Use of recombinant human ocella in the preparation of a medicament for treating choroidal vascular disease

Abstract

The present invention relates to a new use of recombinant human ocella plasmin in the preparation of a medicament for the treatment and / or prevention of choroidal vascular diseases.

Description

Technical Field

[0001] This invention relates to pharmaceutical use for treating or preventing complications of human retinal diseases or conditions. More specifically, this invention relates to the use of truncated plasminogen lysate in the preparation of medicaments for treating or preventing human retinal diseases or complications of such diseases. Background Technology

[0002] Polypoidal choroidal vasculopathy (PCV) is a choroidal vascular disease characterized by orange-red choroidal retinal edema in the macular region of the fundus. Some patients may also exhibit extensive subretinal hemorrhage in the macular region, hard retinal exudates, and indocyanine green fundus angiography (ICGA) revealing polypoidal choroidal vascular lesions, abnormal branching vascular networks (BVN), and terminal aneurysmal dilated nodules (polyps). This leads to recurrent episodes of hemorrhagic and serous macular degeneration and multiple retinal pigment epithelium detachments (PED), severely impacting visual acuity. This disease was first described in the early 1990s and primarily occurs in Asian populations.

[0003] PCV is prone to causing hemorrhage, with 30.0% to 63.6% of patients experiencing subretinal hemorrhage. The location of polypoid lesions also varies; in Asian patients, 92% of polypoid lesions are distributed in the macula, while in European patients, polypoid lesions are evenly distributed around the optic disc and in the macula.

[0004] Currently, no drugs for treating polypoidal choroidal vasculopathy (PCV) have been approved for marketing in China or abroad. The current standard treatment for PCV is photodynamic therapy (PDT) combined with anti-VEGF drugs. However, studies have shown that PDT can lead to recurrent hemorrhages and potentially cause vision loss in a significant proportion of patients (HIRAMI et al. Hemorrhagic complications after photodynamic therapy for polypoidal choroidal vasculopathy. Retina: March 2007, Volume 27, Issue 3, p335-341, doi:10.1097 / 01.iae.0000233647.78726.46). The risk of massive subretinal hemorrhage after PDT has led researchers to consider less invasive medical interventions, especially anti-VEGF therapy; however, the results of anti-VEGF therapy have been mixed. Although one study showed that intravitreal bevacizumab injection caused a temporary reduction in exudation, indocyanine green angiography (ICGA) still detected choroidal vascular abnormalities in 10 out of 11 eyes (F Gomietal. Efficacy of intravitreal bevacizumab for polypoidal choroidal vasculopathy. British Journal of Ophthalmology 2008; 92:70–73. doi:10.1136 / bjo.2007.122283). Although bevacizumab can stabilize vision and reduce exudative retinal detachment, photodynamic therapy (PDT) remains essential for treating polyposis-like lesions (TYY Lai et al. Intravitreal bevacizumab (Avastin) with or without photodynamic therapy for the treatment of polypoidal choroidal vasculopathy. British Journal of Ophthalmology 2008; 92:661–666. doi:10.1136 / bjo.2007.135103). Given the issues with the efficacy and safety of the aforementioned therapies and the significant clinical need arising from the high incidence of PCV, there is an urgent need to develop drugs with new targets or mechanisms of action.

[0005] Human plasmin is composed of 791 amino acids, with a molecular weight of approximately 88 kDa, forming two chains linked by disulfide bonds between the heavy and light chains. Recombinant human octoplasmin, as a recombinant serine protease plasmin that retains only the active enzyme fragment, is the original product. Approved by the European Medicines Agency for the treatment of symptomatic vitreomacular adhesions, vitreomacular traction, and macular holes with a diameter ≤400 μm associated with macular traction in adults, this drug induces posterior vitreous detachment by promoting vitreous liquefaction and reducing vitreomacular adhesions, thus avoiding the drawbacks of surgical posterior vitreous detachment such as large trauma and incomplete detachment. Recombinant human octanoplasticase possesses the proteolytic catalytic activity of plasmin and is more stable than human plasmin.

[0006] In the process of developing recombinant human octofibrinolytic enzyme, the inventors of this application innovatively applied it to the treatment of PCV and were pleasantly surprised to find that octofibrinolytic enzyme had a significant effect on improving PCV-related indicators. It showed advantages in both safety and efficacy, laying the foundation for the development of subsequent indications. Summary of the Invention

[0007] The purpose of this invention is to provide new uses for truncated plasmin that retains the enzyme's active domain, specifically new uses for recombinant human octanoic plasmin.

[0008] Unbound by any theoretical constraints, the application of recombinant human octokinase with preserved enzyme active fragments in the treatment of PCV patients can significantly improve related symptoms. On one hand, it can reduce the osmotic pressure within polypoid vascular clusters, thereby reducing leakage and bleeding; on the other hand, it can dissolve blood clots outside the vascular clusters, promoting the removal of submacular hemorrhage, thus achieving the goal of treating PCV, especially hemorrhagic idiopathic PCV. Therefore, this invention provides the use of truncated plasmin with preserved enzyme active domains in the preparation of drugs for the prevention or treatment of PCV.

[0009] According to the present invention, the truncated plasmin that retains the enzyme's active domain refers to a plasmin protein produced by deleting amino acids 79-791 from one or more human plasminogen esters, and the resulting protein has the catalytic activity of a serine protease. Preferably, it is a recombinant human octoplasmin that retains only the C-terminal 249 amino acids of plasminogen esters, having a heavy chain of 19 amino acids as shown in SEQ ID NO:1 (although this peptide chain is short, it originates from the heavy chain of human plasmin, and is therefore still referred to as the heavy chain), and a light chain of 230 amino acids as shown in SEQ ID NO:2. The heavy chain and light chain are linked by two disulfide bonds, and the light chain also contains four disulfide bonds (e.g., ...). Figure 4 (As shown).

[0010] According to the present invention, "choroidal vascular disease" refers to a general term for lesions such as choroidal ischemia and choroidal hemorrhage caused by various reasons. It is a common ophthalmic disease that can lead to blindness and is characterized by pathological changes such as changes in the morphology of blood vessel walls, arteriosclerosis, leakage, edema, hemorrhage, exudation and neovascularization.

[0011] The term "polypoid choroidal vascular lesion (PCV)" refers to a major abnormality in choroidal circulation. Its characteristic feature is that the internal choroidal vascular network eventually forms polypoid dilatations, presenting as orange-red nodular lesions in the fundus, abnormal branching vascular networks and terminal polypoid vascular dilatations (polyps) on ICGA examination. In addition to polyps, exudative PCV has obvious serous retinal pigment epithelial detachment (PED) and / or retinal detachment, as well as a large amount of yellowish-white lipid exudation. Hemorrhagic PCV is mainly hemorrhagic pigment epithelial detachment or a large amount of subretinal hemorrhage, and vision may be lost suddenly.

[0012] Physiologically, the choroid is a thin, elastic, and vascularized brown membrane located between the retina and the sclera. Its inner side is connected to the pigment epithelium of the retina via a smooth vitreous membrane, while its outer side connects to the sclera via a potential gap. The choroid's primary function is to nourish the outer layer of the retina, and secondly, to block light from penetrating the sclera and entering the eye, thus ensuring clear imaging. The choroid and the retina are completely different structures in the fundus.

[0013] In clinical diagnosis, PCV and AMD present with similar symptoms, but their specific diagnostic and epidemiological characteristics differ significantly, especially in the formation of polypoid structures, as shown in the table below:

[0014]

[0015] A second object of the present invention is to provide a method for preventing or treating PCV, the method comprising administering a drug containing a truncated plasmin containing a retained enzyme active domain to a patient, thereby bringing an effective amount of the truncated plasmin containing the retained enzyme active domain to contact the affected area of ​​the patient's fundus, and producing a therapeutic effect on the PCV patient in a manner that relieves, eliminates or reverses symptoms, wherein the truncated plasmin containing the retained enzyme active domain is preferably recombinant human octanoplasticase.

[0016] In some embodiments, the medicament of the present invention may be a liquid formulation and may be applied to the vitreous body, aqueous humor, sclera, conjunctiva, between the sclera and conjunctiva, retinal choroidal tissue, macula, or other areas within or near the subject's eye that can be delivered via these tissues or periocular routes. In some embodiments, for the prevention, treatment, or delay of PCV onset, different routes of administration may require different effective doses and durations of action.

[0017] In some embodiments, the drug of the present invention may be a liquid formulation, including but not limited to delivery of the drug by: (1) administering the drug by using a conventional syringe or a hypodermic needle; (2) injecting the drug using a specially designed device; (3) delivering the drug by direct injection into the vitreous body or any other relevant tissue via a syringe.

[0018] In some embodiments, the medicament of the present invention may be a liquid formulation, including but not limited to solutions, suspensions, and emulsions. The liquid formulation may optionally further comprise stabilizers, excipients, adjuvants, antioxidants, or combinations thereof. In some embodiments, the w / v concentration (w / v) of the truncated plasmin containing the enzyme's active domain in the liquid formulation is between about 0.01% and 10%, between about 0.05% and 10%, between about 0.05% and 8%, between about 0.05% and 6%, between about 0.05% and 4%, between about 0.05% and 2%, between about 0.05% and 1%, between about 0.05% and 0.5%, between about 0.05% and 0.3%, or between about 0.05% and 0.15%. In some embodiments, the truncated plasmin containing the active enzyme domain in the liquid formulation is recombinant human octokinase, with a w / v concentration of about 0.01%-0.5%. In some embodiments, the w / v concentration of recombinant human octokinase in the liquid formulation is any one of 0.01%, 0.05%, 0.0627%, 0.1%, 0.125%, or 0.15%.

[0019] In some embodiments, the total localized volume of the liquid formulation of the present invention is about 500 μL, about 400 μL, about 300 μL, about 200 μL, about 150 μL, about 100 μL, about 90 μL, about 80 μL, about 70 μL, about 60 μL, about 50 μL, about 40 μL, about 30 μL, about 20 μL, about 10 μL, about 5 μL, about 3 μL, or about 1 μL. In some embodiments, the liquid formulation of the present invention is directly applied to the vitreous body of a patient, and the application volume includes, but is not limited to, about 150 μL, about 100 μL, about 90 μL, about 80 μL, about 70 μL, about 60 μL, about 50 μL, about 40 μL, about 30 μL, about 20 μL, about 10 μL, about 5 μL, about 3 μL, or about 1 μL. In some embodiments, the local single-dose dose of the truncated plasmin containing the retained enzyme active domain in the liquid formulation is about 500 μg, about 400 μg, about 300 μg, about 200 μg, about 100 μg, about 80 μg, about 60 μg, about 40 μg, about 20 μg, about 10 μg, or about 5 μg. In some embodiments, the truncated plasmin containing the retained enzyme active domain in the liquid formulation is recombinant human octoplasticase, and its intravitreal single-dose dose is about 200 μg, about 100 μg, about 80 μg, about 60 μg, about 40 μg, about 20 μg, about 10 μg, or about 5 μg.

[0020] In some embodiments, the unit dosage form is prepared at the concentration to be administered or diluted before administration to a subject. In some embodiments, the liquid formulation provided by the present invention is diluted in an aqueous medium before administration to a subject. In some embodiments, the aqueous medium is an isotonic medium. In some embodiments, the liquid formulation provided by the present invention is diluted in a non-aqueous medium before administration to a subject.

[0021] Beneficial effects

[0022] 1. Tissue-type plasminogen activator (t-PA), as a plasminogen activator, has been explored in previous studies in its potential application in the treatment of PCV. This invention compares and examines the fibrinolytic effects of different concentrations of recombinant human octoplasmic plasmin and t-PA in vitro. The final concentrations of recombinant human octoplasmic plasmin from 0.125 to 0.25 mg / mL were compared with those of t-PA. 125 I-labeled human fibrinogen-prepared rat thrombi showed significant thrombolytic activity, with a certain time- and concentration-dependent relationship, and was superior to t-PA under the same administration conditions.

[0023] 2. This invention compares and examines the posterior vitreous detachment after administration of different concentrations of recombinant human octoplasmic plasmin and different concentrations of t-PA in a cynomolgus monkey PVD model. The experimental results show that, in terms of both the effectiveness and safety in degrading collagen fibers on the surface of the internal limiting membrane, recombinant human octoplasmic plasmin is significantly superior to t-PA.

[0024] 3. This invention compares and examines the retinal vein recanalization levels after administration of different concentrations of recombinant human octoplasticase and different concentrations of t-PA in a New Zealand white rabbit RVO model. The experimental results show that the complete recanalization rates of the different concentrations of recombinant human octoplasticase administration groups were 70% and 90%, respectively, while the complete recanalization rates of the different concentrations of t-PA administration groups were 30% and 50%, respectively.

[0025] 4. Since the application of t-PA for PCV indication is still at the research level and has not been approved, this invention compares the therapeutic effects of recombinant human octoplase with the current conventional treatment for PCV (anti-VEGF+PDT) in the clinical trial design. The results show that, within the same treatment cycle, the recombinant human octoplase treatment group has certain advantages in efficacy compared with the ranibizumab and PDT combination treatment group, including significantly improving patients' vision and reducing subretinal hemorrhage and exudation in the macular region of the fundus. Attached Figure Description

[0026] Figure 1 Different concentrations of recombinant human octoplasmic plasmin and t-PA were used to target 125 Statistical graph of in vitro thrombolysis rate of I-labeled human fibrinogen thrombi;

[0027] Figure 2 Low, medium and high doses of recombinant human octoplasmic plasmin and t-PA were administered to the eyes of cynomolgus monkeys to induce vitreous detachment and OCT imaging.

[0028] Figure 3 Scanning electron micrographs of the inner retinal interface after administration of low, medium and high doses of recombinant human octoplasmic fibrinolytic enzyme to the eyes of cynomolgus monkeys.

[0029] Figure 4 The amino acid sequence of recombinant human octanolytic enzyme and the disulfide bond linkage sites therein are shown. Detailed Implementation

[0030] The following description of preferred embodiments of the present invention provides a detailed explanation but does not limit the scope of the invention.

[0031] Materials / Reagents and Animals (Unless otherwise specified, all materials used in this invention are commercially available).

[0032]

[0033] Example 1: Preparation of Recombinant Human Occlusive Plasmin

[0034] The amino acid sequence of recombinant human octoplasmic plasmin is shown in SEQ ID NO.1 and SEQ ID NO.2, and the disulfide bond linkage sites in its structure are as follows: Figure 4 As shown in the figure, and following the method disclosed in CN114480353A, the required recombinant human octanolytic enzyme was prepared. The preparation steps are briefly described below:

[0035] 1) Process of fermentation of recombinant human plasminogen oxidase

[0036] Using genetic engineering techniques, a synthesized coding gene was cloned into the expression vector pPICZ to construct a recombinant expression vector. The gene fragment has a sense strand as shown in SEQ ID NO:4 and an antisense strand as shown in SEQ ID NO:5. The recombinant expression vector was then transformed into Escherichia coli DH5α for amplification and culture. The recombinant expression vector was extracted using a kit. After linearization and further purification, the recombinant expression vector was transformed into competent yeast host Pichia PastorisX33 by electroporation to obtain recombinant engineered bacteria with the target gene integrated into the yeast genome. Stable and highly expressed engineered bacteria were obtained after screening.

[0037] Take the engineered bacteria, thaw the glycerol seed culture, inoculate it into a shake flask of BMGY medium, and amplify it to obtain OD. 600 The seed culture was prepared with a pH value between 5.0 and 15.0. The seed culture was inoculated into BSM basal medium (10% of the BSM basal medium volume) for fermentation at a temperature of 30.0 ± 1.0 °C, with the pH controlled at 5.0 ± 0.5 and dissolved oxygen (DO) maintained above 10%. When the initial glycerol in the fermentation broth was depleted (at which point the dissolved oxygen in the fermentation broth rose to 100%), 3% of the initial fermentation broth volume of 50% glycerol was added. Glycerol addition was stopped when the cell wet weight in the fermentation broth reached 192 g / L. After the added glycerol was depleted, methanol was added for induction, at 40% of the initial fermentation broth volume. Simultaneously, tranexamic acid was added to a final concentration of 0.1 mol / L, and 2.5% of the initial fermentation broth volume of YP solution was added. Fermentation was induced for 52 hours. The fermentation supernatant was centrifuged at 12000–15000 g at 2–8 °C for 15–30 min. The supernatant was then filtered through a 0.45 μm pore size filter. The filtrate was subjected to mixed-mode chromatography using MMC Mustang as the chromatography packing material. The sample loading parameters were: pH 6.0, conductivity not exceeding 16 mS / cm, and retention time 2 min. After loading, the sample was eluted sequentially with 18% B (180 mmol / L NaCl) for 3–9 cycles, followed by elution with 35% B (350 mmol / L NaCl). The target protein was then collected.

[0038] 2) Preparation of SAK enzyme after digestion

[0039] Add 24 μL of EK enzyme (enterokinase) solution (with an EK enzyme activity of 10 IU / μL) to 8 mL of glucokine fusion protein solution (concentration of glucokine fusion protein is 2 mg / mL), mix well, and incubate in a water bath at 23±2℃ for 17±0.1 hours. Filter to obtain the enzymatically digested SAK enzyme (glucokine) solution, labeled EK-SAK-meiqie-30min-20190107. Collect the eluent containing the enzymatically digested SAK enzyme after NI affinity chromatography and cation chromatography. Then perform mixed-mode chromatography using Bestarose Diamond MIX-A as the chromatography medium and 10 mM CB buffer at pH 5.4±0.1 as the equilibration buffer. Collect the flow-through containing the target component and filter through a 0.22 μm filter to obtain the purified SAK enzyme stock solution (labeled EK-SAK-FT-20190107), and store at -20℃. The amino acid sequence of the purified SAK enzyme after enzyme digestion is shown in SEQ ID NO:3.

[0040] 3) Preparation of recombinant human octoplasmic plasmin

[0041] Take 30 mL of the target protein eluent obtained from fermentation in step 1), add 10 mL of 0.8 M tranexamic acid to make the final concentration of tranexamic acid in the sample 0.2 M. Add 0.53 mL of the digested SAK enzyme obtained in step 2) to the resulting diluent, controlling the mass ratio of recombinant human plasminogen activator to SAK enzyme to be 120:1. Mix the solution well and place it in a water bath at 23 ± 2 °C for enzymatic digestion. Remove the solution after 4 ± 0.1 hours. Dilute the resulting digested solution with a liquid containing 20 mM PPB, 2 M (NH4)2SO4, and 0.2 M tranexamic acid at a volume ratio of 1:1. After filtration and hydrophobic chromatography, obtain the purified recombinant human plasminogen activator solution.

[0042] Example 2 125 I-labeled human fibrinogen in vitro degradation assay

[0043] 1. Experimental objective:

[0044] The in vitro validation of the thrombolytic effect of the recombinant human octofibrinolytic enzyme prepared in Example 1 provides a reference for its subsequent pharmacodynamic evaluation and clinical application.

[0045] 2. Experimental methods:

[0046] 1) 125 Preparation of I-labeled human fibrinogen thrombi ( 125I-labeled human fibrinogen: Labeling was performed by Jiangsu Huayi Technology Co., Ltd., at the Radiation Medicine Laboratory of Soochow University.

[0047] Take fresh rat whole blood and mix it with 3.8% sodium citrate at a ratio of 9:1. Add the following to 0.5 mL of the whole blood in sequence: 125 I-labeled fibrinogen 20 μL, 0.5 mol / L CaCl2 7.5 μL, 100 U / mL human thrombin 7.5 μL, mix rapidly, and proceed as follows: 125 I-labeled fibrinogen requires protection. The protection method refers to the corresponding SOP of Jiangsu Huayi Technology Co., Ltd.: Quickly aspirate the mixture with a PE-200 tube, let it stand at room temperature for 10 minutes, then incubate at 37°C for 1 hour to form a strip-shaped blood clot in the tube. Heat to 60°C and incubate for 30 minutes, then let it stand at room temperature for 10 minutes. Wash with physiological saline 4 times, 2 minutes each time, and let it stand at 4°C overnight to ensure that the blood clot can harden sufficiently while avoiding autolysis of the blood clot due to prolonged standing.

[0048] 2) Detection of in vitro thrombolytic activity of recombinant human octoplasmic fibrinolytic enzyme

[0049] The thrombus strips were cut into 2cm segments, and their total radioactivity was measured. These segments were then added to different volumes of rat plasma, with corresponding volumes of solvent added to the control group, t-PA group, and test samples 1-4 (samples 1-4 were respectively 0.25 mg / mL for the plasmin group, 0.125 mg / mL for the plasmin group, 0.0625 mg / mL for each plasmin group, and 0.03125 mg / mL for the plasmin group, as detailed in Table 1). The samples were then immediately incubated at 37℃. 0.05 mL of the solution was taken at 15 min, 30 min, 1 h, 2 h, and 4 h to measure its radioactivity, and the thrombolysis rate of each group was calculated. Three samples were collected from each group.

[0050] Table 1

[0051]

[0052] Thrombolysis rate for each group = (Radioactivity in the test group - Average radioactivity in the solvent group) ÷ (Total radioactivity of thrombus in the test group - Average radioactivity in the solvent group) × 100%

[0053] Thrombolysis rate in the solvent control group = (Radioactivity in the solvent group ÷ Total radioactivity of the thrombus in the solvent group) × 100%

[0054] 3. Experimental Results

[0055] Thrombolysis rates at different time points for each group are shown below. Figure 1As shown in the figure, the thrombolysis rates of the solvent control group at 15 min, 30 min, 1 h, 2 h, and 4 h were 0.36±0.08%, 0.35±0.04%, 0.55±0.05%, 0.66±0.15%, and 1.39±0.04%, respectively. The low thrombus self-dissolution rate at different time points indicates that the isotope data obtained under the experimental conditions are reliable.

[0056] The thrombolysis rates of the test samples with final concentrations of 0.03125 mg / mL and 0.0625 mg / mL at different time points were similar to those of the solvent control group, but the rates were low. The thrombolysis rate of the test sample with a final concentration of 0.125 mg / mL at 4 h was 5.00 ± 2.24%, with low rates at other time points. The thrombolysis rates of the test sample with a final concentration of 0.25 mg / mL at 2 h and 4 h were 2.59 ± 1.19% and 17.98 ± 5.16%, respectively, with relatively low rates at other time points. The thrombolysis rates of t-PA with a final concentration of 0.25 mg / mL at 2 h and 4 h were 1.95 ± 0.24% and 12.68 ± 3.21%, respectively, significantly lower than those of the octoclopramide test sample group under the same administration conditions.

[0057] Therefore, under the conditions of this experiment, the final concentration of the octoclopramide test sample at 0.125–0.25 mg / mL is effective against [the following conditions]. 125 I-labeled human fibrinogen-prepared rat thrombi showed significant thrombolytic activity, with a certain time- and concentration-dependent relationship, and was superior to t-PA under the same administration conditions.

[0058] Example 3: Recombinant human octoplasmic plasmin-induced complete posterior vitreous detachment (PVD) experiment in cynomolgus monkeys

[0059] 1. Experimental Objective

[0060] Using cynomolgus monkeys as experimental animals, this study investigated the effects of different doses of recombinant human octoplasmic plasmin on the incidence of posterior vitreous detachment and the posterior limiting membrane and its fibers, and verified the degradation effect of recombinant human octoplasmic plasmin on collagen fibers of the limiting membrane in the fundus.

[0061] 2. Experimental Methods

[0062] Experimental animals and grouping: 70 cynomolgus monkeys were randomly divided into 7 groups, namely, ocular balanced salt buffer (BSS buffer), low-medium-high dose octokinase administration group (62.5 μg / eye, 75 μg / eye, 125 μg / eye), and low-medium-high dose t-PA administration group (62.5 μg / eye, 75 μg / eye, 125 μg / eye). The drug was administered to one eye (left eye) once, with an administration volume of 50 μL / eye / time.

[0063] Experimental Procedure: Following the experimental groups described above, the corresponding drugs were administered via intravitreal injection. Optical coherence tomography (OCT) scans were performed 2 days (3 days) after administration and after a 4-week (29 days) recovery period. The optic disc, retinal vascular arch, and posterior pole of the retina were examined. After the observation period, one animal from each dose group of the octanoic plasminogen activator was randomly selected, euthanized, and an eyeball sample was taken. Half of the eyeball was cut into a strip of full-thickness tissue for scanning electron microscopy.

[0064] 3. Experimental Results

[0065] Referring to Table 2, OCT results showed that after unilateral administration, no posterior vitreous detachment was observed in the solvent control group. In the octoclopramide administration group, low (62.5 μg / eye), medium (75 μg / eye), and high (125 μg / eye) doses all promoted posterior vitreous detachment. Two days (3 days) after administration, one cynomolgus monkey in the low-dose octoclopramide group developed complete posterior vitreous detachment, while nine cynomolgus monkeys in the high-dose group did. In contrast, the t-PA administration group failed to induce complete posterior vitreous detachment at either low, medium, or high doses. Furthermore, after a four-week (29-day) recovery period, the octofibrinolytic enzyme administration groups showed a sufficient effect in inducing posterior vitreous detachment at low, medium, and high doses. Only one cynomolgus monkey in the low-dose group failed to achieve complete posterior vitreous detachment. Moreover, no significant adverse reactions were observed in any of the octofibrinolytic enzyme administration groups. In contrast, t-PA was less effective than octofibrinolytic enzyme in inducing posterior vitreous detachment and carried the risk of adverse reactions such as fundus hemorrhage and macular hole. Under the experimental conditions, experimental animals in the low, medium, and high-dose t-PA groups all experienced fundus hemorrhage and / or macular hole. Figure 2 The results of OCT imaging of animals in D29 and the treatment groups are shown. Among them, the Octocin plasmin treatment group showed more complete posterior vitreous detachment. Figure 3 Scanning electron micrographs of D3, octofibrinolytic enzyme at doses of 75 μg / eye and 125 μg / eye were shown. The results showed that octofibrinolytic enzyme could effectively degrade or loosen collagen fibers in the posterior vitreous membrane of cynomolgus monkeys, and almost no collagen fibers were observed on the surface of the internal retinal membrane.

[0066] Table 2. Statistical table of posterior vitreous detachment results in cynomolgus monkeys

[0067]

[0068]

[0069] Note: "++" indicates no posterior vitreous detachment observed; "++" indicates incomplete posterior vitreous detachment with significant adhesion between the inner retinal surface and the vitreous cortex; "+" indicates incomplete posterior vitreous detachment with minor adhesion between the inner retinal surface and the vitreous cortex; "-" indicates complete posterior retinal detachment with a smooth inner retinal limiting membrane.

[0070] Example 4: Thrombolysis Experiment of Recombinant Human Occlusive Plasmin Promoting Retinal Vein Occlusion (RVO) Model in Dutch Rabbits

[0071] 1. Experimental Objective

[0072] Using the Dutch rabbit RVO model, the thrombolytic effect of recombinant human octoplasmin on retinal vein embolism was verified in vivo.

[0073] 2. Experimental Methods

[0074] 1) Photochemical induction of retinal vein occlusion (RVO) model in Dutch rabbits

[0075] The procedure is briefly as follows: Bengal red was diluted with physiological saline to 7.5 g / L, sterilely filtered, and the animal was anesthetized. 1% atropine and pyramide eye drops were alternately instilled into the operated eye to fully dilate the pupil. The animal's head was fixed, and Bengal red was injected via the marginal ear vein at a dose of 10 mg / kg. One minute later, after the Bengal red had circulated into the vein, the main vein to be blocked was irradiated with a 550 nm wavelength xenon laser. Fundus observation and fluorescein angiography were performed at 1 hour, 3 hours, 1 day, 3 days, 7 days, 2 weeks, 3 weeks, and 4 weeks after thrombosis to determine the success of the model.

[0076] 2) Therapeutic effect of recombinant human octoplasmic plasmin on Dutch rabbit RVO model

[0077] Experimental animals and grouping: Fifty successfully modeled Dutch rabbits were selected and randomly divided into 5 groups of 10 each. The groups were: ophthalmic balanced salt buffer (BSS buffer), octocin plasmin administration group (15 μg / eye, 25 μg / eye), and t-PA administration group (15 μg / eye, 25 μg / eye). Single eye administration was performed, with an administration volume of 50 μL / eye / time.

[0078] Experimental procedure: The corresponding drugs were administered via intravitreal injection according to the above experimental groups. Seven days (8 days) after administration, the therapeutic effect of retinal vein thrombosis in the experimental animals was observed by fundus photography and fluorescein fundus angiography.

[0079] 3. Experimental Results

[0080] Referring to Table 3, fundus photography and angiography results showed that after unilateral administration, no vascular recanalization was observed in the solvent control group, and significant hemorrhage was observed in the fundus. Four animals developed retinal detachment. Angiography revealed no fluorescent perfusion in the embolized veins, with peripheral vessels showing significant tortuosity, thickening, increased permeability, and severe fluorescent leakage. The drug-treated groups were able to improve vascular embolism and fluorescent leakage to some extent. The complete vascular recanalization rates in the 15 μg / eye and 25 μg / eye octoplasmic fibrinolytic enzyme groups were 70% and 90%, respectively, significantly reducing fluorescent leakage. In contrast, the complete vascular recanalization rates in the 15 μg / eye and 25 μg / eye t-PA groups were 30% and 50%, respectively, weaker than those in the octoplasmic fibrinolytic enzyme group. In both the 15 μg / eye and 25 μg / eye t-PA groups, some animals exhibited varying degrees of vascular non-patency, which was observed in angiography as vascular tortuosity and fluorescent leakage.

[0081] Table 3. Statistical table of retinal vein occlusion recanalization results in Dutch rabbits.

[0082]

[0083] Example 5: The therapeutic effect of recombinant human octoplasmic plasmin on PCV patients

[0084] 1. Research Objectives

[0085] A randomized, double-blind, parallel-group, positive-controlled clinical trial was conducted to compare the efficacy of combination therapy with anti-VEGF and PDT versus intravitreal injection of recombinant human octokinase for PCV.

[0086] 2. Research Design

[0087] Patient inclusion criteria: 1. Signed informed consent and willing to be followed up according to the trial schedule; 2. Age ≥ 45 years, gender not limited; 3. BCVA of the target eye ≥ 24 letters and ≤ 73 letters; 4. In patients with PCV diagnosed by ICGA, the target eye produces single or multiple focal hyperfluorescent lesions originating from the choroidal circulation within the first 6 minutes after injection of indocyanine green contrast agent, with or without associated branching vascular networks (BVN); 5. No refractive media opacity or pupillary constriction affecting fundus examination.

[0088] Study Design: Twenty patients with PCV who met the inclusion criteria were included (only one eye was selected as the target eye) and randomly assigned to the following two treatment groups in a 1:1 ratio:

[0089] Group 1: Rhabdominis monoclonal antibody + V - In the PDT treatment group, verteporfen was used as a photosensitizer in PDT treatment. Body surface area was calculated based on the subjects' height and weight. Verteporfen was diluted with 5% glucose solution and stored protected from light, at a dose of 6 mg / m². 2 Calculate the drug dosage. After tropicamide was administered to the affected eye for mydriasis and slowly injected via the antecubital vein, the lesion was irradiated with a diode laser 15 minutes later (energy 50 J / cm², power density 600 mW / cm²). 2 The wavelength is 689nm and the duration is 83s. The spot diameter must be more than 1000μm larger than the lesion. After treatment, the patient should be instructed to avoid light for 72 hours. On the 4th day after PDT treatment, ranibizumab is injected intravitreally (0.5mg, 0.05mL).

[0090] Group 2: The recombinant human octoplasticase treatment group received intravitreal injection of recombinant human octoplasticase (concentration of 0.45 mg / 0.3 mL, injection of 100 μL).

[0091] Endpoints: Patients in both groups were followed up on days 14, 28, and 56 after treatment. During follow-up, ophthalmological examinations (including BCVA, OCT, CFP, ICGA, FFA, and LOP) were performed to assess efficacy and safety. The primary endpoints were: 1. Changes in BCVA from baseline on days 14, 28, and 56; 2. Changes in CRT from baseline on days 14, 28, and 56.

[0092] 3. Research Results

[0093] As shown in Table 4, although the mean BCVA improved from baseline in all treatment groups, the administration of octoclopramide significantly improved visual acuity in PCV patients compared to the combination therapy of ranibizumab and PDT. Specifically, the octoclopramide group showed an average improvement of 5.3, 7.2, and 9.3 letters in BCVA from baseline at days 14, 28, and 56, respectively, while the ranibizumab + PDT combination showed an average improvement of 4.2, 5.7, and 7.5 letters from baseline at days 14, 28, and 56. Two patients in the ranibizumab + PDT combination group showed an improvement of 15 or more letters, while four patients in the recombinant human octoclopramide group did. At the last visit (day 56 after treatment), the reduction in central retinal thickness (CRT) in the octoclopramide group was significantly greater than that in the ranibizumab + PDT group. The PDT combination therapy group showed a slight advantage compared to the baseline group, with the former showing a 99 μm reduction in CRT and the latter a 90 μm reduction in CRT. Among patients who underwent ICGA measurements, the area of ​​abnormal branch vascular network in the fundus was reduced or remained relatively stable in the occlusive fibrinolytic enzyme group, while the area of ​​abnormal branch vascular network in the fundus remained relatively stable or slightly increased in the combination therapy group.

[0094] The above results preliminarily demonstrate that recombinant human octocolytic enzyme can reduce or eliminate polypoid lesions in the eyes of PCV patients, and the expected beneficial effects include a reduction in abnormal branched vascular networks and stable or improved vision.

[0095] Table 4. Statistical table of efficacy results of recombinant human octoclozyme in PCV patients.

[0096]

[0097]

[0098] Sequence information:

[0099] SEQ ID NO:1:

[0100] APSFDCGKPQVEPKKCPGR

[0101] SEQ ID NO:2:

[0102] VVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSS YKVILGAH

[0103] QEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVAD RTECF

[0104] ITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDS

[0105] GGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

[0106] SEQ ID NO:3:

[0107] SSSFDKGKYKKGDDASYFEPTGPYLMVNVTGVDSKGNELLSPHYVEFPIKPG TTLTKEKIEYYVEWALDATAYKEFRVVELDPSAKIEVTYYDKNKKKEETKSFPITEKGFVVPDLSEHIKNPGFNLITKVVIEKK

[0108] SEQ ID NO:4:

[0109] GCCCCATCTTTTGATTGTGGTAAACCACAAGTTGAACCTAAGAAGTGTCCAGGTAGAGTT GTTGGTGGTTGTGTTGCTCATCCTCATTCTTGGCCATGGCAAGTTTCTTTGAGAACTAGATTTGGTATGCATTTTTTTGGGTGGTACTTTGATTTCTCCAGAATGGGTTTTGACTGCTGCT CATTGTTTGGAAAAGTCTCCTAGACCATCTTCTTACAAGGTTATTTTGGGTGCTCATCAA GAAGTTAACTTGGAACCACATGTTCAAGAAATTGAAGTTTCTAGGTTGTTTCTGGAACCAACTAGAAAAGATATTGCTTTGTTGAAGTTGTCATCTCCAGCTGTTATTACTGATAAGGTT ATTCCAGCTTGTTTGCCTTCTCCAAACTACGTTGTTGCTGATAGAACTGAATGTTTTATTACTGGTTGGGGTGAAACTCAAGGTACTTTTTGGAGCTGGTTTGTTGAAGGAAGTCTAATTG CCTGTTATTGAAAACAAGGTTTGTAACAGATATGAATTTTTGAACGGTAGAGTTCAATCTACTGAATTGTGTGCTGGTCATTTGGCTGGTGGTACTGATTCTTGTCAAGGTGACTCTGGT GGTCCTTTGGTTTGTTTTGAAAAGGATAAGTACATTTTGCAAGGTGTTACTTCCTGGGGTTTGGGTTGTGCTAGACCTAACAAGCCTGGTGTTTACGTTAGAGTTTCCAGATTTGTTACTTGGATTGAAGGTGTTATGAGAAACAAC

[0110] SEQ ID NO:5

[0111] CGGGGTAGAAAACTAACACCATTTGGTGTTCAACTTGGATTCTTCACAGGTCCATCTCAA CAACCACCAACACAACGAGTAGGAGTAAGAACCGGTACCGTTCAAAGAAACTCTTGATCTAAACCATACGTAAAAACACCACCATGAAACTAAAGAGGTCTTACCCAAAACTGACGACGA GTAACAAACCTTTTCAGAGGATCTGGTAGAAGAATGTTCCAATAAAACCCACGAGTAGTTCTTCAATTGAACCTTGGTGTACAAGTTCTTTAACTTCAAAGATCCAACAAAGACCTTGGT TGATCTTTTCTATAACGAAACAACTTCAACAGTAGAGGTCGACAATAATGACTATTCCAATAAGGTCGAACAAACGGAAGAGGTTTGATGCAACAACGACTATCTTGACTTACAAAATAA TGACCAACCCCACTTTGAGTTCCATGAAAACCTCGACCAAACAACTTCCTTCGAGTTAAC GGACAATAACTTTTGTTCCAAACATTGTCTATACTTAAAAACTTGCCATCTCAAGTTAGATGACTTAACACACGACCAGTAAACCGACCACCATGACTAAGAACAGTTCCACTGAGACCA CCAGGAAACCAAACAAAACTTTTCCTATTCATGTAAAACGTTCCACAATGAAGGACCCCAAACCCAACACGATCTGGATTGTTCGGACCACAAATGCAATCTCAAAGGTCTAAACAATGAACCTAACTTCCACAATACTCTTTGTTG

Claims

1. Use of recombinant human octokinase in the preparation of drugs for the treatment and / or prevention of choroidal vascular diseases, wherein the choroidal vascular disease is a polypoid choroidal vascular lesion, and the heavy chain amino acid sequence of the recombinant human octokinase is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that... The polypoid choroidal vascular lesion is a hemorrhagic idiopathic polypoid choroidal vascular lesion.

3. The use according to claim 1, characterized in that... The recombinant human octoplasmic fibrinolytic enzyme was prepared into a liquid formulation with a mass-to-volume ratio of 0.01%-0.5%.

4. The use according to claim 3, characterized in that... The liquid preparation is applied to the vitreous cavity of the eye.

5. The use according to claim 3 or 4, characterized in that... The single dose of the liquid formulation is 0.15 mg of recombinant human octoplasmic fibrinolytic enzyme.

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