Tacrolimus, also known as fujimycin or FK506, is an immunosuppressive drug used mainly after allogeneic organ transplant to lower the risk of organ rejection. It achieves this by inhibiting calcineurin involved in the production of interleukin-2, a molecule that promotes the development and proliferation of T cells, which are vital to the body’s learned (or adaptive) immune response. Tacrolimus is also used in the treatment of other T cell-mediated diseases such as eczema and psoriasis. (for which it is applied to the skin in a medicated ointment), severe refractory uveitis after bone marrow transplants, exacerbations of minimal change disease, Kimura’s disease, and the skin condition vitiligo.

Chemically it is a 23-membered macrolide lactone that was first discovered in 1987 from the fermentation broth of a Japanese soil sample that contained the bacterium Streptomyces tsukubaensis.

Tacrolimus is also used to treat dry eye syndrome in cats and dogs.^[1]^[2]

Medical uses[edit]

Organ transplantation[edit]

It has similar immunosuppressive properties to ciclosporin, but is much more potent. Immunosuppression with tacrolimus was associated with a significantly lower rate of acute rejection compared with ciclosporin-based immunosuppression (30.7% vs 46.4%) in one study.^[3] Clinical outcome is better with tacrolimus than with ciclosporin during the first year of liver transplantation.^[4]^[5] Long-term outcome has not been improved to the same extent. Tacrolimus is normally prescribed as part of a post-transplant cocktail including steroids, mycophenolate, and IL-2 receptor inhibitors such as basiliximab. Dosages are titrated to target blood levels.

Ulcerative colitis[edit]

In recent years, tacrolimus has been used to suppress the inflammation associated with ulcerative colitis (UC), a form of inflammatory bowel disease. Although almost exclusively used in trial cases only, tacrolimus has shown to be significantly effective in the suppression of flares of UC.^[6]^[7]

Skin[edit]

Tacrolimus 0.1% ointment

As an ointment, tacrolimus is used in the treatment of eczema, in particular atopic dermatitis. It suppresses inflammation in a similar way to steroids, and is equally as effective as a mid-potency steroid. An important advantage of tacrolimus is that, unlike steroids, it does not cause skin thinning (atrophy), or other steroid related side effects.^[8]

It is applied on the active lesions until they heal off, but may also be used continuously in low doses (twice a week), and applied to the thinner skin over the face and eyelids.^{[citation needed]} Clinical trials of up to one year have been conducted. Recently it has also been used to treat segmental vitiligo in children, especially in areas on the face.^[9]

Lupus Nephritis

Tacrolimus has been shown to reduce the risk of serious infection in lupus nephritis, when compared to other agents.^[10]

Contraindications and precautions[edit]

Contraindications and precautions include:^[11]

Breast-feeding
Hepatic disease
Immunosuppression
Infants
Infection
Neoplastic disease, such as:
- Skin cancer
- Lung cancer
Oliguria
Pregnancy
QT interval prolongation
Sunlight (UV) exposure
Grapefruit juice^[12]

Topical use[edit]

Occlusive dressing
Known or suspected malignant lesions
Netherton’s syndrome or similar skin diseases
Certain skin infections^[8]

Side effects[edit]

By mouth or intravenous use[edit]

Side effects can be severe and include infection, cardiac damage, hypertension, blurred vision, liver and kidney problems (tacrolimus nephrotoxicity),^[13] hyperkalemia, hypomagnesemia, hyperglycemia, diabetes mellitus, itching, lung damage (sirolimus also causes lung damage),^[14] and various neuropsychiatric problems such as loss of appetite, insomnia, posterior reversible encephalopathy syndrome, confusion, weakness, depression, vivid nightmares, cramps, neuropathy, seizures, tremors, and catatonia.^[15]

In addition, it may potentially increase the severity of existing fungal or infectious conditions such as herpes zoster or polyoma viral infections.^[11]

Carcinogenesis and mutagenesis[edit]

In people receiving immunosuppressants to reduce transplant graft rejection, an increased risk of malignancy (cancer) is a recognised complication.^[11] The most common cancers are non-Hodgkin’s lymphoma^{[citation needed]} and skin cancers. The risk appears to be related to the intensity and duration of treatment.

Topical use[edit]

The most common adverse events associated with the use of topical tacrolimus ointments, especially if used over a wide area, include a burning or itching sensation on the initial applications, with increased sensitivity to sunlight and heat on the affected areas. Less common are flu-like symptoms, headache, cough, and burning eyes.^[16]

Cancer risks[edit]

Tacrolimus and a related drug for eczema (pimecrolimus) were suspected of carrying a cancer risk, though the matter is still a subject of controversy. The FDA issued a health warning in March 2005 for the drug, based on animal models and a small number of patients. Until further human studies yield more conclusive results, the FDA recommends that users be advised of the potential risks. However, current practice by UK dermatologists is not to consider this a significant real concern and they are increasingly recommending the use of these new drugs.^[17]

Interactions[edit]

Also like cyclosporin, it has a wide range of interactions. Tacrolimus is primarily metabolised by the cytochrome P450 system of liver enzymes, and there are many substances that interact with this system and induce or inhibit the system’s metabolic activity.^[11]

Interactions include that with grapefruit which increases tacrolimus plasma concentrations. As infections are a major cause of morbidity and mortality in the post-transplant patient, the most commonly^{[citation needed]} reported interactions include interactions with anti-microbial drugs. Macrolide antibiotics including erythromycin and clarithromycin, as well as several of the newer classes of antifungals, especially of the azole class (fluconazole, voriconazole), increase tacrolimus levels by competing for cytochrome enzymes.^[11]

Pharmacology[edit]

Mechanism of action[edit]

FKBP12, the target protein of tacrolimus

Tacrolimus is a macrolide calcineurin inhibitor. In T-cells, activation of the T-cell receptor normally increases intracellular calcium, which acts via calmodulin to activate calcineurin. Calcineurin then dephosphorylates the transcription factor nuclear factor of activated T-cells (NF-AT), which moves to the nucleus of the T-cell and increases the activity of genes coding for IL-2 and related cytokines. Tacrolimus prevents the dephosphorylation of NF-AT.^[18]

In detail, tacrolimus reduces peptidylprolyl isomerase activity by binding to the immunophilin FKBP12 (FK506 binding protein), creating a new complex. This FKBP12–FK506 complex interacts with and inhibits calcineurin, thus inhibiting both T-lymphocyte signal transduction and IL-2 transcription.^[19] Although this activity is similar to that of cyclosporin, the incidence of acute rejection is reduced by tacrolimus use over cyclosporin use.^[3] Although short-term immunosuppression concerning patient and graft survival is found to be similar between the two drugs, tacrolimus results in a more favorable lipid profile, and this may have important long-term implications given the prognostic influence of rejection on graft survival.^[20]

Pharmacokinetics[edit]

Oral tacrolimus is slowly absorbed in the gastrointestinal tract, with a total bioavailability of 20 to 25% (but with variations from 5 to 67%) and highest blood plasma concentrations (C_max) reached after one to three hours. Taking the drug together with a meal, especially one rich in fat, slows down resorption and reduces bioavailability. In the blood, tacrolimus is mainly bound to erythrocytes; only 5% are found in the plasma, of which more than 98.8% are bound to plasma proteins.^[11]^[21]

The substance is metabolized in the liver, mainly via CYP3A, and in the intestinal wall. All metabolites found in the circulation are inactive. Biological half-life varies widely and seems to be higher for healthy persons (43 hours on average) than for patients with liver transplants (12 hours) or kidney transplants (16 hours), due to differences in clearance. Tacrolimus is predominantly eliminated via the faeces in form of its metabolites.^[11]^[21]

When applied locally on eczema, tacrolimus has little to no bioavailability.^[11]

Pharmacogenetics[edit]

The predominant enzyme responsible for metabolism of tacrolimus is CYP3A5. Genetic variations within CYP3A5 that result in changes to the activity of the CYP3A5 protein can affect concentrations of tacrolimus within the body. In particular, individuals who are homozygous for the G allele at the single nucleotide polymorphism (SNP) rs776746 (also known as CYP3A5 *3/*3) have a non-functional CYP3A5 protein. The frequency of the G allele varies worldwide, from 4% in some African populations to 80–90% in Caucasian populations.^[22] Across a large number of studies, individuals homozygous for the G allele have been shown to have higher concentrations of tacrolimus and require lower doses of the drug, as compared to individuals who are not homozygous for the G allele. Achieving target concentrations of tacrolimus is important – if levels are too low, then there is a risk of transplant rejection, if levels are too high, there is a risk of drug toxicities. There is evidence to suggest that dosing patients based on rs776746 genotype can result in faster and more frequent achievement of target tacrolimus levels. However, there is a lack of consistent evidence as to whether dosing based on rs776746 genotype results in improved clinical outcomes (such as a decreased risk for transplant rejection or drug toxicities), likely because patients taking tacrolimus are subject to therapeutic drug monitoring.^[23]^[24]^[25]^[26]

Studies have shown that genetic polymorphisms of genes other than CYP3A5, such as NR1I2^[27]^[28] (encoding PXR), also significantly influence the pharmacokinetics of tacrolimus.

History[edit]

Tacrolimus was discovered in 1987;^[29] it was among the first macrolide immunosuppressants discovered, preceded by the discovery of rapamycin (sirolimus) on Rapa Nui (Easter Island) in 1975.^[30] It is produced by a soil bacterium, Streptomyces tsukubaensis.^[31] The name tacrolimus is derived from “Tsukuba macrolide immunosuppressant”.^[32]

Tacrolimus was first approved by the Food and Drug Administration in 1994 for use in liver transplantation; this has been extended to include kidney, heart, small bowel, pancreas, lung, trachea, skin, cornea, bone marrow, and limb transplants.

Available forms[edit]

The branded version of the drug is owned by Astellas Pharma, and is sold under the trade name Prograf, given twice daily. A number of other manufacturers hold marketing authorisation for alternative brands of the twice-daily formulation.^[33]

Once-daily formulations with marketing authorisation include Advagraf (Astellas Pharma) and Envarsus (marketed as Envarsus XR in US by Veloxis Pharmaceuticals and marketed in Europe by Chiesi).^[33] These formulations are intended to reduce pharmacokinetic variation in blood levels and facilitate compliance with dosing.

The topical formulation is marketed by LEO Pharma under the name Protopic.^[33]

Biosynthesis[edit]

The biosynthesis of tacrolimus is hybrid synthesis of both type 1 polyketide synthases (PKS 1) and nonribosomal peptide synthases (NRPS). The research shows the hybrid synthesis consists of ten modules of type 1 polyketide synthase and one module of nonribosomal peptide synthase. The synthetic enzymes for tacrolimus are found in 19 gene clusters named fkb. The 19 genes are fkbQ, fkbN, fkbM, fkbD, fkbA, fkbP, fkbO, fkbB, fkbC, fkbL, fkbK, fkbJ, fkbI, fkbH, fkbG, allD, allR, allK and allA.^[34]

There are several possible ways of biosynthesis of tacrolimus. The fundamental units for biosynthesis are following: one molecule of 4,5-dihydroxycyclohex-1-enecarboxylic acid (DHCHC) as a starter unit, four molecules of malonyl-CoA, five molecules of methylmalonyl-CoA, one molecule of allylmalonyl-CoA as elongation units. However, two molecules of malonyl-CoA are able to be replaced by two molecules of methoxymalonyl CoA. Once two malonyl-CoA molecules are replaced, post-synthase tailoring steps are no longer required where two methoxymalonyl CoA molecules are substituted. The biosynthesis of methoxymalonyl CoA to Acyl Carrier protein is proceeded by five enzymes (fkbG, fkbH, fkbI, fkbJ, and fkbK). Allylmalonyl-CoA is also able to be replaced by propionylmalonyl-CoA.^[34]

The starter unit, DHCHC from the chorismic acid is formed by fkbO enzyme and loaded onto CoA-ligase domain (CoL). Then, it proceeds to NADPH dependent reduction(ER). Three enzymes, fkbA,B,C enforce processes from the loading module to the module 10, the last step of PKS 1. fkbB enzyme is responsible of allylmalonyl-CoA synthesis or possibly propionylmalonyl-CoA at C21, which it is an unusual step of general PKS 1. As mentioned, if two methoxymalonyl CoA molecules are substituted for two malonyl-CoA molecules, they will take place in module 7 and 8 (C13 and C15), and fkbA enzyme will enforce this process. After the last step (module 10) of PKS 1, one molecule of L–pipecolic acid formed from L–lysine and catalyzed through fkbL enzyme synthesizes with the molecule from the module 10. The process of L-pipecolic acid synthesis is NRPS enforced by fkbP enzyme. After synthesizing the entire subunits, the molecule is cyclized. After the cyclization, the pre-tacrolimus molecule goes through the post-synthase tailoring steps such as oxidation and S-adenosyl methionine. Particularly fkbM enzyme is responsible of alcohol methylation targeting the alcohol of DHCHC starter unit (Carbon number 31 depicted in brown), and fkbD enzyme is responsible of C9 (depicted in green). After these tailoring steps, the tacrolimus molecule becomes biologically active.^[34]^[35]^[36]

References[edit]

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These papers does not mention eye issues:

2008 Sep;159(4):942-51. doi: 10.1111/j.1365-2133.2008.08747.x. Epub 2008 Jul 15.

A 4-year follow-up study of atopic dermatitis therapy with 0.1% tacrolimus ointment in children and adult patients.

Tacrolimus
Clinical data
Trade names	Prograf, Advagraf, Protopic, others
Other names	FK-506, fujimycin
AHFS/Drugs.com	Monograph
License data	EU EMA: by INN;
Pregnancy; category	AU: C; US: C (Risk not ruled out);
Routes of; administration	Topical, oral, iv
ATC code	D11AH01 (WHO); L04AD02 (WHO);
Legal status
Legal status	In general: ℞ (Prescription only);
Pharmacokinetic data
Bioavailability	24% (5–67%), less after eating food rich in fat
Protein binding	≥98.8%
Metabolism	Hepatic CYP3A4, CYP3A5
Elimination half-life	11.3 h for transplant patients (range 3.5–40.6 h)
Excretion	Mostly faecal
Identifiers
	IUPAC name[show]
CAS Number	104987-11-3 ;
PubChem CID	6473866;
DrugBank	DB00864 ;
ChemSpider	4976056 ;
UNII	Y5L2157C4J;
KEGG	DG00439 ;
ChEBI	CHEBI:61049;
ChEMBL	ChEMBL269732 ;
CompTox Dashboard (EPA)	DTXSID5046354 ;
ECHA InfoCard	100.155.367
Chemical and physical data
Formula	C44H69NO12
Molar mass	804.018 g/mol g·mol−1
3D model (JSmol)	Interactive image;
	SMILES[show]
	InChI[show]
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Reitamo S1, Rustin M, Harper J, Kalimo K, Rubins A, Cambazard F, Brenninkmeijer EE, Smith C, Berth-Jones J, Ruzicka T, Sharpe G, Taieb A; 0.1% Tacrolimus Ointment Long-term Follow-up Study Group.

Collaborators (46)

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Abstract

BACKGROUND:

For the treatment of a chronic disease like atopic dermatitis, sustained tolerability and efficacy of the applied medication are essential.

OBJECTIVES:

The present open-label, noncomparative study was conducted to obtain information on the long-term safety and efficacy of 0.1% tacrolimus ointment.

METHODS:

Patients aged 2 years or older with an affected body surface area of more than 5%, who previously participated in a clinical trial on tacrolimus ointment, were eligible for this study. The treatment area was defined by the investigator at study entry. Both children and adults applied continuously or intermittently 0.1% tacrolimus ointment twice daily during episodes of active disease plus an additional week after remission over a follow-up period of up to 4 years.

RESULTS:

The intent-to-treat population comprised 782 patients, with a median age of 22 years (range 2-72). Patients remained in the study for up to 4 years. Approximately half of the patients discontinued the study prematurely; the median follow-up was 1422 days. Median tacrolimus ointment use was 31.2 g during the first week; ointment use decreased during the first year and then remained stable for the remainder of the study. The median cumulative tacrolimus use was 271.5 g at month 6, 462.5 g at month 12, 739.9 g at month 24, 1029.3 g at month 36 and 1320.8 g at month 48. Altogether 51.8% of patients discontinued the study prematurely; the main reasons were withdrawal of consent (13.3%), loss to follow-up (11.3%) and lack of efficacy (9.4%). Adverse events led to study discontinuation in 3.7% of the patients. The most frequent application site events were skin burning and pruritus. These events were most often reported in adult patients during the initial treatment period; prevalence decreased after the first week and remained at a low level throughout the study. Nonapplication site events occurred with stable incidences throughout the study period. In general, calculated daily hazard rates did not indicate an increased risk of adverse events with prolonged treatment. The total affected body surface area decreased substantially upon onset of treatment and efficacy of treatment was maintained until the end of the study with smaller but continuous improvements throughout the follow-up period. Overall, 75% of the patients and 76% of the investigators rated their satisfaction with the treatment as excellent, very good or good at the end of the study or at the time of premature discontinuation.

CONCLUSIONS:

The safety profile of intermittent or continuous long-term application of 0.1% tacrolimus ointment for up to 4 years was consistent with that which has been established from shorter studies and gave no reason for concern. In addition, 0.1% tacrolimus ointment demonstrated sustained efficacy as reflected by the expression of high satisfaction with treatment by both patients and investigators.

Journal of the American Academy of Dermatology

Volume 53, Issue 2, Supplement 2, August 2005, Pages S186-S194

https://doi.org/10.1016/j.jaad.2005.04.062 Get rights and content

Objective

This study was designed to evaluate the long-term safety and efficacy of 0.1% tacrolimus ointment in adult and pediatric patients with atopic dermatitis (AD).

Methods

A total of 408 adult and 391 pediatric patients with AD who had participated in a previous clinical trial of tacrolimus ointment were enrolled in this long-term, open-label, noncomparative trial. Tacrolimus ointment 0.1% was applied twice daily either intermittently or continuously to the affected areas. Efficacy and safety assessments included percent body surface area affected, Eczema Area and Severity Index score, individual signs of AD, and the incidence of adverse events.

Results

A total of 799 patients were evaluated, of whom 300 (37.5%) were followed for more than 3 years (maximum 49 months). Improvements in efficacy parameters were observed within 1 week of treatment and continued for the duration of the study. Common adverse events included skin burning, pruritus, skin infection, skin erythema, flu-like symptoms, and headache. The incidence of adverse events, including cutaneous infections, did not increase with time on study.

Conclusion

Tacrolimus ointment therapy is a rapidly effective and safe treatment for the management of AD in pediatric and adult patients for up to 4 years.

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Abstract

Objective. This study was designed to compare the safety and efficacy of tacrolimus ointment 0.03% with vehicle ointment for the treatment of mild to moderate atopic dermatitis (AD) in pediatric patients.

Methods. A total of 317 patients (2–15 years of age) with mild to moderate AD were randomized to receive tacrolimus ointment or vehicle ointment twice daily in a 6-week, multicenter, double-blind study. Efficacy evaluations, including the Investigators’ Global Atopic Dermatitis Assessment, eczema area and severity index, percentage of total body surface area affected, and patient assessment of itch occurred at baseline, day 4, and weeks 2, 4, and 6. Cutaneous adverse events were recorded to evaluate safety.

Results. At the end of study, 50.6% (80 of 158) of the patients were treated successfully with tacrolimus ointment based on Investigators’ Global Atopic Dermatitis Assessment scores, a significant improvement compared with patients treated with vehicle ointment (25.8% [41 of 159]). The percent improvement from baseline in eczema area and severity index scores was also significantly greater in tacrolimus-treated patients (54.8%) compared with vehicle-treated patients (20.8%). There was also a significant improvement in the percentage of total body surface area affected of tacrolimus-treated patients (50.5% reduction from baseline) compared with vehicle-treated patients (16.4%). Patient itch scores were significantly lower in tacrolimus-treated patients (2.1) versus vehicle-treated patients (3.7). Overall, the incidence of cutaneous adverse events reported was similar for both treatment groups. There was no significant difference in the incidence of burning or stinging between treatment groups. Significantly fewer tacrolimus-treated patients prematurely discontinued from the study because of a cutaneous adverse event in the treatment area or experienced increased itching and erythema at the application site.

Conclusion. Monotherapy with tacrolimus ointment 0.03% is a safe and effective treatment alternative for pediatric patients with mild to moderate AD.

Arquivos Brasileiros de Oftalmologia

Print version ISSN 0004-2749On-line version ISSN 1678-2925

Arq. Bras. Oftalmol. vol.81 no.4 São Paulo July/Aug. 2018

https://doi.org/10.5935/0004-2749.20180059

ORIGINAL ARTICLES

Comparison of the efficacy of 0.03% tacrolimus eye drops diluted in olive oil and linseed oil for the treatment of keratoconjunctivitis sicca in dogs

Comparação do colírio tacrolimus 0,03% em óleo de oliva e linhaça no tratamento da ceratoconjuntivite seca em cães

Luís Felipe da Costa Zulim¹

Gisele Alborgetti Nai²

Rogério Giuffrida¹

Carolina Silva Guimarães Pereira¹

Hugo Benguella³

Aline Gutierrez Cruz³

Bruna Toledo Duran Foglia³

Aline da Silveira Batista⁴

Silvia Franco Andrade¹

^¹Animal Science Program, Universidade do Oeste Paulista, Presidente Prudente, SP, Brazil.

^²Department of Anatomy Pathology, Faculdade de Medicina, Universidade do Oeste Paulista, Presidente Prudente, SP, Brazil.

^³Faculty of Veterinary Medicine, Universidade do Oeste Paulista, Presidente Prudente, SP, Brazil.

^⁴Clinical Laboratory, Veterinary Hospital, Universidade do Oeste Paulista, Presidente Prudente, SP, Brazil.

ABSTRACT

Objective:

To compare the efficacy of 0.03% tacrolimus eye drops diluted in two different vehicles (linseed oil and olive oil) for the treatment of keratoconjunctivitis sicca (KCS) in dogs.

Methods:

This study included 60 dogs. Of this group, 20 were healthy and allocated to the control group, and 40 were diagnosed with bilateral KCS and randomly allocated to either the TO (tacrolimus in olive oil) or the TL (tacrolimus in linseed oil) groups. Ophthalmic examinations, Schirmer Tear Test-1 (STT-1), Tear Film Break-up Time (TBUT) and Fluorescein Test (FT) were carried out monthly, along with cytological and histopathological examinations at the beginning and end of the study.

Results:

The clinical signs, corneal ulcers, Schirmer Tear Test-1 values, and Tear Film Break-up Time values improved in both groups after one month of treatment. Cytological examination at the end of the study showed decreased lymphocytes, neutrophil, metaplastic, and squamous cell counts in both groups, while the histopathological analysis showed decreases in lymphocytes and neutrophils and an increase in goblet cell density (cells/mm²). The decreases in neutrophil count were more significant (p<0.05) in the TL group for both types of examination.

Conclusion:

In sum, 0.03% tacrolimus eye drops diluted in olive oil and linseed oil were effective in the treatment of keratoconjunctivitis sicca. None of the evaluated parameters differed significantly between the two groups, except for neutrophil count which was significantly lower in the TL group. Thus, linseed oil may be considered as an alternative diluent for tacrolimus eye drops.

Keywords: Keratoconjunctivitis sicca; Tacrolimus; Olive oil; Linseed oil; Ophthalmic solutions; Animals; Dogs

RESUMO

Objetivo:

Comparar a eficácia do tacrolimus 0,03% colírio, diluído em óleo de linhaça e óleo de oliva, no tratamento de ceratoconjuntivite seca em cães.

Métodos:

Foram utilizados 60 cães; 20 cães saudáveis como grupo controle, e 40 cães com diagnóstico de ceratoconjuntivite seca bilateral, distribuídos aleatoriamente em dois grupos: Tacrolimus em óleo de oliva (TO) e Tacrolimus em óleo de semente de linhaça (TL). Os animais foram avaliados mensalmente com exames oftálmicos, Teste lacrimal de Schirmer-1 (TLS-1), Tempo de ruptura do filme lacrimal (TRFL) e Teste de Fluoresceína (TF), e mensalmente com citologia conjuntival e com exame histopatológico no início e final do estudo.

Resultados:

Nos dois grupos de tratamento os sinais clínicos, Teste lacrimal de Schirmer-1, óleo de semente de linhaça e Tempo de ruptura do filme lacrimal apresentaram melhora após um mês de tratamento. E no final do estudo, na análise citológica, ambos apresentaram diminuição de linfócitos, neutrófilos, células metaplásicas e células escamosas, e na análise histopatológica houve diminuição de linfócitos, neutrófilos e o aumento de células caliciformes. No grupo óleo de semente de linhaça, a diminuição de neutrófilos foi mais significativa (p<0,05) em ambas análises.

Conclusão:

Em suma, tacrolimus 0,03% colírio diluído em óleo de oliva e óleo de linhaça foram eficientes no tratamento de ceratoconjuntivite seca. Nenhum dos parâmetros avaliados diferiu significativamente entre os dois grupos, exceto a contagem de neutrófilos, que foi significativamente menor no grupo TL. Assim, o óleo de linhaça pode ser considerado como um diluente alternativo para o colírio tacrolimus.

Descritores: Ceratoconjuntivite seca; Tacrolimus; Azeite de oliva; Óleo de semente do linho; Soluções oftálmicas; Animais; Cães

INTRODUCTION

Keratoconjunctivitis sicca (KCS) is a chronic, inflammatory ophthalmic disorder that commonly occurs in humans and dogs. It is characterized by a quantitative decrease in the aqueous layer of the tear film and/or a qualitative deficiency in the lipid or mucin layer that leads to a progressive inflammatory process primarily affecting the cornea, conjunctiva, and lacrimal glands. Its origin is usually immunomediated⁽¹^–³⁾.

Canine models are excellent for developing an understanding of this disease as the symptomatology in dogs is quite similar to that observed in humans. The clinical signs of canine KCS include conjunctival hyperemia, chemosis, blepharospasm, photophobia, mucoid and mucopurulent ocular secretion, corneal ulcer, vascularization and pigmentation of the cornea, and loss of vision⁽³^,⁴⁾. Certain canine breeds such as the English Bulldog, Lhasa Apso, and Cocker Spaniel are predisposed to KCS, particularly in females.

Topical therapy for KCS predominantly consists of immunosuppressants, including cyclosporine, tacrolimus, and pimecrolimus, used in association with lubricants. Anti-inflammatory agents, antibiotics and mucolytics may also be used if considered necessary⁽⁴^–¹²⁾. Previous studies have reported benefits associated with the adjunctive use of oral or topical omegas 3 (ω-3) and 6 (ω-6) for the treatment of KCS as they restore the lipid layer, decrease inflammation and apoptosis, and increase tear secretion⁽¹³^–²¹⁾.

Tacrolimus (FK506) is a macrolide antibiotic that is isolated from Streptomyces tsukubaensis. Its effects are similar to cyclosporine, and include a combination of local immunosuppression, goblet cell proliferation, suppression of lacrimal cell apoptosis, and anti-inflammatory action⁽⁹^–¹¹⁾. It is common for tacrolimus or cyclosporine eye drops to be diluted in olive oil or almond oil⁽⁹^,²⁰^–²²⁾, which are rich in essential fatty acids (EFAs), such as ω-3 and ω-6, that act as natural anti-inflammatory agents. Alpha-linolenic acid (ALA), an ω-3 fatty acid, and linoleic acid (LA), an ω-6 fatty acid, compete for metabolism by the enzyme D6-desaturase, and convert into omegas with anti-inflammatory properties. ALA gives rise to the ω-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), while LA gives rise to the ω-6 fatty acids dihomo-gamma-linolenic acid (DGLA), prostaglandin E1 (PGE1), and thromboxane (TXA1)⁽¹³^–¹⁶⁾.

Olive oil, derived from Olea europeae, contains large quantities of monounsaturated fatty acids (MUFAs), 70-80% oleic acids, 10-15% saturated fatty acids (palmitic acid), and a small quantity of polyunsaturated fatty acids (PUFAs) including approximately 5 to 10% of omegas 3, 6, and 9. It has anti-inflammatory, antinociceptive, immunomodulatory, and antimicrobial properties⁽²³^,²⁴⁾. A study comparing the treatment of experimentally induced KCS in rabbits using olive oil alone or in combination with cyclosporine reported that the former exhibited considerable ability to control the symptoms of the disease⁽²¹⁾.

Linseed oil is considered one of the most abundant sources of the EFAs, ω-3 and ω-6, with the ratio of ω-6 to ω-3 being close to ideal at 1:3⁽²⁵⁾. Oral administration of this oil has been recommended as an adjuvant therapy for Sjögren KCS syndrome in human beings⁽¹³^,¹⁴⁾. Moreover, use of linseed oil, both alone (orally, topically or a combination of the two) and in association with tacrolimus and cyclosporine, has also been reported to have improved the symptoms of experimentally induced KCS in rabbits⁽¹⁵^,¹⁹^–²¹⁾.

The objective of this study was to compare the efficacy of tacrolimus 0.03% eye drops diluted in olive oil (commonly used for this purpose) to those diluted in linseed oil, which has not been tested previously as a tacrolimus diluent for the treatment of KCS in dogs.

METHODS

Animals

The study was approved by the Ethics Committee on Animal Use (CEUA) of UNOESTE (protocol no. 1794), and it was undertaken in accordance with the rules of ARVO (Association for Research in Vision and Ophthalmology-Statement for the Use of Animals in Ophthalmic and Visual Research). The study included forty dogs [25 females (62.5%) and 15 males (37.5%)] with bilateral KCS that belonged to clients attending the university veterinary hospital. The mean age of the dogs was 6.7 ± 3.9 years, and the mean weight was 10.3 ± 7.7 kg. The inclusion criteria were presence of typical ophthalmic clinical signs (ocular discharge, conjunctivitis, corneal opacity, and pigmentation), Schirmer Tear Test-1 (STT-1) values that were <10 mm/min, and/or Tear Film Break-up Time (TBUT) values that were <10 sec. The negative control group consisted of twenty healthy mixed breed dogs [9 males (45%) and 11 females (55%)] selected from the university kennel. Their mean age was 3.5 ± 2.4 years and the mean weight was 10.3 ± 7.7 kg. Ophthalmic examination of the dogs included slit-lamp bio-microscopy (SL-15 Kowa, Japan), STT-1, TBUT, and cytological and conjunctival histopathological examination.

Groups

After diagnosis of KCS, the dogs were randomly divided into two groups, as follows: a) TO group (n=20): tacrolimus 0.03% eye drops diluted in olive oil (Ophthalmos Laboratory, São Paulo, Brazil), and b) TL group (n=20): tacrolimus 0.03% eye drops diluted in linseed oil (Ophthalmos Laboratory, Sao Paulo, Brazil). Double-blinding was maintained throughout the course of this study. Treatment in both groups consisted of application of the respective tacrolimus combinations twice daily in both eyes. Both groups also received 1 drop of propylene glycol-based lubricant (Systane^®, Alcon, São Paulo, Brazil) in both eyes twice daily for six months.

Antibiotic eye drops (1 drop 4 times per day for 15 days) were used when cultures of ocular samples secretions from the dogs demonstrated antimicrobial sensitivity and the presence of corneal ulcers or clinical signs such as conjunctivitis and mucopurulent ocular secretions were observed. Anti-inflammatory eye drops containing diclofenac sodium (Still^®, Allergan, São Paulo, Brazil) were only used in case of eye discomfort and ocular hyperemia without corneal ulcers [TO group: 70% (28/40), TL group: 57.5% (23/40)]. Melting ulcers (keratomalacia) were observed in 2.5% (1/40) of the TO group and 2.5% (1/40) of the TL group and were treated using anti-collagenase treatment with equine serum (1 drop 6 times per day for 15 days). The TO and TL combinations were formulated free of preservatives, stored at room temperature, and protected from the sun.

Ophthalmic examinations

Ophthalmic and cytological examinations were performed monthly, with the first day representing baseline (M0) and all subsequent treatments being considered as follow-up time-points (M1 to M6). Histopathological examinations were performed at the time of diagnosis (M0) and study completion (M6). Specific scores for all ophthalmologic clinical signs were assigned by the same examiner (LFCZ), as follows: conjunctivitis (0-none; 1-mild conjunctival hyperemia; 2-moderate to severe conjunctival hyperemia; 3-moderate to severe conjunctival hyperemia and chemosis); ocular discharge (0-none; 1-minor serous discharge; 2-moderate mucoid discharge; 3-marked mucopurulent discharge); corneal opacity (0-none/absent;1-nebula/minor, diffuse and hazy opacity with indistinct borders, less than 25% extension of the cornea; 2-macula/moderately dense opacity with circumscribed border, between 25% and 50% extension of the cornea; 3-leukoma/dense and white opacity, more than 50% extension of the cornea); and corneal pigmentation (0-none; 1-less than 25%; 2-between 25% to 50%; and 3-more than 50%). Measurements in the control group, carried out at M0, were considered to be the “normal” parameters for the ophthalmic examinations conducted in the case group.

The Schirmer Tear Test-1 (STT-1) (Teste de Schirmer^® – Ophthalmos Laboratory, São Paulo, Brazil), used to quantify the tears, was always performed at the same time (between 2 and 3 at pm) and without any anesthetic drops. A score of <10 mm/min was considered diagnostic for KCS ⁽²⁶⁾. The TBUT was used to qualitatively evaluate the tear film. After placing a drop of 1% fluorescein eye drops (Fluoresceína^®; Allergan, Sao Paulo, Brazil) in the lower fornix, a slit-lamp with a bright light setting and a cobalt blue filter was used to measure the time between the last blink and the first appearance of a dark spot on the cornea (formation of a dry area). TBUT values <10 seconds were considered diagnostic for KCS ⁽²⁷⁾.

The fluorescein test (FT) was performed using 1% fluorescein eye drops (Fluoresceína^®; Allergan, São Paulo, Brazil), and the presence or absence of corneal ulcers were evaluated with the help of slit-lamp bio-microscopy (SL-15, Kowa, Japan) using a cobalt blue filter⁽²⁶⁾. Scores were assigned based on the severity, extension, or depth of the ulcer (0-negative, 1- small superficial ulcer, 2- medium superficial ulcer, 3- extensive surface ulcer, 4-small stromal ulcer, 5-medium stromal ulcer, 6-extensive stromal ulcer, 7-descemetocele, and 8-keratomalacia).

Cytological and histological examinations

The cytological examinations were performed by the same person throughout the course of the study. The eye was cleaned with saline and anesthetic eye drops (0.5% proxymetacaine, Anestalcon^®; Allergan, São Paulo, Brazil) were then applied. Samples from the lower palpebral conjunctival cells were harvested using a sterile swab moistened with saline and a microscope glass slide. The samples were then fixed in methanol and stained using the MGG technique (May-Grunwald-Giemsa). Lymphocytes, neutrophils, metaplastic cells, and squamous cells were counted in 10 fields under an optical microscope at 40x objective.

The conjunctival biopsy was performed after application of anesthetic eye drops (0.5% proxymetacaine, Anestalcon^®, Allergan, Sao Paulo, Brazil). Specimens measuring 1-3 mm were harvested from the palpebral portion of the inferior medial conjunctival fornix using forceps and conjunctiva scissors (HR, São Paulo, Brazil). The histological section was placed on a fragment of paper (1×1 cm in size), fixe d in formalin, and then embedded in paraffin (Dinâmica Reagentes Analíticos, São Paulo, Brazil). A rotary microtome was used to create 5-mm sections of the specimen, and these were then stained using hematoxylin and eosin (HE) (Dolles, São Paulo, Brazil) and Periodic acid-Schiff stain (PAS) (Merck, USA). The HE stain was used to count the lymphocytes and neutrophils, while the PAS stain was used to measure the goblet cell density (cells/mm²). A Nikon Eclipse E200 (Tokyo, Japan) optical microscope at 40X objective was used for all cytological and histological cell counting, and the Leica ICC50HD (Wetzlar, Germany) was used for all light microscopy examinations.

Statistical analysis

The two-way analysis of variance (ANOVA) for paired samples with post-hoc Tukey’s test were used to analyze the STT-1 and TBUT variables, including goblet cell density and the number of squamous cells, metaplastic cells, lymphocytes, and neutrophils. With regard to the FT variables, the Friedman’s non-parametric test was used to compare various time points, while the Kruskal-Wallis test with post-hoc Dunn’s test was used to compare the variables between groups. Statistical significance was set at p<0.05, and all analyses were performed using the statistical package R, version 3.2.2. (The R Foundation for Statistical Computing, 2015).

RESULTS

The normal parameters, defined by the negative control group, are shown in table 1. Both treatment groups (TO and TL) exhibited improvements in the clinical signs (Figure 1), and no statistically significant differences (p>0.05) between the groups were observed. However, the findings showed significant differences (p<0.05) between M0 and the other time points. Complete remission of ocular discharge, conjunctivitis, and corneal opacity were observed in both treatment groups at M1, while the median for corneal pigmentation exhibited complete remission by M5.

Figure 1 Median scores of clinical signs observed over time (M0 to M6) in the TO (olive oil) and TL groups (linseed): (A) ocular discharge, (B) conjunctival hyperemia, (C) corneal opacity and (D) corneal pigmentation.

The STT-1 (Figure 2) showed that the TO and TL groups exhibited significant increases at M1 (TO: 18.2 ± 6.6, p=0.9; TL: 15.1 ± 5.5, p=0.5). Moreover, statistically significant differences were observed at M2 (p=0.002) and M3 (p=0.007), with the TO group exhibiting higher values compared to the TL group. Both groups demonstrated statistically significant differences (p<0.05) in STT-1 values between M0 and all other time points although overall the values were similar to those observed in the negative control group (Table 1). The TBUT (Figure 2) showed that the two groups exhibited significant increases at M1 (TO: 16.6 ± 4.3, p=0.48; TL: 15.8 ± 3.4, p=0.6) that continued until M6 (TO: 19.2 ± 3.6, p=0.27; TL: 17.9 ± 2.6, p=0.34), and the values were similar to those observed in the negative control group (Table 1). Although no differences (p>0.05) in TBUT values were observed between the groups, a significant difference was seen when comparing M0 with the other time points.

Table 1 Mean and standard deviation of the Schirmer Tear Test-1 (STT-1) values, Tear Film Break-up Time (TBUT) test values, and conjunctival cytology and histopathology parameters for healthy dogs in the negative control group (n=20)

Ophthalmic examinations
STT-1 (mm/min)	27.3 ±04.1
TBUT (seconds)	23.3 ±05.1
Conjunctival cytology
Lymphocytes*	0.1 ± 0.4
Neutrophils*	0 ± 0
Metaplastic cells*	0.5 ± 2.1
Squamous cells*	0.1 ± 0.3
Conjunctival histopathology
Lymphocytes*	26.7 ± 24.2
Neutrophils*	02.6 ±04.4
Goblet cells (cells/mm²)	28.3 ± 16.0

^*cell count in 10 fields, 40x objective.

¹Values ≤10 mm/min positive for KCS; ²Values ≤10 seconds positive for KCS; *p<0.05 (Tukey’s test to compare time points); ^a,bp<0.05 (Kruskal-Wallis test to compare groups); Two-way analysis of variance (ANOVA) for paired samples with post-hoc Tukey’s test was used to analyze the STT-1 and TBUT variables.

Figure 2 (A) Mean and standard deviation (from M0 to M6) of Schirmer Tear Test (STT-1)¹ values (mm/min) in the TO (tacrolimus diluted in olive oil) and TL groups (tacrolimus diluted in linseed oil); (B) Mean and standard deviation of the Tear Film Break-up Time (TBUT)² values (seconds) (from M0 to M6) in the TO and TL groups.

The FT showed that the ulcers observed in the two groups at M0 differed in terms of severity and extent [TO: 30% (12/40), TL: 42.5% (17/40)]. Excellent healing of all ulcers was observed in both groups at M1 [TO: 91.7% (11/12), TL: 94.1% (16/17)], except for keratomalacia [TO: 8.3% (1/12), TL: 5.9% (1/17)] which only completely resolved in both groups at M2. No statistically significant differences (p>0.05) were observed.

The cytological examination (Figure 3) showed decreases in all cell counts in both groups. No statistically significant differences were observed between the groups, except with regard to the neutrophils, which were significantly lower in the TO group compared to the TL group at time point M1 (p=0.036) and in the TL group compared to the TO group at time-point M6 (p=0.029). The histopathological examination (Figure 4) showed that both groups exhibited decreases in the inflammatory cell counts, with the neutrophil count (p=0.0016) in the TL group being significantly lower than that in the TO group at M6. An overall increase in the number of goblet cells (p=0.012) was observed in both groups, with no statistically significant differences between the two (p=0.6). However, significant differences were seen between time points M0 and M6 (TO: p=0.012, TL: p=0.005).

*p<0.05 (Tukey’s test to compare time points); ^a,bp<0.05 (Kruskal-Wallis test to compare groups).

Figure 3 (A) Mean and standard deviation of the lymphocyte count, observed upon conjunctival cytology examination (from M0 to M6) for the TO and TL groups; (B) Mean and standard deviation of the neutrophil counts (from M0 to M6); (C) Mean and standard deviation of the metaplastic cell counts (from M0 to M6); and (D) Mean and standard deviation of the squamous cell counts (from M0 to M6). Cell counts were performed in 10 fields under an optical microscope using a 40x objective.

*p<0.05 (Tukey’s test to compare time points); ^a,b p<0.05 (Kruskal-Wallis test to compare groups).

Figure 4 (A) Mean and standard deviation of the lymphocyte counts, as shown by conjunctival histopathological examination, at M0 and M6 in the TO and TL groups; (B) Mean and standard deviation of the neutrophil counts at M0 and M6; (C) Mean and standard deviation of the goblet cell counts at M0 and M6.

Therefore, the results showed that both groups exhibited improvements overall, with the observed values being close to the ‘normal’ parameters defined by the negative control group (Table 1). The aspects of some eyes and cytological and histopathological examinations from the control group, TO group and TL group are shown in figure 5.

Figure 5 (A) Control group: right eye; (B) Control group: conjunctival cytology showing presence of a squamous cell (arrow); (C) Control group: histopathological examination showing a large number of goblet cells (arrow); (D) TO group: M0, left eye, keratomalacia and corneal vascularization; (E) TO group: M6, ulcer healing; (F) TO group: conjunctival cytology at M0, presence of neutrophils seen (arrow); (G) TO group: conjunctival cytology, M6, presence of squamous cell (arrow); (H) TO group: histopathology at M0, absence of goblet cells; (I) TO group: histopathology at M6, large quantity of goblet cells seen (arrow); (J) TL group: M0, right eye, mucoid secretion and corneal pigmentation; (K) TL group: clinical evolution at M6; (L) TL group: conjunctival cytology at M0, presence of neutrophils (arrow); (M) TL group: conjunctival cytology at M6, presence of squamous cells (arrow); (N) TL group: histopathology at M0, absence of goblet cells; (O) TL group: histopathology at M6, large quantity of goblet cells (arrow). Cytology: MGG staining (May-Grunwald-Giemsa), 400x magnification; Histopathology: PAS staining, 400x magnification.

DISCUSSION

In this study, both treatment groups demonstrated improvements in the clinical signs of KCS, increased STT-1 and TBUT values, and resolution of corneal ulcers. These improvements could be a result of the proven effects of tacrolimus (local immunosuppression, goblet cell proliferation, suppression of lacrimal cell apoptosis, and anti-inflammatory action)⁽⁹^–¹¹⁾, as well as the possible additional effects of the oils added to the formula. The ω-3 and ω-6 fatty acids present in these oils inhibit the formation of pro-inflammatory eicosanoids and increase the formation of anti-inflammatory mediators such as EPA, DHA, prostaglandin E1 (PGE1), and TXA1⁽¹³^–¹⁸⁾.

Studies examining the treatment of experimentally induced KCS in rabbits using common topical immunosuppressants (cyclosporine and tacrolimus) diluted in vegetable oils (almond, olive oil, and linseed oil), as well as those focusing on the isolated use of these oils, reported that both the immunosuppressants and the oils exhibited efficacy in controlling the symptoms of KCS. This was particularly true with regard to the ω-3 and ω-6 fatty acids, which induced formation of anti-inflammatory mediators such as PGE1 A1 and thromboxane 1 (TXA 1)⁽¹⁹^–²¹⁾. Most of these studies reported better results with linseed oil compared to any of the other oils, including almond⁽²⁰⁾ and olive⁽²¹⁾, possibly due to its higher ω-3 and ω-6 content⁽¹⁹^–²⁵⁾.

Another study examining the treatment of experimentally induced KCS in rabbits using linseed oil through various routes (oral, topical, and oral and topical in combination) reported that it was effective in controlling the symptoms of the disease and increasing the goblet cell counts⁽¹⁹⁾. Although these experimental studies used rabbits in their KCS induction protocols with topical atropine combined or not with third eyelid gland removal, was different from our study, in which KCS was studied in dogs with a natural immune-mediated disease, all of these studies serve as a basis for better understanding this disease.

Beagle dogs treated with tacrolimus diluted in olive oil exhibited an improvement in clinical signs (secretion, pigmentation, and hyperemia). This did not differ significantly from the effects observed with another immunosuppressant, cyclosporine. A previous study also reported a significant increase in the STT-1 values at the first time-point, and this was similar to the findings of the current study⁽⁹⁾.

The STT-1 and TBUT values significantly increased over the study period, although no statistically significant differences between the TO and TL groups were observed. This was in agreement with another study that used 0.02% cyclosporine diluted in olive oil and linseed oil to treat experimentally induced KCS in rabbits⁽²¹⁾. Conversely, another study using 0.03% tacrolimus diluted in almond oil and linseed oil reported significant improvement only in the group with topical application of linseed oil⁽²⁰⁾. Significantly higher STT-1 values were also observed in another study using oral and topical linseed oil for the treatment of KCS in rabbits⁽¹⁹⁾.

In the current study, excellent resolution of corneal ulcers was observed in both groups (TO and TL), and there were no statistically significant differences between the two. This was in agreement with several other studies⁽²⁰^,²¹⁾, including one that reported resolution of keratomalacia (“melting” corneal ulcers) upon oral and topical administration of linseed oil for the treatment of KCS in rabbits⁽¹⁹⁾. The present study also included several other substances such as lubricants, antibiotics and equine serum for the treatment of ulcers, and this differed from the studies focusing on experimentally induced KCS in rabbits, which primarily used immunosuppressive agents and/or vegetable oils only.

The neutrophil count at the end of treatment was seen to differ significantly between the two groups in the current study, with numbers being considerably lower in the TL group compared to the TO group. A previous study also reported a decrease in the number of inflammatory cells following treatment of KCS in dogs using 2% topical cyclosporine⁽²⁸⁾. The literature suggests that the pathogenesis of KCS can be attributed to immune-mediated inflammation and destruction of the lacrimal glands, characterized by inflammatory infiltrates such as lymphocytes and, to a lesser degree, neutrophils⁽¹^,²^,²⁹⁾.

The present study showed a significant increase in the goblet cell density between the start and end of treatment, with no significant differences observed between the groups. This was in agreement with previous studies that also reported an increase in goblet cells following treatment using immunosuppressants⁽²⁰^,²¹⁾ or omega fatty acids⁽¹³^,¹⁹⁾.

In conclusion, the results of this study showed that 0.03% tacrolimus eye drops diluted in olive oil (TO) and linseed oil (TL) was effective for the treatment of KCS in dogs. There were no statistically significant differences between the two treatment groups, except with regard to the neutrophil count which was greater in the TL group. This suggested that linseed oil could be used as an alternative diluent for tacrolimus eye drops.

Funding: No specific financial support was available for this study.

³Approved by the following research ethics committee: Ethics Committee on the Use of Animals (CEUA) of UNOESTE (# 1794).

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Clinical treatment of dry eye using 0.03% tacrolimus eye drops.

Abstract

PURPOSE:

METHODS:

RESULTS:

CONCLUSIONS:

Side effects[edit]

By mouth or intravenous use[edit]

Carcinogenesis and mutagenesis[edit]

Topical use[edit]

Cancer risks[edit]

References

Tacrolimus Ointment for Refractory Posterior Blepharitis.

Abstract

PURPOSE:

METHODS:

RESULTS:

CONCLUSION:

Allergic contact dermatitis from tacrolimus.

Abstract

Tacrolimus

Contents

Medical uses[edit]

Organ transplantation[edit]

Ulcerative colitis[edit]

Skin[edit]

Contraindications and precautions[edit]

Topical use[edit]

Side effects[edit]

By mouth or intravenous use[edit]

Carcinogenesis and mutagenesis[edit]

Topical use[edit]

Cancer risks[edit]

Interactions[edit]

Pharmacology[edit]

Mechanism of action[edit]

Pharmacokinetics[edit]

Pharmacogenetics[edit]

History[edit]

Available forms[edit]

Biosynthesis[edit]

See also[edit]

References[edit]

External links

A 4-year follow-up study of atopic dermatitis therapy with 0.1% tacrolimus ointment in children and adult patients.

Abstract

BACKGROUND:

OBJECTIVES:

METHODS:

RESULTS:

CONCLUSIONS:

Efficacy and safety of tacrolimus ointment treatment for up to 4 years in patients with atopic dermatitis

Objective

Methods

Results

Conclusion

Tacrolimus Ointment 0.03% Is Safe and Effective for the Treatment of Mild to Moderate Atopic Dermatitis in Pediatric Patients: Results From a Randomized, Double-Blind, Vehicle-Controlled Study

Abstract

Print version ISSN 0004-2749On-line version ISSN 1678-2925

Arq. Bras. Oftalmol. vol.81 no.4 São Paulo July/Aug. 2018