New developments continue to drive use of corneal collagen cross-linking

François Malecaze, MD, said corneal cross-linking is now an accepted practice for halting progression of corneal ectasia. Image: Malecaze F

Thirteen years after the first corneal collagen cross-linking procedure was performed, today it is a well-established practice in Europe, with advancements under way.

“If some years ago there were still people who were skeptical about the procedure, the consensus is now quite unanimous. Multicenter studies have brought evidence that cross-linking halts the progression of corneal ectasia, with results that are not conflicting. There is no controversy now, and also safety is no longer a concern,” François Malecaze, MD, said.

In a study of 500 eyes with a follow-up of at least 5 years, Paolo Vinciguerra, MD, proved that the beneficial effects of corneal cross-linking (CXL) not only are maintained, but progress over time.

“Results are beyond expectations, because not only do we have stabilization of the disease, but also a progressive flattening of the ectasia and remarkable subjective visual improvement, more than we can see on topography,” he said.

“I was initially skeptical because of the lack of measurable evidence in the early days. But I was intrigued by the procedure and went to the root of it with basic science research. In addition, more organized trials, not least the U.S. trial, are gathering a high amount of scientific data that confirm the efficacy of cross-linking,” John Marshall, MD, PhD, said.

There are now further developments in cross-linking that should broaden indications, reduce times and costs, and minimize stress and tissue trauma induced by the current procedure. One advance that is gaining much interest is the transepithelial approach, which is already used in most clinical settings and is under scrutiny for efficacy and future potential.

Crossing the epithelial barrier

Current cross-linking protocols, including the Dresden protocol, entail scraping off the epithelium before the procedure. This is performed because the corneal epithelium constitutes a barrier against penetration of molecules with a molar mass of more than 100 g/mol, Dr. Malecaze explained. Riboflavin has a molar mass of 376 g/mol and therefore cannot penetrate the tight junctions of the epithelium.

“However, disepithelialization carries potential complications such as scar tissue formation, ulceration and infection,” he said.

Several laboratories are currently working on a riboflavin formula with modified pharmacokinetics to enhance penetration and allow epithelium-on cross-linking. The new Ricrolin TE (Sooft Italia), currently marketed in Europe, has added to the conventional Ricrolin formula (riboflavin 0.1% and destrane 20%) the amino alcohol TRIS (trometamol) and the EDTA disodium salt, which, in combination, enable transepithelial penetration.

“We tested this new solution within the Cross-Linking Evolution Study at the University of Siena, Italy, in 2009. We now use it in about 20% of our cross-linking procedures,” Aldo Caporossi, MD, said.

In addition to avoiding potential complications related to epithelium removal, an epithelium-on procedure results in better comfort for the patient, with no pain and a faster visual recovery. There is less time involved and the possibility to perform cross-linking outside the operating room, he explained.

The procedure is similar to the conventional method, Dr. Malecaze said. One drop of pilocarpine and one drop of anesthetic are instilled 30 minutes before irradiation. Immediately after, one drop of transepithelial riboflavin is instilled every 2 minutes, for a total of at least 16 drops in half an hour. The eye is then exposed to ultraviolet light for 30 minutes, during which transepithelial riboflavin is administered every 5 minutes for a total of five additional drops.

Shallower diffusion

The problem with the transepithelial procedure, despite the new riboflavin formula, is penetration. All studies so far suggest that cross-linking occurs but only within the first 100 µm of the corneal stroma, which is one-third of the depth of the epi-off procedure.

“By confocal microscopy studies, we were able to see that transepithelial cross-linking is equally safe, as no damage is induced to the corneal endothelium, crystalline lens and macula. However, keratocyte apoptosis was limited to 100 µm of the anterior stroma,” Dr. Caporossi said.

Keratocyte apoptosis is a key step in the treatment, the spark that triggers the cascade of events leading to the cross-linking of collagen fibers, Rita Mencucci, MD, said.

“If there is apoptosis also with the epithelium on, it means that we have crossed the barrier and that we are inducing the process, though not as deep as in the conventional procedure,” she said.

In a study, Dr. Mencucci and colleagues at the University of Florence, Italy, evaluated the effects of ex vivo histological and immunohistochemical methods on transepithelial cross-linking in corneas undergoing transplantation for keratoconus.

“We performed transepithelial cross-linking 2 hours before PK and in some patients 3 months before PK. Normal corneal buttons from healthy donors were used as control. All corneal structures (epithelium, stroma, endothelium and the nerve fibers of the subepithelial plexus), after an initial alteration, regained integrity within 3 months,” she said.

“Keratocyte apoptosis was detected by TUNEL positivity after the treatment at a variable depth, however more superficial than traditional cross-linking. At 3 months, an almost normal expression of CD34 was visible in the keratocytes of the whole stroma, confirming the wellness of this structure. The collagen fibers appeared nicely interwoven, with a pattern that was similar, though not identical, to that of normal corneas,” Dr. Mencucci said.

Researchers at the University of Toulouse, France, were actively involved in assessing whether transepithelial riboflavin guarantees sufficient stromal diffusion for effective UV light penetration and collagen cross-linking.

“We performed an experimental animal study in 40 eyes, half of whom were treated with the conventional epi-off and Ricrolin and half with epi-on technique and Ricrolin TE. High-performance liquid chromatography analysis showed that Ricrolin TE does cross the epithelial barrier, but stromal impregnation is about one-fifth compared to the conventional epi-off technique,” Dr. Malecaze said.

“The next question now is whether this amount of transepithelial diffusion is sufficient to induce cross-linking. We don’t have an answer yet,” he said.

Anterior chamber optical coherence tomography images with the Visante (Carl Zeiss Meditec) after cross-linking in the two groups showed that the demarcation line between cross-linked and non-cross-linked cornea is significantly deeper in eyes treated with the conventional technique.

Epi-on indications

While there is a general consensus that transepithelial cross-linking needs further improvement but is worth pursuing, not all surgeons agree on current usage and indications.

According to David Touboul, MD, until sufficient evidence of efficacy is demonstrated by clinical studies, this approach is not recommended.

“At the University of Bordeaux, we did about 50 cases. Then we stopped because the morphological changes in topography and refraction were frankly poor,” Dr. Touboul said. “The effect is pretty low compared to conventional cross-linking. In very thin corneas it might be better than nothing, but we might as well use hypotonic riboflavin solution to swell the cornea.”

Dr. Mencucci does conventional epi-off cross-linking in routine cases but believes the transepithelial approach could be used in specific cases, such as ultra-thin corneas with progressive keratoconus.

“This technique allows us to maintain the cut-off value of 400 µm in corneas where we would not be able to use the conventional treatment. A weapon of this kind is very important,” she said.

Children and patients with poor compliance, including Down syndrome patients, are also good candidates, in her opinion.

According to Dr. Vinciguerra, thin corneas are not an indication, as they are often correlated with aggressive forms of the disease.

Patients with moderate keratoconus that is still progressing after the age of 30 years are the best candidates and benefit greatly from the treatment, he said.

In a study, Dr. Caporossi evaluated the clinical outcomes of transepithelial cross-linking in 20 patients between the ages of 15 years and 26 years with keratoconus stage 1 and 2.

Aldo Caporossi

“At 6 months, we found a slight improvement of +0.06 Snellen lines, which could be correlated with the reduction of coma aberration produced by the impact of the treatment on the anterior stroma. Mean apical keratometry was reduced by –1.58 D. However, asymmetry indexes increased and corneal pachymetry decreased. Though these changes were not statistically significant, some doubts remain about instability of the posterior surface of the cornea, where keratoconus most likely originates, particularly in younger patients with a more progressive disease,” he said.

Based on its studies and wide experience, the Siena group has drawn indications for transepithelial cross-linking. Absolute indications are progressive keratoconus or ectasia after LASIK in corneas with the thinnest point less than 400 µm as well as mildly progressive keratoconus in patients older than 26 years, independent of corneal thickness. Relative indications are nonprogressive or mildly progressive keratoconus in patients with poor compliance regarding contact lenses, independent of corneal thickness, as well as nonprogressive ectasia after LASIK.

Pediatric cases were excluded from the list of indications.

“We treat a lot of children with cross-linking, but since they need a stable, long-term effect, we don’t believe that the transepithelial approach is suitable,” Dr. Caporossi said.

According to Dr. Vinciguerra, iontophoresis may be the answer. This technique is used for transdermal delivery of drugs, and with modifications, it could be used to bypass the barriers that prevent riboflavin penetration into the intact cornea. Experiments have already been carried out by Dr. Vinciguerra’s group in Milan, Italy.

Accelerated cross-linking

Shortening UV light exposure is another future goal of corneal cross-linking. The preliminary, experimental results of a rapid method of cross-linking, called flash-linking, were published by Rocha and colleagues in 2008. This new method uses a customized photoactive cross-linking agent that, following UVA irradiation, induces an oxygen-independent, non-thermal photochemical, not photosensitizing, reaction. Cross-linking is achieved after a short exposure of only 30 seconds of UVA light exposure, as compared with 30 minutes. So far, only ex vivo experiments have been performed, and animal and human studies will be necessary to evaluate the safety and efficacy of this mechanism.

Other methods to achieve accelerated cross-linking are currently under evaluation, but no studies have been published to date. The Avedro KXL system is based on the assumption, proved in several experiments, that higher powers delivered over shorter time periods can provide the same corneal strengthening as lower power over longer time periods.

“The same total amount of UVA light energy (5.4 J/cm²) is delivered, with a high irradiance of 30 mW/cm² for 3 minutes as compared to 3 mW/cm² for 30 minutes,” Dr. Touboul said.

The safety of short-term, high-UVA irradiance has been proven in animal studies by Werner and colleagues and in cultivated human cells by Dr. Marshall. Avedro is currently in the process of recruiting patients for a phase 3 clinical trial involving 16 centers in the United States to evaluate the safety and efficacy of the 3-minute cross-linking procedure in eyes with keratoconus.

“We had the chance to try the procedure in our department 6 months ago. We only applied it to eight eyes, but results were quite interesting and we would like to purchase the machine in the near future. Only 13 minutes are needed to carry out the entire procedure, from riboflavin administration to the end. Conventional CXL takes a full hour,” Dr. Touboul said.

Dr. Marshall, Frost Professor of Ophthalmology at University College London, is currently working on advances of ultra-fast technology. One of them is a modified Scheimpflug camera system, which can monitor the migration of riboflavin and the cross-linking activity within the corneal tissue.

“We can almost tailor the cross-linking procedure, adjusting the treatment to individual eyes based on feedback information. Accuracy and safety of the treatment can be greatly improved,” he said.

The extensive studies he has conducted on corneal biomechanics suggested that there is no point in going farther than 150 µm depth with cross-linking treatment because the intrinsic strength of the cornea is within the anterior at 150 µm.

“If you go farther back there is a possibility of involving the endothelium. With our system there is no risk of endothelial damage,” he said.

Another innovation Dr. Marshall is working on is a topography-guided cross-linking system with an optimized beam profile for customized treatment.

“We have the ability to cross-link specific areas, tailoring the dose of UV light activation on the z plane (depth) as well as on the x and y planes,” he said. “Topography-guided cross-linking will enable us to cross-link specific areas.”

Dr. Marshall is currently treating his patients with the ultra-fast technique and is happy with the results.

Dr. Malecaze has treated five patients with the same procedure, adjusting the machine setting to deliver UVA over 10 minutes rather than 3 minutes.

“I think 3 minutes is too short. We have a tight margin of error, where 1 minute of accidental additional exposure would cause damage, while 11 minutes instead of 10 would not. We need control, and 10 minutes seems a reasonable time even for the most impatient of surgeons,” he said.

Further studies are necessary before accelerated protocols can be introduced into routine clinical practice.

Assessing results

There are two ways of assessing the effects of cross-linking in the cornea: the demarcation line and biomechanical measurements, Dr. Vinciguerra said.

“Curvature changes, visual acuity and topographic changes are secondary effects of what we primarily want to induce, which is a biomechanical effect of increased resistance. The only way we can truly evaluate the efficacy of CXL is by measuring the biomechanical changes, with normal corneas as a benchmark, and the depth of these changes,” he said.

Research on new methods of measuring biomechanical results is ongoing. The Ocular Response Analyzer (ORA, Reichert) with new 3.01 software enables users, through a complex analysis of morphological parameters such as height, width and curvature gradient, to determine keratoconus severity and, eventually, post-treatment changes.

“Data processing results in two new indexes: the keratoconus match index and the keratoconus match probability. Using the indexes of normal corneas as a benchmark, individual eyes are classified as normal, keratoconus suspect, mild, moderate and severe keratoconus,” Dr. Touboul said.

The novel Corvis ST (corneal visualization Scheimpflug technology) by Oculus is likely to rapidly become the gold standard in assessing corneal properties, according to Dr. Vinciguerra.

Corvis ST is a non-contact tonometer that provides real-time, viewable measurement of corneal biomechanics. A high-speed Scheimpflug camera records the movements of the cornea while it is exposed to an air impulse, delivering more than 4,000 images per second.

The instrument has recently become available in Europe. Dr. Vinciguerra has been using it for more than 1 year to study the progression of cross-linking effects in his patients. He said that the changes induced by the cross-linking treatment in keratoconus eyes were visible and measurable immediately after the treatment, and that further improvements were observed in the following months.

Elastography is another emerging technology for ultrasound assessment of tissue elasticity. Dr. Touboul said two types of elastography are currently under investigation for corneal assessment. OCT elastography provides dynamic micro-scale information on the corneal response to mechanical pressure stimuli. Transient elastography, introduced in medicine for breast palpation, has been modified for ophthalmic use by Dr. Touboul.

The radiation force of a non-contact ultrasonic focalized beam is used to exert pressure on the cornea, generating an elastic sheer wave that propagates through the underlying tissue. Pulse-echo ultrasound imaging is used to follow the propagation, measure its speed and draw a propagation diagram.

A clinical trial will start in Bordeaux, France, in 2012 to demonstrate that collagen cross-linking increases corneal stiffness, leading to an approximately 50% increase of sheer wave speed.

“To our knowledge, sheer wave speed changes occur only with the epithelium-off technique, and this will also be investigated in our study,” Dr. Touboul said.

As science progresses, corneal cross-linking will increasingly become routine practice for an even larger number of patients, reducing the number of corneal transplantations to a small minority of cases.

“Taking into account that 90% of corneal transplantations are for keratoconus, if we systematically treat the disease early enough to stop disease progression, our volume of transplantation surgery will be extremely low in the future,” Dr. Vinciguerra said. – by Michela Cimberle

What is the best solution to improve the visual outcomes of cross-linking?

References:

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• Aldo Caporossi, MD, can be reached at Policlinico Le Scotte, V.le Bracci, 53100 Siena, Italy; +39-0577-233356; fax: +39-0577-233358; email: caporossi@unisi.it.
• François Malecaze, MD, can be reached at CHU de Toulouse-Hôpital Purpan, Place du Docteur Baylac-TSA 40031, 31059 Toulouse Cedex 9, France; email: malecaze.fr@chu-toulouse.fr.
• John Marshall, MD, can be reached at UCL Institute of Ophthalmology, 11-43 Bath St., London EC1V 9EL, UK; +44-20-7608-4033; email: eye.marshall@googlemail.com.
• Rita Mencucci, MD, can be reached at Università degli Studi di Firenze, Clinica Oculistica II, Viale Morgagni 85, 50134 Firenze, Italy; +39-055-411-765; fax: +39-055-437-7749; email: rita.mencucci@unifi.it.
• David Touboul, MD, can be reached at CHU de Bordeaux, site Pellegrin, centre Xavier François Michelet, place Amélie Raba-Léon Bordeaux, 33000 Bordeaux, France; email: toubould@gmail.com.
• Paolo Vinciguerra, MD, can be reached at the Istituto Clinico Humanitas, Milan, Italy; +39-02-55211388; fax: +39-02-57410355; email: paolo.vinciguerra@humanitas.it.
• Disclosures: Drs. Caporossi, Malecaze, Mencucci and Touboul have no relevant financial disclosures. Dr. Vinciguerra is a consultant for Oculus and Nidek. Dr. Marshall is principle scientific adviser for Avedro.