Initial Studies Using Aliphatic β-Nitro Alcohols for Therapeutic Corneal Cross-Linking

Keratoconus has an estimated incidence from 1 in 435 to 2000 in the general population. In its classical form, keratoconus begins at puberty and progresses to the third to fourth decade of life.1 Thus, its overall impact is magnified by virtue of the younger population affected. Although the underlying etiology of keratoconus remains unclear, collagen interlamellar or intralamellar slippage is likely to play a role2 in the ultimate loss of biomechanical strength that occurs.3 Clinically, the disease is marked by progressive thinning of the corneal stroma with resultant bulging and distortion of the thinned, weakened areas. The bulging, distorted cornea creates an optically imperfect surface to the eye that produces increasingly irregular astigmatism and myopia. Current treatment options either mask the surface irregularity with a variety of contact lenses or attempt to improve the surface contour with intracorneal ring segments, lamellar keratoplasty, or excimer laser surgery.4 Corneal collagen cross-linking has recently been introduced as an effective, minimally invasive means to treat progressive keratoconus. The seminal works of Seiler and his coauthors, Spoerl et al.5 and Wollensak et al.,6 in the past decade have outlined the usefulness of photochemical cross-linking with UVA irradiation (λmax = 370 nm) with riboflavin as a photosensitizer (UVAR). This process is described by clinicians as CXL, an abbreviation for collagen cross-linking. Clinical trials are ongoing in Europe7-9 with encouraging progress reports. For example, after 5 years in the Dresden study, participants who have undergone this treatment protocol have shown no progression of keratoconus. With these exciting results, corneal cross-linking therapy has been extended to include patients with related disorders such as postsurgical ectasia, corneal edema, and corneal melting.10 Despite these successes, UVAR therapy poses attendant risks, particularly related to ultraviolet irradiation. As such, this therapy has only recently been granted approval by the Food and Drug Administration for the start of clinical trials in the United States. Because free oxygen radical formation occurs with riboflavin photolysis,11 this cross-linking method has a negative impact on cell viability. Indeed, keratocyte12 and corneal endothelial cell toxicity13 do occur with application of this therapy to the cornea. In lieu of such toxicity, it has been recommended that patients with particularly thin (<400 μm) central corneas not undergo this therapy because the depth of UVA penetration potentially exposes the endothelial cells to toxic photochemical damage. That said, several groups have recently promoted the use of hypo-osmotic riboflavin drops as a means of temporarily swelling the cornea before UVA irradiation. In this way, it may be possible to protect the underlying corneal endothelium during cross-linking of thin (<400a μm) corneas. The possibility of using a topical self-administered compound to produce a comparable degree of collagen cross-linking to UVAR therapy has several potential benefits. First, if no UVA irradiation is necessary, this could lessen the degree of cytotoxicity, allowing ophthalmologists to offer routine cross-linking treatment to patients regardless of corneal thickness (<400 μm). Second, patients would experience less discomfort because epithelial debridement would not be necessary (as it is with UVAR therapy). Third, because the compound could perhaps be self-administered, patients would benefit from the ease of application and the reduced costs. Fourth, dose modulation could supply an effect of controlled magnitude rather that the single effect currently produced with the UVAR procedure. Fifth, more complete cross-linking may be possible, particularly if water-soluble cross-linking compounds that can diffuse easily through the corneal stroma are used. The UVAR method cross-links only the anterior 200 μm of cornea, which correlates with the depth of penetration of UVA irradiation into the riboflavin-soaked cornea.14 Finally, a pharmacologic approach to corneal cross-linking could allow for treatment of the peripheral cornea in processes such as pellucid marginal degeneration. Cross-linking of the limbal region with the UVAR approach is prohibited because of the risk for damage to limbal stem cells. In their early studies, Spoerl,5 Seiler,15 and Wollensak16 explored the possibility of using chemical cross-linking agents for corneal stabilization. They include some well-known cross-linking agents such as glutaraldehyde, formaldehyde, glyceraldehyde, ribose, and glucose.5,15,16 However, this line of investigation appears to have been largely abandoned because of concerns regarding compound toxicity and efficacy. Glyceraldehyde was singled out as a compound that could potentially provide efficacious cross-linking with low toxicity. Indeed, a recent study describes the use of glyceraldehyde for scleral cross-linking administered through retrobulbar injection in live rabbits.17 Our studies indicate that short-chain aliphatic β-nitro alcohols (Fig. 1) may be useful for in vivo tissue cross-linking. Shifts in thermal shrinkage temperature, a general index of nonenzymatic collagen cross-linking, were observed in studies using porcine and human sclera as a collagenous tissue substrate.18 The present study is an extension of those previous studies. Herein, we examine the changes in thermal shrinkage temperature effects of corneal tissue induced by these aliphatic β-nitro alcohols and characterize the effects on light transmission. The results indicate that under conditions of physiologic pH and temperature, corneal tissue cross-linking is feasible and has negligible effects on light transmission. Figure 1 Structures of the short-chain aliphatic β-nitro alcohols (A) 2-nitroethanol, (B) 2-nitro-1-propanol, and (C) 3-nitro-2-pentanol.