CN114028374A - Application of dimethyl fumarate in preparation of medicines for protecting pancreatic beta cell function - Google Patents

Abstract

The invention discloses an application of dimethyl fumarate in preparation of a medicine for protecting pancreatic beta cell functions, wherein the medicine for protecting the pancreatic beta cell functions can be used for preventing and/or treating diabetes, and particularly has a good treatment effect on type 1diabetes. Compared with the common treatment mode of insulin injection for the diabetic, the invention uses the dimethyl fumarate to treat the diabetic through oral medicaments, so that the diabetic can avoid the trouble of daily insulin injection, has lower cost than biological preparations, and can reduce the economic burden of the diabetic. In addition, compared with simple immunotherapy, the dimethyl fumarate can realize the function protection of pancreatic beta cells and protect the pancreatic functions of patients while regulating the immunity.

Description

Application of dimethyl fumarate in preparation of medicines for protecting pancreatic beta cell function

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of dimethyl fumarate in preparation of a medicine for protecting pancreatic beta cell functions.

Background

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia, which are caused by defects in insulin secretion or impaired biological actions thereof, or both. Diabetes mainly includes two types of type 1diabetes and type 2 diabetes, and is exemplified by type 1diabetes, and type 1diabetes (T1 DM) is an organ-specific autoimmune disease mainly characterized by T cell-mediated islet β cell destruction. Patients with T1DM account for approximately 5% -10% of the diabetic population. Based on the large population base of China, it is estimated that about 500 million patients with T1DM exist in China, and the incidence rate of type 1diabetes mellitus is on the trend of rising year by year. The islet beta cells are the only cells that secrete insulin that reduces blood glucose. Currently, mainstream supplemental insulin therapy changes type 1diabetes from acute, lethal disease to chronic disease, but insulin does not block the progression of islet autoimmune attack. Patients eventually develop fragile diabetes mellitus with frequent severe hypoglycemia and huge fluctuations in blood glucose due to complete loss of islet function.

Attack of islet beta cells by T cells is an important pathogenesis of T1 DM. The classical view is that autoreactive T cells can falsely destroy healthy ("innocent") beta cells. In recent years, more and more research has shown that beta cells not only play as "innocent" victims during autoimmune attack, but are actually also a key factor in the development of T1DM disease, i.e., T cells are not the only pathological driver of T1 DM. Evidence that supports this includes (1) that normal individuals possess islet-specific Autoreactive T cells (Danke, n.a., Koelle, d.m., ye, c., Beheray, s., and Kwok, W.W. (2004) Autoreactive T cells in health indusivia. j immune 172, 5967-ion 5972); (2) humanized mice expressing islet antigen TCR did not induce T1DM (Tan, s., Li, y., Xia, J.) after islet antigen stimulationin, C.H., Hu, Z., Duinkerken, G., Li, Y., Khosravi Mahaloei, M., Chavez, E., Nauman, G., Danzl, N., Nakayama, M., Roep, B.O., Sykes, M., and Yang, Y.G, (2017) Type 1diabetes index in humanized micron, Proc Natl Acad Sci U S A114, 10954-; (3) patients with pancreatitis do not suffer from T1DM despite carrying HLA high risk genes (Lampeter, E.F., Seifert, I., Lohmann, D., Heise, J.W., Bertrams, J.J., Christie, M.R., Kolb-Bachofen, V.and Kolb, H. (1994) Inflummatter islet in space bearings HLA-DR 3and/or DR 4haplotypes not T lead to isautoimmumology. diabetes 37, 471-475); (4) immunotherapy does not preserve β -cell function continuously (keymeuleen, b., vandmeulebroucke, e., Ziegler, a.g., Mathieu, c., Kaufman, L., Hale, g., Gorus, f., Goldman, m., Walter, m., Candon, s., Schandene, L., Crenier, L., De Block, c., Seigneurin, j.m., De Pauw, p., Pierard, d., Weets, i, Rebello, p., Bird, p., Berrie, e., freighten, m., meddmann, h., Bach, j.f., pierers, d., chatten, L. (r), r, p., freuder), freighten, m., meddmann, h., Bach, j.f., piuder, d., CD, flux, L. (2005) tissue, r, etc. (35 3) tissue, etc.; (5) most T1DM patients are not characterized by immune modulation abnormalities (Long, S.A., Cerosaletti, K., Bollyky, P.L., Tatum, M., Shilling, H., Zhang, S., Zhang, Z.Y., Pihoker, C., Sanda, S., Greenbaum, C., and Buckner, J.H. (2010) Defects in IL-2R signaling consistent to minor main of FOXP3 expression in CD4(+) CD25(+) regulatory T-cells of type 1 diagnostic subjects. diabetes59, 407-415); (6) islets also exhibit HLA class I molecular upregulation in the absence of inflammation (Coppieters, k.t., Dotta, f., Amirian, n., Campbell, p.d., Kay, t.w., Atkinson, m.a., roap, b.o., and von Herrath, M.G. (2012) monitoring of islet-automatic CD 8T cells in inductive dispersions from the patient set and long-term type 1diabetes tissues, j Exp Med209, 51-60); (7) immune sensitization by islet autoantigen failed to induce T1DM (Ludvigsson, j.,

M.,Hjorth,M.,Axelsson,S.,Chéramy,M.,Pihl,M.,Vaarala,O.,Forsander,G.,Ivarsson,S.,Johansson,C.,Lindh,A.,Nilsson,N.O.,Aman,J.,Ortqvist,E.,Zerhouni,P.,and Casas,R.(2008)GAD treatment and insulin secrecovery in recovery-type 1diabetes. N Engl J Med359, 1909-1920). Thus, the release of autoantigens following beta cell stress or destruction may further facilitate the attack of T cells on the islet beta cells.

To date, several studies have demonstrated that inhibition of autoimmunity alone is not sufficient to achieve long-lasting protection of beta cell function, suggesting the need to combine immune intervention with beta cell protection. In recent years, several studies have evaluated the effect of several therapeutic strategies aimed at intervening T2DM by reducing beta cell stress in T1DM, such as treating T1DM patients with metformin, GLP1 analogs (liraglutide, exendin 4 or sitagliptin) and vilapamide in combination with insulin, showing a certain therapeutic effect (ovale, f., Grimes, T., Xu, g., Patel, a.j., gyson, t.b., Thielen, l.a., Li, p., and Shalev, a. (2018) paramil and beta cell functions in with a receiver-type 1diabetes. nat. med24, 1108-1112). Therefore, reducing the immune attack of autoreactive T cells on beta cells while enhancing protection of islet beta cells is critical to T1DM treatment.

Dimethyl fumarate (DMF) is a derivative of fumarate in the tricarboxylic acid cycle, an immunomodulatory drug. DMF is rapidly hydrolyzed by esterase to monomethyl fumarate (MMF) which, in turn, is further hydrolyzed to Fumaric Acid (FA). Because of its neuroprotective and myelin protective effects, it is commonly used in the treatment of Multiple Sclerosis (MS) and psoriasis (Dubey, d., kiesei, b.c., Hartung, h.p., Hemmer, b., warnenke, c., range, t., Miller-light, w.a., and Stuvee, O. (2015) Dimethyl fumarate in reusing-recovering multiple sclerosis: rationale, mechanism of action, pharmacokinetics, efficacy and safety, expert Rev neuro 15, 339-346). In 2013, DMF was approved by The U.S. Food and Drug Administration (FDA) and The European Drug Administration (EMA) as The first oral first line treatment for relapsing-remitting multiple sclerosis (RRMS) (Saidu, N.E.B., Kavia, N.Leroy, K., Jacob, C., Nicco, C., Batteux, F., and Alexander, J. (2019) Dimethyl fumarate, a wwo-edged Drug: Current status and future references Res Rev39, 1923-1952). Two 24-month randomized controlled phase 3 clinical trials showed that oral doses of 240mg of DMF twice daily and three times daily, respectively, reduced the annual relapse rate of RRMS patients by 44% and 51%, respectively, while reducing the rate of progression of disability and the number of neuropathy in patients (Gold, r., Kappos, l., Arnold, d.l., Bar-Or, a., Giovannoni, g., Selmaj, k., Tornatore, c., Sweetser, m.t., Yang, m., Sheikh, s.i., and Dawson, K.T (2012) Placebo-controlled phase 3study of oral BG-12for relaating multiple sclerosis. n Engl J367, 1098-1107).

DMF has been clearly demonstrated for its anti-inflammatory and immunomodulatory effects. And DMF modulates only activated immune cells, with no effect on resting state immune cells (Kornberg, m.d., bhragova, p., Kim, p.m., Putluri, v., Snowman, a.m., Putluri, n., Calabresi, p.a., and Snyder, S.H. (2018) Dimethyl sulfate targets GAPDH and aerobic glycerol modulation immunity. science360, 449-453). Studies have shown CD8 in DMF treated RMMS patients⁺The number of T cells, Th1 cells, B cells and plasmacytoid Dendritic Cells (DCs) was reduced compared to placebo-treated patients, and at the same time, DMF could regulate maturation of DCs and promote activation of anti-inflammatory DCs by inhibiting maturation of DCs and expression of MHC class II molecules. In addition, DMF has also been found to have antioxidant and anticancer activity. Therefore, DMF as a potential anticancer drug has been extensively studied in disease models of melanoma, breast cancer, cervical cancer, cutaneous T-cell lymphoma, lung cancer, etc., and has been confirmed to have anticancer potential.

At present, the immunoregulation effect of DMF is considered to be mainly shown in that DMF and its dynamic Metabolite Monomethyl Fumarate (MMF) regulate activated immune response by inhibiting nuclear factor kb translocation, up-regulating nuclear factor carotenoid derivative 2-related factor antioxidant pathway and activating hydroxycarboxylic acid receptor 2, and act on various immune cells, thereby generating a therapeutic effect on autoimmune diseases. Numerous studies have shown that DMF not only alters the proportion of individual lymphocytes in the body, but also affects lymphocyte proliferation and induces oxidative stress in T cells.

Immunotherapy for type 1diabetes includes nonspecific immunotherapy and antigen-specific immunotherapy. The current more sophisticated treatments are mainly non-specific immunotherapy. Non-specific immunotherapy mainly involves (i) T cell immunosuppressive agents: polyclonal anti-T cell antibodies, CTLA-4-Ig interfering with T cell co-stimulatory molecules, anti-CD 3 monoclonal antibodies directed against T cell receptors, low dose anti-thymocyte globulin; ② a B cell immunosuppressant: an anti-CD 20 monoclonal antibody directed against a B cell receptor; (iii) anti-inflammatory factor therapy: the tumor necrosis factor antagonist etanercept, low-dose rhIFN-alpha, low-dose IL-2 and IL-1 are blocked.

Although current immunotherapy may alleviate the T1DM autoimmune attack to some extent, the following disadvantages exist: the immunotherapy effect is limited, and the occurrence and development of T1DM cannot be completely prevented; ② because of all immune cell specificity, may have the general immunosuppression, cytokine release syndrome and infection and tumor risk; and the function of islet beta cells cannot be protected.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide the application of dimethyl fumarate in preparing a medicament for protecting the function of islet beta cells.

The purpose of the invention is realized as follows: use of dimethyl fumarate for the manufacture of a medicament for protecting pancreatic β -cell function. Further, the medicine for protecting the function of the islet beta cells can be used for preventing and/or treating diabetes, and particularly has a good treatment effect on type 1diabetes.

As a preferred technical scheme, the medicament for protecting the function of the islet beta cells further comprises pharmaceutically acceptable pharmaceutic adjuvants. Further preferably, the medicament for protecting the function of the islet beta cells is any dosage form in the current pharmaceutical field, including oral preparations and injection preparations; the oral preparation comprises capsules or tablets and the like.

The invention has the beneficial effects that:

compared with the common treatment mode of insulin injection for the diabetic, the invention uses the dimethyl fumarate to treat the diabetic through oral medicaments, so that the diabetic can avoid the trouble of daily insulin injection, has lower cost than biological preparations, and can reduce the economic burden of the diabetic. In addition, compared with simple immunotherapy, the dimethyl fumarate can realize the function protection of pancreatic beta cells and protect the pancreatic functions of patients while regulating the immunity.

Drawings

FIG. 1 is the results of experiments in which NOD mice at 4 weeks of age were observed to have a disease rate of 34 weeks of age after 5 weeks of DMF and placebo treatment, respectively;

FIG. 2 is a graph comparing the degree of pancreatic lymphocyte infiltration (A) and insulitis score (B) at the end of treatment (i.e., 9 weeks of age) in 4-week-old NOD mice given DMF and placebo treatment for 5 weeks, respectively;

FIG. 3 shows the blood glucose (A) and insulin secretion (B) at each time point evaluated by IPGTT after 4-week-old NOD mice were fed DMF and placebo for 5 weeks, respectively, and then stopped;

FIG. 4 shows the effect of MIN6 cells on insulin secretion after 24 hours of CK, 7.5uM DMF + CK, 15uM DMF + CK, 30uM DMF + CK, 60uM DMF + CK, and 120uM DMF + CK treatment;

FIG. 5 shows the effect of primary islets of C57BL/6N mice on insulin secretion after 24 hours of CK treatment with 60uM DMF + CK;

FIG. 6 shows the apoptosis of TC6 cells after CK treatment and DMF treatment at various concentrations;

FIG. 7 shows the proliferation of islet beta cells in NOD mice in DMF-treated and control groups.

Detailed Description

The present invention will be further described with reference to the following examples and drawings so that those skilled in the art can better understand the present invention and can carry out the present invention, but the examples are not intended to limit the present invention.

Some of the materials and some of the experimental methods used in the following examples are presented below:

1) NOD mice: the method selects a spontaneous type 1diabetes mellitus mouse model, namely an NOD mouse, which is the most commonly used mouse model for researching type 1diabetes mellitus at present, and the incidence rate of type 1diabetes mellitus of a female NOD mouse is obviously higher than that of a male NOD mouse, so the female NOD mouse is selected, the NOD mice used by the method are purchased from Nanjing university model animal institute, and are bred in Specific Pathogen Free (SPF) level environment of Nanjing university medical laboratory animal center after being purchased, and food and padding of the NOD mouse are changed every other day to freely drink and eat water. All the operations in the invention meet the requirements of the experimental animal welfare ethical review committee of Nanjing medical university and are approved by the Lung-Cit.

2) Dispensing: preparing DMF solution according to a formula of 0.025g DMF + 10% DMSO + 0.8% Methylcellulose (MC) suspension for treatment group experiment; preparing a placebo according to a formula of 10% DMSO + 0.8% MC suspension for a control group experiment; the prepared medicine is stored in an environment of 4 ℃ for standby.

3) Administration: the treatment group was administered by gavage after calculating the volume of administration per mouse at a dose of 25mg/kg, and the control group was administered with an equivalent volume of placebo. The control group had 30 mice filled with placebo; the treatment group had 30 mice infused with DMF solution.

4) Monitoring blood glucose: cutting off about 1mm mouse tip, bleeding, discarding the first drop of blood, sucking on blood sugar test paper after the second drop of blood flows out, reading, and repeating the blood sugar value for two times to be >13.3mmol/L to be considered as dominant diabetes.

EXAMPLE 1 Effect of dimethyl fumarate on the incidence of type 1diabetes in NOD mice

After 3-week-old NOD mice were randomly divided into a DMF treatment group and a control group (namely a placebo treatment group) and adaptively fed for 1 week, the mice were gavaged every day for 5 weeks from 4 weeks until the NOD mice are 34 weeks old, so as to observe and count the incidence rate of autoimmune diabetes.

The results are shown in FIG. 1, where FIG. 1 shows the results of experiments in which the incidence of 4-week-old NOD mice was observed 5 weeks after administration of DMF and placebo, respectively, to 34 weeks of age of the mice, as can be seen in FIG. 1: NOD mice in the control group developed disease at the earliest of 10 weeks of age and had an overall incidence of 50.0% at the observation endpoint (34 weeks of age); not only did the mice in the DMF treated group had a significant delay in onset time (first onset occurred at 15 weeks of age), but the incidence was much lower than in the control group [ 25% vs (vs) 50%, P <0.05 ]. Therefore, the dimethyl fumarate can obviously reduce the incidence rate of type 1diabetes of NOD mice.

Example 2 dimethyl fumarate significantly ameliorates inflammatory infiltration of pancreatic islets in NOD mouse pancreas

The experimental method comprises the following steps: NOD mice were divided into a treatment group and a control group, and administered once a day by intragastric administration starting from 4 weeks of age, and the pancreas of mice was taken at 9 weeks of age for 5 weeks continuously, and after anesthetizing the mice, the abdominal cavity was opened, the thoracic cavity was opened, and the right auricle was excised. An intravenous blood collection needle was inserted into the left ventricle and perfused with 100ml of saline to wash it, and then perfused with 100ml of 4% paraformaldehyde and fixed. Pancreas specimens were carefully removed from the duodenum and fixed in 15ml centrifuge tubes containing 4% paraformaldehyde to prepare paraffin sections, which were then HE stained for insulitis.

The results are shown in fig. 2, which is a graph of the degree of pancreatic lymphocyte infiltration (a) and insulitis score (B) in 4-week-old NOD mice compared to the treatment endpoint (i.e., 9 weeks of age) after 5 weeks of DMF and placebo treatment, respectively. As shown in fig. 2A, the extent of pancreatic lymphocyte infiltration was significantly reduced in DMF treated mice compared to control mice; as can be seen from fig. 2B, the insulitis score of the DMF treated mice was significantly lower than the control group. Therefore, dimethyl fumarate can be used for protecting the islets of the NOD mice from being attacked by autoimmune cells.

Example 3 experiment of dimethyl fumarate capable of improving the function of islet of NOD mouse

The experimental method comprises the following steps: randomly dividing NOD mice of 4 weeks into two groups, wherein one group is a treatment group and the medicine is DMF solution; the other group was a control group and the medication was placebo. Two groups of mice were administered with the drug for 5 weeks, and then the drug was stopped, and were raised to 20 weeks of age, and islet function of the mice was evaluated by Intraperitoneal glucose tolerance test (IPGTT).

The results are shown in FIG. 3, which is a graph showing the results of blood glucose (A) and insulin secretion (B) at each time point in two groups of mice after intraperitoneal injection of glucose; it can be seen that blood glucose of mice in DMF group was significantly lower than that in control group at each time point (P <0.01 at time points of 5min, 60min, 90min and 120min in FIG. 3A), and insulin secretion was significantly higher than that in control group (P <0.05 at time points of 0min, 30min, 60min and 120min in FIG. 3B). Therefore, dimethyl fumarate can be used for improving the pancreatic islet function of NOD mice.

Example 4 dimethyl fumarate can improve insulin secretion experiments in mice primary islets and MIN6 cells in inflammatory environment

The experimental method comprises the following steps: a C57BL/6N mouse primary islet and MIN6 cell line are used, after 1000U/ml TNF-alpha +100U/ml IL-1 beta +1000U/ml IFN-gamma cytokine (cytokine, CK) is used for treating for 24 hours, a glucose-stimulated insulin secretion experiment (GSIS) is carried out, firstly, sugar-free KRBH liquid is used for starvation for 40 minutes, then, KRBH liquid containing 2mM low-concentration glucose is used for treating for 1 hour, then, supernatant is collected, and finally, total protein correction is extracted after the KRBH liquid containing 20mM high-concentration glucose is used for treating for 1 hour. Cell supernatants were measured for insulin content using ELISA.

The results of GSIS experiments with MIN6 cells are shown in FIG. 4A, and the 7.5uM, 15uM, and 60uM DMF + CK treatment groups all produced increases in the amount of insulin secretion stimulated by high glucose (7.5uM DMF + CK: P < 0.01; 15uM DMF + CK: P < 0.05; 60uM DMF + CK: P < 0.01). Further comparison of the amount of insulin secretion at high glucose concentration versus the doubling of insulin secretion at low glucose concentration between groups shows that the doubling ability of the 7.5uM, 15uM, 30uM DMF + CK treated groups under high glucose conditions was significantly improved compared to the CK group alone (7.5uM DMF + CK: P < 0.0001; 15uM DMF + CK: P < 0.001; 30uM DMF + CK: P < 0.05). We therefore concluded that DMF significantly increased the insulin secretion from MIN6 cells in the inflammatory environment compared to the CK group alone, while 7.5uM, 15uM, 30uM, 60uM DMF treatment increased the insulin secretion from MIN6 cells under high glucose conditions to varying degrees and improved the doubling of the insulin secretion from MIN6 cells under low or high glucose stimulation.

The results of the primary islet experiment of the C57BL/6N mouse are shown in FIG. 5, the insulin secretion capacity of the two groups is not different under the low sugar stimulation, but the insulin secretion content of the 60uM DMF + CK group is obviously increased under the high sugar stimulation. It can be seen that DMF can improve the insulin secretion capacity of mouse primary islets and MIN6 cells in inflammatory environments.

Example 5 dimethyl fumarate reduces islet cell apoptosis and promotes islet cell proliferation in both in vivo and in vitro experiments

Experimental method 1: this study simulated a model of islet immune injury in the progression of autoimmune diabetes in NOD mice by TC6 cells treated with 1000U/ml TNF-. alpha. +100U/ml IL-1. beta. +1000U/ml IFN-. gamma.cytokines (CK). Adding DMF and CK of 7.5uM, 15uM, 30uM, 60uM and 120uM into the cells respectively for in vitro treatment for 24 hours, and then detecting the apoptosis condition of the cells by flow cytometry after Annexin V and PI staining.

The results are shown in fig. 6, fig. 6A shows the apoptosis of TC6 cells after receiving CK and DMF at different concentrations, fig. 6B, 6C, and 6D show the early, late and total islet cell apoptosis rate of mice after different treatments, P <0.05, P <0.01. P < 0.0001; as can be seen from the figure, the mouse islet cell early apoptosis rate, late apoptosis rate and total apoptosis rate after DMF treatment are obviously reduced (2.65 +/-1.70% vs 6.96 +/-5.24%, P <0.01), wherein 60uM DMF in vitro treatment can obviously reduce apoptosis.

Experimental method 2: NOD mice were divided into treatment groups using DMF solution and control groups using placebo. The administration was performed by gavage once a day from 4 weeks of age, and the pancreas of mice was taken at 5 weeks of continuous administration and 9 weeks of age, and after anesthetizing the mice, the abdominal cavity was opened, the thoracic cavity was opened, and the right auricle was excised. An intravenous blood collection needle was inserted into the left ventricle and perfused with 100ml of saline to wash it, and then perfused with 100ml of 4% paraformaldehyde and fixed. Pancreatic specimens were carefully removed from the duodenum and placed into EP tubes containing OCT, and cryosections were prepared for immunofluorescent co-staining with instulin and Ki67, respectively.

The results are shown in FIG. 7, FIG. 7A shows the proliferation of islet beta cells of NOD mice in DMF treatment group and control group, and FIG. 7B shows the expression level of mouse islet Ki 67; it was found that islets Ki67 exhibited high expression (× P <0.001) in mice treated with DMF, indicating that significant proliferation of islets occurred in mice after DMF treatment.

The experiments show that dimethyl fumarate can be used for inhibiting apoptosis of islets of NOD mice and promoting proliferation of the islets of the NOD mice.

In the above examples, the therapeutic effect of dimethyl fumarate on type 1diabetes was clarified using a cell line and a mouse model, and it is known to those skilled in the art that the function of islet β cells also has an effect on type 2 diabetes, and therefore it is inferred that dimethyl fumarate also has a certain therapeutic effect on type 2 diabetes.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (4)

1. Use of dimethyl fumarate for the manufacture of a medicament for protecting pancreatic β -cell function.

2. Use of dimethyl fumarate according to claim 1 for the preparation of a medicament for protecting pancreatic beta cell function, characterized in that: the medicine for protecting the function of the islet beta cells can be used for preventing and/or treating diabetes.

3. Use of dimethyl fumarate according to claim 1 or 2for the preparation of a medicament for protecting pancreatic beta cell function, characterized in that: the medicine for protecting the function of the islet beta cells comprises pharmaceutically acceptable pharmaceutic adjuvants.

4. Use of dimethyl fumarate according to claim 3 for the preparation of a medicament for protecting pancreatic beta cell function, characterized in that: the medicament for protecting the function of the islet beta cells is any dosage form in the current pharmaceutical field, including oral preparations and injection preparations.

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