Immune-mediated rejection remains the leading cause of allograft failure in human high-risk keratoplasty. Cyclosporin A–based immunosuppression has substantially improved corneal allograft survival. 1,2 However, the use of cyclosporin A is limited because it causes dose-dependent hepatotoxicity, nephrotoxicity, and neurotoxicity. 3 This toxicity is at least partially explained by its interference with the calcineurin system. 4
RAD is a new immunosuppressive substance that does not interfere with the calcineurin system. It is an oral rapamycin derivative produced by Novartis Pharma. RAD is derived chemically from rapamycin, which has been obtained by fermentation of an Actinomycetes strain. It has been found that RAD (40-0-[2-hydroxyethyl])-rapamycin is stable in oral formulations and that its efficacy after oral dosing is at least equivalent to that of rapamycin. 5–10 RAD affects growth factor–induced intracellular signaling and thus inhibits the IL-2-stimulated clonal expansion of activated T cells. Thus, the action of RAD is distinct to that of cyclosporin, which acts early during an immune response by inhibiting the antigen-induced activation of T cells and IL-2 production. These different modes of action provide a rationale for expecting synergistic effects of the two compounds. In fact, this synergism has already been shown in murine lung, kidney, and heart transplantation. 11,12
As the goal of multiple immunosuppressive therapy in organ transplantation is to enhance therapeutic efficacy while minimizing the toxicity of individual drugs used in the regimen, the aim of this study was to evaluate the combined effect of RAD and cyclosporin A in the prevention of acute allograft rejection after murine corneal transplantation.
MATERIALS AND METHODS
Two inbred rat strains, Fisher (RTI-I v1) as donors and Lewis (RTI-I e ) as recipients, were used in this study. All animals were females weighing 180 to 220 g (Janvier, France). The animals were obtained and cared for in accordance with the Directives of the European Community and with the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication 85–23, revised 1985).
We conducted an orthotopic perforating keratoplasty according to the technique of Herbort et al. 13 Before surgery, all animals were given phenylephrine hydrochloride 10% eyedrops (Neosynephrin-POS, Ursapharm, Saarbruecken, Germany) to achieve maximal pupil dilation. These eyedrops were administered three times at intervals of 10 minutes before the operation. During an inhalation of anesthesia with diethyl ether, the corneas of the donor rats (Fisher) were obtained by the use of a 3.5-mm trepan. Until their implantation, donor buttons were stored at room temperature for approximately 20 minutes in a conservation medium for corneas (Likorol, Opsias, France).
The recipient animals (Lewis) were pretreated in the same way and, after a brief inhalation anesthesia with diethyl ether, were anesthetized with an intraperitoneal mixed injection of ketamine hydrochloride 100 mg/kg body weight (Ketanest, Parke-Davis, Berlin, Germany), midazolam (Dormicum, Roche, Grenzach-Whylen, Germany) 0.5 mg/kg body weight, and atropine sulfate (Atropinsulfat Braun, B. Braun, Melsungen, Germany) 0.5 mg/kg body weight, and fixed in a dextral lateral position. After the left host cornea had been removed with a 3.0-mm trepan, the donor cornea was transplanted. The transplant was sewn in with eight interrupted sutures (Ethicon 11.0). The anterior eye chamber was restored at the end of the operation by the instillation of balanced salt solution. To protect the transplant, a blepharorrhaphy was performed by means of two interrupted sutures (prolene 6.0) that remained in place for 3 days, and gentamicin (Refobacin, Merck, Darmstadt, Germany) ointment was applied in the palpebral fissure.
The groups were divided as follows: group 1 (n = 13), Lewis/Lewis (syngeneic control); group 2 (n = 13), Fisher/Lewis (allogeneic control); group 3 (n = 10), Fisher/Lewis (RAD 2.5 mg/kg body weight/day); group 4 (n = 11), Fisher/Lewis (cyclosporin A 10 mg/kg body weight/day; Sandimmun optoral, Novartis Pharma); and group 5 (n = 10), Fisher/Lewis (RAD 1.5 mg/kg plus cyclosporin A 5 mg/kg; Sandimmun optoral).
Medication in the therapy groups was applied orally by a feeding gavage, started on the day of operation, and continued daily for the duration of 18 days.
Thirty-seven rats—group 1 (n = 9), group 2 (n = 9), group 3 (n = 6), group 4 (n = 7), and group 5 (n = 6)—were subjected to clinical examination every third day for the duration of at most 100 days. The graft survival was than considered as indefinite because we did not see any immunologic reactions within the graft beyond the 30th postoperative day. Each animal was examined by slit-lamp biomicroscopy during a brief inhalation of anesthesia with diethyl ether. The transplants were evaluated by means of a scoring system, which assessed opacity, edema, and neovascularization.
Opacity was graded as follows: 0, no opacity; 1, slight opacity with details of the iris clearly visible; 2, some details of iris no longer visible; 3, pronounced opacity with the pupil still recognizable; and 4, total opacity. Edema was graded as follows: 0, no edema; 1, mild edema; and 2, pronounced edema with raised transplant. Neovascularization was graded as follows: 0, no vessels; 1, vessels in the periphery; 2, vessels extending deeper; and 3, vessels extending to the center.
The target criterion was complete opacification of the transplant. After 100 days or after complete opacification of the transplant, the recipient animals were killed through the inhalation of CO 2 . After that, the operated eye was enucleated and fixed in a buffered formalin solution (4%). For histologic assessment, the formalin-fixed eyes were cut into 4-μm thick preparations and subjected to hematoxylin and eosin or elastica staining. Histologic evaluation was undertaken to confirm the clinical diagnosis of rejection and to rule out other reasons for graft opacification.
In a second set of experiments, 20 animals were transplanted and treated according to the aforementioned groups (four per group). All rats were killed for immunohistologic evaluation on day 14. Four-micrometer thick frozen sections of each frozen eye were stained by an immunoperoxidase technique using monoclonal mouse antirat antibodies to T-helper cells (CD4, W3/25), cytotoxic T cells (CD8, MRC OX-8), IL-2 receptor (CD25, MRC OX-39), B cells (CD45, MRC OX-29), and intercellular adhesion molecule 1 (CD54, TLD-4C9). All monoclonal antibodies were purchased from Serotec (Serotec, Littlehampton, UK). The cryosections were fixed with chloroform for 30 minutes and then stained using a standard two-layer indirect immunoperoxidase technique. Adjacent frozen tissue sections were stained with hematoxylin and eosin. Positive cells were stained with red. To quantify the degree of graft infiltration by each cell type and to ensure consistency in having the slides read, cells were counted and recorded by a masked examiner in the same anatomic area (i.e., the central zone of each graft).
Time to rejection was calculated as the time to the event from the date of transplant and evaluated with the Kaplan–Meier estimator. The number of cells stained positively by immunohistochemistry was compared between the control and treatment groups using the Student t-test for unequal variances. For all tests a p value less than 0.05 was considered statistically significant. Experimental results are presented as the arithmetic mean ± standard error of the mean for each group.
The interaction between the two drugs, synergism, summation, or antagonism is assessed by the combination index (CI) which has been used in similar models. 14 According to Chou's interpretation, 15 combination indices less than 1.0 suggest synergism, whereas those equal to 1.0 indicate summation, and those above 1.0 show antagonism.
Table 1 shows the transplant survival rate in the control and therapy groups. Corneal transplantation in the syngeneic combination (group 1) led to slight perioperative stromal transplant edema, which was no longer detectable from the sixth postoperative day onward. Throughout the entire period of the examination, the transplants remained clear. Neovascularization was detectable only in the area of the sutures. The average transplant survival rate in the allogeneic combination (group 2) was 12.3 ± 0.3 days. Therapy with RAD 2.5 mg/kg led to a statistically significantly prolonged transplant survival (37.7 ± 12.5 days, p < 0.05). There was no statistically significant difference when compared with cyclosporin A 10 mg/kg monotherapy (39.7 ± 12.5 days). Double drug therapy with RAD 1.5 mg/kg plus cyclosporin A 5 mg/kg resulted in significantly superior graft survival when compared with each monotherapy (87.8 ± 12.2 days, p < 0.05).
Histology and Immunohistology
On the 14th postoperative day, the transplants of the syngeneic control group (group 1) showed only in the area of the interrupted sutures isolated foreign-body giant cells and a low level of mononuclear infiltrate. The center of the transplant showed no cellular infiltration whatsoever, and the configurations of epithelium, stroma, and endothelium were normal. Allografts from control rats (group 2) showed edema characterized by an extreme thickening of the stroma and the development of vacuoles, particularly in the area of the basal membrane of the epithelium, with the histologic picture of a bullous keratopathy. A pronounced mononuclear infiltration was present in all layers of the transplants but was most pronounced in the area of the stroma and the deeper layers of the epithelium. However, corneas taken from animals treated with RAD, cyclosporin A, and the double drug regimen had fewer infiltrating cells, an almost normal corneal thickness, and less neovascularization.
Table 2 summarizes the results of the immunohistochemical studies. In all allogeneic groups, the inflammatory cell population was composed of CD4 + and CD8 + cells (i.e. helper and cytotoxic T cells). Although B cells (CD45 + ) have been observed during rejection, they did not contribute much to the inflammatory process. However, there were significant differences in the number of cells observed. There was a statistically significant reduction in the number of CD4 + , CD8 + , and CD45 + cells in the animals treated with RAD and cyclosporin A when compared with the allogeneic control. When looking at inflammatory markers, expression of IL-2 receptor (CD25 + ) was significantly reduced only in the RAD 2.5 mg/kg group when compared with the allogeneic control. This therapeutic protocol also led to a significant reduction of infiltrating CD4 + cells when compared with the cyclosporin A–treated animals. No statistically significant differences have been found in the expression of intercellular adhesion molecule-1 (CD54) as between the allogeneic control and the treatment groups. Approximately 10% of the infiltrating lymphocytes in the allogeneic control have been CD45 + (expressed only on B lymphocytes), which have been significantly reduced in the animals treated with RAD and cyclosporin A. This significant reduction in infiltrating CD45 + and CD25 + cells has not been found in the double drug therapy.
Compatibility of RAD, Cyclosporin A, and the Double Drug Therapy
All experimental animals have been closely monitored for signs of toxic side effects (e.g., weight loss and emaciation) during the whole follow-up. No side effects in the different treatment protocols have been observed.
The strategy of multiple immunosuppressive therapy in organ transplantation is to enhance therapeutic efficacy while minimizing the toxicity of individual drugs used in the regimen. The current experiments examined the effects of RAD and cyclosporin A monotherapy and combination therapy in prolongation of corneal allograft survival in murines. Our data showed that combined therapy with RAD and cyclosporin A was more effective than RAD or cyclosporin A alone.
The immunosuppressant RAD is a novel rapamycin derivative in which the hydroxyl at position 40 of rapamycin has been alkylated with a 2-hydroxyethyl group. This chemical deviation results in altered physiochemical properties with respect to rapamycin (i.e., markedly enhanced solubility in several organic solvents and galenic excipients). 6 In this study, we were able to prove the immunosuppressive effect of RAD in delaying rejection after allogeneic corneal transplantation. These data are comparable to those in a study by Olsen et al. 16 that focused on monotherapy with rapamycin. Unlike in this study with rapamycin, we did not find any influence of RAD on graft neovascularization. Therapy with RAD 2.5 mg/kg produced a statistically significantly prolonged transplant survival (p < 0.05) as compared with the allogeneic control. This efficacy was comparable with the cyclosporin A 10 mg/kg therapy. The unique finding of this study was that RAD and cyclosporin A combination therapy showed a significant synergistic effect in the prophylaxis of acute allograft rejection after corneal transplantation without a higher incidence of complications related to drug toxicity or overimmunosuppression.
The immunosuppressive efficacy of RAD, cyclosporin A, and the combination thereof was confirmed by histology and immunohistochemistry. There was a significant reduction in the number of infiltrating cells of the CD4 + and CD8 + subsets, which are major mediators of acute rejection in this model. Although there were fewer immunocompetent cells in the graft, we did not find a significant reduction in the expression of IL2-R (CD25 + ), a marker of T-cell activation in the cyclosporin A–treated animals. RAD 2.5 mg/kg reduced the expression of CD25, which suggests that RAD led to significant reduction in the recruitment and activation of inflammatory cells. However, this effect could not be seen in the combination therapy. Although we found a tendency, the monotherapy and combination therapy did not lead to a statistically significant reduction in the expression of ICAM-1 (CD54 + ), a surface molecule involved in the recruitment of cells into the area of inflammation. 17
A main problem in clinical transplantation is the dose-dependent nephrotoxicity, hepatotoxicity, and neurotoxicity of cyclosporin A or FK506, which is considered to be mainly mediated through inhibition of the phosphatase calcineurin. 4 Rapamycin has no effect on calcineurin and hence may not have the potential to induce this toxicity in humans. 4 Furthermore, our data suggest a cyclosporin A dose-sparing effect of RAD when used in a double drug regimen.
In conclusion, our results show that concomitant oral immunosuppression with RAD and cyclosporin A produces synergistic effects in the prevention of acute murine corneal allograft rejection. These results warrant further investigation of RAD in preclinical and clinical high-risk keratoplasty.
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