Introduction

The promising new immunosuppressive drug FK778, a malononitrilamide, is a derivative of the active metabolite of leflunomide A77 1726. Its main mechanism of action is the inhibition of the dehydroorotate dehydrogenase. The resulting reduced capacity of de novo pyrimidine synthesis leads to inhibition of T- and B-cell proliferation.

Efficacy of FK778 in preventing graft rejection in solid organ transplantation has been shown in several animal models.1, 2, 3, 4, 5, 6 The substance has been found to be effective and safe in a phase II study in renal transplantation in humans.7

As the mechanism of graft rejection in solid organ transplantation differs considerably from those in corneal transplantation, it is necessary to evaluate the efficacy of FK778 in preventing corneal graft rejection in an animal model.

The purpose of this study was to prove the efficacy of the new immunosuppressive drug FK778 in preventing immunologic graft rejection in allogeneic orthotopic keratoplasty in rats. Results were compared with the efficacy of mycophenolate mofetil (MMF). MMF is the bioavailability-enhanced morpholino ethyl ester of the active substance mycophenolic acid (MPA), which interferes with the de novo synthesis of guanosine nucleotides by reversibly inhibiting the enzyme inosine monophosphate dehydrogenase.8 MMF has proven its potency in preventing corneal allograft rejection in the rat keratoplasty model,9, 10 as well as in human corneal transplantation.11, 12

Materials and methods

Animals

Sixty-seven orthotopic penetrating keratoplasties were performed using Fisher rats (Rtl-lvl) as donors in the allogeneic groups and Lewis rats (Rtl-lle) as donors in the syngeneic control group. Lewis rats were used as recipients in all groups. All animals were female, weighing 150–200 g. They were approximately 12 weeks old. Animals were obtained from Charles River Deutschland GmbH, (Sulzfeld, Germany). They were obtained and cared for in accordance with the Directives of the European Community as well as with the recommendations of the National Institutes of Health's Guide for the Care and Use of Laboratory Animals, NIH Publication Number 85–23 (revised 1985).

Surgery

Orthotopic penetrating keratoplasties were performed according to the technique of Herbort et al.13

Before surgery (in donors and recipients), mydriasis was achieved by local application of phenylephrine (Neosynephrin-POS 10%; Ursapharm, Saarbrücken, Germany) and tropicamid eyedrops (Mydriatikum Stulln, Pharma Stulln GmbH, Stulln, Germany). Eyedrops were administered three times at intervals of about 5–10 min before the operation. Corneal grafts were obtained using a 3.5 mm trephine after donors were killed by means of CO2 inhalation. The grafts were stored at room temperature in a conservation medium for corneas (Medium 2, Biochrom AG, Berlin, Germany) until transplantation (maximum 2 h).

Recipients underwent short inhalation anaesthesia with isofluran (Forene, ABBOTT GmbH&Co., KG, Wiesbaden, Germany) and were then injected intraperitoneally with a mixture of ketaminhydrochloride 60 mg/kg bodyweight (Ketamin 10%, Essex Tierarznei, Munich, Germany), xylazinhydrochloride 4 mg/kg bodyweight (Rompun 2%, Bayer Vital GmbH, Leverkusen, Germany), and atropinsulphat 0.2 mg/kg bodyweight (Atropinsulfat B.Braun 0.5 mg/ml, B.Braun Melsungen AG, Melsungen, Germany). The recipients were fixed in a dextral lateral position. Central 3.0 mm trephination was performed at the left cornea and the 3.5 mm donor corneal button was transplanted. The graft was fixed with 8–10 interrupted sutures (11.0 Ethilon, Ethicon GmbH, Norderstedt, Germany). The sutures were left in place for the complete follow-up to enhance vascularization and graft rejection. The anterior chamber of the eye was restored at the end of the operation by injection of balanced salt solution. Gentamicin ointment (Refobacin, Merck, Darmstadt, Germany) was applied and a blepharorraphy, which remained in place for 3 days, was performed with one interrupted suture (6.0 Vicryl, Ethicon GmbH, Norderstedt, Germany) to protect the corneal graft.

Groups

The groups were divided as follows:

Group 1 (n=11):

Fisher/Lewis (allogeneic control)

Group 2 (n=12):

Lewis/Lewis (syngeneic control)

Group 3 (n=11):

Fisher/Lewis (MMF, 40 mg/kg bodyweight /day)

Group 4 (n=12):

Fisher/Lewis (FK778, 5 mg/kg bodyweight /day)

Group 5 (n=12):

Fisher/Lewis (FK778, 10 mg/kg bodyweight /day)

Group 6 (n=9):

Fisher/Lewis (FK778, 20 mg/kg bodyweight /day)

We applied the same dosages of FK778 as used in previous animal models, for example the rat cardiac transplantation model.1, 2

The dosage of 40 mg/kg bodyweight/day MMF has shown a strong immunosuppressive effect in several previous studies and is therefore used as control for the high-dosage FK778 group in this study.2, 10, 14, 15

Medication in the therapy groups 3–6 was given orally with a stomach tube, starting on the day of operation and continuing daily for 18 days. MMF was given as a suspension in water and FK778 was dissolved in 1% of carboxymethylcellulosis.

Clinical evaluation

Fourty-three rats (group 1 (n=7), group 2 (n=8), group 3 (n=7), group 4 (n=8), group 5 (n=8), group 6 (n=5) were subjected to clinical examination for a maximum of 100 days.

Examination was perfomed with the surgery-microscope following brief inhalation anaesthesia with isofluran (Forene, ABBOTT GmbH&Co, KG, Wiesbaden, Germany).

The grafts were evaluated every three days by means of a scoring system including opacity, oedema, and vascularisation. Graft rejection was defined as total opacity of the graft. Between days 9 and 12, grafts were evaluated daily to determine the exact day of rejection. To enhance accuracy of graft evaluation, each animal was evaluated by two examiners (FB and CS).

We scored opacity as follows: 0=no opacity; 1=slight opacity (details of iris clearly visible); 2=some details of iris no longer visible; 3=pronounced opacity (pupil still recognizable); 4=total opacity.

Not all of the grafts experienced total opacification in the groups with oral immunosuppression. Some of them reached only opacity level 3 and cleared up afterwards to opacity level 2 or even 1. In such cases, we defined the day of reaching opacity level 3 as the day of rejection.

After 100 days, or following clinically diagnosed, complete opacification of the corneal graft, the recipient animals were killed by CO2 inhalation. The operated eye was enucleated and fixed in a buffered formalin solution (4%). For histological assessment, the formalin-fixed eyes were cut into 4-μm-thick preparations and subjected to hematoxylin–eosin staining.

The animals were closely monitored for signs of toxic side effects (e.g., weight loss) during the entire follow-up.

Immunohistology

Four rats from each group were killed for immunohistological evaluation on day 14. Using a cryostat, 7-μm-thick sections of each frozen eye were prepared. Specimens were allowed to dry 2 h on silanised slides, then fixed in 100% acetone at −20°C for 1 min. Once the acetone had evaporated, the slides were stored in plastic film at −20°C for further use. For staining, the slides were fixed in acetone at −20°C for 30 min under addition of 0.1 ml 30% H2O2 every 10 min. Then the slides were immersed in Tris buffer with calf serum. The primary antibodies were applied to the sections for 1 h at room temperature. Monoclonal mouse anti-rat antibodies to T-helper cells (cluster of differentiation (CD) 4, W3/25, 1:200), cytotoxic T cells (CD8, MRC OX-8, 1:200), interleukin (IL)2-receptor (CD25, MRC OX-39, 1:100), B cells (CD45RA, MRC OX-33, 1:100), natural killer cells (CD161, 10/78, 1:200), macrophages (CD163, ED2, 1 : 500) and dendritic cells (DC, MRC OX-62) were used. CD4, CD8, CD25, and CD161 were purchased from Biozol Diagnostica GmbH (Eching, Germany), CD45RA from Oxford Biotechnology Ltd 2000 (Oxford, UK), CD163 from RDI Research Diagnostics Inc. (Flanders, NJ, USA) and DC from Cedarlane Laboratories Limited (Ontario, Canada). After washing in Tris buffer, we applied the biotinylated rabbit anti-mouse second antibody (Anti-Mouse Immunoglobulins/Biotinylated, Polyclonal Rabbit, Dako Cytomation GmbH, Hamburg, Germany) for 45 min. The slides were washed in Tris buffer and incubated with horseradish peroxidase 1:500 (HRP, Dako A/S, Denmark). 3-Amino-9-ethyl-carbazole was then applied for 10 min to the tissue, followed by counterstaining with hematoxylin for 2 min. One digital photograph was taken for every slide in the central zone of each graft. Cells were counted on the photographs on an area of 1/100 mm2 to quantify the degree of graft infiltration by each cell type. Immunohistological staining was controlled with staining of rat spleen specimens as positive control and corneal grafts without primary antibodies as negative control.

Statistical analysis

Time to rejection was analysed with the Kaplan–Meier survival analysis16 and compared with the log-rank test. The densities of infiltrating immune cells were compared statistically using the non-parametric Mann–Whitney test.

All statistical evaluation was performed using SPSS Windows 12.0 (Microsoft Corp., Redmond, USA).

Results

Clinical evaluation

In the allogeneic control group (group 1) mean graft survival was 11.4 days. We found complete opacification combined with total vascularization and pronounced oedema. Graft rejection was confirmed as cause of graft failure by means of histological examination of the enucleated, formalin-fixed eyes (haematoxylin–eosin staining).

Grafts remained clear during the total follow-up time of 100 days in the syngeneic control group (group 2).

In all grafts of the allogeneic groups, vascularization reached the center of the corneal button. There was no statistically significant difference regarding vascularization between the allogeneic groups. Even in the syngeneic group, we observed a suture-induced vessel ingrowth in the graft, which stopped in the periphery of the grafts.

In group 3 (MMF 40 mg/kg), the mean time from transplantation to complete opacification of the graft was 24.0 days.

In the FK778-treated animals, we observed a dose-related, differential effect: mean survival was 15.7 days in group 4 (FK778 5 mg/kg), 19.1 days in group 5 (FK778 10 mg/kg) and 25.4 days in group 6 (FK778 20 mg/kg).

The pair-wise log-rank test showed a Pvalue smaller than 0.05 for all groups except groups 3 against 6.

Figure 1 shows a Kaplan–Meier survival plot of rejection-free graft survival.

Figure 1
figure 1

Kaplan–Meier survival plot of rejection-free graft survival; MMF: mycophenolate mofetil; pair-wise log-rank test: P<0.05 for all groups except group 3 against group 6.

Immunohistology

We found dense cell infiltration in the allogeneic grafts in group 1 with CD4+ (T-helper cells; mean 22 cells per 1/100 mm2), CD8+ (cytotoxic T cells; mean 22 cells per 1/100 mm2), CD25+ (IL2-receptor; mean 12 cells per 1/100 mm2), CD161+ (natural killer cells; mean 7 cells per 1/100 mm2) and CD163+ (macrophages; mean 29 cells per 1/100 mm2) cells (Figures 2, 3, 4, 5, 6 and 7).

Figure 2
figure 2

Immunohistology of corneal grafts. Density of infiltrating CD4+ cells; P<0.0001, ANOVA.

Figure 3
figure 3

Immunohistology of corneal grafts. Density of infiltrating CD8+ cells; P<0.0001, ANOVA.

Figure 4
figure 4

Immunohistology of corneal grafts. Density of infiltrating CD25+ cells; P=0.001, ANOVA.

Figure 5
figure 5

Immunohistology of corneal grafts. Density of infiltrating CD161+ cells (NK cells); P=0.002, ANOVA.

Figure 6
figure 6

Immunohistology of corneal grafts. Density of infiltrating CD163+ cells (macrophages); P<0.0001, ANOVA.

Figure 7
figure 7

Immunohistology of corneal grafts; CD4+ (T helper cells) and CD163+ (macrophages) staining cells.

The syngeneic grafts were nearly free of immunologic cell infiltration.

Grafts from Group 3 (MMF) showed a statistically significantly decreased infiltration of CD4+, CD8+, CD25+, CD161+, and CD163+ cells (P<0.002, anlysis of variance (ANOVA)).

We found a dose-related reduction in immunologic cell infiltration in all FK778-treated groups, which reached statistical significance in group 6 when compared with the allogeneic control group (P<0.002 ANOVA). Reduction in immune-cell infiltration in the grafts showed no statistically significant difference between treatment groups 3 and 6. Four from eight grafts in the follow-up group 4 were already rejected at day 14 (Figure 1), the remaining a few days later. The four grafts of the immunohistology group 4 were all rejected at day 14.

Only very few DCs (mean 0.5–2 cells per 1/100 mm2) were found in group 1, 3, 4, and 5, and no DCs were found in groups 2 and 6. Owing to the low cell count, this difference is not statistically significant.

No CD45RA+ (B cells) were detected in any of the grafts.

Safety

One animal treated with MMF died on day 17 of emaciation. One animal that received FK778 20 mg/kg bodyweight died of emaciation on day 27. These rats were excluded from the study.

The group 3 animals (MMF 40 mg/kg) experienced a weight loss of 20 g (median, minimum 10 g, maximum 40 g) and those in group 6 (FK778 20 mg/kg) of 5 g (median, minimum 0 g, maximum 15 g) during the period of oral immunosuppression. Once therapy ceased, the rats gained weight constantly. We noted a constant increase in weight in all other groups. In groups 3 and 6, we observed soft stool during the first 18 days.

Discussion

Clear graft survival following normal-risk keratoplasty is superior to graft survival in solid organ transplantation owing to the immune privilege of the cornea and the anterior chamber, even without systemic immunosuppression or human leukocyte antigen matching.17 In high-risk situations, such as vascularization of the recipient's cornea, history of previous keratoplasty, or positioning of the graft close to the limbus, the situation is different. Percentage of grafts that experience opacification owing to immune reaction is up to 75% in high-risk keratoplasties without systemic immunosuppression.18, 19, 20, 21

Systemic immunosuppression following high-risk keratoplasty prolongs clear graft survival significantly.11, 19, 21, 22 In some units, cyclosporine A (CSA) and MMF are used routinely following penetrating high-risk keratoplasty.

As keratoplasty is not a life-saving procedure, the pros and cons of any systemic immunosuppression have to be considered carefully. Side effects might lead to premature withdrawal of the immunosuppressive drug with a resulting higher risk for rejection.12, 23, 24 Therefore, it is desirable to have a broader armamentarium of immunosuppressive agents offering a more favourable side effect profile and comparable efficacy in preventing graft rejection following penetrating high-risk keratoplasty.

The synthetic malononitrilamide FK778 is a derivative of leflunomide, which has high immunosuppressive potential. Leflunomide's potency in preventing graft rejection following rat keratoplasty was shown.25 Today, leflunomide is used clinically only in treating rheumatoid arthritis. Owing its long half-time (1–4 weeks) it is not suitable to prevent graft rejection following organ transplantation.

On the other hand, FK778 could be an interesting agent in organ transplantation, as its efficacy is similar to that of leflunomide, but with significantly shorter half-time. It has been shown that FK778 inhibits acute rejection in experimental solid organ transplantation.1, 2, 3, 4, 5, 6

Furthermore, FK778 shows some mechanisms, which are separate from the blockade of pyrimidine synthesis. FK778 inhibits the DC-derived activation of T-cells via blocking the activation of the transcription factor NF-KB .26, 27 FK778 influences further critical early steps in graft rejection of solid organs: FK778 leads to a reduction of the endothelial adhesion molecule upregulation and attenuates lymphocyte–endothelium interactions. Additionally, FK778 has antiproliferative potency on smooth muscle cells, which may be an important mechanism to inhibit the fibroproliferative lesions of chronic organ rejection.28, 29, 30

Furthermore, FK778 demonstrates inhibition of cytomegalovirus replication in animal and in vitro models.7

In this study, we have shown for the first time that FK778 is safe and effective in preventing immunologic corneal graft rejection in rats.

Efficacy

We found a dose-related, statistically significantly prolonged graft survival in all dosages (5, 10, 20 mg/kg body weight/day) compared with the allogeneic control group (P<0.005).

Graft survival in the 20 mg FK778 group and the 40 mg MMF group was comparable (P=0.24).

The immunosuppressive effect of FK778 was confirmed immunhistologically. A significant dose-dependent reduction in immune-cell infiltration was observed.

There was no significant difference in immune-cell infiltration in the grafts between the high-dosage FK778 group and the MMF group. There might be a special role of the FK778-mediated inhibition of DC activation in preventing graft rejection, as the only groups with no DC are the syngeneic control group and the high-dosage FK778 group. However, as the cell count was relatively low, no statistically significant difference was found.

Safety

We noted a weight loss in the 20 mg/kg FK778 and in the 40 mg/kg MMF groups. One animal in each group died of emaciation.

Conclusions

Systemic immunosuppression with FK778 is safe and effective in prolonging corneal allograft survival in the rat keratoplasty model. We observed a dose-related efficacy of FK778 both, in vivo and immunhistologically. FK778's efficacy and safety (20 mg/kg) is comparable with that of MMF (40 mg/kg) in rat corneal transplantation.

It must be evaluated in clinical studies whether systemic FK778 has the potential to become an alternative immunomodulative drug for treatment following penetrating high-risk keratoplasty.