “Med” represents the background of survival/proliferation of cells cultured in medium only. The “vehicle” control represents stimulation with the specific peptide and the concentration of DMSO corresponding to the DMSO of 30 μM PP-001 dilution. Mean ± SE OD of triplicate cultures are shown. Significant differences are shown in comparison with group receiving vehicle only: *P > 0.05.

Cytokine Secretion of T-Cell Lines

The effects of PP-001 on cytokine and chemokine secretion were then investigated. The highest concentration of the vehicle DMSO (1:1000 dilution) was again included as control. The supernatants were analyzed for a panel of 27 cytokines and chemokines, of which only IFN-γ, IL-17A, IP-10, RANTES/CCL5, MIP-1α, and VEGF were found to be expressed above the detection limit of the assay, determined according to standard curves (Fig. 4). Mean concentrations of cytokines/chemokines from supernatants taken after 24 and 48 hours of antigen stimulation from the second restimulation of the cell lines are shown, except for IFN-γ and IL-17, which were detectable in high amounts after the first restimulation in vitro. Regulation of secretion was seen after the first and second in vitro stimulation. Secretion of IFN-γ and IL-17A was completely abrogated in both T-cell lines when PP-001 was added during the primary restimulation (Fig. 4A). 

Figure 4

 

Inhibition of cytokine secretion by T-cell lines. Culture supernatants of the triplicates from stimulated T-cell lines specific for PDSAg or R14 (as shown in Fig. 3) were collected at 24 and 48 hours after primary and secondary antigen stimulation in vitro. Data are means of cytokine/chemokine concentrations of pooled supernatants from microwell triplicate cultures of both time points. “Vehicle” indicates cultures with solvent of PP-001 (concentration corresponding to 30 μM PP-001) and respective antigen; 3, 10, and 30 μM are the concentrations of PP-001 in cultures with antigen. (A) IFN-γ and IL-17 concentrations in supernatants from the T-cell lines after primary and secondary restimulation. (B) Supernatants from T-cell lines after the second restimulation.

IP-10 was found to be decreased in both T-cell lines with all concentrations of PP-001, whereas secretion of RANTES/CCL5 was efficiently suppressed only in R14-specific T cells with the highest dose of PP-001 given. MIP-1α/CCL3 and VEGF were expressed and/or regulated differently in PDSAg than in R14-specific T cells (Fig. 4B). MIP-1α was expressed only in R14-specific T cells and was decreased with PP-001treatment. By contrast, VEGF was produced only by PDSAg-specific T cells but was also efficiently suppressed by PP-001. 

Prevention of Chorioretinal Neovascularization

Unpublished data from our group have shown that PDSAg-induced uveitis leads to the growth of new vessels from the choroid into the retina, starting at approximately 4 weeks after immunization (Diedrichs-Möhring M, Wildner G, unpublished data, 2005). The effect of PP-001 on the initiation of neovascularization was compared to that of treatment with CyA, a therapeutic drug currently used for uveitis in humans. Rats were immunized with PDSAg-CFA as described earlier, and groups of animals were then fed daily with PP-001 or CyA, either from onset of clinical signs of uveitis (day 9) or from day 15, when the intraocular inflammation had reached its peak. Control groups received the respective vehicles for PP-001 or CyA daily from day 9 until the experiment was terminated at day 40. The late onset of treatment (day 15 [Fig. 5A]) had no significant effect on the maximal uveitis scores compared to the vehicle controls, because the treatment was initiated after the peak of the clinical disease (“clinical”), when the retinal destruction was already underway (“histology”). In contrast, early treatment with PP-001 or CyA (day 9) significantly reduced clinical signs and tissue destruction (Fig. 5). In addition to the mean uveitis scores, treatment with PP-001 and CyA also reduced the incidence of EAU preferentially in the early treatment groups (Table 2), whereas all animals in the vehicle groups developed uveitis. Groups treated with PP-001 and CyA from day 9 had similar rates of clinical uveitis (four of six rats in each group), whereas histology showed all CyA-treated animals had retinal destruction. Some rats developed a mild disease 3 to 4 days after onset of early treatment with PP-001, and four of six rats in the respective CyA-treated group developed mild signs of uveitis from days 10 to 16, despite daily treatment. 

Table 2

 

Incidence of PDSAg-Induced EAU After Treatment With PP-001 or CyA

Parameter	Vehicle PP-001		PP-001 Day 9		PP-001 Day 15		Vehicle CyA		CyA Day 9		CyA Day 15
	Clinical Score	Histological Score	Clinical Score	Histological Score	Clinical Score	Histological Score	Clinical Score	Histological Score	Clinical Score	Histological Score	Clinical Score	Histological Score
Positive rats	6	6	4	4	6	5	6	6	4	6	6	5
%	100	100	67	67	100	83	100	100	67	100	100	83
Positive eyes	12	12	5	7	11	9	11	11	7	10	9	8
%	100	100	50	58	92	75	92	92	58	83	75	67

 “Positive rats” and “positive eyes” are the numbers of rats or eyes with positive clinical–histological scores and the percentage positive by clinical or histological score (n = 6 rats or 12 eyes, respectively).

To be able to correlate chorioretinal neovascularization with the overall intensity of uveitis according to retinal destruction, the number of newly formed vessels was determined from the tissue sections used for histological grading. Compared to vehicle-treated control groups, the mean number of choroidal neovascularization was significantly reduced in the eyes from rats treated with PP-001 or CyA from day 9 (Figs. 5B, 6A, 6B, 6D, 6E). Although late initiation of treatment (day 15) with PP-001 still resulted in significantly less neovascularization than that in the control group (Figs. 5B, 6C), late treatment with CyA did not prevent the growth of new vessels (Fig. 6F). 

Figure 5

 

Prevention of retinal neovascularization. Rats immunized with 15 μg PDSAg in 50 μL CFA were fed daily with 25 mg/kg BW PP-001 or 10 mg/kg BW cyclosporin A in corn oil/PBS, either from day 9 (onset of EAU) or day 15 (peak disease) after immunization until day 39. The control groups received the respective vehicle (Veh) orally and daily from day 9. Uveitis was graded clinically and histologically for n = 12 eyes per group as described. (A) Mean ± SE maximal clinical and mean histological uveitis scores of each eye are shown for each group. (B) The mean (±SE) numbers of areas of neovascularization were determined for each treatment group as described in text. P values are shown only for significantly different groups.

Figure 6

 

Cryosections of rat eyes with neovascularization. Representative hematoxylin-stained cryosections from rat eyes 40 days after immunization with PDSAg-CFA (same experiment as described in Fig. 5). Animals were fed daily until day 39 as indicated. Control groups received the respective vehicles from day 9. (A) Control for PP-001; (B, C) rats fed with 25 mg/kg BW PP-001 as indicated; (D) control for cyclosporin A, corn oil; (E, F) rats fed with 10 mg/kg BW cyclosporin A in corn oil as indicated. Areas of neovascularization are marked with arrows.

Discussion

Targeting DHODH has been shown to be a successful therapeutic approach in several models of T cell–mediated autoimmune diseases.¹¹ To date, the best characterized DHODH inhibitor is leflunomide,⁷ which is used for the treatment of rheumatoid arthritis. Effective treatment of experimental autoimmune neuritis¹² and S-antigen-¹³ and R14-induced¹⁴ experimental autoimmune uveitis with leflunomide has also been described. 

In addition to blocking de novo pyrimidine biosynthesis, leflunomide also has the off-target effect of inhibiting tyrosine phosphorylation by inhibiting tyrosine kinase activity.¹⁵ PP-001 does not bind tyrosine kinase, and side effects of leflunomide, like weight and hair loss, as described in the treatment of Lewis rats,¹⁴ were not observed in our studies during oral PP-001 application. 

When we investigated the effect of PP-001 on the prevention of EAU by daily oral gavage, from immunization to termination of the experiment, we found differences between the effects of PDSAg-induced chronic disease and those of R14-induced relapsing-remitting disease. In both cases, the high dose (30 mg/kg/d) of PP-001 dramatically reduced intraocular inflammation (clinical score) and retinal destruction (determined by histology). The dose of 18 mg PP-001 suppressed intraocular PDSAg-mediated inflammation to approximately 50%, with mild clinical signs of uveitis appearing with a delay of 7 days, whereas retinal destruction was only slightly ameliorated. In contrast, this dose had no effect on R14-induced EAU, except for a slight delay of onset by 2 days. This difference could have been due the distinct roles of Th1 and Th17 cells in the pathogenesis of uveitis.¹⁶ The investigation of intraocular T cells during the course of PDSAg-induced monophasic/chronic rat EAU and R14-mediated relapsing-remitting disease revealed different population dynamics in the two types of disease. IFN-γ-producing T cells played a major role in relapsing disease, whereas intraocular IL-17-producing cell populations increased during monophasic disease and decreased during the primary course of R14-induced uveitis.⁴ 

The spontaneously relapsing-remitting model of uveitis enabled investigation of the effect of PP-001 on relapses. Oral PP-001 application was initiated individually in rats after the resolution of the primary course of intraocular inflammation. A daily dose of 25 mg/kg BW PP-001 reduced the number and intensity of relapses, the latter determined as area under the curve. Here, treatment was initiated 7 to 9 days after onset of uveitis, at an advanced stage of the efferent immune response, which parallels the situation seen in patients who usually receive treatment when the disease is already active, with the aim of preventing relapses or chronicity. PP-001 proved to be a highly suitable drug for this purpose. 

When PP-001 was applied to antigen-specific T-cell lines in vitro, a suppression of proliferation and metabolic activity was observed with all doses. A more pronounced effect was observed with the highest dose of 30 μM in all three cycles of in vitro antigen restimulation of the T cells. Interestingly, this pattern was not reflected by changes in the secretion of cytokines. During primary in vitro restimulation, even the lowest dose of PP-001 completely abrogated the secretion of IFN-γ and IL-17A in both the PDSAg- and R14-specific T cells, whereas during the second in vitro restimulation, only the highest dose of PP-001 led to complete suppression of IFN-γ and IL-17A secretion. These data indicate that decreased cytokine secretion is not just a matter of reduced metabolic activity or viability of the cells. Inhibition of IFN-γ and IL-17A production by targeting DHODH has been reported in animal models and in patients.^17,18 

With respect to T-cell proliferation and cytokine secretion, we demonstrated the dual effects of DHODH inhibition by PP-001: Blocking de novo pyrimidine synthesis not only impedes DNA replication and proliferation of T cells by deprivation of cytosine and thymine but also RNA synthesis and, thus, could inhibit cytokine production via blockade of de novo synthesis of uracil. In addition, other mechanisms could be effective. Other DHODH inhibitors have been shown to also have an impact on the protein expression of IL-17, independent of the pyrimidine pool.¹⁷ PP-001 may act in a similar way, because suppression of cytokine secretion was observed earlier than the anti-proliferative effect. DHODH is a multifunctional enzyme whose action is not limited to the de novo pyrimidine pathway. Recent evidence has shown that DHODH is also an important player in transcriptional elongation of transcripts involved in cell growth and development.¹⁹ The cytokine inhibition seen may be due to the inhibition of transcriptional elongation of central transcription factors such as STAT3 and NFκB. 

The prodrug leflunomide, but not its DHODH-binding metabolite, can bind the aryl hydrocarbon receptor AhR, a cytoplasmic transcription regulator also expressed in T cells, which has been shown to mediate antiproliferative effects in melanoma cells.²⁰ The various effects of leflunomide are dose-dependent, for example, DHODH blocking occurs at low concentrations, whereas tyrosine kinase inhibition requires 10-fold higher concentrations of the active metabolite of leflunomide.²¹ To date, we do not have any information on potential PP-001 binding to AhR or whether it can block other kinases. It is possible that, like leflunomide, PP-001 may have effects in addition to DHODH inhibition. 

The two T-cell lines revealed striking differences in the production of some cytokines/chemokines, especially with respect of VEGF, which was produced only by PDSAg-specific T cells. Vascular endothelial growth factor promotes the formation of new vessels and functions as a chemotactic factor for certain T-cell populations.²² Thus, reducing VEGF can also have implications for T-cell activation and migration.³ VEGF was originally discovered as a vascular permeability factor²³ and also functions as an inflammatory cytokine.²⁴ 

MIP-1α/CCL3 is a proinflammatory chemokine, which recruits inflammatory cells such as monocytes, DCs, and NK cells from the circulation to inflamed tissues.²⁵ The increased expression of MIP-1α/CCL3 by R14-specific T cells is thought to promote the recurrent intraocular infiltration of inflammatory cells like monocytes and macrophages, leading to the clinical picture of relapsing uveitis. 

Data suggest that additional mechanisms may exist by which PP-001 interferes with the regulation of cytokine secretion. 

Although PDSAg-induced uveitis presents with only one course of clinically visible intraocular inflammation, structural changes appeared in the retina within 2 to 3 weeks after resolution of clinical uveitis, including formation of chorioretinal neovascularization (Diedrichs-Möhring M, Wildner G, unpublished data, 2005). Formation of new blood vessels in the choroid and retina is a rare but severe sight-threatening complication in chronic posterior uveitis. This is thought to be induced by VEGF, as blocking VEGF has an ameliorating effect.^5,26 Many different cell types are known to produce VEGF, including vascular endothelia, macrophages, and tumor cells. T lymphocytes as a source of VEGF have only recently been described.²⁷ Neovascularization is a major problem in several chronic diseases affecting the retina (e.g., diabetic retinopathy, retinal vein occlusion, age-related macular degeneration, and chorioretinal vein occlusion). The underlying stress leading to the induction of new vessel growth is mainly ischemia, which causes the induction of VEGF by local cells.^28,29 Here we showed that autoreactive T lymphocytes infiltrating the retina in experimental rat uveitis can also produce VEGF. 

Also, IL-17A can promote angiogenesis, either directly^30,31 or by stimulating VEGF production,^32,33 whereas IFN-γ can inhibit VEGF.³⁴ Because both T-cell lines produced IL-17 as well as IFN-γ but differed in VEGF production, VEGF production by T cells is suggested to be the pivotal factor in PDSAg-induced postuveitis neovascularization, as neovascularization was observed only in the retinas of subjects with EAU induced by PDSAg but not by R14. 

The effect of PP-001-treatment on the prevention of retinal neovascularization in PDSAg-induced EAU was compared to treatment with CyA, which is an approved drug for the treatment of autoimmune uveitis in Germany. 

When starting treatment with onset of clinical disease, we expected a suppression of general T-cell activity and diminished production of VEGF. Oral PP-001 was initiated at the peak of disease, where PDSAg-specific T-cell activation had already reached its maximum with respect to production of IFN-γ and IL-17.⁴ Because ocular infiltration of autoreactive T cells generally precedes intraocular inflammation by 3 days,³⁵ we concluded that the clinical (inflammation) score largely reflected the previous T-cell activity; at “peak disease” with the highest inflammation score, there might already be “resolution” on the T-cell level. 

Newly formed vessels can be found in histological sections approximately 10 days after resolution of PDSAg-induced uveitis (Diedrichs-Möhring M, Wildner G, unpublished observations, 2005), suggesting that this process is initiated at the end of clinically visible uveitis as the tissue tries to recover from inflammatory destruction. Autoreactive T lymphocytes can remain in the retinal tissue for an extended time, even after the resolution of inflammation.³ Thus VEGF-secreting PDSAg-specific T cells remaining in the retina after resolution of clinical signs of uveitis may promote vessel growth. In comparison with CyA treatment, which had no inhibitory effect on neovascularization in advanced disease (treatment from day 15), PP-001 significantly suppressed the growth of new vessels in the later stage of uveitis. 

In conclusion, PP-001 is a new candidate for the treatment of autoimmune uveitis, which prevents inflammation and relapses as well as late complications like chorioretinal neovascularization and influences cytokine production. 

Acknowledgments

We thank Svetlana Hamm for cytokine assay and Stephan Thurau and Peter Nelson for critically reviewing the manuscript. 

Supported by grants from 4SC Discovery GmbH and Panoptes Pharma GmbH. 

Disclosure: M. Diedrichs-Möhring, None; J. Leban, 4SC Discovery GmbH (E); S. Strobl, 4SC Discovery GmbH (E); F. Obermayr, Panoptes Pharma GmbH (E); G. Wildner, 4SC Discovery GmbH (F), Panoptes Pharma GmbH (F)