N-oleoylethanolamine and e. rectale for the treatment of diarrhea-predominant irritable bowel syndrome

Abstract

N-oleoylethanolamine (OEA) or a microbiota producing OEA, such as Eubacterium rectale (ATCC 33656) Eubacterium rectale for use in the treatment of irritable bowel syndrome (IBS).

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 562,293, filed March 7, 2024, which is incorporated herein by reference in its entirety. References to sequence lists

[0002] The sequence list, identified as Sequence_Listing_P25963PCT.xml, which is 15,122 bytes in size and created on February 26, 2025, and submitted with this document, is incorporated into this document in its entirety by reference. Technical Field

[0003] This disclosure relates to N-oleoylethanolamide (OEA) or the microbial community that produces OEA (such as Escherichia coli). Eubacterium rectale Used to treat irritable bowel syndrome (IBS). Background Technology

[0004] Diarrhea-predominant irritable bowel syndrome (IBS-D) is a gastrointestinal disorder characterized by frequent diarrhea, abdominal pain, and bloating. Conventional medications for IBS-D can relieve symptoms of diarrhea and abdominal pain, but are not effective in treating the condition itself. One physiological factor of interest in the treatment of IBS-D is the regulation of peripheral serotonin (5-hydroxytryptamine, 5-HT), as peripheral 5-HT is involved in regulating intestinal motility, fluid secretion, and visceral sensitivity. Disorders of peripheral 5-HT signaling can lead to dysfunction of intestinal motility and visceral sensitivity. Developing novel therapies to regulate 5-HT signaling (e.g., the regulation of peripheral 5-HT levels discussed in this paper) is of great significance for the treatment of IBS-D.

[0005] Approximately 90% of peripheral serotonin is produced by enterochromaffin (EC) cells in the gut. One of the factors regulating EC cell production of peripheral serotonin is the gut microbiota. Recent findings and our previous research have shown that the gut microbiota participates in the pathogenesis of IBS-D by inducing diarrhea-like phenotypes through stimulating serotonin production, suggesting the great potential of modulating the gut microbiota in regulating serotonin levels and treating IBS-D. Although our previous studies identified a series of gut microbial metabolites that induce diarrhea-like phenotypes by stimulating peripheral serotonin biosynthesis, it remains unclear whether there are gut microbial metabolites that can inhibit serotonin production and improve diarrhea-like phenotypes by inhibiting peripheral serotonin signaling. Summary of the Invention

[0006] In a first aspect, this disclosure provides a method for treating irritable bowel syndrome (IBS) in a subject of need, the method comprising: administering a therapeutically effective amount of N-oleoylethanolamine (OEA) to the subject.

[0007] In some implementations, IBS is the diarrheal subtype (IBS-D).

[0008] In some implementations, the OEA is separate and essentially pure.

[0009] In some implementations, the amount of OEA administered is sufficient to prolong gastrointestinal transit time, reduce fecal water content, reduce defecation frequency, reduce serum serotonin concentration, or a combination thereof, in subjects.

[0010] In some implementations, the subject has a lower serum OEA concentration compared to the average serum OEA concentration of healthy subjects who do not have IBS.

[0011] In some embodiments, the method further includes providing a sample obtained from the subject, wherein the sample is a stool sample or a serum sample; determining the amount of one or more biomarkers selected from the group consisting of OEA or serotonin in the sample; and determining that the subject has IBS based on the amount of the one or more biomarkers in the sample prior to the step of administering OEA to the subject.

[0012] In some implementations, the subjects are humans, non-human primates, rodents, canines, felines, bovines, or equines.

[0013] In some implementations, the subjects are humans.

[0014] In some implementations, the OEA is administered orally.

[0015] In a second aspect, this article provides a method for treating irritable bowel syndrome (IBS) in a subject in need, the method comprising administering to the subject a therapeutically effective amount of N-oleoylethanolamine (OEA)-producing bacteria.

[0016] In some implementations, IBS is the diarrheal subtype (IBS-D).

[0017] In some embodiments, the bacteria are *E. rectum* (…). Eubacterium rectale ).

[0018] In some implementations, the amount of intestinal bacteria administered is sufficient to prolong gastrointestinal transit time, reduce fecal water content, reduce defecation frequency, reduce serum serotonin concentration, or a combination thereof, in the subject.

[0019] In some implementations, the subject has a lower serum OEA concentration compared to the average serum OEA concentration of healthy subjects who do not have IBS.

[0020] In some embodiments, the method further includes providing a sample obtained from the subject, wherein the sample is a fecal sample or a serum sample; determining the amount of one or more markers selected from the group consisting of OEA or serotonin in the sample; and determining that the subject has IBS based on the amount of the one or more markers in the sample prior to the step of administering the bacteria to the subject.

[0021] In some implementations, the bacteria are administered orally or via fecal microbiota transplantation.

[0022] In some implementations, the subjects are humans, non-human primates, rodents, canines, felines, bovines, or equines.

[0023] In some implementations, the subjects are humans. Brief description of the attached diagram

[0024] The above and other objects and features of this disclosure will become apparent from the following description of the disclosure, taken in conjunction with the accompanying drawings.

[0025] Figure 1 The results showed that OEA levels were significantly reduced in serum and fecal samples from both germ-free and normal mice. Data (wild-type mice n=3, germ-free mice n=6) are presented as mean values. + SEM was used to represent the results. Significant differences were analyzed using a t-test.

[0026] Figure 2 The results showed that serotonin levels were significantly reduced in fecal samples from both germ-free and normal mice. Data (wild-type mice n=3, germ-free mice n=6) are presented as mean values.+ SEM was used to represent the results. Significant differences were analyzed using a t-test. A significance test was performed between the wild-type group and the sterile group. p Value comparison.

[0027] Figure 3 The results showed that serum OEA levels were decreased in IBS-D patients and were negatively correlated with serum serotonin, diarrhea, and abdominal pain. (A) Decreased serum OEA in IBS-D patients. (BD) Nonparametric Spearman correlation analysis (n=94) between serotonin, abdominal pain, Bristol score, and OEA levels in healthy controls and IBS-D patients. HC: Healthy controls; r: Correlation coefficient; p p-value.

[0028] Figure 4 The results showed that physiological doses of OEA treatment improved the diarrhea-like phenotype in TNBS-treated mice. (AC) Fecal water content, GI transit time, and defecation frequency in mice treated with or without OEA. Data (n=6) are presented as mean values. + SEM was used to represent the results. Significant differences were analyzed using one-way ANOVA. Significance was assessed among the control group, model group, and different OEA treatment groups. p Value comparison.

[0029] Figure 5 The results showed that in TNBS-treated mice, treatment with physiological doses of OEA alleviated the abdominal pain-like phenotype. (AE) Visceral motor and electromyographic responses to colorectal distension with or without OEA treatment. Data (n=6) are presented as mean. + SEM was used to represent the results. Significant differences were analyzed using one-way ANOVA. Significance was assessed among the control group, model group, and different OEA treatment groups. p Value comparison.

[0030] Figure 6 The results showed that physiological doses of OEA treatment suppressed serotonin levels in serum and ileum in TNBS-treated mice. (AB) Serum and ileum serotonin concentrations in TNBS-treated mice. Data (n=6) are presented as mean values. + SEM was used to represent the results. Significant differences were analyzed using one-way ANOVA. Significance was assessed among the control group, model group, and different OEA treatment groups. p Value comparison.

[0031] Figure 7The results showed that treatment with physiological doses of OEA suppressed serotonin levels in QGP1 cells in a dose-dependent manner. (AB) QGP1 cells were treated with OEA at concentrations of 1 μM, 5 μM, and 10 μM for 4 h and 24 h. Data (n=3) are presented as mean. + SEM was used to represent the results. Significant differences were analyzed using one-way ANOVA. Significance was assessed among the control group, model group, and different OEA treatment groups. p Value comparison.

[0032] Figure 8 The results showed that in TNBS-treated mice, OEA treatment regulated serotonin biosynthesis and reuptake genes (including...). Tph1 and Sert mRNA expression of ). Data (n=6) are presented as average. + SEM was used to represent the results. Significant differences were analyzed using one-way ANOVA. Significance was assessed among the control group, model group, and different OEA treatment groups. p Value comparison.

[0033] Figure 9 The results showed that OEA treatment regulated serotonin biosynthesis and reuptake genes (including) in QGP1 cells. TPH1 and SERT mRNA expression of ). Data (n=3) are presented as average. + SEM was used to represent the results. Significant differences were analyzed using one-way ANOVA. Significance was assessed between the control group and the treatment groups with different concentrations of OEA. p Value comparison.

[0034] Figure 10 The results showed that *E. rectum* colonization improved the diarrhea-like phenotype in TNBS-treated mice. (AC) Fecal water content, GI transit time, and defecation frequency in TNBS-treated mice with or without *E. rectum* colonization. Data (n=6) are presented as mean values. + SEM was used to represent the results. Significant differences were analyzed using one-way ANOVA. Significance was assessed among the control group, model group, and *E. rectum* colonization group. p Value comparison.

[0035] Figure 11 The results showed that colonization of *E. rectum* improved the abdominal pain-like phenotype in TNBS-treated mice. (AE) Visceral motor and electromyographic responses to colorectal distension in the presence or absence of *E. rectum* colonization. Data (n=6) are presented as mean. + SEM was used to represent the results. Significant differences were analyzed using one-way ANOVA. Significance was assessed among the control group, model group, and *E. rectum* colonization group. p Value comparison.

[0036] Figure 12 The colonization of *E. rectum* increased OEA levels in TNBS-treated mice. (AC) OEA levels in fecal samples and colonic tissue from TNBS-treated mice. Data (n=6) are presented as mean. + SEM was used to represent the results. Significant differences were analyzed using one-way ANOVA. Significance was assessed among the control group, model group, and *E. rectum* colonization group. p Value comparison.

[0037] Figure 13 The results show that *E. rectum* suppresses serum serotonin levels in the TNBS mouse model. Data (n=6) are presented as mean values. + SEM was used to represent the results. Significant differences were analyzed using one-way ANOVA. Significance was assessed among the control group, model group, and *E. rectum* colonization group. p Value comparison.

[0038] Figure 14 Showing Escherichia coli ( E. coli ) ereT+ and E. coli 载体 Concentration of OEA produced. (AB) In vitro OEA levels with or without IPTG induction. Data are expressed as mean. + SEM was used to represent the results. Significant differences were analyzed using a t-test. In *E. coli* ereT+ colonization group and Escherichia coli 载体 Significance between planting groups p Value comparison.

[0039] Figure 15 The results showed that in mice treated with TNBS, Escherichia coli ereT+ The colonization increased OEA levels. Data (n=8) are presented in mean... + SEM was used to represent the results. Significant differences were analyzed using one-way ANOVA. This was performed in the control group, model group, and *E. coli* group. ereT+ and E. coli 载体 Significance between planting groups p Value comparison.

[0040] Figure 16 The results showed that in mice treated with TNBS, Escherichia coli ereT+ Colonization improved the diarrhea-like phenotype. (AC) in the presence or absence of Escherichia coli ereT+ Fecal water content, GI transport time, and defecation frequency in TNBS-treated mice under colonization conditions. Data (n=8) are presented as mean + SEM. Significant differences were analyzed using one-way ANOVA. (Comparison was conducted in the control group, model group, and E. coli group.) ereT+ and E. coli载体 Significance between planting groups p Value comparison. Detailed Implementation

[0041] definition Throughout this specification, unless the context otherwise requires, the word “comprise” or variations such as “comprises” or “comprising” should be understood to imply inclusion of the stated integer or group of integers, but not to exclude any other integer or group of integers. It should also be noted that in this disclosure, particularly in the claims and / or paragraphs, terms such as “comprises,” “computed,” or “comprising” may have the meanings conferred upon them under U.S. patent law; for example, they may mean “includes,” “included,” or “including,” etc.; and terms such as “consisting essentially of” and “consists essentially of” have the meanings conferred upon them under U.S. patent law, for example, they allow for elements not explicitly stated, but exclude elements found in the prior art or affecting the essential or novel features of the invention.

[0042] Furthermore, throughout this specification and claims, unless the context otherwise requires, the word "include" or variations such as "includes" or "including" should be understood to imply inclusion of the said integer or group of integers, but not to exclude any other integer or group of integers.

[0043] As used herein, the terms “treat,” “treating,” “treatment,” etc., refer to reducing or improving a condition / disease and / or its associated symptoms. It should be understood that treating a disease or condition does not require the complete elimination of the disease, condition, or its associated symptoms, but this is not excluded. In some implementations, treatment includes preventing a condition or condition and / or its associated symptoms. As used herein, the term “prevention” refers to any action that inhibits or at least delays the development of a condition, condition, or its associated symptoms. Prevention can include primary, secondary, and tertiary levels of prevention, wherein: a) primary prevention avoids the development of disease; b) secondary prevention activities aim to treat the disease early, thereby increasing the opportunity for intervention to prevent disease progression and the onset of symptoms; and c) tertiary prevention reduces the negative effects of an identified disease by restoring function and reducing disease-related complications.

[0044] As used herein, the term "subject" refers to an animal, typically a mammal or a human, that will be or has been the subject of treatment, observation, and / or experimentation. When this term is used in conjunction with the administration of the compounds described herein, then the subject becomes the object of treatment, observation, and / or administration of the compounds described herein.

[0045] As used herein, the term "therapeutic effective amount" refers to the amount of a compound or agent that, as sought by researchers, veterinarians, clinicians, or physicians, induces a biological and / or medical response (including relief of symptoms of the disease, condition, or symptom being treated) in cell cultures, tissue systems, subjects, animals, or humans.

[0046] As used herein, the term “isolated” in relation to the compounds described herein means that the compound is separated from other components of natural origin (such as plants or cells) or synthetic organic reaction mixtures, for example, by conventional techniques.

[0047] As used herein, the term "substantially pure" in relation to a sample of the compound described herein means that the sample contains at least 60% by weight of the compound. In some embodiments, the sample contains at least 70% by weight of the compound; at least 75% by weight of the compound; at least 80% by weight of the compound; at least 85% by weight of the compound; at least 90% by weight of the compound; at least 95% by weight of the compound; or at least 98% by weight of the compound.

[0048] Unless otherwise expressly stated, the use of the singular in this document includes the plural (and vice versa). Furthermore, when the term “about” is used before a quantitative value, this teaching also includes the specific quantitative value itself unless otherwise expressly stated. As used herein, unless otherwise specified or inferred, the term “about” means a variation of ±10%, ±7%, ±5%, ±3%, ±1%, or ±0% from the nominal value.

[0049] This article provides a method for treating IBS in a subject in need, the method comprising: administering a therapeutically effective amount of OEA or OEA-producing bacteria to the subject.

[0050] IBS can be classified into four basic subtypes: diarrhea-predominant subtype (IBS-D), constipation-predominant subtype (IBS-C), mixed bowel subtype (IBS-M), and unclassified subtype (IBS-U). The IBS can be any subtype. In some implementations, the IBS is the diarrhea-predominant subtype (IBS-D).

[0051] When OEA is administered to a subject, the OEA may be isolated and / or the OEA may be substantially pure.

[0052] The bacteria that produce OEA can be naturally occurring bacteria or recombinant bacteria. In some embodiments, the bacteria that produce OEA are *E. rectum*. In some embodiments, the recombinant bacteria that produce OEA contain recombinant bacteria. ereS , ereT , ereA and ereC Gene.

[0053] Administering subjects with sufficient amounts of OEA or OEA-producing bacteria can prolong gastrointestinal transit time, reduce fecal water content, decrease defecation frequency, reduce serum serotonin concentration, or achieve a combination of the above effects.

[0054] Following administration of OEA or OEA-producing bacteria, the gastrointestinal transit time of the subjects may be prolonged by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. Following administration of OEA or OEA-producing bacteria to the subjects, the gastrointestinal transit time of the subjects may be prolonged by 5-100%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, or 5-10%. In some implementations, gastrointestinal transit time was prolonged by 36.6% and 53.2% after administration of OEA at doses of 50 mg / kg and 100 mg / kg, respectively, and by 79.5% after administration of OEA-producing bacteria to the subjects.

[0055] Following administration of OEA or OEA-producing bacteria to subjects, the fecal water content of subjects may decrease by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. Following administration of OEA or OEA-producing bacteria, the fecal water content of subjects may decrease by 5-100%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, or 5-10%. In some implementations, fecal water content decreased by 3.2% and 2.3% after administration of OEA at doses of 50 mg / kg and 100 mg / kg, respectively, and by 6.4% after administration of OEA-producing bacteria to the subjects.

[0056] Following administration of OEA or OEA-producing bacteria to subjects, the frequency of bowel movements in subjects may decrease by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. Following administration of OEA or OEA-producing bacteria to subjects, the frequency of bowel movements in subjects may decrease by 5-100%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, or 5-10%. In some implementations, after administration of OEA at doses of 50 mg / kg and 100 mg / kg, the frequency of defecation in subjects decreased by 27.7% and 26.1%, respectively, and by 30.0% after administration of OEA-producing bacteria to subjects.

[0057] Following administration of OEA or OEA-producing bacteria to a subject, the subject's serum serotonin concentration may decrease by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. Following administration of OEA or OEA-producing bacteria to a subject, the subject's serum serotonin concentration may decrease by 5-100%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, or 5-10%. In some implementations, serotonin concentrations in the serum of subjects were reduced by 17.6% and 13.4% after administration of OEA at doses of 50 mg / kg and 100 mg / kg, respectively, and by 11.1% after administration of OEA-producing bacteria to the subjects.

[0058] In some implementations, the subject has a lower serum OEA concentration compared to the average serum OEA concentration of healthy subjects who do not have IBS.

[0059] In some embodiments, serum or stool samples obtained from said subjects contain lower amounts of one or more biomarkers selected from OEA and serotonin compared to the average amount of one or more biomarkers in stool and / or serum samples obtained from healthy controls (e.g., subjects without IBS).

[0060] In some embodiments, the method further includes providing a sample obtained from the subject, wherein the sample is a fecal sample or a serum sample; determining the amount of one or more markers selected from the group consisting of OEA and serotonin in the sample; and determining that the subject has IBS based on the amount of the one or more markers in the fecal sample prior to the step of administering OEA or OEA-producing bacteria to the subject.

[0061] The step of determining that a subject has IBS based on the amount of one or more biomarkers in the sample may include: comparing the measurement of one or more biomarkers in the sample with the average amount of one or more biomarkers in a sample obtained from a healthy control (e.g., a subject who does not have IBS).

[0062] One or more biomarkers may be measured using any method known to those skilled in the art. In some embodiments, the steps of measuring one or more biomarkers may involve using one or more analytical tools, such as high-performance liquid chromatography (LC), ultra-high-performance liquid chromatography (UPLC), liquid chromatography-mass spectrometry (LCMS), liquid chromatography-tandem mass spectrometry (LCMS / MS), microscopy, Gram staining, examination of bacterial morphology, nucleic acid analysis (e.g., using restriction enzymes, hybridization, polymerase chain reaction (PCR) amplification and / or sequencing), screening for nutritional requirements and / or antimicrobial susceptibility of cultures, analysis of fatty acid distribution and / or testing of antigens, or combinations thereof.

[0063] For therapeutic use of OEA or OEA-producing bacteria, the mode of administration may be any suitable route of delivery of the agent to the host, such as parenteral administration, for example, intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary administration; transmucosal administration (oral, intranasal, vaginal, rectal); formulations in the form of tablets, capsules, solutions, suspensions, powders, gels, or granules; and contained in syringes, implantable devices, osmotic pumps, cartridges, micropumps; or other means known in the art to those skilled in the art. Site-specific administration can be achieved via, for example, intra-articular, intrabronchial, intra-abdominal, intra-cystic, intra-cartilaginous, intracavitary, intra-body cavity, intracerebellum, intravenous, intracolonic, intracervical, intragastric, intrahepatic, intracardiac, intraosseous, intrapelvic, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intrabladder, intralesional, vaginal, rectal, fecal microbiota transplantation, buccal, sublingual, intranasal, or percutaneous delivery. In some embodiments, OEA is administered orally. In some embodiments, OEA-producing bacteria are administered orally or via fecal microbiota transplantation.

[0064] Subjects typically refer to humans, but can also include other animals, such as non-human primates, canines, equines, felines, sheep, pigs, rodents, etc. In some implementations, subjects are humans.

[0065] Materials and methods 1. Reagents 2,4,6-Trinitrobenzenesulfonic acid (TNBS, Sigma, catalog number p2297), ethanol (Honeywell, catalog number 32221-2.5L), isoflurane (Piramal, lot number N0791K11), N-oleoylethanolamine (OEA, CHENGDU MUST BIO-TECHNOLOGY, CAS.111-58-0), and Enterobacter rectosa (catalog number 33656) were purchased from ATCC. Carmine (Macklin, catalog number C805213, CAS. 1390-65-4), Sodium Carboxymethyl Cellulose (Chem Scene, catalog number CS-0015715, CAS. 9004-32-4), PrimeScript RT Master Mix (Takara, catalog number RR036A), SYBR Premix Ex TaqII (Thermo Fisher Scientific, catalog number A46109), RPMI Medium 1640 Powder (gibco, catalog number 31800-022), pET-22b (miaoling, P0017), pET28a (miaoling, P0023), BL21(DE3) (miaoling, T0041), pCDFDuet-1 (miaoling, P0052), pCOLADuet-1 (miaoling, P0430), M9 Minimal Salt Medium (Sigma, M6030), MgSO4 (Sigma, 230391-25g), TransTaq high-fidelity DNA polymerase (HiFi) (Transgene, AP131-02), NdeI (NEB, R0111S), XhoI (NEB, R0146M), EcoRI (NEB, R0101S), SacI-HF (NEB, R3156S), LB Broth Base (Invitrogen, 12795-027), LB agar (Invitrogen, 22700-025), ampicillin (MCE, HY-B0522), streptomycin (Beyotime, ST2717-25g), kanamycin (MCE, HY-16566), Escherichia coli BAP1 (Biobw, Bio-114617), isopropyl-β-d-thiogalactopyranoside (IPTG, Sigma, I6758), sodium oleate (MCE, HY-N1446B), DH5α competent cells (TIANGEN, CB101-01), 2X Phusion Master Mix (NEB, M0531L).

[0066] 2. Methods 2.1 Animals Male C57 / 6J mice (6-8 weeks old) were purchased from Gem Pharmatech Company (Jiangsu Province, China). Mice were housed in animal units at room temperature under a 12-hour light / 12-hour dark cycle, with free access to standard rodent diet and water. To induce the PI-IBS model, C57BL / 6J mice were fasted overnight but allowed access to water containing 5% glucose. After weighing and anesthetizing the mice, 100 μL of TNBS solution containing 30% EtOH was slowly injected into the colon using a 1-mL syringe and a 3.5-F catheter. The catheter was then removed, and the mouse was placed head-down for 5 minutes. Subsequently, the catheter was gently removed from the colon, and the mouse was held head-down in a vertical position for 5 minutes. A control group was treated with saline. Mice were weighed on days 1-5, 14, and 21 after TNBS administration.

[0067] For OEA processing All PI-IBS mice were randomly divided into four groups for 19 days: 1) Control group: mice (n=6) were administered 300 μL of control medium and 0.5% CMC-Na solution; 2) Model group: mice (n=6) were administered 300 μL of control medium and 0.5% CMC-Na solution; 3) Low-dose OEA group: mice (n=6) were administered 300 μL of OEA (50 mg / kg) dissolved in 0.5% CMC-Na solution; 4) High-dose OEA group: mice (n=6) were administered 300 μL of OEA (100 mg / kg) dissolved in 0.5% CMC-Na solution.

[0068] Gastrointestinal (GI) transit, fecal water content, and defecation frequency were measured on day 21 after administration of TNBS solution, and on days 7 and 14 after treatment, to determine whether OEA administration affected diarrhea-like symptoms in an IBS-D-like mouse model.

[0069] Gastrointestinal (GI) transport Mice were placed separately in sterile cages and observed using a 6% carmine solution (containing 0.5% methylcellulose, w / v). At the start of the experiment, each mouse that had not been fasted beforehand was administered 300 μL of carmine solution by gavage at 9:00 AM. The time from gavage to the first appearance of carmine in the feces was recorded as the GI transport time for that mouse.

[0070] Assessment of defecation frequency and fecal moisture content Mice were placed separately in sterile cages, and the cumulative number of fecal particles for each mouse over 120 minutes was recorded as the corresponding number of defecations. Fresh fecal particles were collected from each mouse immediately after defecation. The wet and dry weights of each fecal particle were recorded before and after oven drying (at 105°C for 24 h). The fecal moisture content (%) was calculated using the following formula: .

[0071] Colorectal dilatation (visceral sensitivity) Visceral hypersensitivity scores were measured on day 8 to investigate the analgesic effect of OEA in an IBS-D mouse model. Mice were anesthetized (1% isoflurane), the transmitter was inserted into the peritoneum, and the electrode was passed through the abdominal wall. Colonic and rectal dilatation was measured as abdominal spasm induced by a dilatation catheter (volume 30 to 90 μL, Fogarty catheter, 4F, used for arterial embolization). The EMG response to rectal dilatation was assessed by dividing the area under the curve (AUC) of electromyography (EMG) during 20 s dilatation by the AUC of EMG recorded at baseline 20 s before each dilatation and multiplying by 100, as shown in the formula below: .

[0072] Data were calculated at expansion pressures of 30, 60, and 90 μL.

[0073] 2.2 In vitro studies QGP-1 cells (human-derived) were grown in RPMI-1640 culture medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin). Cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. When 80% confluence was reached, cells were cultured at a rate of 5 x 10⁻⁶ cells / mL. 5 Cells were seeded at a density of / wells into 12-well plates. After 24 h, cells were incubated with different concentrations (1 μM, 5 μM, and 10 μM) of OEA for 4 h and 24 h to determine serotonin production in the culture medium.

[0074] 2.3 Bacterial Research Select bacteria containing four biosynthetic genes from E. rectum. ereS , ereT , ereA and ereC The *Ere* bacterial gene cluster is used for the heterologous production of OEA. The enzymes encoded by the gene cluster are annotated as follows: ereS Sterol carrier protein-2 sterol transfer family protein; ereT Acyl carrier protein; ereA : Long-chain acyl-CoA synthase (AMP formation); and ereC Nonribosomal peptide synthase.

[0075] Using primer pair 5'-GGC GAATTC GATGAAAACCCGCAAAGGCCA-3' (SEQ ID NO: 17) and 5'-C GAGCTC TTACACCAGTTTGATAATTT-3' (SEQ ID NO: 18) ereC The gene was amplified by PCR. The PCR product was purified using a DNA gel purification kit and then... EcoRI and SacI ( EcoRI and SacI Restriction sites (underlined) were digested. The digestion product was purified using a DNA gel purification kit and ligated to the pCDFduet-1 cloning site via T4 DNA ligation to generate pCDFduet-1 overnight at 16°C. ereC Add the ligation product to DH5α competent cells and place the mixture on ice for 30 minutes. Heat shock at 42°C for 90 seconds and place the mixture on ice for 2 minutes. Add 900 µl of LB medium to the test tube. Incubate the test tube at 37°C for 60 minutes. Shake vigorously (240 rpm). Heat the selection plate (streptomycin, 50 ng / ml) to 37°C. Centrifuge the DH5α competent cells and spread them onto the selection plate, then incubate overnight at 37°C. Pick single colonies into LB medium containing 50 ng / ml streptomycin. Extract pCDFduet- using the TIANprep Mini Plasmid Kit (TIANGEN). ere C plasmid. After digestion, the plasmid was validated by Sanger sequencing.

[0076] Using primer pair 5'-GGAATTC CATATG CTGTTTCATACCATTCC-3' (SEQ ID NO: 19) and 5'-CCG CTCGAG TTAGCTGCGTTTGATTTTAC-3' (SEQ ID NO: 20) ereA Genes were amplified by PCR. Using... NdeI and XhoI ( NdeI and XhoI Restriction sites are shown underlined. The PCR product was digested and cloned into pCDFDuet- via T4 DNA ligation. ereC In this process, pCDFDuet- is generated overnight at 16°C. ereC - ereA The ligation product was added to DH5α competent cells to generate pCDFDuet- ereC - ereA The plasmid was validated using Sanger sequencing.

[0077] Using primer pair 5'-GGCG AATTC GATGACCTATGCCGATATGTT-3' (SEQ ID NO: 21) and 5'-C GAGCTC TTAGGCTTTTTTGCTATCGA-3' (SEQ ID NO: 22) ereS Genes were amplified by PCR. Using... EcoRI and SacI ( EcoRI and SacI (Restriction sites are underlined) The PCR product was digested overnight at 37°C. The digested product was purified using a DNA gel purification kit and ligated to the pETDuet-1 cloning site using T4 DNA ligase to generate pETDuet-1 overnight at 16°C. ereS The ligation product was added to DH5α competent cells to generate pETDuet- ereS The plasmid was validated using Sanger sequencing.

[0078] Using primer pair 5'-GGAATTC CATATG TTCGATGAACTGGTGGA-3' (SEQ ID NO: 23) and 5'-CCG CTCGAG TTAGGCATCTTTCAGGGTTT-3' (SEQ ID NO: 24) pair ereT Genes were amplified by PCR. Using... NdeI and XhoI ( NdeI and XhoI Restriction sites are underlined) digested PCR products and cloned into pETDuet via T4 DNA ligation. ereS In this process, pETDuet- is generated overnight at 16°C. ereS - ereT The ligation product was added to DH5α competent cells to generate pETDuet- ereS - ereT The plasmid was validated using Sanger sequencing.

[0079] Inoculate 10 ml LB with a single *E. coli* BAP1 colony. Incubate the culture overnight at 37°C with shaking at 240 rpm. The next morning, inoculate 20 ml LB with 0.2 ml of saturated overnight culture. Incubate the culture at 37°C with shaking at 240 rpm until the OD600 reaches 0.6 (approximately 3 h). Centrifuge at 5000 rpm for 10 min at 4°C to pellet the bacterial cells and discard the supernatant. Place the pellet on ice and resuspend the cells in 5 mL of cold 0.1 M CaCl2 solution. (This process is repeated three times in the original text.) Prepare equal aliquots (100 μl) of competent cells for later use.

[0080] Add pCDFduet to E. coli BAP1 competent cells ereC - ereA (1 µg) and pETDuet- ereS - ereT (1 µg). Place the mixture on ice for 30 minutes. Heat shock at 42°C for 90 seconds. Place the mixture on ice for 2 minutes. Add 900 µL of room temperature LB medium to the test tube. Incubate the test tube at 37°C for 60 minutes. Shake vigorously (240 rpm). Heat the selective plates (streptomycin, 50 ng / ml and ampicillin, 100 ng / ml) to 37°C. Centrifuge E. coli BAP1 and spread E. coli BAP1 onto the plates. After incubating overnight at 37°C, pick single colonies into LB medium supplemented with 50 ng / ml streptomycin and 100 ng / ml ampicillin.

[0081] 2.4 In vitro studies of OEA-engineered bacteria E. coli ereT+ and E. coli 载体 The medium was incubated overnight at 37°C and 240 rpm in a shaker. 2 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and 5 mM sodium oleate were added to LB medium, and the mixture was incubated for 48 h at 25°C and 240 rpm in a shaker. After incubation, the medium was centrifuged, and 100 μL of the supernatant was collected for OEA concentration determination using LC-MS / MS.

[0082] 2.5 In vivo studies of OEA-engineered bacteria For E. coli ereT+ and E. coli 载体 PlantingAll PI-IBS mice were randomly divided into four groups for 19 days: 1) Control group: mice (n=8) were administered 200 μL of control medium and sterile PBS; 2) Model group: mice (n=8) were administered 200 μL of control medium and sterile PBS; 3) Escherichia coli ereT+ Colonization group: 200 μL of Escherichia coli dissolved in sterile PBS was administered to mice (n=8). ereT+ (1×10 9 CFU); 4) Escherichia coli 载体 Colonization group: 200 μL of Escherichia coli dissolved in sterile PBS was administered to mice (n=8). 载体 (1×10 9 CFU); for Escherichia coli ereT+ and E. coli 载体 The transplanted group was given 10 mM IPTG in drinking water.

[0083] Gastrointestinal (GI) transit, fecal water content, and defecation frequency were measured on day 21 after administration of TNBS solution and on days 7 and 14 after intervention to determine whether colonization with OEA-engineered bacteria affected diarrhea-like symptoms in an IBS-D-like mouse model.

[0084] 2.6 RNA extraction and quantification using real-time polymerase chain reaction Total RNA was isolated from the ileum and distal colon of the PI-IBS mouse model using Trizol reagent (TAKARA) and purified using a total RNA extraction kit (Solarbio, catalog number R1200). QGP1 cells were treated with different concentrations of OEA for 4 h and 24 h. At the end of the experiment, cells were washed with cold PBS, and total RNA was extracted using a total RNA extraction kit (Solarbio, catalog number R1200). The concentration of total RNA was measured using NanoDrop-ONEc. Total RNA was reverse transcribed using PrimeScript RT MasterMix (Perfect Real Time) (TAKARA, catalog number rr036a). Quantitative real-time PCR was performed using SYBR Green MasterMix and completed using a QuantStudio 7 Flex Real-Time System (AppliedBiosystems, Thermo Fisher Scientific, USA). The values ​​of target genes TPH1, AADC, ALDH, MAO, and SERT were normalized to β-actin, and relative expression was shown as a fold change relative to the control group.

[0085] Table 1. Primer Information 2.7 Quantitative analysis of metabolites An Agilent 1290 Infinity II UPLC system coupled with a triple quadrupole (QQQ) 6470 mass spectrometer was used for targeted metabolomics profiling. A Waters BEH2.1 3 50 mm C18 1.7 mm column with pre-column was used. The mobile phases for serotonin detection in LC-MS-QQQ were solution A (water containing 0.1% formic acid) and solution B (acetonitrile containing 0.1% formic acid). The gradients were set as follows: 2% B (0–0.5 min), 2%–30% B (0.5–4 min), 30%–100% B (4–6 min), 100% B (6–8 min), 100%–2% B (8–8.5 min), and maintained at 2% B (8.5–10 min). The mobile phases used in OEA detection in LC-MS-QQQ were solution A (water containing 0.1% formic acid) and solution B (acetonitrile containing 0.1% formic acid). The gradients were set to 70% B (0 min), 70-75% B (0-6 min), 75%-80% B (6-9 min), 100% B (9-11 min), 100%-70% B (11 min), and maintained at 70% B (11-14 min). MS data were collected and processed using Agilent software. The list of standards, MRM transitions, fragmentation voltages, and impact energies are listed in Table 2.

[0086] Table 2. MRM transitions and parameters related to STAR for metabolite quantification 2.8 Statistics Data were analyzed using GraphPad Prism 9.0, and results are expressed as mean ± SEM. Significance was calculated using t-tests or one-way ANOVA. p Values ​​less than 0.05 p The value is considered statistically significant.

[0087] result OEA is mainly produced by the gut microbiota. It was unclear whether OEA was a gut microbial metabolite. We then identified changes in gut microbial metabolites in germ-free mice. Figure 1 As shown, serum and fecal OEA levels were significantly reduced in germ-free mice. We further determined the serotonin levels in germ-free mice. Figure 2As shown, fecal OEA levels in germ-free mice were significantly reduced, indicating that OEA in the gut is mainly produced by the gut microbiota.

[0088] IBS-D patients have reduced OEA We subsequently found that serum OEA levels were significantly reduced in IBS-D patients and were negatively correlated with serotonin levels and the index of diarrhea and abdominal pain symptoms in IBS-D patients. Figure 3 ).

[0089] OEA improved the diarrhea and abdominal pain-like phenotype in TNBS-treated mice. We used a TNBS-induced IBS-D animal model to investigate the effects of OEA on diarrhea and abdominal pain. Notably, we demonstrated that OEA significantly improved the diarrhea-like and abdominal pain-like phenotypes in TNBS-treated mice (n=6), as evidenced by improved GI transport, defecation frequency, fecal water content, and abdominal electromyography (EMG). OEA prolonged GI transport time, reduced fecal water content, and decreased defecation frequency in a dose-dependent manner. Figure 4 ).

[0090] We also demonstrated that OEA improved the abdominal pain-like phenotype in TNBS-treated mice (n=6). Figure 5 As shown, OEA treatment reduced the severity of abdominal pain, which was measured by EMG using colorectal distension.

[0091] OEA inhibits serotonin levels in TNBS-treated mice. Targeting serotonin signaling for the treatment of diarrhea-predominant irritable bowel syndrome (IBS-D) has been a focus of research and drug development. Based on the improvement of IBS-D mouse model by OEA, we further examined peripheral serotonin levels in mice treated with TNBS. Figure 6 As shown, peripheral serotonin levels increased in TNBS-treated mice, while OEA treatment suppressed serotonin levels in the serum and ileum of TNBS-treated mice, demonstrating that OEA inhibits peripheral serotonin levels in vivo.

[0092] OEA inhibits serotonin production in vitro. To investigate whether OEA, as a gut microbial metabolite, regulates serotonin levels, we first determined the effect of OEA on serotonin production in QGP-1 cells, an in vitro cell model for serotonin production. We demonstrated that physiologically appropriate levels of OEA inhibit serotonin production in vitro in a dose-dependent manner. Figure 7 ).

[0093] OEA inhibits serotonin biosynthesis and reuptake both in vivo and in vitro. We found that OEA inhibits peripheral serotonin in vivo and in vitro. Based on this finding, we further determined the mRNA expression levels of serotonin biosynthesis and reuptake, including tryptophan hydroxylase-1 (…). Tph1 ), serotonin reuptake transporter Sert Monoamine oxidase ( Mao ), aromatic L-amino acid decarboxylase ( Aadc ).like Figure 8 As shown, in TNBS-treated mice, OEA inhibited the activity of OEA in the ileum and distal colon tissues. Tph1 and Aadc Gene expression and upregulation Mao and Sert Gene expression was downregulated in the QGP1 cell line. Consistent with in vivo q-PCR results, we demonstrated that OEA was downregulated in the QGP1 cell line. TPH1 Gene expression and upregulation SERT Gene expression ( Figure 9 This indicates that OEA can regulate serotonin levels by inhibiting serotonin biosynthesis and reuptake.

[0094] OEA-producing bacteria improved the diarrhea and abdominal pain-like phenotype in TNBS-treated mice. We then used a bacterium named *Eubacterium rectum* that produces OEA to further investigate its effect on alleviating IBS-D-related symptoms. Transplantation of *Eubacterium rectum* for 19 days significantly improved the diarrhea-like phenotype in TNBS-treated mice (n=6), including GI transit time, defecation frequency, and fecal water content. Figure 10 As shown, *E. rectum* significantly reduced defecation frequency, prolonged GI transport time, and decreased fecal water content.

[0095] To investigate the effect of *E. rectum* on abdominal pain in TNBS-treated mice, colonic and rectal dilatation was measured as previously described. Our results showed that *E. rectum* significantly improved visceral hypersensitivity in TNBS-treated mice. Figure 11 ).

[0096] OEA-producing bacteria inhibit serotonin levels in TNBS-treated mice. We transplanted *E. rectum* into the colon of TNBS-treated mice via daily oral gavage and demonstrated a significant increase in OEA levels in the distal colon and ileum. Figure 12 This indicates that *E. rectum* successfully colonizes the mouse intestine and produces OEA. We also demonstrate that *E. rectum* inhibits peripheral serotonin in TNBS-treated mice. Figure 13 ).

[0097] Engineering-modified E. coli ereT+ OEA produced in vitro To study engineered Escherichia coli ereT+ Can OEA be generated in vitro, with or without 2 mM MIPTG and 5 mM sodium oleate, in the engineered E. coli? ereT+ and E. coli 载体 Incubate at 25°C for 48 hours. We demonstrate that the engineered E. coli... ereT+ IPTG in vitro can significantly increase OEA production. Figure 14 ).

[0098] Engineering-modified E. coli ereT+ Increase OEA concentration in TNBS-treated mice We will kill E. coli through daily oral gavage. ereT+ and E. coli 载体 Transplanted into the colon of TNBS-treated mice, and demonstrated a significant increase in fecal OEA levels ( Figure 15 This indicates that the engineered E. coli ereT+ It successfully colonized the mouse intestine and successfully produced OEA.

[0099] Engineering-modified E. coli ereT+ Improving the diarrhea-like phenotype in TNBS-treated mice We then used engineered E. coli ereT+ To further investigate its effect on improving diarrhea and visceral hypersensitivity in TNBS-treated mice. Colonized *Escherichia coli* ereT+ Eighteen days significantly reduced the diarrhea-like phenotype in TNBS-treated mice (n=8), including GI transit time, defecation frequency, and fecal water content. Figure 16 As shown, Escherichia coli ereT+ It significantly reduced defecation frequency, prolonged GI transport time, and reduced fecal water content.

Claims

1. A method for treating irritable bowel syndrome (IBS) in a subject of need, the method comprising: The subject was given a therapeutically effective amount of N-oleoylethanolamine (OEA).

2. The method according to claim 1, wherein the IBS is a diarrheal subtype (IBS-D).

3. The method of claim 1, wherein the OEA is isolated and the OEA is substantially pure.

4. The method of claim 1, wherein the amount of OEA administered is sufficient to prolong gastrointestinal transit time, reduce fecal water content, reduce defecation frequency, reduce serum serotonin concentration, or a combination thereof, in the subject.

5. The method of claim 1, wherein the subject has a lower serum OEA concentration relative to the average serum OEA concentration of a healthy subject who does not have IBS.

6. The method of claim 1, further comprising providing a sample obtained from the subject, wherein the sample is a stool sample or a serum sample; determining the amount of one or more biomarkers in the sample, the one or more biomarkers being selected from the group consisting of OEA or serotonin; and determining that the subject has IBS based on the amount of the one or more biomarkers in the sample prior to the step of administering OEA to the subject.

7. The method according to claim 1, wherein the subject is a human, a non-human primate, a rodent, a canine, a feline, a bovine, or an equine.

8. The method of claim 1, wherein the subject is a human.

9. The method of claim 1, wherein the OEA is administered orally.

10. A method for treating irritable bowel syndrome (IBS) in a subject of need, the method comprising: The subjects were given a therapeutically effective amount of bacteria that produce N-oleoylethanolamine (OEA).

11. The method of claim 10, wherein the IBS is a diarrheal subtype (IBS-D).

12. The method according to claim 10, wherein the bacteria is *E. rectum* (…). Eubacterium rectale ).

13. The method of claim 10, wherein the amount of intestinal bacteria administered is sufficient to prolong gastrointestinal transit time, reduce fecal water content, reduce defecation frequency, reduce serum serotonin concentration, or a combination thereof, in the subject.

14. The method of claim 10, wherein the subject has a lower serum OEA concentration relative to the average serum OEA concentration of a healthy subject who does not have IBS.

15. The method of claim 10, further comprising providing a sample obtained from the subject, wherein the sample is a fecal sample or a serum sample; determining the amount of one or more biomarkers in the sample, the one or more biomarkers being selected from the group consisting of OEA or serotonin; and determining that the subject has IBS based on the amount of the one or more biomarkers in the sample prior to the step of administering the bacteria to the subject.

16. The method of claim 10, wherein the bacteria are administered orally or via fecal microbiota transplantation.

17. The method of claim 10, wherein the subject is a human, a non-human primate, a rodent, a canine, a feline, a bovine, or an equine.

18. The method of claim 10, wherein the subject is a human.

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