December 2012
Volume 53, Issue 13
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Retina  |   December 2012
Pharmacokinetics of Ocriplasmin in Vitreous
Author Affiliations & Notes
  • Marc D. de Smet
    From the Department of Ophthalmology, University of Amsterdam, Amsterdam, The Netherlands; the
    Retina and Inflammation Center, MIOS, Lausanne, Switzerland;
  • Bart Jonckx
    Research and Development, ThromboGenics NV, Heverlee, Belgium; and the
  • Marc Vanhove
    Research and Development, ThromboGenics NV, Heverlee, Belgium; and the
  • Joachim van Calster
    Department of Ophthalmology, University Hospitals Leuven, Belgium.
  • Peter Stalmans
    Department of Ophthalmology, University Hospitals Leuven, Belgium.
  • Jean Marie Stassen
    Research and Development, ThromboGenics NV, Heverlee, Belgium; and the
  • Corresponding author: Marc D. de Smet, Retina and Inflammation Center, MIOS, Chemin des Allinges 10, Lausanne, Switzerland 1001; mddesmet1@mac.com
Investigative Ophthalmology & Visual Science December 2012, Vol.53, 8208-8213. doi:10.1167/iovs.12-10148
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      Marc D. de Smet, Bart Jonckx, Marc Vanhove, Joachim van Calster, Peter Stalmans, Jean Marie Stassen; Pharmacokinetics of Ocriplasmin in Vitreous. Invest. Ophthalmol. Vis. Sci. 2012;53(13):8208-8213. doi: 10.1167/iovs.12-10148.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Ocriplasmin contains the active moiety of plasmin enzyme. At a physiologic pH, ocriplasmin is highly proteolytic and autolytic, limiting its duration of activity. Specific inhibitors of plasmin are present in the vitreous under normal and disease conditions and could affect its activity. Each may contribute to its mode of action.

Methods.: Degradation characteristics were determined in porcine, human vitreous, and PBS under reducing conditions with different incubation periods between 0 and 24 hours on SDS-PAGE Tris-glycine gels. Residual activity was determined by spectrophotometry of p-nitroaniline release through hydrolysis of l-pyroglutamyl-l-phenylalanyl-l-lysine-p-nitroaniline hydrochloride. The presence of endogenous inactivators of ocriplasmin in human vitreous was determined in a series of vitreous samples using an ELISA specific for alpha(2)-antiplasmin, antithrombin, and antitrypsin.

Results.: Degradation productions from autolysis are similar between vitreous and PBS with a significant prolongation of the effect in vitreous. Both follow a nonlinear pattern over time. The degradation corresponds best to a second-order kinetic process. The resulting rate constants were 207 ± 60 M−1 s−1 in PBS, 81 ± 15 M−1 s−1 in porcine vitreous, and 195 M−1 s−1 in human vitreous natural inhibitors were identified in samples of donor vitreous. Amounts differed significantly between samples, which may help explain the observed variability in human subjects.

Conclusions.: Ocriplasmin is autolytic in vitreous. Biologic activity extends to several days following injection. The exact duration will vary based on the presence and concentration of serine protease inhibitors.

Introduction
Ocriplasmin (Microplasmin; ThromboGenics NV, Leuven, Belgium), is being developed for the pharmacologic induction of a posterior vitreous detachment in patients with vitreomacular traction (VMT), including patients whose VMT was complicated by macular hole. It is a recombinant protein containing the active enzymatic site of plasmin in a truncated form. 1 It catalyzes the cleavage of peptide bonds on the carboxyterminal side of lysine and arginine protein substrates. When ocriplasmin is administered into the vitreous cavity, a dose- and time-dependent posterior vitreous detachment is observed. 25  
Early in the development process of ocriplasmin, it was recognized that pH-neutral solutions of ocriplasmin were highly autolytic, leading to a rapidly reduced enzymatic activity. 6 A similar autolytic process is quite likely to occur in the vitreous following an intravitreal injection of ocriplasmin but has yet to be characterized. In addition, both plasmin and ocriplasmin have natural inhibitors in serum, including alpha(2)-antiplasmin, antithrombin III, and alpha(1)-antitrypsin, which significantly influence their biologic activity. 7,8 These were also shown to be present in both human and porcine vitreous. 911 Such serine protease inhibitors could limit the enzyme's overall activity as a vitreolytic agent. 
Determining the pharmacokinetic characteristics of ocriplasmin in human vitreous is critical to understand its mode of action. If the enzymatic activity is of short duration, choosing the site of injection in the vitreous cavity relative to a specific retinal pathology may influence the clinical outcome, because diffusion through formed vitreous is a relatively slow, time-dependent process. 2 The presence of significant amounts of inhibitors would decrease the activity of ocriplasmin in vitreous and cause considerable variability and unpredictability in the clinical response. To answer these questions, inhibitors of ocriplasmin were quantitated in human vitreous samples, and the pharmacokinetic parameters were characterized in vitreous from freshly slaughtered pigs as well as in pooled human vitreous samples obtained at the onset of vitrectomy surgery. 
Materials and Methods
Vitreous Preparation
The porcine vitreous was extracted from fresh eyes obtained from a local slaughter house and transported on ice to the laboratory within 1 to 2 hours. A scleral incision was made with a carbon steel surgical blade at a distance of 5 to 7 mm from the corneal limbus after cleaning the eyes of surrounding tissue debris. The top of the eye was cut off, keeping the distance of 5 to 7 mm distal from the corneal limbus, and the eye was turned upside down over a new petri dish (60 × 15 mm). The vitreous fluid was gently squeezed into the petri dish, transferred to a 13-mL round-bottom tube (Sarstedt AG, Nümbrecht, Germany), and kept on ice. The porcine vitreous was homogenized by flushing it twice through a 5-mL syringe and flushing it thereafter three to five times through a 5-mL syringe fitted with an 18 gauge × 1–1½″ needle until the extracellular matrix was broken. The vitreous fluid was divided equally into two safe-lock tubes (Eppendorf International, Hamburg, Germany) and centrifuged for 5 minutes at 12,000g at 4°C. The supernatants were collected and transferred to a 13-mL polystyrene round-bottom tube (Sarstedt AG) and stored on ice or frozen until further analysis. 
Human vitreous was obtained from routine vitreo–retinal surgery procedures, kept on ice, and transported to the laboratory for further processing within 1 hour of collection. The vitreous was homogenized as described above in porcine vitreous. Samples from the various patients were pooled, aliquoted, and stored at −20°C until required. 
Determination of Degradation Characteristics of Ocriplasmin in PBS and Vitreous
Phosphate-buffered saline (PBS, 300 μL) was placed in a 1.5-mL microtube. A 27-μL aliquot was removed and mixed with 3 μL of 10−2 M d-Val-Phe-Lys chloromethyl ketone, dihydrochloride in 10−3 M HCl, a potent irreversible inhibitor of ocriplasmin. This was the blank PBS aliquot, stored frozen until analyzed. To the remaining PBS, 5 μL of ocriplasmin was added and incubated at 37°C. At time points 0, 30 minutes, and 1, 2, 3, 6, and 24 hours additional 27-μL samples were obtained and mixed with 3 μL of 10−2 M d-Val-Phe-Lys chloromethyl ketone, dihydrochloride in 10−3 M HCl before storing at −20°C until analyzed. An identical procedure was carried out on a sample of homogenized porcine vitreous prepared as described above. 
SDS-PAGE and Western blotting were carried out as described elsewhere. 6  
Briefly, to each thawed sample, 30 μL Novex Tris-glycine sample buffer (2×) (LC2676; Invitrogen/Life Technologies Corp., Carlsbad, CA) and 6 μL reducing agent (10×) (NP0009; Invitrogen/Life Technologies Corp.) was added. The samples were heated for 10 minutes at 70°C and centrifuged for 1 minute at 13,000 rpm before loading 25 μL on 4–12% Bis-Tris gel (NuPAGE, NP0335BOX; Invitrogen/Life Technologies Corp.). The gels were run for approximately 50 minutes at 200 V, using a MOPS running buffer (NP0001, NuPAGE MOPS SDS Running Buffer; Invitrogen/Life Technologies Corp.) with the addition of 0.5 mL antioxidant (NP0005; Invitrogen/Life Technologies Corp.) to the running buffer located in the inner chamber. The gel was removed from its cassette and blotted with a commercial blotting System (IB1001EU, iBlot Dry Blotting system, onto IB3010‐02, iBlot Transfer Stacks Nitrocellulose, Mini; Invitrogen/Life Technologies Corp.). 
The blot was incubated overnight at 4°C in PBS blocking buffer (37516 SuperBlock T20 Blocking Buffer; Thermo Fisher Scientific, Inc., Waltham, MA) and the next day freshly prepared primary antibody solution (2.7 mg/mL) (7H11A11; ThromboGenics NV): 7.4 μL + 20 mL SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific, Inc.), was put on the membrane for 1 hour at room temperature (RT). After washing, the membrane was incubated with 20 mL of goat anti-mouse (H+L) secondary antibody (1:1000 dilution in incubation buffer, 32430; Thermo Fisher Scientific, Inc.) for 1 hour at RT before developing with an enhanced chemiluminescence (ECL) substrate (SuperSignal West Dura; Thermo Fisher Scientific, Inc.). Image capture was performed with an imaging system (UVP 97‐0400‐02, BioSpectrum 600 Imaging System; Ultra-Violet Products, Upland, CA). 
For Coomassie blue staining, 30 μL of the sample was loaded on a 10% Bis-Tris gel (NP0301BOX, NuPAGE; Invitrogen/Life Technologies Corp.). The gels were run for approximately 50 minutes at 200 V using MES running buffer (NP0002, NuPAGE MES SDS Running Buffer; Invitrogen/Life Technologies Corp.) with the addition of 0.5 mL antioxidant (NP0005; Invitrogen/Life Technologies Corp.) to the running buffer located in the inner chamber. The gel was removed from its cassette and incubated for 1 hour in a Coomassie stain (LC6060, SimplyBlue SafeStain; Invitrogen/Life Technologies Corp.) and afterward destained in distilled water at RT before imaging (BioSpectrum 600 Imaging System). 
Determination of the Residual Ocriplasmin Activity in Porcine Vitreous
The residual concentration of active ocriplasmin was determined via hydrolysis of l-pyroglutamyl-l-phenylalanyl-l-lysine-p-nitroaniline hydrochloride (S-2403, 822254‐39; Chromogenix, Milan, Italy) leading to a release of p-nitroaniline and an increased spectrophotometric absorption at 405 nm (UV-Vis 96-well plate reader, SpectraMax 190 Microplate Reader; Molecular Devices Corp., Sunnyvale, CA). The rate of formation of this chromagen (i.e., the increase in absorbance) per second was proportional to the enzymatic activity of ocriplasmin measured by microplate spectrophotometry at 5 minutes following the addition of substrate to the incubation mixture. 
Ocriplasmin concentrations in porcine vitreous fluid or PBS were calculated via a conversion factor representing the ΔmAbs/min value that corresponds to the ΔmAbs/min value by 1 nM ocriplasmin. For determination of the conversion factor, ocriplasmin solutions of 10, 20, and 40 nM (equivalent to 272, 545, and 1090 ng/mL) were prepared by dilution of ocriplasmin 4.0 mg/mL with NaCl-citrate buffer (n = 3 per ocriplasmin vial). Standard solutions (30 μL each) were added to 240 μL of preincubated (for at least 5 minutes) Tris buffer (37°C) placed on 96-well plates. After equilibration, 30 μL of substrate solution (3 mM S-2403) were added and gently mixed. The absorbances at 405 nm were measured every 20 seconds with a microplate spectrophotometer up to 5 minutes and transformed into values expressing the increase of absorbance/min (ΔmAbs/min) using the software associated with the microplate reader (Molecular Devices Corp.). The increase in absorption was measured four times and the mean values were used in the calculation of the ΔmAbs/min values corresponding to a concentration of 1 nM ocriplasmin (equivalent to 27.2 ng/mL). 
The experimental samples were obtained as follows: 100 μL of each formulation of 0.5, 1.25, 1.75, and 2.50 mg/mL ocriplasmin (n = 3) were added to the supernatant of homogenized porcine vitreous (2.1–3.0 mL), resulting in concentrations of 16 to 114 μg/mL and incubated at 37°C. Immediately after and 15, 30, and 45 minutes as well as 1, 2, 3, 4, 5, 6, 7, and 24 hours postaddition of ocriplasmin, the concentrations of active ocriplasmin were determined as follows. The vitreous fluid samples were diluted with NaCl-citrate buffer as follows: 1:50: 125, 175, and 250 μg dose groups, and 1:12.5: 50 μg dose group. Each diluted vitreous fluid (30 μL) was added to 240 μL of preincubated (for at least 5 minutes) Tris buffer (37°C) placed on 96-well plates. After equilibration, 30 μL of substrate solution (3 mM S-2403) was added and gently mixed. The absorbances at 405 nm were measured every 20 seconds with a microplate spectrophotometer (Molecular Devices Corp.) up to 5 minutes and transformed into values expressing the increase of the absorbance/min (ΔmAbs/min) using the software associated with the microplate reader (Molecular Devices Corp.). The increase in absorption was measured eight times and the mean values were divided by the conversion factor and multiplied by 125 (50-μg dose group) and 500 (125-, 175-, and 250-μg dose groups) to compensate for the respective dilutions during the overall procedure. Finally, these values were multiplied by the molecular weight to obtain the recovery expressed in μg/mL. 
Determination of the Residual Activity of Ocriplasmin in Human Vitreous
Ocriplasmin (125 μg) was added to a final concentration of 40.3 μg/mL of pooled vitreous in accordance with the proposed human clinical dosing strategy. The sample was vortexed and incubated at 37°C for up to 72 hours. Samples were taken immediately after 5, 15, 30, and 45 minutes and 1, 2, 3, 4, 5, 24, 48, and 72 hours after addition of ocriplasmin. The samples were diluted in 5 mM CAM-NaCl buffer as follows: 1:16 at time points 0, 45, 30, and 45 minutes and 1, 2, 3, 4, and 5 hours; 1:6 at time point 24 hours; 1:3 at time point 48 hours; 1:1.5 at time point 72 hours. The concentrations of active ocriplasmin were determined via the bioassay methodology described above. Experiments were repeated independently twice on the same pool of human vitreous. 
Determination of the Presence of Endogenous Inactivators of Ocriplasmin in Human Vitreous
ELISAs specific for alpha(2)-antiplasmin, antithrombin, and antitrypsin were developed (ThromboGenics NV) using in-house–generated monoclonal antibodies. Briefly, the primary antibody was coated overnight at 4°C in PBS. A human vitreous sample was applied and incubated for 2 hours at RT before incubating with a matched horseradish peroxidase (HRP)–labeled secondary antibody against the protein of interest. Colorimetric detection was performed using OPD. 
Results
We first compared the rate of degradation of ocriplasmin in PBS versus homogenized porcine vitreous. On SDS-PAGE gels stained with Coomassie blue, the nondegraded ocriplasmin could be observed around 27.2 kDa. Over time at +37°C and at pH 7.2, the intact ocriplasmin band decreased in intensity under reducing conditions, whereas the two main cleavage products of a lower molecular weight of approximately 16 and 9 kDa, respectively, were formed by cleavage of peptide bond 155 Leucine–156 Lysine. 12 Cleavage of ocriplasmin peptide bond 155 Leucine–156 Lysine fully inactivates the enzyme (data not shown). A very low intensity product could be seen at approximately 3 kDa, most likely corresponding to the 19 amino acids released from ocriplasmin's heavy chain under reducing conditions. Because these cleavage products are kept together by disulfide bridges, these bands could be detected only under such conditions. In Western blot using the monoclonal antibody 7H11A11, only the nondegraded ocriplasmin and the degradation band at 16 kDa could be identified. Smaller fragments were not seen on the Western blot, likely due to the loss of the recognition epitope for the primary antibody. Decrease of intensity of the intact ocriplasmin band followed a nonlinear pattern over time in both PBS and porcine vitreous (Fig. 1). Although the degradation fragments observed on Coomassie-stained gels were identical in both matrices, the rate of degradation in vitreous was significantly prolonged compared with that of PBS. 
Figure 1. 
 
Western blot of ocriplasmin inactivation samples under reducing conditions taken at various time points after addition to PBS (upper lane) or porcine vitreous. Time points following addition of ocriplasmin are indicated above. Staining is provided by the monoclonal antibody 7H11A11.
Figure 1. 
 
Western blot of ocriplasmin inactivation samples under reducing conditions taken at various time points after addition to PBS (upper lane) or porcine vitreous. Time points following addition of ocriplasmin are indicated above. Staining is provided by the monoclonal antibody 7H11A11.
To measure the rate of inactivation of ocriplasmin, a bioassay was developed where the release via hydrolysis of the chromogenic substrate S-2403 was analyzed. Upon interaction of ocriplasmin with S-2403 during an incubation at 37°C, the chromogen p-nitroaniline is released in direct proportion to the concentration of the enzyme, leading to an increase of the absorbance at 405 nm. The mean increase of the absorbance (ΔmAbs/min) at 405 nm by 1 nM ocriplasmin calculated from the values obtained from standard solutions containing 10, 20, and 40 nM ocriplasmin (n = 3) was 22.123 ± 2.533. The concentration of active ocriplasmin decreased rapidly in PBS, leading to a residual concentration at the end of a 24-hour incubation period of between 1% and 5% of the initial concentration (Fig. 2A). Similarly, the activity of residual ocriplasmin in vitreous was determined. Figure 2B shows the ocriplasmin [M] (mean ± SEM) versus time profile in porcine vitreous fluid for different starting concentrations of ocriplasmin. The concentration of active ocriplasmin decreased more slowly than that in PBS, leading to a residual concentration at the end of 24 hours of between 8% and 24%. 
Figure 2. 
 
Concentrations of active ocriplasmin in PBS (A) or porcine vitreous fluid (B) [M] and data fits following the addition of 0, 50, 125, 175, and 250 μg ocriplasmin and incubation at 37°C (means ± SEM, n = 3).
Figure 2. 
 
Concentrations of active ocriplasmin in PBS (A) or porcine vitreous fluid (B) [M] and data fits following the addition of 0, 50, 125, 175, and 250 μg ocriplasmin and incubation at 37°C (means ± SEM, n = 3).
The best fit for linearization of the concentration curve over time is obtained by using a concentration−1 versus time plot, indicating that inactivation of ocriplasmin is subject to a second-order kinetic process. 12 The resulting rate constant is 207 ± 60 M−1 s−1 in PBS (n = 3), whereas in vitreous it is 81 ± 15 M−1 s−1 (n = 3). 
The kinetics of inactivation of ocriplasmin in homogenized pooled human vitreous was determined in a similar fashion at a fixed ocriplasmin concentration. We chose to study the effect of a concentration equivalent to an intravitreal injection of 125 μg, which would be of highest clinical relevance. As in the pig, the inactivation in human vitreous followed a second-order rate constant. The concentration of active ocriplasmin decreased to a residual concentration at 24 hours of 2%, further decreasing to <0.6% of the initial concentration at the end of the incubation period (72 hours). The average rate constant in human vitreous was calculated at 195 M−1 s−1 (n = 2) (Fig. 3). 
Figure 3. 
 
Concentrations of active ocriplasmin in pooled human vitreous fluid following the addition of 125 μg ocriplasmin and incubation at 37°C (mean ± SEM, n = 2).
Figure 3. 
 
Concentrations of active ocriplasmin in pooled human vitreous fluid following the addition of 125 μg ocriplasmin and incubation at 37°C (mean ± SEM, n = 2).
Some preliminary experiments indicated that the initial loss of activity might be related to the presence of natural inhibitors within human vitreous fluid. To explore this further, quantifications by ELISA of antiplasmin, antithrombin III, and alpha(1)-antitrypsin levels were obtained in human donor vitreous. Levels for alpha(2)-antiplasmin were found to range between 0 and 520 nM (mean ± SEM: 64 ± 39 nM); antithrombin 1 and 2187 nM (371 ± 176 nM), and antitrypsin 21 and 11,718 nM (1490 ± 940 nM) (Table). Western blots of the vitreous solution admixed with ocriplasmin demonstrated the presence of higher-molecular-weight inhibitor–ocriplasmin complexes (not shown). 
Table. 
 
Quantification by ELISA of Natural Inhibitors of Ocriplasmin in Human Donor Vitreous
Table. 
 
Quantification by ELISA of Natural Inhibitors of Ocriplasmin in Human Donor Vitreous
Sample
ID
Vitreous Concentration, nM
Antiplasmin Antithrombin Antitrypsin
2 102 431 914
11 1 11 60
13 2 34 160
14 1 138 1,257
15 60 532 1,233
16 0 1
17 0 12 29
18 0 21
19 36 314 11,718
20 2 20 137
21 77 555 891
22 20 214 857
24 520 2187 602
Average concentration 64 371 1,490
SEM 39 176 940
Discussion
Ocriplasmin is being developed for use in retinal diseases where it plays an important role in inducing vitreous separation from the retinal surface. At the vitreo–retinal interface collagen fibers run parallel to the retinal surface. They are anchored by means of binding proteins including laminin and fibronectin to the inner limiting lamina of the retina, and thus play a major role in vitreoretinal attachment. 1315 Serine proteases, including ocriplasmin are able to degrade a variety of structural and binding proteins including laminin and fibronectin from cell basement membranes. 16,17 Ocriplasmin was shown in both preclinical models and clinical studies to induce separation of the vitreous, allowing even a nonsurgical resolution of focal vitreomacular traction in a number of patients (see de Smet MD, et al. IOVS 2009;50:ARVO E-Abstract 6237). 3,5,18 All serine proteases are highly autolytic; therefore, knowledge of the kinetics in vitreous is essential in predicting and assessing its clinical effect. 
The activity of ocriplasmin is pH dependent. An acid environment prevents serine deprotonation, which in turn limits interactions with potential substrates. 19 At a neutral pH, it will direct its activity against any protein including itself. The difference in activity between saline and liquefied vitreous gel is in this respect not surprising. In the presence of substrate other than itself, the rate of autolysis will be decreased as a function of the availability of substrate. Thus, ocriplasmin in PBS has a rate constant of 207 ± 60 M−1 s−1, whereas in vitreous the rate constant is 81 ± 15 M−1 s−1. The activity in PBS provides the lower limit of the possible enzymatic activity that can be expected with ocriplasmin in a clinical setting. This would correspond to its effect when injected at the end of a vitrectomy procedure in a PBS filled, or balanced salt solution–filled eye. 
Plotting concentration−1 versus time linearizes the slope of the decay curve. Such linearization is classically associated with chemical reactions respecting second-order kinetics. Autoproteolytic reactions such as the decay of ocriplasmin are best represented by a second-order kinetic process. 12,20,21 From a practical standpoint, second-order kinetics imply a concentration-dependent inactivation, with faster rates of degradation being observed at higher concentrations. Although there may be no practical way to reduce the equilibrium between folded and unfolded ocriplasmin in the vitreous, the presence of a high concentration of vitreous substrates will reduce the rate of autolysis. In addition, if ocriplasmin can diffuse quickly away from the site of injection, autolysis will be reduced. Diffusion in formed vitreous is relatively slow, but will be more important in patients that have partial or complete vitreous schisis. 2  
A direct comparison between human and porcine vitreous is not possible in the current set of experiments. The protein concentrations between the two solutions were not standardized. Differences in rates may also be due to inherent variations in the protein sequence of vitreous proteins and their affinity for ocriplasmin. Differences in viscosity, thus integrity of the vitreous, may also influence the rate of autolysis because the pooled vitreous came from diseased, relatively aged individuals rather than healthy young eyes as is found in the slaughtered pigs. At best, we can state that the process in both settings follows the same kinetic process, as might be expected. 
In young individuals, baseline levels of plasmin are in the order of 1 nM, whereas above 50 years of age, these increase to 4 nM. 22 Based on our measured kinetic constant of 195 M−1 s−1 in human vitreous, second-order kinetics, and an average vitreous volume of 4 mL, a 125-μg dose of ocriplasmin can be expected to maintain biologic activity between 16 and 42 days. The former figure is close to the observed period of clinical benefit. Although many of the patients included in the clinical trials were more than 50 years of age, the presence of macular traction might indicate a lower concentration of vitreous serine proteases than that of similar patients without traction, or possibly the presence of higher levels of serine protease inhibitors within the vitreous cavity. Studies will be required to elucidate this further. 
Inhibitors of serine proteases are present in human vitreous. The presence of alpha(1)-antitrypsin (AAT), a member of the serpin family of protease inhibitors, was previously identified in human vitreous. 911 We confirm the presence of alpha(2)-antiplasmin and antithrombin III within human vitreous. The ability of these to inhibit ocriplasmin activity was not assessed in the current study. To assess the role of inhibitors, a representative number of samples taken from patients with target vitreo–retinal pathologies would be required. The levels of inhibitors reported in this study, pooled from eyes with unspecified ocular pathology, such as macular holes, puckers, and diabetic retinopathy, and most likely not representative of the normal state, 11 demonstrated that the level of AAT upregulation clustered with specific vitreo–retinal disease entities. For nonproliferative diabetic retinopathy, AAT overexpression compared with control was 4.64; in proliferative diabetic retinopathy = 4.84; in rhegmatogenous retinal detachment = 8.95; and in proliferative vitreoretinopathy = 15.59. A proteomic analysis of vitreous comparing diabetics with or without proliferative retinopathy demonstrated that there were significant changes in the type and degree of protein upregulation. In nonretinopathy patients, both plasminogen and serine protease inhibitors were upregulated by the same order of magnitude. However, in diabetic retinopathy patients, the upregulation of the inhibitors is up to 3-fold higher than the upregulation of plasminogen, resulting in a relative reduction of vitreous plasminogen levels. 10  
Of all the inhibitors mentioned in the Table, only alpha(2)-antiplasmin is known to effectively and completely inhibit ocriplasmin. 23 However, the amount of alpha(2)-antiplasmin present (64 nM) is sufficient to inhibit only approximately 4% of the ocriplasmin injected (1500 nM). AAT and antithrombin are described as slow (progressive) inhibitors of plasmin. 24 The concentrations we report in the vitreous, 370 and 1480 nM, respectively, are sufficient to potentially inhibit a large portion of the administered ocriplasmin. However, the total amount of inhibition observed in this study was limited because the initial activity of ocriplasmin in vitreous was assessed at between 93% to 104%, suggesting that antitrypsin and antithrombin are not potent inhibitors of ocriplasmin under the tested conditions. Thus, although we have shown the presence of potential inhibitors of ocriplasmin, more extensive analysis of vitreous would be required to determine their exact effect on the activity of ocriplasmin. 
In conclusion, ocriplasmin has a high autolytic activity in vitreous. The presence of partially liquefied vitreous may reduce the rate of autolysis. Attempting to place the enzyme deep inside the vitreous cavity, preferably in an area that has already undergone liquefaction, may prolong the duration of action of the enzyme and allow a more complete diffusion into the vitreous cavity. Injecting the enzyme close to the site of vitreomacular traction may also enhance the biologic effect. However, caution should be taken to assess the potential benefit of deep vitreal placement versus the potential to elicit more (prolonged) side effects. Biologic activity in vitreous appears to last between 16 to 42 days. The duration of biologic activity may be dependent on endogenous levels of serine protease inhibitors, which will require more elaborate studies to elucidate. 
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Footnotes
 A portion of this work has been published previously as an ARVO abstract: de Smet MD, et al. IOVS 2009;50:ARVO E-Abstract 6237.
Footnotes
 Disclosure: M.D. de Smet, ThromboGenics NV (F, I, C, R), P; B. Jonckx, ThromboGenics NV (I, E); M. Vanhove, ThromboGenics NV (I, E); J. van Calster, ThromboGenics NV (F, I, E); P. Stalmans, ThromboGenics NV (F, R); J.M. Stassen, ThromboGenics NV (I, E)
Figure 1. 
 
Western blot of ocriplasmin inactivation samples under reducing conditions taken at various time points after addition to PBS (upper lane) or porcine vitreous. Time points following addition of ocriplasmin are indicated above. Staining is provided by the monoclonal antibody 7H11A11.
Figure 1. 
 
Western blot of ocriplasmin inactivation samples under reducing conditions taken at various time points after addition to PBS (upper lane) or porcine vitreous. Time points following addition of ocriplasmin are indicated above. Staining is provided by the monoclonal antibody 7H11A11.
Figure 2. 
 
Concentrations of active ocriplasmin in PBS (A) or porcine vitreous fluid (B) [M] and data fits following the addition of 0, 50, 125, 175, and 250 μg ocriplasmin and incubation at 37°C (means ± SEM, n = 3).
Figure 2. 
 
Concentrations of active ocriplasmin in PBS (A) or porcine vitreous fluid (B) [M] and data fits following the addition of 0, 50, 125, 175, and 250 μg ocriplasmin and incubation at 37°C (means ± SEM, n = 3).
Figure 3. 
 
Concentrations of active ocriplasmin in pooled human vitreous fluid following the addition of 125 μg ocriplasmin and incubation at 37°C (mean ± SEM, n = 2).
Figure 3. 
 
Concentrations of active ocriplasmin in pooled human vitreous fluid following the addition of 125 μg ocriplasmin and incubation at 37°C (mean ± SEM, n = 2).
Table. 
 
Quantification by ELISA of Natural Inhibitors of Ocriplasmin in Human Donor Vitreous
Table. 
 
Quantification by ELISA of Natural Inhibitors of Ocriplasmin in Human Donor Vitreous
Sample
ID
Vitreous Concentration, nM
Antiplasmin Antithrombin Antitrypsin
2 102 431 914
11 1 11 60
13 2 34 160
14 1 138 1,257
15 60 532 1,233
16 0 1
17 0 12 29
18 0 21
19 36 314 11,718
20 2 20 137
21 77 555 891
22 20 214 857
24 520 2187 602
Average concentration 64 371 1,490
SEM 39 176 940
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