Pharmacokinetics and Posterior Segment Biodistribution of ESBA105, an Anti–TNF-α Single-Chain Antibody, upon Topical Administration to the Rabbit Eye

Abstract

purpose. The purpose of this study was to characterize local distribution and systemic absorption of the tumor necrosis factor (TNF)-α inhibitory single-chain antibody fragment (scFv) ESBA105 following topical administration to the eye in vivo.

methods. Rabbits received ESBA105 as topical eye drops in two dosing regimens. First, pharmacokinetics after the topical route of administration was compared to the intravenous (i.v.) route by means of applying the identical cumulative daily dose of ESBA105. In a second study rabbits received five eye drops daily for six consecutive days in a lower frequency topical dosing regimen. Kinetics and biodistribution of ESBA105 in ocular tissues and fluids as well as in sera were determined in all animals.

results. After topical administration to the eye, ESBA105 quickly reaches therapeutic concentrations in all ocular compartments. Systemic exposure after topical administration is 25,000-fold lower than exposure after i.v. injection of the identical cumulative daily dose. ESBA105 levels in vitreous humor and neuroretina are significantly higher on topical administration than after i.v. injection. Absolute and relative intraocular biodistribution of ESBA105 is different with topical and systemic delivery routes. Compared to its terminal half-life in circulation (7 hours), the vitreal half-life of ESBA105 is significantly enhanced (16–24 hours).

conclusions. On topical administration, ESBA105 is efficiently absorbed and distributed to all compartments of the eye, whereby systemic drug exposure is very low. Based on its unique intraocular biodistribution and pharmacokinetics and the absolute intraocular levels reached, topical ESBA105 appears highly attractive for treatment of various ophthalmological disorders.

Antibody-based therapies have dramatically changed treatment options for patients suffering from exudative AMD and other diseases affecting the back of the eye. ¹ Anti-VEGF antibodies are used to inhibit choroidal neovascularization in patients with AMD. ^{1 2 3} The Fab fragment ranibizumab (Lucentis; Genentech USA, Inc.) is approved for subfoveal neovascular AMD. The IgG bevacizumab (Avastin; Genentech, San Diego, CA), which is approved for intravenous (i.v.) application in colon cancer; is also used for the eye, although it is not approved for use in this application. However, antibodies need to be delivered by (repeat) intravitreal injection, a laborious administration procedure associated with the potential for serious adverse events. ^{2 3} Thus, feasibility of topical self-administration of antibody-based therapeutics in an outpatient setting would represent a major innovation in ophthalmology. However, conventional antibodies, due to their large molecular weight of 150 kDa, do not penetrate into the inner of the eye on topical administration. ^{4 5} Single-chain antibody fragments (scFv) consist of the variable domains of monoclonal antibodies interconnected by a peptide linker. Based on their smaller size of only 26 kDa, scFvs have the potential for penetration through corneal epithelium in vivo, as shown previously. ^{5 6} However, due to the typical biophysical limitations of common scFvs, topical formulations for scFvs appeared to be an absolute requirement. ^{5 6} Unfortunately, excipients enhancing transcorneal transport have a significant potential to induce ocular toxicity, likely because they affect the integrity of interepithelial tight junctions and/or epithelial membrane function. ^{7 8 9 10} In particular, the most effective penetration enhancer identified for scFv, ^{5 6} sodium caprate, is toxic to the cornea ¹¹ and is not approved for topical use by the FDA. 

Tumor necrosis factor (TNF)-α is an attractive, emerging target in several ophthalmological disorders (for review see Ref. ¹² ). ESBA105, a potent scFv directed against TNF-α, was designed for excellent biophysical properties (unpublished results, 2006). Our work with ESBA105 presented in the accompanying paper ¹¹ revealed efficient delivery into aqueous and vitreous after topical administration onto the ocular surface in absence of any penetration enhancer. In particular, high solubility and monomeric state of the protein appear to be prerequisites for efficient intraocular delivery by topical application. In addition to the finding of high ESBA105 levels in the anterior chamber, our data suggested that a unique translimbal/intrascleral migration route exists for ESBA105 in vivo, by which the molecule is efficiently delivered to vitreous and retina after topical administration. Surprisingly, absence of a penetration enhancer in the formulation further favored delivery of ESBA105 to posterior compartments via translimbal/intrascleral migration in vivo. In contrast, transcorneal penetration of ESBA105 to the anterior chamber (but also systemic exposure) was higher with a penetration enhancer in the topical formulation. Due to the extended half-life of 25 hours in vitreous ¹¹ after topical administration to the eye, ESBA105 steady state levels build up in the vitreous. Thus, our studies also suggested that the vitreous could act as a ‘reservoir’ for ESBA105, from which it is then continuously released to both anterior chamber and retina. 

Quickly obtaining therapeutic drug levels in the eye following treatment initiation is a critical therapeutic success factor in the treatment of highly inflammatory disease states such as acute anterior uveitis, ¹³ for which a topical TNF-α inhibitor may be suited. To achieve the first goal of maximizing intraocular ESBA105 levels within the first day of treatment, a high frequency dosing regimen was used here in a first set of in vivo studies (Table 1) . Intraocular distribution patterns and systemic exposure for ESBA105 dosed topically or systemically were compared. In addition, also intra- and interocular migration pathways for ESBA105 were characterized. 

Whereas in acute inflammatory conditions it is important to reach high intraocular drug levels quickly, for chronic diseases requiring long-term treatment it is essential to identify dosing regimens convenient for patients. Therefore, a multi-day dosing regimen of ESBA105 over six consecutive days with lower dosing frequency was evaluated (Table 1) . Again, intraocular distribution patterns and systemic exposure for ESBA105 dosed topically were assessed. 

Materials and Methods

ESBA105

ESBA105 was expressed in and purified from Escherichia coli, as described previously. ¹¹ Asymmetrical flow field-flow fractionation (FFF) of the protein samples was performed in a trapezoidal channel, 26.5 cm in length and 350 μm in height, connected to a protein separation system (Eclipse F; Wyatt Technology Europe, Dernbach, Germany). The bottom of the channel was lined with a polyether sulfone membrane (5 kDa cutoff; Microdyn-Nadir GmbH, Wiesbaden, Germany). Nine μg ESBA105 in 20 mM sodium citrate buffer (115 mM NaCl, pH 6.0) were injected in 1 μL into the system and eluted with the same buffer. The channel flow was set to 1 mL/min and the injection flow to 0.2 mL/min. The elution duration was 9.5 minutes with an applied cross-flow of 4.5 mL/min. Each run was preceded by a conditioning step and by a rinsing step with the cross flow set to 0 mL/min. A multi-angle light scattering detector (Dawn EOS; Wyatt Technology, Santa Barbara, CA) and a UV detector (Agilent Technologies Schweiz AG, Basle, Switzerland) were coupled in-line with the FFF channel. The light scattering detector was equipped with a GaAs laser (wavelength 690 nm) and 18 detectors. Scattered light was collected at defined angles between 14° and 163°. Sample concentration was determined by UV absorbance at 280 nm. Data were collected and analyzed (Astra software, version 5.1.9.1; Wyatt Technology). 

For stability studies, ESBA105 was incubated in biological fluids at various temperatures. Samples were withdrawn at defined time points and analyzed by ELISA (see below) for ESBA105 content and activity. 

The topical formulation for one-day treatment contained 10 mg/mL ESBA105 in 20 mM sodium citrate buffer (115 mM NaCl, pH 6.0). Sterile-filtered ESBA105 was filled into drop-counting flasks (Remy & Geiser, Anhausen, Germany) and stored at 4°C. The volume of each drop was approximately 50 μL Due to non-precise volume the first two drops of each flask always were discarded. The topical formulation for multi-day treatment contained 9.6 mg/mL ESBA105 in 0.15 M sodium phosphate (pH 6.0). The i.v. formulation consisted of 20 mg/mL ESBA105 in 20 mM sodium citrate buffer (115 mM NaCl, pH 6.0). 

Topical Administration of ESBA105

All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

One-day treatment (Table 1) : Female New Zealand White rabbits (∼3 kg, Elevage des Pins, Epeigne sur Deme, France) were treated topically with single drops of ESBA105 in hourly intervals for up to 10 hours. Drops were placed under the upper eyelid of the rabbit’s right eye. The contralateral eye remained untreated. After each instillation, animals were manually maintained still for 30 seconds to allow distribution of ESBA105 on the ocular surface. At 0, 1, 5, 10, and 33 hours, five animals each were anesthetized by intramuscular injection of ketamine 1000 (0.1 mg/kg) + xylazine (Rompun, 0.1 mL/kg); when animals were deeply asleep, blood samples were drawn cardially. Thereafter, rabbits were euthanatized by 1 gram of pentobarbital sodium (Pentothal; Abbott, Rungis, France) in 10 mL of 0.9% NaCl intracardially injected to each rabbit. Then, both eyeballs were surgically removed, washed in PBS, wiped dry, and dissected on ice. Aqueous and vitreous humor, neuroretina, and RPE-choroid were collected. Blood samples were left to coagulate 1 hour at room temperature and 4°C overnight. Then, serum was separated by centrifugation and stored at −80°C until analysis. Samples were frozen in dry ice and stored at −80°C until analysis. 

Multi-day treatment: New Zealand White rabbits received one topical eye drop onto both eyes at five occasions per day in three-hour intervals up to six days. A volume of 50 μL was applied under the upper eyelid of the rabbit’s eye as above. On days 1, 3, and 6, two animals per time point were euthanatized (see above), always 1 hour after the second administration of ESBA105. Rabbits treated with buffer only received five daily applications and were euthanatized on day 6 one hour after the second instillation and served as background controls. 

Intravenous Application of ESBA105

New Zealand White rabbits received a single bolus injection of 5 mg ESBA105 via the marginal ear vein. Blood samples were collected from ear veins under local anesthesia 5 minutes, and 0.5, 1, 2, 6, 11, and 25 hours post injection. The final blood sample of each animal was taken intracardially at the time of kill. For this, rabbits were anesthetized and euthanatized as above. One, 10, and 25 hours after the injection, three animals were killed and ocular tissues/fluids (see above) and blood collected. Naïve control blood samples were obtained from animals not treated with ESBA105. Sera were prepared as above. 

Tissue Preparation

One hundred microliters of lysis buffer (10 mM Tris, pH 7.4, 0.1% SDS, with proteinase inhibitor cocktail; Roche Diagnostics, Rotkreuz, Switzerland) were added per 15 mg of neuroretina or RPE-choroid tissue. Tissues were sonicated (Sonopuls; Bandelin, Berlin, Germany), centrifuged, and the supernatants were stored at −80°C until analysis. 

Quantification of ESBA105 Concentrations in Liquids and Tissues

Predilutions of each sample were prepared in dilution buffer (PBS, 0.2% Tween20, 1% BSA, with 10% of the respective matrix added: aqueous humor, vitreous humor, neuroretina, RPE-choroid, or serum). Standard reference dilutions series (1500–50 pg/mL) of ESBA105 were prepared in dilution buffer containing 10% matrix. Ocular fluids and tissue extracts used for predilutions were isolated from rabbit eyes obtained from Metzgerei Schönbächler, Wädenswil, Switzerland. 

ESBA105 concentrations were determined in triplicate by ELISAs developed at ESBATech. ESBA105 concentrations in all samples obtained from the high frequency topical dosing study and the i.v. injected rabbits were measured in a sandwich ELISA, with polyclonal rabbit anti-ESBA105 antibody (AK3A; ESBATech, Schlieren, Switzerland) for capture and a biotinylated monoclonal mouse anti-ESBA105 antibody (31-5-2, ESBATech, Schlieren, Switzerland) for detection. The lower limit of quantitation (LOQ) was in the range from 0.03 to 0.45 ng/mL. ESBA105 concentrations in ocular tissue extracts and fluids of the multi-day animal study were measured in a direct ELISA using human TNF-α (Peprotech, London, UK) as capturing agent and a biotinylated polyclonal rabbit anti-ESBA105 antibody (AK3A) for detection. The minimum quantifiable concentration of ESBA105 was 2.3 ng/mL. ESBA105 levels in serum samples were determined in a sandwich ELISA with a polyclonal rabbit antibody to ESBA105 (AK2A; ESBATech) used for capture and a different polyclonal rabbit anti-ESBA105 antibody (AK3A), conjugated to biotin, for detection. The LOQ was in the range of 0.5 to 2 ng/mL. Undiluted samples that resulted in signals below the LOQ were set to zero for mathematical evaluation but excluded from graphs with logarithmic scales. 

Pharmacokinetics

Pharmacokinetics (PK) analyses were performed with values from individual animals averaged for a group. Calculations were done using PK data analysis software (PK Solutions 2.0; Summit Research Services, Montrose, CO) as well as add-ins (Allergan, Irvine, CA, for Microsoft Excel 2007). 

Results

Completely Monomeric ESBA105 in the Formulations Used for In Vivo Administration

Before use of ESBA105 in vivo, the monomer content (a critical parameter for efficient intraocular delivery) and stability in biological fluids was assessed. Monomer content was shown by field-flow fractionation (FFF). ESBA105 was injected into the FFF channel using a standard separation method. The main peak had a molecular weight of 26 kDa. Only 0.3% of low molecular weight aggregates could be detected in the applied formulation of ESBA105 (Fig. 1) . These findings were confirmed by analyzing the same sample without applying any focusing step or cross flow. Under these conditions, the separation does not take place on the FFF channel. The sample of ESBA105 eluted in one broad peak within two minutes and the light scattering signal indicated that the protein was under its monomeric form (data not shown). To ensure stability and structural integrity of ESBA105 incubations in biological fluids at various concentrations, temperatures and durations were performed. In short, ESBA105 is stable for at least one week at 37°C in serum, aqueous, and vitreous humor as assessed by ELISA (data not shown). 

High Frequency One-Day Treatment with Topical ESBA105

As described elsewhere, ESBA105 on administration to the ocular surface penetrates into the rabbit eye in vivo even in absence of a penetration enhancer. ¹¹ Herein, it was first evaluated how rapidly and at which levels ESBA105 reached various compartments of the eye in rabbits in vivo after high dosing frequency topical or i.v. bolus application (Table 1 , Fig. 2 ). Striking differences of ESBA105 levels were observed in individual ocular compartments when comparing topical and i.v. dosing. After one-day topical application, levels of ESBA105 were above 10 ng/mL in aqueous and above 100 ng/mL in vitreous humor, neuroretina, and RPE-choroid. After systemic administration, ESBA105 concentrations were highest in sera as well as in the highly vascularized RPE-choroid and lowest in vitreous humor and neuroretina. Comparing distribution patterns, the anterior chamber was preferably reached by i.v. injection, while after topical treatment higher ESBA105 levels were reached in the more posterior compartments (vitreous and neuroretina). 

Efficient Delivery of ESBA105 to Vitreous, Neuroretina, and RPE-Choroid

ESBA105’s biodistribution and kinetics in tissues from the above study were analyzed. With topical application of ESBA105, delivery was most efficient to more posterior compartments like vitreous, neuroretina, and RPE-choroid (Fig. 2) . Peak levels (C _max) in vitreous humor at 295 ng/mL were almost 25-fold above ESBA105 levels in aqueous in this study (Fig. 2) . Most importantly, when comparing C _max after topical or i.v. dosing, ESBA105 levels in vitreous humor were approximately 4.7-fold higher with topical dosing. To measure ESBA105 levels in retina, neuroretina was separated from the underlying retinal pigment epithelium (RPE) and choroidea during necropsy. After high frequency topical dosing, concentrations in both neuroretina (214 ng/mL) and RPE-choroid (263 ng/mL) were higher than in aqueous but similar to levels in vitreous humor (Fig. 2) . Intravenous ESBA105 resulted in lower concentrations in the avascular neuroretina compared to the levels obtained after topical application (Fig. 2) . In contrast, in the RPE-choroid layer, systemic application led to seemingly higher tissue concentrations of ESBA105. As animals were not perfused before necropsy, the higher ESBA105 levels in RPE-choroid measured after i.v. injection might be artificial caused by ESBA105 from within blood vessels in the highly vascularized choroidea. In aqueous, ESBA105 concentrations reached maximal levels of 12 ng/mL after 10 hours of high frequency topical dosing (Fig. 2) . Intravenous administration led to approximately 15-fold higher ESBA105 concentrations in aqueous with a C _max of 175 ng/mL (Fig. 2) . After high frequency topical one-day dosing, C _max of ESBA105 in sera was below 1 ng/mL, a level strikingly lower than in any of the analyzed ocular compartments (Fig. 2) . In extreme contrast, after i.v. injection of ESBA105 C _max in sera was 89,000 ng/mL (Fig. 2) . 

Biodistribution to the Untreated, Contralateral Eye after High Frequency Topical Administration

ESBA105, on topical administration to the right eye, also resulted in considerable peak levels in the contralateral eye (Fig. 3) . C _max of ESBA105 measured in ocular compartments of the contralateral eye was 4 to 15 times lower than in the treated eye. Importantly, ESBA105 levels in the contralateral eye mirror intraocular distribution in the treated eye, though at a significantly reduced level and with a delay (Figs. 3 4 5 6 7) . In contrast, i.v. bolus administration of the identical cumulative daily dose as given topically results in an entirely different intraocular distribution pattern for ESBA105, associated with very high systemic exposure (Fig. 2) . These results demonstrate two important points. First, they strongly suggest that on topical administration, ESBA105 is directly delivered from the ocular surface to the vitreous humor and the retina of the topically treated eye and is not indirectly delivered via the systemic circulation. Second, these data suggest that topical ESBA105 migrates through a local migration pathway to the contralateral eye rather than indirectly via the systemic circulation. 

ESBA105 Pharmacokinetics after High Frequency One-Day Treatment

During the first 5 to 10 hours, a steady increase in ESBA105 concentrations was observed both in treated as well as untreated, contralateral eyes (Figs. 4 5 6 7) . Pharmacokinetics analysis shows crucial characteristics of intraocular availability after topical administration of ESBA105. Even after application of only the first single drop to the ocular surface, ESBA105 concentrations reached 98 ng/mL in vitreous humor (Fig. 5) . In contrast, i.v. injection resulted in significantly lower local concentrations of ESBA105 at any time point in vitreous humor than after topical application (Fig. 5) . Local half-lives of ESBA105 in posterior compartments of the eye were clearly longer than the 6 or 7 hour half-life of ESBA105 in aqueous or serum (Figs. 4 5 6 7 8and Table 2 ). Specifically, half-lives in vitreous humor, neuroretina, and RPE-choroid were 16, 27, and 14 hours, respectively. The longer half-life in the stagnant vitreous humor is consistent with results from our earlier studies where a prolonged local elimination half-life of 25 hours was observed for ESBA105 after intravitreal injection. ¹¹  

To compare efficiency of delivery by i.v. and topical application, local bioavailabilities of ESBA105 were calculated (F _local = local AUC_0-inf /systemic AUC_0-inf), where local AUC_0-inf is the area under the concentration-time curve at the local site and systemic AUC_0-inf is the area under the concentration-time curve in circulation after i.v. injection (see Table 2 ). Local bioavailabilities of ESBA105 in posterior ocular segments after high frequency topical dosing compared to i.v. administration were 4.1- and 3.7-fold higher in vitreous humor and neuroretina, respectively. This suggests efficient drug delivery from the ocular surface to the inner of the eye, particularly to the more posterior segments. In contrast, at least short term after topical application, quantitative delivery to aqueous on topical administration was less efficient, with a bioavailability coefficient of 0.13. Again, systemic bioavailability was extremely low after topical administration, when compared to i.v. injection, with a relative bioavailability of 0.00004. In fact, five minutes after i.v. injection, ESBA105 levels in serum were at 89,000 ng/mL (Fig. 8)and declined in a multiphase manner thereafter. Total systemic exposure to ESBA105, based on AUC_0-inf calculation, was nearly 25,000-fold lower after topical treatment than after i.v. injection (Table 2) . 

Multi-day Topical Dosing of Rabbits

To explore dosing regimens convenient for treatment of chronic diseases located in the back of the eye, we performed a multi-day topical dosing study in rabbits. ESBA105 was administered at five drops per day topically onto both eyes over a period of six consecutive days (Table 1) . Both eyes were treated to mirror clinical situations where both eyes are affected. In addition, we were interested to evaluate systemic drug exposure under maximal topical dosing during multi-day treatment. ESBA105 was present at all time-points at significant levels in all ocular compartments analyzed (aqueous, vitreous humor, neuroretina, RPE-choroid) but to much lower extent in sera (Fig. 9A) . Indeed, during the course of the experiment, ESBA105 levels rose continuously in all ocular compartments to reach steady state concentrations above 300 ng/mL in retina and above 500 ng/mL in vitreous humor. These data are in good accordance with modeled values that were calculated based on data from the above high frequency dosing one-day study (see Fig. 9B ). T _max was reached in all but one ocular compartments after day six, which is in line with the expected duration to reach steady state (about five half-lives; i.e., 5 days based on an elimination half-life of about one day in vitreous). C _max of ESBA105 in the various ocular fluids/tissues are lowest in anterior chamber (153 ng/mL) and highest in RPE-choroid (1298 ng/mL) with concentrations in vitreous humor (580 ng/mL) and neuroretina (917 ng/mL) in between (Table 3) . In sera, C _max of ESBA105 did not exceed 3 ng/mL and was not detectable at 3 and 6 days. Even with parallel treatment of both eyes, systemic exposure to ESBA105, based on AUC, was 80 to 1000 times lower than exposure in the individual ocular compartments (Table 3) . This again confirms very low systemic exposure after topical administration of ESBA105. 

Discussion

We have shown elsewhere that on topical administration, ESBA105 penetrates cornea and sclera most probably by passive diffusion through the interstitial space. ¹¹ This occurs in a concentration-dependent manner, suggesting a non-saturable penetration mechanism for ESBA105. As also shown elsewhere, with high solubility—and consequently high applicable concentrations of the scFv—there is no need for a penetration enhancer in the topical formulation. ¹¹ Delivery to the back of the eye was found to be even more pronounced without penetration enhancer. ¹¹ Consequently, for scFvs such as ESBA105, it appears more advisable for therapeutic purposes to increase the concentration in the formulation rather than adding absorption-enhancing excipients, which carry risk of eye irritation and increased absorption to systemic circulation. 

The data presented here show that topical application of ESBA105 in a simple formulation without any penetration enhancer results in higher levels in vitreous and neuroretina than in aqueous. This may be explained on one hand by the faster turnover rate of the anterior chamber fluid compared to vitreous and on the other hand by a lower penetration rate through cornea epithelium than through limbus/sclera. Unlike aqueous humor, which is continuously replenished, vitreous is more gelatinous and stagnant ¹⁴ which may be one reason for the prolonged elimination half-life of ESBA105 in the vitreous. Most importantly, the prolonged intravitreal half-life results for vitreous to act as a reservoir for ESBA105 in vivo. Prolonged half-lives for other macromolecules in vitreous have also been described elsewhere. ¹⁵  

The fact that ESBA105 reaches very high levels in vitreous humor as soon as one hour after topical administration of the first drop (Fig. 5)strongly suggests that there is an efficient migration pathway for ESBA105 to the vitreous bypassing cornea and anterior chamber. It is likely that ESBA105, on diffusion through the limbus, migrates within the sclera, directly to vitreous humor and retina. Alternatively, it may be readily absorbed through conjunctiva to migrate within sclera. In agreement with the latter hypothesis, quantitative autoradiography experiments with ¹²⁵I-ESBA105 indicate that ESBA105 may indeed penetrate through the conjunctiva and then distribute in the entire eye socket within less than one hour (data not shown). From the eye socket, ESBA105 may be absorbed through the sclera, which in contrast to cornea is lacking an epithelial barrier. In any case, the relatively short half-life in the anterior chamber does not appear to be limiting for ESBA105 absorption into the posterior eye segment, which is in strong contrast to most small molecules applied topically. ¹⁶  

The higher ESBA105 levels in treated eyes compared to the untreated, contralateral eyes in combination with the extremely low concentrations found in the circulation after topical application clearly speak in favor of a direct ESBA105 migration from the ocular surface through ocular epithelia to the inner of the eye. Our data strongly argue against indirect ESBA105 migration by systemic absorption first and subsequent delivery of ESBA105 via the bloodstream to the different compartments of the eye. 

ESBA105 concentrations in treated eyes were considerably higher than in untreated, contralateral eyes. However, levels in untreated, contralateral eyes were still higher than in sera at any time point measured. Therefore, ESBA105 migration from one eye to the contralateral eye is unlikely to occur via central circulation but rather via other routes connecting the eyes. Comparable considerations were reported based on findings with topically applied insulin. ^{17 18} However, it remains for future studies to elucidate the specific migration pathway of ESBA105 to the contralateral eye. 

No striking differences in ESBA105 concentrations were observed between vitreous humor, neuroretina, and RPE-choroid after topical application, suggesting efficient penetration throughout all posterior ocular compartments. After i.v. injection, high levels of ESBA105 were measured not only in serum but also in RPE-choroid. As the choroidea is a highly vascularized tissue, high ESBA105 levels in choroidea were not unexpected. On the other hand, ESBA105 levels in neuroretina were considerably lower than in RPE-choroid after systemic administration, presumably due to neuroretina being an avascularized tissue and only low amounts of ESBA105 penetrating the blood-retina barrier. Most interestingly, our data presented here and in the accompanying publication ¹¹ indicate that there is both direct intrascleral delivery of ESBA105 to the retina as well as indirect delivery from the vitreal reservoir to the retina after topical administration. 

The extent of absorption of topically applied conventional low molecular weight ophthalmic drugs is between 1% and 10%. ^{19 20 21 22 23} This is considerably higher than what we report here for the 26 kDa ESBA105 molecule. However, in a therapeutic setting this lower extent of absorption should be compensated for by the higher potency and specificity of antibody therapeutics. For example, a 16-fold excess of ESBA105 over TNF-α was sufficient to block 90% of TNF-α-induced effects in vivo (manuscript in preparation). In addition, ESBA105 levels in all compartments of the rabbit eye were found to be significantly above the reported concentrations of TNF-α in various human ocular disorders (Table 4) . As an example, TNF-α concentrations in the aqueous of uveitis patients were reported to be 15 pg/mL. ²⁴ In our study, ESBA105 concentrations in aqueous reached more than 100 ng/mL during steady state conditions. This corresponds to a 10,000-fold excess over TNF-α on a weight basis (w/w) or a 6500-fold excess on a molar basis (Table 4) . Similarly, a 3600-fold excess (w/w) was reached over reported TNF-α concentrations in vitreous humor in proliferative diabetic retinopathy (Table 4) . As scFv antibodies consist of only the variable domains of natural antibodies all their amino acids are either directly or indirectly (by influencing the three-dimensional structure of the binding domain) involved in binding of the target protein. Thus, if a scFv was degraded or its structure was compromised, binding to the target would be negatively affected, which could be detected by ELISA. Therefore, in all likelihood, ESBA105 measured in ocular fluids and tissues on topical administration to the eye represents fully stable and functional scFv protein, as it appears very unlikely that instability of the scFv would not result in loss of binding activity in ELISA. Thus, in contrast to most small molecule therapeutics, ESBA105 seems to be particularly suited for the topical therapy of disorders of the back of the eye. Furthermore, in clinical settings the high local concentrations reached rapidly after starting topical administration of ESBA105, in combination with low systemic exposure, promise immediate onset of therapeutic effects combined with a low probability for adverse drug reactions due to systemic depletion of TNF-α. 

In conclusion, topical application of ESBA105 or other highly soluble and monomeric scFvs may present a safer and more efficacious therapeutic modality for as many ocular diseases as the current standard of care represents. Indeed, modeling based on our rabbit data (see also Fig. 9 ) suggest that 2, 3, 5, or 10 daily eye drops result in vitreal steady state levels of ESBA105 of 120, 200, 300, or 450 ng/mL, respectively. This illustrates that ESBA105 levels in all compartments of the eye can easily be adjusted by varying the dosing interval and/or drug concentration of eye drops. In acute inflammatory situations, the observed “depot effect” due to the prolonged half-life of ESBA105 in vitreous suggests that after an initial day with more frequent dosing (comparable to a “loading dose”) can be prolonged to dosing intervals for maintenance therapy to regimens convenient for longer-term use for patients. Similarly, the depot effect of the vitreous should allow pausing treatment over night. 

Here we have presented the results of in vivo studies of topically applied ESBA105, designed to evaluate high frequency and multi-day topical dosing regimens in rabbits. As described, for treatment of highly inflammatory conditions it is important to establish therapeutically effective drug concentrations quickly. ¹³ At high dosing frequency of 10 drops per day topically within a few hours therapeutic levels of ESBA105 are reached in all ocular compartments. Of equal importance, due to efficient penetration to and prolonged half-life in vitreous high steady state levels of ESBA105 in all disease-relevant ocular compartments can be reached after only a few days of treatment. With both, high frequency and multi-day topical treatment with ESBA105, systemic exposure was found to be very low. As ESBA105 levels are several hundred to a thousand-fold above the reported local TNF-α levels in ocular human disease (Table 4) , ESBA105 by topical delivery appears a highly attractive compound for both acute and chronic TNF-α–driven ocular conditions. As of May 2008 a first clinical trial with topical ESBA105 is ongoing. 

 Submitted for publication June 3, 2008; revised July 17 and August 5, 2008; accepted December 30, 2008.

 Disclosure: E. Furrer, ESBATech AG (E); M. Berdugo, None; C. Stella, None; F. Behar-Cohen, None; R. Gurny, None; U. Feige, ESBATech AG (I, E, C); P. Lichtlen, ESBATech AG (E, P); D.M. Urech, ESBATech AG (E, P)

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Summary of Dosing Protocols in Rabbits

	Route of Administration (Duration)	Rabbits per Time Point	Treated Eyes	Dosing Frequency (Duration)	ESBA 105 Concentration (Cumulative Dose)
High frequency dosing	Topical, one-day	n = 5	One	1 drop/hour (up to 10 hours)	10 mg/mL (up to 5 mg/day)
Systemic dosing	Intravenous, once	n = 3	None	1 × bolus	20 mg/mL (5 mg/day)
Multi-day dosing	Topical, multi-day	n = 2	Both	5 drops/day (up to 6 days)	9.6 mg/mL (up to 15 mg/6 days)

Figure 1.

 

ESBA105 is a monomer in solution. FFF analysis of ESBA105 (9 mg/mL) was performed in citrate buffer (pH 6.0). Molecular weight (red line) is superimposed to absorbance at 280 nm (dotted line). The monomer peak at 8.5 minutes shows a molecular weight of 26 kDa.

Figure 2.

 

Peak levels (C _max) of ESBA105 in ocular compartments and serum after i.v. injection or high frequency topical application. For comparison of the two routes of application the cumulative daily dose applied topically (5 mg) was injected i.v. Mean and SD are shown, C _max is given in ng/mL ESBA105. Note the logarithmic scale used for ease of data presentation.

Figure 3.

 

Comparison of peak ESBA105 concentrations (C _max) in different ocular compartments of treated eye and untreated contralateral eye. Treatments as in Figure 2 . Mean and SD are shown, C _max is given in ng/mL ESBA105.

Figure 4.

 

Comparison of ESBA105 concentrations in aqueous humor after high frequency topical or i.v. application. Treatments as in Figure 2 . Note the logarithmic scale used for ease of data presentation.

Figure 5.

 

ESBA105 concentrations in vitreous humor after high frequency topical or i.v. application. Treatments as in Figure 2 . Note the logarithmic scale used for ease of data presentation.

Figure 6.

 

ESBA105 concentrations in the neuroretina after high frequency topical or i.v. application. Treatments as in Figure 2 . Note the logarithmic scale used for ease of data presentation.

Figure 7.

 

Comparison of ESBA105 concentrations in the RPE-choroid after high frequency topical or i.v. application. Treatments as in Figure 2 . Note the logarithmic scale used for ease of data presentation.

Figure 8.

 

Comparison of ESBA105 concentrations in serum after high frequency topical or i.v. application. Treatments as in Figure 2 . Note the logarithmic scale used for ease of data presentation.

Table 2.

 

Pharmacokinetic Parameters in Different Ocular Compartments and Serum after High Frequency Topical or Systemic Administration of ESBA105

	Aqueous	Vitreous	Neuroretina	RPE-Choroid	Serum
Topical*
C max (ng/mL)	12	295	214	263	1
T max (h)	10	5	5	5	1
t 1/2 (h)	5.87	15.88	26.87	14.11	6.56
AUC(0-33) (ng-h/mL)	201	2561	2231	3779	11
AUC(0-inf) (ng-h/mL)	208	3017	3420	4644	11
Bioavailability, † (%)	0.08	1.14	1.29	1.75	0.004
Systemic
C max (ng/mL)	175	63	66	2690	89283
T max (h)	1	1	1	1	0.083
t 1/2 (h)	6.85	23.88	23.39	9.97	7.23
AUC(0-25) (ng-h/mL)	1482	492	582	23865	260898
AUC(0-inf) (ng-h/mL)	1588	735	923	27231	271176
Bioavailability, † (%)	0.60	0.28	0.35	10.26	100

*  For comparison of the two routes of application, the cumulative daily dose applied topically (5 mg) was injected intravenously. PK parameters after topical application are based on the values of the respective ocular compartment of treated eyes.

†  Based on the AUC.

Figure 9.

 

ESBA105 concentrations in different ocular compartments and serum after multi-day topical administration. (A) ESBA105 levels. At time point 0, ESBA105 was detected in very low amounts in the vitreous humor but not in the other analyzed compartments. *Serum: At time points 3 and 6 days, ESBA105 was below LOQ in sera. (B) Comparison of modeled and measured ESBA105 concentrations in vitreous humor. Modeling was performed in a spreadsheet program (Microsoft Office Excel; Microsoft Corporation) using the pharmacokinetic parameters identified in the in vivo short-term treatment study. Briefly, topically applied ESBA105 reaches the vitreous humor after a lag period of one hour following application and is eliminated from there with an elimination half life of approximately 25 hours. ¹¹ For serial applications, concentration curves of single drops were superimposed. Bioavailability based on AUC was 0.0114 (1.14%).

Table 3.

 

Pharmacokinetic Parameters in Different Ocular Compartments and Serum after Multi-day Topical Administration of ESBA105

	Aqueous	Vitreous	Neuroretina	RPE-choroid	Serum
C max (ng/mL)	153	580	917	1298	3
T max (days)	6	6	3	6	1
AUC(0-6) (ng-day/mL)	389	1361	3093	4810	5

 Steady state levels of ESBA105 achievable within 3 to 6 days suggest that more prolonged dosing intervals can be applied during long-term maintenance dosing/therapy.

Table 4.

 

Reported Concentrations of TNF-α in Different Ocular Compartments of Patients Suffering from Various Ocular Diseases Compared to ESBA105 Levels Reached in Corresponding Ocular Compartments of Rabbits Following Topical Dosing

Compartment	Ocular Disease	TNF-α (ng/mL)	Reference	ESBA105* (ng/mL) (Molar excess ESBA105 vs. TNF-α)
								High Frequency Topical Dosing 1 Day Study	Multi-day Topical Dosing 6 Day Study
Aqueous humor	Uveitis	0.015	24 25	12 (520-fold)	153 (6,500-fold)
Vitreous humor	PDR	0.160	26	295 (1,200-fold)	580 (2,340-fold)
Epiretinal membrane	PDR, PVR	∼0.500	27	214 (270-fold)	917 (1,200-fold)

 PDR, proliferative diabetic retinopathy; PVR, proliferative vitreoretinopathy.

*  ESBA105 concentration reached at the end of the study (Table 1) .