Medicine for treating diabetic foot ulcer

Abstract

The invention provides a medicine for treating diabetic foot ulcer, and the medicine for treating diabetic foot ulcer provided by the invention is an M2 type macrophage polarization inducer. The M2 type macrophage polarization inducer comprises but is not limited to IL-4, IL-13, IL-10, retinoic acid, a mitogen-activated protein kinase kinase (MEK) inhibitor, a histone deacetylase (HDAC) inhibitor, a granulocyte / macrophage colony stimulating factor and the like, and can be used independently or in a mixed manner. The composition can effectively promote healing of diabetic foot ulcer wounds and pathological improvement of wound inflammation, and the curative effect is obviously better than that of a treatment method in the prior art.

Description

[0001] This patent application claims the priority of Chinese patent application with application number 202410166855.5 filed with the State Intellectual Property Office on February 6, 2024 and invention name “A drug for treating diabetic foot ulcers”, the entire contents of which are incorporated by reference into this patent application. Technical Field

[0002] The invention belongs to the field of medicine and medical science, and relates to a medicine for treating diabetic foot ulcers. Background Art

[0003] Diabetic wounds are one of the most serious complications of diabetic patients. According to statistics, 19%-34% of diabetic patients may eventually develop diabetic foot ulcers (DFU) [see: Armstrong DG, Boulton AJM, Bus SA. (2017) Diabetic Foot Ulcers and Their Recurrence. The New England journal of medicine. 376 (24): 2367-75.]. The amputation rate of DFU patients is 10%-20%, and the disease is closely related to the mortality rate of diabetic patients [see: Moulik PK, Mtonga R, Gill GV. (2003) Amputation and mortality in new-onset diabetic foot ulcers stratified byetiology. Diabetes Care. 26: 491-494.]. In addition, diabetic wounds also cause huge economic, social and public health burdens. According to statistics, the treatment of diabetic wounds accounts for about one-third of the total expenditure on diabetes [see: Bommer C, Heesemann E, Sagalova V, Manne-Goehler J, Atun R, Barnighausen T, et al. (2017) The global economic burden of diabetes in adults aged 20-79years: a cost-of-illness study. The lancet Diabetes&endocrinology. 5(6): 423-30.].

[0004] The first-line conventional treatment of diabetic wounds mainly includes internal and external surgical debridement, wound decompression, infection control, and revascularization [see: Edwards J, Stapley S. (2010) Debridement of diabetic footulcers. Cochrane Database Syst Rev. 2010: CD003556.]. However, these methods do not all achieve sufficiently ideal therapeutic effects, and the cost of treatment is high. Compared with ordinary wounds, diabetic wounds take longer to heal. In patients who do not undergo surgical intervention, the median time for wound healing is about 12 weeks [see: Morbach S, Furchert H, Groblinghoff U, et al. (2012) Long-term prognosis of diabetic foot patients and their limbs: amputation and death over the course of a decade. Diabetes Care. 35: 2021-2027]. A large cross-sectional study in China found that the average healing time for diabetic wounds was 76 days for men and 117 days for women [see: Jiang Y, Huang S, Fu X, Liu H, Ran X, Lu S, et al. (2011) Epidemiology of chronic cutaneous wounds in China. Wound repair and regeneration: official publication of the Wound Healing Society [and] the European Tissue Repair Society. 19 (2): 181-8.]. About 20% of patients' wounds fail to heal within 1 year.

[0005] Wound healing can be divided into three consecutive stages, including the first stage - the initial stage of inflammation, the second stage - the stage of tissue formation, and the third stage - the stage of tissue reorganization [see: Gurtner GC, Werner S, Barrandon Y, Longaker MT. (2008) Wound repair and regeneration. Nature. 453: 314-321.]. During the stage of tissue formation, the inflammatory response is weakened, and angiogenesis, fibroblasts and keratinocytes migrate and proliferate to help the wound close.

[0006] Unlike normal tissue healing, clinical and experimental evidence shows that diabetic wounds do not follow these three consecutive stages. Excessive inflammation of the wound is a very important factor [see: Blakytny R, Jude E. (2006) The molecular biology of chronic wounds and delayed healing in diabetes. Diabet Med. 23: 594-608.], which makes the wound surface always in the first stage, that is, the initial stage of inflammation, and cannot transition to the second stage, resulting in angiogenesis disorders and delayed epithelialization. Despite more than a century of research, there is no drug that can effectively help and promote the healing of diabetic wounds. Therefore, exploring new and effective treatment options for diabetic foot ulcers is an urgent task to solve this clinical problem. Summary of the invention

[0007] The task of the present invention is to provide a medicine for treating diabetic foot ulcers to overcome the deficiencies of the prior art.

[0008] The technical solution for realizing the present invention is:

[0009] The drug for treating diabetic foot ulcer provided by the present invention is an M2 macrophage polarization inducer, and the M2 macrophage polarization inducer includes but is not limited to IL-4, IL-13, IL-10, retinoic acid, mitogen-activated protein kinase kinase (MEK) inhibitor, histone deacetylase (HDAC) inhibitor, and granulocyte / macrophage colony-stimulating factor (Granulocyte / macrophage colony-stimulating factor, GM-CSF). The M2 macrophage polarization inducer can be used alone, or two or more M2 macrophage polarization inducers can be selected and used in combination.

[0010] The present invention discloses that an M2 macrophage polarization inducer is used to treat diabetic foot ulcers, can promote wound healing and improve wound inflammation pathology, and has a therapeutic effect that is significantly better than existing treatment methods.

[0011] The present invention has found that the M2 macrophage polarization inducer can be used to treat diabetic foot ulcers in a mouse wound model, and the efficacy is significantly better than the existing treatment methods. One embodiment of the present invention explored the mechanism of M2 macrophage polarization inducer in treating diabetic foot ulcers in a mouse model. We used the M2 macrophage polarization inducer GM-CSF to treat diabetic foot ulcers, and performed western blot detection on mouse wound tissues. It was found that on the 5th day, the expression of the M2a marker YM-1+2 in the GM-CSF group was significantly increased compared with the control group, proving that GM-CSF intervention can indeed significantly promote the transformation of macrophages from M1 to M2, and M2 is mainly type a, exerting an anti-inflammatory effect. Vascular endothelial growth factor (VEGF) is a marker for M2d. On the 10th day, VEGF expression increased significantly, indicating that the performance of macrophages at this time is mainly M2d ( Figure 6 ). Our animal experiments have demonstrated for the first time that the use of GM-CSF in wounds can promote macrophage transformation, and have identified the specific subtypes of transformation. Based on the above research foundation, we used GM-CSF in the DFU population to clinically verify its effectiveness. This clinical evidence provides a basis for the use of GM-CSF in patients with diabetic wounds, helps to increase the treatment options for diabetic wounds, and also reveals the effectiveness of M2 macrophage polarization inducers in the treatment of diabetic foot ulcers. During treatment, M2 macrophage polarization inducers, such as GM-CSF, play a regulatory role on macrophages, transforming the pro-inflammatory M1 into the anti-inflammatory M2, and then promoting angiogenesis and epithelialization, thereby accelerating the healing of diabetic wounds.

[0012] In one embodiment of the present invention, a case of observation of the efficacy of treating diabetic foot ulcers with the M2 macrophage polarization inducer GM-CSF is disclosed. Case 1: Patient, 69 years old, male, diagnosed with diabetic foot ulcer (right calf), which did not heal after 3 months of conventional treatment. The wound was infiltrated with medication (2.0 μg / cm2), covered with a Defu antibacterial hydrocolloid dressing once every three days, and after 5 GM-CSF treatments, the wound was basically healed. Case 2: Patient, 52 years old, male, diagnosed with diabetic foot ulcer (right ankle), which did not heal after 1 and a half months of conventional treatment. The wound was infiltrated with medication (2.0 μg / cm2), covered with a Defu antibacterial hydrocolloid dressing once every three days, and after 5 GM-CSF treatments, the wound was completely healed.

[0013] The M2 macrophage polarization inducer is used as an active ingredient, and a pharmaceutically acceptable additive, excipient or / carrier is added to prepare a pharmaceutical preparation for treating diabetic foot ulcers, specifically, it can be prepared into emulsions, ointments, solutions, creams, gels and other dosage forms. A dressing containing the M2 macrophage polarization inducer for treating diabetic foot ulcers can also be prepared. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Flow chart of the study on the efficacy of GM-CSF in the treatment of diabetic foot ulcers.

[0015] Figure 2 GM-CSF promotes wound healing

[0016] Figure 3 :GM-CSF promotes the improvement of wound inflammatory pathology.

[0017] Figure 4 :A case study of GM-CSF treatment of diabetic foot ulcers. Figure (A) shows the efficacy of GM-CSF in treating diabetic foot ulcers. Case 1: Patient, 69 years old, male, diagnosed with diabetic foot ulcer (right calf), which did not heal after 3 months of conventional treatment. Wound infiltration administration (2.0μg / cm 2 ), covered with Defu antibacterial hydrocolloid dressing, once every three days, after 5 GM-CSF treatments, the wound was basically healed. Figure (B) is the observation of the efficacy of GM-CSF in the treatment of diabetic foot ulcers Case 2: Patient, 52 years old, male, diagnosed with diabetic foot ulcer (right ankle), did not heal after 1 and a half months of conventional treatment. The wound was infiltrated with medication (2.0μg / cm2), covered with Defu antibacterial hydrocolloid dressing, once every three days, after 5 GM-CSF treatments, the wound was completely healed.

[0018] Figure 5 :To explore the effectiveness of GM-CSF in promoting wound healing in animal models. Among them: Figure (A) is a representative picture of wound healing in mice treated with GM-CSF. The head side is a control wound (top), with a hydrogel dressing every day (the same volume of sterile saline was dripped into the wound). The tail side is a GM-CSF-treated wound (bottom), with a hydrogel dressing every day (GM-CSF was dripped into the wound). Figure (B) is a curve chart of the relative area of ​​the wound.

[0019] Figure 6 :The mechanism by which GM-CSF promotes wound healing was explored in animal models, wherein: Figure (A) is a western blot detection of M1 and M2 cell markers in mouse wound tissue on the 5th day; Figure (B) is a western blot detection of M1 and M2 cell markers in mouse wound tissue on the 10th day.

[0020] Figure 7:GM-CSF promotes wound healing in diabetic foot patients (50 cases have been included so far).

[0021] Figure 8 :The mechanism by which GM-CSF promotes wound healing in human wounds was explored, wherein: Figure (A) is an immunofluorescence analysis of M1 cell markers in human wound tissue on the 12th day; Figure (B) is an immunofluorescence analysis of M2 cell markers in human wound tissue on the 12th day. DETAILED DESCRIPTION

[0022] Example 1: GM-CSF and wound healing mechanism (see Figure 5 ,6)

[0023] We first investigated the therapeutic efficacy of GM-CSF, an M2 macrophage polarization inducer, and the regulation of GM-CSF in a mouse wound model. We prepared two wounds on the back of each mouse using a sterile 6-mm skin biopsy punch. Figure 5 Figure (A). Control wound (upper, head side): hydrogel dressing every day (the same volume of sterile saline was dripped into the wound). GM-CSF treatment group (treated wound, tail side): hydrogel dressing every day (GM-CSF was dripped into the wound). We found that at the end of the observation period of day 10, the wound area in the GM-CSF group was 7.1±22.5% of the initial wound area; while the control group was only 37.7±6.6% of the initial wound area (p<0.05), see Figure 5 Figure (B).

[0024] Furthermore, we explored the mechanism in the mouse model to see whether the use of GM-CSF can promote the transformation of macrophage phenotype from M1 to M2. We performed western blot detection on mouse wound tissue and found that on the 5th day, the expression of M2a marker YM-1+2 in the GM-CSF group was significantly increased compared with the control group, indicating that GM-CSF intervention can indeed significantly promote the transformation of macrophages from M1 to M2, and M2 is mainly type a, exerting an anti-inflammatory effect. Vascular endothelial growth factor (VEGF) is a marker for M2d. On the 10th day, VEGF expression increased significantly, indicating that the expression of macrophages at this time is mainly M2d ( Figure 6 Our animal experiments demonstrated for the first time that the use of GM-CSF in wounds can promote macrophage transformation and identify the specific subtypes of transformation.

[0025] Based on the above research foundation, we conducted clinical verification of GM-CSF in the DFU population and proved its effectiveness, which provided a basis for the application of GM-CSF and M2 macrophage polarization inducers in patients with diabetic wounds, revealed the effectiveness of M2 macrophage polarization inducers in the treatment of diabetic foot ulcers, and provided an effective treatment for diabetic wounds.

[0026] Example 2: Study on the efficacy of GM-CSF in treating diabetic foot ulcers. GM-CSF promotes wound healing and improves wound inflammation pathology (see Figure 1 , Figure 2 , Figure 3 )

[0027] After the first use of GM-CSF to treat diabetic foot ulcers, 1 case (3.03%) of ulcers achieved complete epithelialization; 7 cases (21.2%) of ulcers had an ulcer healing rate of >75%; after the fourth use of medication, 12 days after treatment, 10 cases (30.3%) of ulcers achieved complete epithelialization, and 21 cases (63.6%) had an ulcer healing rate of >75%. There were statistical differences in the overall average healing rate (p < 0.001) and the percentage of wound surface to initial volume (p < 0.01) between the two groups. Studies have confirmed that GM-CSF can promote wound healing and improve wound inflammation pathology in diabetic foot ulcers.

[0028] Example 3: A case study on the treatment of diabetic foot ulcers with GM-CSF.

[0029] Case 1: Patient, 69 years old, male, diagnosed with diabetic foot ulcer (right calf), which did not heal after 3 months of conventional treatment. Wound infiltration medication (2.0 μg / cm 2 ), covered with antibacterial hydrocolloid dressing every three days. After 5 GM-CSF treatments, the wound was basically healed. Figure 4 Figure (A).

[0030] Case 2: Patient, 52 years old, male, diagnosed with diabetic foot ulcer (right ankle), which did not heal after 1.5 months of conventional treatment. The wound was infiltrated with medication (2.0μg / cm2) and covered with Defu antibacterial hydrocolloid dressing once every three days. After 5 treatments of GM-CSF, the wound healed completely. Figure 4 Figure (B).

[0031] Example 4: GM-CSF promotes wound healing in diabetic foot patients (see Figure 7 , 50 cases have been included so far, Figure 7 for Figure 2 (Supplementary data).

[0032] This study has currently included 50 patients with DFU (the clinical study has been registered with the National Center for Clinical Trials: ClinicalTrials.gov, identifier: NCTChiCTR2400081658). The regimen adopted was to change the dressing every 3 days, and the GM-CSF dose was 2.0 μg / cm 2 .

[0033] The enrolled patients were randomly divided into groups. During the trial, 4 patients were divided into the treatment group and 3 patients in the control group withdrew from the trial for various reasons. Currently, there are 21 patients in the GM-CSF treatment group and 22 patients in the control group who have completed the study, with a total of 43 DFU patients. The average age of the treatment group was 60.8±10.5 years old; the average age of the males in the control group was 59.0±9.6 years old. The duration of ulcer before admission in the treatment group was 1.5 months, while that in the control group was 1.9 months. The SINBAD score of the treatment group was 2-4 points, and the Wagner score was 1-3 points. The baselines of the two treatment groups and the control group were the same.

[0034] Analysis of the ulcer area showed that the relative values ​​of the ulcer area in the GM-CSF treatment group at each time point were always smaller than those in the conventional treatment control group ( Figure 7 ). During the observation period, after 2-8 GM-CSF treatments, 18 patients (85.7%) achieved complete wound epithelialization, with a median number of treatments of 5. The average healing time in the GM-CSF treatment group was 15.5±6.8 days, and the overall wound healing rate was 0.38±0.31cm 2 In the control group, only 3 cases (13.6%) achieved complete epithelialization during the observation period, and the rest of the patients had not healed at the end of the observation period. The overall wound healing rate was 0.19±0.15cm 2 / d, which was significantly lower than that in the GM-CSF treatment group. The above results show that rhGM-CSF can significantly accelerate the healing of ulcer wounds and improve the overall healing level.

[0035] Example 5: GM-CSF and wound healing mechanism (see Figure 8 )

[0036] We further explored whether GM-CSF can promote the transformation of macrophage phenotype from M1 to M2 in a human population trial. We collected samples on the 12th day and detected the expression levels of M1 (CD68+CD86+) macrophages and M2 (macrophages). It was found that after GM-CSF treatment, compared with the control group, the M1 in the treatment group was significantly reduced, while the M2 was significantly increased (see Figure 8 ). This is consistent with the conclusion of animal experiments.

Claims

1. Application of M2 macrophage polarization inducer in the preparation of drugs for treating diabetic foot ulcers.

2. The use according to claim 1, characterized in that: The M2 macrophage polarization inducer is one of the following substances, or a mixture of two or more thereof: IL-4, IL-13, IL-10, retinoic acid, mitogen-activated protein kinase kinase (MEK) inhibitor, histone deacetylase (HDAC) inhibitor, granulocyte / macrophage colony stimulating factor (GM-CSF).

3. A pharmaceutical preparation for treating diabetic foot ulcers, characterized in that: The invention is a pharmaceutical preparation which takes an M2 macrophage polarization inducer as an active ingredient and is added with pharmaceutically acceptable additives, excipients or / carriers.

4. The pharmaceutical preparation according to claim 3, characterized in that The M2 macrophage polarization inducer is one of the following substances, or a mixture of two or more thereof: IL-4, IL-13, IL-10, retinoic acid, mitogen-activated protein kinase kinase (MEK) inhibitor, histone deacetylase (HDAC) inhibitor, granulocyte / macrophage colony stimulating factor (GM-CSF).

5. The pharmaceutical preparation according to claim 3 or 4, characterized in that The pharmaceutical preparation is an emulsion, an ointment, a solution, a cream or a gel.

6. A dressing for treating diabetic foot ulcers, characterized in that: The dressing contains an M2 macrophage polarization inducer.

7. The dressing for treating diabetic foot ulcer according to claim 6, characterized in that: The M2 macrophage polarization inducer is one of the following substances, or a mixture of two or more thereof: IL-4, IL-13, IL-10, retinoic acid, mitogen-activated protein kinase kinase (MEK) inhibitor, histone deacetylase (HDAC) inhibitor, granulocyte / macrophage colony stimulating factor (GM-CSF).

8. A dressing for treating diabetic foot ulcers, characterized in that: A pharmaceutical preparation comprising the pharmaceutical preparation according to claim 3 or 4.

9. A dressing for treating diabetic foot ulcers, characterized in that: A pharmaceutical preparation comprising the pharmaceutical preparation according to claim 5.

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