Posterior vitreous detachment with microplasmin alters the retinal penetration of intravitreal bevacizumab (Avastin) in rabbit eyes.

PURPOSE: Intravitreal bevacizumab (BV) (Avastin, Genentech Inc., South San Francisco, CA) is frequently used for the treatment of age-related macular degeneration. Previous studies have demonstrated full-thickness retinal penetration. Intravitreal recombinant microplasmin (MP) has been shown to successfully induce a posterior vitreous detachment (PVD) and vitreous liquefaction in animals. It has been suggested that a PVD may alter the retinal penetration of molecules in the vitreous cavity. The aim of this study was to compare BV retinal penetration in rabbit eyes with and without an MP-induced PVD. METHODS: Twelve adult rabbits were injected with 0.1 mL (0.4 mg) of MP into the vitreous cavity of 1 eye. One week later, the rabbits were injected with 0.05 mL (1.25 mg) of BV into both eyes. Both eyes of 3 rabbits were harvested at 6 hours, 12 hours, 24 hours, and 72 hours after the BV injection. Frozen retinal cross sections were prepared, and BV retinal penetration was evaluated with immunohistochemistry using a fluorescence-labeled antibody against BV. Two eyes from one rabbit were not injected with either agent and used as controls to compare the background autofluorescence. Peripapillary retinal sections were recorded with a digital camera, and intraretinal BV fluorescence-labeled antibody was measured by qualitative photographic interpretation. Two additional rabbits received an intravitreal injection of 0.1 mL of MP in 1 eye. One week later, both eyes from each rabbit were enucleated, and frozen retinal sections were prepared and analyzed with light microscopy to evaluate histologic damage. RESULTS: Full-thickness BV retinal penetration was observed throughout the retina in both eyes of each rabbit. All the MP-injected eyes exhibited increased antibody labeling in retinas evaluated at 6 hours, 12 hours, and 24 hours after BV injection when compared with the contralateral non-MP-injected eyes. By 3 days after BV injection, all eyes demonstrated decreased antibody labeling compared with earlier periods. At 3 days, 1 rabbit showed increased antibody labeling in the retina of the non-MP-injected eye compared with the contralateral MP-injected eye, and 2 rabbits exhibited similar antibody labeling in both eyes. When compared with control eyes, light microscopy demonstrated normal retinal histologic findings in eyes injected only with MP. CONCLUSION: Increased BV retinal penetration is observed initially in eyes with an MP-induced PVD, and the mechanism is likely multifactorial. By 3 days, retinal penetration is similar in eyes with and without a PVD. Although it is difficult to directly extrapolate to humans, our study suggests that a PVD may alter the retinal penetration of BV.

A disposable system for rapid purification of autologous plasmin as an adjunct to vitrectomy - performance and safety profile.

BACKGROUND: The generation of an atraumatic posterior vitreous detachment (PVD), a common goal in vitreoretinal surgery, is a challenge particularly in children and young trauma patients. Plasmin has been proposed as a surgical adjunct to enzymatically generate a PVD. This study assesses the performance and safety of a new system for rapid purification of plasmin as an adjunct to vitrectomy. METHODS: Plasminogen was isolated from human plasma by affinity chromatography using a disposable rapid purification kit, and activated to plasmin with streptokinase. Activities were assessed spectrophotometrically. For safety studies, 38 rabbits received intravitreal injections of one of the following compounds in 0.1 ml respectively: 4.7, 12.7 and 24 IU plasmin, 15 mg dextran, 4,100 U streptokinase, 500 µg ε-aminocaproic acid, 0.1 M potassium phosphate or balanced salt solution (BSS). Thirty min after injection, a two-port vitrectomy was performed. Rabbits were followed clinically and with bright flash electroretinography (ERG) for up to 9 months. The eyes were investigated by light and transmission electron microscopy. RESULTS: The specific plasmin activity obtained from blood of healthy volunteers averaged 42.3 ± 6.6 IU/ml (range 21.6 IU/ml to 54.5 IU/ml). The identity and purity of the enzyme was confirmed by several methods. Clinically, a mild to moderate inflammatory response was seen in most eyes on day 1, but had disappeared by day 7. ERG showed moderate depressions of a- and b-wave amplitudes on day 2, particularly in the potassium phosphate (a: -29.16 ± 4.56, b: -21.23 ± 6.31), 4.7 (a: -34.38 ± 6.64, b: -26.66 ± 6.06) and 24 IU (a: -38.25 ± 4.05, b: -23.38 ± 4.29) plasmin groups, but also in the BSS- (a: -11.19 ± 21.78, b: -11.41 ± 15.47) and dextran- (a: -17.86 ± 14.18, b: -6.67 ± 18.14) treated eyes. ERG changes recovered during follow-up. One rabbit each from the 12.7 and the 24 IU plasmin groups showed a minimal discoloration of one medullary ray after 9 months. Histology did not reveal morphologic signs of toxicity. CONCLUSION: The isolation system generated plasmin with a high degree of purity. A failure-mode analysis did not reveal significant risks of toxicity. A single preparation can provide a maximum dose of 10.9 IU/200 µl, the likely target clinical dose being 1.88 IU. Plasmin doses of at least 12.7 IU appear be safe when injected into rabbit eyes, followed by vitrectomy.

Nonclinical safety and pharmacokinetics of intravitreally administered human-derived plasmin in rabbits and minipigs.

The use of plasmin for pharmacologic vitreolysis and the creation of a posterior vitreous detachment offers several potential advantages over surgery. The nonclinical pharmacokinetics and safety of human-derived plasmin was evaluated following single or multiple intravitreal injections to rabbits and minipigs. Single intravitreal injections of plasmin at 45-900 microg resulted in a no adverse effect level (NOAEL) of 45 microg in both species; effects at higher doses included chemosis, mucopurulent discharge, mononuclear cell infiltrates in the iris-ciliary body, and reversible changes in electroretinogram waveforms and parameters. No retinal histopathology abnormalities were observed. Following 4 weekly intravitreal injections at 4-423 microg, a NOAEL of 4 microg was identified. Effects at the higher doses included myosis, iritis, iridolenticular synechiae, and changes in electroretinogram waveforms and parameters that were generally not reversible in the present investigation. Vitreal plasmin concentrations were highest at 30 min after dosing and decreased rapidly; measurable concentrations remained, in some animals, at 24 h. Intravitreal plasmin exposure increased in a less-than-dose-proportional manner and tended to be lower in minipigs than in rabbits. The current findings demonstrate acceptable nonclinical safety and pharmacokinetics of intravitreal human plasmin in rabbits and minipigs and support the clinical development of plasmin for ocular diseases.

Comparative effects of microplasmin and tissue-type plasminogen activator (tPA) on cerebral hemorrhage in a middle cerebral artery occlusion model in mice.

BACKGROUND: Thrombolytic therapy of ischemic stroke with tissue-type plasminogen activator (tPA) improves clinical outcome which may, however, be partially offset by significant intracerebral bleeding (ICB). OBJECTS: The comparative effects of microplasmin (microPli) and tPA on ICB were evaluated in a thrombotic middle cerebral artery (MCA) occlusion model in mice. METHODS: A dose of microPli (5 mg kg(-1)) or tPA (4 mg kg(-1)) which are comparably effective for reduction of brain damage, or a double dose (10 or 8 mg kg(-1), respectively) or the microPli excipient as a control were intravenously administered as a bolus at 30 min or 4 h after MCA occlusion. ICB was measured at 24 h by hemoglobin assay of exsanguinated brain extracts. Bleeding time was measured by tail cutting. RESULTS: In controls given solvent at 4 h, ICB was on average 8.8 micro L, which was significantly increased with 10 mg kg(-1) microPli and with 4 and 8 mg kg(-1) tPA to 12-13 micro L (P < 0.05 each vs. controls, n = 7-9), whereas 5 mg kg(-1) microPli did not affect bleeding (8.5 micro L P = NS vs. controls, n = 7). When given at 30 min, neither microPli nor tPA altered ICB (6.3-6.8 micro L, mean; n = 7-9). tPA but not microPli increased bleeding time; from 2.4 min in controls to 5.9 min (median, P < 0.05 vs. controls) and 8.7 min (P < 0.01 vs. controls) with 4 and 8 mg kg(-1), respectively, and to 2.3 and 2.9 min with 5 and 10 mg kg(-1) microPli, respectively (n = 10). CONCLUSIONS: microPli at a dose comparably effective as tPA for brain damage reduction induced significantly less ICB, and less bleeding time prolongation in mice with thrombotic MCA occlusion.

Distinct dose-dependent effects of plasmin and TPA on coagulation and hemorrhage.

All thrombolytic agents in current clinical usage are plasminogen activators. Although effective, plasminogen activators uniformly increase the risk of bleeding complications, especially intracranial hemorrhage, and no laboratory test is applicable to avoid such bleeding. We report results of a randomized, blinded, dose-ranging comparison of tissue-type plasminogen activator (TPA) with a direct-acting thrombolytic agent, plasmin, in an animal model of fibrinolytic hemorrhage. This study focuses on the role of plasma coagulation factors in hemostatic competence. Plasmin at 4-fold, 6-fold, and 8-fold the thrombolytic dose (1 mg/kg) induced a dose-dependent effect on coagulation, depleting antiplasmin activity completely, then degrading fibrinogen and factor VIII. However, even with complete consumption of antiplasmin and decreases in fibrinogen and factor VIII to 20% of initial activity, excessive bleeding did not occur. Bleeding occurred only at 8-fold the thrombolytic dose, on complete depletion of fibrinogen and factor VIII, manifest as prolonged primary bleeding, but with minimal effect on stable hemostatic sites. Although TPA had minimal effect on coagulation, hemostasis was disrupted in a dose-dependent manner, even at 25% of the thrombolytic dose (1 mg/kg), manifest as rebleeding from hemostatically stable ear puncture sites. Plasmin degrades plasma fibrinogen and factor VIII in a dose-dependent manner, but it does not disrupt hemostasis until clotting factors are completely depleted, at an 8-fold higher dose than is needed for thrombolysis. Plasmin has a 6-fold margin of safety, in contrast with TPA, which causes hemorrhage at thrombolytic dosages.

Endothelial cell injury induced by plasmin in vitro.

We investigated the effect of plasmin on the integrity and function of endothelial cells to elucidate the mechanism by which bleeding or rethrombosis may be induced in thrombolytic therapy. When incubated with cultured human umbilical vein endothelial cells (HUVECs), plasmin increased the endothelial permeability to serum albumin 10 minutes after the incubation. Plasmin damaged the cell membranes 30 minutes after the incubation, detached the cells from the matrix 3 hours after the incubation, and finally induced cell lysis. Such damaging effects on HUVECs were not observed with plasminogen or plasmin pretreated with aprotinin and alpha 2-plasmin inhibitor, suggesting that the catalytic function of plasmin plays an important role in inducing this damage. Sulfur 35-labeled glycosaminoglycans (35S-GAGs) of HUVECs were decreased 1 hour after the incubation of plasmin with HUVECs, and the thrombomodulin (TM) activity of HUVECs measured by protein C activation capacity was decreased 6 hours after the incubation. Neither 35S-GAGs nor the TM activity of HUVECs was decreased after the incubation of plasmin pretreated with aprotinin and alpha 2-plasmin inhibitor. These findings suggest that the nonthrombogenic properties of endothelial cells can be damaged by the proteolytic action of plasmin. Our findings, taken together, suggest that plasmin damages the endothelial barrier function, endothelial cell integrity, and nonthrombogenic properties. These damaging effects of plasmin on endothelial cells may be related to the pathophysiology of bleeding or rethrombosis observed in patients undergoing high-dose thrombolytic therapy for thrombosis.