Use of glutamate carboxypeptidase ii in the preparation of a medicament for the treatment of gastric metaplasia

Abstract

The application discloses application of glutamate carboxypeptidase II in preparation of a medicine for treating gastric mucosal intestinal metaplasia, reveals that an endogenous metabolite N-acetyl aspartyl glutamic acid is a key pathological factor driving gastric mucosal intestinal epithelial metaplasia, and glutamate carboxypeptidase II can efficiently degrade the metabolite, block downstream signal cascade reactions mediated by the metabolite, and significantly down-regulate the expression level of an intestinal specific marker CDX2 from a molecular root, so that the treatment purpose of effectively reversing or delaying the progress of gastric mucosal intestinal metaplasia is achieved. The application clearly proposes a new treatment strategy of taking glutamate carboxypeptidase II as a precise molecular target, directly supplementing an active protease in the form of an injection preparation to remove pathogenic metabolites, and effectively solves technical problems of low bioavailability of existing oral medicines, easy enzymatic destruction in a digestive tract, and unknown target of existing intervention means and insufficient reversal rate.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of glutamate carboxypeptidase II in the preparation of drugs for treating gastric mucosal intestinal metaplasia. Background Technology

[0002] Gastric cancer is the fifth most common malignant tumor worldwide, with a low early diagnosis rate and high mortality rate, posing a serious threat to human health. The development of intestinal-type gastric cancer follows the classic Correa cascade process: from chronic non-atrophic gastritis, chronic atrophic gastritis, gastric intestinal metaplasia (GIM), dysplasia, and finally invasive adenocarcinoma. Among these precancerous lesions, gastric intestinal metaplasia is considered a key "irreversible point," significantly increasing the risk of malignant transformation. Elucidating its molecular mechanisms and achieving effective intervention is crucial for reducing gastric cancer-related mortality.

[0003] Currently, clinical treatment strategies for gastric mucosal intestinal metaplasia still suffer from significant technical deficiencies and unmet needs. Existing treatments primarily focus on symptomatic management, such as eradicating Helicobacter pylori, suppressing acid, and protecting the gastric mucosa, lacking specific drugs that can directly reverse the pathological changes of intestinal metaplasia. While some literature reports the use of folic acid, morotan, celecoxib, metformin, and lamb stomach extract vitamin B12 for intervention in gastric mucosal intestinal metaplasia, their reversal rates have all fallen below 60%, far from meeting clinical needs. Furthermore, many of these drugs are "repurposed drugs," with their mechanisms of action not fully understood and lacking clear molecular targets, resulting in a lack of theoretical guidance for drug screening, optimization, and individualized clinical medication. In addition, some existing drugs pose safety risks with long-term use; for example, celecoxib may increase the risk of cardiovascular events, and metformin may cause lactic acidosis and vitamin B12 deficiency, limiting their widespread use in non-target populations. From an overall treatment perspective, current clinical management of precancerous lesions of the stomach primarily relies on regular endoscopic follow-up and lesion resection. There are currently no effective and safe methods to reverse the molecular nature of intestinal metaplasia, and standard care focuses only on monitoring disease progression. Therefore, there is an urgent need in this field to develop novel therapeutic drugs with clear targets that can efficiently and safely reverse gastric mucosal intestinal metaplasia. Summary of the Invention

[0004] The present invention aims to provide a novel use of glutamate carboxypeptidase II (GCPII), namely, the application of GCPII in the preparation of drugs for treating gastric mucosal intestinal metaplasia, in order to solve the problem of the lack of effective drugs to reverse gastric mucosal intestinal metaplasia in the prior art.

[0005] This invention provides a novel pharmaceutical use for glutamate carboxypeptidase II, specifically relating to its application in the preparation of drugs for treating gastric mucosal intestinal metaplasia. Glutamate carboxypeptidase II (GCPII) is a type II transmembrane glycoprotein encoded by FOLH1, which promotes neurotransmission of glutamate and absorption of folic acid. In the field of tumor research, this protein is known as prostate-specific membrane antigen (PSMA); in the field of intestinal research, it is known as folic acid hydrolase-1 (FOLH1); and in the field of nervous system research, it is known as N-acetyl-L-aspartate-L-glutamate peptidase I (NAALA Dase I).

[0006] In the technical solution of this invention, gastric mucosal intestinal metaplasia specifically refers to gastric mucosal intestinal epithelial metaplasia, a pathological change in which gastric mucosal epithelial cells are replaced by intestinal-type epithelial cells, representing a key stage in precancerous lesions of the stomach. The drug contains glutamate carboxypeptidase II as its active ingredient. This enzyme can effectively block downstream signaling pathways mediated by N-acetylaspartate glutamate by hydrolyzing its substrate, thereby significantly reducing the expression levels of gastric mucosal intestinal metaplasia markers and achieving the therapeutic goal of reversing or delaying the progression of gastric mucosal intestinal metaplasia. The gastric mucosal intestinal metaplasia markers include CDX2 and MUC2. CDX2 is an intestinal-specific transcription factor and a key gene regulating intestinal epithelial cell differentiation; its ectopic expression in the gastric mucosa is considered the most hallmark molecular event of gastric mucosal intestinal metaplasia. MUC2 is an intestinal-specific mucin and a major component of goblet cells; its expression marks the appearance of intestinal-type secretory cells. Glutamate carboxypeptidase II fundamentally intervenes in the molecular nature of intestinal metaplasia by reducing the expression level of CDX2.

[0007] Building upon the aforementioned applications, this invention further specifies that the drug is formulated as an injectable preparation. Since glutamate carboxypeptidase II is a biologically active protein macromolecule, it is readily degraded by proteases in the gastrointestinal tract and loses its activity when administered orally. Furthermore, large protein molecules are difficult to fully absorb by crossing the gastrointestinal mucosal barrier. Therefore, preparing it as an injectable preparation is crucial to ensure that the drug enters the systemic circulation in its intact active form, thereby exerting its therapeutic effect. Injectable preparations can bypass the gastrointestinal digestive system, directly delivering the active protein into the body, significantly improving its bioavailability and avoiding the first-pass effect and enzymatic degradation problems associated with oral administration.

[0008] The aforementioned injectable formulations can be further formulated into injectable solutions or lyophilized powder for injection. Injectable solutions are ready-to-use liquid formulations, offering the advantage of ease of use; they can be directly injected without reconstitution, making them suitable for applications requiring rapid drug delivery. Lyophilized powder for injection is a solid block or powder formulation produced by freeze-drying liquid formulations under vacuum at low temperatures. Its advantages include significantly enhanced stability of the active protein components during storage, effectively slowing down physical and chemical degradation processes, and significantly extending the product's shelf life, making it particularly suitable for pharmaceutical products requiring long-term storage and transportation. When using, lyophilized powder for injection only needs to be reconstituted with a prescribed amount of sterile solvent to restore it to a clear injectable solution.

[0009] The medicament used in this invention comprises a therapeutically effective amount of glutamate carboxypeptidase II and a pharmaceutically acceptable carrier.

[0010] The therapeutically effective dose (TAD) refers to the dose of glutamate carboxypeptidase II that, when administered to a subject, is sufficient to produce the desired therapeutic effect in the subject. This dose effectively reduces the expression level of gastric mucosal intestinal metaplasia markers, reverses or delays the pathological process of gastric mucosal intestinal metaplasia, and does not cause serious toxic side effects. A TAD of 0.8-1.6 μg / mL is preferred. The specific TAD can be determined by clinicians based on common sense, taking into account factors such as the subject's age, weight, sex, health status, disease severity, route of administration, and administration regimen. Pharmaceutically acceptable carriers refer to non-toxic excipients added to pharmaceutical preparations, in addition to the active ingredient, to ensure the physicochemical properties, biological properties, formability, stability, and usability of the drug. These carriers are harmless to the subject at the concentrations and doses used and do not react adversely with the active ingredient glutamate carboxypeptidase II, thereby affecting its therapeutic activity.

[0011] For pharmaceutically acceptable carriers, a buffer may be included, configured to maintain the pH of the drug between 5.0 and 8.0. Glutamate carboxypeptidase II, as an active protein molecule, exhibits a highly dependent three-dimensional conformation and biological activity on the pH of its solution. Excessively acidic or alkaline environments can lead to conformational changes, subunit depolymerization, or chemical bond breakage of the protein molecule, resulting in loss of activity or even the formation of immunogenic aggregates. Strictly controlling the pH of the formulation within the range of 5.0 to 8.0 provides a mild and stable physicochemical microenvironment for glutamate carboxypeptidase II, maximizing the preservation of its native conformation and enzymatic catalytic activity. The buffer used to achieve this purpose can be selected from at least one of phosphate buffers, histidine buffers, or citrate buffers. Phosphate buffers, such as a mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate, are classic buffer systems widely used in biopharmaceuticals, possessing good pH buffering capacity and physiological compatibility. Histidine buffer not only possesses excellent buffering capacity, but as an amino acid, it also stabilizes proteins and can form complexes with certain metal ions, further inhibiting metal-catalyzed protein oxidative degradation. Citrate buffers, such as systems containing citric acid and sodium citrate, have a good ability to chelate metal ions, effectively shielding trace metal ions from their adverse effects on protein stability. Those skilled in the art can flexibly select one or combine multiple buffer systems based on the characteristics of the active pharmaceutical ingredient and the specific requirements of the formulation process.

[0012] The pharmaceutically acceptable carrier may also contain stabilizers to prevent protein aggregation and denaturation. During purification, formulation, storage, and transportation, proteins are susceptible to various external physicochemical stresses, such as temperature, shear force, and interfacial effects, leading to changes in their spatial conformation, exposing internal hydrophobic groups, and causing aggregation into visible particles or invisible oligomers due to intermolecular hydrophobic interactions. This aggregation and denaturation not only significantly reduces the therapeutic potency of the drug but may also trigger the production of drug-resistant antibodies, leading to allergic reactions or even severe immunogenic reactions. Adding stabilizers to the formulation is a key strategy to address this problem. The stabilizer is selected from at least one of the following groups: sucrose, trehalose, mannitol, polysorbate 80, polysorbate 20, and EDTA. Sucrose, trehalose, and mannitol are sugar and polyol stabilizers. Their mechanism of action mainly involves stabilizing the native conformation of proteins through preferential hydration or the glassy state hypothesis. This means they are preferentially excluded from the protein molecule surface, forming a dense hydration layer that makes the protein more likely to maintain its compact native state. Trehalose, due to its high glass transition temperature, can form an amorphous glassy matrix during freeze-drying, physically embedding and isolating protein molecules, effectively preventing denaturation and aggregation caused by molecular motion. Polysorbate 80 and 20 are nonionic surfactant stabilizers. They preferentially occupy the gas-liquid or liquid-solid interface of the solution, effectively protecting protein molecules from denaturation and aggregation caused by interfacial effects during shaking, freeze-thaw cycles, or reconstitution. EDTA is a chelating agent that can chelate trace amounts of metal ions in solution, eliminating oxidative damage to sensitive amino acid residues in protein molecules caused by metal ions and their catalytic reactive oxygen species, thereby maintaining the structural integrity of the protein.

[0013] The pharmaceutically acceptable carrier may also contain an isotonic adjuster. The osmotic pressure of human body fluids is approximately 300 mOsm / L. If the osmotic pressure of an injectable drug formulation differs significantly from this, it can lead to local tissue irritation, pain, redness, and swelling at the injection site, and even serious adverse reactions such as hemolysis. By adding an isotonic adjuster, the osmotic pressure of the drug formulation is adjusted to a range that is isotonic or nearly isotonic with human body fluids, significantly improving patient injection tolerance and medication adherence, and ensuring safe and effective drug delivery. The isotonic adjuster is selected from at least one of sodium chloride or glucose. Sodium chloride is the most physiologically accepted isotonic adjuster, widely used in infusions and injections to provide a sodium and chloride ion environment consistent with body fluids. Glucose has the dual functions of isotonic regulation and providing energy; its isotonic concentration is approximately 5%, making it a very safe isotonic adjusting substance.

[0014] When the drug is formulated as a lyophilized powder for injection, the pharmaceutically acceptable carrier preferably contains a lyophilization protectant. During lyophilization, proteins undergo two harsh physical processes: low-temperature freezing and vacuum dehydration. This can easily lead to loss of activity due to low-temperature stress, ice crystal formation, and exposure of the hydrophobic core after water removal. The addition of a lyophilization protectant can form hydrogen bonds with protein molecules during the freezing stage, replacing hydration and maintaining their natural folded structure. During the drying stage, it forms a high-viscosity glassy matrix, encapsulating the protein molecules and restricting their molecular movement, thus imparting excellent long-term storage stability in the dry state. The lyophilization protectant is selected from at least one of sucrose, trehalose, or mannitol. Sucrose and trehalose are both non-reducing disaccharides with excellent hydrogen bond-forming ability with protein molecules, perfectly replacing the stabilizing effect of water molecules on the protein surface. Furthermore, they are chemically inert and will not undergo Maillard reactions with the active groups of the protein. Mannitol is commonly used as a filler, which can provide freeze-dried products with a good appearance and mechanical skeleton, prevent the freeze-dried blocks from collapsing and shrinking, and play a certain protective role in the dry state.

[0015] When the drug is formulated as a multi-dose formulation requiring multiple aspirations, the pharmaceutically acceptable carrier may also contain a preservative. After the first aspiration of a multi-dose injection or a reconstituted multi-dose lyophilized powder, the needle hole on the stopper can become a potential channel for external microorganisms to enter the formulation. Furthermore, environmental microorganisms may be introduced during aspiration, leading to microbial growth and posing serious safety risks for subsequent use. The addition of a preservative effectively inhibits the growth and reproduction of bacteria and fungi that may invade the formulation, ensuring the microbiological safety of the formulation during multiple aspirations and protecting patient safety. The preservative is selected from at least one of benzyl alcohol or m-cresol. Benzyl alcohol is a long-established preservative used in parenteral drug formulations and has broad-spectrum antibacterial activity. m-Cresol is more widely used in multi-dose protein drug formulations such as insulin, exhibiting good protein compatibility and excellent preservative efficacy. The added concentration must be above the minimum concentration required to produce an effective antibacterial effect while remaining safe for the patient.

[0016] From a biological mechanism perspective, in the application described in this invention, glutamate carboxypeptidase II blocks downstream signaling pathways by hydrolyzing N-acetylaspartate glutamate, thereby reducing the expression levels of gastric mucosal intestinal metaplasia markers. The research team of this application, through systematic research, discovered that during the development of gastric mucosal intestinal metaplasia, chenodeoxycholic acid (CDCA) upregulates the transcriptional expression of carbamoyl phosphate synthase 1 (CPS1) through the farnesoid X receptor (FXR) signaling pathway, leading to the abnormal accumulation of its downstream metabolite, N-acetylaspartate glutamate (NAAG). The abnormally elevated N-acetylaspartate glutamate, by affecting the epigenetic regulatory mechanisms related to high-mobility group A1, ultimately drives the ectopic overexpression of intestinal metaplasia markers such as the gut-specific transcription factor CDX2, thereby promoting the transformation of the gastric mucosa into an intestinal phenotype. Glutamate carboxypeptidase II is a zinc-dependent metalloproteinase that efficiently hydrolyzes N-acetylaspartate glutamate. It specifically cleaves N-acetylaspartate glutamate molecules, breaking them down into N-acetylaspartate and glutamate. Exogenous supplementation with glutamate carboxypeptidase II, which possesses hydrolytic activity, can significantly reduce the level of N-acetylaspartate glutamate accumulated in diseased tissues, fundamentally relieving its continuous activation of downstream intestinal metaplasia signaling pathways, and ultimately shutting down the abnormal transcription of effector molecules such as CDX2, achieving precise targeted reversal at the molecular level. This mechanism is completely different from traditional drugs that only treat the local inflammatory environment of the gastric mucosa, achieving causal intervention on the pathogenesis of intestinal metaplasia. Attached Figure Description

[0017] Figure 1 Heatmap of differential metabolites after CPS1 knockdown and CDCA treatment of GES-1 cells.

[0018] Figure 2 The image shows the results of detecting the mRNA expression levels of intestinal metaplasia markers in GES-1 cells after NAAG treatment.

[0019] Figure 3 The image shows the results of detecting the protein expression levels of intestinal metaplasia markers in GES-1 cells after NAAG treatment.

[0020] Figure 4 The figure shows the results of detecting the mRNA expression levels of intestinal metaplasia markers in GES-1 cells after treatment with different concentrations of GCPII and NAAG.

[0021] Figure 5 The image shows the results of detecting the mRNA expression levels of intestinal metaplasia markers in GES-1 cells after treatment with GCPII, NAAG, and CDCA.

[0022] Figure 6This image shows the immunofluorescence results of intestinal metaplasia markers in mouse gastric organoids after treatment with GCPII, NAAG, and CDCA.

[0023] Figure 7 The image shows the detection results of mRNA markers of intestinal metaplasia in mouse gastric organoids after treatment with GCPII and NAAG.

[0024] Figure 8 This image shows the immunofluorescence results of intestinal metaplasia markers in human gastric mucosa organoids after GCPII treatment. Detailed Implementation

[0025] This invention is based on a novel molecular pathological discovery: N-acetylaspartate glutamate is a key endogenous metabolite driving gastric mucosal intestinal metaplasia, and glutamate carboxypeptidase II, as its specific hydrolase, can reverse or delay the progression of gastric mucosal intestinal metaplasia by degrading this metabolite and blocking downstream pathological signaling cascades. Based on this, this invention provides the application of glutamate carboxypeptidase II in the preparation of medicaments for the treatment of gastric mucosal intestinal metaplasia. The invention is further illustrated below with specific pharmacological experimental examples and formulation preparation examples, but the invention is not limited to these examples.

[0026] Pharmacological Experiment Example 1: Metabolite Screening and Initial Activity Screening

[0027] The aim of this experiment was to screen and identify differentially expressed metabolites that play a key driving role in the development of gastric mucosal intestinal metaplasia. Previous studies have established that chenodeoxycholic acid (CDCA) can affect the transcription of carbamoyl phosphate synthase I by acting on farnesol X receptors.

[0028] Taking this as a starting point, this experiment divided human gastric mucosal epithelial cells GES-1 into four groups: blank control group (treated by transfection with non-specific control siRNA, i.e., siNC, and adding dimethyl sulfoxide solvent to a final concentration of 0.1%), chenodeoxycholic acid treatment group (treated by adding chenodeoxycholic acid to a final concentration of 100 μM after transfection with siNC), carbamoyl phosphate synthase I knockdown group (transfection with siRNA specifically targeting carbamoyl phosphate synthase I to reduce its expression), and carbamoyl phosphate synthase I knockdown combined with chenodeoxycholic acid treatment group (treated by adding 100 μM chenodeoxycholic acid after transfection with the above specific siRNA).

[0029] After collecting cells from each group, intracellular metabolites were rapidly extracted using a pre-cooled mixed solvent of acetonitrile and methanol in a 1:1 volume ratio. Targeted metabolomics analysis was then performed using a liquid chromatography-tandem mass spectrometry system equipped with a BEH amide column.

[0030] The screening criteria for differentially expressed metabolites were strictly set as follows: variable projection importance value (VIP value) > 1.0, fold change in expression between groups > 1.5, and p-value < 0.05 for statistical tests. Metabolomics analysis was performed by Novogene Ltd.

[0031] The filtering results are as follows Figure 1 As shown in the analysis, among the many regulated metabolites, N-acetylaspartoylglutamate (NAAG) showed the most significant changes. It accumulated significantly in the chenodeoxycholic acid treatment group and decreased significantly in the carbamoyl phosphate synthase I knockdown group, thus identifying it as the number one candidate pathological metabolite.

[0032] In further functional validation, GES-1 cells were treated with N-acetylaspartate at concentrations of 100 μM and 200 μM for 48 h, and then detected by real-time quantitative polymerase chain reaction and Western blotting. Figure 2 and Figure 3 As shown, the results indicated that the mRNA level of CDX2, a core marker of intestinal metaplasia, as well as the protein expression levels of CDX2 and MUC2, were significantly increased in a dose-dependent manner compared to the control group.

[0033] This result strongly demonstrates that N-acetylaspartoylglutamate is a direct metabolite that induces intestinal metaplasia-like phenotype changes in gastric mucosal epithelial cells, and also provides a clear molecular target for subsequent precision targeted therapy, namely, blocking the pathological process by clearing N-acetylaspartoylglutamate.

[0034] Pharmacological Experiment Example 2: Evaluation of the therapeutic effect of glutamate carboxypeptidase II on N-acetylaspartate-glutamate-induced GES-1 cell intestinal metaplasia model

[0035] This experiment aims to systematically verify the therapeutic effect of glutamate carboxypeptidase II at the in vitro cellular level. Human gastric mucosal epithelial cell line GES-1 (purchased from iCell Bioscience) was used as the experimental subject. Cells were cultured in RPMI-1640 complete medium (purchased from Gibco) containing 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated at 37°C in a 5% carbon dioxide incubator. Cells in the logarithmic growth phase were harvested and cultured at 2 × 10⁶ cells per well. 5 The cells were evenly seeded at a density in a six-well cell culture plate and cultured overnight until the cells were fully adhered.

[0036] Cells were randomly divided into a blank control group, a model group, a low-dose treatment group, and a high-dose treatment group. The blank control group (NC) received 0.1% dimethyl sulfoxide (DMSO) as a solvent. The model group received 200 μM N-acetylaspartate glutamate (NAAG, purchased from MedChemExpress) and was cultured for 24 hours. The low-dose treatment group received 200 μM NAAG and was cultured for 24 hours, followed by the addition of recombinant glutamate carboxypeptidase II protein (GCPII, purchased from Solarbio) at a final concentration of 0.8 μg / mL, and was cultured for another 48 hours. The high-dose treatment group received 200 μM NAAG and was cultured for 24 hours, followed by the addition of GCPII at a final concentration of 1.6 μg / mL, and was cultured for another 48 hours. After culture, the culture medium was discarded, and the cells were washed twice with pre-cooled phosphate buffer. Cells were then collected for various assays.

[0037] Cells were lysed using TRIZOL reagent and total RNA was extracted. After the concentration and purity of the extracted RNA were determined by UV spectrophotometry, 1 μg of total RNA was taken and reverse transcribed into complementary DNA using a reverse transcription kit. Using this complementary DNA as a template, the mRNA expression level of the intestinal metaplasia marker CDX2 was detected using SYBR Green quantitative PCR reagent and specific primers on a real-time quantitative PCR instrument. The primer sequences used are shown in Table 1. GAPDH was used as an internal reference gene, and 2... -ΔΔCt The relative expression levels of each gene can be calculated.

[0038] Table 1. qPCR primer sequences

[0039]

[0040] Test results as follows Figure 4 As shown, the CDX2 mRNA expression level in the model group (NAAG) was significantly higher than that in the blank control group (NC), indicating that the model was successfully constructed. The CDX2 mRNA level in the high-dose treatment group (GCPII 1.6 μg / mL) was statistically significantly downregulated compared to the corresponding model group, indicating that 1.6 μg / mL is the effective concentration of GCPII for treating intestinal metaplasia.

[0041] After determining the optimal concentration of GCPII, cells were randomly divided into a blank control group, model group 1, treatment group 1, model group 2, and treatment group 2, with three replicates in each group. The blank control group (NC) was treated with 0.1% dimethyl sulfoxide solvent; model group 1 was treated with 200 μM N-acetylaspartate glutamate (NAAG, purchased from MedChemExpress) and cultured for 24 hours; treatment group 1 was treated with 200 μM N-acetylaspartate glutamate and cultured for 24 hours, followed by the addition of 1.6 μg / mL recombinant glutamate carboxypeptidase II protein (GCPII, purchased from Solarbio), and cultured for another 48 hours; model group 2 was treated with 100 μM chenodeoxycholic acid and cultured for 24 hours; treatment group 2 was treated with 100 μM chenodeoxycholic acid and cultured for 24 hours, followed by the addition of 1.6 μg / mL recombinant glutamate carboxypeptidase II protein, and cultured for another 48 hours. After the culture was completed, the culture medium was discarded, and the cells were washed twice with pre-cooled phosphate buffer. The cells were then collected for various tests.

[0042] Test results as follows Figure 5 As shown, the CDX2 mRNA expression levels in model group 1 (NAAG) and model group 2 (CDCA) were significantly higher than those in the blank control group (NC), indicating successful model construction. The CDX2 mRNA levels in treatment group 1 (NAAG+GCPII) and treatment group 2 (CDCA+GCPII) were statistically significantly downregulated compared to their corresponding model groups. This experiment clearly demonstrates in a cell model that exogenous supplementation with glutamate carboxypeptidase II can efficiently hydrolyze accumulated N-acetylaspartate glutamate, thereby directly downregulating the expression of CDX2, a core transcription factor for intestinal differentiation, at the transcriptional level, achieving effective reversal of the intestinal metaplasia phenotype. This solves the problem of the lack of drugs in existing technologies that directly target pathological metabolites and intervene in molecular mechanisms at the root.

[0043] Pharmacological Experiment Example 3: Therapeutic Effect of Glutamate Carboxypeptidase II in an Isolated Mouse Gastric Organoid Intestinal Metaplasia Model

[0044] This experiment further validated the efficacy of GCPII by constructing a three-dimensional gastric organoid model that more closely resembles the in vivo microenvironment. Healthy 6-week-old male C57BL / 6 mice, weighing 20-25g, were selected, sacrificed after fasting for 12 hours, and their entire stomachs were isolated under sterile conditions. The stomachs were cut open along the greater curvature, rinsed clean in ice-cold PBS, and the surface mucus and contents were scraped off.

[0045] Cut the stomach tissue into pieces approximately 1-2 mm in size. 3Tissue blocks were digested using a digestive solution containing 2 mg / mL collagenase I in a 37°C shaker for 30 minutes with continuous shaking. The digested material was then filtered through a 70 μm pore size cell filter, and the filtrate rich in gastric glands was collected and centrifuged. The supernatant was discarded, and the resulting gastric gland precipitate was gently and evenly mixed with Matrigel on ice. 30 μL of this mixture was seeded into the center of each well of a 24-well plate and incubated at 37°C for 30 minutes to allow it to solidify. 500 μL of preheated mouse gastric organoid complete culture medium (Advanced DMEM / F12 based) was added to each well, containing 50 ng / mL epidermal growth factor, 100 ng / mL Noggin, 500 ng / mL R-spondin 1, 1×B27 supplement, and 1×N2 supplement. The medium was replaced with fresh medium every 3 days. After five to seven days of culture, mature cystic gastric organoids formed.

[0046] Organoids were randomly divided into five groups: control group, model group 1, treatment group 1, model group 2, and treatment group 2. The control group received 0.1% dimethyl sulfoxide (DMSO) solvent; model group 1 received N-acetylaspartate glutamate at a final concentration of 200 μM; treatment group 1 received 200 μM N-acetylaspartate glutamate and recombinant glutamate carboxypeptidase II protein at a final concentration of 1.6 μg / mL; model group 2 received chenodeoxycholic acid at a final concentration of 100 μM; and treatment group 2 received 100 μM chenodeoxycholic acid and recombinant glutamate carboxypeptidase II protein at a final concentration of 1.6 μg / mL. Each group had three replicates and was incubated at 37°C in a 5% CO2 incubator for 72 hours.

[0047] After processing, organoids were collected from each group, fixed with 4% paraformaldehyde, and then routinely embedded in paraffin and frozen sectioned. After dewaxing, rehydration, and high-temperature antigen retrieval, non-specific sites were blocked with goat serum. Rabbit anti-CDX2 primary antibody diluted 1:200 was added, and the sections were incubated overnight in a humidified chamber at 4°C. The next day, after thorough washing with PBS, Alexa Fluor 488-labeled goat anti-rabbit fluorescent secondary antibody was added, and the sections were incubated at room temperature in the dark for 1 hour. After washing, the sections were counterstained with a fluorescent quenching mounting medium containing 4',6-diamidinyl-2-phenylindole (DAPI), mounted, and observed and photographed under a fluorescence microscope.

[0048] The intensity of CDX2 green fluorescence in each organoid slice was quantitatively analyzed using ImageJ software. The results are as follows: Figure 6As shown, compared with the control group (NC), the organoid epithelium of model group 1 (NAAG) and model group 2 (CDCA) showed diffuse and bright CDX2 green fluorescence, clearly indicating the occurrence of intestinal type differentiation; while the CDX2 positive area of ​​treatment group 1 (NAAG+GCPII) and treatment group 2 (NAAG+GCPII) was significantly reduced, and the fluorescence intensity was statistically significantly reduced compared with the model group.

[0049] At the same time, such as Figure 7 As shown, qPCR detection of extracted organoid RNA revealed that CDX2 mRNA levels were also significantly inhibited in the treatment group. This experimental case, in an organoid model, confirmed that glutamate carboxypeptidase II can effectively degrade N-acetylaspartate glutamate in the tissue microenvironment, successfully alleviating gastric mucosal intestinal metaplasia. This indicates that the protein drug has the potential to penetrate mucosal structures and exert local therapeutic effects, overcoming the limitations of existing drugs that simply reduce inflammation or repair the membrane, directly targeting and reversing the nature of intestinal metaplasia lesions.

[0050] Pharmacological Experiment Example 4: Therapeutic Effect of Glutamate Carboxypeptidase II on Organoids Derived from Human Intestinal Metaplasia Tissue

[0051] This experimental case represents the most decisive evidence prior to clinical translation. The study protocol has received full approval from the ethics committees of the collaborating hospitals, and informed consent was obtained from all patients before enrollment. Gastric mucosal tissue was obtained from patients with a definitive pathological diagnosis of moderate to severe intestinal metaplasia (Sydney score not lower than 2) in the antrum or body of the stomach, accompanied by bile reflux, through endoscopic biopsy of the lesion site. Enrolled patients were between 40 and 70 years of age, and those with concurrent gastric cancer, high-grade intraepithelial neoplasia, or severe cardiac, hepatic, or renal insufficiency were excluded.

[0052] Biopsies were taken from patients with moderate to severe intestinal metaplasia in the gastric antrum or body accompanied by bile reflux. Freshly obtained biopsy tissue blocks were immediately immersed in pre-cooled tissue preservation solution at 4°C and delivered to the laboratory for processing within 2 hours. In a biosafety cabinet, the tissue samples were repeatedly rinsed with sterile PBS containing high concentrations of antibiotics, then minced and transferred to a mixed digestion solution consisting of 1 mg / mL collagenase IV and 0.1 mg / mL deoxyribonuclease I, and digested at 37°C for 30 minutes.

[0053] Digestion products were filtered through a 70 μm filter, and cells were collected by low-speed centrifugation of the filtrate. The cell pellet was mixed with Matrigel and seeded at 30 μL per well, then solidified. Preheated human gastric organoid complete medium was then added. This human gastric organoid complete medium, based on Advanced DMEM / F12, was supplemented with 1×B27, 1×N2, 50 ng / mL epidermal growth factor, 100 ng / mL Noggin, 500 ng / mL R-spondin1, 10 μmol / L Y-27632 (ROCK pathway inhibitor), 10 μmol / L SB202190 (p38 MAPK inhibitor), and 1×Primocin antibiotic. The medium was incubated at 37°C in a 5% CO2 incubator, with medium changes every 2-3 days. After approximately 7-10 days of culture, the formation of human intestinal metaplasia-derived organoids (hGIM organoids) with typical structures was observed.

[0054] Human intestinal metaplasia organoids that have undergone passage culture and are in good condition were randomly divided into a control group (addition of double-distilled water) and a treatment group (addition of 1.6 μg / mL glutamate carboxypeptidase II). Each group had three replicates, and the drug was continuously administered for 72 hours.

[0055] The expression of CDX2 was detected using the same immunofluorescence staining procedure as in pharmacological experiment example 3. The results are as follows: Figure 8 As shown, compared with the control group (GIM), the treatment group (GIM+GCPII) showed a significant decrease in the immunofluorescence intensity of CDX2, the core marker of intestinal metaplasia, in hGIM organoids treated with 1.6 μg / mL GCPII for 72 hours, with the positive area almost disappearing. This experiment, using a patient-derived organoid model, strongly demonstrates that GCPII can reverse intestinal metaplasia lesions in human gastric mucosa, providing sufficient evidence for its potential as a new candidate drug for the treatment of gastric mucosal intestinal metaplasia. Glutamate carboxypeptidase II, as a biopharmaceutical, possesses powerful efficacy in fundamentally treating and reversing existing human intestinal metaplasia tissue. This directly addresses the urgent clinical need for drugs that can substantially regress this precancerous lesion of gastric mucosal intestinal metaplasia, filling the gap in current technology for specific, highly effective, and low-toxicity reversal drugs.

[0056] Formulation Preparation Example 1: Preparation of Glutamate Carboxypeptidase II Injection

[0057] A ready-to-use glutamate carboxypeptidase II injection solution suitable for intravenous or local injection was prepared. First, under strictly aseptic conditions, the excipient solutions required for the formulation were prepared. An appropriate amount of water for injection was weighed and placed in a preparation container. Disodium hydrogen phosphate and sodium dihydrogen phosphate, as buffers, were added sequentially to a final concentration of 20 mM, and the mixture was stirred until completely dissolved. The pH of the solution was measured using a calibrated pH meter and finely adjusted to 7.2 to maintain the higher-order structural stability and enzyme activity of the glutamate carboxypeptidase II protein.

[0058] Subsequently, sucrose as a stabilizer was added to a final concentration of 5% by volume, and polysorbate 80 as a nonionic surfactant was added to a final concentration of 0.01% by volume. The mixture was gently stirred to ensure complete dissolution. These two substances work synergistically to prevent the protein from denaturing and agglomerating due to interfacial effects during preparation, filtration, and storage.

[0059] Next, sodium chloride, acting as an isotonic adjuster, was added to adjust the osmotic pressure of the solution to approximately 300 mOsm / L per liter, which is isotonic with human blood plasma. The prepared excipient solution was then sterilized by filtering through a 0.22 μm pore size filter membrane. In another container, the pre-purified and qualified recombinant glutamate carboxypeptidase II stock solution was diluted with a small amount of the above sterilized excipient solution, and the amount was calculated and quantitatively added to achieve a final glutamate carboxypeptidase II protein concentration of 1.6 mg / mL. After gentle mixing, the solution was aseptically filled into cleaned and sterilized vials, which were then capped and corked.

[0060] The entire preparation process was carried out under Class C cleanroom conditions with localized Class A laminar flow protection. The resulting injection solution has a pH precisely controlled between 7.0 and 7.4, and the protein exists in monomeric and active native conformation forms, making it directly injectable. This significantly improves the convenience of medication and patient compliance, while avoiding the degradation and destruction of protein drugs by gastrointestinal proteases during oral administration, thus ensuring efficacy.

[0061] Formulation Preparation Example 2: Preparation of Lyophilized Powder for Glutamate Carboxypeptidase II Injection

[0062] To provide a dosage form with a longer shelf life and better transport stability, this embodiment prepares a lyophilized powder for injection. Based on the excipient formulation of Example 1, the isotonicity adjuster sodium chloride is omitted, and the protein protection system is enhanced. L-histidine and L-histidine hydrochloride monohydrate, used as buffers, are accurately weighed and dissolved in water for injection to prepare a histidine buffer with a final concentration of 20 mM and a pH of 6.5. This buffer not only provides a stable pH environment, but its histidine molecules themselves can also stabilize protein structure through multiple mechanisms.

[0063] Subsequently, trehalose, acting as a freeze-drying protectant and stabilizer, was added to the solution to achieve a final concentration of 8% by mass-volume ratio. Trehalose is an excellent freeze-drying protectant; during freezing, it forms a strong hydrogen bond network with the hydration layer on the protein surface and, after dehydration, forms a high-viscosity glassy matrix, physically immobilizing protein molecules and preventing their denaturation and aggregation.

[0064] In addition, polysorbate 20 at a final concentration of 0.005% by volume was added to protect the protein from damage caused by ice-water interface stress during freezing and drying. After complete dissolution, the solution was filtered through a 0.22 μm filter membrane for sterilization. High-purity glutamate carboxypeptidase II stock solution was diluted with this excipient solution to a protein concentration of 1.6 mg / mL, and the solution was brought to volume and mixed thoroughly with gentle stirring. Under Class 100 laminar flow protection, the solution was precisely filled into 10 mL molded vials at 2.0 mL per vial, and partially stoppered. The filled products were then quickly transferred to the shelves of a freeze dryer pre-cooled to 5°C.

[0065] The freeze-drying process is as follows: First, the product is pre-frozen at -40℃ for 2 hours to ensure that the moisture is completely frozen into homogeneous ice crystals. Then, the vacuum pump is turned on to reduce the chamber pressure to below 15Pa, and the temperature is raised to -20℃ within 2 hours and maintained at this temperature for 10 hours for primary drying, allowing most of the free ice to sublimate directly. Next, the shelf temperature is gradually increased to 25℃ over 4 hours and maintained under high vacuum for 6 hours for desorption drying to completely remove bound water. The drying endpoint is determined by the convergence of readings from the Pirani vacuum gauge and the capacitive pressure gauge. After the freeze-drying process is completed, the equipment automatically performs full plugging and then capping under sterile nitrogen gas.

[0066] Before use, simply reconstitute it with water for injection containing 0.9% sodium chloride or 5% glucose injection. The lyophilized powder cake will dissolve rapidly within one minute, forming a clear, transparent injection solution without visible particles. This lyophilized powder injection formulation is extremely stable during long-term storage at 2-8°C, effectively avoiding the risk of degradation and inactivation of liquid protein formulations during storage and transportation. This significantly reduces the cold chain maintenance costs of the medical system and provides a highly effective treatment option that can be stored for a long time for patients with gastric mucosal intestinal metaplasia.

[0067] Based on all the above pharmacological experiments, this invention constructs a complete and interconnected chain of evidence, from the discovery of metabolic driving factors and the verification of in vitro molecular mechanisms to the evaluation of mouse organ models simulating the in vivo environment, and finally to the direct treatment of patient-derived diseased organoids that most closely resemble the pathological essence of humans. This demonstrates that glutamate carboxypeptidase II can serve as a fundamental drug for treating gastric mucosal intestinal metaplasia. Its therapeutic mechanism is clearly distinct from any symptomatic treatment or intervention with unclear mechanisms in existing technologies. It precisely hydrolyzes the pathological metabolite N-acetylaspartate glutamate, directly cutting off the upstream signals that trigger ectopic transcription of intestinal-type genes such as CDX2, fundamentally blocking the process of irreversible malignant differentiation of gastric mucosal epithelial cells into the intestinal type. This reversal strategy based on precise molecular mechanisms overcomes the shortcomings of existing drugs, such as unclear drug targets and the inability to reverse the underlying pathology, which can only alleviate symptoms. At the same time, as a supplement to naturally occurring metabolic regulatory enzymes in the body, it not only demonstrates the advantages of high efficiency and low toxicity through scientific evidence, but also fills the gap in the field of precise targeted therapy for gastric mucosal intestinal metaplasia. It provides a solution that can substantially reverse the pathological process for clinically challenging precancerous lesions of the stomach, meeting a long-standing unmet clinical need and demonstrating extremely significant medical value and broad application prospects. It avoids the safety issues of existing chemical drugs such as celecoxib causing cardiovascular events or metformin causing lactic acidosis and other serious toxic side effects.

[0068] The specific embodiments described in this invention are merely illustrative of the invention and are not intended to limit it. Those skilled in the art can make modifications to these embodiments without contributing any inventive step after reading this specification, but such modifications are protected by patent law as long as they fall within the scope of the claims of this invention.

Claims

1. Application of glutamate carboxypeptidase II in the preparation of drugs for the treatment of gastric mucosal intestinal metaplasia.

2. The application according to claim 1, characterized in that, The drug is formulated as an injectable preparation.

3. The application according to claim 2, characterized in that, The injectable formulation is prepared as an injection solution or a lyophilized powder for injection.

4. The application according to claim 1, characterized in that, The drug contains a therapeutically effective amount of glutamate carboxypeptidase II and a pharmaceutically acceptable carrier.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable carrier comprises a buffer configured to maintain the pH of the drug between 5.0 and 8.

0.

6. The application according to claim 5, characterized in that, The buffer is selected from at least one of phosphate buffer, histidine buffer, or citrate buffer.

7. The application according to claim 4, characterized in that, The pharmaceutically acceptable carrier contains a stabilizer to prevent protein aggregation and denaturation, the stabilizer being selected from at least one of sucrose, trehalose, mannitol, polysorbate 80, polysorbate 20, or EDTA.

8. The application according to claim 4, characterized in that, The pharmaceutically acceptable carrier comprises an isotonic regulator selected from at least one of sodium chloride or glucose.

9. The application according to claim 4, characterized in that, The drug is formulated as a lyophilized powder for injection, and the pharmaceutically acceptable carrier contains a lyophilization protectant selected from at least one of sucrose, trehalose, or mannitol.

10. The application according to claim 1, characterized in that, The glutamate carboxypeptidase II blocks downstream signaling pathways by hydrolyzing N-acetylaspartate glutamate, thereby reducing the expression level of gastric mucosal intestinal metaplasia markers; the gastric mucosal intestinal metaplasia markers include CDX2 and / or MUC2.

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