Application of bacteroides ovale protein DUF3109 in preparation of antitumor drugs

Abstract

The invention discloses an application of a DUF3109 protein in preparation of an antitumor drug. The DUF3109 protein is a bacteroides ovatus DUF3109 protein. The DUF3109 protein can promote the formation of a three-level lymph structure in a tumor by regulating CXCL13 secreted by CD4 + T cells, so that the progress of liver cancer is inhibited. In a specific embodiment, the DUF3109 protein shows that the DUF3109 protein has a remarkable inhibition effect on liver in-situ tumors and remarkably promotes CD4 < + > T lymphocytes and Jurkat cells to secrete CXCL13. According to the technical scheme, the application of the DUF3109 protein is expanded, a potential innovative solution is provided for tumor treatment, and a new research direction is provided for development of anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of Bacteroides ovatus protein DUF3109 in the preparation of anti-tumor drugs. Background Art

[0002] Hepatocellular carcinoma (HCC) is the third most lethal malignant tumor worldwide, with a five-year survival rate of less than 20%. Existing treatments for advanced HCC face multiple challenges, including high tumor heterogeneity, active vascular invasion, and complex immune escape mechanisms. Although immunotherapy, represented by PD-1 / PD-L1 inhibitors, has made some progress, the objective response rate of patients is still only 12-25%, and there is a 6.2% risk of hyperprogression. Therefore, there is an urgent need to develop new immune regulatory strategies targeting the tumor microenvironment to reconstruct the dynamic balance of the anti-tumor immune response.

[0003] Tertiary lymphoid structures (TLS) are hubs of immune activation within the tumor microenvironment, recruiting CD4+ T cells and B cells to form germinal center microdomains through CXCL13 / CCL21. Their anti-tumor mechanisms include promoting effector T cell differentiation, enhancing neoantigen presentation, and inducing plasma cells to produce targeted antibodies. Clinical studies have shown that liver cancer patients with high TLS density have significantly improved five-year survival rates and response rates to PD-1 inhibitors.

[0004] Existing methods for promoting TLS formation include phototherapy and nanomaterials, cytokines and chemokines, chemotherapy, and radiotherapy. However, phototherapy and nanomaterials have limited effectiveness in cold tumors due to insufficient laser penetration depth; cytokines and chemokines can trigger systemic inflammation; and chemotherapy and radiotherapy are associated with significant side effects. While these methods have some effectiveness, they also have limitations. Therefore, identifying new therapeutic agents that target TLS formation is crucial for improving the current state of liver cancer immunotherapy and developing new and effective immunotherapies. Summary of the Invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention aims to provide a use of Bacteroides ovatus protein DUF3109 in the preparation of anti-tumor drugs.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides the use of DUF3109 protein in the preparation of drugs for preventing and / or treating tumors.

[0008] In some embodiments of the present invention, the amino acid sequence of the DUF3109 protein is shown in SEQ ID NO: 1.

[0009] In some embodiments of the present invention, the amino acid sequence of the DUF3109 protein includes the amino acid sequence shown in SEQ ID NO: 1 modified at the N-terminus, C-terminus, amino acid backbone and / or amino acid side chain groups to obtain a polypeptide derivative with the same function.

[0010] In some embodiments of the present invention, the modification comprises one or more of glycosylation, phosphorylation, N-methylation, myristoylation, palmitoylation, biotinylation, fluorescent labeling, polyethylene glycol modification, multimeric antigen peptide, prenylation cyclization, acetylation or amidation.

[0011] In some embodiments of the invention, the tumor comprises melanoma, breast cancer, non-small cell lung cancer, liver cancer, or ovarian cancer.

[0012] In some embodiments of the present invention, the dosage form of the drug includes tablets, injections, powders, oral solutions or injections.

[0013] In some embodiments of the present invention, the drug achieves tumor treatment by promoting the formation of tertiary lymphoid structures in tumors and / or promoting the secretion of CXCL13 by T lymphocytes.

[0014] In some embodiments of the present invention, the T lymphocytes include CD4+ T lymphocytes.

[0015] In some embodiments of the present invention, the CD4+ T lymphocytes include Jurkat cells.

[0016] The second aspect of the present invention is the use of a biomaterial in preparing a drug for preventing and / or treating tumors, characterized in that the biomaterial comprises:

[0017] (1) A nucleic acid molecule encoding the DUF3109 protein described in the above aspects;

[0018] (2) an expression cassette containing the nucleic acid molecule described in (1);

[0019] (3) a recombinant vector containing the nucleic acid molecule (1) or the expression cassette (2);

[0020] (4) A recombinant cell containing (1) the nucleic acid molecule, (2) the expression cassette, (3) the recombinant vector or the DUF3109 protein described in the above aspects.

[0021] The third aspect of the present invention provides a pharmaceutical composition, which comprises the DUF3109 protein described in the above aspects and at least one of the biological materials.

[0022] In some embodiments of the present invention, the DUF3109 protein includes modified or unmodified DUF3109 protein.

[0023] In some embodiments of the present invention, the modification comprises adding a tag sequence to the N-terminus or C-terminus of the amino acid sequence of the DUF3109 protein shown in SEQ ID NO: 1.

[0024] In some embodiments of the present invention, the tag sequence includes at least one of a signal peptide, a targeting peptide, a tag peptide, a fluorescent protein, or a transmembrane peptide.

[0025] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0026] In some embodiments of the present invention, the excipients include one or more of a diluent, a stabilizer, an osmotic pressure regulator, a pH regulator, a preservative, and an antioxidant, but are not limited thereto.

[0027] In some embodiments of the present invention, the pH adjuster includes at least one of citric acid / sodium citrate, glucose, sodium hydroxide and sodium bicarbonate, but is not limited thereto.

[0028] In some embodiments of the present invention, the antioxidant includes at least one of vitamin C, sodium metabisulfite, and sodium sulfite / sodium bisulfite, but is not limited thereto.

[0029] In some embodiments of the present invention, the dosage of the pharmaceutical composition is: administering the pharmaceutical composition in an amount of 1 to 5 mg of DUF3109 protein per kg of patient body weight.

[0030] The beneficial effects of the present invention are:

[0031] The present invention provides the use of the DUF3109 protein in the preparation of anti-tumor drugs. The DUF3109 protein inhibits the progression of liver cancer by regulating the secretion of CXCL13 by CD4+ T cells, promoting the formation of tertiary lymphoid structures within tumors. In the examples, compared with the control group, the DUF3109 protein exhibited a significant inhibitory effect on liver tumors in situ and significantly promoted the secretion of CXCL13 by CD4+ T lymphocytes and Jurkat cells. The technical solutions of the present invention not only expand the uses of the DUF3109 protein, but also provide a potential innovative solution for tumor treatment and a new research direction for the development of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the construction of the recombinant plasmid of Example 1 of the present invention.

[0033] Figure 2This is a graph showing the expression and purification results of the DUF3109 protein identified by SDS-PAGE electrophoresis and Coomassie blue staining in Example 1 of the present invention, wherein M is the standard molecular weight protein, 1 represents the protein before induction, 2 represents the protein after induction, 3 represents the sample flow-through, and 4-6 represent the purified protein.

[0034] Figure 3 Figure 1 shows the inhibitory effect of the DUF3109 protein on a liver tumor in situ model. A is a schematic diagram of the experimental steps; B is a live imaging result of mouse tumor tissue after DUF3109 protein intervention in a liver tumor in situ model, where PBS is a blank control group; C is a bar graph comparing infrared radiation intensity in Figure B, *: p < 0.05; D is a multiple immunofluorescence staining assay for detecting intratumoral TLS levels, where the expression level of CD3 represents the density of T cells in the tumor tissue (red fluorescence), the expression level of CD19 represents the density of B cells in the tumor tissue (yellow fluorescence), and blue fluorescence represents DAPI-stained cell nuclei; E is a bar graph comparing the ratio of TLS area to total area in Figure D, **: p < 0.01; F is a bar graph comparing the number of TLS per square millimeter in Figure D, **: p < 0.01.

[0035] Figure 4 Figures 1 and 2 show flow cytometry results demonstrating the effect of the DUF3109 protein described herein on CXCL13 expression in Jurkat cells and human CD4+ T cells. A shows flow cytometry results for CXCL13 expression in human CD4+ T cells; B shows flow cytometry results for CXCL13 expression in Jurkat cells. Both groups used PBS as a blank control group. ****: p < 0.0001, ***: p < 0.001.

[0036] Figure 5 This is an ELISA result of the DUF3109 protein of the present invention on the expression of CXCL13 in mouse CD4+ T cells, wherein PBS is a blank control group, ***: p<0.001. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below by way of specific examples. Unless otherwise specified, the raw materials, reagents, or devices used in the examples are all commercially available or can be obtained by conventional methods. Unless otherwise specified, all experiments or testing methods are conventional methods in the art.

[0038] Example 1 Purification of DUF3109 recombinant protein

[0039] This example provides a method for purifying a recombinant DUF3109 protein, wherein the amino acid sequence of the DUF3109 protein is: MIQIGDVVVSLDVFQEKFLCDLGACKGACCIEGDAGAPVELDEVMELEEVLPVIWDELAPEARAVIEKQGVVYTDQEGDLVTSIVNNKDCVFTCYDENGCCYCAIEKAYRAGKTAFYKPVSCHLYPIRIGDYGPYKAVNYNRWDICKAAVLLGKKENLPVYQFLKEPLIRKFGEEWYKELVTVAEELKKQQYI (SEQ ID NO: 1). The specific experimental steps are as follows.

[0040] (1) The sequence encoding the DUF3109 protein was digested with PaeR7I and XhoI endonucleases and then inserted into the commercially available pET-42b vector to obtain a recombinant plasmid. After correct sequencing, the recombinant plasmid was transformed into the host bacterium Escherichia coli to obtain a monoclonal bacterium.

[0041] (2) Prepare sterile kanamycin (KAN) solid LB medium plates, spread the monoclonal bacteria obtained in (1), and culture overnight.

[0042] (3) Take the plate after culture in (2), select the single clone colony and culture it in LB liquid medium (10 mL) supplemented with KAN (50 μg / mL) for 24 h.

[0043] (4) The bacterial solution obtained in (3) was expanded and cultured in 200 mL of LB liquid medium supplemented with KAN (50 μg / mL), cultured at 37°C until the OD600 value reached about 0.6-0.8, and IPTG was added to a final concentration of 1 mM. The culture was induced at 37°C for 6 h.

[0044] (5) The bacterial solution induced in (4) was centrifuged at 8000 g for 10 min, the cells were collected, washed once with PBS, the supernatant was removed, and 10 mL of PBS, 100 μL of protease inhibitor (Solarbio, P6730) and lysozyme (1 mg / mL) were added to the precipitate and incubated on ice for 30 min.

[0045] (6) Place the solution from step (5) on ice and use an ultrasonic lysator to treat the solution. Set the ultrasonic power to 225W, sonicate for 5 seconds each time, with 10 seconds intervals, for a total of 20 minutes, until the solution is clear.

[0046] (7) Centrifuge the clear solution obtained in (6) at 4°C and 12000g for 10 min, aspirate the supernatant, sieve it (0.45 μm) and transfer it to a new centrifuge tube. Add 4 mL of 50% BeyoGoldTM His-tag Purification Resin (reduction-resistant chelating type) was shaken slowly on a shaker at 4°C for 60 min.

[0047] (8) The solution obtained in (7) was added to an empty chromatography column tube (Biyuntian, FCL06), the sample was slowly loaded, the load flow-through was collected, and the DUF3109 protein was eluted with a gradient solution of 50, 200 and 500 mM Imidazole to obtain the protein.

[0048] (9) The obtained DUF3109 protein was subjected to SDS-PAGE (10%) electrophoresis and Coomassie blue staining to verify the size of DUF3109. At the same time, a 14 kDa dialysis bag was immersed in PBS dialyzate for 16 h for desalting. The protein concentration was measured using a BCA protein concentration assay kit (used according to the instructions), and the protein was freeze-dried overnight and stored at -80°C.

[0049] The constructed recombinant plasmid map is as follows Figure 1 The experimental results are shown in Figure 2 As shown, the purified DUF3109 protein was obtained in this example.

[0050] Example 2 Inhibitory Effect of DUF3109 Protein on Hepatic Tumor in Situ Model

[0051] This example provides a test for the inhibitory effect of DUF3109 protein on a liver tumor in situ model. The experimental steps are as follows: Figure 3 As shown in Figure A, one experimental cycle lasted ten days. Treatment began on the third day after the mice were inoculated with tumor cells. The mice were divided into two groups: a control group (administered with PBS buffer) and a treatment group (administered with DUF3109 protein). The specific experimental steps are as follows.

[0052] (1) Construction of orthotopic liver transplantation tumor mouse model: Mouse hepatocellular carcinoma cells Hepa1-6 (Wuhan Pronocell Life Science Co., Ltd.) in the logarithmic growth phase were prepared into a single cell suspension and washed three times with PBS buffer; female C57BL / 6J mice, 6-8 weeks old, were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution at a standard rate of 5 mL / kg. The abdominal skin of the mice was cut open with sterile scissors to expose the internal organs. The liver was found and 20 μL (5 × 10 5 cells) cell suspension.

[0053] (2) Starting from the third day after inoculation of liver cancer cells, DUF3109 protein in PBS buffer was injected into the liver orthotopic transplanted tumor mouse model through the tail vein every day. The amount of DUF3109 protein used was DUF3109 protein: mouse = 4 mg / kg. The control group was injected with the same amount of PBS buffer.

[0054] (3) Ten days after the establishment of the liver orthotopic transplant tumor model, the mice were anesthetized with 1% sodium pentobarbital and D-luciferin potassium salt solution was injected intraperitoneally. After 5 minutes, the fluorescence level was detected using a small animal live imaging device to record the growth of the mouse liver orthotopic tumor.

[0055] (4) At the same time, after euthanizing the mice, free liver cancer tissue was obtained and paraffin sections were prepared. The sections were placed in fresh xylene for dewaxing for 10 minutes, repeated 3 times, and then gradually hydrated with gradient ethanol (100%, 95%, 70%) for 5 minutes, 5 minutes and 2 minutes respectively. The sections were washed with PBS buffer 3 times, each for 1 minute. The sections were placed in EDTA antigen retrieval solution (Solerbo, C1034), heated in a microwave oven at high heat (700W) until boiling, then turned to low heat (150W) for 15 minutes, and then taken out and naturally cooled to room temperature. The sample area was circled with an immunohistochemistry pen, 5% BSA blocking solution was added, and the sections were kept moist and shaken at room temperature for 10 minutes. After removing the blocking solution, the diluted CD3 primary antibody solution (1:1000, abcam, ab237721) was added and incubated at 37°C for 2 hours. The sections were washed with TBST buffer for 3 minutes, repeated once. Fluorescently labeled secondary antibody (1:600, Yazyme, LF102) was added dropwise and incubated at room temperature for 30 minutes. After washing with TBST again, TSA + enhancer (five-color multiple fluorescent staining kit, GuduoBiological, HYDS0045) (diluted at 1:100) was added dropwise and moisturized and incubated for 15 minutes. Wash with TBST buffer 3 times, 3 minutes each time. The above steps were then repeated and a second round of staining was performed using CD19 primary antibody solution (1:1000, abc am, ab317335). After staining, the slides were sealed with anti-fluorescence quenching sealing fluid (containing DAPI) (Biyuntian, P0131) and the expression of CD3 and CD19 was observed and photographed using a Nikon inverted fluorescence microscope.

[0056] The experimental results are as follows Figure 3 As shown, Figure 3 Middle B shows that the tumor volume of mice treated with DUF3109 was significantly reduced compared with the control group, and there was a significant difference ( Figure 3 Middle C), Figure 3 Middle D shows the abundance of TLS in tumor tissue after treatment of mouse liver orthotopic transplanted tumor model with DUF3109 protein and PBS buffer by multiple immunofluorescence. The expression level of CD3 represents the density of T cells in tumor tissue (red represents positive); the expression level of CD19 represents the density of B cells in tumor tissue (yellow represents positive). The area where both cells are enriched can be defined as TLS structure. The abundance of TLS in tumors of mice treated with DUF3109 was significantly increased compared with that in the control group ( Figure 3 E and F).

[0057] Example 3 DUF3109 protein promotes the expression of CXCL13 in CD4+ T cells

[0058] This example provides information on the effects of DUF3109 protein on the expression of the chemokine CXCL13 in Jurkat cells and human CD4+ T lymphocytes. PBS buffer was used as a control group, and an enzyme-linked immunosorbent assay (ELISA) was used to examine the effect of DUF3109 protein treatment on the secretion of CXCL13 in mouse CD4+ T lymphocytes for 48 hours. The specific experimental steps are as follows.

[0059] 1. Mouse CD4+ T cell extraction and culture

[0060] (1) After euthanizing mice, the spleens were removed in a sterile environment and gently ground in PBS buffer containing 2% FBS to prepare a mononuclear cell suspension;

[0061] (2) The cell suspension was treated with red blood cell lysis buffer at room temperature for 10 min to remove red blood cells. After lysis, the cells were washed by centrifugation (300 g, 5 min), the lysis buffer was removed, and then the cells were resuspended in PBS buffer;

[0062] (3) Filter the cell suspension obtained in (2) through a 70 μm cell strainer to remove tissue debris;

[0063] (4) using a mouse CD4+ T cell magnetic bead sorting kit to sort CD4+ T cells from the cell suspension obtained in (3);

[0064] (5) Mouse CD3 stimulator (BioGems, 05112-25) and CD28 stimulator (BioGems, 10312-25-500) were added to the CD4+ T cells obtained in (4) to a final concentration of 3 μg / mL. Mouse IL-2 cytokine (PEPROTECH, 212-12-5) was also added to a final concentration of 20 nM in the solution. The cells were then placed in a 37°C, 5% CO2 cell culture incubator for continued culture.

[0065] 2. Sorting and culturing human CD4+ T lymphocytes

[0066] (1) Collect fresh anticoagulated human peripheral blood, dilute it with PBS buffer at a ratio of 1:1, and mix it by inverting;

[0067] (2) Add 5 mL of lymphocyte separation medium to a 15 mL centrifuge tube, gently cover 10 mL of the diluted blood in (1) on the lymphocyte separation medium, and centrifuge at 400 g for 30 min at room temperature with an acceleration of 0;

[0068] (3) After centrifugation, the tube was taken out smoothly, and the lymphocyte layer was carefully aspirated using a Pasteur pipette. PBS buffer was added and centrifuged at 300 g for 5 min at room temperature to collect and extract peripheral blood mononuclear cells.

[0069] (4) using a human CD4+ T lymphocyte isolation kit according to the instructions to separate CD4+ T lymphocytes from the cells obtained in (3);

[0070] (5) The CD4+ T lymphocytes obtained in (4) were washed once by centrifugation with PBS solution (300g, 5min), resuspended in RPMI-1640 medium containing 10% FBS, and 25μL of CD3 / CD28 combined activator (Thermo Fisher, 11161D) and mouse IL-2 cytokine (PEPROTECH, 212-12-5) were added to make the final concentration in the solution 20nM. The cells were placed in a cell culture incubator at 37°C and 5% CO2 for further culture.

[0071] 3. Flow cytometry experiments

[0072] (1) Human CD4+ T lymphocytes (1×10 6 ) and Jurkat cells (1×10 6 100 μg of the DUF3109 protein was added to each of the cells (Wuhan Punosai Life Science Technology Co., Ltd., catalog number CL-0129) and treated for 24 h, and then centrifuged at 300 g and 4 °C for 10 min to collect the cells for subsequent antibody staining steps;

[0073] (2) The cells were incubated with anti-CXCL13 antibody (Abmart, PC2553S) at 4°C for 1 h, washed twice with PBS, and then incubated at 4°C for 30 min with CoraLite594–conjugated Goat Anti-Rabbit IgG (H+L) diluted 1:200. The cells were washed twice with PBS, collected, fixed with 4% paraformaldehyde, and detected by flow cytometry.

[0074] 4. Enzyme-linked immunosorbent assay

[0075] The enzyme-linked immunosorbent assay (ELISA) kit (MM-45471M2) was used for the ELISA. The specific experimental steps are as follows.

[0076] In the above obtained 1×10 7100 μg of DUF3109 was added to CD4+ T lymphocytes of each mouse and cultured for 24 hours. The cell culture medium was collected and centrifuged at 300g for 10 minutes at room temperature. The supernatant of the culture medium was collected and diluted with sample diluent. The standard solution was diluted and the standard solution, sample to be tested, negative control, and positive control were added in sequence. The plates were incubated at 37°C for 30 minutes. The plates were washed 5 times and the enzyme-labeled reagent was added and incubated at 37°C for 30 minutes. The plates were washed 5 times and the color developing solutions A and B were added. The plates were reacted at 37°C in the dark for 15 minutes. The stop solution was added and the OD value of the sample wells was detected using a microplate reader within 15 minutes.

[0077] The experimental results are as follows Figure 4 and 5 As shown, Figure 4 As shown in Figure A, in human CD4+ T lymphocytes, DUF3109 promoted the positive rate of cell expression of CXCL13 to 52.8%, which was significantly higher than that of the control group (26.5%). Figure 4 Middle B shows that in Jurkat cells, the negative control promoted the positive rate of CXCL13 expression in cells to 9.16%, while DUF3109 promoted the positive rate of CXCL13 expression in cells to 52.1%, which was significantly higher than that in the control group; Figure 5 It was shown that DUF3109 protein had a significant stimulating effect on the secretion of CXCL13 by mouse CD4+ T lymphocytes.

[0078] In summary, the DUF3109 protein described in the present invention promotes the formation of intratumoral TLS and inhibits the progression of liver cancer by regulating the secretion of CXCL13 by CD4+ T cells. This protein exhibits significant anti-tumor activity and has broad application potential. It can be used to develop anti-tumor protein drugs, providing an innovative therapeutic approach for the current field of tumor treatment.

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of DUF3109 protein in the preparation of a drug for preventing and / or treating tumors, characterized in that: The amino acid sequence of the DUF3109 protein is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The tumor includes melanoma, breast cancer, non-small cell lung cancer, liver cancer or ovarian cancer.

3. The use according to claim 1, characterized in that The dosage form of the drug includes tablets, injections, powders, oral solutions or injections.

4. The use according to claim 1, characterized in that The drug achieves tumor treatment by promoting the formation of tertiary lymphoid structures in tumors and / or promoting T lymphocytes to secrete CXCL13.

5. The use according to claim 4, characterized in that The T lymphocytes include CD4+ T lymphocytes; the CD4+ T lymphocytes include Jurkat cells.

6. Use of biomaterials in the preparation of drugs for preventing and / or treating tumors, characterized in that: The biological material includes: (1) A nucleic acid molecule encoding the DUF3109 protein of claim 1; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant vector containing the nucleic acid molecule (1) or the expression cassette (2); (4) A recombinant cell containing (1) the nucleic acid molecule, (2) the expression cassette, (3) the recombinant vector or the DUF3109 protein according to claim 1.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises at least one of the DUF3109 protein according to claim 1 and the biological material according to claim 6.

8. The pharmaceutical composition according to claim 7, characterized in that The DUF3109 protein includes modified or unmodified DUF3109 protein; the modification includes adding a tag sequence to the N-terminus or C-terminus of the amino acid sequence of the DUF3109 protein shown in SEQ ID NO: 1; the tag sequence includes at least one of a signal peptide, a targeting peptide, a tag peptide, a fluorescent protein or a transmembrane peptide.

9. The pharmaceutical composition according to claim 7 or 8, characterized in that The pharmaceutical composition further includes pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that The auxiliary materials include one or more of diluents, stabilizers, osmotic pressure regulators, pH regulators, preservatives and antioxidants.

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