Use of a tnF-a inhibitor for the preparation of a medicament for the treatment of psoriasis comorbidities

Abstract

The application discloses application of a TNF-alpha inhibitor in preparation of a medicine for treating psoriasis nervous system comorbidity, and relates to the technical field of biological medicine. The application specifically proposes application of the TNF-alpha inhibitor in preparation of a medicine for treating psoriasis nervous system comorbidity, wherein the psoriasis nervous system comorbidity is one or more of depression, anxiety and cognitive impairment; the application of the TNF-alpha inhibitor is explicitly extended to the field of nervous system comorbidity of psoriasis for the first time, a new pathological mechanism-based medicine selection is provided for clinical treatment of the refractory comorbidity, peripheral and central TNF-alpha levels are reduced, blood-brain barrier tight junction structures are protected, brain inflammatory factor infiltration and neural cell apoptosis are reduced, and thus nervous system dysfunction is reversed.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of TNF-α inhibitors in the preparation of drugs for treating neurological comorbidities of psoriasis. Background Technology

[0002] Psoriasis is a systemic, immune-mediated, chronic inflammatory skin disease with a global prevalence of approximately 2-3%, severely impacting patients' quality of life. Recent studies have shown that psoriasis is not only a skin disease but also a systemic inflammatory state, often accompanied by multiple comorbidities, including psoriatic arthritis, cardiovascular disease, metabolic syndrome, and neurological dysfunction.

[0003] Among psoriasis patients, the incidence of depression, anxiety, and cognitive impairment is significantly higher than in the general population. Epidemiological studies show that approximately 30-50% of psoriasis patients experience varying degrees of depression or anxiety symptoms, and their risk of suicidal ideation and behavior is also significantly increased. Furthermore, multiple clinical studies suggest that psoriasis is associated with cognitive decline and even the risk of dementia. These neurological comorbidities severely impact patients' quality of life, treatment adherence, and long-term prognosis, and have become a crucial issue that urgently needs to be addressed in the comprehensive management of psoriasis. Summary of the Invention

[0004] In order to comprehensively address the above problems, the present invention aims to provide the use of TNF-α inhibitors in the preparation of medicaments for treating neurological comorbidities of psoriasis.

[0005] The first aspect of this invention provides the use of TNF-α inhibitors in the preparation of medicaments for treating neurological comorbidities of psoriasis.

[0006] Preferably, the neurological comorbidities of psoriasis include one or more of depression, anxiety, and cognitive impairment.

[0007] Preferably, the TNF-α inhibitor is InVivo MAb anti-mouse TNF-α.

[0008] A second aspect of the present invention provides a medicament for treating neurological comorbidities of psoriasis, said medicament comprising a TNF-α inhibitor.

[0009] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0010] Preferably, the dosage form of the drug includes any one of granules, tablets, capsules, and injections.

[0011] Compared with the prior art, the advantages of this invention are as follows: This invention is the first to explicitly extend the application of TNF-α inhibitors to the field of neurological comorbidities of psoriasis, providing a novel drug option based on pathological mechanisms for the clinical treatment of this type of refractory comorbidity. It was verified that TNF-α inhibitors can simultaneously improve skin inflammation and neuropsychiatric symptoms of psoriasis through the same mechanism of action, achieving dual effects with one drug, improving treatment efficiency, and improving the overall prognosis of patients; By reducing peripheral and central TNF-α levels, protecting the tight junctions of the blood-brain barrier, and reducing the infiltration of inflammatory factors and neuronal apoptosis in the brain, abnormal nervous system function can be reversed. Attached Figure Description

[0012] Figure 1 The images show the changes in the appearance of the skin on the trunk of mice in each group on days 3 and 6 after drug administration.

[0013] Figure 2 The following are PASI scores for the severity of skin lesions on the trunk of mice in each group of mice in this application; where A is the erythema score from day 1 to day 6 of drug administration; B is the scaling score from day 1 to day 6 of drug administration; C is the thickness score from day 1 to day 6 of drug administration; and D is the PASI score from day 1 to day 6 of drug administration.

[0014] Figure 3 This is a schematic diagram showing the movement trajectories of mice in each group in the open field on days 3 and 6 during the open field test of this application.

[0015] Figure 4 This is a schematic diagram comparing the total distances moved by mice in each group on days 3 and 6 during the open field test of this application.

[0016] Figure 5 This is a schematic diagram showing the comparison of the cumulative time mice in each group spent in the central area on days 3 and 6 during the open field test of this application.

[0017] Figure 6 This is a schematic diagram showing the comparison of the number of times mice in each group entered the central region on days 3 and 6 during the open field test of this application.

[0018] Figure 7 The following are statistical graphs showing the time of complete immobility of mice in each group during the tail suspension test in this application; where A is the statistical graph of the time of complete immobility on day 3; and B is the statistical graph of the time of complete immobility on day 6.

[0019] Figure 8 This is a schematic diagram comparing the movement trajectories of mice in the Y-maze on days 3 and 6 in the Y-maze experiment of this application.

[0020] Figure 9The graphs show the time spent in the new and different arms of mice in each group in the Y maze according to this application; where A is the time spent in the new and different arms of mice in each group on day 3; and B is the time spent in the new and different arms of mice in each group on day 3.

[0021] Figure 10 This is a schematic diagram showing the localization of Dextean cells in the brain tissue of mice in each group on days 3 and 6, as detected by immunofluorescence staining in this application.

[0022] Figure 11 This is a schematic diagram showing the levels of Dextran in the brain tissue of mice in each group as detected by immunofluorescence staining in this application; where A is a schematic diagram of the Dextran levels in the brain tissue on day 3; and B is a schematic diagram of the Dextran levels in the brain tissue on day 6.

[0023] Figure 12 This is a schematic diagram showing the changes in the expression levels of TNF-α, IL-17, and IL-6 in the peripheral blood of mice in each group as detected by the ELISA experiment in this application; where A is a schematic diagram showing the changes in the expression levels of TNF-α, IL-17, and IL-6 in the peripheral blood on day 3; and B is a schematic diagram showing the changes in the expression levels of TNF-α, IL-17, and IL-6 in the peripheral blood on day 6. Detailed Implementation

[0024] The following is in conjunction with embodiments of the present invention. Figures 1-12 It should be understood that the preferred embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0025] The raw material information involved in this invention is as follows: C57BL / 6 mice, male, 6-8 weeks old, sourced from Guangdong Provincial Medical Laboratory Animal Center, were housed in an SPF animal room. During the rearing period, the room temperature was controlled at 20-24℃ and the humidity at 50%-70%. The light and dark environment was alternated every 12 hours. Standard feed and drinking water were provided regularly. Experiments were conducted after 1 week of feeding adaptation.

[0026] All solvents used in this invention are commercially available analytical grade reagents. Among them, the monoclonal antibody InVivo MAbanti-mouse TNF-α (Catalog#BE0058, Clone:XT3.11, BioXcell) and the ELISA kit were purchased from Abcam.

[0027] Mice were randomly divided into a control group (Ctrl), an IMQ group, and an IMQ+anti-TNF-α treatment group (IMQ+anti-TNF-α), with 8 mice in each group.

[0028] After shaving the backs of mice in the control group, Vaseline was applied daily for 6 consecutive days.

[0029] IMQ mice were used to establish a psoriasis-like dermatitis model. After shaving the back of the mice, 62.5 mg of imiquimod (IMQ) cream (5%, w / w) was evenly applied to an area of ​​about 2 cm × 3 cm daily for 6 consecutive days.

[0030] After topical application of imiquimod, as Figure 1 The IMQ group mice shown exhibited psoriasis-like characteristics starting on day 3, manifesting as plaque formation and scaly skin lesions, peaking on day 6; Figure 2 The results showed that the erythema, scaling, thickening, and PASI score of the IMQ group mice gradually increased from day 3, and there were significant differences compared with the control group from day 3 to day 6. Therefore, a stable psoriatic dermatitis model was successfully established in the IMQ group. No abnormalities were observed in the skin of the control group mice.

[0031] In the IMQ+ anti-TNF-α treatment group, mice were treated with IMQ and then injected intraperitoneally with 300 μg of anti-TNF-α antibody on days 0, 2 and 4 after IMQ administration.

[0032] Experimental Analysis 1. Measurement of erythema, scaling, and thickness of mouse skin lesions Mice in the control group, IMQ group, and IMQ+anti-TNF-α treatment group had skin lesions of erythema, scaling, and thickness on the trunk from day 1 to day 6. PASI scores were then used to assess these lesions. Erythema, scaling, and thickness were scored from 0 to 4, with 0 representing no lesion and 4 representing the most severe lesion. The PASI score was calculated by weighting the scores of erythema, scaling, and thickness and taking into account the affected body surface area. Higher scores indicated more severe skin lesions. Specific criteria are shown in Table 1. The PASI score for each group of mice was calculated according to Table 1, i.e., the total score of (I+E+D), and a curve was plotted based on the average PASI score of each group, as shown in the figure. Figure 2 ,according to Figure 1 and Figure 2 The results showed that the erythema, scaling, and thickness of skin lesions in the IMQ+ anti-TNF-α treatment group were significantly lower than those in the IMQ group.

[0033] 2. Detection of anxiety behavior in mice Open field experiments were conducted on mice in each group. The distance the mice traveled, the time spent in the central area, and the number of times they entered the central area were analyzed to reflect their anxiety behavior. The experiment was conducted in a dark field under 50cm×50cm×50cm square open field conditions, with the central area defined as a 25cm×25cm square. The experimental procedures are as follows: One hour before the experiment, the mice were brought into the experimental room to acclimatize to the environment; The mice were placed in the center of an open field, and their movement was recorded using a camera system over a period of 5 minutes. After each experiment, wipe the open area with 75% alcohol to remove any residual odor. The activity trajectory of the mice was analyzed using EthoVision XT version 9.0 software, including the time spent in the central area and the distance traveled in other areas.

[0034] according to Figures 3-6 The results showed significant differences in total movement distance, central region exploration time, and number of entries into the central region among mice in the control group, IMQ group, and IMQ+ anti-TNF-α treatment group on days 3 and 6. Compared with the IMQ group, the total movement distance of mice in the anti-TNF-α antibody treatment group increased, suggesting an improvement in their overall activity level. Furthermore, the central region exploration time and number of entries were also higher in the anti-TNF-α antibody treatment group than in the IMQ group, but still lower than in the control group. Anti-TNF-α antibody treatment can partially alleviate anxiety behavior, but it does not fully restore it to normal levels.

[0035] 3. Detection of depressive-like behavior in mice The tail suspension test was performed on mice in each group. The degree of depressive-like behavior was inferred from the immobility exhibited by the animals under unavoidable stress. During the experiment, approximately one-third of the mouse's tail was fixed to a support using transparent tape, with its head about 30 cm above the ground, ensuring the mouse could not reach the ground or other objects. Hollow cylinders were placed around the base of the tail to prevent the mice from climbing. The specific experimental steps are as follows: One hour before the experiment, the mice were brought into the experimental room to acclimatize to the environment; The mouse's tail was fixed to a horizontal bar, and the time the mouse remained still during the 6-minute experiment was recorded. The time spent completely stationary was quantitatively analyzed using EthoVision XT version 9.0 software.

[0036] like Figure 7 The tail suspension test results showed that on day 6 of the mouse tail suspension test, the immobility time of the IMQ group was significantly longer than that of the control group, while the immobility time of the anti-TNF-α antibody treatment group was significantly shorter than that of the IMQ group, suggesting that anti-TNF-α antibody treatment can partially alleviate depressive-like behavior. This indicates that, in addition to TNF-α, other inflammatory factors or factors may also be involved in the development of psoriasis-related depressive-like behavior.

[0037] 4. Detection of Spatial Working Memory Impairment in Mice The Y-maze experiment was conducted on mice in each group to analyze their cognitive function, learning ability, and anxiety level. The Y-maze consists of three arms of equal length (approximately 35 cm long, 5 cm wide, and 15 cm high), with the arms forming a Y-shape at a 120° angle. The Y-maze experiment included a training period and a testing period, with the following specific steps: Training period: One arm of the Y maze (the novel arm) was closed off with a partition, and the mice were allowed to explore freely in the other two arms (the starting arm and the other arms) for 6 minutes; Testing period: After 4 hours, the partition of the new variant arm was removed, and the mice were allowed to explore freely in all three arms for 5 minutes; the number of times and the time of entry into each arm were recorded, with particular attention paid to the exploration of the new variant arm.

[0038] After each experiment, the maze was cleaned with 75% alcohol to eliminate odor interference. All data were analyzed using EthoVision XT version 9.0 software.

[0039] according to Figures 8-9 The Y-maze test results showed significant differences in the time spent in the open arm among the three groups of mice. Mice in the IMQ group spent a significantly shorter time in the open arm than the control group and the anti-TNF-α treatment group, suggesting that IMQ-induced psoriasis model mice have impaired spatial learning and memory abilities. Anti-TNF-α antibody treatment can improve the spatial learning and memory abilities of IMQ model mice.

[0040] 5. Dextran tracer immunofluorescence detection Each group of mice was injected with 10-kDa Dextraran, and the specific steps were as follows: First, heat the mouse tail with a warm lamp. Once the mouse tail vein dilates, slowly inject 100 μL of Dextran tracer through the tail vein. After injection, keep the needle in place for 5 minutes to ensure tracer circulation in the blood vessels and prevent premature outflow. After removing the needle, apply pressure to the injection site for 10 minutes. Remove the mouse brain tissue using the same method and place it in a 4% formaldehyde solution. Fix the tissue overnight at 4°C. After fixation, remove the brain tissue from the formaldehyde solution and dehydrate it. During dehydration, sequentially... The brain tissue was immersed in 10%, 20%, and 30% sucrose solutions for 2-3 days until it settled to the bottom of the solution, at which point it was removed. After dehydration, the brain tissue was embedded in the coronal plane using an OCT compound in a cryostat (set to -20°C) for 30 minutes. After embedding, the sample was fixed on a microtome, cut into 30 μm thick sections, and washed three times with PBS buffer for 10 minutes each time. The sections were then placed in a solution containing 5% bovine serum albumin (BSA) and 0.5% Triton X-ray. The slides were incubated overnight at 4°C in a solution of X-100 and diluted with anti-CD31 antibody (1:200 dilution). The slides were then removed and washed with PBS three times for 10 min each time. The slides were placed in a goat anti-mouse secondary antibody (green fluorescent label) containing 5% BSA and 0.5% Triton X-100 and incubated at room temperature for 2 h. The slides were washed with PBS three times for 10 min each time. The slides were then observed under a confocal fluorescence microscope.

[0041] 6. Dextran tracer immunofluorescence quantitative detection 100 μL of Dextran solution was slowly injected into the mouse tail vein; the needle was kept in place for 5 minutes to promote the circulation of the tracer in vivo and prevent it from being lost from the injection site; the injection site was pressed for 10 minutes to further ensure the uniform distribution of the tracer; mouse brain tissue was removed and weighed according to the above method; the brain tissue was mixed with PBS buffer for homogenization; the processed tissue sample was placed in an ultrasonic cell tissue homogenizer and sonicated twice until visible precipitate was observed; after sonication, the sample was centrifuged in a low-temperature centrifuge pre-cooled to 4°C at 12000 r / min for 10 minutes; the supernatant was collected after centrifugation and transferred to a 96-well plate; the 96-well plate was placed in a microplate reader, and the distribution and concentration of the tracer were accurately measured using a fluorescence intensity quantitative detection method with an excitation wavelength of 555 nm and a receiving wavelength of 585 nm.

[0042] Location and quantitative detection results, as follows Figures 10-11As shown, on day 3, compared with the IMQ group, the leakage of Dextran tracer in the brain of mice treated with anti-TNF-α antibody was significantly reduced, indicating that anti-TNF-α antibody treatment effectively protected the integrity of the blood-brain barrier. On day 6, there was no significant difference in the amount of Dextran tracer leakage in the brain of mice in each group.

[0043] 7. ELISA testing Mouse eyeballs were removed with forceps, and blood was dripped into pre-prepared coagulation tubes. After standing at room temperature for 20 minutes, the blood was centrifuged for 20 minutes at 12000 rpm in a pre-cooled 4°C centrifuge. The supernatant from each group was aspirated and transferred to EP tubes, and the levels of corresponding cytokines were detected using an ELISA kit. A standard curve was established using the standards in the kit. 50 μL of sample was added to each well of a 96-well plate, with a sub-well for each sample. The prepared mixed antibody was added to each well, and the plate was incubated at room temperature for 1 hour. After washing three times with washing buffer, 100 μL of TMB was added, and stop solution was added after 10 minutes. The plate was then read and analyzed at 450 nm using a spectrometer.

[0044] according to Figure 12 The ELISA results showed that on days 3 and 6, compared with the IMQ group, the expression levels of TNF-α, IL-17 and IL-6 in the brains of mice treated with anti-TNF-α antibody were significantly reduced, indicating that TNF-α antibody treatment can comprehensively reduce the level of inflammation in the central nervous system and improve the immune environment in the brain.

[0045] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. Application of TNF-α inhibitors in the preparation of drugs for the treatment of neurological comorbidities of psoriasis.

2. The application according to claim 1, characterized in that, The neurological comorbidities of psoriasis include one or more of depression, anxiety, and cognitive impairment.

3. The application according to claim 1, characterized in that, The TNF-α inhibitor is InVivo MAb anti-mouse TNF-α.

4. A drug for treating neurological comorbidities of psoriasis, characterized in that, The drugs include TNF-α inhibitors.

5. The medicament for treating neurological comorbidities of psoriasis according to claim 4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. The medicament for treating neurological comorbidities of psoriasis according to claim 4, characterized in that, The dosage form of the drug includes any one of granules, tablets, capsules, and injections.

Images