CN115671036A - Gel medicine for treating fundus and intraocular diseases - Google Patents
Gel medicine for treating fundus and intraocular diseases Download PDFInfo
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Abstract
The present invention provides a gel drug for treating fundus and intraocular diseases, which comprises an injectable hydrogel and a drug for treating choroidal neovascularization. The gel drug provided by the invention can reduce the dosage of the anti-VEGF drug, prolong the action time of the anti-VEGF drug and reduce the administration frequency of the anti-VEGF drug.
Description
Technical Field
The invention relates to a medicine, in particular to a medicine for treating fundus and intraocular diseases, and particularly relates to a gel medicine for treating choroidal neovascular diseases.
Background
The fundus choroid is a highly vascularized tissue that serves primarily to provide adequate nutrients for the retinal photoreceptors and retinal pigment epithelium, and may serve to clear the retina of metabolic waste products. Choroidal disorders are primarily characterized by progressive dysfunction of retinal pigment epithelial cells, and by abnormalities in choroidal vascular structure and function that cause choroidal vascular disease, such as Polypoidal Choroidal Vasculopathy (PCV), central serous chorioretinopathy, and age-related macular degeneration-related Choroidal Neovascularization (CNV). A large number of studies indicate that the choroidal vascular disease has a wide range of effects and the incidence rate increases exponentially. The pathological processes of choroidal vascular diseases mainly include increased vascular permeability and neovascularization, leading to severe impairment of visual function and even irreversible blindness. CNV refers to the growth of new blood vessels from the choroid to the neural retina under the macula, causing macular edema, exudation, hemorrhage, photoreceptor injury, and eventually the formation of the final stage fibrotic scar-currently, methods of treatment for CNV include laser photocoagulation, vitrectomy, and intravitreal injection of anti-Vascular Endothelial Growth Factor (VEGF). However, laser photocoagulation and vitrectomy procedures have limited effectiveness and these procedures can damage the retina and blood vessels. Although currently effective in anti-VEGF therapy, a number of problems continue to exist, for example, frequent intravitreal injections of anti-VEGF drugs can lead to cataracts, glaucoma, endophthalmitis, uveitis, and retinal detachment. Importantly, the burden of frequent intravitreal injections can significantly reduce patient compliance. Furthermore, anti-VEGF has no effect on fibrotic scarring and may even impair the long-term viability of the choroid and retina. Therefore, intensive research into the pathogenesis of CNV, finding new effective therapeutic approaches and solutions, and increasing the anti-angiogenic therapeutic level of CNV is urgently needed.
Although the etiology is unclear, inflammation plays a crucial role in the development of CNV. In particular, previous studies have shown that proinflammatory cytokines such as interleukin-6 (IL-6) and interleukin-8 (IL-8) generally promote the development and/or progression of CNV. In addition, oxidative stress-induced damage is considered to be a key factor in Retinal Pigment Epithelium (RPE) degeneration, which is another important factor in the progression of CNV. Studies have shown that excessive inflammation-associated Reactive Oxygen Species (ROS) and oxidized lipoproteins lead to protein misfolding, aggregation and chronic activation of the innate immune response, positively correlated with pathological angiogenesis of CNV. In addition, ROS significantly promotes the expression of Vascular Endothelial Growth Factor (VEGF) in the retina. Thus, anti-inflammatory therapy and effective scavenging of ROS may be key strategies for CNV treatment.
Ranibizumab (trade name Lucentis) is a monoclonal antibody Fragment (FAB) that is obtained from the same parent murine antibody as bevacizumab. The ranibizumab is a typical anti-VEGF drug, can inhibit the vascular endothelial growth factor A in a targeted manner, and can delay the progress of neovascular diseases by inhibiting the proliferation of vascular endothelial cells and the formation of new blood vessels. At present, the mode of treating CNV by using ranibizumab is to directly inject the ranibizumab into vitreous body, and the ranibizumab has large dosage, short action time, needs to be administered once per month and is frequently administered.
Disclosure of Invention
In view of the above-mentioned defects of the prior art, the present invention provides a drug gel mixture for treating fundus and intraocular diseases, and aims to solve the technical problems of reducing the dosage of an anti-VEGF drug, increasing the action time of the anti-VEGF drug, and reducing the administration frequency of the anti-VEGF drug.
In order to solve the problems, the invention adopts the technical scheme that: a gel medicament for the treatment of ocular fundus and intraocular diseases, the gel medicament comprising an injectable hydrogel and a medicament for the treatment of choroidal neovascular diseases.
Preferably, the drug for treating choroidal neovascular disorders is an anti-VEGF drug.
Preferably, the drug for treating choroidal neovascular disorders is ranibizumab.
Preferably, in the gel medicament, the mass content of the ranibizumab is more than 1%.
Preferably, the method of preparing the injectable hydrogel comprises: the betamethasone disodium phosphate and the calcium chloride are mixed into a solution so that the mixed solution is converted into a gel.
Preferably, the ratio of the molar concentrations of betamethasone phosphate disodium and calcium chloride is 0.9-1.1.
Preferably, the molar concentrations of the betamethasone phosphate disodium and the calcium chloride are respectively more than 0.01M.
Preferably, the molar concentrations of the betamethasone phosphate disodium and the calcium chloride are respectively below 0.025M.
Preferably, the gel medication is configured to be injected with a 30G syringe and the liquid state returns to the gel state when the stress is removed.
The invention has the beneficial effects that: the invention provides a drug for treating choroidal neovascularization diseases, which has good biocompatibility, simple preparation and long efficacy, can continuously release an anti-VEGF drug for a long time to inhibit Choroidal Neovascularization (CNV) generation, can also remove Reactive Oxygen Species (ROS), and reduces local inflammation. The effective time of the traditional anti-VEGF drug treatment is remarkably prolonged. All components of the hydrogel can be applied to clinical application which can be easily converted into choroidal neovascular treatment by adopting clinically approved drugs, and can replace the current anti-VEGF drug treatment.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is a photograph showing the appearance of the hydrogel (0.0125M-BetP-Gel) formed in example 1, when the molar concentration of betamethasone disodium phosphate and calcium chloride reached 0.0125M.
FIG. 2 is a photograph showing the appearance of hydrogels formed by disodium betamethasone phosphate and calcium chloride at different molar concentrations (0.005M, 0.01M,0.0125M,0.025, 0.05M) in example 1.
FIG. 3 shows a transmission electron microscope (TEM image) of 0.0125M-BetP-Gel in example 1.
Figure 4 shows the rheological behaviour of 0.0125M-BetP-Gel in example 1.
FIG. 5 shows an element mapping image of 0.0125M-BetP-Gel in example 1.
FIG. 6 shows 0.0125M-BetP-Gel and Ca in example 1 2+ Photographs of the results of chelating agent ethylenediaminetetraacetic acid (EDTA) incubation.
FIG. 7 shows fluorescence images of IgG-loaded betamethasone phosphate hydrogel (IgG @ BetP-Gel) in example 2. Wherein, the left image is a bright field image, the middle image is a fluorescence image, and the right image is a combined image.
FIG. 8 shows the rheological behavior of IgG @ BetP-Gel in example 2. Wherein, G': storage modulus; g': loss modulus.
FIG. 9 shows the cumulative release profile of IgG after incubation of IgG @ BetP-Gel in PBS at 37 ℃ for 72h in example 2.
FIG. 10 shows photographs of the degradation of gG @ BetP-Gel in example 2 in PBS solution (left) and deionized water (right).
FIG. 11 shows the retention of IgG-Cy5.5 in vivo at various time points after intravitreal injection of free IgG-Cy5.5 or IgG-Cy5.5@ BetP-Gel in example 2.
FIG. 12 shows the results of the qPCR analysis of ZO-1, RPE65 and recovein mRNA expression levels in choroidal tissues in example 4, where the left bar data are untreated mouse (untreated) results, the middle bar data are control CNV mouse results, and the right bar data are gel drug injected CNV mouse results.
FIG. 13 shows the results of the western blot analysis of ZO-1, RPE65 and recovein mRNA expression levels in choroidal tissues in example 4, with untreated mice (untrained) results on the left, control CNV mice in the middle and gel drug injected CNV mice on the right.
Fig. 14 shows fluorescein angiographic images of CNV mice treated with four agents in example 5, where representative Infrared (IR) fundus and FA images show choroidal neovascular leakage before and after injection. Wherein, the four reagents are respectively: physiological Saline (Saline), 0.0125M-BetP-Gel prepared in example 1, anti-VEGF ranibizumab (Anti-VEGF, 10 mg/ml), and the Gel drug prepared in example 3 (Anti-VEGF @ BetP-Gel).
Figure 15 shows images of IB4 (biomarker of neovascularisation) staining of RPE/choroidal patches following treatment of CNV mice with the four agents of example 5, where IB4 staining in differently treated eyes revealed the CNV region.
Fig. 16 shows the quantitative results of the CNV region of the image shown in fig. 15.
Detailed Description
Example 1 preparation and characterization of hydrogels
After the applicant finds out that when the ratio of the molar concentration of the betamethasone disodium phosphate to the molar concentration of the calcium chloride is 0.9-1.1-0.9, hydrogel (BetP-Gel) can be formed in a few seconds. Particularly, when the ratio of the molar concentration of the betamethasone disodium phosphate to the molar concentration of the calcium chloride is 1. The lowest gelation concentration is about 0.01M each of betamethasone disodium phosphate and calcium chloride; when the molar concentration of the betamethasone disodium phosphate and the calcium chloride reaches 0.0125M, the formed gel is convenient for vitreous cavity injection, has no toxic effect on tissues, and can achieve the purpose of gel slow release. FIG. 1 shows a photograph of the appearance of the Gel (0.0125M-BetP-Gel) formed when the molar concentration of betamethasone disodium phosphate and calcium chloride reached 0.0125M. FIG. 2 shows photographs of the appearance of gels formed by betamethasone phosphate disodium with calcium chloride at different molar concentrations (0.005M, 0.01M,0.0125M,0.025, 0.05M). FIG. 3 shows a transmission electron microscope (TEM image) of 0.0125M-BetP-Gel, from which it can be seen that the hydrogel formed is a nanofiber structure. Figure 4 shows the rheological behavior of 0.0125M-BetP-Gel, which further confirms the formation of a hydrogel that can be injected with a 30G syringe and which returns to the Gel state when the stress is removed. Through experiments, the applicant finds that when the molar concentration of the betamethasone phosphate disodium and the calcium chloride is 0.01M-0.025M, the formed hydrogel can be easily injected by a 30G syringe, and the liquid state is recovered to be a gel state after the stress is removed. Fig. 5 shows an element mapping image of 0.0125M-BetP-Gel, from which betamethasone phosphate and calcium ions are uniformly distributed in the network structure.
The inventor of the application further proves that the forming mechanism of the nanofiber hydrogel is phosphate and Ca through FTIR spectrum and XRD spectrum of the hydrogel 2+ Form coordinate bonds between them. If 0.0125M-BetP-Gel is mixed withCa 2+ Incubation with chelating agent ethylenediaminetetraacetic acid (EDTA) (FIG. 6) found that BetP-Gel rapidly transformed from hydrogel morphology to solution, further validating phosphate and Ca 2+ The synergy between them is responsible for the formation of the hydrogel network.
EXAMPLE 2 hydrogel ability to deliver antibodies
And mixing the betamethasone phosphate disodium, the IgG antibody and the calcium chloride in the solution to form the IgG-loaded betamethasone phosphate hydrogel (IgG @ BetP-Gel). Wherein, the molar concentration of the betamethasone disodium phosphate and the calcium chloride is 0.0125M, and the mass content of the IgG antibody is 1 percent, namely 10mg/g.
Goat anti-rabbit IgG H&L(Alexa 
488 Confocal imaging of antibody-stained IgG @ BetP-Gel frozen sections showed that IgG was evenly distributed among the formed IgG @ BetP-Gel (as shown in FIG. 7). The rheological properties of IgG-loaded BetP-Gel and IgG-unloaded BetP-Gel were similar, indicating that encapsulation of the antibody had no significant effect on hydrogel formation (as shown in figure 8). In addition, due to the phosphate under physiological conditions and Ca in the hydrogel 2+ There is a competitive interaction between them, and the encapsulated antibody can be gradually released as the BetP-Gel is degraded. In Phosphate Buffered Saline (PBS), betP-Gel degraded and released about 50% of the antibody continuously over 3 days (as shown in fig. 9, fig. 10).
To further test the potential of BetP-Gel for intraocular antibody delivery, applicants injected Cyanine 5.5 labeled IgG (free IgG-cy 5.5) or BetP hydrogel encapsulated IgG-Cy5.5 (IgG-Cy5.5 @BetP-Gel) intravitreally and monitored mice using a live fluorescence imaging system (Perkinelmer IVIS Lumina III) at various time points post injection. After 14 days of free IgG-Cy5.5 injection, the intraocular Cy5.5 signal disappeared, indicating that free antibody can rapidly diffuse from the outside of the eye. In sharp contrast, the fluorescent signal of IgG-Cy5.5 remains in the eye after injection of IgGCy5.5@ BetP-Gel. On day 14, intense fluorescent signals were still observed in IgGCy5.5@ BetP-Gel-treated mice (as shown in FIG. 11).
EXAMPLE 3 preparation of gel drug
Considering the rapid sol-gel transformation and shear dilution and self-healing capabilities of the hydrogels, applicants loaded anti-VEGF therapeutic drug ranibizumab in the BetP hydrogel for the treatment of the laser-induced mouse CNV model. Only needs to mix BetP solution containing antibody with CaCl 2 The antibody loading can be easily realized by mixing the solutions.
In this example, a hydrogel was formed as a Gel drug for choroidal neovascularization (Anti-VEGF @ betp-Gel) by mixing a clinical Anti-inflammatory drug, betamethasone disodium phosphate approved by the national drug administration for clinical use, a gold-standard Anti-neovascularization drug for CNV treatment (Anti-VEGF) ranibizumab, and calcium chloride in a solution. Wherein, the molar concentration of the betamethasone phosphate disodium and the calcium chloride is 0.0125M, and the mass content of the ranibizumab is 1%.
Example 4 protective Effect of gel drug on laser-induced mouse CNV model
Applicants tested the protective effect of the gel drug prepared in example 3 on a laser-induced mouse model of choroidal neovascularization. Seven days after the laser-induced model was established, the vitreous chamber of the left eye of the same mouse was injected with physiological saline, the right eye was injected with the gel drug prepared in example 1, and 28 days after administration, the choroids of different groups of mice were collected and further subjected to qPCR and western blot experiments (the experimental results are shown in fig. 12 and 13). Experiments prove that the expression levels of ZO-1, RPE65 and recovery mRNA and protein are obviously reduced after laser photocoagulation, and the expression levels of ZO-1, RPE65 and recovery mRNA and protein are obviously reversed after the intravitreal injection of the BetP-Gel, thereby powerfully indicating that the BetP-Gel has a protective effect on a laser-induced mouse CNV model.
Example 5 Long-term therapeutic Effect of gel drugs on laser-induced mouse CNV model
Four agents were injected intravitreally into CNV model mice and CNV leakage was detected at weeks 1, 2, and 4 post-treatment. Wherein, the four reagents are respectively: physiological Saline (Saline), 0.0125M-BetP-Gel prepared in example 1, anti-VEGF ranibizumab (Anti-VEGF, 10 mg/ml), and the Gel drug prepared in example 3 (Anti-VEGF @ BetP-Gel).
Figure 14 shows fluorescein angiographic images (FFA) of CNV mice treated with four agents. The Anti-vegf group showed the best effect in the initial stage of treatment in the vascular leakage area, but the effect gradually decreased in the later stage with the passage of time. In contrast, CNV mice treated with Anti-VEGF @ BetP-Gel showed a gradually increasing therapeutic effect with the degradation of BetP-Gel and the sustained release of Anti-VEGF drugs.
Applicants also assessed CNV lesion areas by IB4 (biomarker for neovasculature) staining of RPE/choroidal patches (results are shown in fig. 15). The results showed that, consistent with FFA results, CNV lesion area was minimal at week 2 in the anti-VEGF group. But as the anti-VEGF effect disappeared, the CNV lesion area increased again. In contrast, after intravitreal injection of Anti-VEGF @ BetP-Gel, the thickness and length of the CNV lesion gradually decreased after 4 weeks, although the treatment effect of CNV was not as good as Anti-VEGF at week 2. It is noteworthy that the therapeutic effect of Anti-VEGF @ BetP-Gel was significantly better than Anti-VEGF from week 2 onwards (results are shown in FIG. 16).
The research results jointly prove that Anti-VEGF treatment time of a mouse CNV model induced by laser can be effectively prolonged by significantly reducing vascular leakage and weakening CNV through Anti-VEGF @ BetP-Gel treatment. Anti-VEGF @ betp-Gel achieves good therapeutic effect for a long period of time, and has important clinical transformation value, because it can significantly reduce the administration times and dosage of Anti-VEGF, thereby greatly reducing the risk of side effects of current Anti-VEGF therapy CNV.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (9)
1. A gel medicament for the treatment of ocular fundus and intraocular diseases, characterized in that it comprises an injectable hydrogel and a medicament for the treatment of choroidal neovascularization.
2. The gel drug for the treatment of ocular fundus and intraocular diseases according to claim 1, characterized in that said drug for the treatment of choroidal neovascular diseases is an anti-VEGF drug.
3. The gel medication for the treatment of fundus and intraocular diseases according to claim 1, wherein said medication for the treatment of choroidal neovascular diseases is ranibizumab.
4. The gel drug for the treatment of ocular fundus and intraocular diseases according to claim 1, wherein the mass content of ranibizumab in the gel drug is 1% or more.
5. The gel drug for the treatment of fundus and intraocular diseases according to claim 1, characterized in that the injectable hydrogel is prepared by a method comprising: the betamethasone disodium phosphate and the calcium chloride are mixed into a solution so that the mixed solution is converted into a gel.
6. The gel medication for the treatment of fundus and intraocular diseases according to claim 5, characterized in that the ratio of the molarity of betamethasone disodium phosphate and calcium chloride is 0.9-1.1.
7. The gel pharmaceutical for the treatment of ocular fundus and intraocular diseases of claim 5, wherein said betamethasone phosphate disodium and calcium chloride have molar concentrations above 0.01M, respectively.
8. The gel pharmaceutical for the treatment of ocular fundus and intraocular diseases of claim 5, wherein said betamethasone phosphate disodium and said calcium chloride are present in a molar concentration of 0.025M or less, respectively.
9. The gel medication for treating fundus and intraocular diseases of claim 5, wherein said gel medication is configured to be injected with a 30G syringe and when the stress is removed the liquid state returns to gel state.
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