Use of a proanthocyanidin for the preparation of a medicament for the treatment of sjogren's syndrome

Abstract

This invention discloses the application of geraniol in the preparation of a drug for treating Sjögren's syndrome. In vivo experiments using a NOD mouse model of Sjögren's syndrome demonstrated that geraniol treatment significantly improved salivary secretion disorders and reduced abnormally high water intake in the mice; significantly reduced lymphocyte infiltration in salivary gland tissue, improved glandular pathological damage, downregulated the levels of characteristic autoantibodies (Anti-SSA / Ro and Anti-SSB / La) in serum, upregulated the expression levels of AQP5 and AR, and reduced organ indices in salivary glands, spleen, and thymus tissue, comprehensively improving dry mouth symptoms in mice. In vitro experiments also showed that geraniol treatment upregulated the expression levels of AQP5 and AR. Therefore, this invention provides the application of geraniol in the preparation of a drug for treating Sjögren's syndrome, which has the advantages of significant efficacy and addressing both the symptoms and root cause, and possesses good clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of geraniol in the preparation of drugs for the treatment of Sjögren's syndrome. Background Technology

[0002] Sjögren's syndrome (SS) is a chronic, systemic autoimmune disease characterized by lymphocytic infiltration and dysfunction of exocrine glands (particularly salivary and lacrimal glands). Clinical manifestations include dry mouth and dry eyes; in severe cases, it can lead to difficulty eating, rampant dental caries, and corneal damage, significantly impairing patients' quality of life. It is explicitly classified as an "autoimmune exocrine gland disease" or "connective tissue disease" by rheumatological criteria (such as the ACR / EULAR classification). Epidemiological studies show that Sjögren's syndrome has a significant age-related prevalence, primarily affecting middle-aged and elderly women over 45 years of age. Furthermore, with the increasing aging of the global population, its incidence is rising annually, making it a significant public health issue.

[0003] The exact etiology and mechanism of Sjögren's syndrome remain incompletely understood, but it is generally believed to result from the interaction of multiple factors, including genetic susceptibility, environmental factors, infection triggers, and abnormal immune regulation. Currently, clinical treatment strategies for Sjögren's syndrome mainly focus on symptom relief and inflammation control, lacking specific drugs that can fundamentally reverse glandular dysfunction. For treating dryness symptoms, clinical practice often employs replacement therapies such as artificial saliva and artificial tears, or cholinergic receptor agonists (such as pilocarpine and cevimeline). However, these drugs are not very effective, and long-term use can easily cause systemic adverse reactions such as excessive sweating, nausea, and abdominal pain. For systemic treatment, clinical practice often uses glucocorticoids or immunosuppressants. Although these can suppress immune inflammation to some extent, restoring glandular function (such as salivary and lacrimal glands) remains an unsolved problem, and there are potential risks such as secondary infections and bone marrow suppression. Therefore, developing new, highly effective, and low-toxicity therapeutic drugs, especially those that can fundamentally improve salivary gland secretion function and repair acinar epithelial cells, has become an urgent need and important direction in the current research field of Sjögren's syndrome.

[0004] In recent years, natural plant active ingredients (such as flavonoids) have received widespread attention due to their significant anti-inflammatory, antioxidant, and immunomodulatory activities. Among them, diosmetin (DIOS, chemical formula C600000) is particularly noteworthy. 16 H 12O5 (CAS: 520-34-3), as a flavonoid compound, exhibits good therapeutic potential. Studies have shown that geraniol has significant effects in antioxidation, anti-inflammation, and antiviral activity, and can play a protective role in the skin, digestive system, and nervous system. In chronic autoimmune diseases such as rheumatoid arthritis, geraniol has been found to exert antiproliferative and anti-inflammatory effects through the protein kinase B and nuclear factor κB signaling pathways.

[0005] Patent WO2022257986A1 discloses the application of CYP450 inhibitors in inhibiting or killing mites and treating dry eye syndrome, wherein the CYP450 inhibitor can be selected from geraniol. However, the dry eye syndrome in this patent is caused by Demodex mites phagocytosing lipids, leading to meibomian gland dysfunction, which is different from Sjögren's syndrome. Although geraniol has shown therapeutic potential in other autoimmune diseases and mite-induced dry eye syndrome, its specific therapeutic effect on Sjögren's syndrome and its effect on improving salivary gland secretion function have not been clearly reported or applied in the prior art. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an application of geraniol in the preparation of a drug for the prevention and / or treatment of dry mouth caused by Sjögren's syndrome.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] This invention systematically evaluated the in vivo therapeutic effect of geraniol (DIOS) on dry mouth caused by Sjögren's syndrome in a classic NOD mouse model. Experimental results showed that geraniol significantly improved salivary secretion disorders and reduced abnormally high water intake in Sjögren's syndrome model mice. Simultaneously, it significantly reduced lymphocyte infiltration in salivary gland tissue, improved glandular pathological damage, downregulated the levels of characteristic autoantibodies (Anti-SSA / Ro and Anti-SSB / La) in serum, upregulated the expression levels of AQP5 and AR, and reduced organ indices in salivary glands, spleen, and thymus. In vitro cell experiments also demonstrated that geraniol treatment upregulated the expression levels of AQP5 and AR. Furthermore, geraniol showed no significant toxic side effects on vital organs such as the heart, liver, brain, lungs, and kidneys at effective therapeutic doses, exhibiting good biocompatibility. Therefore, the geraniol provided by this invention, as a natural Chinese medicine monomer, has a wide range of sources, clear components, and controllable quality. It also has excellent biocompatibility and significant therapeutic effects, providing a safe and effective new drug option for the clinical treatment of Sjögren's syndrome. It has important development value and application prospects.

[0009] Therefore, the present invention provides the use of geraniol in the preparation of medicaments for the prevention and / or treatment of Sjögren's syndrome.

[0010] Furthermore, the drug achieves treatment by improving salivary secretion disorders in the subject.

[0011] Furthermore, the drug achieves treatment by reducing organ indices in the salivary glands, spleen, and thymus tissues of the subjects. This invention demonstrates that geraniol not only significantly increases salivary flow in SS mice but also effectively reduces the area of ​​inflammatory infiltration foci in the salivary glands and lowers autoantibody levels, achieving a dual therapeutic effect on Sjögren's syndrome.

[0012] Furthermore, the drug achieves treatment by reducing the number and extent of lymphatic infiltration foci in the subject's salivary gland tissue.

[0013] Furthermore, the drug achieves treatment by reducing the levels of anti-SSA / Ro and anti-SSB / La antibodies in the subject.

[0014] Furthermore, the drug achieves treatment by protecting and / or repairing the subject's salivary gland epithelial cells.

[0015] Furthermore, the drug achieves treatment by upregulating the expression levels of AQP5 and AR in the subjects.

[0016] Furthermore, the effective dose range of geraniol in the drug is 20–60 mg / kg body weight / day.

[0017] Furthermore, the effective dose range of geraniol in the drug is 25–50 mg / kg body weight / day.

[0018] Furthermore, the subjects were selected from mammals.

[0019] Furthermore, the mammals mentioned are selected from rats, cats, dogs, pigs, cattle, horses, sheep, monkeys, and humans.

[0020] Preferably, the effective dose range of geraniol in the drug is 50 mg / kg body weight / day. The determination of the dosage needs to comprehensively consider factors such as the severity of the patient's condition, weight, and route of administration, and can be administered in a single dose or multiple doses. This invention has shown good efficacy using oral gavage, suggesting that this drug is suitable for development into an oral formulation for long-term use by patients.

[0021] Furthermore, the drug also includes other pharmaceutically acceptable carriers.

[0022] Furthermore, the carrier is a diluent or excipient.

[0023] Furthermore, the dosage form of the drug is an injection, an oral preparation, an eye drop, or an oral spray.

[0024] Furthermore, the oral preparation method is as follows: 0.5 g of phylloxera, 0.1 g of sodium carboxymethyl cellulose, 5.0 g of sorbitol, and 0.02 g of sodium benzoate are thoroughly stirred and dissolved, and then diluted to 100 mL with purified water.

[0025] Furthermore, the dosage form of the drug is either a capsule or a gel.

[0026] Specifically, the oral preparation is prepared by dispersing geraniol micropowder in a medium containing a suspending agent (such as sodium carboxymethyl cellulose or xanthan gum), a wetting agent (such as glycerin), and a preservative. For example, geraniol can be mixed with a filler (such as microcrystalline cellulose or lactose) and a lubricant (such as magnesium stearate), and then processed into tablets or filled into capsules using wet granulation or direct compression processes to obtain tablets or capsules.

[0027] The injection is made by dissolving geraniol in sodium carboxymethyl cellulose solution, filtering it under sterile conditions, and adjusting it to the required concentration, for intravenous injection, intraperitoneal injection, or local intraglandular injection.

[0028] The eye drops are prepared by dissolving micronized geraniol in a sterile aqueous matrix containing boric acid and antibacterial agents to directly improve dryness of the ocular surface and corneal and conjunctival damage.

[0029] Geraniol, a flavonoid compound derived from a natural plant, exhibits excellent biocompatibility. Detailed in vivo toxicological evaluations have rigorously validated its safety, demonstrating no significant toxic side effects at effective doses. Its safety profile is superior to many chemically synthesized drugs and immunosuppressants. Furthermore, geraniol maintains its physical stability and biological activity over extended periods at 2-8°C, which is beneficial for storage, transportation, and clinical application.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This invention provides the application of geraniol in the preparation of drugs for treating Sjögren's syndrome. Experimental results show that geraniol treatment significantly improves salivary secretion disorders and reduces abnormally high water intake in Sjögren's syndrome model mice. Simultaneously, it significantly reduces lymphocyte infiltration in salivary gland tissue, improves glandular pathological damage, downregulates serum levels of characteristic autoantibodies (Anti-SSA / Ro and Anti-SSB / La), upregulates AQP5 and AR expression levels, reduces organ indices in salivary glands, spleen, and thymus, and improves dry mouth symptoms in mice, indicating that geraniol can be used to treat Sjögren's syndrome. In vitro experiments also show that geraniol treatment upregulates AQP5 and AR expression levels. Furthermore, geraniol, at effective therapeutic doses, has no significant toxic side effects on vital organs such as the heart, liver, brain, lungs, and kidneys, demonstrating good biocompatibility. Therefore, this invention provides a safe and effective new drug option for the clinical treatment of Sjögren's syndrome, with significant efficacy and the advantage of addressing both the symptoms and the root cause, showing promising clinical application prospects. Attached Figure Description

[0032] Figure 1 HE staining images (scale bar = 100 μm) of major organs (heart, liver, brain, lungs, kidneys) and results of hematological tests.

[0033] Figure 2 This study evaluates the efficacy of geraniol in NOD mice. Among other things, Figure 2 In the figures, A represents the water intake and salivary secretion index of mice in each group after treatment; B represents the Hematologic and Escherichia coli (H&E) maps of the salivary glands in each group; C represents the pathological scoring of salivary gland infiltration foci in each group; D represents the Anti-SSA / Ro level; E represents the Anti-SSB / La level; F represents the salivary gland organ index; G represents the spleen organ index; and H represents the thymus organ index. P <0.05,** P <0.01, *** P <0.001.

[0034] Figure 3 Geraniol upregulated AQP5 expression in the salivary glands of NOD mice. Figure 3 In the table, A represents the relative expression level of AQP5 mRNA in mouse salivary glands detected by RT-qPCR; B represents the expression level and quantitative analysis of AQP5 in mouse salivary glands detected by immunohistochemistry. Note: Scale bars: 50 μm and 20 μm; **P<0.01, ***P<0.001.

[0035] Figure 4 Geraniol upregulated AR expression in the salivary glands of NOD mice. Figure 4In the table, A represents the relative expression level of AR mRNA in mouse salivary glands detected by RT-qPCR; B represents the expression level and quantitative analysis of AR protein in mouse salivary glands detected by Western blotting; and C represents the expression level and quantitative analysis of AR in mouse salivary glands detected by immunohistochemistry. Note: Scale bars: 50 μm and 20 μm; *P<0.05, **P<0.01, ***P<0.001.

[0036] Figure 5 Geraniol upregulated the expression of AR and AQP5 in NS-SV-AC cells. Figure 4 In the table, A represents the relative expression levels of AR and AQP5 mRNA in NS-SV-AC detected by RT-qPCR; B represents the expression levels and quantification of AR and AQP5 proteins in NS-SV-AC detected by Western blotting; C represents the expression level and quantification of AR in NS-SV-AC detected by IF assay; and D represents the expression level and quantification of AQP5 in NS-SV-AC detected by IF assay. Note: Scale bar: 20 μm, *P<0.05, **P<0.01, ***P<0.001.

[0037] Figure 6 To verify the efficacy of geraniol oral suspension. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0039] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0040] Geranin:

[0041] .

[0042] Example 1: In vivo safety evaluation of geraniol (DIOS)

[0043] Eight-week-old female NOD mice (classic SS model) and ICR mice were used as experimental animals. Mice were randomly divided into three groups (n=6): Model group (NOD mice, SS model): administered sodium carboxymethyl cellulose solution by gavage once daily for 8 weeks; Treatment group (NOD mice, SS+DIOS): administered geraniol (50 mg / kg body weight, dissolved in sodium carboxymethyl cellulose solution) by gavage once daily for 8 weeks; Control group (ICR mice): administered sodium carboxymethyl cellulose solution by gavage once daily for 8 weeks. During the observation period, all mice showed normal mental status, activity, and appetite, and no deaths occurred. After the last administration, organs were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed blinded by a pathologist; hematological parameters were also measured.

[0044] The results are as follows Figure 1 As shown, HE staining results indicated that no significant pathological changes related to drug administration were found in the geraniol-treated group; hematological parameters showed no difference in relative alanine aminotransferase (ALT), relative aspartate aminotransferase (AST), relative ALT / AST ratio, and relative serum creatinine compared to the control group after administration. This indicates that geraniol does not exhibit significant toxic side effects at effective doses and has high safety.

[0045] Example 2: Therapeutic effect of geraniol on SS model mice

[0046] I. Experimental Methods

[0047] Eight-week-old female NOD mice (classic SS model) were used as experimental animals, and ICR mice were used as healthy controls. The NOD mice were randomly divided into two groups of six each.

[0048] (1) NOD mouse group: The mice were treated with sodium carboxymethyl cellulose by gavage once a day for 8 weeks.

[0049] (2) Treatment group (NOD+50mg / kg DIOS): The same volume of sodium carboxymethyl cellulose containing geraniol (50mg / kg / d) was administered by gavage once daily for 8 weeks.

[0050] (3) ICR healthy control group: The same volume of sodium carboxymethyl cellulose was administered by gavage once a day for 8 weeks.

[0051] After 8 weeks of continuous administration, the following tests were performed:

[0052] (1) Water intake measurement: The mice were weighed and their water intake was measured every 2 weeks before and after gavage. The average water intake was calculated by comparing the ratio of water intake (mL) / body weight (g) of the mice every 2 weeks. Finally, the average water intake of each group of mice was obtained.

[0053] (2) Saliva secretion measurement: The saliva flow rate of mice was measured every 2 weeks. The specific operation method is as follows: the mice were anesthetized by intraperitoneal injection of 0.7% sodium pentobarbital (10 mg / kg), and pilocarpine (0.1 mg / kg) was injected subcutaneously to stimulate the mice to secrete saliva. The mice were fixed in a head-down position, and the saliva secretion of the mice was collected within 10 minutes using a capillary tube. The average saliva secretion index of each group of mice was obtained by calculating the saliva volume (mg) / body weight (g).

[0054] (3) Detection of inflammatory infiltration of mouse salivary glands: After an 8-week drug administration experiment, salivary gland tissues of mice in each group were collected and subjected to tissue H&E staining.

[0055] (4) Detection of anti-SSA / Ro and anti-SSB / La antibodies: Mouse serum was used to perform ELISA experiments to detect the expression of anti-SSA / Ro and anti-SSB / La antibodies, which are SS serum markers.

[0056] (5) Determination of organ index in mice: Weigh the mice, sacrifice the mice and collect salivary gland, spleen and thymus tissues, weigh the organs, and calculate the organ index by comparing the ratio of organ weight (g) / body weight (g) to obtain the organ index of each group of mice.

[0057] (6) AQP5 is a marker protein for changes in SS disease, and its level decreases after the onset of the disease. AR is a key functional regulatory protein in SS, and its expression decreases and its function is impaired after the onset of the disease, which is the core upstream reason for the downregulation of AQP5. The expression levels of AQP5 and AR in mouse salivary glands were detected by real-time quantitative polymerase chain reaction (RT-qPCR), Western blot (WB), and immunohistochemistry.

[0058] All experimental data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using GraphPad Prism 8.0 software. Student's t-test was used for comparisons between two groups, and one-way ANOVA, followed by Tukey's post hoc test, was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

[0059] II. Experimental Results

[0060] (1) Results of water intake measurement are as follows Figure 2 As shown in A, geraniol can significantly reduce water intake in NOD mice.

[0061] (2) Results of saliva secretion measurement are as follows Figure 2 As shown in A, geraniol can significantly improve salivary secretion disorders in NOD mice.

[0062] (3) Results of inflammatory infiltration of mouse salivary glands as follows Figure 2 As shown in BC, the number and extent of lymphatic infiltration foci in the salivary gland tissue were reduced in the treatment group (p<0.01).

[0063] (4) Results of anti-SSA / Ro and anti-SSB / La antibody detection are as follows Figure 2 As shown in the DE, the treatment group had a significantly lower level than the NOD group, and the level was close to that of the ICR group (p<0.05).

[0064] (5) Results of mouse organ index measurement are as follows Figure 2 As shown in the FH, the organ indices of the salivary glands, spleen, and thymus tissues in the treatment group were the lowest among the three groups.

[0065] (6) Results of AQP5 qRT-PCR and immunohistochemical staining are as follows: Figure 3 As shown, the AQP5 expression level in the treatment group mice was significantly higher than that in the disease group (p<0.001). AR qRT-PCR, WB, and immunohistochemical staining results are shown below. Figure 4 As shown, the AR expression level in the treatment group mice was significantly higher than that in the disease group.

[0066] Example 3: In vitro therapeutic effects of geraniol

[0067] I. Experimental Methods

[0068] Interferon-γ (IFN-γ) was used to treat human immortalized salivary gland acinar cells (Normal Sallivary gland-SV40 transformed-Acinar Cell line, NS-SV-AC) for 24 hours as an in vitro disease model of salivary gland spondylitis (SS). The next day, geraniol (10... -6Treatment with the AR agonist dihydrotestosterone (DHT) (10 nM) for 24 h. Cellular AR expression changes were verified at the cellular level using real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting (WB), and immunofluorescence (IF).

[0069] II. Experimental Results

[0070] In RT-qPCR experiments ( Figure 5 In the Western blot (WB) experiment, compared with the normal control group, the expression levels of AR and AQP5 mRNA were downregulated in the IFN-γ group and upregulated in the IFN-γ+DIOS group (P<0.05). Figure 5 (B) The results showed that, compared with the normal control group, the expression levels of AR and AQP5 proteins were downregulated in the IFN-γ group, while the expression levels of AR and AQP5 proteins were upregulated in the IFN-γ+DIOS group, and the expression levels were similar to those in the normal control group and the IFN-γ+DHT group (P<0.05). In the immunofluorescence staining experiment ( Figure 5 The results showed that, compared with the normal control group, the expression levels of AR and AQP5 proteins were downregulated in the IFN-γ group, while the expression levels of AR and AQP5 proteins were upregulated in the IFN-γ+DIOS group. This indicates that geraniol can upregulate the expression of AR and AQP5 in NS-SV-AC, and has a certain therapeutic effect on Sjögren's syndrome.

[0071] Example 4: Preparation and efficacy verification of geraniol oral suspension

[0072] Take 0.5 g of geraniol raw material, micronize it (pass through a 200-mesh sieve), and mix it evenly with 0.1 g of sodium carboxymethyl cellulose (thickener / suspending agent), 5.0 g of sorbitol (flavoring agent and humectant), and 0.02 g of sodium benzoate (preservative). Add an appropriate amount of purified water and grind evenly. Then, make up the volume to 100 mL with purified water, stir thoroughly, and dispense into brown oral liquid bottles to obtain geraniol oral suspension (each 1 mL contains 5 mg of geraniol). This product needs to be shaken well before use and can be administered orally or via tube feeding.

[0073] Eight-week-old female NOD mice were randomly divided into two groups (n=6): a model control group (administered daily by gavage with an equal volume of blank matrix, i.e., the above-mentioned excipient solution without geraniol) and a geraniol oral suspension treatment group (administered daily by gavage with the above-mentioned oral suspension at a dose of 50 mg / kg body weight based on geraniol). Saliva flow was measured after 8 weeks of continuous administration.

[0074] The results are as follows Figure 6 As shown, the salivary flow rate of mice in the geraniol oral solution treatment group was significantly higher than that in the model control group (p<0.01), indicating that this oral suspension can effectively improve salivary secretion disorders in Sjögren's syndrome model mice. This preparation method is simple and has good patient compliance, providing a clinical translational basis for geraniol treatment of Sjögren's syndrome.

Claims

1. The use of geraniol as the sole active ingredient in the preparation of drugs for the prevention and / or treatment of dry mouth caused by Sjögren's syndrome.

2. The application according to claim 1, characterized in that, The drug achieves its therapeutic effect by improving salivary secretion disorders in the subject.

3. The application according to claim 1, characterized in that, The drug achieves its therapeutic effect by reducing organ indices in the subject's salivary glands, spleen, and thymus tissue.

4. The application according to claim 1, characterized in that, The drug achieves its therapeutic effect by reducing the number and extent of lymphatic infiltration foci in the subject's salivary gland tissue.

5. The application according to claim 1, characterized in that, The drug achieves treatment by reducing the levels of anti-SSA / Ro and anti-SSB / La antibodies in the subject.

6. The application according to claim 1, characterized in that, The drug achieves treatment by protecting and / or repairing the subject's salivary gland epithelial cells.

7. The application according to claim 1, characterized in that, The drug achieves its therapeutic effect by upregulating the expression levels of AQP5 and AR in the subjects.

8. The application according to claim 1, characterized in that, The effective dose range of the geraniol is 20–60 mg / kg body weight / day.

9. The application according to claim 1, characterized in that, The drug also includes other pharmaceutically acceptable carriers.

10. The application according to claim 1, characterized in that, The dosage form of the drug is injection, oral administration, eye drops, or oral spray.

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