WO2023061154A1 - 脆弱拟杆菌在防治癌症相关腹泻中的应用 - Google Patents

脆弱拟杆菌在防治癌症相关腹泻中的应用 Download PDF

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WO2023061154A1
WO2023061154A1 PCT/CN2022/119521 CN2022119521W WO2023061154A1 WO 2023061154 A1 WO2023061154 A1 WO 2023061154A1 CN 2022119521 W CN2022119521 W CN 2022119521W WO 2023061154 A1 WO2023061154 A1 WO 2023061154A1
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bacteroides fragilis
group
diarrhea
inactivated
animals
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French (fr)
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刘洋洋
郑丽君
常秀娟
梁德宝
王晔
智发朝
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广州知易生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the application of Bacteroides fragilis, in particular to the application of Bacteroides fragilis in preventing and treating diarrhea associated with cancer treatment.
  • CTID Cancer treatment-induced diarrhea
  • CTID is a common gastrointestinal toxic side effect caused by chemotherapy in cancer patients.
  • CTID will not only reduce the quality of life of patients, but also lead to water and electricity disturbance, dehydration, infection, and even shock and death in severe cases.
  • CTID is mainly a secretory diarrhea (Rutledge DN, Engelking C. Cancer-related diarrhea: selected findings of a national survey of oncology nurse experiences [J]. Oncol Nurs Forum, 1998, 25(5): 861-73), It usually occurs within 24 to 96 hours after chemotherapy or targeted drug infusion.
  • CTID tumor therapy-associated mucositis plays a dominant role in the occurrence of CTID.
  • Tumor therapy-related mucositis leads to intestinal flora imbalance, intestinal mucosal barrier damage (intestinal mucosal layer destruction and intestinal epithelial shedding), damage to the reabsorption function of microvilli cells, and cause diarrhea.
  • CTID Drugs that can cause CTID include the following classes: fluoropyrimidines (eg, 5-fluorouracil and the highly active compound capecitabine), topoisomerase inhibitors (eg, irinotecan, topotecan), and others (eg, o saliplatin, paclitaxel, cisplatin, cytarabine), among which itirican (CPT-11) and 5-fluorouracil (5-FU) are the most common; in addition, among emerging targeted small molecule drugs, also Similar symptoms exist.
  • fluoropyrimidines eg, 5-fluorouracil and the highly active compound capecitabine
  • topoisomerase inhibitors eg, irinotecan, topotecan
  • others eg, o saliplatin, paclitaxel, cisplatin, cytarabine
  • itirican CPT-11
  • 5-fluorouracil 5-fluorouracil
  • Symptomatic treatment is the main treatment for CTID.
  • Conventional drugs for CTID include non-specific morphine analgesics, opioid receptor inhibitor loperamide, somatostatin analogue octreotide, and other antidiarrheal agents, such as montmorillonite powder;
  • loperamide is the first-line drug
  • octreotide is the first-line drug for patients with CTID score above 3.
  • both loperamide and octreotide have significant side effects.
  • Side effects of loperamide include severe constipation, abdominal pain, dizziness, rash and worsening of pre-existing bloating, nausea and vomiting, etc.
  • Side effects of octreotide include slow and/or uneven heartbeat, severe constipation, stomach pain, goiter Big, vomiting, nausea, headache and dizziness etc.
  • montmorillonite powder has little effect on severe diarrhea.
  • Probiotics are a type of active microorganisms that are beneficial to the host by colonizing the human body and changing the composition of the flora in a certain part of the host.
  • the probiotic preparations commonly used in the treatment of CTID are Zhengchangsheng (Bifidobacterium Triple Capsules), Siliankang (Bifidobacteria Quadruple Tablets) and their combination with other probiotic preparations, such as Zhengchangsheng (Bacillus licheniformis) use.
  • Co-probiotics are defined as "non-viable microbial cells which, when administered orally or topically in sufficient quantities, confer benefits to the user". This definition includes microbial cells with incomplete morphology and microbial cells with complete morphology.
  • the mainstream research point of view believes that broken microbial cells can release functional molecules better, so they have better effects; but for intact microbial cells that have been inactivated, there is no A unified view can explain why it is superior to live bacteria in some applications.
  • the research on secondary probiotics is concentrated on the first-generation probiotics such as Lactobacillus and Bifidobacterium, and the research direction is relatively fixed; therefore, it is necessary to develop new types of secondary probiotics and expand the scope of indications of secondary probiotics.
  • the present invention provides the application of Bacteroides fragilis in the preparation of a composition for preventing and treating cancer treatment-related diarrhea.
  • the Bacteroides fragilis is selected from Bacteroides fragilis ZY-312 with a deposit number of CGMCC No.10685.
  • the present invention further provides the use of Bacteroides fragilis in the preparation of a composition for preventing and/or treating side effects of chemotherapy or targeted drugs.
  • the chemotherapy drugs are selected from one or more of cytotoxic chemotherapy drugs, taxanes, platinums, fluorouracils and camptothecins; and/or, the targeting The drug is selected from Anti-EGFR, Anti-HER-2, Anti-BRAF, Anti-MEK, Anti-EML4/ALK, Anti-VEGF, Multi-targeted TKI, Anti-mTOR, Anti-CDK4/6 and Anti-PARP one or more.
  • the chemotherapeutic drug is selected from one or more of docetaxel, oxaliplatin, cisplatin, fluorouracil, capecitabine and irinotecan; and/or, the Targeted drugs are selected from the group consisting of afatinib, osimertinib, neratinib, vemurafenib, dabrafenib, cobitinib, trametinib, crizotinib, aflibercept, One of patinib, pyrotinib, imatinib, pazopanib, everolimus, sirolimus, palbociclib, ribociclib, olaparib, and rucaparib one or more species.
  • said Bacteroides fragilis has a selection as indicated above.
  • the above-mentioned Bacteroides fragilis is inactivated, attenuated, low infectivity or non-infectious, and contains bacterial protein components of natural structure, which can effectively reduce the expression of exhaustion molecules in T cells and enhance T cell immunity.
  • the Bacteroides fragilis is one or more of the following: live Bacteroides fragilis; Bacteroides fragilis that has undergone inactivation, genetic recombination, transformation or modification, attenuation, chemical treatment, physical treatment or inactivation; Bacteroides fragilis Bacteroides lysate; and/or Bacteroides fragilis liquid culture supernatant.
  • the inactivated Bacteroides fragilis is an inactivated bacterium with a complete form and/or an inactivated bacterium with an incomplete form, preferably an inactivated bacterium with a complete form.
  • the above-mentioned Bacteroides fragilis is killed by any one or more methods of dry heat, moist heat, filtration, organic solvents, chemical reagents, ultraviolet or infrared rays, fermentation, freeze-drying, genetic recombination, genetic modification or transformation. Live.
  • inactivated bacterial powder is obtained by freeze-drying.
  • the composition may be any one of pharmaceutical composition, food, health product or food additive.
  • the invention also provides a pharmaceutical composition prepared by Bacteroides fragilis for preventing and treating diarrhea associated with cancer treatment.
  • the pharmaceutical composition contains Bacteroides fragilis ZY-312 with the preservation number CGMCC No.10685.
  • Bacteroides fragilis is an inactivated bacterium obtained by freeze-drying.
  • the pharmaceutical composition further contains a pharmaceutically acceptable carrier and/or adjuvant.
  • the dosage form of the pharmaceutical composition is pill, tablet, granule, capsule, powder, suspension, oral liquid or enema.
  • the pharmaceutical composition is administered orally or enemaly.
  • the administration period of the pharmaceutical composition is intermittent administration, periodic administration, continuous administration or long-term administration.
  • the present invention also provides a health product composition for preventing and treating cancer treatment-associated diarrhea, wherein the health product composition contains Bacteroides fragilis ZY-312 with a preservation number of CGMCC No. 10685.
  • the present invention also provides a method for preventing and treating cancer treatment-associated diarrhea, comprising administering a therapeutically effective amount of the above-mentioned Bacteroides fragilis or the composition to a patient.
  • prevention includes prevention and/or treatment.
  • the present invention unexpectedly finds that inactivated Bacteroides fragilis, especially the inactivated Bacteroides fragilis ZY-312 with the preservation number CGMCC No. 10685, not only has the function of living bacteria adhering to intestinal epithelial cells, but also protecting the intestinal epithelium. Function; reduce inflammatory cytokines such as TNF- ⁇ , IL-1 ⁇ , IFN- ⁇ , IL-6, and reduce intestinal inflammation through the interaction with Toll-like receptors (TLR) and the subsequent activation of NF- ⁇ B signaling pathway , restore the effect of intestinal mucosal barrier function, and also have better resistance to gastric acid and bile salts;
  • TLR Toll-like receptors
  • Fig. 1 is the colony characteristic figure of the Bacteroides fragilis ZY-312 of embodiment 1;
  • Fig. 2 is the microscopic observation figure after Gram staining of Bacteroides fragilis ZY-312 of embodiment 1;
  • Fig. 3 is a morphological diagram of inactivated cells of Bacteroides fragilis in Example 2.
  • Embodiment 1 Fermentation culture of Bacteroides fragilis
  • Bacteroides fragilis ZY-312 was cultured on a blood plate for 48 hours, and it appeared round, slightly convex, translucent, white, smooth, non-hemolytic, and the diameter of the colony was between 1-3 mm, see Figure 1.
  • Bacteroides fragilis ZY-312 was examined by Gram staining. It is a Gram-negative bacterium with a typical rod shape, blunt rounded ends and dense staining. The uncolored part in the middle of the bacteria is like a vacuole. figure 2.
  • Colony characteristics Bacteroides fragilis ZY-312 is cultured on a blood plate for 48 hours, showing a round slightly convex, translucent, white, smooth surface, and no hemolysis. The diameter of the colony is 1-3mm, see Figure 1.
  • Enrichment Select a single colony from step 1) and inoculate in TSB (tryptone soybean broth, containing 5% fetal bovine serum) for enrichment culture, and the obtained bacterial solution is preserved for future use.
  • TSB tryptone soybean broth, containing 5% fetal bovine serum
  • Viable Bacteroides fragilis solution The bacteria solution prepared in step 1) was used to measure the number of bacteria with a McFarland turbidimetric tube, diluted with normal saline to 10 7 CFU/ml and 10 9 CFU/ml, and stored for later use.
  • Bacteroides fragilis inactivated bacterial liquid the inactivated Bacteroides fragilis liquid is prepared by high temperature or ultraviolet irradiation. Specific steps: Take 5 mL of 10 7 cell/mL and 10 9 cell/mL live Bacteroides fragilis solution prepared in step 2) respectively and put them in a beaker, and place the beaker containing the bacteria solution in a constant temperature water bath at 100°C Inactivated Bacteroides fragilis can be prepared for 20 to 30 minutes or placed in an ultraviolet environment for 30 to 60 minutes.
  • Bacteroides fragilis lysate The Bacteroides fragilis lysate was prepared by ultrasonic disruption. Specific steps: use 5 mL of live Bacteroides fragilis solution prepared in step 2), and lyse it with an ultrasonic instrument for 30 minutes, On for 10 seconds, and Off for 10 seconds. Centrifuge at °C, filter with a 0.22 ⁇ m filter head and set aside.
  • step (3) Centrifuge the inactivated bacteria liquid obtained in step (2) to collect the inactivated bacteria sludge.
  • step (3) Add excipients to the inactivated bacteria slime collected in step (3) to make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain the inactivated bacteria stock solution;
  • step (4) Vacuum freeze-dry the stock solution of inactivated bacteria obtained in step (4), pre-freeze at -40 ⁇ 2°C for 1 to 3 hours, then pre-freeze at -20 ⁇ 2°C for 0.5 to 1 hour, and finally refreeze at -40 ⁇ 2°C Pre-freeze for 0.5-2 hours, and prepare inactivated bacterial powder by drying once (-5 ⁇ 2°C and 0 ⁇ 2°C) and analyzing and drying (35 ⁇ 2°C) under 0.25mbar vacuum degree, and the bacterial count of the bacterial powder reaches 1 ⁇ More than 10 11 Cell/g.
  • Example 3 Effect of Bacteroides fragilis liquid on diarrhea in mice caused by irinotecan
  • mice after the quarantine period, were randomly divided into A. normal group, B. model group, C. implementation 2 ZY-312 live bacteria solution (10 7 CFU/mL), D. embodiment 2 ZY-312 live bacteria solution (10 9 CFU/mL), E. Example 2 ZY-312 live bacteria lysate, F. Example 2 ZY-312 inactivated bacteria solution (10 7 cell/mL), G. Implementation Example 2 ZY-312 inactivated bacterial liquid (10 9 cell/mL), H. positive drug group BEFICOL group (344mg/kg), 8 groups in total, 11 rats in each group.
  • mice except the normal group were intraperitoneally injected with irinotecan hydrochloride solution (20 mL/kg) at a dose of 80 mg/kg, once a day, to establish a diarrhea model.
  • irinotecan hydrochloride solution 20 mL/kg
  • groups A and B were given 10 mL/kg of NS orally as controls.
  • Diarrhea of experimental mice was tested every day. The experiment was finished at 9 days, fasted for more than 12 hours in advance, and the mice in each group were killed by cervical dislocation.
  • Diarrhea record and scoring standard Record the diarrhea situation of each experimental mouse every day, and the scoring standard of diarrhea refers to the diarrhea scoring method in the study of Kurita A et al. 0 points: normal or no stool; 1 point: mild diarrhea, slightly wet and soft stool; 2 points: moderate diarrhea, wet and shapeless stool, and mild perianal coloring; 3 points: severe diarrhea, Watery stools with heavy perianal pigmentation.
  • the dispersion liquid of the suspension in the following examples is physiological saline.
  • mice select 100 BALB/c mice, half male and half male, and randomly divide them into 10 groups according to the sex and body weight of the animals, namely blank group, model group, positive 1 (whole intestine raw 500mg/kg), positive group 2 ( Imodium 4mg/kg), NCTC 9343/ZY-312 inactivated bacterial powder low-dose group (2 ⁇ 10 5 per bird), NCTC 9343/ZY-312 inactivated bacterial powder medium-dose group (2 ⁇ 10 7 /bird), NCTC9343/ZY-312 inactivated bacterial powder high-dose group (2 ⁇ 109 /bird), 10 in each group, half male and half male.
  • NCTC 9343 is the type strain derived from the American population.
  • the animals in other groups were intraperitoneally injected with irinotecan 70mg/kg (0.14mL/10g) once a day for 5 consecutive days to prepare the diarrhea model; from 3 days before the model establishment, the animals in the corresponding groups were intragastrically administered (0.1mL/10g body weight) give whole intestine raw suspension, imodium suspension, NCTC 9343/ZY-312 bacterial powder suspension of different concentrations (high, medium and low doses), each morning and afternoon every day For 11 consecutive days, the blank group and the model group were given the same amount of normal saline, and euthanized after the last administration.
  • the animals were generally observed and recorded every day, including the appearance and signs of the animals, behavioral activities, respiration, gland secretion, and feces, etc.
  • the diarrhea of each animal was observed and recorded, and scored.
  • the feces of each animal were collected for weighing and determination of water content; about 0.5 mL of blood was collected from the orbital venous plexus of all surviving animals after isoflurane anesthesia, and the serum was collected after centrifugation and used for liquid chip reagents respectively.
  • IL-1 ⁇ , IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, TNF- ⁇ , INF- ⁇ and other cytokines were detected by kits, and ICAM-1 was measured by ELISA kits; After euthanasia, the whole intestinal tract was collected, fixed with 10% formaldehyde solution, and stained with HE for pathological examination.
  • Diarrhea record and scoring standard Record the diarrhea situation of each experimental mouse every day, and the scoring standard of diarrhea refers to the diarrhea scoring method in the study of Kurita A et al. 0 points: normal or no stool; 1 point: mild diarrhea, slightly wet and soft stool; 2 points: moderate diarrhea, wet and shapeless stool, and mild perianal coloring; 3 points: severe diarrhea, Watery stools with heavy perianal pigmentation.
  • Intestinal histopathological examination After euthanasia of all surviving animals, the entire intestinal tract was taken, fixed with 10% formaldehyde solution, and pathological tissue sections were prepared, stained with HE, and examined under a microscope for intestinal histopathological examination.
  • Table 2 shows the food intake of animals in each group.
  • the animals in the model group and each administration group began to have diarrhea on the 4th day (D7). Compared with the blank group, the diarrhea score during the test period was significantly increased, and the diarrhea score increased with time, indicating that the modeling was successful.
  • the diarrhea scores of positive group 1, positive group 2 and each dose group of NCTC 9343 also showed an upward trend, but they were all lower than the model group.
  • Each group of ZY-312 showed the effect of maintaining the stability of the diarrhea score, and showed a better effect of reducing the increase of the diarrhea score, all of which were lower than those of the NCTC 9343 group.
  • n is the number of animals; compared with the model group, * indicates significant difference p ⁇ 0.05, ** indicates extremely significant difference p ⁇ 0.01.
  • the inflammatory factors TNF- ⁇ , IL-1 ⁇ , IL-5, IL-6, IL-17, INF- ⁇ and ICAM-1 proteins in the model group were significantly increased; the anti-inflammatory factors IL-4, IL- 10.
  • IL-13 decreased significantly.
  • the positive drug group has a down-regulation effect on the level of inflammatory factors, and an up-regulation effect on the watermark of anti-inflammatory factors.
  • Each group of Bacteroides fragilis also showed the regulatory effect on cytokines; among them, the regulatory effect of ZY-312 was better than that of NCTC 9343. There were no significant dose differences within the B. fragilis strain group.
  • n is the number of animals; compared with the model group, * indicates significant difference p ⁇ 0.05, ** indicates extremely significant difference p ⁇ 0.01.
  • NCTC9343 groups and ZY-312 all showed therapeutic effects, and the therapeutic effect of ZY-312 groups was superior. In each group of NCTC 9343.
  • NCTC 9343 group showed the effect of curbing the deterioration of this condition, and ZY-312 also showed a therapeutic effect, and the degree of improvement was better than that of the NCTC 9343 group.
  • B. fragilis strain group there were no significant dose differences.
  • the inactivated Bacteroides fragilis powder (ZY-312/NCTC 9343) can treat cancer therapy-related diarrhea in mice caused by irinotecan, and the therapeutic effect of ZY-312 is better than that of NCTC 9343.
  • Example 5 Curative effect of inactivated Bacteroides fragilis powder on 5-fluorouracil (5-FU)-induced mouse cancer treatment-related diarrhea model
  • mice 100 BALB/c mice were selected for the experiment, half male and half male, and randomly divided into 10 groups according to the sex and body weight of the animals, namely blank group, model group, positive group 1 (whole intestine raw 500mg/kg), positive group 2 (Imodium 4mg/kg), NCTC9343/ZY-312 inactivated bacterial powder low-dose group (2 ⁇ 10 5 pcs/bird), NCTC 9343/ZY-312 inactivated bacterial powder medium-dose group (2 ⁇ 10 7 each), NCTC 9343/ZY-312 inactivated bacteria powder high-dose group (2 ⁇ 10 9 mice/rat), 10 rats in each group, half male and half male.
  • positive group 1 whole intestine raw 500mg/kg
  • positive group 2 Imodium 4mg/kg
  • NCTC9343/ZY-312 inactivated bacterial powder low-dose group (2 ⁇ 10 5 pcs/bird
  • NCTC 9343/ZY-312 inactivated bacterial powder medium-dose group (2 ⁇ 10
  • the animals in the other groups were intraperitoneally injected with 5-FU 45mg/kg (0.1mL/10g) once a day for 5 consecutive days to prepare the diarrhea model; starting from 3 days before the model establishment, the animals in the corresponding groups were intragastrically administered (0.1mL/10g body weight) give whole intestine raw suspension, imodium suspension, NCTC 9343/ZY-312 bacterial powder suspension of different concentrations (high, medium and low doses), each morning and afternoon every day For 11 consecutive days, the blank group and the model group were given equal volumes of normal saline (0.1 mL/10 g body weight), and euthanized on the second day after the last administration.
  • the animals were observed and recorded in general every day, including the appearance and signs of the animals, behavioral activities, respiration, glandular secretion, feces, etc.
  • the diarrhea of each animal was observed and recorded, and scored.
  • the feces of each animal were collected for weighing and determination of water content; about 0.5 mL of blood was collected from the orbital venous plexus of all surviving animals after isoflurane anesthesia, and the serum was collected after centrifugation and used for liquid chip reagents respectively.
  • IL-1 ⁇ , IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, TNF- ⁇ , INF- ⁇ and other cytokines were detected by kits, and ICAM-1 was detected by ELISA kits; After euthanasia, the whole intestinal tract was collected, fixed with 10% formaldehyde solution, and stained with HE for pathological examination.
  • Diarrhea record and scoring standard Record the diarrhea situation of each experimental mouse every day, and the scoring standard of diarrhea refers to the diarrhea scoring method in the study of Kurita A et al. 0 points: normal or no stool; 1 point: mild diarrhea, slightly wet and soft stool; 2 points: moderate diarrhea, wet and shapeless stool, and mild perianal coloring; 3 points: severe diarrhea, Watery stools with heavy perianal pigmentation.
  • Table 7 shows the food intake of animals in each group.
  • Table 8 shows the statistics of the moisture content of animal feces in each group.
  • the Bacteroides fragilis groups increased the wet weight of feces and decreased the water content of feces. Among them, the effect of each group of ZY-312 was better than that of each group of NCTC9343; in the Bacteroides fragilis strain group, there was no obvious dose dependence.
  • Diarrhea score the diarrhea scores of animals in each group are shown in Table 9.
  • the animals in the model group and each administration group began to have diarrhea after the 4th day of modeling (D8), and the diarrhea score showed an increasing trend with the test time; compared with the blank group, the animals in the model group and each test group After modeling (D8-12), the diarrhea scores were all significantly increased (P ⁇ 0.01 or 0.05), indicating that the modeling was successful. In the model group, the diarrhea score decreased naturally on the 12th day.
  • Serum inflammatory factors the scores of serum inflammatory factors of animals in each group are shown in Table 10.
  • the inflammatory factors TNF- ⁇ , IL-1 ⁇ , IL-5, IL-6, IL-17, INF- ⁇ and ICAM-1 proteins in the model group were significantly increased; the anti-inflammatory factors IL-4, IL- 10.
  • IL-13 decreased significantly. It shows that the positive drug group has a down-regulation effect on the level of inflammatory factors and an up-regulation effect on the level of anti-inflammatory factors.
  • Each group of Bacteroides fragilis also showed the regulating effect on cytokines; among them, the regulating effect of ZY-312 was better than that of NCTC9343. There were no significant dose differences within the B. fragilis strain group.
  • Pathological examination select jejunum, ileum and colorectal tissue, and HE staining to evaluate the degree of pathological damage.
  • Bacteroides fragilis inactivated bacteria powder (ZY-312/NCTC 9343) can treat cancer treatment-related diarrhea in mice caused by 5-FU, and the therapeutic effect of ZY-312 is better than that of NCTC 9343.
  • mice 90 female SD rats were selected and randomly divided into 10 groups according to body weight intervals, namely blank group, model group, positive group (Imodium 1.5mg/kg), low dose of NCTC9343/ZY-312 inactivated bacterial powder group (10 6 /bird), NCTC9343/ZY-312 inactivated bacterial powder medium-dose group (10 8 /bird), NCTC9343/ZY-312 inactivated bacterial powder high-dose group (10 10 /bird), each Group 10.
  • the animals in the other groups were given 20mg/kg (0.01mL/10g body weight) of afatinib, once a day, for 14 consecutive days to prepare the diarrhea model; Gastrointestinal administration (0.1mL/10g body weight) of Imodium suspension and different concentrations (high, medium and low doses) of NCTC 9343/ZY-312 bacterial powder suspension, twice a day, once in the morning and once in the afternoon,
  • the blank group and the model group were given the same amount of normal saline, and euthanized after the last administration. Animals were generally observed and recorded every day, expressed in terms of body weight, and the diarrhea situation of each animal was observed and recorded in particular, and scored.
  • all the intestines of the surviving animals were euthanized, fixed with 10% formaldehyde solution, and stained with HE for pathological examination.
  • Body weight The body weight of the animals was weighed and recorded daily using an electronic balance.
  • Diarrhea record and scoring standard Record the diarrhea situation of each test mouse every day, and the scoring standard of diarrhea is as follows:
  • Intestinal histopathological examination After euthanasia of all surviving animals, the entire intestinal tract was taken, fixed with 10% formaldehyde solution, and pathological tissue sections were prepared, stained with HE, and examined under a microscope for intestinal histopathological examination.
  • Body weight the body weight of animals in each group is shown in Table 12.
  • the body weights of the animals in each group were similar before the start of the experiment (D0) and before the modeling (D3), and the body weights of the animals in the blank group, the model group and each administration group increased at a similar rate. Since D3, the body weight of the animals in the model group showed a downward trend, and the body weight changes of the animals in each administration group were similar to those in the model group. Among them, the average body weight of the model group remained at 220g, and D13 dropped to 210g; the average body weight of the positive group was similar to that of the model group, and D13 dropped significantly to less than 210g.
  • NCTC9343 administration groups began to decline from D6, and remained at around 210g from D9 to D13; the body weight of the ZY-312 administration groups did not change much, and remained at around 220g from D6-13.
  • the body weight indexes were not significantly different among the administration groups.
  • Table 13 shows the diarrhea score of animals in each group.
  • NCTC 9343 was similar to that of the positive drug, and the effect of ZY-312 in each group Better than NCTC 9343 group.
  • the inactivated Bacteroides fragilis powder (ZY-312/NCTC 9343) can treat afatinib-induced weight loss, chemotherapy-induced diarrhea, intestinal mucosal injury, mucosal hyperemia, inflammatory cell infiltration and glandular injury in rats. body injury, and the therapeutic effect of ZY-312 is better than that of NCTC 9343.

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Abstract

本发明提供了脆弱拟杆菌在防治癌症治疗相关性腹泻中的应用，特别是，脆弱拟杆菌在癌症治疗相关性腹泻中的应用。同时，本发明还提供了脆弱拟杆菌在制备预防和/或治疗化疗或靶向药物副作用的组合物中的用途，如摄食量变化、体重的变化、腹泻、炎性因子增加、肠黏膜损伤、黏膜充血、炎细胞浸润和/或腺体损伤等。

Description

脆弱拟杆菌在防治癌症相关腹泻中的应用
本申请要求享有2021年10月12日向中国国家知识产权局提交的，专利申请号为202111188989.X，发明名称为“脆弱拟杆菌在防治癌症相关腹泻中的应用”的在先申请的优先权权益。所述在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明涉及一种脆弱拟杆菌的应用，具体而言，涉及脆弱拟杆菌在防治癌症治疗相关性腹泻中的应用。
背景技术
癌症治疗相关性腹泻(cancer treatment-induced diarrhea,CTID)是肿瘤患者化疗引起的一种常见消化道毒副反应。CTID不仅会降低患者的生活质量，还会导致水电紊乱、脱水、感染，严重可致休克、死亡。CTID主要是一种分泌性腹泻(Rutledge DN,Engelking C.Cancer-related diarrhea:selected findings of a national survey of oncology nurse experiences[J].Oncol Nurs Forum,1998,25(5):861-73)，通常发生在化疗或靶向药物输注后24到96小时内。
CTID的病理生理学是复杂的，目前认为在CTID发生过程中占主导性地位的是肿瘤治疗相关性黏膜炎。肿瘤治疗相关性黏膜炎导致肠道菌群失调，肠黏膜屏障损坏(肠道黏膜层破坏和肠上皮脱落)，破坏微绒毛细胞的重吸收功能，引起腹泻。能够引发CTID的药物包括以下类型：氟嘧啶(例如，5-氟尿嘧啶和高活性的化合物卡培他滨)、拓扑异构酶抑制剂(如伊立替康，拓扑替康)和其他类型(例如奥沙利铂、紫杉醇、顺铂、阿糖胞苷)，其中伊替立康(CPT-11)和5-氟尿嘧啶(5-FU)最为常见；此外，在新兴的靶向小分子药物中，也存在类似的症状。以表皮生长因子受体(EGFR)靶向抑制剂(吉非替尼、阿法替尼等)为例，从皮疹到黏膜炎都是其副作用。根据药物的不同，肠黏膜屏障功能受损的时间也有所不同。
CTID以对症治疗为主，常规治疗CTID的药物包括非特异性的***类止痛剂，阿片类受体抑制剂洛哌丁胺、生长抑素类似物奥曲肽和其他止泻剂，如蒙脱石散；其中，洛哌丁胺为一线用药，奥曲肽为CTID评分3级以上患者的一线用药。然而，洛哌丁胺和奥曲肽均有明显的副作用。洛哌丁胺的副作用包括严重的便秘，腹痛，头晕，皮疹以及已经存在的腹胀，恶心和呕吐的恶化等，奥曲肽的副作用有缓慢和/或不均匀的心跳，严重的便秘，胃痛，甲状腺肿大，呕吐，恶心，头痛和头晕等。而蒙脱石散对严重的腹泻作用甚微。
共生细菌在肠道稳态中起着重要作用，并对肠道完整性具有一定的保护作用。因此，采用益生菌治疗CTID成为近年来研究的热点。益生菌是通过定植在人体内，改变宿主某一部位菌群组成的一类对宿主有益的活性微生物。目前常用于治疗CTID的益生菌制剂为整肠生(双歧杆菌三联胶囊)、思连康(双歧杆菌四联片)及它们与其他益生菌制剂，如整肠生(地衣芽胞杆菌)联合使用。
针对上述问题，副益生菌(Paraprobiotic)应运而生。副益生菌被定义为“无活力的微生物细胞，如果口服或局部使用足够的数量，即可为使用者带来好处”。该定义包括形态不完整的微 生物细胞和形态完整的微生物细胞，主流研究观点认为，破碎的微生物细胞能够更好地释放功能分子，因此具有更好的效果；但对于完整的灭活微生物细胞，没有统一的观点能够说明其在某些应用中优于活菌的原因。目前针对副益生菌的研究集中在乳杆菌、双歧杆菌等一代益生菌上，研究方向相对固定；因此，有必要开发新型副益生菌，扩展副益生菌的适应症范围。
发明内容
针对上述问题，本发明提供了脆弱拟杆菌(Bacteroides fragilis)在制备防治癌症治疗相关性腹泻组合物中的应用。根据本发明的实施方案，所述脆弱拟杆菌选自保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312。
本发明还进一步提供了脆弱拟杆菌在制备预防和/或治疗化疗或靶向药物副作用的组合物中的应用。
根据本发明的实施方案，所述化疗药物选自细胞毒类化疗药物、紫杉烷类、铂类、氟尿嘧啶类和喜树碱类中的一种或多种；和/或，所述靶向药物选自Anti-EGFR、Anti-HER-2、Anti-BRAF、Anti-MEK、Anti-EML4/ALK、Anti-VEGF、Multi-targeted TKI、Anti-mTOR、Anti-CDK4/6和Anti-PARP的一种或多种。
根据本发明的实施方案，所述化疗药物选自多西他赛、奥沙利铂、顺铂、氟尿嘧啶、卡培他滨和伊立替康中的一种或多种；和/或，所述靶向药物选自阿法替尼、奥希替尼、奈拉替尼、维莫非尼、达拉非尼、考比替尼、曲美替尼、克唑替尼、阿帕西普、拉帕替尼、吡咯替尼、伊马替尼、帕唑帕尼、依维莫司、西罗莫司、帕博西尼、瑞博西尼、奥拉帕尼和芦卡帕尼中的一种或多种。
根据本发明的实施方案，所述脆弱拟杆菌具有如上文所示的选择。上述脆弱拟杆菌是灭活的、减毒的、低感染性或非感染性的，并且含有天然结构的细菌蛋白组分，能够有效降低T细胞表达耗竭分子从而增强T细胞免疫。所述脆弱拟杆菌是以下中的一种或多种：脆弱拟杆菌活菌体；经过灭活、基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌液体培养上清液。
优选地，所述灭活的脆弱拟杆菌为形态完整的灭活菌和/或形态不完整的灭活菌，优选为形态完整的灭活菌。
优选地，上述脆弱拟杆菌是通过干热、湿热、过滤、有机溶剂、化学试剂、紫外或红外射线、发酵、冻干、基因重组、基因修饰或改造的方法中的任意一种或多种灭活的。比如，通过冻干方法得到灭活菌粉。
上述的应用和/或用途中，所述组合物可以是药物组合物、食品、保健品或食品添加剂中的任意一种。
本发明还提供了一种脆弱拟杆菌制备防治癌症治疗相关性腹泻的药物组合物。其中，所述药物组合物含有保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312。
在一种实施方案中，所述脆弱拟杆菌为通过冻干方法得到的灭活菌。
根据本发明的实施方案，所述药物组合物还含有药学上可接受的载体和/或辅料。
根据本发明的实施方案，所述药物组合物的剂型为丸剂、片剂、颗粒剂、胶囊、散剂、混悬剂、口服液或灌肠剂。
优选地，所述药物组合物通过口服或灌肠的形式给药。
根据本发明的实施方案，所述药物组合物的给药周期为间歇给药、周期性给药、持续给药或长期给药。
本发明还提供了一种用于防治癌症治疗相关性腹泻的保健品组合物，其中，所述保健品组合物含有保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312。
本发明还提供一种防治癌症治疗相关性腹泻的方法，包括向患者施用治疗有效量的上述脆弱拟杆菌或组合物。
其中，所述“防治”包括预防和/或治疗。
本发明的有益效果：
本发明出人预料地发现，灭活脆弱拟杆菌，特别是保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312灭活菌，不仅具有活菌粘附肠道上皮细胞，保护肠道上皮的作用；通过与Toll样受体(TLR)的相互作用及随后的NF-κB信号通路激活，减少TNF-α、IL-1β、IFN-γ、IL-6等炎性细胞因子，减轻肠道炎症，恢复肠黏膜屏障功能的效果，还具有更好的耐胃酸、胆盐性质；
安全性高、易储存、稳定性强等优点。
附图说明
图1为实施例1的脆弱拟杆菌ZY-312的菌落特征图；
图2为实施例1的脆弱拟杆菌ZY-312进行革兰氏染色后的显微镜观察图；
图3为实施例2脆弱拟杆菌灭活细胞形态图。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
实施例1.脆弱拟杆菌的发酵培养
将脆弱拟杆菌ZY-312菌种划线接种于血平皿，厌氧培养48h。观察菌落形态特征、染色特性、大小、球杆状和分布情况等。
菌落特征：脆弱拟杆菌ZY-312在血平皿上培养48h后，呈现圆形微凸、半透明、白色、表面光滑、不溶血，菌落直径在1-3mm之间，参见图1。
显微镜下形态：脆弱拟杆菌ZY-312进行革兰氏染色镜检，为革兰阴性细菌，呈现典型的杆状，两端钝圆而浓染，菌体中间不着色部分形如空泡，参见图2。
选取单个菌落接种于培养基中进行发酵培养8小时(温度为37℃)，所得菌液离心沉淀，转速3000r/min，离心15min，去上清，收集沉淀物，即得脆弱拟杆菌ZY-312菌泥。
实施例2.样品制备
1.脆弱拟杆菌活菌液、灭活菌液及裂解液的制备
1)1)将菌种划线接种于血平皿，37℃、厌氧培养48h。
菌落特征：脆弱拟杆菌ZY-312在血平皿上培养48h后，呈现圆形微凸、半透明、白色、表 面光滑、不溶血，菌落直径在1-3mm，参见图1。
2)增菌：从步骤1)中选取单个菌落接种于TSB(胰蛋白胨大豆肉汤，含5％胎牛血清)中进行增菌培养，所得菌液保存备用。
3)脆弱拟杆菌活菌液：将步骤1)制备的菌液，用麦氏比浊管做菌数测定，用生理盐水稀释至10 7CFU/ml和10 9CFU/ml，保存备用。
4)脆弱拟杆菌灭活菌液：采用高温或紫外照射的方法制备脆弱拟杆菌灭活菌液。具体步骤：分别取步骤2)制备的10 7cell/mL，10 9cell/mL的脆弱拟杆菌活菌液5mL置于烧杯中，并将盛有菌液的烧杯置于100℃的恒温水浴锅中20～30min或置于紫外线环境30～60min，便可制备脆弱拟杆菌灭活菌液。
5)脆弱拟杆菌裂解液：通过超声破碎的方法制备脆弱拟杆菌裂解液。具体步骤：采用步骤2)制备的脆弱拟杆菌活菌液5mL，经超声仪裂解30分钟，On 10秒，Off 10秒，该体系裂解菌体效率达到99％，裂解后经6000rpm、10min、4℃离心，0.22μm滤头过滤后备用。
2.脆弱拟杆菌灭活菌粉的制备
(1)取实施例1制得的脆弱拟杆菌发酵液对发酵液进行离心处理，收集湿菌体(即菌泥)，按菌体：生理盐水＝1:(10～30)(m:v)比例加入生理盐水对菌泥进行重悬洗涤，再次离心收集洗涤后的菌体；
(2)对步骤(1)所得菌体加入5％麦芽糊精+0.9％氯化钠混合成的赋形剂，按菌体：赋形剂＝1:(5～15)(m:m)比例进行添加，搅拌分散后在(70～100)±5℃热灭活(20～40)±5分钟，得灭活菌液；
(3)将步骤(2)所得的灭活菌液离心收集灭活菌泥。
(4)在步骤(3)收集的灭活菌泥中加入赋形剂使总重量与灭活前菌液重量一致，搅拌完全溶解，得灭活菌原液；
(5)将步骤(4)所得的灭活菌原液进行真空冷冻干燥，-40±2℃预冻1～3小时后，-20±2℃预冻0.5～1h，最后-40±2℃再预冻0.5～2h，0.25mbar真空度下经一次干燥(-5±2℃和0±2℃)、解析干燥(35±2℃)制备成灭活菌粉，菌粉的菌数达到1×10 11 Cell/g以上。
实施例3.脆弱拟杆菌菌液对伊利替康所致小鼠腹泻影响
(1)试验方法
BALB/c小鼠88只，经过检疫期，根据体重随机分为A.正常组，B.模型组，C.实施2 ZY-312活菌液(10 7CFU/mL)，D.实施例2 ZY-312活菌液(10 9CFU/mL)，E.实施例2 ZY-312活菌裂解液，F.实施例2 ZY-312灭活菌液(10 7cell/mL)，G.实施例2 ZY-312灭活菌液(10 9cell/mL)，H.阳性药组培菲康组(344mg/kg)，共8组，每组11只。实验1～4d，除正常组外其余小鼠按80mg/kg剂量腹腔注射盐酸伊立替康溶液(20mL/kg)，每天1次，建立腹泻模型。1～8d，各组小鼠按设定剂量及给药方式进行药物治疗，每天给药1次；A、B组口服10mL/kg的NS作对照。每天测实验鼠腹泻情况。9d结束实验，提前禁食12h以上，颈椎脱臼处死各组小鼠。
腹泻记录及评分标准：每天记录每只试验鼠腹泻情况，腹泻的评分标准参照Kurita A等研究中的腹泻评分方法。0分：大便正常或没有；1分：轻度腹泻，大便可见轻微湿软；2分：中度腹泻，大便较湿且不成形，并且有轻度的肛周着色；3分：重度腹泻，水样便并伴有重度肛 周着色。
表1 ZY-312菌株对腹泻评分的影响
Figure PCTCN2022119521-appb-000001
注：与模型组比较，*：P<0.05，**：P<0.01。
表1所示的腹泻评分结果：与正常组比较，模型组5～8d腹泻评分显著增加(P<0.01)。
实验5d，与模型组比较，样品4组的腹泻评分显著降低(P<0.05)。实验6～8d，与模型组比较，各药物组均能降低腹泻评分，但各药物组之间无显著差异，其中灭活菌液高剂量组显示相对较好的治疗的效果。
以下实施例中的混悬液的分散液均为生理盐水。
实施例4脆弱拟杆菌灭活菌粉对伊立替康所致小鼠迟发型腹泻模型的疗效
(1)试验方法
试验设计：选用BALB/c小鼠100只，雌雄各半，按动物性别和体重区间随机分为10组，即空白组、模型组、阳性1(整肠生500mg/kg)、阳性组2(易蒙停4mg/kg)、NCTC 9343/ZY-312灭活菌粉低剂量组(2×10 5个/只)、NCTC 9343/ZY-312灭活菌粉中剂量组(2×10 7个/只)、NCTC9343/ZY-312灭活菌粉高剂量组(2×10 9个/只)，每组10只，雌雄各半。NCTC 9343为美国人群来源的模式株。除空白组外，其余各组动物每天腹腔注射伊立替康70mg/kg(0.14mL/10g)1次，连续5天，以制备腹泻模型；从造模前3天开始，相应组动物分别灌胃(0.1mL/10g体重)给予整肠生混悬液、易蒙停混悬液、不同浓度(高、中、低剂量)的NCTC 9343/ZY-312菌粉混悬液，每天上下午各1次，连续11天，空白组和模型组给予等量生理盐水，末次给药后进行安乐死。给药后每天对动物进行一般观察和记录，包括动物的外观体征、行为活动、呼吸、腺体分泌、粪便情况等，重点观察并记录每只动物的腹泻情况，并进行评分。在造模结束、试验结束时，收集各动物的粪便进行称重及测定含水量；所有存活动物在异氟烷麻醉后眼眶静脉丛采集血液约0.5mL，离心后取血清分别用液相芯片试剂盒测定IL-1β、IL-4、IL-5、IL-6、IL-10、IL-13、IL-17、TNF-α、INF-γ等细胞因子，用ELISA试剂盒测定ICAM-1；安乐死后分别取全部肠道，用10％甲醛溶液固定，HE染色后进行病理学检查。
检测方法与频率：
摄食量：采用电子天平进行饲料添加和剩余量的称量操作，在给药前(D0)、D4分别记录加入量，分别在D4、D8、D11(试验结束)称剩余量1次，并记录，按公式计算：日平均摄食量(g)＝(加入量-剩余量)/动物数/天数。
腹泻记录及评分标准：每天记录每只试验鼠腹泻情况，腹泻的评分标准参照Kurita A等研究中的腹泻评分方法。0分：大便正常或没有；1分：轻度腹泻，大便可见轻微湿软；2分：中度腹泻，大便较湿且不成形，并且有轻度的肛周着色；3分：重度腹泻，水样便并伴有重度肛周着色。
肠道组织病理学检查：所有存活动物安乐死后取全部肠道，用10％甲醛溶液固定，制备病理组织切片，采用HE染色，在显微镜下进行肠道组织病理学检查。
数据统计与分析：对体重、摄食量、腹泻评分和病理学检查结果等数据以均数±标准差(Mean±SD)表示，使用SPSS统计软件25.0进行统计学分析。
(2)试验结果
1)摄食量：各组动物摄食量如表2。
表2各组动物的摄食量统计结果(均数±标准差，n＝10)
Figure PCTCN2022119521-appb-000002
注：与模型组比较，*表示差异显著p<0.05；**表示差异极显著p<0.01。
在D4(即造模前)，空白组、模型组及各给药组动物的日均摄食量均无明显差异(p>0.05)；
与空白组相比，在D8、D11即造模后，模型组雌雄动物的日均摄食量均显著下降(p<0.01)。
D8时，各给药组与模型组摄食量差异不大。D11时，阳性组2摄食量相比模型组显著下降；阳性组1、NCTC 9343各组摄食量与模型组相似；ZY-312各剂量组摄食量高于模型组。
2)粪便湿重、含水量和腹泻评分：
各组动物粪便湿重和含水量如表3。
表3各组动物的粪便湿重和含水量统计结果(均数±标准差，n＝10)
Figure PCTCN2022119521-appb-000003
注：与模型组比较，*表示差异显著p<0.05，**表示差异极显著p<0.01。
在粪便湿重及粪便含水量上，空白组没有明显差异。造模结束后，各造模组粪便湿重没有明显差异，但模型组粪便含水量相对空白组明显升高，但各给药组相对模型组粪便含水量呈下降趋势。其中，脆弱拟杆菌各组有效遏制了粪便含水量升高的趋势，但ZY-312组效果优于NCTC9343组。
各组动物腹泻评分如表4。
表4各组动物的腹泻评分统计结果(均数±标准差，n＝5)
Figure PCTCN2022119521-appb-000004
Figure PCTCN2022119521-appb-000005
注：与模型组比较，*表示差异显著p<0.05；**表示差异极显著p<0.01。
模型组及各给药组动物在第4天(D7)开始出现腹泻，与空白组相比，试验期间的腹泻评分显著升高，且腹泻评分随时间增高，说明造模成功。
对于雄性动物而言，阳性组1、阳性组2及NCTC 9343各剂量组的腹泻评分也是呈上升趋势，但是均低于模型组。ZY-312各组显示出维持腹泻评分稳定的效果，显示出较优的降低腹泻评分上升的效果，且均低于NCTC 9343组。
对于雌性动物而言，上述药效基本相似，ZY-312各组效果自始优于阳性药和NCTC 9343各组。
3)炎症因子
各组动物炎症因子如表5。
表5各组动物的炎症因子统计结果(均数±标准差)
Figure PCTCN2022119521-appb-000006
Figure PCTCN2022119521-appb-000007
续表
Figure PCTCN2022119521-appb-000008
注：n为动物数；与模型组比较，*表示差异显著p<0.05，**表示差异极显著p<0.01。
相对于空白组，模型组炎症因子TNF-α、IL-1β、IL-5、IL-6、IL-17、INF-γ及ICAM-1蛋白 显著升高；抗炎因子IL-4、IL-10、IL-13显著下降。阳性药组对炎性因子水平具有下调作用，对抗炎因子水印具有上调作用。脆弱拟杆菌各组同样体现出对细胞因子的调节作用；其中，ZY-312的调节效果优于NCTC 9343。脆弱拟杆菌菌株组内没有明显的剂量差异性。
4)病理检查：选取空肠、回肠和结直肠组织，HE染色，进行病理学评价。结果如表6。
表6各组动物病理检查结果(均数±标准差)
Figure PCTCN2022119521-appb-000009
Figure PCTCN2022119521-appb-000010
注：n为动物数；与模型组比较，*表示差异显著p<0.05，**表示差异极显著p<0.01。
空肠部位，相对于空白组，只有模型组出现了不明显的黏膜出血，且模型出现了明显的腺体扩张、肠黏膜损伤、炎细胞浸润、黏膜充血等病理学损伤；NCTC 9343各组菌粉在腺体扩张、肠黏膜损伤、炎细胞浸润方面体现了一定的治疗效果，而ZY-312各组体现出对腺体扩张、肠黏膜损伤、炎细胞浸润、黏膜充血各个病理损伤的治疗效果。
对于回肠部位，模型组出现了一定的黏膜出血，脆弱拟杆菌各组没有出现这种情况。阳性药组在剩余的腺体扩张、肠黏膜损伤、炎细胞浸润、黏膜充血方面，有明显的治疗效果；NCTC9343各组和ZY-312均显示出治疗效果，ZY-312各组的治疗效果优于NCTC 9343各组。
对于结直肠部位，各组均没有发生肠黏膜损伤。此外，模型组出现了腺体扩张、炎性细胞浸润、一定的黏膜充血和出血状况。NCTC 9343组表现出了遏制这种情况恶化的作用，ZY-312同样表现出治疗效果，且改善程度优于NCTC 9343组。在脆弱拟杆菌菌株组内，不存在明显的剂量差异性。
综上所述，脆弱拟杆菌灭活菌粉(ZY-312/NCTC 9343)能够治疗伊立替康所致的小鼠癌症治疗相关性腹泻，且ZY-312的治疗效果优于NCTC 9343。
实施例5.脆弱拟杆菌灭活菌粉对5-氟尿嘧啶(5-FU)所致小鼠癌症治疗相关性腹泻模型的疗效
(1)试验方法
试验设计：试验选用BALB/c小鼠100只，雌雄各半，按动物性别和体重区间随机分为10 组，即空白组、模型组、阳性组1(整肠生500mg/kg)、阳性组2(易蒙停4mg/kg)、NCTC9343/ZY-312灭活菌粉低剂量组(2×10 5个/只)、NCTC 9343/ZY-312灭活菌粉中剂量组(2×10 7个/只)、NCTC 9343/ZY-312灭活菌粉高剂量组(2×10 9个/只)，每组10只，雌雄各半。除空白组外，其余各组动物每天腹腔注射5-FU 45mg/kg(0.1mL/10g)1次，连续5天，以制备腹泻模型；从造模前3天开始，相应组动物分别灌胃(0.1mL/10g体重)给予整肠生混悬液、易蒙停混悬液、不同浓度(高、中、低剂量)的NCTC 9343/ZY-312菌粉混悬液，每天上下午各1次，连续11天，空白组和模型组给予等体积生理盐水(0.1mL/10g体重)，末次给药后第2天进行安乐死。每天对动物进行一般观察和记录，包括动物的外观体征、行为活动、呼吸、腺体分泌、粪便情况等，重点观察并记录每只动物的腹泻情况，并进行评分。在造模结束、试验结束时，收集各动物的粪便进行称重及测定含水量；所有存活动物在异氟烷麻醉后眼眶静脉丛采集血液约0.5mL，离心后取血清分别用液相芯片试剂盒测定IL-1β、IL-4、IL-5、IL-6、IL-10、IL-13、IL-17、TNF-α、INF-γ等细胞因子，用ELISA试剂盒测定ICAM-1；安乐死后分别取全部肠道，用10％甲醛溶液固定，HE染色后进行病理学检查。
检测方法与频率：
粪便含水量测定：分别在D8下午(造模结束)、D11下午采用单笼收集4h每只小鼠粪便并称湿重，然后放置80℃烘箱3h将粪便烘干后称干重并记录。按公式计算：粪便含水量(％)＝(粪便湿重－粪便干重)/粪便湿重×100。
腹泻记录及评分标准：每天记录每只试验鼠腹泻情况，腹泻的评分标准参照Kurita A等研究中的腹泻评分方法。0分：大便正常或没有；1分：轻度腹泻，大便可见轻微湿软；2分：中度腹泻，大便较湿且不成形，并且有轻度的肛周着色；3分：重度腹泻，水样便并伴有重度肛周着色。
血清样品收集与测定：存活动物在异氟烷麻醉后眼眶静脉丛采血约0.5mL。血液不抗凝，在室温静置30～50min，待血清析出后3000r/m离心15min，分离所有血清。用液相芯片试剂盒测定细胞因子IL-4、TNF-α。
数据统计与分析：对体重、摄食量、腹泻评分和病理学检查结果等数据以均数±标准差(Mean±SD)表示，使用SPSS统计软件25.0进行统计学分析。
(2)试验结果
1)摄食量：各组动物摄食量如表7。
表7各组动物摄食量统计结果(均数±标准差)
Figure PCTCN2022119521-appb-000011
Figure PCTCN2022119521-appb-000012
在造模前(D4)，各组小鼠摄食量没有明显差别。造模结束后(D8)，相对于空白组，各组小鼠摄食量均有所下降。至试验结束(D11)，与空白组相比，各组小鼠摄食量明显下降。D8时，模型组及阳性药组、NCTC 9343低中高剂量组摄食量相似，ZY-312低中高剂量组摄食量高于上述组；D11时，脆弱拟杆菌组小鼠的摄食量增加了，其中ZY-312各组增加的幅度大于NCTC9343各组。脆弱拟杆菌菌株组内，没有明显的剂量差异性。
2)粪便含水量和腹泻评分：各组动物粪便含水量统计如表8。
表8各组动物粪便含水量检查结果(均数±标准差，n＝10)
Figure PCTCN2022119521-appb-000013
注：与模型组比较，*表示差异显著p<0.05；**表示差异极显著p<0.01。
从实验开始到试验结束，空白组小鼠粪便湿重和含水量没有明显差异。造模结束后，各组 小鼠粪便湿重显著低于空白组小鼠，粪便含水量显著高于空白组小鼠。造模后模型组小鼠及各给药小鼠粪便湿重及粪便含水量相似。试验结束时，模型组小鼠粪便湿重自然恢复，但粪便含水量没有明显变化；阳性组1和阳性组2上调了小鼠的粪便湿重。相对于阳性药组，脆弱拟杆菌各组提高了粪便湿重，降低了粪便含水量。其中，ZY-312各组的效果优于NCTC9343各组；脆弱拟杆菌菌株组内，不存在明显的剂量依赖性。
腹泻评分：各组动物腹泻评分如表9。
表9各组动物腹泻评分(均数±标准差，n＝5)
Figure PCTCN2022119521-appb-000014
Figure PCTCN2022119521-appb-000015
注：与模型组比较，*表示差异显著p<0.05；**表示差异极显著p<0.01。
对于雄性动物，模型组及各给药组动物在造模第4天后(D8)开始出现腹泻，且腹泻评分随试验时间呈增长趋势；与空白组相比，模型组及各受试物组动物在造模后(D8-12)的腹泻评分均显著升高(P<0.01或0.05)，说明造模成功。模型组在第12天，腹泻评分自然下降。
对于雄性动物而言，阳性组1和阳性组2均降低了腹泻评分。脆弱拟杆菌各组呈现相似的趋势，但ZY-312各组下调腹泻评分的速度较快，且幅度大于NCTC9343各组。各脆弱拟杆菌菌株组内，没有明显的剂量依赖性。对于雌性动物而言，各给药组效果与雄性相似。
3)血清炎症因子：各组动物血清炎症因子评分如表10。
表10各组动物血清炎症因子评分(均数±标准差，n＝10)
Figure PCTCN2022119521-appb-000016
续表
Figure PCTCN2022119521-appb-000017
Figure PCTCN2022119521-appb-000018
注：与模型组比较，*表示差异显著p<0.05；**表示差异极显著p<0.01。
相对于空白组，模型组炎症因子TNF-α、IL-1β、IL-5、IL-6、IL-17、INF-γ及ICAM-1蛋白显著升高；抗炎因子IL-4、IL-10、IL-13显著下降。说明阳性药组对炎性因子水平具有下调作用，对抗炎因子水平具有上调作用。脆弱拟杆菌各组同样体现出对细胞因子的调节作用；其中，ZY-312的调节效果优于NCTC9343。脆弱拟杆菌菌株组内没有明显的剂量差异性。
5)病理检查：选取空肠、回肠和结直肠组织，HE染色评价病理学损伤程度。
表11各组动物肠道病理学损伤程度评分(均数±标准差，n＝10)
Figure PCTCN2022119521-appb-000019
Figure PCTCN2022119521-appb-000020
Figure PCTCN2022119521-appb-000021
对于各肠道部位，相对于空白组，模型组出现了肠黏膜损伤、明显的腺体扩张、炎细胞浸润、黏膜充血等病理学损伤，阳性组1和阳性组2显示出了治疗作用。脆弱拟杆菌各组显示出了治疗效果，而ZY-312各组的效果优于NCTC 9343各组。在脆弱拟杆菌菌株组内，不存在明显的剂量差异性。
脆弱拟杆菌灭活菌粉(ZY-312/NCTC 9343)能够治疗5-FU所致的小鼠癌症治疗相关性腹泻，且ZY-312的治疗效果优于NCTC 9343。
实施例6脆弱拟杆菌灭活菌粉对阿法替尼所致大鼠癌症治疗相关性腹泻模型的疗效
(1)试验方法
试验设计：选用雌性SD大鼠90只，按体重区间随机分为10组，即空白组、模型组、阳性组(易蒙停1.5mg/kg)、NCTC9343/ZY-312灭活菌粉低剂量组(10 6个/只)、NCTC9343/ZY-312灭活菌粉中剂量组(10 8个/只)、NCTC9343/ZY-312灭活菌粉高剂量组(10 10个/只)，每组10只。除空白组外，其余各组动物灌胃阿法替尼20mg/kg(0.01mL/10g体重)，每天1次，连续14天，以制备腹泻模型；从造模前3天开始，相应组动物分别灌胃(0.1mL/10g体重)给予易蒙停混悬液、不同浓度(高、中、低剂量)的NCTC 9343/ZY-312菌粉混悬液，每天2次，上下午各一次，连续14天，空白组和模型组给予等量生理盐水，末次给药后进行安乐死。每天对动物进行一般观察和记录，以体重表示，重点观察并记录每只动物的腹泻情况，并进行评分。在试验结束时所有存活动物在安乐死后分别取全部肠道，用10％甲醛溶液固定，HE染色后进行病理学检查。
检测方法与频率：
体重：采用电子天平每日称量动物体重并记录。
腹泻记录及评分标准：每天记录每只试验鼠腹泻情况，腹泻的评分标准如下：
Figure PCTCN2022119521-appb-000022
肠道组织病理学检查：所有存活动物安乐死后取全部肠道，用10％甲醛溶液固定，制备病理组织切片，采用HE染色，在显微镜下进行肠道组织病理学检查。
数据统计与分析：对体重、摄食量、腹泻评分和病理学检查结果等数据以均数±标准差(Mean±SD)表示，使用SPSS统计软件25.0进行统计学分析。
(2)试验结果
1)体重：各组动物体重如表12。
表12各组动物体重(均数±标准差，n＝10)
Figure PCTCN2022119521-appb-000023
注：与模型组比较，*表示差异显著p<0.05；**表示差异极显著p<0.01。
试验开始前(D0)及造模前(D3)各组动物体重相似，且空白组、模型组及各给药组动物的体重以相似速度上升。自D3开始，模型组动物体重呈下降趋势，各给药组动物体重变化与模型组相似。其中模型组体重均值维持在220g，D13下滑至210g；阳性组体重均值与模型组相似，D13大幅下降，不足210g。NCTC9343各给药组自D6开始下滑，D9到D13均维持在210g上下；ZY-312各给药组体重变化不大，D6-13维持在220g上下。体重指标各给药组相差不大。
2)腹泻评分：各组动物腹泻评分如表13。
表13各组动物腹泻评分(均数±标准差，n＝10)
Figure PCTCN2022119521-appb-000024
Figure PCTCN2022119521-appb-000025
注：与模型组比较，*表示差异显著p<0.05；**表示差异极显著p<0.01。
各组动物在造模后第2天(D4)开始出现腹泻，与空白组相比，模型组及各给药组试验期间的腹泻评分显著升高，且腹泻评分随时间增高。阳性组及NCTC9343各组腹泻评分上升的趋势类似于模型组；ZY-312表现出维持腹泻评分稳定的能力。在D6腹泻评分即不明显地小于其他各组；低剂量和中剂量的遏制趋势保持到D13，且高剂量的腹泻评分在D13显著低于模型组；中剂量的遏制趋势维持至D11。因此，各给药组均呈现出遏制腹泻评分上升的能力，其中ZY-312效果优于阳性组和NCTC 9343组。
3)肠道组织病理学检查：选取空肠、回肠和结直肠组织，评价黏膜充血和损伤程度。结果如表14。
表14各组动物病理检查结果(均数±标准差，n＝10)
Figure PCTCN2022119521-appb-000026
Figure PCTCN2022119521-appb-000027
肠道部位，相对于空白组，模型组出现了明显的腺体扩张和黏膜充血，各给药组对上述损伤有治疗效果，NCTC 9343的治疗效果类似于阳性药，ZY-312各组的效果优于NCTC 9343组。
综上所述，脆弱拟杆菌灭活菌粉(ZY-312/NCTC 9343)能够治疗阿法替尼所致的大鼠体重降低、化疗性腹泻、肠黏膜损伤、黏膜充血、炎细胞浸润和腺体损伤，且ZY-312的治疗效果优于NCTC 9343。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 脆弱拟杆菌在制备预防和/或治疗癌症治疗相关性腹泻组合物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述脆弱拟杆菌选自保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312。
  3. 脆弱拟杆菌在制备预防和/或治疗化疗或靶向药物副作用的组合物中的应用。
  4. 根据权利要求3所述的应用，其特征在于，所述脆弱拟杆菌选自保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312。
  5. 根据权利要求4所述的应用，其特征在于，所述化疗药物选自细胞毒类化疗药物、紫杉烷类、铂类、氟尿嘧啶类和喜树碱类中的一种或多种；和/或，所述靶向药物选自Anti-EGFR、Anti-HER-2、Anti-BRAF、Anti-MEK、Anti-EML4/ALK、Anti-VEGF、Multi-targeted TKI、Anti-mTOR、Anti-CDK4/6和Anti-PARP的一种或多种；
    优选地，所述化疗药物选自多西他赛、奥沙利铂、顺铂、氟尿嘧啶、卡培他滨和伊立替康中的一种或多种；和/或，所述靶向药物选自阿法替尼、奥希替尼、奈拉替尼、维莫非尼、达拉非尼、考比替尼、曲美替尼、克唑替尼、阿帕西普、拉帕替尼、吡咯替尼、伊马替尼、帕唑帕尼、依维莫司、西罗莫司、帕博西尼、瑞博西尼、奥拉帕尼和芦卡帕尼中的一种或多种。
  6. 根据权利要求1-3任一项所述的应用，其特征在于，所述脆弱拟杆菌是以下中的一种或多种：脆弱拟杆菌活菌体，经过灭活、基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌，脆弱拟杆菌裂解物，脆弱拟杆菌液体培养上清液。
  7. 根据权利要求6所述的应用，其特征在于，所述灭活的脆弱拟杆菌为形态完整的灭活菌和/或形态不完整的灭活菌，优选为形态完整的灭活菌。
  8. 根据权利要求7所述的应用，其特征在于，所述脆弱拟杆菌是通过干热、湿热、过滤、有机溶剂、化学试剂、紫外或红外射线、发酵、冻干、基因重组、基因修饰或改造的方法中的任意一种或多种灭活的；
    优选地，通过冻干方法得到灭活菌粉。
  9. 根据权利要求1-3任一项所述的应用，其特征在于，所述组合物是药物组合物、食品、保健品或食品添加剂中的任意一种。
  10. 根据权利要求9所述的应用，其特征在于，所述药物组合物含有药学有效剂量的，保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312灭活菌；和/或，所述药物组合物还包括药学上可接受的载体和/或辅料；和/或，所述药物组合物通过口服或灌肠的形式给药。
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