Background/aims: Loss of corneal sensation results in the development of persistent corneal epithelial defects. The combination of a substance P-derived peptide (FGLM-amide) and an insulin-like growth factor-1 (IGF-1)-derived peptide (SSSR) stimulates rabbit corneal epithelial migration in vitro and rabbit corneal epithelial wound closure in vivo. The clinical efficacy of eye-drops containing FGLM-amide and SSSR for the treatment of persistent corneal epithelial defects in individuals with neurotrophic keratopathy was examined in a prospective open study.
Methods: Twenty-five consecutive patients (26 eyes) with persistent corneal epithelial defects associated with neurotrophic keratopathy were treated by administration of eye-drops containing FGLM-amide and SSSR. The course of epithelial healing was monitored by slit-lamp examination.
Results: Epithelial defects resurfaced completely in 19 of the 26 eyes (73%) within 4 weeks after treatment initiation. Complete resurfacing of epithelial defects was apparent in 18 of 22 (82%) or in one of four (25%) eyes without or with limbal stem cell deficiency, respectively. No adverse effects of treatment were observed in any subject.
Conclusion: Eye-drops containing FGLM-amide and SSSR induced the rapid resurfacing of persistent epithelial defects in stem cell-positive individuals with neurotrophic keratopathy.
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The corneal epithelium is a stratified squamous epithelium with a thickness of ∼50 μm that serves as a barrier to the external environment.1 The persistence of epithelial defects of the cornea often results in the development of corneal ulcers, potentially leading to perforation and loss of vision. Epithelialisation is a critical step of corneal wound healing. Corneal epithelial defects are resurfaced as a result of migration of the remaining epithelial cells, and they heal within 2–3 days in most individuals with normal corneal sensation and without conditions that might affect the healing process, such as diabetes mellitus, familial dysautonomia (Riley–Day syndrome), and recent neurosurgery or penetrating keratoplasty. However, patients whose corneal epithelial defects persist and are refractory to treatment are often encountered in clinical practice.2 3 Many such patients have a reduced corneal sensitivity or have a deficiency of the limbal stem cells that give rise to corneal epithelial cells. The corneal defects of individuals with such a stem cell deficiency are sometimes covered by migrating conjunctival cells, resulting in an irregular corneal surface and epithelial opacity.
The cornea is innervated by the ophthalmic branch of the fifth cranial (trigeminal) nerve as well as by sympathetic nerves. It contains a high density of sensory nerve endings and is the most sensitive tissue in the body.4 Corneal innervation is important for maintenance of the normal structure and function of the cornea. Loss of such innervation or corneal sensation thus often results in various types of corneal epithelial disorder, including superficial punctate keratitis, persistent epithelial defects, and corneal ulcer. Neurotrophic keratopathy is a degenerative disease of the cornea that results from corneal denervation. It is characterised clinically by reduced corneal sensitivity and impaired corneal epithelial wound healing, and its fundamental cause is injury to or dysfunction of the trigeminal nerve. The neurotransmitter substance P is present in the sensory nerve fibres of the corneal epithelium,5 and both substance P and its metabolites have been detected in tear fluid.6 The concentration of substance P in tear fluid is decreased for eyes affected by corneal hypesthesia.7
We have previously shown that substance P and insulin-like growth factor-1 (IGF-1) synergistically stimulate both corneal epithelial migration in an organ culture system of the rabbit cornea in vitro8 as well as corneal epithelial wound closure in rabbits in vivo.9 Substance P is a peptide of 11 amino acids and plays a role in a wide variety of physiological processes from miosis to neurogenic inflammation. To develop a substance P-based drug devoid of substantial adverse effects, we identified the minimal essential sequence of substance P required for synergism with IGF-1 in stimulation of corneal wound healing. We found that the peptide FGLM-amide, corresponding to the amino acid sequence at the carboxyl terminus of substance P, mimicked the function of the full-length molecule in organ culture.10 In contrast to substance P,11 this peptide did not induce miosis.10 We subsequently achieved the successful treatment of persistent epithelial defects of the cornea in patients with neurotrophic keratopathy by administration of eye-drops containing either substance P and IGF-112 or FGLM-amide and IGF-1.13 14 We also showed that the combination of FGLM-amide and IGF-1 promoted the regeneration of corneal nerve fibres.15 IGF-1 consists of 70 amino acids and exhibits various biological activities including the stimulation of cell proliferation and the inhibition of apoptosis. We recently found that the peptide SSSR, corresponding to a four-amino acid sequence in the C domain of IGF-1, is the minimal essential sequence for the synergistic stimulation with substance P of corneal epithelial migration.16–18
On the basis of our laboratory studies, we set out to develop a new mode of treatment for persistent epithelial defects associated with neurotrophic keratopathy with the use of eye-drops containing both the FGLM-amide and SSSR peptides. We have now evaluated the clinical efficacy of such eye-drops. We also examined the indication for this treatment with regard to the presence or absence of limbal stem cells.
SUBJECTS AND METHODS
FGLM-amide and SSSR were obtained from Protein Research Foundation (Osaka, Japan), and eye-drops containing FGLM-amide (1 mg/ml) and SSSR (0.05629 μg/ml, trifluoroacetate form) were prepared in phosphate-buffered saline and sterilised by ultrafiltration under sterile conditions in the Department of Pharmacy at Yamaguchi University Hospital. The eye-drops were administered one drop per eye four times a day for 28 days. The enrolment criteria for the study subjects included (1) the presence of persistent epithelial defects resistant to conventional treatments such as lubrication with artificial tears, wearing of an eye patch, or application of eye ointment; (2) the absence of signs of active infection; and (3) a reduced corneal sensation of <40 mm as measured with a Cochet–Bonnet aesthesiometer. We defined stem cell deficiency on the basis of the occurrence of conjunctivalisation of the cornea associated with Stevens–Johnson syndrome, ocular pemphigoid, graft-versus-host disease, alkali burns, trachoma, radiation keratopathy or toxic keratopathy.
Twenty-five individuals (17 men, eight women), with a mean (SD) age of 65.7 (16.2) years (range 28–84 years), were enrolled in the study between January 2004 and March 2007 (table 1). In one female patient (case nos 14 and 16), epithelial defects were apparent in both eyes. Thirteen eyes manifested hypolacrimation (<10 mm per 5 min), and 13 eyes showed normal tear secretion in the Schirmer test; the mean value for all eyes was 14.7 (13.4) mm per 5 min. The mean duration of epithelial defects treated with previous therapies was 3.2 (4.3) months (range, 2 days to 15 months), and the mean corneal sensation was 4.8 (9.2) mm (Cochet–Bonnet aesthesiometer). The cornea was examined with a slit-lamp microscope and fluorescein staining before as well as 1, 2, 3, 5, 7, 14 and 28 days after treatment initiation. The period required for complete healing was recorded. We also evaluated patients for possible adverse effects of treatment such as ocular pain, hyperaemia of the eye, intraocular inflammation and increased ocular discharge. The mean follow-up time was 476 (297) days (range 63–1246 days). Eyes were classified as responders or non-responders on the basis of whether the epithelial defect had completely resurfaced or not within 28 days from the initiation of treatment.
Data are presented as means (SD) and were analysed by the paired t test or Fisher exact test. A p value of <0.05 was considered statistically significant.
Of the 26 eyes with persistent epithelial defects, 19 eyes (73%) responded to treatment with eye-drops containing the peptides FGLM-amide and SSSR, with complete epithelial resurfacing being achieved within 4 weeks. However, seven eyes (27%) did not achieve complete epithelial resurfacing within 4 weeks (table 1). For the 19 responder eyes, complete epithelial resurfacing was achieved in 10.5 (8.1) days, with a median of 7 days and range of 1–25 days.
Among the 22 eyes positive for limbal stem cells, 18 eyes (82%) were responders, and four eyes (18%) were non-responders. Among the four eyes negative for stem cells, however, one eye (25%) was a responder, and three eyes (75%) were non-responders (table 1). This difference in the response to treatment between eyes with or without stem cells was statistically significant (p = 0.047, Fisher exact test).
Visual acuity improved by >2 lines in 17 of the total of 26 eyes (65%), worsened as a result of thinning of the cornea and after cataract in two eyes (8%), and remained unchanged (change of <1 line) in seven eyes (27%) (fig 1).
Corneal sensation showed a significant increase after treatment in all subjects as well as in responders (table 2). In contrast, tear secretion did not differ significantly between before and after treatment in all subjects or in responders (table 3). None of the study subjects manifested any ophthalmic or systemic adverse effects during or after treatment.
Slit-lamp images of representative cases are shown in figs 2–6. A persistent epithelial defect due to herpes infection was observed in a 61-year-old woman (case no. 12); the duration of the defect was 2 months, but it had disappeared within 21 days of treatment with eye-drops containing FGLM-amide and SSSR (fig 2). A 79-year-old man (case no. 13) underwent penetrating keratoplasty because of infectious corneal ulcer, and a corneal epithelial defect appeared ∼6 months after the surgery; the duration of the defect was 1 month, but it had decreased markedly in size by 1 day after treatment initiation and had disappeared within 6 days (fig 3). A 78-year-old woman (case no. 19) underwent vitrectomy, and a corneal epithelial defect appeared ∼5 days after the surgery; the duration of the defect was 2.5 months, but it had disappeared within 7 days after treatment initiation (fig 4). A persistent epithelial defect developed after corneal infection in a 79-year-old woman (case no. 26); the duration of the defect was 1.2 months, but it had disappeared within 25 days after treatment initiation (fig 5). Finally, a persistent epithelial defect due to ocular pemphigoid was observed in a 78-year-old man (case no. 23); the duration of the defect was 5 months, and it was still apparent at 25 days after treatment initiation (fig 6).
We have shown here that eye-drops containing the substance P-derived peptide FGLM-amide and the IGF-1-derived peptide SSSR are clinically effective for the resurfacing of epithelial defects in eyes with a low corneal sensation and without limbal stem-cell deficiency. Three-quarters of the treated eyes responded, as defined by complete resurfacing of the corneal epithelial defects, within an average of 10.5 days. The mean duration of the epithelial defects in all eyes was 3.2 months before experimental treatment onset. Furthermore, we did not observe any adverse effects of the treatment regimen in any of the study subjects.
Whereas substance P induces miosis, we previously showed that FGLM-amide does not have such an effect in rabbits.10 Substance P is also readily degraded and inactivated by neuropeptidases such as carboxypeptidases and endopeptidases, whereas FGLM-amide is likely more stable. Furthermore, whereas injection of IGF-1 into the rabbit corneal stroma induced neovascularisation, injection of SSSR did not.18 Small and stable peptides derived from substance P and IGF-1 that retain the desired biological effect are thus preferable to the intact molecules for clinical use. Our present data show that such peptides are both safe and clinically efficacious.
Substance P is the primary neurotransmitter of sensory nerves in the cornea, which is the most sensitive tissue in the body. The concentration of substance P and its degradation products is decreased in tear fluid of patients with persistent corneal epithelial defects.6 These observations are thus consistent with our present clinical finding that the combination of FGLM-amide and SSSR promotes the resurfacing of such defects in eyes with a low corneal sensation. The effectiveness of this treatment might thus be due in part to the supplementation of endogenous substance P with FGLM-amide.
Corneal epithelial wound healing also depends on the presence of limbal stem cells. Patients with limbal stem-cell deficiency, such as those with Stevens–Johnson syndrome, ocular pemphigoid, graft-versus-host disease, alkali burns, trachoma, radiation keratopathy or toxic keratopathy,19 thus manifest a delay in corneal epithelial resurfacing as well as migration of conjunctival epithelial cells over the cornea. The presence of limbal stem cells is important for the successful treatment of persistent epithelial defects, regardless of whether or not such treatment involves agents that stimulate the migration of corneal epithelial cells. Indeed, we have now shown that eye-drops containing FGLM-amide plus SSSR did not manifest clinical efficacy in most cases of persistent epithelial defects associated with a deficiency of limbal stem cells.
Conventional treatments for persistent corneal epithelial defects include ocular lubricants, soft contact lenses and tarsorrhaphy. However, some cases, especially those associated with a reduced corneal sensation, are refractory to such conventional approaches. Eye-drops containing nerve growth factor (NGF)20 or autologous serum21 have been shown to be effective for treatment of corneal epithelial disorders. The local application of NGF was thus clinically efficacious for treatment of neurotrophic keratopathy. This treatment regimen is based on the trophic and regenerative effects of NGF on sensory nerves. Whereas NGF might stimulate the regeneration of nerve fibres in the cornea, the combination of the substance P-derived peptide (FGLM-amide) and SSSR likely targets corneal epithelial cells directly, bypassing nerve fibres and triggering epithelial migration through activation of intracellular signal transduction systems. This effect of substance P is mediated by the NK-1 subtype of tachykinin receptor.22 The promotion of corneal epithelial migration by substance P and IGF-1 appears to be mediated, at least in part, by activation of the integrin, focal adhesion kinase, and paxillin system, which is responsible for cell adhesion to the fibronectin matrix.23–25 With the use of a confocal biomicroscope, we also observed regeneration of corneal nerve fibres in a patient with neurotrophic keratopathy treated with FGLM-amide plus IGF-1.15
Confirmation of the therapeutic efficacy of eye-drops containing the two peptides FGLM-amide and SSSR for persistent corneal epithelial defects associated with neurotrophic keratopathy will require double-blinded multicentre clinical trials. Trials may also be warranted to compare the efficacy of this treatment regimen with that of eye-drops containing NGF.
We thank Makoto Asashima and Satoshi Ebina of the ICORP Organ Regeneration Project, Japan Science and Technology Agency, for helpful discussion on this manuscript.
Competing interests: None.
Ethics approval: The open non-randomised clinical study was approved by the Institutional Review Board of Yamaguchi University Hospital.
Patient consent: Written informed consent was obtained from all subjects.
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