Intraocular pharmacotherapy: specificity and safety issues

Completion of the Human Genome Project in 2003 catalyzed the development of several sophisticated analytical technologies and genome-wide investigations. Since then, we have gained valuable insights into the pathophysiology of several retinal diseases and developed new treatment modalities. This is particularly true for age-related macular degeneration (AMD), which still remains the leading cause of blindness among the elderly in the Western world [1]. Over the last 5 years, an increasing number of new therapeutic possibilities targeting pathological signaling pathways in AMD have been developed. VEGF was the first cytokine to be targeted, owing to its central role in the development of subretinal neovascularization. Intravitreal anti-VEGF antibodies [2] (bevacizumab and ranibizumab), anti-VEGF aptamer (pegaptanib) [3], VEGF-trap [4] and siRNAs [5] have all been developed to antagonize the proangiogenic activity of VEGF. Furthermore, triamcinolone [6] and anecortave acetate [7] have been used to neutralize the biological effects of VEGF. Several other revolutionary treatments, such as gene therapy with adenoviral vector-delivered pigment epithelium-derived factor, are also in the pipeline [8].

At the dawn of this new era, it may be useful to remember that the basic principle of medicine is not to harm the patient. This principle becomes quite handy considering that the discovery of each new drug costs approximately US$800 million and takes 10–12 years to develop. Thus, it is vital to give extreme caution to the safety of a xenobiotic during the development stages. Anti-VEGF agents are a good example of this, as VEGF has vital importance for wound healing and collateral formation in the vital organs, such as the heart and brain. Possible systemic side effects of VEGF-targeting become a serious concern considering that patients with AMD already have a three-times higher rate of incident coronary heart disease [9] and nearly a two-times higher rates of stroke [10]. Systemic VEGF-blocking with bevacizumab in patients treated for metastatic cancer resulted in a two-times higher rate of stroke [11]. Early trials with intravitreal use of ranibizumab and pegaptanib did not show a statistically significant increase in systemic vascular complications. However, in these studies establishing a causal relationship with systemic vascular complications and intravitreal injection of the drug was left to the discretion of the participating physician. This often raises questions about the validity of the collected data. Also, these studies were conducted with a limited number of selected patients and their power is not sufficient to demonstrate the impact of these drugs on relatively infrequent complications, such as systemic vascular events. For example, extrapolating the Minimally Classic/Occult Trial of the Anti-VEGF Antibody Ranibizumab in the Treatment of Neovascular AMD (MARINA) study data [12] to the whole exudative AMD population in the USA, which consists of 1.25 million patients [13], indicates 20,000 more coronary vascular events and stroke. Such concerns have been fueled by a recent report from Genentech that indicated a significant increase in stroke rate with a higher dose of ranibizumab (Lucentis™; 1.2 vs 0.3%) in the still ongoing Safety Assessment of Intravitreal Lucentis For AMD (SAILOR) study. Despite such concerns, ranibizumab was approved by the US FDA for the treatment of exudative AMD in July 2006 leaving in many curious minds some questions, such as whether this will turn into another Phen-Fen, Redux, Vioxx or Avandia fiasco.

There is no doubt that intravitreal anti-VEGF drugs leak into the systemic circulation. The fact that systemically absorbed amounts of these drugs can exert biological effects on other vascular beds was clearly demonstrated by observing the fellow eyes. A similar synchronous effect was observed in the fellow eye [14]. The biological effects of this ‘leaking’ amount on the cerebral and cardiac vascular beds are still unknown. However, within 6 months of the publication of the first report on the intravitreal injection of bevacizumab, 5% of members of the American Society of Retina Specialists responding to a poll stating that they had experienced local and/or systemic thromboembolic events related to bevacizumab [101]. There may be several ways to avoid such possible side effects of systemic VEGF neutralization. The safest approach would be not to consider VEGF blocking for patients that have active coronary artery disease symptoms (e.g., angina and shortness of breath) or are at high risk for stroke (i.e., uncontrolled systemic hypertension and diabetes) or have a recent history of transient ischemic attacks. Care should be given to patients with diabetes who may not have classical cardiac symptoms but who are more prone to develop systemic vascular complications. Intravitreal steroids alone, or in combination with photodynamic treatment, are a reasonable alternative for these patients. For the remainder of patients, a safer approach is to decrease the intravitreal dose. The real dose–effect relationship for these drugs is not known, since only two doses of ranibizumab and pegaptanib were studied in clinical trials, however, it is quite possible that a similar benefit can be obtained by injecting significantly lower doses. One way to achieve similar, or better, therapeutic value is to combine anti-VEGF treatments with steroids (triamcinolone or dexamethasone) and/or photodynamic therapy. Combined treatment also lowers the need for frequent intravitreal injections [14].

Avoiding systemic adverse side effects will be a result of modern biological research that has already produced promising therapeutic alternatives. Increasing the efficacy and safety margin of ocular drugs will require the following steps:

•	Better understanding of gene and protein expression abnormalities that play a role in pathophysiological pathways;
•	Discovery of disease-specific diagnostic and prognostic biomarkers that may be used for early diagnosis and monitoring the response to treatment;
•	Identification of cell-specific molecular markers for drug targeting;
•	Optimizing the chemical structure of the drug to obtain the highest therapeutic effect on targeted tissue without any harm to the off-target tissue;
•	Developing carrier molecules that can increase the penetrance of the drugs into intraocular and intracellular compartments to decrease the applied dose and eliminate risky and uncomfortable ocular injections;
•	Mapping genes involved in drug sensitivity and resistance;
•	Identifying the factors that play a role in individual response differences among different phenotypes of the disease.

Several promising steps have already been taken to reach these goals. Hypoxia-inducible factor, integrins, angiopoetins, erythropoietin and pigment epithelium-derived growth factor are among the considered targets for AMD pharmacotherapy [15]. It is obvious that the list of targeted pathways and cytokines will be increasing as more powerful proteomic and genomic analyses are employed in AMD research. Progress in bioinformatics and the development of automated tests for high-throughput screening of thousands of compounds will fuel the discovery of new and effective drugs. A good example is the recent discovery of hemoglobin expression in the mammalian retinal pigment epithelium [16]. Hemoglobin is the main oxygen carrier in the body and exhibits an allosteric oxygen-binding property. Hemoglobin is abundant in the macula within the retinal pigment epithelium and Bruch’s membrane and its spatial expression pattern implies its role in oxygen delivery to the outer retina. Age-related decrease in hemoglobin expression of retinal pigment epithelial cells may constitute the reason for outer retinal hypoxia and subsequent VEGF upregulation leading to age-related cellular and structural changes. Iron bound to hemoglobin may also be a critical mediator of cell death in AMD through the production of highly reactive hydroxyl radicals via the Fenton reaction [17]. Although the source of iron in the outer retina is not known, hemoglobin is a possible candidate since more than two-thirds of the body’s iron is bound to hemoglobin [18]. Pharmacologic modulation of retinal pigment epithelial hemoglobin expression is a new and promising avenue for AMD treatment.

Targeting xenobiotics to ocular tissues requires tissue-specific markers. Potential molecules for targeted delivery of anti-angiogenic drugs to proliferating vascular endothelia include tissue factor, α5β3 integrins [19], endoglin [20], EN 7/44 antigen [21], endothelial ectopeptidases [22], intercellular adhesion molecule (ICAM) [23] and other cell-adhesion molecules expressed on activated endothelial cells, and transferrin receptor [24]. Among them, tissue factor has a high specificity for proliferating endothelial cells [25]. It has been targeted with a recombinant antibody-like molecule, called an immunoconjugate (ICON), which is composed of Factor VII, the natural ligand for tissue factor, conjugated to the Fc domain of a human IgG1. Factor VII domain of this chimeric protein binds with high affinity and specificity to tissue factor, and the Fc-effector domain recruits natural killer cells and activates the complement cascade, initiating a powerful cytolytic response against cells that express tissue factor. Treating mice and pigs with ICON by intravenous or intravitreal injection of the purified fusion protein or an adenoviral vector encoding the ICON resulted in almost complete destruction of the choroidal neovascularization without associated local or systemic complications [25,26].

Conventional invasive delivery methods such as subtenon and intravitreal injections are uncomfortable for the patients and carry serious risks, such as endophthalmitis, retinal tears and bleeding. The most convenient route for drug intake is by mouth. However, oral or intravenous drugs distribute systemically and their access to ocular compartments is blocked by blood–ocular barriers. Therefore it is necessary to conjugate these drugs with molecules that will facilitate their diffusion through ocular barriers and direct them to the targeted cell. Several small peptides with cell membrane translocation properties have been identified over the last decade to facilitate drug delivery into cells. Tat, transportan, VP22-derived peptide and antennapedia-derived peptides are some of the most commonly used members of this group that are collectively called ‘cell-penetrating peptides’ [27]. Efficacy of targeting and intraocular delivery of cell-penetrating peptides has been tested recently using a multifunctional tetraazacyclo-dodecanetetra-acetic acid (DOTA)-TAT carrier core to which drugs and cell-specific targets can be linked. Initial animal experiments with this multifunctional DOTA-TAT carrier core showed increased drug penetrance and therapeutic efficacy [28]. Cyclodextrins [29] and engineered homing peptides [30] are other promising carrier molecules that can be used for the same purpose. Once the drugs are delivered into the cells they are also prone to be degradated by the endosomal/lysosomal or ubiquitin/proteosomal system, which also necessitates employing endosomal escape mechanisms for a better therapeutic efficacy [31].

While the hype of anti-VEGF therapy is rapidly affecting the current management of exudative AMD, one point should always be kept in mind: the therapeutic value of the currently available anti-angiogenic drugs is limited only to a temporary increase in vision due to cessation of the leakage through incompetent vasculature of the subretinal neovascular complex and the possible limitation of the extent of the final subretinal scar. Thus, their benefit may not be sufficient to restore the central vision of the patients who had already reached the late stages of this devastating disease. A possible remedy for such patients will be to reconstruct the damaged macula using tissue engineering and cell-replacement techniques – a process collectively termed as ‘maculoplasty’ [32]. Initial maculoplasty attempts have been accomplished by transplanting retinal cells obtained from fetal and adult human donors. Results of these early trials indicated that structural and molecular alterations of the aged Bruch’s membrane create a hostile environment for the survival of transplanted cells. Molecular rejuvenation of Bruch’s membrane has been shown to enhance the survival of transplanted cells [33] and in vivo application of these techniques are currently under development. On the other hand, conventional treatments, such as laser photocoagulation and photodynamic treatment, have been almost abandoned owing to their destructive and palliative nature, and poorer visual outcome compared with anti-angiogenic medications. A recent survey conducted among the members of American Society of Retina Specialists demonstrated that less than 1% of the responding retina specialists prefer either of these treatments [101]. Thus, in this era of transformation, where we abandon old treatments and walk towards more sophisticated and effective treatment alternatives, pharmacotheraphy will dominate the field. Aiming for specific targeting, better delivery methods and safety should be our prime goal.

Similar principles should also be applied for developing intraocular drugs for conditions other than AMD. One good example is the emerging field of pharmacological vitreolysis. Enzymatic separation of posterior hyaloid from the retina can shorten the surgical time and decrease the complications during vitrectomy. It also has the potential to avoid blinding complications of diabetic retinopathy and improve macular edema in several macular diseases, such as vein occlusions, diabetes and macular degeneration. It can also be therapeutic, by relieving the mechanical traction in conditions such as taut posterior hyaloid, vitreomacular traction syndrome and macular hole. The prime goal in accomplishing enzymatic posterior vitreous detachment (syneresis) should be to cleave the vitreoretinal attachments without digesting the vitreous itself. Otherwise prematurely liquefied vitreous will collapse, pull the retina and create iatrogenic retinal tear. This fact was best demonstrated by the doubling of the retinal tear rate during the safety trials of intravitreal hyaluronidase used to clear vitreous hemorrhage [34]. Therefore, enzymes used for pharmacological vitreolysis should act primarily on the molecules that mediate the attachment of internal limiting membrane to cortical vitreous to avoid such complications. Internal limiting membrane is a three-layered basal lamina with an electron dense lamina densa flanked by two electron-lucent lamina rara [35]. Lamina densa is mainly composed of type IV collagen and during the natural process of posterior vitreous detachment cleavage occurs at this plane [36]. Dispase is the only enzyme that has a high specificity for type IV collagen [37] among several other enzymes currently at different development stages for inducing posterior vitreous detachment. Early studies showed that it can reproducibly cleave the internal limiting membrane at the lamina densa–lamina rara interna junction and expose the Muller cell endplates without inducing vitreous liquefaction [37]. It also fulfills the critical requirement of creating a uniform enzymatic cleavage without any collagen debris that may otherwise act as a scaffold for subsequent cellular proliferation and epiretinal membrane formation. The clinical importance of cleaving the internal limiting membrane at this level has recently been verified in a report where anatomical closure rate of macular holes was increased from 81 to 92% and the reopening rate decreased from 7 to 0.6% [38]. Currently, the development of dispase is at the purification and recombinant production stage. Among several other enzymes, plasmin and its recombinant catalytic domain (microplasmin) are the closest for clinical use. The ability of microplasmin to induce posterior vitreous detachment prior to vitrectomy is currently under trial (Phase IIb) in the USA. In another trial in Europe (Phase II) its ability to treat diabetic macular edema has been tested. Both plasmin and microplasmin are serine esterases that directly or indirectly activate several enzymes and can result in vitreous liquefaction and separation. Unfortunately, plasmin and microplasmin have no direct effect on type IV collagen [39]. They can act on laminin and fibronectin and induce posterior vitreous detachment through the liquefaction and subsequent collapse of the vitreous body [39]. This may result in retinal tears and detachment as occurred 2 h after the injection of the enzyme during the initial clinical trials in 40 patients [40]. Inability of plasmin and microplasmin to cleave type IV collagen often results in patches of collagen fibers on the internal limiting membrane [41]. Such island of collagen can act as a scaffold for subsequent cellular proliferation. Also, the enzymatic activity of plasmin and microplasmin can be inhibited by α-2 antiplasmins, which are abundant in the vitreous of patients with vitreoretinal diseases due to blood–retinal barrier breakdown [42]. This fact itself raises concerns about their efficacy and reproducibility in inducing posterior vitreous detachment.

In conclusion, the accelerated pace of scientific and technological breakthroughs have increased our ability to explore complex pathophysiological pathways in many vitreoretinal disorders. In the near future we shall witness the development of new and more effective therapies. In this fascinating era of enthusiasm and revolutionary spirit, safety and specificity of the drug should always be kept in mind. Overall, the physician’s first duty is not to harm.

Financial & competing interests disclosure

The author is a major shareholder of RegenaSight, LLC., a biotechnology company developing Dispase for enzymatic induction of posterior vitreous detachment. Dr Tezel’s research was supported in part by the NIH (KO8EY0416120) and a Career Development Award from Research to Prevent Blindness, Inc, NY, USA. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.