Use of lanosterol derivatives for the preparation of medicaments for the treatment of retinal degenerative diseases

Abstract

The application discloses application of lanosterol derivatives with the following structure in preparation of drugs for treating retinal degenerative diseases. A novel compound, lanosterol derivative L7, is synthesized by reasonably modifying a lanosterol mother nucleus structure, and the bioavailability and retinal targeting property of the lanosterol derivative L7 are significantly improved, and the lanosterol derivative L7 can significantly improve retinal degeneration and shows better safety and effectiveness.

Description

Technical fields:

[0001] This invention relates to the field of biomedical technology, specifically to the application of lanosterol derivatives in the preparation of drugs for treating retinal degenerative diseases. Background technology:

[0002] Retinal degenerative diseases, including age-related macular degeneration (AMD), diabetic retinopathy (DR), and retinitis pigmentosa (RP), are the leading causes of irreversible vision loss and blindness worldwide.

[0003] A growing body of research indicates that abnormal lipid metabolism plays a crucial role in the development and progression of age-related macular degeneration (AMD), and is considered one of its main pathogenic mechanisms. A typical clinical feature of AMD is the formation of drusen, structures primarily composed of lipid droplets (LDs) accumulating beneath the retinal pigment epithelium (RPE). As the disease progresses, drusen continue to enlarge and accumulate, eventually leading to RPE cell dysfunction and atrophy, resulting in irreversible visual impairment. Based on its clinical characteristics, AMD is classified into two types: "wet" (accompanied by choroidal neovascularization) and "dry" (primarily characterized by RPE dysfunction and lipid accumulation). Current clinical treatments mainly focus on anti-VEGF drugs, such as ranibizumab (Lucentis) and aflibercept (Eylea), for the intervention of wet AMD; however, there are currently no approved effective drugs for dry AMD, especially for retinal degeneration lesions related to lipid metabolism disorders.

[0004] Besides AMD, diabetic retinopathy (DR), one of the most common microvascular complications of diabetes, has also been shown in recent years to be closely related to lipid metabolism disorders (see X. Zhang et al., “Dysregulated Serum Lipid Metabolism Promotes the Occurrence and Development of Diabetic Retinopathy Associated With Upregulated Circulating Levels of VEGF-A, VEGF-D, and PlGF,” Front. Med., vol. 8, p. 779413, 2021, doi:10.3389 / fmed.2021.779413.). Furthermore, lipid droplets and lipofuscin also show abnormal accumulation in diabetic models, further suggesting that lipid metabolism disorders are an important driving factor in the development of DR. Although several studies have linked lipid-lowering therapy with the delay of DR, there is currently a lack of specific therapeutic drugs targeting lipid metabolism regulation.

[0005] The retinal pigment epithelium (RPE) is a crucial component of the blood-retinal barrier, performing a variety of key physiological functions. Its proper functioning depends on a precisely regulated lipid homeostasis network with photoreceptor cells, including the uptake of exogenous lipids from the circulation, the phagocytosis of cholesterol-rich photoreceptor outer segment fragments, and the synthesis of necessary lipids through endogenous synthetic pathways.

[0006] Recent studies (see RH Guymer and TGCampbell, Age-related macular degeneration, Lancet Lond. Engl., vol. 401, no. 10386, pp. 1459–1472, Apr. 2023, doi: 10.1016 / S0140-6736(22)02609-5.) have shown that lipid metabolism imbalance, cholesterol homeostasis disturbance, and lipid droplet and lipofuscin accumulation in RPE cells are closely related to various retinal degenerative diseases such as AMD. However, current intervention strategies targeting lipid metabolism pathways are relatively scarce, limiting the effective blocking of related pathological mechanisms.

[0007] Lanosterol is a key intermediate in steroid biosynthesis and has been reported (L. Lim et al., "Lanosterol induces mitochondrial uncoupling and protects dopaminergic neurons from cell death in a model for Parkinson's disease," Cell Death Differ., vol. 19, no. 3, pp. 416–427, Mar. 2012, doi: 10.1038 / cdd. 2011. 105.) to have various potential biological activities, including protein folding regulation, mitochondrial homeostasis maintenance, and steroid metabolism regulation. Zhao et al. (L. Zhao et al., “Lanosterol reverses protein aggregation in cataracts,” Nature, vol. 523, no. 7562, pp. 607–611, Jul. 2015, doi: 10.1038 / nature14650.) reported that lanosterol can reverse lens protein aggregation in a canine cataract model, opening up opportunities for its application in ophthalmology. However, due to its limited water solubility and biomembrane permeability, the drug development of lanosterol still faces significant challenges. CN 119708108 A discloses a lanosterol derivative, its preparation method, and its application. The structural formula of the lanosterol derivative is shown in Formula I or Formula II.

[0008]

[0009] Y1, Y2, and Y3 are independently hydrogen, deuterium, C1-C4 alkyl, or unsaturated hydrocarbon groups; X is independently oxygen, sulfur, or an NR1 group, wherein R1 in the NR1 group is hydrogen or an alkyl group; R is independently hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl; when X is oxygen, R and Y1, Y2, and Y3 are not simultaneously hydrogen. The results of the examples show that the provided lanosterol derivative has significantly higher water solubility than lanosterol and exhibits significant cataract treatment effects in in vivo animal experiments.

[0010] Currently, there are no reports in publicly available literature or patent databases regarding the application of structurally modified derivatives with systematically optimized pharmacokinetic properties in retinal diseases. Summary of the Invention:

[0011] The purpose of this invention is to provide lanosterol derivatives and their use in the preparation of drugs for treating retinal degenerative diseases.

[0012] This invention is achieved through the following technical solutions:

[0013] A lanosterol derivative, with the following structure:

[0014]

[0015] The synthetic reaction route is as follows:

[0016]

[0017] Includes the following steps:

[0018] a) Compound 1 reacts with succinic anhydride to give intermediate 2;

[0019] b) Compound 7a reacts with silver nitrate to give nitrate ester intermediate 7b;

[0020] c) Intermediate 7b and intermediate 2 are esterified to obtain lanosterol derivative L7.

[0021] This invention significantly improves the solubility, bioavailability, and retinal targeting of lanosterol by rationally modifying its core structure. The resulting lanosterol derivative L7 can significantly improve retinal degeneration and shows better safety and efficacy compared with existing compounds.

[0022] Therefore, this invention also protects the use of the lanosterol derivative L7 in the preparation of drugs for treating retinal degenerative diseases.

[0023] The aforementioned drug contains pharmaceutically usable excipients and is formulated into various dosage forms, including liquids and solids. These dosage forms include tablets, capsules, oral liquids, lozenges, granules, powders, pills, powders, ointments, elixirs, suspensions, powders, injections, suppositories, sprays, drops, or patches.

[0024] The concentration of lanosterol derivative L7 in the drug is 9-11 mg / mL, preferably 10 mg / mL.

[0025] The beneficial effects of this invention are as follows:

[0026] 1. This invention designs and synthesizes a novel compound, referred to as lanosterol derivative L7, by rationally modifying the core structure of lanosterol. This significantly improves its bioavailability and retinal targeting, and can significantly improve retinal degeneration, showing better safety and efficacy.

[0027] 2. This invention is the first to discover the application of lanosterol derivative L7 in the preparation of drugs for treating retinal degenerative diseases. In the constructed animal model of retinal degenerative diseases with lipid metabolism disorders, lanosterol derivative L7 showed good tissue protection and functional recovery effects, providing an innovative treatment idea and a transferable candidate drug for lipid homeostasis-regulated retinal diseases, with good application prospects. Currently, clinical and research focus on anti-angiogenic therapy (such as anti-VEGF), while there is a lack of effective intervention methods for retinal degenerative changes caused by lipid metabolism disorders. Attached image description:

[0028] Figure 1 Lanosterol derivative L7 1 H NMR spectrum (CDCl3303KAV-300).

[0029] Figure 2 Lanosterol derivative L7 13 C NMR spectrum (CDCl3303KAV-300).

[0030] Figure 3 After knocking down LSS in ARPE19 cells, administration of lanosterol derivatives significantly increased lanosterol content.

[0031] Figure 4 :Lss flox / flox BEST1-Cre mice were administered lanosterol derivatives intravitreally and iodate modeled, resulting in a reduction of fundus lesions.

[0032] Figure 5 :Lss flox / flox BEST1-Cre mice were administered lanosterol derivatives intravitreally and then modeled with sodium iodate, resulting in improved visual function. The figure shows Lss. fl / fl For Lss flox / flox The abbreviation is , where A represents the overall waveform of dark-adapted ERG; B represents the quantitative statistical results of the amplitude of dark-adapted ERG Ga wave; C represents the quantitative statistical results of the amplitude of dark-adapted ERG b wave; D represents the overall waveform of ERG c wave; and E represents the quantitative statistical results of the amplitude of ERG c wave. Detailed implementation method:

[0033] The following is a further description of the invention, but not a limitation thereof.

[0034] Example 1: Synthesis of Lanosterol Derivative L7

[0035]

[0036] Includes the following steps:

[0037] a) Lanosterol (1707 mg, 4 mmol, 1 equiv), succinic anhydride (440 mg, 4.4 mmol, 1.1 equiv), and DMAP (489 mg, 4 mmol, 1 equiv) were dissolved in 20 mL of DMF and reacted at room temperature for 24 h. The reaction progress was monitored by TLC. After the reaction was complete, most of the DMF was removed by rotary evaporation. 50 mL of water was added to the reaction flask, and a large amount of white solid was observed to precipitate. After filtration, the filter cake was washed with methanol (3 × 10 mL) and then dried under vacuum to obtain intermediate 2.

[0038] b) Compound 7a (1240 mg, 1.0 equiv) was dissolved in acetonitrile (0.1 mmol / mL), followed by the addition of silver nitrate (4175 mg, 2.5 equiv), and heated under reflux overnight in the dark. The reaction was monitored by TLC, and phosphomolybdic acid was observed to develop color (nitrate esters appear yellow). Acetonitrile was removed by rotary evaporation, and excess saturated saline and EA were added. The mixture was stirred and allowed to stand. The supernatant was transferred to a separatory funnel, and the solid in the flask was washed three times with an equal volume of water and EA. The solid was then transferred to a separatory funnel for extraction. The organic phases were combined, washed three times with saturated saline, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain intermediate 7b.

[0039] c) Intermediate 7b (107 mg, 1.0 equiv) was dissolved in 10 mL of DMF with EDCI·HCl (477.5 mg, 2.5 equiv), DMAP (13 mg, 0.1 equiv), and intermediate 2 (526 mg, 1 equiv) and reacted overnight at room temperature. The reaction progress was monitored by TLC (EA / MeOH = 40:1), and the compound was finally purified by column chromatography to obtain compound L7. 1 HNMR(300MHz,Chloroform-d)δ5.10(s,1H),4.70–4.64(m,2H),4.53(dd,J=11.2,4.8Hz,1H),4.43–4.35(m,2H),2.66(s,4H),2.03(s,4H), 1.70(q,J=13.0,11.4Hz,7H),1.58(d,J=15.0Hz,5H),1.51–1.20(m,9H),1.17(s,1H),1.13(s,1H),1.00(s,3H),0.88(s,14H),0.69(s,3H). 13CNMR (75MHz, Chloroform-d) δ 171.91, 171.60, 134.60, 134.27, 130.80, 125.27, 81.52, 70.31, 60.38, 50.58, 49.84, 44.54, 37.88, 36.93, 35.27, 31.02, 30.84, 29.38, 29.02, 28.17, 27.89, 26.39, 25.64, 24.94, 24.23, 24.15, 22.76, 21.03, 19.15, 18.63, 18.13, 17.57, 16.51, 15.76. Its NMR spectrum is shown in [reference needed]. Figure 1 and Figure 2 .

[0040] Example 2: Drug Efficacy Verification Experiment

[0041] I. Construction of the mouse model:

[0042] Referring to the method described in CN120513907A for establishing an animal model of age-related retinopathy caused by lipid metabolism disorder, this study used CRISPR / Cas9 gene editing technology to specifically knock out the Lanosterolsynthase (Lss) gene in retinal pigment epithelial cells, thus establishing an Lss model. flox / flox BEST1-Cre mouse model.

[0043] In constructing an ideal experimental animal model of age-related retinopathy (LSS) flox / flox Based on the BEST1-Cre mouse model, further retinal oxidative stress damage was induced by tail vein injection of sodium iodate to construct an animal model of lipid metabolism disorder and retinal degenerative disease (LSS). flox / flox BEST1-Cre mice (+SI) were used to simulate retinal degeneration caused by lipid metabolism disorders, providing a reliable model basis for efficacy verification.

[0044] II. Lipid metabolomics detection

[0045] 1. RNA interference

[0046] Human retinal pigment epithelial cells (ARPE-19) were used. ARPE-19 cells were purchased from the China Center for Type Culture Collection. The cells were cultured at 37°C in an incubator containing 5 vol.% carbon dioxide, and in DMEM-F12 medium supplemented with 10 wt.% fetal bovine serum and 1 wt.% penicillin / streptomycin. Once the cells reached 60% confluence, they were divided into four groups. RNA interference experiments were performed using liposome transfection. LSS small interfering siRNA (siRNA-LSS) or blank control small interfering siRNA (siRNA-NC) was transfected into two groups of cells respectively, as follows:

[0047] 1) Dissolve siRNA (siRNA-LSS or siRNA-NC) in nuclease-free water to a final concentration of 20 nM;

[0048] 2) Mix 2 μl of the siRNA obtained in step 1) with 2 μl of Lipofectamine TM RNAiMAX transfection reagent was added to 100 μl of serum-reduced medium Opti-MEM (Thermo Fisher Scientific GIBCO, 500 ml), and then the two were mixed to form a mixture.

[0049] 3) After incubating at room temperature for 15 minutes, add the mixture to the cells.

[0050] Step 1) involves purchasing siRNA from Gemma Biotechnology, and its sequence is as follows:

[0051]

[0052] 2. Drug treatment

[0053] 24 hours after siRNA transfection of ARPE-19 cells, the transfection solution was discarded, fresh culture medium was replaced, and the cells were then treated with drugs.

[0054] 1) NC+DMSO group: Cells transfected with blank control small interfering siRNA (siRNA-NC) were added with an equal volume of DMSO as a solvent control;

[0055] 2) NC+L7 group: Cells transfected with blank control small interfering siRNA (siRNA-NC) were then treated with lanosterol derivative L7 (final concentration 20 μM);

[0056] 3) siLSS+DMSO group: Cells transfected with LSS small interfering siRNA (siRNA-LSS) were given an equal volume of DMSO.

[0057] 4) siLSS+L7 group: Cells transfected with LSS small interfering siRNA (siRNA-LSS) were given lanosterol derivative L7 (final concentration 20μM).

[0058] The lanosterol derivative L7 is completely soluble in the solvent.

[0059] 3. Extraction of metabolites (operated entirely on ice) steps are as follows:

[0060] 1) After 48 hours of drug treatment, discard the culture medium, add 1 ml of pre-cooled 0.9 wt.% physiological saline to wash the cells, and repeat 2-3 times;

[0061] 2) Add 1 ml of a mixture that has been pre-cooled at -20°C for 4-6 hours in advance. The mixture is made of mass spectrometry grade hexane and mass spectrometry grade isopropanol in a volume ratio of 3:2.

[0062] 3) Quickly scrape the cells and transfer them to a 1.5ml EP tube, vortex for 3 min; centrifuge at 12000g for 10 min at 4℃; remove all organic layers and blow dry with nitrogen, store at -80℃;

[0063] 4. Liquid Chromatography-Mass Spectrometry (LC-MS): Experiments were performed using an Agilent 1290 Infinity II LC system coupled with an Agilent 6495A triple quadrupole LC-MS system. Mass spectrometry employed an atmospheric pressure chemical ionization (APCI) source in multiple reaction monitoring (MRM) mode for qualitative and quantitative detection. The ions tested by mass spectrometry are shown in the table below:

[0064]

[0065] The results are as follows Figure 3 As shown, the results indicated that L7 supplementation significantly increased lanosterol levels in the control group (NC) cells; while in the LSS knockdown group (siLSS) cells, lanosterol levels were significantly decreased, but after L7 administration, lanosterol levels recovered significantly and increased substantially. Furthermore, the lanosterol derivative L7 was completely soluble in the solvent. These results demonstrate that the lanosterol derivative L7 can effectively compensate for the reduction in lanosterol caused by LSS deficiency.

[0066] III. Fundus Examination

[0067] 6-8 week old mice were randomly divided into 4 groups: Lss flox / flox +SI group (control group with NaIO3 modeling), Lss flox / flox +SI+L7 group (control group treated with NaIO3 modeling), Lss flox / flox BEST1-Cre+SI group (experimental modeling group), Lss flox / floxBEST1-Cre+SI+L7 treatment group (experimental modeling treatment group). Among them, Lss flox / flox +SI group is in Lss flox / flox In mice, retinal oxidative stress damage was induced by tail vein injection of sodium iodate (NaIO3). flox / flox +SI+L7 group is in Lss flox / flox Based on the mouse model, a retinal oxidative stress injury model was established by tail vein injection of sodium iodate. Then, L7 (10 mg / ml, 1 μl) was injected into the vitreous cavity of the mice using a microinjector to evaluate the effect of L7 in control mice.

[0068] Lss flox / flox The BEST1-Cre+SI group was an ideal experimental animal model of age-related retinopathy (LSS) constructed in this group. flox / flox Based on the BEST1-Cre mouse model, we further induced retinal oxidative stress damage by injecting sodium iodate into the tail vein to construct an animal model of retinal degenerative diseases caused by lipid metabolism disorders.

[0069] Lss flox / flox The BEST1-Cre+SI+L7 administration group was used in the construction of an ideal experimental animal model of age-related retinopathy (LSS). flox / flox Based on the BEST1-Cre mouse model, retinal oxidative stress damage was further induced by tail vein injection of sodium iodate, and L7 (10 mg / ml, 1 μl) was further injected into the vitreous cavity of the mice using a microinjector.

[0070] AMD model was induced in mice by tail vein injection of NaIO3. One week later, fundus images of the mice were acquired and retinal structures were assessed using the fundus photography module of the Phoenix MICRON IV small animal retinal imaging system. The specific steps are as follows:

[0071] 1. Anesthesia: Anesthesia was administered via intraperitoneal injection of 1 wt.% sodium pentobarbital (dose: 70 mg / kg);

[0072] 2. Mydriasis and topical anesthesia: After the mice are anesthetized for about 3-5 minutes, compound tropicamide eye drops (mydriatic) and acecaine eye drops (topical anesthetic) are instilled into both eyes to dilate the pupils and perform topical anesthesia.

[0073] 3. Animal fixation and imaging preparation: After the pupils are fully dilated (about 5 minutes), place the mouse on the mouse holder of the small animal retinal imaging system to expose the eyeball;

[0074] 4. Open the fundus photography software, acquire retinal images, and drip 0.9 wt.% physiological saline onto the ocular surface to keep the mouse's ocular surface moist and prevent corneal dryness from affecting image quality;

[0075] 5. Image Acquisition and Saving: Launch the Phoenix fundus photography software, adjust the field of view, and acquire color fundus images and OCT (Optical Coherence Tomography) images. After image acquisition, save and export the images for subsequent analysis.

[0076] 6. Fundus examination results are shown below. Figure 4 : Figure 4 Showing Lss flox / flox Mice (top left) and Lss flox / flox ; Fundus lesions in BEST1-Cre mice (top right) induced by sodium iodate (NaIO3) and the therapeutic effects of lanosterol derivative L7 drug intervention (bottom left, bottom right). Figure 4 As shown, after NaIO3 treatment, Lss flox / flox In BEST1-Cre mice, extensive yellowish-white lesions were observed in the retina, indicating severe retinopathy of prematurity (RPE) damage and retinal degeneration. In contrast, the lesion area and yellowish-white lesion region were significantly reduced in the lanosterol derivative L7 treatment group, suggesting that lanosterol derivative L7 has a potential role in alleviating lesions. Figure 4 As shown, OCT imaging revealed that NaIO3-induced Lss flox / flox BEST1-Cre mice exhibited disordered retinal structure, significant atrophy of the RPE layer, and extensive loss of subretinal deposits, the outer nuclear layer (ONL), and the photoreceptor layer. In contrast, the lanosterol derivative L7 treatment group showed a more complete retinal layer structure, significantly reduced subretinal deposits, and better preservation of the ONL thickness and photoreceptor layer, suggesting that lanosterol derivative L7 can protect retinal structure to some extent and delay tissue degeneration.

[0077] IV. Visual function examination

[0078] The electroretinograms (ERGs) of mice were recorded using the Diagnosys Celeris rodent ERG device. The specific steps are as follows:

[0079] 1. Dark adaptation: Mice were placed in a dark environment for at least 6 hours in advance to allow for dark adaptation;

[0080] 2. Anesthesia: Mice were administered an intraperitoneal injection of 1 wt.% sodium pentobarbital (prepared with 0.9 wt.% physiological saline);

[0081] 3. Mydriasis and local anesthesia: Topiramate eye drops and Alcaine eye drops were instilled into the ocular surface of mice;

[0082] 4. Lubrication: Apply hydroxymethyl cellulose to the ocular surface of mice;

[0083] 5. ERG detection: Dark adaptation (scotopic) testing was performed on mice. Mice were placed on a control table, and electrodes were attached to the corneas. The stimulus was white light at 0.03 cd·s / m². 2 ERGa and beta waves in mice were recorded under stimulation light intensity of 150.0 cd·s / m 2 The ERG c-wave of mice was recorded under the stimulation light intensity. After the test, tobramycin eye ointment was applied to the experimental eyes, and the mice were placed on a heating pad for resuscitation. The entire experiment was conducted in a dark environment.

[0084] 6. Results of visual function in mice are shown below. Figure 5 :

[0085] like Figure 5 As shown, from a visual function perspective, sodium iodate creates a model (Lss) flox / flox ;BEST1-Cre+SI) seven days after modeling, Lss flox / flox ; a wave in BEST1-Cre mice ( Figure 5 B), b wave ( Figure 5 C) C wave ( Figure 5 D) was significantly decreased, indicating that photoreceptor function, bipolar cell function, and RPE function were all impaired, meaning the degree of visual impairment in mice was aggravated. In contrast, the lanosterol derivative L7 treatment group (Lss) showed a significant decrease. flox / flox BEST1-Cre+SI+L7) administered intravitreally to mice α-wave ( Figure 5 B), b wave ( Figure 5 C) C wave ( Figure 5 E) has rebounded somewhat, especially wave b ( Figure 5 A) C wave ( Figure 5 D) Significant improvement, indicating visual function recovery, suggesting that lanosterol derivative L7 can alleviate oxidative stress-induced visual impairment and has a certain protective or restorative effect on visual function.

[0086] in conclusion:

[0087] 1. In ARPE-19 cells, LSS expression was interfered with by siRNA and the lanosterol derivative L7 was administered. Lipid metabolomics (LC-MS) was used to confirm that the lanosterol derivative L7 can regulate lipid homeostasis.

[0088] 2. In a mouse model of NaIO3-induced RPE oxidative stress injury, the effects of intravitreal injection of lanosterol derivative L7 on retinal structure and function were observed by combining fundus photography, OCT imaging and ERG electrophysiological detection.

Claims

1. A lanosterol derivative, characterized in that, The structure is as follows: 。 L7 2. The method for preparing the lanosterol derivative according to claim 1, characterized in that, The synthetic reaction route is as follows: ； Includes the following steps: a) Compound 1 reacts with succinic anhydride to give intermediate 2; b) Compound 7a reacts with silver nitrate to give nitrate ester intermediate 7b; c) Intermediate 7b and intermediate 2 are condensed by esterification to obtain lanosterol derivative L7.

3. The use of the lanosterol derivative L7 of claim 1 in the preparation of a medicament for treating retinal degenerative diseases.

4. The application according to claim 3, characterized in that, The drug contains pharmaceutically usable excipients and is formulated as a liquid or solid dosage form.

5. The application according to claim 4, characterized in that, The dosage forms include tablets, capsules, oral liquids, lozenges, granules, powders, pills, ointments, suspensions, powders, injections, suppositories, sprays, drops, or patches.

6. The application according to claim 3, characterized in that, The concentration of lanosterol derivative L7 in the drug is 9-11 mg / mL.

7. The application according to claim 3, characterized in that, The concentration of lanosterol derivative L7 in the drug is 10 mg / mL.

Images