Use of vitamin b12 to enhance dendritic cell function

Abstract

The application discloses a use of vitamin B12 for enhancing functions of dendritic cells, and belongs to the field of biological medicines. The application discloses that vitamin B12 can enhance the maturation and antigen presentation capacity of dendritic cells, so that the immune response mediated by the dendritic cells can be enhanced, and further provides a new use of vitamin B12 as an immunotherapy reagent and a drug application of vitamin B12 in improving the effect of tumor immunotherapy. The discovery realizes a new use of an old drug, can greatly shorten the research and development cost and risk, and meets the medical needs of enhancing the functions of dendritic cells in various clinical scenarios such as tumors, chronic infections, immune aging, vaccine immunization, allergic diseases and immune reconstruction after radiotherapy and chemotherapy.

Description

Technical Field

[0001] This invention relates to reagents and methods for use in immunotherapy, belonging to the field of biomedicine, and specifically to the application of vitamin B12 in enhancing the function of dendritic cells (DCs). Background Technology

[0002] Dendritic cells (DCs) are the most potent professional antigen-presenting cells in the body, playing a central role in initiating, regulating, and maintaining specific immune responses. Immature DCs efficiently take up and process antigens in peripheral tissues, then migrate to secondary lymphoid organs upon stimulation by maturation signals. They highly express major histocompatibility complex (MHC) molecules and co-stimulatory molecules, presenting antigenic peptides to naive T cells and inducing their activation, proliferation, and differentiation. Therefore, the functional state of DCs directly determines the strength and quality of the body's anti-infection and anti-tumor immune responses.

[0003] Numerous studies have confirmed that in the tumor microenvironment, chronic viral infections (such as chronic hepatitis B and human immunodeficiency virus infection), intracellular bacterial or parasitic infections (such as Mycobacterium tuberculosis and Toxoplasma gondii infection), and aging-induced immunosenescence, local or systemic dendritic cells (DCs) often exhibit varying degrees of functional decline or tolerance. Specifically, this manifests as weakened antigen uptake capacity, insufficient expression of co-stimulatory molecules (such as CD80 and CD86), reduced secretion of pro-inflammatory cytokines (such as IL-12), and even induction of T cell anergy or regulatory T cell generation. This DC dysfunction directly leads to the body's inability to effectively initiate and maintain protective T cell immune responses against pathogens or tumor cells, and is a key common mechanism contributing to tumor immune escape, persistent chronic infections, increased susceptibility to infection in the elderly, and elevated vaccine immunization failure rates. Furthermore, after tumor radiotherapy or chemotherapy, the function of residual DCs is generally suppressed, becoming an important reason for delayed immune reconstitution, increased opportunistic infections, and increased risk of tumor recurrence.

[0004] Furthermore, even when DC function is not significantly impaired, further enhancing its antigen-presenting capacity and co-stimulatory signal output still has significant therapeutic value. For example, in immune checkpoint inhibitor therapy, the activation efficiency of functional DCs is a key factor determining the response rate; in vaccination, enhancing DC antigen uptake and presentation can directly improve vaccine immunogenicity. Therefore, finding drugs that can safely and easily enhance DC function in vivo is not only a practical need to correct DC dysfunction, but also has broad and urgent medical significance for improving the efficacy of various immunotherapeutic strategies.

[0005] Although some studies have explored the ability of certain agents / drugs to exert immunomodulatory effects by enhancing dendritic (DC) function, such as biologics like GM-CSF or CD40 agonist antibodies, these studies are prone to inducing serious adverse events such as cytokine storms or autoimmune reactions, resulting in a narrow safety window. Vitamin B12 (also known as cobalamin) is an essential micronutrient for maintaining hematopoietic function, nervous system integrity, and DNA synthesis; however, its potential immunomodulatory mechanisms remain unclear. Currently, there is no guidance or inspiration to use vitamin B12 as an actively pharmacologically active substance to directly enhance DC function in a non-deficiency state to treat or prevent the aforementioned immune-related diseases. Summary of the Invention

[0006] The purpose of this invention is to find drugs that can enhance dendritic cell (DC) function and thus exert immunomodulatory effects. Through in-depth research into the direct effects and molecular mechanisms of vitamin B12 in the immune system, a novel drug application for vitamin B12—safe, convenient, and highly effective—that directly enhances dendritic cell function was unexpectedly discovered. This application meets the urgent medical needs for enhancing DC function in various clinical scenarios, including oncology, chronic infections, immunosenescence, vaccine immunization, allergic diseases, and immune reconstitution after radiotherapy and chemotherapy. This discovery breaks through the traditional understanding of vitamin B12 as merely a nutritional supplement, opening up a completely new application direction for it as an active immunomodulator.

[0007] Specifically, enhancing dendritic cell function refers to achieving one or more of the following pharmacological effects:

[0008] (a) Upregulates the expression of MHC-I and MHC-II molecules on the surface of dendritic cells;

[0009] (b) Upregulate the expression of co-stimulatory molecules on the surface of dendritic cells, such as CD80 and / or CD86;

[0010] (c) Promotes the secretion of pro-inflammatory factors such as IL-12, IL-6, and TNF-α by dendritic cells;

[0011] (d) Enhance the antigen-presenting ability of dendritic cells;

[0012] (e) Enhance antigen specificity CD4 + T cells and / or CD8 + T cell activation and proliferation.

[0013] Furthermore, this invention provides the use of vitamin B12 in the preparation of medicaments for treating and / or preventing diseases by enhancing dendritic cell function, not limited to the pathological state of "dendritic cell dysfunction." In many clinical scenarios, although the host dendritic cell function does not show obvious defects, significant immunotherapeutic gains can still be obtained by further enhancing its activation level and antigen-presenting capacity. Specifically, the pharmaceutical uses of vitamin B12 provided by this invention cover the following two application scenarios:

[0014] The first category involves correcting existing dendritic cell dysfunction. This includes, but is not limited to: malignant tumors (e.g., for tumor immunotherapy); infectious diseases, including acute infections, chronic viral infections (e.g., chronic hepatitis B, HIV infection), intracellular bacterial infections (e.g., tuberculosis), and parasitic infections; age-related immune decline (immunoregeneration); allergic diseases, especially those mediated by Th2-dominant immune disorders, such as allergic asthma, allergic rhinitis, and atopic dermatitis; and immunosuppressive states following tumor radiotherapy and chemotherapy, used to promote immune reconstitution.

[0015] The second category involves further enhancing the function of dendritic cells, building upon their normal function, to improve the effectiveness of immunotherapy. Typical applications include improving vaccine immunogenicity and enhancing the quality of in vitro prepared dendritic cell vaccines. These vaccines can be prophylactic or therapeutic, such as subunit or inactivated vaccines for influenza, hepatitis B, coronavirus, and human papillomavirus, in which vitamin B12 regulates the immune response by enhancing dendritic cell function.

[0016] This invention also provides the use of vitamin B12 in the preparation of reagents for enhancing dendritic cell function in vitro. Further, this reagent is used to induce or activate dendritic cells (DCs) in vitro to prepare DC vaccines. Preferably, in the reagent for enhancing dendritic cell function in vitro, the concentration of vitamin B12 is 250–1000 μM.

[0017] Furthermore, the drug or reagent uses vitamin B12 as the active ingredient, and the vitamin B12 includes different forms such as methylcobalamin, cyanocobalamin, adenosylcobalamin, and hydroxycobalamin, or their derivatives.

[0018] The drug or reagent may be used alone or in combination with other therapeutic agents in the form of a composition. The other therapeutic agents refer to any drug or treatment that can provide therapeutic benefit by enhancing dendritic cell function, including but not limited to chemotherapy drugs (such as platinum-based drugs, taxanes, antimetabolites, etc.), radiotherapy, immune checkpoint inhibitors (such as anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies), tumor vaccines, therapeutic antibodies, cytokines, or other immunomodulators.

[0019] The drug can be formulated into oral, injectable, mucosal, or topical dosage forms and administered via systemic or local routes. The drug also includes pharmaceutically acceptable excipients and / or pharmaceutically acceptable carriers. Preferably, the carrier includes, but is not limited to, liposomes, nanoparticles, microneedles, microspheres, and gels to achieve sustained-release, controlled-release, or targeted delivery of the drug.

[0020] In the application of vitamin B12 in inhibiting tumor growth and activating immunity in tumor-bearing mouse models, the treatment routes and dosages are as follows: subcutaneous injection of 0.6, 1.8, and 5.4 mg / kg every three days. Based on the conversion factor of 10 (9.1–12.3):1 between mice and humans, and the conversion factor of 2 (1.5–2.5):1 between oral and subcutaneous injection, and calculated for an adult weighing 60 kg, this is equivalent to an oral dose of 2.4 mg / day, 7.2 mg / day, and 21.6 mg / day (calculated by evenly distributing to daily doses). This dosage is within the known clinical safety range of vitamin B12 and is far below the potential risk threshold reported in the literature (such as oral doses exceeding 1000 mg per day).

[0021] In some embodiments of the present invention, vitamin B12 can effectively inhibit tumor growth, prolong the median survival of mice in tumor-bearing mouse models, and increase the proportion of mature dendritic cells (DCs) in tumor tissue and draining lymph nodes, as well as activate CD8+. + The proportion of T cells was also observed. Simultaneously, no abnormalities were found in blood biochemistry and routine blood tests in mice after treatment, no systemic or local inflammatory responses were induced, and no pathological changes were observed in major organs and skin tissues, indicating good in vivo safety. Furthermore, the combination of vitamin B12 and immune checkpoint inhibitors (ICIs) further enhanced DC maturation and T cell activation compared to ICIs alone, significantly upregulated the secretion levels of pro-inflammatory factors in the tumor microenvironment, and improved the anti-tumor therapeutic efficacy of ICIs. Moreover, in vitro mechanism studies demonstrated that vitamin B12, by increasing methyl donors within DCs and enhancing Setd2-mediated trimethylation of histone H3 at position 36 (H3K36me3) at the Jak2 gene locus, epigenetically activated the JAK-STAT signaling pathway, driving DC activation and antigen presentation. Compared with existing technologies, the novel pharmaceutical use of vitamin B12 in this invention has the following outstanding beneficial effects:

[0022] (1) High safety: Long-term clinical experience with vitamin B12 shows that it has no significant toxicity, no risk of cytokine storm, and does not induce excessive systemic inflammatory response, far exceeding existing biological immune agonists.

[0023] (2) Convenient and low-cost administration: It can be administered orally, by injection or locally. Its chemical structure is stable and easy to produce and store, which greatly reduces the burden on patients and medical costs.

[0024] (3) Direct pharmacological enhancement of DC: For the first time, it was revealed that vitamin B12 can directly and significantly enhance multiple key immune functions of DC in a non-deficient state, breaking the previous technical prejudice that it was only regarded as a "nutritional supplement";

[0025] (4) Broad range of indications: Based on the common mechanism of enhancing DC function, it can simultaneously cover multiple medical fields such as tumor immunotherapy, anti-infection, antiviral, immune aging, vaccine adjuvant, immune shift in allergic diseases and immune reconstitution of radiotherapy and chemotherapy. Attached Figure Description

[0026] Figure 1 The antitumor effects of different forms of vitamin B12 in mice bearing B16F10 subcutaneous melanoma in Example 1 are shown, where: A is the tumor growth curve of mice after treatment with four different forms of vitamin B12 (adenosylcobalamin, methylcobalamin, cyanocobalamin, and hydroxocobalamin); B is the survival curve of mice after treatment with four different forms of vitamin B12 (adenosylcobalamin, methylcobalamin, cyanocobalamin, and hydroxocobalamin).

[0027] Figure 2 The results of Example 2 show the antitumor effects of different doses of vitamin B12 in mice bearing B16F10 subcutaneous melanoma. In this example, A shows the tumor growth curves of mice after treatment with three different concentrations of methylcobalamin (0.6 mg / kg, 1.8 mg / kg, and 5.4 mg / kg); and B shows the survival curves of mice after treatment with three different concentrations of methylcobalamin (0.6 mg / kg, 1.8 mg / kg, and 5.4 mg / kg).

[0028] Figure 3 The results of routine blood tests and blood biochemistry analysis of mice treated with methylcobalamin (0.6, 1.8, and 5.4 mg / kg) in Example 2 are as follows. The measured parameters include: A. Alanine aminotransferase (ALT); B. Aspartate aminotransferase (AST); C. Creatinine (CREA); D. Creatine kinase (CK); E. Lactate dehydrogenase (LDH); F. White blood cell (WBC) count; G. Hemoglobin (HGB) content; H. Red blood cell (RBC) count.

[0029] Figure 4The figures for Example 2 are: serum and injection site cytokine levels in mice after treatment with PBS or 5.4 mg / kg mecobalamin. A is a heatmap showing the levels of multiple cytokines (IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, and TNFα) in mouse serum, quantitatively determined using Luminex multifactor detection technology. B is a heatmap showing the relative mRNA expression levels of local inflammatory cytokines (IL-1β, IL-6, IL-12p70, and TNFα) in the subcutaneous injection site skin tissue, detected by RT-qPCR.

[0030] Figure 5 The immunological effects in mice treated with PBS or 5.4 mg / kg mecobalamin in Example 3 included: A. CD80 in tumor tissue. + / CD86 + DC ratio; B. H-2Kb in tumor tissue + DC ratio; C. CD80 in lymph nodes + / CD86 + DC ratio; D. H-2Kb in lymph nodes + DC ratio; E. IFN-γ in tumor tissue + CD8 + T cell percentage; F. IFN-γ in lymph nodes + CD8 + T cell ratio.

[0031] Figure 6 The results of Example 4 demonstrate the synergistic enhancement of anti-MC38 tumor effects of mecobalamin and immune checkpoint therapy, where: A represents the tumor growth curves of mice bearing MC38 subcutaneous tumors treated with PBS, mecobalamin, aPD-1, and mecobalamin combined with aPD-1, respectively; B represents the tumor weight of each treatment group at the experimental endpoint; C represents the overall survival (OS) curve of the mice; and D represents the ex vivo tumor tissue at the experimental endpoint.

[0032] Figure 7 The evaluation of the immune effects of methylcobalamin combined with immune checkpoint therapy in mice bearing MC38 subcutaneous tumors in Example 4 included: A. CD80 in tumor tissue. + / CD86 + B. The proportion of DCs; C. CD80 in tumor draining lymph nodes (TDLNs) + / CD86 + The proportion of DCs; C. IFN-γ in tumor tissue + CD8 + The proportion of T cells; D. IFN-γ in tumor-draining lymph nodes + CD8 +The proportion of T cells.

[0033] Figure 8 The effects of different concentrations of methylcobalamin (250 μM, 500 μM, 1000 μM) on bone marrow-derived dendritic cells (BMDC) in Example 5 after 24 hours of treatment are shown. In this example, A represents the proportion of cells that are positive for co-stimulatory molecules CD80 and CD86 by flow cytometry; B represents the proportion of cells that are positive for the antigen-presenting complex SIINFEKL-H-2Kb; and C, D, and E represent the secretion levels of cytokines TNF-α (C), IL-6 (D), and IFN-β (E) in the BMDC supernatant as detected by ELISA.

[0034] Figure 9 The mRNA expression level (A) and protein expression level (B) of JAK-STAT and antigen presentation-related genes on BMDC after treatment with methylcobalamin in Example 6 are shown.

[0035] Figure 10 The optimization of the formulation parameters of the methylcobalamin drug-loaded liposome (LipoMeCbl) in Example 7 included: different molar ratios of the main lipid material to cholesterol (n... HSPC : n Chol The effects of ratios of 0.5:1, 1:1, 1.5:1, and 2:1 on liposome particle size (A), polydispersity index (PDI) (B), encapsulation efficiency (EE%) (C), and drug loading (DL%) (D) were investigated; at a fixed n... HSPC : n Chol Under the premise of 1:1, we examine different drug-lipid molar ratios (n lipo : n vb12 Effects of ratios 1.5:1, 3:1, 6:1 and 12:1 on liposome particle size (E), PDI (F), EE% (G) and DL% (H).

[0036] Figure 11 In Example 11, the content of cobalt (Co) in BMDC after treatment with free methylcobalamin (MeCbl) or methylcobalamin liposomes (LipoMeCbl) was quantitatively detected by inductively coupled plasma mass spectrometry (ICP-MS).

[0037] Figure 12 For the flow cytometry quantitative analysis of CD11c in BMDCs treated with PBS, empty liposomes (LipoBlank), free methylcobalamin (MeCbl), or methylcobalamin liposomes (LipoMeCbl) for 24 h in Example 12, + CD80 in BMDCs + CD86 + (A) CD40 +(B) The ratio of the cell subsets to SIINFEKL-H-2Kᵇ (specific antigen peptide-MHC class I molecular complex) and (C) the cell subsets.

[0038] Figure 13 The efficacy of methylcobalamin liposomes in the B16F10 tumor model in Example 13 is shown, where: A is the tumor growth curve after treatment with PBS, empty liposomes (LipoBlank), free methylcobalamin (MeCbl, 5.4 mg / kg), or methylcobalamin liposomes (LipoMeCbl, 1 mg / kg); B is the Kaplan-Meier survival curve of mice in each group during the 20-day observation period; C is the average weight and individual distribution of ex vivo tumors in each group at the experimental endpoint; D is a photograph of representative ex vivo tumors in each group at the end of the experiment (scale bar = 2 cm).

[0039] Figure 14 The immunological effects of methylcobalamin liposomes in the B16F10 tumor model are shown in Example 13, where: A represents mature DCs (CD80) in tumor tissue. + CD86 + B represents the proportion of mature DCs (CD80) in the draining lymph nodes; B represents the proportion of mature DCs (CD80) in the draining lymph nodes. + CD86 + The proportion of DC); C is the IFN-γ in tumor tissue. + CD8 + The proportion of T cells; D represents the IFN-γ concentration in the draining lymph nodes. + CD8 + The proportion of T cells.

[0040] Figure 15 The efficacy of hydroxycobalamin liposomes in the B16F10 tumor model in Example 14 is shown, where: A is the tumor growth curve after treatment with PBS, doxorubicin liposomes (Liposome Dox, 3 mg / kg), hydroxycobalamin liposomes (Liposome OHCbl, 10 mg / kg), or a combination of doxorubicin liposomes (Liposome Dox, 3 mg / kg, and hydroxycobalamin liposomes (Liposome OHCbl, 10 mg / kg); B is the average weight of the ex vivo tumors of mice in each group.

[0041] Figure 16 The immunological effects of hydroxycobalamin liposomes in the B16F10 tumor model are shown in Example 14, where: A represents mature dendritic cells (CD80) in lymph nodes. + CD86 + The proportion of CD8 in the DC); B is the proportion of CD8 in the draining lymph nodes. + The proportion of T cells; C represents mature DCs (CD80) in tumor tissue. + CD86+ The proportion of CD8+ in the tumor tissue; D represents the proportion of CD8+ in the tumor tissue. + The proportion of T cells. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the implementation of the present invention is not limited thereto.

[0043] In addition, unless otherwise specified, the techniques used in the following examples are conventional methods well known to those skilled in the art, and specific operations can be found in molecular biology guidelines or product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0044] Example 1

[0045] Antitumor effects of four different forms of vitamin B12 (VB12) in a mouse B16F10 tumor model

[0046] Six- to eight-week-old C57BL / 6 mice were injected with murine melanoma B16F10 cells, and 100 μL of cell suspension (i.e., 2 × 10⁶ cells per mouse) was collected. 5 (1 cell) was subcutaneously inoculated into the right groin of mice. The tumor volume was increased to approximately 80 mm. 3 Tumor-bearing mice were randomly divided into 5 groups (n=6 per group): PBS group, mecobalamin, adenosylcobalamin, cyanocobalamin, and hydroxocobalamin group. Each group was administered the drug at a dose of 5.4 mg / kg, with 100 μL injected subcutaneously into each mouse. Subcutaneous administration was performed on days 0, 3, 6, and 9, for a total of 4 administrations. The long and short diameters of the tumor were measured every two days using electronic calipers (tumor volume calculation formula = 0.5 × long diameter × short diameter). 2 The tumor volume was calculated and a tumor growth curve was plotted. When the tumor volume was ≥1500 mm... 3 Or, severe health deterioration (such as decreased activity >50% or weight loss >20%) was used as the survival endpoint (mice were euthanized). Kaplan-Meier survival curves were plotted and the differences in survival rates between groups were analyzed using the Log-rank test.

[0047] Result: As Figure 1 As shown in Figure A, at the tumor growth monitoring endpoint, all VB12 treatment groups showed significant tumor growth inhibition compared to the PBS control group. The tumor inhibition rate was 48% in the adenosylcobalamin (AdoCbl) group, 59% in the mecobalamin (MeCbl) group, 49% in the cyanocobalamin (CNCbl) group, and 50% in the hydroxycobalamin (OHCbl) group. Furthermore, survival data are shown below. Figure 1The results showed that the median survival of mice in the PBS group was 14 days, while the median survival of mice in the adenosylcobalamin (AdoCbl) group and the cyanocobalamin (CNCbl) group was 18 days. The median survival of mice in the mecobalamin (MeCbl) group and the hydroxycobalamin (OHCbl) group was extended to 20 days. These results indicate that all four forms of VB12 can effectively inhibit the growth of B16F10 tumors and prolong the survival of tumor-bearing mice.

[0048] Example 2

[0049] Antitumor efficacy and in vivo safety of different doses of mecobalamin in a mouse B16F10 tumor model

[0050] A mouse model of subcutaneous melanoma was established according to Example 1, and the tumor volume was increased to approximately 80 mm. 3 Tumor-bearing mice were randomly divided into 5 groups: PBS group, mecobalamin 0.6, 1.8 and 5.4 mg / kg group, and each mouse was subcutaneously injected with 100 μL on days 0, 3, 6 and 9, for a total of 4 administrations. Tumor growth curves were plotted and mouse survival curves were recorded.

[0051] For in vivo safety testing: Complete blood count and blood biochemistry tests were performed on day 12 after administration. Additionally, 50 μL serum samples were collected from both the PBS group and the high-dose group (5.4 mg / kg) to detect serum cytokine levels, following the instructions of the Luminex Protein Multifactor Assay Kit. 1 cm of skin was collected from the injection site of both the PBS group and the high-dose group (5.4 mg / kg). 2 After grinding, collect the sample into a centrifuge tube, centrifuge at 12000 rpm at 4℃ for 10 minutes, and take the supernatant to detect the cytokines IL-1β, IL-12p70, IL-6 and TNF-α, according to the ELISA kit instructions.

[0052] Result: As Figure 2 The results from Figure A show that methylcobalamin's inhibitory effect on B16F10 tumor growth exhibits a significant dose-dependent effect. Mouse survival data are as follows... Figure 2 As shown in Figure B, this further confirms the dose-dependent advantage of methylcobalamin in prolonging the survival of tumor-bearing mice. Blood biochemistry and complete blood count results are as follows... Figure 3 The results showed that, compared with the PBS control group, there were no statistically significant differences in liver and kidney function indicators (ALT, AST, CREA, CK, LDH, etc.) and myocardial injury indicators (WBC, RBC, HGB, etc.) among the different doses of mecobalamin administration groups, and no significant hematological toxicity was observed. Further analysis of cytokine levels in mouse serum and skin at the injection site after administration of high-dose mecobalamin (5.4 mg / kg) was performed using Luminex technology. Figure 4The results showed that the secretion levels of several key inflammatory factors in the treatment group remained consistent with those in the control group, with no significant changes, suggesting that high-dose methylcobalamin did not induce systemic or local skin inflammatory reactions. These results indicate that different doses of methylcobalamin have good safety in vivo.

[0053] Example 3

[0054] Evaluation of the immunological effects of methylcobalamin in a mouse B16F10 tumor model

[0055] A subcutaneous melanoma mouse model was established according to Example 1. Subcutaneous administration was performed on days 0, 3, 6, and 9 (PBS group and mecobalamin 5.4 mg / kg group, respectively). Mice were sacrificed by cervical dislocation on day 12, and single-cell suspensions of tumor tissue and tumor-draining lymph node tissue were prepared. The prepared single-cell suspensions were seeded into 100 μL per well of a U-bottom 96-well plate (cell density 1×10⁻⁶). 6 Add 0.8 μL of Fc blocking antibody to each well (cells / mL), mix gently, and incubate at room temperature for 10 min to block nonspecific binding. Except for the blank control wells, add 0.7 μL of Fixable Viability Dye eFluor™ 506 to each well, mix well, and incubate at 4°C in the dark for 30 min. Centrifuge at 4°C, 450 × g for 5 min, discard the supernatant, wash with PBS, and resuspend. Then, stain lymph node and tumor tissue DCs and CD8 cells according to the flow cytometry antibody staining instructions. + T cell surface markers were stained. The proportion of immune cells in tumor tissue and draining lymph nodes was detected by flow cytometry.

[0056] Result: As Figure 5 As shown, flow cytometry analysis of DC subsets revealed that, compared to the PBS group, CD80 levels in tumor tissue were significantly higher after mecobalamin administration. + CD86 + The proportion of DCs increased by 52% compared to the control group, while CD11c + H-2Kb + The proportion of DCs increased by 182%. In draining lymph nodes, CD80... + CD86 + The proportion of DCs increased by 140%, CD11c + H-2Kb + The 10% increase in the proportion of dendritic cells (DCs) indicates that mecobalamin can simultaneously promote the maturation of DCs in both the tumor site and draining lymph nodes, enhancing their antigen-presenting capacity. Furthermore, after mecobalamin administration, IFN-γ levels in tumor tissue increased. + CD8 + The proportion of T cells increased by 155% compared to the control group, and IFN-γ was found in draining lymph nodes (LNs).+ CD8 + The proportion of T cells increased by 217%, suggesting that methylcobalamin can effectively activate CD8. + T cells enhance the anti-tumor immune response.

[0057] Example 4

[0058] In vivo antitumor efficacy and immunological effects of methylcobalamin combined with PD-1 antibody in MC38 mouse tumor model

[0059] Mouse-derived colorectal cancer MC38 cells were administered at a rate of 8 × 10⁸ cells per mouse. 5 Cells were subcutaneously inoculated at a density of 1000 mcg into the right groin of C57BL / 6 mice. The tumor volume was increased to approximately 80 mm. 3 Tumor-bearing mice were randomly divided into 5 groups: PBS group, mecobalamin group, aPD-1 group, and mecobalamin combined with aPD-1 group, with 6 mice in each group. The mecobalamin group was administered 5.4 mg / kg via subcutaneous injection (100 μL); the aPD-1 group was administered 200 μg / mouse via intraperitoneal injection (100 μL). Administered medication on days 0, 3, 6, and 9, once every 3 days, for a total of 4 times. Tumor growth curves and mouse survival were recorded. On day 13, mice were sacrificed by cervical dislocation, and the weight of the excised tumor was measured. Single-cell suspensions of tumor tissue and draining lymph nodes were prepared, and the proportion of immune cells in the tumor was detected by flow cytometry.

[0060] Result: As Figure 6 As shown in Figures A and B, compared with the aPD-1 monotherapy group, the combination therapy group of mecobalamin and aPD-1 showed a further significant reduction in tumor volume of 58% and a significant reduction in mean tumor weight of 55%. Meanwhile, the survival curve results are as follows... Figure 6 In the combined treatment group, the survival time was significantly prolonged, and ex vivo tumor tissue was observed. Figure 6 The results from the study further confirmed the stronger tumor-suppressing effect of the combined therapy.

[0061] To elucidate the immunological mechanism underlying the synergistic antitumor effect of the aforementioned combined therapy, flow cytometry was used to analyze the infiltration and activation status of immune cells. For example... Figure 7 As shown in Figures A and B, compared with aPD-1 alone, the combination therapy group showed greater maturation of dendritic cells (CD11c) in draining lymph nodes and the tumor microenvironment. + CD80 + CD86 + The proportions of ) were increased by 339% and 100% respectively. The maturation of DC led to CD8 + The proliferation and activation of T cells. For example... Figure 7 Figures C and D show that the combination therapy group had locally invasive IFN-γ tumors. + CD8 +The proportion of T cells increased by 90% compared to the aPD-1 monotherapy group, and activated CD8+ cells were observed in the draining lymph nodes. + The proportion of T cells also increased by 58%. This suggests that vitamin B12 supplementation may be a potential combination strategy with immune checkpoint inhibitor therapy.

[0062] Example 5

[0063] The effect of methylcobalamin on promoting dendritic cell maturation and antigen presentation in vitro.

[0064] Bone marrow-derived dendritic cells (BMDCs) from cultured mice were extracted and seeded into 12-well culture plates. OVA was added to each well to a final concentration of 10 μg / mL. 257-264 Polypeptide antigens were then added. Equal volumes of PBS and different concentration gradients (250 μM, 500 μM, 1000 μM) of mecobalamin were added, and after 24 h of culture, the cell supernatant was collected for cytokine ELISA detection. The cell pellet was washed once with pre-chilled PBS and resuspended in 100 μL of PBS. 4 μL of flow cytometry antibody-drug mixture (containing FITC-aCD11c, APC-aCD86, PE / Cyanine7-aCD80, and PE-aSIINFEKL-H-2Kb) was added to each sample tube, mixed thoroughly, and incubated at room temperature in the dark for 1 h. After staining, an appropriate amount of PBS was added, and the cells were washed once by centrifugation to remove unbound free antibodies, and the cell pellet was resuspended. Finally, flow cytometry was used for data acquisition, first identifying CD11c. + Cell populations were analyzed, and subsequently, maturation-related surface markers (CD11c) of BMDCs were analyzed. + CD80 + CD86 + ) and specific antigen-presenting complex (CD11c + The proportion of cells in the SIINFEKL-H-2Kb cell population.

[0065] Result: As Figure 8 As shown in Figure A, compared with the PBS group (14.9%), the CD80 values ​​in the methylcobalamin treatment groups at 250 μM (17.1%), 500 μM (19.1%), and 1000 μM (19.4%) were significantly lower. + CD86 + The percentage of cell population increased significantly. Furthermore, Figure 8 SIINFEKL-H-2Kb in B + The percentage of cell population also increased in a concentration-dependent manner, reaching 16.8%, 21.3%, and 28.5% at 250 μM, 500 μM, and 1000 μM, respectively, compared to the PBS group (12.7%). Meanwhile, Figure 8ELISA results in CE showed that, compared with the PBS group, the mecobalamin treatment group promoted the secretion of pro-inflammatory cytokines by BMDCs in a concentration-dependent manner. Specifically, at 1000 μM, the levels of TNF-α, IL-6, and IFN-β increased by 3.2-fold, 20.8-fold, and 6.3-fold, respectively, compared with the PBS group. These results indicate that mecobalamin can significantly promote DC maturation, antigen presentation, and the secretion of pro-inflammatory cytokines, effectively enhancing the immunostimulatory function of DCs.

[0066] Example 6

[0067] JAK-STAT pathway-related mRNA and protein expression levels on BMDCs after methylcobalamin treatment

[0068] The expression levels of JAK-STAT pathway-related mRNAs in BMDCs after mecobalamin treatment were detected by RT-qPCR. In addition, the expression levels of related proteins in BMDCs after mecobalamin treatment were detected by Western blot.

[0069] Result: As Figure 9 As shown in Figure A, compared with the PBS control group, mecobalamin treatment significantly upregulated the mRNA expression levels of JAK2 and downstream key transcription factors (IRF1, NLRC5) and antigen presentation-related genes (MHC-I, CD86, Tap1, β2m) in BMDCs, but had no significant effect on JAK1 mRNA. Figure 9 Western blot analysis of the samples from the middle B sample further confirmed that mecobalamin treatment significantly upregulated the protein expression levels of JAK2, NLRC5, IRF1, CD86, and MHC-I in a dose-dependent manner (250–1000 μM). Mecobalamin dose-dependently promoted STAT1 phosphorylation (pSTAT1), while the total STAT1 protein level did not change significantly. This result clearly suggests that mecobalamin not only upregulated the expression of downstream effector molecules but also directly triggered the phosphorylation cascade activation of the JAK-STAT signaling pathway.

[0070] Example 7

[0071] Construction of methylcobalamin liposome formulation

[0072] Mecobalamin-loaded liposomes were prepared using a reverse evaporation method. Formulations were screened based on two factors: the molar ratio of the host lipid to cholesterol and the drug-to-liposome ratio. Hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, and distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG2000) were weighed at different molar ratios and dissolved thoroughly in chloroform to obtain the organic phase. Mecobalamin was dissolved in PBS buffer to form the aqueous phase; the volume ratio of organic phase to aqueous phase was 3:1. The molar ratio of the host lipid to cholesterol (n...) was then used for screening. HSPC :n Chol The ratios were 0.5:1, 1:1, 1.5:1, and 2:1, respectively, representing the molar ratios of lipids (HSPC + cholesterol + DSPE-PEG) to methylcobalamin (n). lipo :n vb12 The ratios of organic and aqueous phases were 1.5:1, 3:1, 6:1, and 12:1, respectively. After mixing the organic and aqueous phases, a colostrum was prepared using probe sonication. Chloroform was removed by rotary evaporation at 55°C and 500 mbar. The mixture was then hydrated at 60°C for 30 min, sonicated again using a probe, and dialyzed overnight in PBS at 4°C to obtain a mecobalamin liposome suspension. The particle size and polydispersity index (PDI) of the lipid nanoparticles were determined using Malvern Zetasizer Pro with dynamic light scattering. Free, unencapsulated mecobalamin was collected by ultrafiltration centrifugation, and the encapsulation efficiency and drug loading of mecobalamin were determined by HPLC. Chromatographic conditions: Column: Waters Atlantic T3 C18 column (4.6 mm × 250 mm, 5 μm); Mobile phase: acetonitrile-methanol (0.01% formic acid) = 15:85 (v / v); Flow rate: 1 mL / min; UV detection wavelength: 254 nm; Column temperature: 30℃; Injection volume: 10 µL. The concentration of methylcobalamin in the sample was calculated using the external standard method after preparing standards.

[0073] Result: As Figure 10 This shows that when the molar ratio (n) HSPC :n Chol At a molar ratio of 1:1, the encapsulation efficiency and drug loading were significantly higher than other ratio groups (p < 0.05), the average liposome particle size was approximately 170 nm, and the polydispersity index was less than 0.3. At a fixed molar ratio (n... HSPC :n Chol Given that n = 1:1, when n lipo :n vb12 When the ratio is 3:1, the liposome particle size is about 150 nm, the polydispersity index is maintained below 0.3, the encapsulation efficiency reaches a significant maximum, and the drug loading is maintained at a high level.

[0074] Example 8

[0075] Construction of soluble microneedles encapsulating vitamin B12

[0076] A two-step centrifugal casting method was employed: First, the drug-loaded layer solution for the needle tip was prepared under light-protected conditions. Vitamin B12 (5%) was dissolved in deionized water, followed by the addition of trehalose (5%) and hyaluronic acid (10 kDa) to a final concentration of 5%, stirred until completely dissolved and defoamed. 50–80 μL of this solution was dropped into the center of a PDMS microneedle mold (600 μm high, 10×10 array, pre-vacuum degassed), and centrifuged at 3500 × g for 15 min using a horizontal rotor centrifuge to ensure the high-viscosity drug solution fully filled the micropore tips. Excess solution was then removed from the mold surface with a scraper and pre-dried at room temperature in the dark for 30 min. Next, the needle matrix layer was cast. 100 μL of a solution containing 20% ​​hyaluronic acid (10 kDa) and 10% polyvinylpyrrolidone (PVP K30) was used to cover the entire mold array, and centrifuged at 3000 × g for 5–10 minutes. The needle body layer and the needle tip layer are tightly bonded together. Finally, the backing layer is cast, and 200–300 μL of an ethanol-water mixture containing 40% PVP K90 is added. The mixture is then evenly coated to a thickness of about 0.5–1 mm. The mold is then transferred to a light-proof desiccator and allowed to dry at 20–25°C and relative humidity below 30% for 24–48 h. After the backing layer has completely hardened, it is carefully peeled off to obtain a vitamin B12 soluble microneedle patch with sharp needle shape and a drug loading of about 50 μg / array at the needle tip.

[0077] Example 9

[0078] Construction of polylactic-co-glycolic acid (PLGA) sustained-release microspheres encapsulating vitamin B12

[0079] The double emulsion-solvent evaporation method (W / O / W) was employed: 200 µL of the inner aqueous phase (containing 5% vitamin B12 + 2% gelatin) was injected into 2 mL of the oil phase (containing 10% PLGA (50:50, molecular weight 30 kDa) + 0.1% Span 80 in dichloromethane solution) under ice bath conditions. Emulsification was performed using a probe-type ultrasonic cell disruptor or a high-speed shearing machine until a homogeneous, viscous promulgation was formed. The promulgation was then rapidly injected into 20 mL of the outer aqueous phase (containing 1% polyvinyl alcohol (PVA) + 5% NaCl, pre-cooled to 4°C) to form a stable double emulsion. The double emulsion was quickly transferred to a beaker containing 100 mL of the outer aqueous phase (also containing 0.5% PVA + 5% NaCl), and the organic solvent was evaporated by stirring. The microspheres were collected by centrifugation and lyophilized for storage. A single subcutaneous injection of PLGA microspheres can continuously release methylcobalamin for 2-4 weeks, enhancing immune memory.

[0080] Example 10

[0081] Construction of oral chitosan / pectin nanogel microspheres encapsulating vitamin B12

[0082] A 1% (w / v) aqueous solution of low-methoxyl pectin was slowly added to vitamin B12 powder under stirring in a 40°C water bath. The mixture was stirred for 20 min in the dark to ensure uniform dispersion of VB12. A separate 0.8% (w / v) chitosan solution (dissolved in 1% v / v acetic acid, pH 5.5) containing 0.5% (w / v) sodium tripolyphosphate as an ionic crosslinking aid and 2% (w / v) mannitol as a lyophilization protectant was prepared. Under gentle sonication using an ultrasonic cell disruptor probe (20 kHz, 40 W), the pectin-VB12 mixture was dropwise added to the chitosan-sodium tripolyphosphate solution at a 1:2 volume ratio. After sonication for 3 min, the system was gently magnetically stirred at room temperature for 30 min to solidify the nanogel. The resulting nanogel suspension was centrifuged at 14,000 rpm for 30 min to collect the precipitate, which was then washed three times with deionized water to remove unencapsulated free VB12. Finally, the precipitate was resuspended in a protective solution containing 2% mannitol, and then freeze-dried to obtain VB12-chitosan / pectin nanogel microspheres.

[0083] Example 11

[0084] Uptake of methylcobalamin liposome formulations at BMDC

[0085] The uptake levels of methylcobalamin and methylcobalamin liposomes by BMDC were detected by ICP-MS, wherein the methylcobalamin liposomes were the preferred type selected in Example 7 at a fixed molar ratio n. HSPC :n Chol Given a ratio of 1:1, when n lipo :n vb12 Mecobalamin liposomes were obtained at a ratio of 3:1. BMDCs were treated with 250 μM free mecobalamin and 250 μM mecobalamin liposomes for 24 h, respectively. The suspended cells were then collected into centrifuge tubes containing approximately 1 × 10⁻⁶ methylcobalamin liposomes. 6 -5×10 6 The cell pellet was transferred to a quartz digestion tube and freeze-dried. 0.5 mL of UP grade HNO3 and 0.5 mL of UP grade H2O2 were added, gently mixed, and the digestion vessel was sealed.

[0086]

[0087] Perform the microwave digestion according to the procedure in Table 1. After digestion, transfer the digest to a 15 mL centrifuge tube, rinse the tube several times with ultrapure water and combine the solutions, then bring the volume to 10 mL. Perform the analysis according to the parameters in Table 2. Simultaneously process a blank reagent (0.5 mL HNO3 + 0.5 mL H2O2) without cells to subtract background contamination.

[0088]

[0089] Result: As Figure 11 As shown, the Co content in BMDCs of the LipoMeCbl-treated group was 1.6 times that of the free MeCbl group (p<0.01), suggesting that liposome delivery can significantly promote the uptake efficiency of methylcobalamin by BMDCs, which is beneficial to promoting BMDC maturation and enhancing antigen presentation function.

[0090] Example 12

[0091] Effects of methylcobalamin liposome formulation on BMDC maturation and antigen presentation capabilities

[0092] Immature BMDCs were 5×10 5 Cells were seeded at a density of 10 μg / mL in 12-well culture plates, and OVA was added to each well. 257-264 Polypeptide antigen. Subsequently, equal volumes of PBS, blank liposomes, 250 μM mecobalamin, and 250 μM mecobalamin liposomes (same as in Example 11) were added, and the cells were cultured in a constant temperature cell culture incubator for 24 h. The suspended cells from each group were then collected into centrifuge tubes and centrifuged at 4°C and 1200 ×g for 5 min. The cell pellet was washed once with pre-cooled PBS and resuspended in 100 μL of PBS. Flow cytometry antibody mixtures containing FITC anti-mouse CD11c, APC anti-mouse CD86, PE / Cyanine7 anti-mouse CD80, PE / Dazzle 594 anti-mouse CD40, and PE anti-mouse SIINFEKL-H-2Kb were prepared in advance according to the experimental design. 5 μL of the above antibody mixture was added to each sample tube, mixed thoroughly, and incubated at room temperature in the dark for 1 h. After staining, an appropriate amount of PBS was added, and the cells were washed once by centrifugation to thoroughly remove unbound free antibodies, and the cell pellet was resuspended. Finally, flow cytometry was used for data acquisition, first delineating CD11c. + Cell populations were analyzed, and subsequently, maturation-related surface markers (CD11c) of BMDCs were analyzed. + CD80 + CD86 + and CD11c +CD40 + ) and specific antigen presentation complex (CD11c + SIINFEKL-H-2Kb + The proportion of expression.

[0093] Result: As Figure 12 As shown in Figure A, the expression levels of maturation marker molecules in BMDCs treated with mecobalamin liposomes were significantly upregulated, with the proportion of double-positive cell subsets of co-stimulatory molecules CD80 and CD86 being 1.3 times that of the free mecobalamin group (p<0.001). Figure 12 The proportion of CD40-positive cell subsets in the B group was twice that in the free mecobalamin group (p < 0.0001). Figure 12 The results showed that the proportion of SIINFEKL-H-2Kb positive cells was 1.9 times that of the free mecobalamin group (p < 0.0001). These data indicate that liposome encapsulation significantly increased the abundance of co-stimulatory molecules and antigenic peptide-MHC class I molecule complexes on the cell surface of BMDCs, thereby enhancing the antigen presentation capacity of BMDCs.

[0094] Example 13

[0095] Evaluation of the efficacy and immunological effects of methylcobalamin liposome formulation in the B16F10 tumor model

[0096] In the B16F10 tumor mouse model, the mecobalamin group received a subcutaneous injection of 100 μL at a dose of 5.4 mg / kg; the mecobalamin liposome group (same as in Example 11) received a subcutaneous injection of 100 μL at a dose of 1 mg / kg. The dosing regimen was as follows: administration on days 0, 3, 6, and 9, once every 3 days, for a total of 4 administrations. Tumor growth curves and mouse survival rates were monitored during treatment. After the completion of the dosing cycle, tumor tissue and draining lymph nodes were isolated, and single-cell suspensions were prepared for immunophenotypic identification by flow cytometry.

[0097] Result: As Figure 13 As shown in Figure A, tumors grew rapidly in the PBS and LipoBlank groups, while the LipoMeCbl group showed the most significant tumor-suppressing effect, significantly stronger than the free MeCbl group (p<0.05). Survival curve analysis results are shown below. Figure 13 As shown in Figure B, the median survival of mice in the PBS and LipoBlank groups was 9 days, the median survival of mice in the free MeCbl group was 15 days, while the median survival of mice in the LipoMeCbl group was significantly prolonged to 18 days, indicating a significant increase in mouse survival. Endpoint tumor weight and solid image are shown below. Figure 13 As shown in the CD, the tumor weight in the LipoMeCbl group was reduced by about 70% compared with the PBS group and by about 44% compared with the free MeCbl group, and the differences were statistically significant (p<0.001).

[0098] In addition, such as Figure 14 As shown in Figure AB, compared with the free MeCbl group, the LipoMeCbl group had more mature DCs (CD80) in the tumor local area and draining lymph nodes. + CD86 + The proportion of DCs has increased significantly, while Figure 14 CD shows IFN-γ + CD8 + The proportion of T cells was also significantly increased. These results indicate that mecobalamin liposomes (1 mg / kg) significantly delayed tumor growth, reduced tumor weight, and prolonged survival in B16F10 melanoma mice, with a significantly better tumor-suppressing effect than high-dose free mecobalamin (5.4 mg / kg). Mechanistically, mecobalamin liposomes significantly increased IFN-γ levels by promoting the maturation of dendritic cells (DCs) in tumors and draining lymph nodes. + CD8 + The proportion of effector T cells activates a potent anti-tumor immune response.

[0099] Example 14

[0100] Evaluation of the efficacy and immunological effects of hydroxycobalamin liposomes combined with doxorubicin liposomes in a B16F10 tumor model

[0101] In a B16F10 melanoma mouse model, the PBS group received an equal volume of PBS; the doxorubicin liposome group received intravenous injection of Liposome Dox at a dose of 3 mg / kg; the hydroxycobalamin liposome group received subcutaneous injection of Liposome OHCbl at a dose of 10 mg / kg; and the combination therapy group received treatment with both doxorubicin liposomes (Liposome Dox, 3 mg / kg, intravenous injection) and hydroxycobalamin liposomes (Liposome OHCbl, 10 mg / kg, subcutaneous injection). Tumor volume changes were monitored during treatment, and tumor tissue and draining lymph nodes were isolated at the experimental endpoint. The weight of the isolated tumor was measured, and a single-cell suspension was prepared for flow cytometry analysis of mature dendritic cells (DCs) and CD8+. + T cell ratio.

[0102] Result: As Figure 15 As shown in Figure A, the combined drug administration group exhibited the most significant tumor-suppressing effect, superior to the hydroxycobalamin liposome monotherapy group (p = 0.0178). Figure 15 As shown in Figure B, the endpoint ex vivo tumor weight result is consistent with the tumor growth curve. Compared with the PBS group, the doxorubicin liposome group, the hydroxycobalamin liposome group, and the combination drug group all significantly reduced tumor weight, with the combination drug group showing the lowest tumor weight and the strongest tumor-suppressing effect.

[0103] Further analysis of the immunological effects is needed. For example... Figure 16As shown in Figure AB, compared with the PBS group, both the doxorubicin liposome group and the hydroxycobalamin liposome group increased the number of mature DCs (CD80) in the draining lymph nodes. + CD86 + DCs) and CD8 + The proportion of T cells was significantly increased in the combination therapy group, suggesting that combination therapy can enhance the immune activation state in draining lymph nodes. Figure 16 As shown in the CD10 data, both the hydroxycobalamin liposome group and the combined drug administration group increased the proportion of mature dendritic cells (DCs) in tumor tissue; simultaneously, the doxorubicin liposome group, the hydroxycobalamin liposome group, and the combined drug administration group all increased intratumoral CD8. + T-cell infiltration was most significantly increased in the combination therapy group. These results suggest that hydroxycobalamin liposomes can enhance the antitumor immune response of doxorubicin liposome therapy.

[0104] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Use of vitamin B12 in the preparation of medicines for the treatment and / or prevention of disease by enhancing dendritic cell function.

2. The use as described in claim 1, characterized in that, The disease is associated with impaired dendritic cell function or a need to improve dendritic cell function.

3. The use as described in claim 1, characterized in that, The drug is used to treat and / or prevent malignant tumors, infectious diseases, age-related immune decline, allergic diseases, and immunosuppression after radiotherapy and chemotherapy for tumors.

4. The use as described in claim 3, characterized in that, The malignant tumor is melanoma or colorectal cancer.

5. The use as described in claim 1, characterized in that, The drug is an oral dosage form, an injectable dosage form, a mucosal dosage form, or a topical dosage form, and the drug includes pharmaceutically acceptable excipients and / or carriers.

6. The use as described in claim 1, characterized in that, The active ingredient of the drug is vitamin B12 or a combination of vitamin B12 and other drugs, wherein the combination includes vitamin B12 and one or more of the following drugs: chemotherapy drugs, immune checkpoint inhibitors, tumor vaccines, therapeutic antibodies, cytokines, and immunomodulators.

7. The use of vitamin B12 in the preparation of vaccine adjuvants, wherein the vaccine is a preventive or therapeutic vaccine, characterized in that, Vitamin B12 regulates the immune response by enhancing the function of dendritic cells.

8. Use of vitamin B12 in the preparation of reagents that enhance the function of dendritic cells in vitro.

9. The use as described in claim 8, characterized in that, The reagent is used to induce or activate dendritic cells in vitro to prepare dendritic cell vaccines.

10. The use as described in any one of claims 1 to 9, characterized in that, The vitamin B12 is selected from at least one of the following forms: methylcobalamin, cyanocobalamin, adenosylcobalamin, hydroxycobalamin, or derivatives thereof.

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