Drug application and screening method of trigonelline
By screening the small molecule drug trigonelline as an IL-8 antagonist, the side effects and instability of existing IL-8 antibody drugs have been resolved, achieving a highly efficient and safe IL-8 antagonistic effect and providing a new treatment approach.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-08
- Publication Date
- 2026-03-31
AI Technical Summary
Existing IL-8 antibody drugs have problems such as side effects, treatment risks, instability and poor pharmacokinetic properties, making it difficult to effectively antagonize the effects of IL-8.
By using high-throughput screening of the FDA drug library, trigonelline, a small molecule drug, was identified as an IL-8 antagonist. Its binding ability to IL-8 was utilized to verify its antagonistic efficiency in vitro, and trigonelline with high antagonistic efficiency was selected.
Trigonelline, as a small molecule drug, has low toxicity, few side effects, stable structure, and good pharmacokinetic properties. It can effectively antagonize IL-8 and provides a new approach to treating IL-8-related diseases.
Smart Images
Figure CN121754543A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to the drug application of trigonelline and its screening method. Background Technology
[0002] Interleukin-8 (IL-8), also known as chemokine CXCL8, is a small protein composed of 72 amino acid residues with a molecular weight of approximately 8 kDa. Its structure contains typical CXC chemokine characteristics, namely, two cysteine residues separated by an amino acid. IL-8 is an important inflammatory cytokine, primarily produced by non-immune cells such as endothelial cells, epithelial cells, and fibroblasts, as well as immune cells such as monocytes, macrophages, and neutrophils, under inflammatory stimulation. IL-8 expression is strictly dependent on inflammatory signaling regulation; its expression is extremely low under normal physiological conditions, and rapidly upregulated after inflammatory stimulation.
[0003] IL-8, a key chemokine, specifically binds to CXCR1 / 2 receptors on the surface of target cells, activating downstream signaling pathways and recruiting and activating neutrophils to participate in host defense and tissue repair processes, playing a crucial role in inflammatory responses and diseases. When the body is infected or injured, damaged cells rapidly secrete IL-8, causing neutrophils to migrate rapidly to inflamed tissues and activating various effector functions of neutrophils, enhancing the body's ability to clear pathogens and kill bacteria. During inflammation resolution and tissue repair, IL-8 can promote the proliferation and migration of vascular endothelial cells, participating in angiogenesis, regulating fibroblast activity, promoting collagen synthesis, and participating in tissue repair processes.
[0004] In autoimmune inflammatory diseases, IL-8 participates in tissue damage by excessively recruiting neutrophils. In rheumatoid arthritis patients, IL-8 levels are significantly elevated in the synovial fluid and tissue, and are positively correlated with the degree of neutrophil infiltration. IL-8-mediated neutrophil infiltration can release large amounts of proteases (such as matrix metalloproteinases) and reactive oxygen species, leading to the destruction of articular cartilage and bone tissue, exacerbating joint inflammation and functional impairment. In psoriatic lesions, IL-8 expression is significantly upregulated, participating in the formation of psoriatic plaques by recruiting neutrophils to the epidermis, promoting abnormal proliferation of keratinocytes, and maintaining the chronic inflammatory state of the lesions.
[0005] Given the crucial role of IL-8 in disease, biologics primarily composed of IL-8 inhibitors are an important component of IL-8 antagonists. Antibody drugs alleviate disease symptoms by blocking the interaction between IL-8 and CXCR1 / 2, inhibiting the accumulation of immune cells and inflammatory factors. Antibody therapy can replace hormone therapy; when combined with glucocorticoids, it can reduce the dosage of hormones and decrease the incidence of adverse drug reactions. Inflammatory cytokines, cell surface molecules, and their mediated signaling pathways participate in the pathological process of autoimmune diseases; targeted biologics targeting cytokines, receptors, and signaling molecules have seen rapid development in recent years.
[0006] However, biologic antibody drugs carry certain side effects or treatment risks. Acute reactions following monoclonal antibody injection can be caused by various mechanisms, including acute anaphylactic and anaphylactic-like reactions against the monoclonal antibody, serum sickness, tumor lysis syndrome, and cytokine release syndrome. Clinical manifestations include local skin reactions at the injection site, fever, and flu-like syndrome. Humanization of antibody drugs does not completely eliminate immunogenicity; even the CDR region of humanized antibodies retains strong immunogenicity, generating anti-idiotype antibodies and leading to drug inactivation.
[0007] IL-8 plays a crucial role in many diseases, but current antibody drug treatments targeting IL-8 still have certain limitations.
[0008] (1) Antibody drugs have certain side effects.
[0009] Acute reactions following antibody drug injection can be caused by a variety of mechanisms, including acute anaphylactic and anaphylactic-like reactions against monoclonal antibodies, serum sickness, tumor lysis syndrome, and cytokine release syndrome. Clinical manifestations include local skin reactions at the injection site, fever, and flu-like syndrome.
[0010] (2) Antibody drugs carry certain treatment risks.
[0011] Humanization of antibody drugs does not completely eliminate immunogenicity. Even the CDR region of humanized antibodies still has strong immunogenicity, producing anti-idiotype antibodies and causing the drug to become ineffective.
[0012] (3) Antibody drugs are unstable and their structure is prone to change.
[0013] Biological macromolecules such as monoclonal antibodies possess unique three-dimensional structures. The complex balance of interactions between amino acid functional groups and the external environment, as well as intramolecular and intermolecular interactions, determines the folded structure. Because the folded structure is in a dynamic equilibrium, any factor that alters this equilibrium can lead to structural changes, resulting in an unstable state for the macromolecule. Since the tertiary structure of biopharmaceuticals is susceptible to environmental physical stresses, structural changes in monoclonal antibodies can occur at any stage of the production process, from initial protein expression to processing and storage.
[0014] (4) The pharmacokinetic properties of antibody drugs are poor.
[0015] Most monoclonal antibodies are large in size and have limited ability to penetrate and accumulate in tissues, likely confining themselves to the interstitial space after injection. Biotherapies can reach the bloodstream via two pathways: through capillaries or lymphatic vessels. However, capillary absorption has been reported to rely on passive transport and is limited to compounds with molecular weights below 16 kDa. Therefore, most biotherapeutic drugs cannot be transported via the capillary route and instead rely on the lymphatic system. Furthermore, enzymatic degradation mechanisms in vivo further reduce the pharmacokinetic properties of most types of protein drugs; proteins and monoclonal antibodies are prone to enzymatic degradation.
[0016] Therefore, there is an urgent need to find a small molecule drug that is stable in composition, has few side effects, and can antagonize IL-8. Summary of the Invention
[0017] Therefore, it is necessary to provide methods for the drug application and screening of trigonelline.
[0018] One aspect of this invention provides the use of trigonelline in the preparation of interleukin-8 antagonists.
[0019] Preferably, the interleukin-8 antagonist contains trigonelline as an active ingredient.
[0020] On the other hand, a method for screening interleukin-8 antagonists is provided, comprising the following steps:
[0021] S1. Customize a specific interleukin-8 as a target and customize an FDA drug library as a drug molecule library for backup.
[0022] S2. High-throughput molecular screening of drug molecules using targets to screen specific drug molecules that can bind to targets from the drug molecule library.
[0023] S3. Detect the antagonistic effect of the specific drug molecule on the target, and evaluate the efficacy of the specific drug molecule in antagonizing the target.
[0024] Preferably, in step S1, 3067 FDA-approved compound drugs are prepared and dissolved in DMSO at 1 mg / ml; an IL-8 protein target is prepared, the purity of which is determined to be 95% by SDS-PAGE; the IL-8 protein target is diluted with deionized water in a gradient concentration; wherein, the amino acid sequence of the IL-8 protein target is shown in SEQ ID NO: 1.
[0025] Preferably, in step S2, the FDA drug library is assembled into a microfluidic chip and installed in a PlexArray HT SPRi microarray analyzer; then, the IL-8 target to be tested is injected sequentially according to the concentration gradient; finally, the collected signal data is analyzed using the data analysis software Data Processor Stand Alone, and the results are sorted by molecular affinity to select the molecular compounds that meet the requirements from the drug molecules that can bind to the target.
[0026] Preferably, the drug molecule screened by the screening method is trigonelline.
[0027] Preferably, before sequentially injecting the IL-8 target to be tested according to the concentration gradient, a positive sample is used to pre-test the microfluidic chip, and the chip quality is verified based on whether a positive response signal of the positive sample is detected.
[0028] Preferably, the positive sample is rapamycin.
[0029] Preferably, in step S3, the antagonistic effect of trigonelline on IL-8 is detected using a human interleukin-8 (IL-8) ELISA kit. The antagonistic effect is evaluated by detecting the absorbance using an ELISA reader, establishing a standard curve, calculating the detection concentration, and calculating the antagonistic rate based on the detection concentration. The antagonistic rate is the quotient of the detection concentration and the initial concentration.
[0030] On the other hand, a pharmaceutical composition is also provided, comprising trigonelline as the active ingredient of claim 1, and further comprising one or more pharmaceutically acceptable carriers or excipients.
[0031] This invention provides a method for high-throughput screening of drug molecules that can bind to IL-8 in the FDA drug library using IL-8 as a target, and for verifying their antagonistic efficiency at the in vitro molecular level. Trigonelline, a small molecule drug that can antagonize IL-8, was screened out, providing a new approach for studying the function of IL-8. Attached Figure Description
[0032] The above and other objects, features, and advantages of the invention will become clearer through a more detailed description of the preferred embodiments illustrated in the accompanying drawings. The same reference numerals denote the same parts throughout the drawings, and the drawings are not intentionally drawn to scale with actual dimensions; the focus is on illustrating the gist of the invention.
[0033] Figure 1 The graph shows the trigonelline signal curves collected when IL-8 protein gradient concentrations were 2000 nM, 1000 nM, 500.0 nM, 250.0 nM, 125.0 nM, 62.5 nM, 31.2 nM and 15.6 nM.
[0034] Figure 2 Here is the molecular structure diagram of trigonelline;
[0035] Figure 3 The graph shows the dose-effect analysis of trigonelline at concentrations of 0 pg / L, 7.78 pg / L, 38.79 pg / L, 193.94 pg / L, 969.71 pg / L, 4848.54 pg / L, and 24242.71 pg / L. Detailed Implementation
[0036] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.
[0037] like Figures 1-3 As shown, the drug application and screening methods of trigonelline, the screening method of trigonelline as an interleukin-8 antagonist includes the following steps:
[0038] Step 1: 3067 FDA-approved chemical drugs were customized, dissolved and stored in DMSO at 1 mg / ml, and a drug molecule library with specific composition was established. Simultaneously, interleukin-8 was customized as the IL-8 protein target, with a purity of 95% as determined by SDS-PAGE. IL-8 was serially diluted with deionized water to 2000 nM, 1000 nM, 500.0 nM, 250.0 nM, 125.0 nM, 62.5 nM, 31.2 nM, and 15.6 nM. The amino acid sequence of the IL-8 target is shown in SEQ ID NO: 1.
[0039] The amino acid sequence of IL-8 is as follows:
[0040] SEQ ID NO: 1:
[0041] 1AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPK
[0042] 61 ENWVQRVVEKFLKRAENS
[0043] Step 2: Perform high-throughput molecular screening in the drug molecule library using the target to screen for specific drug molecules that can bind to the target.
[0044] High-throughput molecular screening targeting IL-8: FDA drug library chips were assembled into microfluidic chips and installed in a PlexArray HT SPRi microarray analyzer. The chip quality was verified using rapamycin as a positive sample. Target proteins were injected sequentially according to the concentration gradient described above. Finally, the collected signal data were analyzed using Data Processor StandAlone software, and the results were ranked by molecular affinity.
[0045] Step 3: Detect the antagonistic effect of this specific drug molecule on the target, and evaluate the efficacy of this specific drug molecule in antagonizing IL-8. The screened drug molecule is trigonelline.
[0046] The screened drug molecules were validated at the molecular level: the antagonistic effect of trigonelline on IL-8 was evaluated using a Human Interleukin-8 (IL-8) ELISA kit (Shenzhen Zike). Absorbance was measured using an ELISA reader, and the antagonism rate was calculated based on the detected concentration: Antagonism rate = 100% - (detected concentration / initial concentration).
[0047] like Figure 1 and 2 As shown, trigonelline has a novel application as a human interleukin-8 antagonist.
[0048] In a preferred embodiment, high-throughput screening of the FDA drug library chip is performed using IL-8 as the target, and the steps are as follows:
[0049] (1) Take out the prepared FDA drug library chip, place it in 1% BSA solution and seal it for 2 h, wash the chip with pure water and rinse it with inert gas and dry it before assembling it into a microfluidic chip.
[0050] (2) 1 mg / ml of interleukin-8 (IL-8) protein was serially diluted with PBST buffer.
[0051] (3) The microfluidic chip was installed in the PlexArray HT SPRi microarray analyzer. First, a positive sample of FKBP12 with a concentration of 100 nM was injected at a flow rate of 1 μl / sec for 300 sec. The positive response signal of rapamycin was detected to verify the chip quality.
[0052] (4) Wash with PBST buffer at a flow rate of 1 μl / s for 300 sec. Inject the IL-8 protein to be tested sequentially according to the gradient concentration at a flow rate of 1 μl / sec for 300 sec. Dissociate with PBST buffer at a flow rate of 1 μl / s for 300 sec. Repeat the above steps until all concentrations of IL-8 protein have been injected. Collect signal data.
[0053] (5) The chip was regenerated and stored after binding for 120 s and dissociating for 180 s with Gly-HCl buffer (pH=2.0) at a flow rate of 1 μl / s.
[0054] (6) The collected signal data were analyzed using the data analysis software Data Processor Stand Alone, and the results were sorted by molecular affinity. The small molecule compound trigonelline was screened out.
[0055] As attached Figure 1 As shown, with the increase of IL-8 protein concentration, the data collected by the DataProcessor Stand Alone analysis software became stronger. Trigonelline ranked second in affinity among all small molecules, making it a candidate molecule for further research. Figure 3 The molecular structure of trigonelline.
[0056] In a preferred embodiment, the efficacy analysis was performed using trigonelline to antagonize IL-8, and the steps are as follows:
[0057] (1) Add 50 μl of 10 μg / ml IL-8 protein to an EP tube, add different concentrations of trigonelline, and incubate at 37℃ for 1 h.
[0058] (2) Add 100 μl of protein small molecule mixture or standard and 100 μl of HRP-labeled IL-8 antibody to the pre-coated microwells, incubate at 37°C for 1 h, discard the solution, wash 5 times, and develop TMB color.
[0059] (3) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, establish a standard curve, and then calculate the detection concentration.
[0060] The materials and reagents used were as follows: FDA drug library chip purchased from Selleck Chemicals; interleukin-8 purchased from MCE; PBST buffer purchased from Thermo Fisher Scientific; Gly-HCl buffer purchased from Thermo Fisher Scientific; Human interleukin-8 (IL-8) ELISA kit purchased from Shenzhen Zike Biotechnology Co., Ltd.; and trigonelline purchased from MedChemexpress Biotechnology Co., Ltd. (USA).
[0061] The antagonism rate was calculated based on the detected concentration: Antagonism rate = 100% - (detected concentration / initial concentration). The results are shown in Table 1 below.
[0062] Table 1. Antagonism rates of trigonelline against interleukin-8 at different concentrations
[0063]
[0064] (Note: Based on the molecular weight of trigonelline being 137.14, convert to mass units.)
[0065] refer to Figure 2 Analysis of the results showed that the antagonistic effect of trigonelline on IL-8 protein was 26.70% when the drug concentration was 24.24 μg / L.
[0066] In summary, this invention targets the IL-8 protein, performs high-throughput screening of drug molecules in the FDA drug library that can bind to IL-8, and verifies their antagonistic efficiency at the in vitro molecular level. A molecule that can antagonize IL-8 is screened out, and the screened trigonelline has a significantly higher antagonistic efficiency than ordinary antagonists.
[0067] Trigonelline is a plant alkaloid, a pyridine derivative with a quaternary ammonium salt structure, widely found in plants such as coffee and fenugreek seeds. The highest content is found in fenugreek seeds, at about 0.5%-2%. It has the effects of warming the kidneys, dispelling cold, and relieving pain. The Chinese Pharmacopoeia lists it as a major drug for treating kidney deficiency due to cold, lower abdominal pain due to cold, and cold-damp beriberi.
[0068] Trigonelline also exhibits significant pharmacological effects in diabetes, anti-tumor activity, and liver damage. Studies have found that trigonelline can lower blood sugar levels by promoting insulin secretion, enhancing insulin sensitivity, inhibiting intestinal glucose absorption, and regulating liver glycogen metabolism, thus having an adjunctive regulatory effect on type 2 diabetes. It can also exert anti-tumor effects by inducing tumor cell apoptosis, inhibiting tumor cell proliferation and metastasis, and promoting hepatocyte repair and improving liver damage.
[0069] In inflammatory diseases, trigonelline can alleviate inflammatory responses by blocking inflammatory signaling pathways such as NF-κB and MAPK, inhibiting the activity of pro-inflammatory enzymes (such as COX-2 and iNOS), and reducing the synthesis and release of inflammatory factors (such as IL-6 and TNF-α). In neurodegenerative disease models such as Alzheimer's disease and Parkinson's disease, trigonelline can protect neurons and improve cognitive function by inhibiting neuroinflammatory responses (reducing the release of pro-inflammatory factors), combating oxidative stress damage (scavenging free radicals and increasing the activity of antioxidant enzymes), and regulating neurotransmitter balance.
[0070] Fenugreek is a traditional Chinese medicine. It is sweet and cold in nature, and enters the spleen, stomach, and small intestine meridians. It has the effects of clearing heat and detoxifying, promoting diuresis and reducing swelling. It can be used to treat symptoms such as boils, carbuncles, scrofula, jaundice, and edema.
[0071] This invention targets IL-8, performs high-throughput screening of drug molecules in the FDA drug library that can bind to IL-8, and verifies their antagonistic efficiency at the in vitro molecular level. Trigonelline, a small molecule drug that can antagonize IL-8, was screened out. The screened small molecule drug trigonelline provides a new approach for studying the function of IL-8.
[0072] Trigonelline is a pyridine derivative and a plant alkaloid mainly isolated from fenugreek seeds. It can be used as an antagonist of IL-8. Compared with existing biological agents, it has the following advantages: it can replace antibody drugs to antagonize IL-8; it has low toxicity and few side effects; the drug molecule structure is stable; and it has good pharmacokinetic properties.
[0073] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. Any equivalent structural or procedural transformations made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.
Claims
1. Use of trigonelline in the preparation of an interleukin-8 antagonist.
2. The use of trigonelline according to claim 1 for the preparation of an interleukin-8 antagonist, characterized in that, The interleukin-8 antagonist comprises trigonelline as an effective component.
3. A method of screening for an interleukin-8 antagonist, comprising contacting a test agent with an interleukin-8 and determining whether the test agent inhibits the binding of interleukin-8 to a receptor. The interleukin-8 antagonist is obtained by the following screening method: S1, customizing a specific interleukin-8 as a target, and customizing an FDA drug library as a drug molecule library, standby; S2, using the target to perform high-throughput molecular screening in the drug molecule library, and screening specific drug molecules capable of combining with the target from the drug molecule library; S3, detecting the antagonistic effect of the specific drug molecules on the target, and performing dose-effect evaluation of the specific drug molecules on the target.
4. The screening method according to claim 3, wherein In step S1, 3067 kinds of FDA-approved chemical drugs are customized and preserved by dissolving in DMSO at 1 mg / ml; the IL-8 protein target is customized, and its purity is determined to be 95% by SDS-PAGE; the IL-8 protein target is diluted by gradient concentration with deionized water; wherein the amino acid sequence of the IL-8 protein target is shown as SEQ ID NO:
1.
5. The screening method of claim 3, wherein, In step S2, the FDA drug library is assembled into a microfluidic chip and installed into a PlexArray HT SPRi microarray analyzer; then, the IL-8 target to be tested is sequentially sampled according to the concentration gradient, and finally, the collected signal data is analyzed by using the data analysis software Data Processor Stand Alone, and the results are sorted by molecular affinity size, and the required molecular compounds are screened from the drug molecules capable of combining with the target.
6. The screening method of claim 3, wherein, The drug molecule screened by the screening method is trigonelline.
7. The screening method of claim 3, wherein, Before the IL-8 target to be tested is sequentially sampled according to the concentration gradient, the microfluidic chip sampling detection is performed in advance using a positive sample, and whether the positive response signal of the positive sample is detected, thereby verifying the quality of the chip.
8. The screening method of claim 3, wherein, The positive sample is rapamycin.
9. The screening method of claim 3, wherein, In step S3, the antagonistic effect of trigonelline on IL-8 is detected by using a human interleukin-8 (IL-8) ELISA detection kit, the antagonistic effect is evaluated, the absorbance is detected by using an enzyme label instrument, the detection concentration is calculated after establishing a standard curve, and the antagonistic rate is calculated according to the detection concentration, and the antagonistic rate is the quotient value of the detection concentration and the initial concentration.
10. A pharmaceutical composition, characterized by, The trigonelline of claim 1 is contained as an effective active ingredient, and one or more pharmaceutically acceptable carriers or excipients are further contained.