Application of fluorinated modified peptide in preparation of medicine for treating dry age-related macular degeneration

Abstract

The invention discloses an application of a fluorinated modified peptide in preparation of a medicine for treating dry age-related macular degeneration, the fluorinated modified peptide FNBP can effectively pass through a retinal barrier and exert antioxidant and neuroprotective effects in RPE cells, and the dry age-related macular degeneration is treated by activating a Keap1 / Nrf2 pathway. The new application of the fluorinated modified peptide in dry AMD treatment is provided for the first time, and a new strategy is provided for medicine development of posterior ocular diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of a fluorinated modified peptide in the preparation of a drug for treating dry age-related macular degeneration. Background Technology

[0002] Age-related macular degeneration (AMD) is one of the leading causes of irreversible blindness in the elderly population worldwide. AMD is divided into two types: wet and dry. Existing anti-angiogenic drugs are only applicable to wet AMD, while there are currently no effective treatments for dry AMD, which accounts for 90% of all cases.

[0003] The main pathological mechanisms of dry AMD include the accumulation of oxidative stress, chronic inflammation, and retinal pigment epithelial (RPE) cell apoptosis. Excessive production of reactive oxygen species (ROS) leads to protein misfolding, lipid peroxidation, and persistent activation of the immune system, thereby inducing RPE cell damage and photoreceptor degeneration. Clinical research evidence suggests that oxidative stress is a core pathogenic factor for RPE dysfunction, and antioxidant therapy may bring significant benefits to patients with dry AMD. Early intervention targeting RPE dysfunction is considered an effective strategy to prevent retinal degeneration with minimal side effects. In recent years, the nuclear factor E2-related factor 2 (Nrf2) pathway has been shown to play a central role in regulating cellular redox homeostasis. Nrf2 upregulates the expression of antioxidant genes such as heme oxygenase-1 (HO-1) and glutathione S-transferase M (GSTM) by activating downstream antioxidant response elements, thereby alleviating retinal oxidative stress and inflammation.

[0004] Under normal circumstances, Nrf2 is inactive due to the regulation of its cytoplasmic repressor protein Keap1. Keap1 binds to Nrf2 and mediates its ubiquitination and degradation, thereby inhibiting Nrf2 signaling pathway activation. Therefore, intervening in the interaction between Keap1 and Nrf2 is considered an upstream, precise, and efficient antioxidant regulation strategy. Currently, although small molecule inhibitors targeting the Keap1 / Nrf2 pathway have been developed, their significant off-target effects due to non-specific binding sites limit their clinical application. In contrast, peptide-based inhibitors have higher biocompatibility and target specificity. Among them, the peptide molecule NBP (sequence LQLDEETGEFLPIQ) can activate Nrf2 by selectively binding to the Kelch domain of Keap1. However, peptide molecules have poor in vivo stability and weak cell penetration, especially difficulty crossing the internal limiting membrane of the retina, limiting their application in ophthalmic diseases. Summary of the Invention

[0005] The purpose of this invention is to provide the application of a fluorinated modified peptide (FNBP) in the preparation of a drug for treating dry age-related macular degeneration (AMD). This invention, combined with fluorination modification technology, effectively improves the delivery efficiency of peptide molecules and enhances their stability in vivo. The fluorinated modified peptide FNBP provided by this invention can effectively cross the retinal barrier and exert antioxidant and neuroprotective effects within RPE cells, treating dry AMD by activating the Keap1 / Nrf2 pathway. Therefore, this invention proposes for the first time a novel use of fluorinated modified peptides in the treatment of dry AMD, providing a new strategy for drug development for posterior segment eye diseases.

[0006] The objective of this invention is achieved through the following means: This invention provides the application of fluorinated modified peptide (FNBP) in the preparation of drugs for treating dry age-related macular degeneration.

[0007] The fluorinated modified peptides of this invention achieve transretinal delivery and efficient cytoplasmic entry through fluorinated alkylation modification, thereby regulating the Keap1 / Nrf2 signaling pathway and reducing oxidative stress, inflammatory response and apoptosis. Therefore, they can be used in the preparation of drugs that reduce oxidative stress, inflammatory response and apoptosis.

[0008] The fluorinated modified peptide was prepared by a disulfide bond exchange reaction between a polypeptide with the amino acid sequence shown in SEQ ID No:1 and a compound of formula I.

[0009] The chemical bond connecting the compound of formula I to the polypeptide is -SS-.

[0010] The compound of formula I was prepared by the following method: 1H,1H,2H,2H-perfluoro-1-octylthiol was dissolved in methanol, and 2,2'-dithiodipyridine was dissolved in methanol. The 2,2'-dithiodipyridine solution was slowly added dropwise to the 1H,1H,2H,2H-perfluoro-1-octylthiol solution at 4°C. The mixture was stirred for 12-16 hours. After the reaction was completed, the solvent was removed by rotary evaporation to obtain the crude product. The crude product was further purified by column chromatography using dichloromethane:methanol 30:1 (v / v) as the eluent to obtain the light yellow oily target product.

[0011]

[0012] The fluorinated modified peptide (i.e., a polypeptide with a fluorinated alkyl chain) comprises a polypeptide with the amino acid sequence shown in SEQ ID No:1. The polypeptide with the amino acid sequence (i.e., polypeptide NBP) has the following sequence: SEQ ID No:1: LQLDEETGEFLPIQ. The method for preparing the fluorinated modified peptide via disulfide exchange reaction is described in the method of the embodiment in patent ZL2020103306033.

[0013] Specifically: The peptide NBP was dissolved in methanol; separately, tag 13F (molar ratio of NBP 3:1) was dissolved in dichloromethane. Under nitrogen protection at 4°C, the tag 13F solution was slowly added dropwise to the NBP solution, and the reaction was continuously stirred for 6-8 hours. After the reaction was completed, the mixture was slowly added to cold diethyl ether to quench the reaction. The white precipitate obtained by centrifugation or filtration was collected as the crude FNBP product, with the fluorinated alkyl chain connected to the peptide by the chemical bond -SS-. Subsequently, the crude product was purified using a preparative high-performance liquid chromatography-mass spectrometry system. A C18 reversed-phase column was used with water-acetonitrile as the mobile phase, water:acetonitrile 95:5-20:80 (linear gradient elution for 30 minutes), and the target component was collected to obtain the FNBP product.

[0014]

[0015] FNBP, through fluoroalkylation modification, enhances its lipophilicity and transmembrane capacity, enabling it to self-assemble into stable nanoparticles that cross the retinal barrier and target RPE cells. Once inside the cell, FNBP binds to the Kelch domain of the Keap1 protein, blocking Keap1-mediated Nrf2 degradation. This leads to Nrf2 phosphorylation and entry into the nucleus, activating the expression of downstream antioxidant genes HO-1, GSTM, NQO1, and GCLM. Consequently, FNBP significantly reduces retinal oxidative damage, inhibits the upregulation of inflammatory factors (IL-6 and IL-8), and reduces retinal cell apoptosis.

[0016] The NBP backbone is modified with an N-terminal fluoroalkyl group to form FNBP, which is then linked to a perfluoroalkyl group via a disulfide bond. This allows it to self-assemble into monodisperse nanoparticles (approximately 100 nm in diameter, PDI < 0.3). The fluorinated modified peptide (FNBP) described in this invention can be administered via intravitreal injection to target and deliver it to retinal pigment epithelial cells, thereby activating the Keap1 / Nrf2 pathway.

[0017] Compared with the prior art, the fluorinated modified peptide of the present invention has the following advantages: Significantly improved delivery efficiency: It can effectively cross multiple retinal structures and be taken up by RPE cells; Significant antioxidant and anti-inflammatory effects: significantly reduces oxidative stress levels and decreases the expression of pro-inflammatory factors; Superior neuroprotective effect: Protects the integrity of the RPE and photoreceptor structures, and maintains retinal function; High safety profile: No cytotoxicity or systemic toxicity. This provides a new targeted therapeutic approach for dry AMD and has clear clinical translational potential. Attached Figure Description

[0018] Figure 1 Results of physicochemical property testing of FNBP Figure 2 Results of FNBP delivery in retinal cells Figure 3 To achieve endosome escape results for FNBP in ARPE19 cells Figure 4 Results of FNBP endocytosis pathway in ARPE19 cells Figure 5 Distribution of FNBP in the mouse retina Figure 6 Establishing cell models Figure 7 FNBP activates the Nrf2 signaling pathway in ARPE-19 cells. Figure 8 FNBP reduces reactive oxygen species levels in ARPE-19 cells Figure 9 FNBP reduces the level of pro-inflammatory cytokines in ARPE-19 cells. Figure 10 Establishing animal models Figure 11 FNBP reduces retinal reactive oxygen species levels and inhibits retinal cell apoptosis. Figure 12 FNBP reduces the level of pro-inflammatory cytokines in the retina Figure 13 FNBP protects retinal morphology and structure Figure 14 FNBP protects the molecular structure of retinal cells Figure 15 Evaluation of retinal safety and biocompatibility of FNBP Figure 16 Evaluation of the systemic safety and biocompatibility of FNBP Detailed Implementation

[0019] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0020] In the specific embodiments of the present invention, unless otherwise specified, the reagents and materials used are all conventional commercially available products in the art and can be obtained through commercial channels. Example 1: Preparation of FNBP

[0021] According to the method described in the example of ZL2020103306033, 200 mg (approximately 0.45 mmol) of 1H,1H,2H,2H-perfluoro-1-octylthiol (PFOT) was dissolved in 1.0 mL of methanol, and 347.7 mg (approximately 1.57 mmol) of 2,2'-dithiodipyridine (DTPY) was dissolved in 2.5 mL of methanol. The DTPY solution was slowly added dropwise to the PFOT solution at 4°C, and the reaction was stirred for 12 hours. After the reaction was complete, the solvent was removed by rotary evaporation to obtain the crude product. Further purification was performed by column chromatography using dichloromethane:methanol = 30:1 (v / v) as the eluent to obtain the pale yellow oily target product tag 13F, with a yield of 76.2% (150.3 mg).

[0022] The peptide NBP (commercial Nrf2 peptide, English name: Keap1-Nrf2-IN-16, CAS No.: 1362661-40-2, 10 mg, purchased from Shanghai Chutai Biotechnology Co., Ltd.) was dissolved in 2.0 mL of methanol; separately, tag 13F (molar ratio to NBP: 3:1) was dissolved in 1.0 mL of dichloromethane. Under nitrogen protection at 4℃, the tag 13F solution was slowly added dropwise to the NBP solution, and the reaction was continuously stirred for 6 hours. The reaction process was monitored by thin-layer chromatography or high-performance liquid chromatography. After the reaction was completed, the mixture was slowly added to 30 mL of cold diethyl ether to quench the reaction. The white precipitate obtained by centrifugation or filtration was collected as crude FNBP product, and the chemical bond between the fluorinated alkyl chain and the peptide was -SS-. The crude product was then purified using a preparative high-performance liquid chromatography-mass spectrometry system. A C18 reversed-phase column was used with water-acetonitrile as the mobile phase, and the target component was collected by linear gradient elution at a ratio of 95:5 to 20:80 for 30 minutes to obtain the FNBP product.

[0023] The purity of FNBP was verified to be 98% by high-performance liquid chromatography (HPLC) and energy-in-saturation mass spectrometry (ESI-MS), with a molecular weight of 2227.13 g / mol. HPLC showed that the retention time of FNBP was significantly prolonged compared with that of the unmodified peptide NBP, confirming that its hydrophobicity was enhanced by the introduction of the fluorinated alkyl chain. ESI-MS analysis further confirmed the successful synthesis of each target product, with FNBP being detected. Characteristic ion peaks were detected by FITC-FNBP. Characteristic ion peaks were observed, and all results were consistent with the theoretical molecular weight. Dynamic light scattering results showed that FNBP self-assembled in aqueous solution to form uniformly sized nanoparticles with a hydrodynamic diameter of approximately 364 nm and a polydispersity index of 0.22. Transmission electron microscopy images clearly showed that FITC-FNBP could self-assemble into regularly shaped and uniformly distributed spherical nanoparticles. (See...) Figure 1 ) The FNBP obtained in Example 1 was subjected to the following tests: Experiment 1: Cellular accumulation of FNBP in RPE cells.

[0024] ARPE-19 cells were treated with FITC-labeled FNBP (FITC-FNBP) and NBP (FITC-NBP) at progressively increasing concentrations (5 μM, 10 μM, 20 μM) for 6 hours, or with 10 μM FITC-FNBP for different durations (1, 2, 4, 6, 8 hours). Cells were fixed with paraformaldehyde and observed under a fluorescence microscope or by flow cytometry to detect cell uptake. Fluorescence microscopy showed that, at the same concentration and treatment time, the intracellular fluorescence intensity and distribution in the FITC-FNBP group were significantly higher than those in the FITC-NBP group, and that FITC-FNBP uptake was time- and concentration-dependent. Flow cytometry results of the positive uptake rate were consistent with those of fluorescence microscopy, both indicating that FITC-FNBP uptake was dose- and time-dependent (see [link to relevant documentation]). Figure 2 ARPE-19 cells were incubated with 10 μM FITC-FNBP and red dyes labeling late endosomes and lysosomes for 2 h. Fluorescence microscopy revealed no colocalization of FITC-FNBP with the red acidic organelles, thus confirming that FNBP enables endosome escape (see [link to article]). Figure 3 Furthermore, FITC-FNBP uptake was observed after pretreatment with various endocytosis inhibitors for 0.5 h prior to 10 μM FITC-FNBP treatment. It was found that FITC-FNBP uptake was significantly inhibited at 4°C or in the presence of the lipid raft disruptor methyl-β-cyclodextrin (MβCD), while the presence of the caveolin-mediated endocytosis inhibitor Filipin, the clathrin inhibitor chlorpromazine (CPZ), and the tubulin pathway inhibitor colchicine had only a marginal effect on cellular uptake positivity (see [link to article]). Figure 4 Furthermore, in in vivo experiments, by injecting FITC-FNBP into the vitreous cavity of mice, and then, on the fourth day after injection, removing the mouse eyeballs and preparing frozen sections, retinal pigment epithelial cells were stained with RPE65 antibody. Under a fluorescence microscope, FITC-FNBP was observed to co-localize with the retinal pigment epithelium, confirming that FNBP has the ability to penetrate the retina and can be successfully delivered to the RPE cell layer (see...). Figure 5 ).

[0025] Experimental Example 2: In vitro cell experiment A sodium iodate-induced oxidative stress model was established by co-incubating the human RPE cell line ARPE-19 with 10 mM sodium iodate for 12 h. It was observed that the intracellular reactive oxygen species (ROS) level (H2DCFDA marker) significantly increased with increasing sodium iodate concentration, and ARPE-19 cell viability decreased with increasing sodium iodate concentration. Compared with the control group, the 10 mM sodium iodate treatment group showed a 3-fold increase in ROS content and a 30% decrease in ARPE-19 cell viability (see...). Figure 6 In a sodium iodate-induced oxidative stress model, treatment with 10 μM FNBP peptide significantly reduced ROS levels (by 70%) as shown by reactive oxygen species (ROS) staining (H2DCFDA labeling). Western blotting confirmed that FNBP promoted the expression of p-Nrf2 (2-fold) and HO-1 (5-fold) proteins. qPCR analysis showed upregulation of antioxidant genes HO-1 (2-fold) and GSTM (3-fold), and downregulation of inflammatory factors IL-6 (50% decrease) and IL-8 (70% decrease) (see [link to PCR analysis]). Figure 7-9 ).

[0026] Experimental Example 3: In vivo efficacy test A 5 mg / ml sodium iodate stock solution was prepared in vitro and then diluted with PBS buffer according to the body weight of C57BL / 6 mice. The sodium iodate was then injected intravenously at a rate of 20 mg / kg (200 μL) into the mice using an insulin injection syringe to establish a sodium iodate-induced dry AMD model. Fundus photography and HE staining of retinal sections were performed on mice at 1, 4, and 7 days post-injection. Fundus photography showed large areas of retinal atrophy and degenerative changes over time. HE staining showed that the thickness of the inner nuclear layer (INL) decreased by 20%, 30%, and 50% at 4, 7, and 14 days, respectively, while the thickness of the outer nuclear layer (ONL) decreased by 50%, 50%, and 80% at 4, 7, and 14 days, respectively (see [link to relevant documentation]). Figure 10 In some mice, FNBP was administered via intravitreal injection at a dose of 0.2 nmol / g body weight. Compared with the untreated group, the FNBP-treated group showed an 80% decrease in retinal reactive oxygen species (ROS) levels in retinal DHE staining and a 70% reduction in the number of apoptotic retinal cells in TUNEL assay (see [link to article]). Figure 11 The levels of pro-inflammatory inflammatory factors decreased by 60% (see...) Figure 12 In terms of fundus structure, compared with the untreated group, the FNBP-treated group of mice showed a significant reduction of retinal atrophy by 25%, and the overall retinal thickness and the outer nuclear layer thickness were significantly preserved to 80% of their original thickness. H&E staining showed that the retinal structure was intact (see [link to original text]). Figure 13 Immunofluorescence showed the restoration of expression of ZO-1, RPE65, Rhodopsin, and Recoverin, specific functional proteins of retinal photoreceptor cells and retinal pigment epithelium (see [link to study]). Figure 14 ).

[0027] Test Example 4 Safety Evaluation After instilling mydriatic drugs into the eyes of anesthetized mice, a needle was inserted at a 30-45° angle 1 mm posterior to the limbus. The syringe was then smoothly and slowly advanced, injecting 1 μL of FNBP solution over 5-10 seconds. The FNBP injection dose was 0.2 nmol / g body weight. On the 7th day post-injection, fundus photography was performed to observe the fundus structure of the mice, electroretinography was conducted, and tissue samples from major organs were prepared for HE staining. No abnormalities were found in any of these examinations. Peripheral blood samples were collected for liver and kidney function indicators (ALT, CREA, BUN, ALB), and no significant differences were found compared with the control group, indicating that FNBP has good safety and biocompatibility (see...). Figure 15-16 ).

Claims

1. Use of a fluorinated modified peptide in the preparation of a medicament for treating dry age-related macular degeneration.

2. Use according to claim 1, characterized in that The fluorinated modified peptide is prepared by a disulfide exchange reaction between a polypeptide with an amino acid sequence as shown in SEQ ID No: 1 and a compound of Formula I: 。 3. Use according to claim 2, characterized in that The chemical bond between the compound of Formula I and the polypeptide is -S-S-.

4. Use according to claim 2, characterized in that The compound of Formula I is prepared by the following method: 1H, 1H, 2H, 2H-perfluoro-1-octanethiol is dissolved in methanol, and 2,2'-dithiodipyridine is also dissolved in methanol, then the 2,2'-dithiodipyridine solution is slowly added dropwise into the 1H, 1H, 2H, 2H-perfluoro-1-octanethiol solution at 4°C, and the reaction is stirred for 12-16 hours, after the reaction is completed, the solvent is evaporated to obtain a crude product, which is further purified by column chromatography using dichloromethane:methanol 30:1 as the eluent to obtain the target product as a light yellow oil.

5. Use according to claim 1, characterized in that The fluorinated modified peptide is used in the preparation of a medicament for reducing oxidative stress, inflammatory response and cell apoptosis.

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