Purpose
To examine efficacy and safety of dual sirolimus and mycophenolate mofetil systemic immunosuppression as allograft rejection prophylaxis after penetrating keratoplasty in patients at high rejection risk.
Design
Prospective, interventional case series.
Methods
settings: Single-center subspecialty clinic. patients: Six penetrating transplant recipients at high rejection risk and with no confounding additional cause for high risk of graft failure. All transplant recipient eyes had good visual potential. intervention: Treatment with oral mycophenolate mofetil in combination with sirolimus for 1 year, and sirolimus alone for 2 further years after keratoplasty at doses used in prophylaxis after cadaveric kidney transplantation. main outcomes measures: Interval to first rejection episode, transplant survival, and significant drug adverse effects. Minimum follow-up interval was 13 months after transplantation.
Results
Rejection episodes occurred in 3 patients, one of which led to transplant failure. Of the 6 transplants, 5 remained clear at latest follow-up. Hepatotoxicity required discontinuation of mycophenolate in 1 patient, and both drugs were otherwise free of significant adverse effects.
Conclusions
Sirolimus and mycophenolate mofetil in combination are effective in extending corneal transplant survival in most but not all high rejection risk patients and generally are well tolerated. Results justify further evaluation of this regimen in a larger controlled study.
Immune rejection remains the leading cause of corneal graft failure. Of all graft recipients, among those with the worst prognosis for graft survival is a subset that, before transplantation, can be identified to be at high risk of rejection. These are patients with 1 or multiple rejected previous ipsilateral grafts, corneal vascularization in 2 or more quadrants, and inflammation at the time of transplantation. Standard topical immunosuppression in this patient group is associated graft failure resulting from rejection in a very high percentage. Most of the prospective clinical studies in this patient group have examined the benefit of HLA matching, with the 2 largest randomized studies showing a small or no benefit. This strategy in high rejection risk corneal patients is used only in some European centers. Other than avoidance of repeat transplantation and implantation of a Boston keratoprosthesis, an option in use in some North American centers, use of systemic immunosuppression is an alternative approach in patients at high risk of graft rejection. Systemic rather than local administration is justified by evidence in experimental models that alloantigen immunization does not occur in the eye, but that transported corneal alloantigens lead to clonal expansion of alloreactive T cells in regional lymph nodes and possibly spleen.
There is a striking absence of any randomized trial in corneal transplantation of systemic immunosuppressive drugs, considering that these are used as prophylaxis in many centers in patients at high rejection risk. Various protocols have been used in the few reports of systemic immunosuppression after corneal transplantation, almost all using one of the calcineurin antagonists, cyclosporine and tacrolimus, summarized in Table 1 . By comparison with renal transplant recipients treated with oral immunosuppressants, evident reasons for poor prophylaxis outcomes in corneal patients with these drugs almost certainly include (1) low drug doses, (2) short duration rather than lifelong prophylaxis, and (3) the narrow spectrum of activity within the alloreactive cell phenotypes of monotherapy. With regard to the latter, it is noteworthy that all renal transplantation protocols at least commence with dual- or triple-agent prophylaxis, including typically prednisolone, mycophenolate, and a calcineurin antagonist. One possible further reason for poor outcomes in corneal transplant recipients treated only with calcineurin antagonists, which block T-lymphocyte clonal expansion by interference with interleukin-2 gene transcription, is that most graft-reactive cells in the anterior chamber after rejection onset are not lymphocytes, but are CD14+ cells of monocyte or macrophage lineage.
| Author(s) | Study Type | No. of Patients at High Rejection Risk | HLA Matching | Drug and Dose | Prophylaxis Duration (mos) | Graft Outcomes |
|---|---|---|---|---|---|---|
| Hill | Prospective, nonrandomized series | 43 | No | CsA trough, 130 to 170 μg/L | 4 to 6 (n = 29); 12 (n = 14) | 67% 2-yr survival; 92% 2-yr survival |
| Reinhard and associates | Retrospective, nonrandomized series | 29 | No | CsA trough, 100 to 150 μg/L; fluocortolone in 29/29 | ≥6; mean, 9 | 91% 2-yr survival |
| Poon and associates | Retrospective, case-control series | 49 | Class I, 8/49 patients | CsA trough, 75 to 180 μg/L; prednisolone in 21/49 | ≥12 | Failure 29%, not significantly different from unimmunosuppressed controls |
| Rumelt and associates | Retrospective, nonrandomized series | 28 | No | CsA mean, 325 μg/L a | 1 to 41; mean, 12 | 32% failure because of rejection |
| Joseph and associates | Noncomparative case series | 43 b | Class I, all patients | Tacrolimus trough mean, 1 to 12 μg/L | 18 | 30/39 (77%) 2-yr survival |
| Reinhard and associates | Randomized, prospective trial | 56 | No | CsA trough, 120 to 150 μg/L; MMF, 2 g/day; fluocortolone in 56/56 | 6 | 69% 3-year CsA-treated graft survival; 26% 3-yr MMF-treated graft survival |
| Birnbaum and associates | Prospective, nonrandomized series | 10 | Class I + II, 10/10 patients | Sirolimus trough, 4 to 10 μg/L; fluocortolone in 10/10 | 6 | No rejection-induced failure at 2 yrs |
a Not specified if trough level.
b Series included patients with disorders unrelated to rejection (e.g., glaucoma) or contraindications in this study (e.g., herpes simplex virus keratitis, chemical injury).
Sirolimus (rapamycin) is a microbial macrolide and inhibitor of the mammalian target of rapamycin, rather than calcineurin, and prevents G1 to S phase progression in the T cell division cycle. Accordingly, like calcineurin antagonists, sirolimus is active against T cells; however, sirolimus also is active against dendritic cells, B cells, and monocytes and macrophages. One further property of sirolimus is an antiangiogenic effect in cornea, observed in a rat corneal transplantation study that also demonstrated delayed graft rejection after systemic administration. Sirolimus more recently was reported to prevent corneal transplantation rejection in 78% at 1 year after monotherapy for 6 months. These known attributes and reports of sirolimus justified its further evaluation in corneal transplantation. To address the question of whether insufficient immunosuppression had been a reason for poor corneal graft survival in previous reports, we examined the effectiveness in prophylaxis of sirolimus, but also the purine synthesis inhibitor mycophenolate mofetil (MMF), which blocks proliferation of T and B cells. The drugs were prescribed in combination at doses and blood levels commonly used in renal transplantation recipients.
Methods
Patients and Surgery
Six high rejection risk corneal transplant recipients were managed with a standardized immunosuppression protocol as rejection prophylaxis. Five underwent penetrating keratoplasty and 1 underwent penetrating keratoplasty combined with cataract extraction. Surgery was undertaken by a single surgeon (D.F.P.L.) in a single subspecialty center. All patients except one had very poor vision in the contralateral eye. Factors conferring high rejection risk in these patients were presence of more than 1 quadrant of deep stromal vascularization, a failed previous ipsilateral graft resulting from rejection, or both. Exclusion criteria were (1) any risk factor for graft failure other than rejection, (2) herpetic keratitis, to avoid confusion of herpetic recurrence with rejection, and (3) any noncorneal eye disease significantly limiting visual potential. Patients all had normal general health with baseline hematologic and biochemistry results within normal limits. Preoperative patient features are summarized in Table 2 and are discussed further below. Patients underwent transplantation with donor cornea that was not HLA matched, was preserved in organ culture, and had estimated endothelial cell density of at least 2000 cells /mm 2 at issue for surgery. Patients with failed previous grafts had donor cornea of the same diameter transplanted after full excision.
| Patient No. | Age (years) | Primary Diagnosis | Failed Previous Graft Because of Rejection | Contralateral Corrected Visual Acuity | Quadrants of Recipient Cornea Vascularization | |
|---|---|---|---|---|---|---|
| Ipsilateral | Contralateral | |||||
| 1 | 65 | Macular dystrophy | 2 | 1 | 2/60 | 1 |
| 2 | 51 | Idiopathic lipid keratopathy | 0 | 0 | Hand movements | 4 |
| 3 | 27 | Pseudophakic decompensation, juvenile uveitis | 1 | 2 | 6/60 | 0 |
| 4 | 29 | Keratoconus | 1 | 1 | 6/18 | 4 |
| 5 | 43 | Congenital hereditary endothelial dystrophy | 3 | 1 | 6/9 | 0 |
| 6 | 50 | Post-laser in situ keratomileusis ectasia | 2 | 0 | 20/30 | 0 |
After transplantation, interrupted sutures were left in situ for at least 12 months. In addition to oral immunosuppressants (see below), all patients received topical dexamethasone 0.1% every 2 hours for 2 weeks, reduced thereafter to 1 to 2 times daily as an indefinite maintenance dose. Patients were examined for graft function, signs of rejection, and drug adverse effects monthly for the first 2 years and every 3 months thereafter.
Systemic Immunosuppression and Monitoring
Oral immunosuppression was commenced on the first postoperative day. This comprised (1) sirolimus (Rapamune; Wyeth, Maidenhead, United Kingdom) 2 to 6 mg daily to yield whole blood 16-hour trough concentrations in the range 12 and 20 ng/mL, and (2) MMF (CellCept; Roche, Welwyn Garden City, United Kingdom) 2 g daily. Sirolimus was administered for 3 years and MMF for 1 year only. Whole blood predose levels of sirolimus were measured at least 16 hours after the last sirolimus dose by liquid chromatography-tandem mass spectrometry. Trough levels initially were measured 8 days after commencing sirolimus, more than 5 days after any dose change, and every 3 months if the dose was stable at an appropriate trough concentration. Serum levels of MMF were not measured. Full blood count, renal and liver biochemistry results, and fasting serum lipid results were monitored monthly for the first 3 months after transplantation, then every 3 months thereafter.
Graft Rejection Diagnosis and Treatment
Rejection was diagnosed by identification of anterior chamber inflammation associated with an endothelial rejection line, keratic precipitates in a previously transparent cornea, or both. Rejection treatment was commenced by administration of hourly dexamethasone 0.1% drops and a subconjunctival injection of betamethasone 2 mg, followed in the absence of clinical response by oral prednisolone 1 mg/kg daily or intravenous methylprednisolone 0.5 g. Treatment was continued until signs of inflammation subsided, and then topical dexamethasone frequency was reduced gradually. Graft failure was defined as an irreversible loss of corneal transparency.
Statistical Analysis
Interval to first rejection episode and transplant survival were recorded for each patient. Minimum follow-up interval was 13 months after transplantation. Actuarial rejection-free survival and graft survival were plotted as Kaplan-Meier curves. In analysis of rejection-free survival, time to rejection was calculated as the interval between the date of transplant and either (1) the first day on which the first rejection episode was diagnosed on examination or (2) the most recent follow-up examination date in those patients in whom rejection did not occur. In analysis of transplant survival, trial time was calculated as the interval between the date of transplantation and either (1) the date on which transplant failure was diagnosed, or (2) the most recent follow-up examination date in those patients with functioning transplants.
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