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Topical 0.03% tacrolimus preventing rejection in high-risk corneal transplantation: a cohort study
  1. Otavio A Magalhaes1,
  2. Diane R Marinho2,
  3. Sergio Kwitko2
  1. 1Department of Ophthalmology, Surgery Postgraduate Program—Federal University of Rio Grande do Sul, Clinical Hospital of Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
  2. 2Department of Ophthalmology, Clinical Hospital of Porto Alegre, Porto Alegre, Brazil
  1. Correspondence to Dr Otavio Magalhaes, Department of Ophthalmology, Surgery Postgraduate Program—Federal University of Rio Grande do Sul, Clinical Hospital of Porto Alegre, 333 Mostardeiro Street, Office 503, Porto Alegre, RS 90430-001, Brazil; otaviomaga{at}


Background/aims The present study aims to identify the rate of rejection and safety of 0.03% tacrolimus eye drops associated with 1% prednisolone in a topical formulation, comparing them with the use of 1% prednisolone eye drops alone in patients with high-risk corneal transplantation.

Methods Retrospective cohort study with 72 patients (72 eyes) who underwent more than one penetrating keratoplasty (PK) in the same eye or had severe chemical burn between 2004 and 2011 in the department of cornea and external disease of the Clinical Hospital of Porto Alegre, Brazil. We compared the records of 36 patients that performed unilateral PK and received only 1% prednisolone eye drops between May 2004 and July 2008, with 36 patients that received 0.03% tacrolimus eye drops in addition to 1% prednisolone between August 2008 and August 2011.

Results The mean follow-up of the group exposed to tacrolimus was 23.1 months and 24.0 in the prednisolone alone group. The demographics, intraoperative and initial indications for first PK were similar between groups, as well as the number of regrafts performed. Intraocular pressure (IOP) was not statistically different among groups. Regarding irreversible rejections, topical tacrolimus showed greater protection: only seven grafts (19.4%) lost transparency against 16 (44.4%) in the 1% prednisolone alone group (p <0.05).

Conclusions Topical 0.03% tacrolimus was effective in preventing irreversible rejection in patients with high-risk corneal transplantation without increasing IOP.

  • Cornea
  • Eye (Tissue) Banking
  • Immunology

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Corneal transplantation has been performed for more than 100 years with a graft survival rate of approximately 75% in 5 years.1 In 2011, 14 696 corneal grafts were performed in Brazil according to the Brazilian Association of Organ Transplants. Most of these grafts happen as first transplant in low-risk recipients, including patients with keratoconus and bullous keratopathy. These conditions show a success rate above 90% in 2 years.2 The major challenge occurs in chemical burns, autoimmune diseases, previous history of rejection and vascularised corneas.3 Other risk factors for rejection include herpes simplex virus keratitis,4 chemical burn5 and corneal trephination diameter exceeding 8.25 mm.6 ,7 Despite advances in anti-inflammatory therapy, glaucoma, surgical techniques and postoperative care, rejection rates remain high in this group of patients.8

Tacrolimus (also named FK506) and cyclosporine (CsA) are calcineurin-blocking drugs that inhibit clonal expansion of T lymphocytes through binding of intracellular proteins called immunophilins.9

Topical corticosteroids are the agents traditionally prescribed for all patients undergoing transplantation to prevent and treat rejection, although its use presents several chronic side effects.10 There is no consensus regarding rejection prevention of high-risk recipients. Among the pharmacological alternatives to decrease this risk, corticosteroids associated with the use of systemic immunosuppressant or, more recently, topical 0.5% and 2% cyclosporine A are options available for ophthalmologists.11 Despite its apparent benefit, there is no scientific evidence that any medication is superior to traditional treatment with topical 1% prednisolone.11 ,12 Systemic approach is also a subject of research, with varying results and uncertain benefits due to the significant incidence of side events.13–21

Almost 20 years ago, an experimental study in rabbits demonstrated significant efficacy of topical 0.3 mg/mL tacrolimus in preventing allograft rejection in experimental corneal transplantation.22 A recent case report with tacrolimus eye drops demonstrated good safety and tolerability, preventing new episodes of rejection in four high-risk transplants when associated with topical steroids.23 The available research has a low level of evidence and no control group in humans. Our study evaluates the rate of rejection and safety of tacrolimus associated with prednisolone (both in a topical formulation), comparing them to the use of topical prednisolone alone in patients at high risk for graft rejection.


The study was approved by the ethics committee of the Clinical Hospital of Porto Alegre, Brazil (protocol number 11-0432) and conducted in accordance with the Helsinki Declaration. All data entered here followed the recommendation on observational studies (STROBE).24

We conducted a retrospective cohort study that evaluated the records of patients considered high-risk for rejection who underwent unilateral penetrating keratoplasty (PK) in the cornea department of our hospital. All procedures were performed in our centre with the same technique (interrupted sutures) and undertaken by different surgeons. Corneas were stored in Optisol-GS solution. Our objective was to assess the clinical outcome of patients treated with sterile olive oil diluted 0.03% tacrolimus monohydrate eye drops (Ophthalmos Pharma; Sao Paulo, Brazil) concurrently with the use of 1% prednisolone, comparing them with 1% prednisolone eye drops alone (control group).

All patients of any age who performed more than one corneal transplant in the same eye for any clinical indication (rejection history with graft failure) or chemical burn with 360° peripheral vascularity from May 2004 to August 2011 were included (consecutive sampling). Patients with previous primary donor failure, incomplete documentation or anterior segment congenital malformation were excluded.

Data were collected on patient characteristics and surgical procedure, such as age (at the period of the last transplantation), gender, follow-up period since the last regraft, size of the recipient cornea trephination, extent of preoperative superficial and deep corneal peripheral vascularisation prior to the last PK (ambulatory slit-lamp examination—360° divided into 4 quadrants), intraocular pressure (IOP) measured by applanation tonometer at 1, 3, 6, 12, 24 and 36 months after the last operation, presence of A, B, O or AB blood groups compatibility (ABO) compatibility, first keratoplasty clinical indication and number of transplants performed in the same eye.

All patients received traditional treatment with 1% prednisolone acetate eye drops for 6 months (prescribed every 3 h in the first month and then gradually tapered over equally in both groups). From August 2008, all patients undergoing high-risk PK initiated in addition to 1% prednisolone, topical 0.03% tacrolimus twice daily (instilled into the lower conjunctival sac every 12 h) as adjunctive immunosuppressive therapy on day 15 postgraft. This medication was maintained through the entire study follow-up period and discontinued in case of irreversible rejection confirmation. All rejection episodes were treated in the same way in both groups, using 1% prednisolone eye drops hourly for 7 days and tapering weekly until its suspension. In addition to that, oral 1 mg/kg prednisone daily was also prescribed for 7 days tapering 10 mg weekly until its full suspension in both groups. In patients using tacrolimus, this medication was maintained in the same dosage (twice daily) during the entire rejection episode. We quit follow-up at 3 years in the control group to allow a reliable comparison between groups.

Rejection episode signs were characterised by sudden cornea oedema with epithelial or endothelial line, subepithelial infiltrates, mild-moderate anterior chamber reaction or keratic precipitates. Persistent cornea oedema and cloudiness with loss of transparency after 2 months of rejection treatment was considered secondary failure (irreversible rejection). At this point, the only possible treatment indication was a new corneal transplantation or keratoprothesis to restore transparency.

The primary outcome was the rate of irreversible rejection (loss of graft transparency). We also analysed the rate of rejection episodes in each group and its IOP. Subgroup analysis was performed based on ABO compatibility.


Proportions were used for categorical data. Mean and SD or median and IQR were used for continuous data. The association between categorical variables was evaluated with the χ2 test (with Yates correction for 2×2 tables). Proportions and OR were calculated with a 95% CI. To evaluate the comparability of groups at baseline, the association between continuous and categorical variables was analysed by Student t test. Multiple t tests comparisons were adjusted by Bonferroni correction for statistic significance (modified by Finner). For these analyses, we used SPSS V.18.0.25

Estimating 45% of irreversible rejection in patients treated with prednisolone alone and 15% in patients treated with tacrolimus and corticosteroid, 35 patients are required in each intervention group to obtain a study power of 80% and an α of 0.05.


We identified 81 consecutive patients who underwent more than one PK in the same eye or had severe chemical burn between 2004 and 2011. Of these, 41 patients (41 eyes) performed the last transplant between May 2004 and July 2008, receiving only 1% prednisolone eye drops. Forty patients (40 eyes) between October 2008 and August 2011 received 1% prednisolone and 0.03% tacrolimus eye drops. Three patients instilling only prednisolone, and two using corticosteroid plus tacrolimus, were excluded because they had incomplete data. Each group had one patient with anterior segment malformation and one case of primary donor failure as cause of second graft. These patients were excluded. Ultimately, 36 patients filled criteria in each group. The characteristics among the groups were similar (details summarised in table 1). The mean follow-up of the tacrolimus group was 23.1±8.4 months, while in the control group (only corticosteroid) was 24.0±12.4 months. Clinical indications for first PK demonstrated similar findings between groups (described in table 1). The number of regrafts in each group showed as well an important parity, most of the patients performed two grafts. Two cases submitted to only one PK were due to chemical burn with 360° limbal neovascularisation.

Table 1

Patients data

Regarding irreversible rejections, tacrolimus plus prednisolone presented greater protection: only seven grafts (19.4%) showed loss of clearness due to immunological rejection compared with 16 (44.4%) that used only prednisolone. We observed a smaller rate of rejection episodes with combined therapy (38.8% vs 61.1%), however, without reaching statistical significance (see table 2).

Table 2

Rejection data

Intraoperative characteristics, such as graft trephination size and peripheral vascularisation (superficial and deep), were similar in both groups (see table 3).

Table 3

Intraoperative characteristics

There was no significant difference in IOP among groups in the analysed periods (see table 4).

Table 4

Mean IOP (mm Hg ±SD)

ABO compatibility was identified in 14 patients of the tacrolimus plus prednisolone group and in seven of the control group. This difference was not statistically significant (table 5).

Table 5

ABO compatibility


In our study with high-risk corneal grafts, the use of topical 0.03% tacrolimus significantly decreased the incidence of irreversible rejection (19.4% vs 44.4% control group). There were less rejection episodes in the tacrolimus group (38.8% vs 61.1% control group) with an important absolute risk reduction (22.2%), although the difference was not statistically significant. The most important result was the preservation of transparency in 80.6% of the patients who used tacrolimus eye drops, which represented a 50% recovery of the grafts that had been submitted to an initial rejection episode. This result was statistically significant with an absolute risk reduction of 25%. About one in every four patients will benefit from the tacrolimus rejection prophylaxis (number needed to treat in order to prevent one irreversible rejection). Increasing the number of patients may also demonstrate the effectiveness of tacrolimus topical immunosuppression in rejection episodes.

Since systemic immunosuppressive therapy presents serious side effects, topical administration should be preferred. Thus, clinical trials with cyclosporine A eye drops at a concentration of 0.5% and 2% were carried out to define its immunosuppressive effect. The results were not favourable in the high-risk group.26 Traditional treatment with topical 1% prednisolone monotherapy also showed unsatisfactory results: in a research with 116 patients who underwent regrafts, the survival rate was only 63.9% in 2 years.14 This rate is similar to the 36 patients who used only corticosteroid in our study (58.3%).

We established two groups with similar high-risk characteristics, and analysed the trephination size of the recipient cornea and the limbal vascular bed. Peripheral vascularisation seems to be the main factor associated with rejection, according to several studies.3 ,4 We found no statistical difference in any of these variables between groups.

As already demonstrated in other studies, there was no increase in IOP related to the use of tacrolimus.27 Both groups had equal mean IOP.

Our main limitation is due to the fact that this is a retrospective study. However, there were very few documentation losses. The role of ABO compatibility is difficult to measure in this study. Even though no statistical difference was found regarding ABO, this feature constitutes a selection bias that we tried to minimise enlisting patients with the same characteristics in both groups. It is necessary to deepen research in well-selected high-risk recipients to define its real contribution in rejection prevention.

There are still questions in other studies about the tolerance of topical tacrolimus.20 In 0.05% concentration, its distribution and in vivo stability presented good results and no alteration in aqueous humour or accumulation.21 In our cohort, only four patients (11.1%) reported mild discomfort after topical instillation of tacrolimus (warm sensation). However, none of these discontinued this medication due to intolerance or ocular surface disease (no evidence of epithelial erosion or keratitis). In Brazil, tacrolimus is manipulated with olive oil as a vehicle and presents good tolerance.28 ,29 Our rationale for commencing tacrolimus at postgraft day 15 was epithelium recovery.

This original study suggests that tacrolimus associated with topical prednisolone increases corneal allograft survival in high-risk recipients during moderate follow-up with low side effects. Our results point to the protection of graft clearness greater than or equal compared to systemic immunosuppression reported in other studies.14–18 ,30 Randomised clinical trials are necessary to establish greater effectiveness of this medication.



  • Contributors OAM contributed to conception and design, acquisition of data and analysis, interpretation of data, drafting the article and revising it critically, and final approval of the version to be published. DRM contributed to conception and design, analysis and interpretation of data, revising it critically, and final approval of the version to be published. SK contributed to conception and design, analysis and interpretation of data, revising it critically, and final approval of the version to be published.

  • Competing interests None.

  • Ethics approval Ethics Committee of the Clinical Hospital of Porto Alegre, Brazil.

  • Provenance and peer review Not commissioned; externally peer reviewed.