PreTA inhibitor and application thereof

Abstract

The invention specifically discloses a PreTA inhibitor and application thereof, and relates to the technical field of biological medicines. The inhibitor provided by the invention can be used for effectively inhibiting the activity of PreTA, so that the anti-tumor effect of 5-fluorouracil (5-FU) is recovered.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an inhibitor of PreTA and its application. Background Technology

[0002] Fluoropyrimidine-based treatment regimens, particularly 5-fluorouracil (5-FU) and its oral prodrug capecitabine, are cornerstone drugs for first-line treatment of metastatic pancreatic cancer. However, the development of drug resistance continues to lead to poor patient survival outcomes. The five-year survival rate for pancreatic cancer patients is only 13%, highlighting the urgent need to improve treatment strategies to overcome treatment resistance.

[0003] Recent studies have demonstrated the presence of intratumoral microbiota in solid tumors. For example, Enterobacteriaceae and Pseudomonas orders have been identified as resident microbiota in pancreatic cancer tissue. The presence of intratumoral microbiota may be closely related to response to chemotherapy drugs. Studies have shown that intratumoral microbiota can mediate chemotherapy resistance by directly modifying drug structures, secreting specific metabolites, or virulence factors. Of particular clinical significance is that bacteria within pancreatic tumors can express a long isoform of bacterial cytidine deaminase (CDD). L The enzyme metabolizes gemcitabine, converting it into an inactive metabolite, which leads to chemotherapy resistance. Therefore, drug metabolism may be an important perspective for understanding chemotherapy resistance.

[0004] The pharmacological activity of fluoropyrimidine drugs (especially 5-FU) is closely related to dihydropyrimidine dehydrogenase (DPD), with approximately 20% of administered 5-FU being converted into an active metabolite that inhibits DNA synthesis. However, the majority (80%) of 5-FU is metabolized by the host's DPD into inactive dihydrofluorouracil (DHFU). Therefore, inhibiting 5-FU metabolism to increase its exposure level is a clinically validated strategy to improve clinical efficacy. For example, S-1 is an oral combination drug for the treatment of gastrointestinal tumors, consisting of tegafur (a prodrug of 5-FU), gimeracil (Gim, a dihydropyrimidine dehydrogenase inhibitor), and oteracil potassium. Gim is intentionally added to this formulation to inhibit the metabolic breakdown of 5-FU, thereby increasing 5-FU levels and clinical efficacy. Notably, certain bacteria contain... preTA The operon encodes a functional homolog of human DPD, enabling it to convert 5-FU to DHFU. This intermediate product can be further metabolized into α-fluoro-β-ureapoizolic acid (FUPA) and α-fluoro-β-alanine (FBAL) by bacterial dihydropyrimidinease (DHP) and β-ureidopropionase (BUP) homologs, respectively. This mechanism is similar to that of intratumoral Escherichia coli (E. coli) E. coliPancreatic cancer is associated with resistance to 5-FU in colorectal malignancies. Although the influence of intratumoral bacteria on drug metabolism has become a potential factor determining treatment efficacy, key knowledge gaps remain regarding the pancreatic tumor microbiota. Summary of the Invention

[0005] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide an inhibitor of PreTA. The inhibitor provided by this invention can effectively inhibit the activity of PreTA, thereby restoring the antitumor effect of 5-FU.

[0006] (II) Technical Solution To achieve the above objectives, the present invention provides an inhibitor of PreTA, the inhibitor comprising gimidazine, enirapexine, thymidine, uridine, and / or uracil.

[0007] In one embodiment, the inhibitor is gimeracil (Gim).

[0008] In another aspect, the present invention provides the use of the above-mentioned inhibitor in the preparation of drugs for the prevention and / or treatment of pancreatic cancer.

[0009] In one embodiment, the pancreatic cancer is drug-resistant.

[0010] In one embodiment, the pancreatic cancer is resistant to fluoropyrimidine drugs.

[0011] In one embodiment, the fluoropyrimidine drug includes fluorouracil, capecitabine, tegafur, tegafur, deoxyfluorouracil, tegafur / uracil, and / or trifluuridine teppirimidine.

[0012] In one embodiment, the fluoropyrimidine drug is fluorouracil (5-FU).

[0013] In another aspect, the present invention provides a pharmaceutical composition comprising: (1) A therapeutically effective dose of the above-mentioned inhibitors; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0014] In one embodiment, the pharmaceutical composition further includes a fluoropyrimidine drug.

[0015] In one embodiment, the fluoropyrimidine drug includes fluorouracil, capecitabine, tegafur, tegafur, deoxyfluorouracil, tegafur / uracil, and / or trifluuridine teppirimidine.

[0016] In one embodiment, the fluoropyrimidine drug is fluorouracil (5-FU).

[0017] In one embodiment, the pharmaceutical composition comprises: (1) A therapeutically effective dose of the above-mentioned inhibitors; (2) Pharmaceutically or immunologically acceptable carriers or excipients; (3) 5-Fluorouracil.

[0018] In another aspect, the present invention also provides a pharmaceutical formulation of the above-described pharmaceutical composition.

[0019] In another aspect, the present invention also provides pharmaceutical products, including the pharmaceutical compositions and / or pharmaceutical preparations described above.

[0020] In another aspect, the present invention also provides the use of the above-described pharmaceutical compositions, pharmaceutical preparations and / or pharmaceutical products in the preparation of drugs for the prevention and / or treatment of pancreatic cancer.

[0021] (III) Beneficial Effects This invention provides an inhibitor of PreTA and its application. Compared with the prior art, it has the following advantages: 1. Gimetidine can effectively inhibit bacterial PreTA activity, thereby restoring the antitumor effect of 5-FU. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram illustrating the association between intratumoral citrate bacteria and poor survival in pancreatic cancer patients.

[0024] Figure 2 This is a diagram analyzing the microbial composition and diversity of the tumor and adjacent normal tissues in pancreatic cancer patients.

[0025] Figure 3 It involves targeted isolation and identification of clinically relevant samples from pancreatic cancer. C. freundii Schematic diagram of the strain.

[0026] Figure 4 yes C. freundii A pan-genome analysis diagram of the model strain and clinical isolates.

[0027] Figure 5 yes C. freundii Schematic diagram of the in vitro inhibition of the antitumor activity of 5-FU.

[0028] Figure 6 yes C. freundii A schematic diagram illustrating the reduction of 5-FU's antitumor activity in vivo.

[0029] Figure 7 yes C. freundii Planting analysis diagram.

[0030] Figure 8 yes C. freundii Schematic diagram of 5-FU metabolism via PreTA.

[0031] Figure 9 These are the secondary spectra of 5-FU and FBAL.

[0032] Figure 10 yes C. freundii The characteristics of the mediated 5-FU metabolic mechanism and the analysis diagram of the PreTA structure and function.

[0033] Figure 11 This is a schematic diagram illustrating Gim's inhibition of PreTA metabolism of 5-FU.

[0034] Figure 12 This is a graph showing the inhibitory effects of inhibitors on Escherichia coli PreT and PreA. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Terms and Definitions As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0037] As used herein, the terms "inhibitor" or "inhibitor of PreTA or its encoding nucleic acid molecule" are used interchangeably and refer to a substance that can reduce the level or activity of PreTA or its encoding nucleic acid molecule. Inhibitors that can be used in this disclosure include, but are not limited to, antibodies against PreTA or nucleic acid molecules encoding the protein, siRNA, miRNA, antisense oligonucleotides, antagonists, and blocking agents.

[0038] The inhibitor disclosed in this invention can inhibit PreTA, thereby enabling its further use in the prevention or treatment of diseases and / or related symptoms associated with pancreatic cancer.

[0039] As used herein, the term "pharmaceutical composition" refers to a composition comprising an inhibitor of PreTA formulated with one or more pharmaceutically acceptable carriers.

[0040] The formulation of a pharmaceutical composition can be tailored to the application. In particular, pharmaceutical compositions can be formulated using methods known in the art to provide rapid, continuous, or delayed release of the active ingredient upon administration to mammals. For example, the formulation can be selected from any of the following: plasters, granules, lotions, liniments, lemonade, aromatic water, powders, syrups, eye ointments, liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, eye drops, tablets, suppositories, injections, alcoholic preparations, capsules, creams, lozenges, tinctures, pastes, pills, and soft or hard gelatin capsules.

[0041] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse reactions (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0042] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

[0043] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption-delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin, are possible. Other examples of carriers include culture media, such as DMEM or RPMI; and cryogenic storage media containing components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations to balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0044] The pharmaceutical compositions of the present invention can be administered using any known method. One of a variety of methods known to those skilled in the art can be used to administer the substance, compound, or agent to a subject using the terms "give" or "apply".

[0045] For example, compounds or agents can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). Compounds or agents can also be suitably introduced via rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or formulations) that provide prolonged, slowed, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more prolonged periods.

[0046] As used herein, the term “therapeutic effective dose” refers to a dose sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors known in the medical field.

[0047] As used herein, the term “treatment” for a symptom or patient refers to steps taken to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, symptom, or condition; substantially improving or alleviating the clinical or aesthetic symptoms of a symptom; substantially preventing the clinical or aesthetic symptoms of a disease, symptom, or condition; and avoiding harmful or unpleasant symptoms. Treatment also refers to achieving one or more of the following: (a) reducing the severity of the symptom; (b) limiting the development of characteristic symptoms of the symptom being treated; (c) limiting the exacerbation of characteristic symptoms of the symptom being treated; (d) limiting the recurrence of the symptom in patients who previously had the symptom; and / or (e) limiting the recurrence of symptoms in patients who previously did not have symptoms of the symptom.

[0048] The term "prevention" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0050] Example 1: Analysis of factors affecting poor survival in pancreatic cancer patients: 1. Two groups of pancreatic cancer patients were collected: one group (64 cases) of paraffin-embedded (FFPE) tissue samples for identification of tumor-resident bacteria, and the other group of fresh surgical tumor specimens (10 cases) for bacterial isolation and culture. All patient samples were obtained from Zhejiang Cancer Hospital and collected under an Institutional Review Board (IRB) approved protocol (IRB-2021-146), with informed consent obtained from all participants.

[0051] FFPE tissue samples from 64 pancreatic cancer patients (including 64 tumor (T) samples and 56 paired adjacent normal (N) tissue samples) were used for bacterial detection using fluorescence in situ hybridization (FISH) and a multiplex 16S rDNA sequencing protocol that amplifies five short regions on the 16S rRNA gene (referred to as the 5R 16S rDNA sequencing method). There was no significant difference in gender distribution among the analysis groups.

[0052] 2. The intratumoral microbiota of pancreatic cancer was visualized using EUB338, a specific probe targeting bacterial 16S ribosomal RNA (rRNA).

[0053] The results showed that a rich and individual-variable bacterial community existed within the tumor, and their localization within the tumor was confirmed by comparison with H&E staining. Figure 1 A and Figure 2 (A in the middle).

[0054] 3. The overall microbiome within pancreatic cancer tumors was further characterized using 5R 16S ribosomal DNA (rDNA) sequencing: Tissue (40–70 mg) was extracted from FFPE samples using the CTAB method. Negative controls were included throughout the workflow, including sampling, DNA extraction, and template-free PCR amplification, to minimize the risk of contamination. Multiple regions of five pairs of 16S rRNA genes were amplified and multiplexed. Sequencing was performed using the Illumina NovaSeq 6000 platform.

[0055] After sequencing, the fragmented reads were filtered and mapped to their respective amplification regions based on primer sequences. The short multi-region framework (SMURF) approach was used to integrate the read counts from each region, and a coherent microbial profile was generated through maximum likelihood estimation. GreenGenes was used as the reference database.

[0056] Potential contamination was identified and excluded using established filters, specifically: a) Sample / library preparations containing <1000 readings were discarded; b) Relative abundances <10 -4 Taxonomic groups were filtered out; c) Taxonomic groups prevalent in >30% of the negative controls were removed. Bacterial abundance was quantified as the proportion of sequencing reads assigned to each taxonomic group relative to the total number of reads in the sample. 5R 16S rDNA sequencing of paired tumor and adjacent normal tissues from 64 patients revealed highly similar bacterial profiles at both the phylum and family levels, with Pseudomonas and Bacteroidetes being the most abundant taxa in both tumor and adjacent normal tissues. Figure 2 (B and C in the original text). α diversity index (Chao1, Shannon, and Simpson; all p>0.05) Figure 2 The D) and β diversity analyses (p=0.483) were performed. Figure 2 E) indicates that no significant differences were observed between tumor tissue and adjacent normal tissue in terms of microbial richness, evenness, or community structure.

[0057] 4. Survival Association Analysis: To improve biological relevance and reduce interference from low-abundance taxa, genus-level data were first screened, retaining genera with a mean relative abundance ≥0.05% and a prevalence ≥15% across all samples. Unclassified genera were excluded, resulting in 113 genera for downstream analysis. In the survival association analysis, patients were divided into high-abundance and low-abundance groups based on the median relative abundance of each genus in the cohort. Kaplan-Meier survival analysis was then performed to assess the association between each genus and overall patient survival. Specifically, for the genus *Citrobacter* (… CitrobacterUsing 0% (median relative abundance) as the cutoff value, 64 pancreatic cancer patients were divided into a high abundance group (n=13) and a low abundance group (n=51). Furthermore, a subgroup analysis was performed on 9 patients receiving fluorouracil-based chemotherapy (including capecitabine or FOLFIRINOX) to further explore the potential impact of microorganisms on treatment response (n=9). Specifically, for Citrobacter Based on a cutoff value of 0.19% (median relative abundance), these patients were divided into a high abundance group (n=4) and a low abundance group (n=5). Citrobacter The basic characteristics of patients grouped by median abundance are shown in Tables 1 and 2.

[0058] Table 1 χ²: Chi-square test; -: Fisher exact; Citrobacter The median abundance was 0. Table 2 When calculating survival time, the assessment time for deceased patients is from the date of surgery to the date of death, while for surviving patients, the cutoff time is the date of the last follow-up.

[0059] Seven of these genera are associated with overall survival (OS). Figure 1 Significant associations were found in B) of the data, for example, *Porphyromonas* spp. (HR=4.34, 95% CI=1.75–10.777, p=0.0006) and Citrobacter (HR=3.34, 95%CI=1.17-9.54, p=0.0017, Figure 1 (D in the middle).

[0060] Notably, in a subgroup of nine patients who received 5-fluorouracil chemotherapy, intratumoral... Citrobacter Or microbacteria spp. are strongly associated with significantly worse survival outcomes ( Figure 1 The hazard ratios (HRs) for C and E in the tumor were 8.21 (95% CI = 1.27–53.11, p = 0.0223) and 8.21 (95% CI = 1.27–53.11, p = 0.0223), respectively. This indicates that the tumor... Citrobacter This may be a potential microbial factor that reduces the effectiveness of chemotherapy for pancreatic cancer, suggesting the need for targeted isolation and further functional studies.

[0061] Example 2: Pancreatic cancer tumor Citrobacter Separation and identification: 1. A targeted separation method was used to separate fresh tumor tissues and their paired adjacent normal tissues from 10 pancreatic cancer patients. CitrobacterSeparation: Take 0.2 g of fresh tissue fragments, homogenize them in 1 mL of sterile PBS using a glass homogenizer, and incubate for 5 minutes. PBS serves as a negative control. After precipitation, take 100 μL of the supernatant and dilute it to 10⁻⁶ in PBS. -1 Up to 10 -6 Subsequently, 100 μL of each dilution was inoculated onto Columbia blood agar, nutrient agar, Salmonella-Shigella agar, and chromogenic agar plates. The plates were incubated aerobically at 37°C with 5% CO2 for 72 hours. Different colonies were transferred to liquid culture medium and cultured for 1–3 days. Purification was performed using the streak plating method. Single colonies were selected for liquid culture and then, following the previously described method, underwent DNA extraction, PCR amplification, sequencing, and BLAST analysis, followed by identification using Sanger sequencing.

[0062] Colonies selected based on morphological similarity to the model strain (ATCC 43864) on various chromogenic agar media ( Figure 3 B in the sample was confirmed by Sanger sequencing as... C. freundii Table 3.

[0063] Table 3 Clinical Isolation C. freundii Sanger strain sequencing results 2. Genome Draft Sequencing: To extract genomic DNA, the glycerol-preserved bacterial strain was thawed and cultured in 300 mL of nutrient broth (NB) to the logarithmic growth phase. The bacterial pellet was harvested by centrifugation (4200 rpm, 15 min, 4°C), washed with PBS, and then genomic DNA was extracted using SDS lysis and column purification. Sequencing libraries were prepared using the VAHTS® Universal Plus DNA Library Prep Kit (Vazyme, ND627-01 / 02) and sequenced on a NovaSeq 6000 platform (Illumina). High-quality reads were filtered, assembled de novo, and annotated using standard procedures.

[0064] for C. freundii In sequence 2387001, genome assembly produced 40 contigs with a total length of 5,159,576 base pairs, a GC content of 51.66%, an N50 of 957,981 bp, and a maximum contig length of 1,728,195 bp. The average sequencing depth exceeded 400×, indicating high assembly integrity.

[0065] Further sequencing of the genome draft revealed that C. freundiiThe average nucleotide similarity (ANI) between the clinical isolates and the type strain exceeded 98%, supporting their classification as the same species. Figure 3 (C in the middle).

[0066] Paired fresh tumor (T) and adjacent normal tissue (N) samples from 10 pancreatic cancer patients were used for targeted isolation of intratumoral bacterial strains, and 3 clinical isolates were successfully obtained, all from the same patient. C. freundii Strains 2387001, 2387002, and 2387003. No culturable bacteria were isolated from any adjacent non-tumor tissues.

[0067] 3. Pan-genome analysis was performed on three clinical strains and three reference strains obtained from NCBI (ATCC 43864, MSB1_1H, and CCUG 26829) using the IPGA tool: Of the 6418 orthologous gene clusters, 3936 (61.33%) were core genes, while only 1239 (19.31%) were unique genes belonging to different genomes. Figure 3 D and Figure 4 A in the text). Clinical isolates. C. freundii Strains 2387001, 2387002, and 2387003 have 1, 1, and 7 unique genes, respectively. Figure 3 (D in the text). Furthermore, with... C. freundii ATCC 43864 (435) or C. freundii Compared to CCUG 26829 (234 strains), strains in public databases C. freundii MSB1_1H has more unique genes (561) Figure 3 (D in the text). In addition, for clinical isolates... C. freundii The genome of 2387001 was visualized and presented in a genome helix map. Figure 3 The KEGG pathway annotation showed that this clinical isolate lacked several motility-related genes present in the model strain ATCC 43864, such as E). flgD Conversely, this clinical isolate specifically carried... mtaD This gene is involved in sulfur metabolism and amino acid biosynthesis. Figure 3 F in GO-based analysis ( Figure 4 B) further indicates that the clinical isolate is enriched with genes related to cellular components.

[0068] Example 3 C. freundii In vitro inhibition of the antitumor activity of 5-FU: 1. After co-culturing with 5-FU C. freundii Activity assay: 5-FU (0, 16, 32, 64, 128, and 256 μM) was mixed with... C. freundii (0, 2) 10 8 4 10 8 and 8 10 8 CFU / mL was incubated for 48 hours in DMEM or RPMI-1640 medium containing 10% fetal bovine serum. Bacterial growth was assessed by measuring optical density (OD) at 600 nm. 600 ).

[0069] 2. C. freundii Effects on the in vitro antitumor efficacy of 5-FU: By C. freundii (0, 2) 10 8 4 10 8 and 8 10 8 (CFU / mL) was incubated with 5-fluorouracil (0, 16, 32, 64, 128, and 256 μM) in DMEM / RPMI-1640 medium containing 10% serum for 48 hours. After centrifugation (4200 rpm, 15 min) and filtration through a 0.22 μm filter, the supernatant was diluted 4-fold.

[0070] 3. CCK8 assay: Panc-1 or AsPC-1 cells (5 × 10⁶ cells per well) were added to the wells. 3 Cells were seeded into 96-well plates and cultured overnight at 37°C and 5% CO2 to allow them to adhere. The diluted supernatant was then added to Panc-1 or AsPC-1 cell cultures (100 μL / well). After 48 hours of incubation, cell viability was assessed using CCK-8 (10% v / v, incubation 2–3 hours), and absorbance was measured at 450 nm.

[0071] 4. Cell scratch assay: The scratch assay was performed using a 4-well culture plate. Panc-1 and AsPC-1 cells (1×10⁻⁶) were placed in the plate. 5 Cells (cells / well) were seeded into the wells. After 24 hours, the culture plate was removed, and cells treated with 5-FU (0 and 64 μM) were added. C. freundii Diluted supernatant (0, 2 and 4) 10 8 CFU / mL). Images of the cell scratch assay were taken under a microscope at specific time points (Panc-1 cells: 0, 12, and 24 hours; AsPC-1 cells: 0, 6, and 12 hours). The remaining scratch area was measured using ImageJ software, and the scratch closure rate was calculated.

[0072] 5. Cell migration and invasion assays: Cells treated with 5-FU (0, 64, and 128 μM) were used. C. freundii (0 and 4) 10 8 The supernatant (CFU / mL) was prepared. In vitro migration and invasion assays were performed using Transwell chambers. Cells suspended in serum-free medium were mixed with co-culture supernatant containing 0, 16, or 64 μM 5-fluorouracil (3:1 volume ratio).

[0073] For the migration assay, 200 μL of cell suspension (Panc-1: 6 × 10⁻⁶) was used. 5 / hole); AsPC-1: 5 × 10 5 Add the medium to the upper chamber ( / well), and add 800 μL of medium containing 20% ​​fetal bovine serum to the lower chamber. After incubation (Panc-1: 96 hours; AsPC-1: 72 hours), fix the membrane (4% paraformaldehyde), stain with crystal violet, remove unmigrated cells by wiping, and count the migrating cells under a microscope.

[0074] For invasion assays, chambers were pre-coated with matrix gel (Panc-1: 9 × 10⁻⁶ cells) at a higher cell density. 5 / hole, 96 hours; AsPC-1: 8×10 5 / hole, 72 hours).

[0075] Experiments have shown that: C. freundii Growth is not affected by 5-FU ( Figure 5 (B in the text). Individual. C. freundii The supernatant does not alter the viability of Panc-1 and AsPC-1 cells. Figure 5 (C in the middle). C. freundii 5-FU significantly reduced the effect of 5-FU on the growth of Panc-1 and AsPC-1 cells. Figure 5 D in the middle), migration ( Figure 5 EF) and invasion ( Figure 5 The inhibitory effect of G in the middle.

[0076] After treatment with 16 μM 5-FU, exposure to C. freundii Significantly improved the viability of Panc-1 and AsPC-1 cells (from 34.56% to 81.40% and from 45.86% to 80.50%, respectively); OD 600 =0 compared to 0.4; Figure 5 (D in the middle).

[0077] 6. Subcutaneous pancreatic cancer model in BALB / c nude mice (Panc-1 cells) and bacterial intervention: BALB / c nude mice (4 weeks old) were subcutaneously injected with Panc-1 cells (3 × 10⁻¹⁰ cells) one week after acclimatization.6 (The tumor was dissolved in 200 μL PBS). When the tumor grew to about 1 cm, it was removed, cut into small pieces of 1-3 mm, and transplanted into BALB / c nude mice. When the tumor volume approached 100 mm, the tumor was removed. 3 Mice were randomly divided into four groups (n=6 per group). The control group received a tail vein injection of saline followed by intratumoral injection of PBS. The second group received intratumoral injection of PBS suspended in saline. C. freundii The concentration is 4 × 10 8 Mice in the third group received intravenous injections of 5-FU (75 mg / kg) dissolved in saline and intratumoral injections of PBS. Mice in the fourth group received intravenous injections of 5-FU (75 mg / kg) dissolved in saline and intratumoral injections of PBS. 8 CFU / ml C. freundii (50 μL per mouse), administered twice weekly. Treatment continued for three weeks in all groups, with tumor size and body weight monitored three times weekly. On day 22 post-treatment, mice were sacrificed and tumors harvested for further analysis.

[0078] Experiments have shown that intratumoral injection C. freundii The therapeutic effect of 5-FU was reversed. Figure 6 (B) Compared with the 5-FU group, 5-FU+ C. freundii The tumor weight, volume, and growth rate in this group increased by 63%, 39%, and 109%, respectively. Figure 6 (CE in the text).

[0079] It is worth noting that, C. freundii Group combined with 5-FU C. freundii No significant differences were observed between the groups in terms of tumor weight, volume, and growth rate. Figure 6 (CE in the image), indicating exposure to C. freundii It will weaken the anti-tumor efficacy of 5-FU.

[0080] Furthermore, qPCR analysis confirmed that C. freundii In the two intervention groups C. freundii The copy numbers were similar, but all were higher than those of the 5-FU group used alone ( Figure 6 (F in the text). After homogenization of the tumor and subsequent plate culture, the following can be observed: C. freundii Its growth confirms that it is alive. C. freundii It has been colonized within the tumor, including 5-FU combined with C. freundii The colony count in the group was significantly higher than that in the 5-FU group ( Figure 6 (G in the text). Single colonies were subsequently isolated and sequenced, further confirming they were *Citrobacter freundii* strains. Compared to the 5-FU group, the 5-FU combined with... C. freundii Tumor cell shrinkage and necrosis were reduced in the group, Ki-67 expression was increased, and apoptosis was enhanced. Figure 6 (H in the text).

[0081] 7. Orthotopic pancreatic cancer model in germ-free C57BL / 6 mice (KPC-LUC cells) and bacterial intervention: Germ-free C57BL / 6 mice (6-8 weeks old) were housed and maintained in flexible membrane isolators (Nanjing Jicui Pharmaceutical Co., Ltd., China) under a 12-hour light / dark cycle (lights on at 08:00). Animals had free access to autoclaved water and were fed sterile feed irradiated with gamma rays (50 kGy) (Nanjing Xietong Biotechnology Co., Ltd., China), with bedding changed weekly. Routine screening confirmed the absence of bacterial, viral, and fungal contamination. Eight mice were randomly assigned to a control group (n=3) and an experimental group (n=5). The control group received no treatment. The experimental group received in situ pancreatic inoculation with mouse KPC-LUC cells (3 × 10⁶ cells per mouse). 4 Each cell was dissolved in 40 μL of PBS containing matrix gel. During the 4-day post-operative recovery period, mice in the experimental group received the bacterial suspension via gavage (0.6 × 10⁶ cells). 9 CFU / ml (dissolved in 100 μL PBS), the bacterial solution was administered by gavage every two days for two weeks. Fecal bacterial load was monitored weekly. At the end of the experiment, mice were transported to the laboratory overnight under sterile conditions for immediate necropsy. Heart, liver, spleen, lungs, kidneys, pancreas, pancreatic tumor, colon, and cecal contents were collected. The tissues were divided into two aliquots: one for immediate bacterial culture and the other stored at –80°C for subsequent analysis.

[0082] Tumor homogenate inoculation culture and qPCR analysis confirmed bacterial colonization in the gut. Figure 7 (B, D in the text), notably, were detected in tumor tissue. C. freundii ( Figure 7 (C, D in the original text). Colonies of this bacterium were not isolated from other organs (heart, liver, spleen, lungs, and kidneys), indicating that... C. freundii It may exhibit tropism towards or be selectively enriched within the tumor microenvironment. Figure 7 (E in the text).

[0083] Example 4 C. freundii 5-FU is metabolized via PreTA: The pharmacological activity of 5-FU is closely related to dihydropyrimidine dehydrogenase (DPD). Notably, some bacteria carry functional homologs of human DPD, which encode... preTAWithin the operon, they are able to convert 5-FU to DHFU. This intermediate can be further cleaved by bacterial dihydropyrimidine enzyme (DHP) and β-ureapyruvase (BUP) homologs into α-fluoro-β-ureapyruvate (FUPA) and α-fluoro-β-alanine (FBAL). Intratumoral E. coli The mechanism by which induced 5-FU resistance in colorectal malignancies has been revealed.

[0084] Clinical isolates C. freundii Genome mapping analysis of strain 2387001 Figure 3 E in the text clarifies preTA Location of the operon. Comparative genomic analysis showed that both clinical isolates and type strains carried it. preTA .

[0085] Compare C. freundii and E. coli The AlphaFold2 structures of PreT and PreA proteins were obtained to assess their structural homology. preTA The operon consists of the preT and preA genes encoding the PreTA protein. Multiple sequence alignment (MSA) local distance difference test (LDDT) scores were 0.983 and 0.993, respectively, indicating functional similarity of the PreTA protein between the two species. Figure 10 (G in). Furthermore, it was found that... C. freundii PreTA shares some homology with human DPD, with MSALDDT at 0.562 ( Figure 10 (H in the text).

[0086] 1. Quantitative analysis of 5-FU and its bacterial-derived metabolites by non-targeted metabolomics: 5-FU (40 μM) was mixed with... C. freundii (4 10 8The sample (CFU / mL) was incubated in 600 μL of nutrient broth for 24 hours (n=3), with a blank control group included. After incubation, 600 μL of ice-cold acetonitrile was added to terminate the reaction. After vortex mixing, 200 μL of the sample was mixed with an equal volume of acetonitrile and precipitated at -20°C for 1 hour. The sample was then vortexed again, centrifuged (12,500 rpm, 15 min, 4°C), and 100 μL of the supernatant was collected for quantitative analysis using an ultra-high performance liquid chromatography (UPLC) Orbitrap-Exploris-120-MS / MS instrument with a Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm). The mobile phase consisted of NH4HCO3 (6.5 mM) (A) and acetonitrile (B) at a flow rate of 0.3 mL / min. The gradient elution program was set as follows: 0–1 min, 2% B; 1–3 min, 2–60% B; 3–5 min, 60% B; 5–7 min, 60–100% B; 7–8 min, 100% B; 8–8.1 min, 100–2% B; 10 min, 2% B. Samples (injection volume 10 μL) were separated and analyzed at a column temperature of 40°C. Data acquisition was performed using Xcalibur software.

[0087] Mass spectrometry analysis was performed using full scan (resolution 60,000, m / z range 60–900) and data-dependent (MS2) (dd-MS; resolution 15,000) modes. Ion source parameters included: 2500 V spray voltage (negative ion mode), sheath / auxiliary gas of 50 / 10 arb, and transfer tube temperature of 300°C. Full scan mass spectrometry (Full-MS) used a maximum injection time of 100 ms and standard AGC target; dd-MS2 used auto-injection time and stepped HCD collision energies (20%, 40%, 60%). Retention times for 5-FU FBAL were 0.666 min and 0.687 min, respectively. Data processing used MS-DIAL (ppm calculation), Sirius (validation), and MZmine (MS2). 2 Export).

[0088] After co-incubation with wild-type clinical isolates for 24 hours, 5-FU levels decreased significantly by 3.5-fold. m / z =129.0113, ppm=5.66), while the FBAL level increased significantly from undetectable to 9×10 6 ( m / z =106.0316, ppm=5.42), compared with the 0-hour control group ( Figure 8 B, C, D and Figure 9 Dihydropyrimidine dehydrogenase (DPD) plays a dominant role in 80% of the host's 5-FU metabolism.

[0089] Further molecular docking of the wild-type clinical isolate with 5-FU showed a binding energy of -3.74 ( ). Figure 10 (I) In addition, polymerase chain reaction (PCR) analysis showed that, compared with the group that did not receive 5-FU treatment, preT and preA The expression of was significantly upregulated ( Figure 10 (J in the middle), which further confirms C. freundii The strain metabolizes 5-FU via PreTA.

[0090] 2. Quantitative analysis of 5-FU and its bacterial-derived metabolites using targeted metabolomics: 40 μM 5-FU was mixed with 4... 10 8 CFU / mL preT Gene knockout or wild type C. freundii Clinical isolates were co-incubated in 600 μL of nutrient broth (NB) medium. Simultaneously, gimidazolium, enirapex, thymidine, uridine, and uracil (each at a concentration of 40 μM) were added.

[0091] preT Construction of gene knockout strains: A 20bp spacer sequence (5′-TGAGCCGTAAGACATACCTG-3′) was designed using a CRISPR / Cas9-mediated genome editing protocol to target... preT The gene was extracted and inserted into the pTargetF plasmid using primers pTpreT1-F / pTpreT1-R. C. freundii Using the reference genome of 2387001 as a template, primers pTpreT-F / pTpreT-R and pTpreT3-F / pTpreT3-R were used to amplify the genome. preT Homologous arms of approximately 500 bp flanking the coding sequence were identified. These fragments were fused using overlap extension PCR and cloned into pTargetF, which had been digested with SpeI / SalI, using a seamless cloning technique. The resulting recombinant vector was named pTargetF-preT and amplified in E. coli DH5α after sequencing verification.

[0092] By culturing in LB medium C. freundii 2387001 to OD 600 =0.8, then centrifuged and washed three times with 10% glycerol in an ice bath to prepare electrocompetent states. C. freundii 2387001. pCas plasmid was introduced into competent cells via electroporation, and transformants were screened on LB agar containing kanamycin. Transformants were selected when the culture reached OD... 600At a concentration of 0.3, 30 mM L-arabinose was added to induce the expression of the red recombinase. Subsequently, pTargetF-preT was electroporated into competent cells expressing the recombinase. Colonies resistant to both spectinomycin and kanamycin were screened by PCR to identify candidate strains for successful recombination.

[0093] To remove the edited plasmid, cells were first cultured in LB medium supplemented with 1 mM IPTG to remove the pTargetF-preT plasmid—IPTG can induce sgRNA-mediated cleavage and degradation of the pTargetF plasmid; then cells were cultured at 42°C to remove the pCas plasmid, as the replication origin of this plasmid is temperature sensitive.

[0094] Colonies that could not grow on the corresponding antibiotic selection plates were further verified by PCR and Sange sequencing, and the resulting strains were named... C. freundii ΔpreT 2387001.

[0095] In all the above experiments, the incubation time points were set at 0 hours, 6 hours, and 24 hours (n=3), and a blank control group was included. At the end of each incubation, 600 μL of ice-cold acetonitrile was added to terminate the reaction. After vortexing, 200 μL of each sample was taken, and an equal volume of acetonitrile and 10 μL of the internal standard 5-chlorouracil were added. After vortexing, the sample was centrifuged at 12,500 rpm for 10 minutes at 4 °C. The supernatant (100 μL) was collected, diluted with 4 times its volume of pure water, vortexed, and centrifuged again. The resulting supernatant was collected for subsequent analysis.

[0096] Metabolic profiling and cytotoxicity assays showed that, compared to the wild-type strain, preT Gene knockout strains cannot degrade 5-FU ( Figure 8 The F in the text does not weaken its cytotoxic effect on pancreatic cancer cells (F). Figure 8 (G in the middle).

[0097] The experiment revealed significant differences in the metabolic profiles of the different groups, with adenosine, inosine, and guanosine consistently being abundant. Figure 10 (AB in the text). These nucleosides did not alter the cytotoxicity of 5-FU in vitro. Figure 10 (CF in the middle).

[0098] Gim and Enilaser significantly inhibited C. freundii Strain-mediated 5-FU metabolism ( Figure 11 (A) Gim was selected for further cell efficacy experiments. C. freundii Strains (0, 2 10 8 4 10 8 and 8 10 8 Incubate Panc-1 or AsPC-1 cells (5 × 10³ cells / well) with 5-fluorouracil (0, 16, 32, 64, 128, and 256 μM) and Gim (1:1 molar ratio with 5-FU) in DMEM / RPMI-1640 medium containing 10% serum for 48 hours to prepare a conditioned supernatant. Centrifuge (4200 rpm, 15 min) and filter through a 0.22 μm filter, then dilute the supernatant 4-fold. Seed Panc-1 or AsPC-1 cells (5 × 10³ cells per well) into 96-well plates and incubate overnight at 37°C and 5% CO₂ to allow them to adhere. Then add the diluted supernatant to the Panc-1 or AsPC-1 cell culture (100 μL / well). After incubation for 48 hours, assess cell viability using CCK-8 (10% v / v, incubation 2–3 h) and measure absorbance at 450 nm.

[0099] Gim was found to significantly reverse C. freundii Mediated 5-FU metabolism ( Figure 11 (B in the text). This verified the effect of Gim on the antitumor activity of 5-FU ( Figure 11 (C) Under aseptic conditions, Gim does not interfere with the efficacy of 5-FU (in the context of C). Figure 11 (D in the text), and regardless of the presence or absence of bacteria, it has no cytotoxic effect on Panc-1 or AsPC-1 cells (D). Figure 11 In addition, bacterial culture supernatant alone does not affect cell viability (E). Figure 11 The F in the formula). CCK-8 assay results confirmed that Gim retained the cytotoxicity of 5-FU in Panc-1 and AsPC-1 cells (F). Figure 11 (G in the middle).

[0100] Example 6: Extended verification of the inhibitory effect of Gim on PreTA: Compare C. freundii and E. coli The AlphaFold2 structures of PreT and PreA proteins were determined to assess their structural homology. preTA Manipulator preT and preA The genes encode the PreTA protein. The multiple sequence alignment (MSA) local distance difference test (LDDT) score for PreT is 0.983, while that for PreA is 0.993, indicating that the PreTA protein has functional similarity between the two species.

[0101] Twenty-five candidate inhibitors were screened from a drug and food homology database and FDA-approved compounds. The screening criteria included solubility in methanol and no obvious toxic side effects. The verification method was the same as in Example 4.

[0102] Of the compounds screened, only Marmesin, Licochalcone B, Nordihydroguaiaretic acid, and Flavin mononucleotide inhibited the metabolism of 5-FU in *E. coli*. The inhibition rates of Marmesin, Licochalcone B, Nordihydroguaiaretic acid, and Flavin mononucleotide on 5-FU metabolism in *E. coli* were approximately 29.7%, 47.1%, 44.5%, and 48.6%, respectively, but all were lower than the inhibition of PreT and PreA proteins in *E. coli* by Gimeracil. Figure 12 ).

[0103] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0104] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of an inhibitor of PreTA for the manufacture of a medicament for the prevention and / or treatment of pancreatic cancer, characterized in that, The inhibitors include gemcitabine, eniluracil, thymidine, uridine and / or uracil.

2. Use according to claim 1, characterized in that, The inhibitors are gemcitabine.

3. Use according to claim 1, characterized in that, The pancreatic cancer is resistant to fluoropyrimidines.

4. Use according to claim 3, characterized in that, The fluoropyrimidines include fluorouracil, capecitabine, tegafur, tegafur / gimeracil / oteracil, doxifluridine, tegafur / uracil and / or turofexuridine.

5. Use according to claim 4, characterized in that, The fluoropyrimidines are fluorouracil.

6. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises: (1) a therapeutically effective amount of an inhibitor of PreTA; (2) a pharmaceutically or immunologically acceptable carrier or excipient.

7. The pharmaceutical composition of claim 6, wherein, The pharmaceutical composition further comprises a fluoropyrimidine.

8. A pharmaceutical preparation, characterized by, The pharmaceutical composition of claim 6 or 7.

9. A pharmaceutical product, characterized in that, The pharmaceutical composition of claim 6 or 7 and / or the pharmaceutical preparation of claim 8.

10. Use of the pharmaceutical composition of claim 6 or 7, the pharmaceutical preparation of claim 8 and / or the pharmaceutical product of claim 9 in the preparation of a medicament for preventing and / or treating pancreatic cancer.

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