Patent Application
20250082713
This non-provisional application claims priority under 35 U.S.C. § 119(a) on Patent Application No. 202310921375.0 filed in China on Jul. 26, 2023, the entire contents of which are hereby incorporated by reference.
The present invention belongs to the field of medical technology, Especially involving An active ingredient group for treating UC.
The inventor of this application submitted a US invention patent application on Feb. 11, 2023, titled A Pharmaceutical Composition For Preventing And Treating Inflammatory Bowel Disease, with patent application Ser. No. 18/167,842. In the inventor's previous patent application (application number 18/167842), the drug combination and clinical medical effects of BUCD2103 were recorded in detail. The publicly available drug combination BUCD2103 is referred to as CDD-2103 in this application.
Ulcerative Colitis (UC) is a chronic recurrent gastrointestinal disease. The standard treatment for UC remission is often unsatisfactory. CDD-2103 is a traditional Chinese medicine preparation that has been proven to be effective in treating UC. In fact, our pharmacodynamic studies have shown that CDD-2103 alleviates ulcer symptoms and colon damage in DSS induced chronic colitis mouse models. However, the mechanism of this effect is not yet clear, and further research is needed on the target and active ingredients of CDD-2103 to improve the accuracy and effectiveness of UC disease diagnosis and treatment, and provide further theoretical support for the medical treatment and prevention of UC.
In view of this, this application provides an active ingredient group for treating UC to solve some or all of the technical problems defined in the background technical section of this application.
The active ingredient group for treating UC provided in this application to address its technical issues are:
An active ingredient group for treating UC, characterized by:
Preferably, the active ingredient group can act on the key target combination of UC, which includes MA0A, MAPK14, AHR, PTGS2, PLA2G1B, and ALOX5.
Preferably, the active ingredient group can regulate tryptophan metabolism, glycerolphospholipid metabolism, and linoleic acid metabolism.
Preferably, there is a strong binding between the active ingredient group and the key target combination of UC.
Preferably, the biomarkers for treating UC using the active ingredient group are KA, PC, and AA.
Preferably, the active ingredient group comes from CDD-2103.
Preferably, CDD-2103 comprises:
The active ingredient group for treating UC provided in this application can effectively prevent and treat UC by regulating tryptophan metabolism, glycerophospholipid metabolism and linoleic acid metabolism by establishing strong combination between the active ingredient group and the key target combination of UC. At the same time, the biomarkers for the diagnosis and treatment of UC have also been disclosed.
The following is a detailed introduction to the technical solution and effects of this application, combined with the accompanying drawings and specific implementation methods of the specification.
The specific validation experiments and theoretical studies using CDD-2103 as a UC treatment drug will provide a detailed explanation of the active ingredient group for treating UC disclosed in this application.
Dextran sulfate sodium (DSS; MW: 36-50 kDa) was bought from MP Biomedicals (USA). Sulfasalazine (SASP) was acquired from Europharm Laboratoires Company Limited (China). The hematoxylin-eosin (H&E) staining solution was obtained from Sigma-Aldrich (USA). The reference standards of berberine hydrochloride, epiberberine, coptisine chloride, palmatine, morroniside, loganin, and curcumin were purchased from Meilunbio (China). Militarine, glycyrrhizin ammonium salt, jatrorrhizine, columbamine, liquiritin, demethoxycurcumin, bisdemethoxycurcumin, berburrubine, and magnoflorine were purchased from CFW Laboratories (China). The purity of all standards was ≥98.0% (HPLC).
The powdered extract of CDD-2103 (batch no. J-220606-01) was provided by Beijing Increasepharm Corporation Limited (China). All herbs were authenticated by Pony Testing International Group (China) according to the Chinese Pharmacopoeia (version 2020) or Chinese Herbal Medicine Quality Standard (Hebei Province, version 2018). Briefly, HL, JH, and SY were extracted together with 8-fold 70% ethanol for 1 h three times, under reflux, and another six herbs in CDD-2103 were extracted together with water. The extracts were combined, filtered, and then concentrated by rotary evaporation. The freeze-dried powder was then stored at −20° C. until further use.
The animal experiment was approved by the Committee on the Use of Human and Animal Subjects in Teaching and Research of Hong Kong Baptist University (REC/19-20/0301). Male C57BL6J mice aged 6-8 weeks were purchased from The Chinese University of Hong Kong (China). The mice were kept at a controlled temperature with a 12 h light/dark cycle with free access to water and food. On Day 13, the mice were divided into six groups (n=12/group) as follows: CTR, control group; DSS, group administered with DSS; SASP, group administered with sulfasalazine and DSS; and 3 groups one each administered with low (5.8 g/kg), medium (8.8 g/kg), and high (11.7 g/kg) of CDD-2103 and DSS. Mice in DSS, SASP group and CDD-2103 groups with different dosages were subjected to three cycles of 1.8% DSS to induce chronic colitis during the 49-day experimental period.
The CDD-2103 and SASP groups were orally administered with the CDD-2103 extract and SASP (500 mg/kg), respectively, once per day starting at Day 13. The dosages from the low, medium, and high CDD-2103 groups were based on the clinical effective dosage of CDD-2103 (0.8 g raw herbs/kg/day) experienced in UC patients. They were, 5.8, 8.8, and 11.7 g of raw herbs/kg/day, respectively, (equivalent to 0.8, 1.1 and 1.5 g of raw herbs/kg/day respectively of clinical dosage). The CTR and DSS groups were orally administered with an equal volume of water as in SASP and CDD-2103 groups. On Day 48, fecal samples of all groups were collected and kept at −80° C. until further use. On Day 49, mice were sacrificed, and serum and colon samples were collected.
The severity of colitis in mice was scored according to weight loss, the scoring of bloody stool, and stool consistency as reported previously with a minor adjustment. Body weight change was calculated as the difference between the initial body weight on Day 1, and rated as follows: 0, <1% no loss; 1, 1-5% loss; 2, >5-10% loss; 3, >10-15% loss; 4, >15% loss. Hemoccult SENSA kits (USA) were used for bloody stool detection. Results were rated as follows: 0, no blood (brown); 1, some bleeding high in the gastrointestinal tract (slightly blue); 2, significant bleeding high in the gastrointestinal tract (blue); 3, slight bleeding (slightly red); 4, significant bleeding (red). Stool consistency was rated as follows: 0, normal; 1, soft but formed; 2, soft; 3, very soft and wet; 4, watery. Colon length was determined by measuring from the ileocecal junction to the anus.
After mice sacrifice, distal colons were rinsed and soaked in 4% paraformaldehyde at 4° C. overnight. Colonic tissues were sequentially dehydrated and finally embedded in paraffin blocks. Tissues were segmented into 4 m slices and stained (H&E) for the observation of colonic injuries.
The freeze-dried feces were extracted with 30-fold volume of 80% methanol (m/v), then mixed with steel beads and processed with Tissue Lyser to obtain a fecal slurry. After incubation at −80° C. for 4 hours, the homogenate was centrifuged (18,000×g, 4° C., 10 min) and the supernatant was collected. Serum was extracted with a 4-fold volume of methanol; the mixture was then vortexed for 1 min and centrifuged (18,000×g, 4° C., 10 min). The supernatant was dried at low temperature under vacuum. The dried samples were reconstituted with equal volume of 50% methanol, vortexed for 5 min, and centrifuged (18,000×g, 4° C., 10 min). The supernatant was used for ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) analysis. A combined quality control (QC) sample was prepared by mixing equal amount of each sample.
An Agilent 1290 Infinity II UPLC coupled with a 6546 Q-TOF/MS system was used to obtain the fecal and serum metabolomics profiles. A 100 mm×2.1 mm Acquity UPLC HSS C18 1.7 m column was used for analysis. The chromatographic conditions were as follows: mobile phase A, water with 0.1% formic acid; mobile phase B, acetonitrile with 0.1% formic acid; a linear gradient, 0-1 min (1% B), 1-12.5 min (1-100% B), 12.5-14.5 min (100% B), 14.5-14.7 min (100-1% B), 14.7-17.7 min (1% B). MS analysis was carried out in both positive and negative ion modes. The electrospray ionization (ESI) source had the following parameters: gas temperature, 300° C.; drying gas, 8 L/min; nebulizer, 45 psi; sheath gas temperature, 350° C.; sheath gas flow, 8 L/min; voltage of capillary, 3.0 kV. MS1 full scan range was m/z 80-1000; MS2 full scan method was as follows: full scan range: m/z 40-1000; collision energy was set at 10, 20, and 40 eV.
The acquired MS1 raw data was converted to mzXML format by using MSConvert GUI software from the ProteoWizard toolset. The intensities were corrected for batch effect and signal drift by fitting a locally quadratic (loess) regression model to the median intensity of combined QC samples. For multivariate statistical analysis, supervised partial least squares discrimination analysis (PLS-DA) was processed with SIMCA-P software. The features with fold change (FC)>1.2 or <0.8, and p-value<0.05 were considered to be potentially differential compounds. Identification of these compounds was first performed by comparing the accurate MS and MS/MS (ppm<5) with Human Metabolome Database (HMDB). Some of them was verified by comparison with authentic standards. Heat maps were created using the heatmap package in R. Metabolic pathway analysis was enriched by MetaboAnalyst 5.0. For the quantitative analysis of tryptophan metabolites, the sample preparation method was the same as above, using the quantification instrument and method as described in a previous report.
Sprague Dawley rats (200±20 g) were purchased from The Chinese University of Hong Kong (China). Ten male rats were randomly divided into two groups (n=5). After overnight fasting, they were orally administered with 4.7 g raw herbs/kg of CDD-2103 (equivalent to 0.8 g of raw herbs/kg/day of clinical dose used in human). Serum was collected from one group, and feces were collected from the second group. Before administered with CDD-2103, feces, and blood samples were collected from each rat for the self-control study. At 0.25, 0.5, 1.0, and 2.0 h after oral administration of CDD-2103, blood samples (0.2 mL) of rats were collected from the ophthalmic veins. Blood was centrifuged (4000×g, 4° C., 10 min); the supernatant was collected and stored at −80° C. until analysis. At 8.0 and 12.0 h after administration, fecal samples were collected and were directly stored at −80° C. until further use.
For serum analysis, a 5-fold volume of methanol was added to each sample; it was vortexed for 30 s, centrifuged (18,000×g, 4° C., 10 min), and then collected the supernatant. Fecal samples were weight, extracted with 50-fold volume of methanol, and subjected to ultrasonic 30 min, then centrifuged (18,000×g, 4° C., 10 min). The supernatant was used for UPLC-Q-TOF/MS analysis. For phytochemical analysis of both serum and feces samples, the gradients were set as 5-10% B for 0-2 min, 10-15% B for 2-8 min, 15-17% B for 8-10 min, 17-20% B for 10-16 min, 20-50% B for 16-21 min, 50-54% B for 21-24 min, 54-100% B for 24-25 min, 100% B for 25-26 min, and 100-5% B for 26-28 min. The compound database of CDD-2103 was constructed according to the HERB database and the literature. Components in serum and feces were identified based on the accurate m/z, fragmentation data, or by comparison with reference standards.
The HERB database was used to manually search for the molecular targets of the components (Cx) identified from the serum and feces samples of rats administered with CDD-2103. The drug similarity search tool in Therapeutic Targets Database (TTD) was used to recognize drugs' similarity with Cx. Only drugs with a high structural similarity score (>0.70, described as “moderately similar” to “very similar”) were selected. The therapeutic targets of the recognized drugs were also considered to be the potential targets of Cx. The biological targets of UC were collected by searching on the keywords “ulcerative colitis”, “gut immune modulation” and “colon mucosa protection” in GeneCards. The intersection genes of Cx with disease genes were constructed by a Venn diagram. Protein-protein interactions (PPI) of the intersection targets were constructed in a STRING database. We considered the overlapping targets to be therapeutic targets for CDD-2103 in the treatment of UC. Functional enrichment analysis of the intersection genes was carried out by DAVID database. Cytoscape 3.7.2 was utilized to create the herb-compound-gene-pathway-disease network.
To reveal the altered key metabolites, related pathways, targets and active compounds, integrated networks were constructed, and analyzed. First, the differential metabolites identified by metabolomics and the predicted genes predicted by network pharmacology were imported into the Cytoscape equipped with MetScape to produce a compound-reaction-enzyme-gene network. This construction depicted the interactions among the metabolites, pathways, enzymes, and genes for further analysis. Second, the herbs, active compounds, genes, pathways, and metabolites were imported into Cytoscape to obtain the network. Third, the key metabolites, genes and active compounds were recognized in the two integrated networks.
Docking of the key active compounds and genes was explored using CB-Dock, an online molecular docking tool. PDP files of receptors were downloaded from the RCSB Protein Data Bank, and the active ingredients SDF files were downloaded from the PubChem database. All the prepared files were uploaded into CB-Dock. After determining the docking pocket coordinates, molecular docking and conformational scoring were performed using CB-dock. The lower the Vina scores, the more stable is the ligand binding to the receptor; thus, Vina scores were, used for preliminary evaluation of the binding activity of the compounds to the targets.
Statistical analysis of t-test, and one-way ANOVA were performed using GraphPad Prism 8.0 (USA). A p-value<0.05 was considered statistically significant.
As shown in
A total of 17,735 features were determined in all the fecal samples and 7,702 features in all the serum samples. The stability and repeatability of metabolomics were evaluated by QC samples. 96.5% and 96.4% of features in fecal and serum samples, respectively, had an RSD %<30%. The total ion chromatograms (TICs) showed that QC samples behaved stably during the process. These data suggested the high stability of the instrument and the repeatability of the metabolomics method.
To further investigate the differences among the six groups of mice, we performed PLS-DA analysis. As shown in
Based on FC>1.2 and p<0.05, according to HMDB, 37 and 15 metabolites were identified in feces and serum, respectively (Tables 1 and 2). To visualize the variation in metabolites among the six groups, we plotted heat maps.
To further investigate the metabolic pathways of CDD-2103 in DSS mice, we imported these differential metabolites to MetaboAnalyst 4.0. As shown in
Studies have revealed that tryptophan metabolites are involved in immune regulation by modulating intestinal microbiota composition and homeostasis. Our untargeted metabolomics study showed the significant changes in the tryptophan metabolites profiles, demonstrating the important role of tryptophan in the pathogenetic development of UC. Therefore, we carried out targeted quantification analysis of the metabolites involved in tryptophan metabolism (
The peaks detected in the biological samples and not in the blank sample were interpreted as corresponding to the absorbed or dis-absorbed compounds in the intestinal. A total of 14 prototypes and 2 metabolites were identified in rat serum, and 15 prototypes compounds and 3 metabolites were identified in rat feces; 11 prototypes were detected in both serum and feces (
23 compounds, including prototypes and metabolites, identified in the serum-feces pharmacochemistry study were subjected to network pharmacology study. According to HERB and TTD database, a total of 505 targets were derived from these compounds. From GeneCards, a total of 2106 genes related to UC were obtained; of these, 310 overlapped with the CDD-2103 targets in the Venn diagram analysis (
To identify the functions and mechanisms of CDD-2103 in UC remission, GO and KEGG pathway enrichment analyses were carried out based on the 310 overlapping targets. For GO-term analysis, CDD-2103 against UC mainly involved in apoptosis, inflammatory response, and positive regulation of NF-kappaB transcription factor activity (
To further elucidate the relationship between herbs, compounds, genes, pathways, and diseases, an herb-compound-gene-pathway-disease network was constructed. This comprised 7 herbs, 21 compounds, 310 targets, and 10 pathways from the above prediction (
To obtain a comprehensive view of the mechanisms of CDD-2103 against UC, we constructed two integrated interaction networks based on metabolomics analysis and network pharmacology and matched them by Cytoscape. First, based on the differential metabolites and the potential targets, the compound-reaction-enzyme-gene network showed that MA0A, AHR, MAPK, PLA2, ALOX5, and PTGS2 might be the key targets, and that, KA, phosphatidylcholine (PC), and arachidonic acid (AA) might be the key metabolites regulated by CDD-2103 (
To further investigate the possibility of interaction between CDD-2103 and the key targets, we performed molecular docking studies (
UC is a chronic relapsing gastrointestinal disease that is difficult to treat. Although several anti-inflammatory drugs are applied in UC patients, these medications cannot successfully maintain long-lasting remission in UC patients. This demonstrates the urgent need to discover a new medication for UC rescue. TCM formulas are generally composed of combined herbs and compounds; according to traditional principles and practice, multiple herbs increase efficacy, counteract side effects, and moderate effects. It is generally believed, biochemically, that these activities are achieved via multiple targets and pathways. This study found that a TCM formulation CDD-2103 could significantly attenuate ulcerous symptoms and colonic injuries in the DSS-induced chronic colitis mice model. We used a novel strategy, involving metabolomics, network pharmacology, serum-feces pharmacochemistry and molecular docking to identify its active compounds, regulating targets, and pathways that are involved in its effect on UC.
In the present work, 23 compounds, including phototypes and metabolites, were identified in the serum and feces of rats after CDD-2103 administration. We understood these as active components exhibiting biological functions via either entering the circulation or modulating the gut microbiota. These identified compounds were subjected to network pharmacology for target prediction. By integrating metabolomics and serum-feces pharmacochemistry-based network pharmacology, we uncovered that the five major active compounds (curcumin, berberine, atractylenolide III, liquiritin, and militarine) from the herbs strongly bind to six key targets (MAOA and MAPK14, AHR, PTGS2, PLA2G1B, and ALOX5, respectively), regulating related metabolic pathways (tryptophan metabolism, glycerophospholipid metabolism, and linoleic acid metabolism) to alleviate UC, and that the key metabolites (KA, PC, and AA) can serve as biomarkers for diagnosis and treatment. These findings provide a specific biochemical framework for understanding the functional nature of CDD-2103 in UC remission (
Tryptophan metabolites comprise host-derived metabolites, such as kynurenic acid (KA) from the kynurenine pathway, and serotonin (5-HT) from the 5-HT pathway, as well as bacteria-derived metabolites, including indole derivatives and tryptamine (TM) from the indole pathway. Most of the tryptophan metabolites can bind with aryl hydrocarbon receptor (AhR) to aggravate or prevent the inflammatory responses in IBD. For instance, on the one hand, KA activates G protein-coupled receptor 35 (GPR35) in the bowel wall, which may aggravate DSS-induced chronic inflammation by affecting nod-like receptor protein 3 (NLRP3). On the other hand, KA also plays an anti-inflammatory role by regulating oxidative stress and inhibiting LPS-induced pro-inflammatory cytokines, such as IL-1 and IL-6. 5-HT, a key metabolite from the 5-HT pathway, can also alter numerous immune cells and modulates the gut microbiota composition through binding with AHR. Suppression of 5-HT production in the intestine has been proven to reduce inflammation in DSS-treated mice. Indole carboxaldehyde (IC), derived from the gut bacterial metabolism of tryptophan, leads AHR-dependent IL-22 production and mucosal protection. Studies showed that serum IC was decreased in DSS-induced acute colitis mice. In the current study, the up-regulated levels of both KA and IC in DSS-treated mice were reversed by CDD-2103 treatment in the serum samples, although KA and IC levels in fecal samples of DSS mice were down-regulated, CDD-2103 could also reverse its metabolic perturbation. Moreover, the up-regulated 5-HT levels in the serum and fecal samples of DSS-induced colitis mice could also be reversed by CDD-2103. Therefore, CDD-2103 comprehensively exhibited its therapeutic effect on kynurenine, 5-HT, and indole pathways of tryptophan metabolism, and KA, 5-HT, and IC could be understood as the corresponding metabolites that CDD-2103 targeted in both feces and serum samples. Based on network analysis, berberine, and curcumin predicted the binding activity with AHR, and it was verified by molecular docking. This might be another mechanism by which CDD-2103 prevents the inflammation of DSS-treated mice by regulating tryptophan metabolism.
Lipids are in cell membranes, where they are responsible for mucus production, barrier integrity, and intra- and intercellular signaling. Phosphatidylcholine (PC), a metabolite of dietary choline, is the major bioactive phospholipid for mucus composition. The increasing activity of phospholipase A2 (PLA2), one of the degradation enzymes of PC, leading to the production of lysophosphatidylcholine (LPC) and arachidonic acid (AA), is deduced as the cause of PC content decreasing in UC patients. LPC exhibits proinflammatory effects via binding with dendritic cells to produce IFN-γ, resulting in greater tissue destruction. AA is the essential bioactive molecule enriched in linoleic acid metabolism and can be metabolized to pro-inflammatory eicosanoids such as prostaglandin E2 (PGE2). Phosphatidylethanolamine (PE) is a key constituent of lipid rafts. Mitogen-activated protein kinase (MAPK) is discovered in lipid rafts to sense the inflammatory cytokines such as TNF-α and IL-lip, then mediate inflammatory responses. In the present study, CDD-2103 significantly increased PC and PE levels in the serum samples, while the increasing level of LPC was reversed by CDD-2103 in the fecal samples of DSS-treated mice. The binding effect of liquirtin on PLA2G1B (the first member of the PLA2 family) might be the possible mechanism by which CDD-2103 adjusted the LPC/PC ratio to alleviate the disease severity. Moreover, the MAPK signaling pathway was enriched in network pharmacology studies, and MAPK14 was predicted as the hub gene. The biological function of PE related to this pathway, alkaloids from HL, and curcuminoids from JH could be considered as the active compounds from CDD-2103 acting on this part. These results supported the possible hypothesis that CDD-2103 protects intestinal mucosa by regulating glycerophospholipid metabolism.
Inflammation in IBD is correlated with a high level of production of eicosanoids from linoleic acid metabolism. Metabolites from this metabolic pathway are mainly composed of ω-6 fatty acid, and arachidonic acid (AA); the metabolite of linolenic acid (LA), is the primary precursor for eicosanoids' production. In the early stage of inflammation, the expression of ALOX5 and PTGS2 increases, leading to the production of PGE2, and other eicosanoids from AA. PGE2 can act as an irritant in the gut lumen, to relax the colonic muscles and contribute to diarrhea. Furthermore, MA0A (a metabolic enzyme of 5-HT and TM), is an intestinal stem cell marker that was inhibited by PGE2 following TNF stimulation in intestinal organoids. 5-aminosalicylic compounds, such as SASP used in the current study as the positive drug, can suppress PGE2 levels to exhibit the anti-colitis effect in a dose-dependent manner. In our study, LA and AA were increased in the serum samples of the DSS-treated mice, whilst CDD-2103 could reverse this condition. These findings revealed that CDD-2103 compounds, including atractylenolide III, and militarine might exhibit its anti-colitis effect by regulating ALOX5 and PTGS2, to inhibit the production of eicosanoids derived from linoleic acid metabolism.
It can be seen from the above description that, based on the comprehensive analysis of metabolomics and network pharmacology based on serum-feces pharmacochemistry, this application finds that several active compounds in CDD-2103 can regulate MA0A, AHR, MAPK14, PLA2G1B, ALOX5 and PTGS2, and regulate the metabolism of tryptophan, glycerolphospholipid and linoleic acid. This comprehensive strategy has been proven to have a powerful role in effectively discovering active compounds and mechanisms of action in traditional Chinese medicine prescriptions. Therefore, the UC medical model and medical method based on CDD-2103 provided in this application can effectively prevent and treat UC by regulating tryptophan metabolism, glycerolphospholipid metabolism and linoleic acid metabolism by establishing a strong combination between the active ingredient group of CDD-2103 and the key target combination of UC. This application also discloses the use of CDD-2103 as a biomarker for the diagnosis and treatment of UC, which can be used for the diagnosis and monitoring of UC prevention and treatment.
However, the active ingredient group disclosed in this application that can effectively treat UC may not only come from CDD-2103, but may also come from one or more other drug formulations. Thus, as long as the drug contains both the active ingredient group claimed for patent protection in this application, and the active ingredient group can establish a strong connection with the UC key target combination disclosed in this application, thereby preventing and treating UC by regulating tryptophan metabolism, glycerolphospholipid metabolism, and linoleic acid metabolism. All fall within the scope of protection of this application.
The above provides a detailed explanation of the technical solution and effects of the present application in conjunction with the accompanying drawings and specific embodiments in the specification. It should be noted that the specific embodiments disclosed in the specification are only the preferred embodiments of the present application, and technical personnel in the field can also develop other embodiments based on this; Any simple deformation and equivalent replacement that does not deviate from the innovative concept of this application are covered by this application and fall within the scope of protection of this patent.
