CN114668764A

CN114668764A - Application of compound in preparation of external medicine for treating diabetic foot

Info

Publication number: CN114668764A
Application number: CN202210360897.3A
Authority: CN
Inventors: 项荣; 赵美方; 王芳; 黄皓; 盛月; 刘宇星
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2022-04-07
Filing date: 2022-04-07
Publication date: 2022-06-28

Abstract

The invention relates to an external medicine for treating diabetic foot. The invention discloses an application of a compound in preparing an external medicine for treating diabetic foot, wherein the molecular structural formula of the compound is as follows:

wherein R1 includes:

r2 includes:

Description

Application of compound in preparation of external medicine for treating diabetic foot

Technical Field

The invention belongs to the field of medicines, and particularly relates to an external medicine for treating diabetic foot.

Background

The diabetic foot is one of serious complications of diabetes, is lower limb infection, ulcer or deep tissue ulceration caused by combined neuropathy and lower limb vascular lesions of various degrees, and brings great pain and heavy economic burden to patients. A common clinical manifestation is chronic ulcers, with the most serious outcome being amputation and even death. The key of the current treatment principle of the diabetic foot disease lies in preventing and reducing the amputation rate of diabetic patients, and is to prevent diabetic foot ulcer, reasonably treat foot ulcer and prevent ulcer recurrence.

At present, the treatment modes of the diabetic foot are mainly divided into comprehensive treatment and local treatment. The comprehensive treatment generally comprises metabolic control, blood vessel expansion, blood circulation activation and blood stasis removal, application of antibiotics and the like, and the main purposes are to reduce blood sugar and improve vascular tension and nerve function. From the perspective of reducing blood sugar, the complications are relieved from worsening, and wound healing is promoted, but comprehensive treatment generally has the pain point that the treatment period is long and life-long cooperation treatment is needed. The local treatment mainly aims at the treatment of local wounds, and partially converts infected or polluted wounds into clean wounds so as to be beneficial to wound healing, the treatment mode mainly adopts sterilization and anti-inflammation, the blood sugar level of a patient is not influenced, the general local treatment comprises debridement for cutting necrotic tissues, wound treatment for sterilizing and diminishing inflammation of the wounds by changing medicines every day and the like, and the treatment is mainly aiming at the wounds, but the local treatment also has a plurality of problems, for example, the sterilization and anti-inflammation treatment has partial effects but cannot completely cure; the effect is slow; the price is high; poor comfort for patients during treatment, etc. The treatment process of the diabetic foot always has high cost, the technical means is not mature enough, the clinical data is insufficient, and the like, so that the treatment effect is difficult to satisfy. Some diabetic foot patients can only prevent the condition from expanding and deteriorating by means of amputation, and the cost is heavy.

Therefore, it would be of great significance to develop early prevention and treatment of foot problems in diabetic patients.

It has been found that compounds of formula 1 (including 6- [4- (2-piperidine-1-yl-ethoxy) -phenyl ] -3-pyridine-4-yl-pyrrazolo [1,5-a ] pyrimidine,6- [4- (2-piperidin-1-yl-ethoxy) -phenyl ] -3-pyridin-4-yl-pyrazolo [1,5-a ] pyrimidine, also known as Dorsomorphin doxororphine and 4- [6- [ 4-isopropoxyphenyl ] pyrazolo [1,5-a ] pyrimidin-3-yl ] quinoline), are AMPK inhibitors, which are useful in the inhibition of hyperferremia, in the promotion of cardiomyocyte differentiation in mouse and human pluripotent stem cells (Hao et al, Kattman et al), in the promotion of adipocyte differentiation, inhibiting osteogenic differentiation of human mesenchymal osteoblasts. The compound is a pyrazolopyrimidine derivative, has a strong inhibitory effect on AMPK, and is an AMPK inhibitor widely applied.

Disclosure of Invention

The invention aims to provide the application of the compound in preparing the external medicine for treating diabetic foot, and the treatment mode is convenient and easy to operate and does not influence the life quality of the diabetic foot.

In order to solve the technical problems, the technical scheme of the invention is as follows:

the application of a compound in preparing an external medicine for treating diabetic foot is disclosed, wherein the molecular structural formula of the compound is shown as the formula (I):

wherein R1 includes:

r2 includes:

preferably, the compound has the structural formula

Preferably, the external medicine is any one of plaster, gel, lotion, cream and spray.

Preferably, in order to better improve the absorption effect of the medicament, shorten the onset time of the medicament, particularly reduce the irritation of the medicament to the skin, reduce the discomfort of partial allergic skin patients and improve the medication compliance, thereby better improving the treatment effect, the plaster also comprises auxiliary materials and absorbents commonly used in the field.

Preferably, the auxiliary material and the absorbent are titanium dioxide, polyvinyl alcohol, glycerol or menthol. The dosage of the traditional Chinese medicine is generally 5-15%, preferably about 5-10% of the weight of the raw materials of the plaster.

Preferably, the gel is prepared from the medicine and appropriate auxiliary materials, and the auxiliary materials comprise one or more of the auxiliary materials which can be accepted in the common skin external preparation, such as a humectant, a solubilizer and gel.

Preferably, the lotion comprises one or more of a surfactant, a skin care agent, a preservative, a bactericide and a solvent in addition to the drug.

Preferably, the cream comprises, in addition to the medicament, one or more of a moisturizer, an emollient, a solvent, a preservative and a fragrance.

Preferably, the spray comprises one or more of a surfactant, a cooling component, a solvent and a stabilizer in addition to the drug.

Existing topical anti-inflammatory treatments have partial effects, such as aspirin, but cannot be completely cured; has slow effect. None of the prior art discloses that AMPK inhibitors can be used in the treatment of diabetic feet.

The compound shown in the formula (I) can reduce inflammatory factors taking ICAM-1, VCAM-1, iNOS/NO, NLRP3, NOS2, IL-1 beta, IL-6, TNF alpha, MCP-1 and the like as targets, wherein ICAM-1 and VCAM-1 are inflammasome related to cardiovascular diseases, and IL-1 beta is an important inflammasome of type II diabetes.

The currently known action mechanism of aspirin and diclofenac is that platelet cyclooxygenase (COX-1) is inhibited in vivo, and then thromboxane A2 is inhibited, so that the production of inflammasome such as prostaglandin and thromboxane is reduced (the body generates symptoms such as fever and fever due to the large secretion of PGE 2).

Compared with other medicines, the compound plaster shown in the formula (I) has the advantages of higher treatment speed and better effect in treating diabetic feet, because the compound shown in the formula (I) has a wide anti-inflammatory spectrum, and compared with common anti-inflammatory medicines such as aspirin and diclofenac, the compound shown in the formula (I) has a wider anti-inflammatory spectrum. In our previous studies, it was found that the compound of formula (I) has a blood sugar level-lowering effect in the mouse body after it is ingested into high-fat-fed mice. After the insulin is injected into a mouse, the drug is found to relieve the insulin resistance of the mouse at the same time, and the compound shown in the formula (I) is more advantageous to the treatment of diseases related to hyperglycemia compared with other drugs. In the process of treating diabetic feet by using the compound plaster, the compound shown in the formula (I) is contacted with the surface of a wound, the wound surface is sterilized and is anti-inflammatory so as to achieve a medical effect, and the infiltrated medicine can be combined with a blood vessel related inflammatory body near the wound to slow down the vascular lesion, so that the curative effect is more obvious compared with other medicines. Compared with common anti-inflammatory drugs, the compound proved by ELISA has better capability of eliminating related inflammasome.

The invention firstly explains that the compound shown in the formula (I) has exact treatment effect on diabetic foot, has good anti-inflammatory and anti-infection effects, provides a good lead compound for developing novel diabetic foot medicaments, and has wide application prospect.

The compound shown in the formula (I) has a remarkable inhibiting effect on bacterial infection of diabetic feet, the healing time of ulcer or lesion after bacterial infection of the diabetic feet is remarkably shortened, meanwhile, the treatment mode belongs to local treatment, and compared with the traditional local treatment, the external medicine has the remarkable advantages of being friendly to people in price, convenient to use and remarkable in curative effect, and does not show side effects.

Drawings

Fig. 1 shows a first finished product of the plaster of the present invention.

Fig. 2 shows a second patch application product of the present invention, the patch uses a patch matrix as a carrier, and the compound is heated and melted, and then taken out, and the mixture is coated on a blank patch to be solidified and formed.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings, which are for illustrative purposes only and are not intended to limit the scope of the invention. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention can be embodied in many different forms than those herein described and modified by those skilled in the art without departing from the spirit and scope of the invention and it is intended that the invention not be limited to the specific embodiments disclosed below.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example 1

1. Preparation of plaster

50mg of doxorphine and 4- [6- [ 4-isopropoxyphenyl ] pyrazolo [1,5-a ] pyrimidin-3-yl ] quinoline powder were dissolved in 21mL of sterile PBS to prepare a 5mM stock solution of the compound. 1mL of a stock solution of doxorphine or 4- [6- [ 4-isopropoxyphenyl ] pyrazolo [1,5-a ] pyrimidin-3-yl ] quinoline was diluted with a sterile PBS solution to prepare working solutions of 100uM, 80uM, 60uM, 40uM, and 20uM, respectively.

50ul of working solution with different concentrations is respectively sucked by a pipette gun and titrated on a sponge of a blank woundplast without medicament to prepare the plaster 1 as shown in figure 1.

Respectively sucking a small amount of working solution with different concentrations by using a pipette, dripping the working solution on a blank plaster substrate, heating to melt the substrate, stirring to fully mix the compound solution and the substrate uniformly to prepare plaster with 100uM concentration, then respectively taking about 1-5 g of the substrate by using a medicine spoon, uniformly coating the substrate on the blank plaster, coating the substrate on the blank plaster to form plaster with different areas so as to be suitable for wound surfaces with different areas, waiting for the plaster to solidify, and preparing plaster 2 with different areas, as shown in figure 2.

The blank plaster is prepared by sucking equal amount of distilled water and a sponge of the blank woundplast or a matrix of the blank plaster.

The patches 1 or 2 were used for the following animal experiments.

2. Establishment of diabetic foot rat model

The tested animals are 30 large SPF SD male rats in two months, the weight of the tested animals is 220-240 g, the tested animals are fed with water freely, the room temperature of a breeding room is kept at 25 +/-1 ℃, and the light and shade period is 12 hours.

After 3 days of adaptive feeding of SD rats, weighing and measuring the blood sugar of the rats, wherein the blood sugar meter is a trinor Jinshun plus blood sugar meter (the maximum range is 33mmol/L), Streptozotocin (STZ) is injected into the abdominal cavity, the injection amount is 50mg/kg, the blood sugar of the rats is measured after the rats are continuously fed with high-fat feed for one week, the blood sugar level is higher than 16.7mmol/L, and the conditions of polyphagia, polydipsia, polyuria and weight loss are accompanied, so that the model building of the diabetic rats is considered to be successful. After 2 weeks of streptozotocin administration, a wound was created and this day was defined as day 0. Wounds were created in the right hind paw (foot) of each rat. Each rat was anesthetized with 10% chloral hydrate (0.3ml/100g) by intraperitoneal injection. A rectangular pattern was marked on the back surface of the right hind paw (foot) using a flexible clear plastic template, and then the full thickness skin of about 2mm x 5mm standard area was removed. After the diabetic wounds are induced, a blank plaster is given to rats for treatment, which is recorded as a diabetic foot wound surface group, the rest 3 groups are given with plasters with different concentrations for treatment, and the other 3 groups are also given with two groups of positive control groups, wherein 1 group of positive control groups are given with sodium prostaglandins combined with aspirin plaster for treatment, and each plaster contains 0.5mg of aspirin and 0.6ug of sodium prostaglandins; 1 group of positive control groups were treated with diclofenac sodium lidocaine plaster, which contained 10ul of diclofenac sodium lidocaine solution per day.

The plaster comprises the following components: a high-concentration doxorphine treatment group, a medium-concentration doxorphine treatment group and a low-concentration doxorphine treatment group. The high-concentration doxorphine treatment group was intervened with a patch at a concentration of 100 μ M doxorphine, the medium-concentration doxorphine treatment group was intervened with a patch at a concentration of 60uM doxorphine, and the low-concentration doxorphine treatment group was intervened with a patch at a concentration of 20uM doxorphine.

The preparation method of the STZ solution comprises the following steps: weighing 1.05g of citric acid, adding 50ml of sterile water, and uniformly mixing to obtain solution A; weighing 1.47g of sodium citrate, adding 50ml of sterile water, and mixing uniformly to obtain solution B. Solution a and solution B1: 1, and adjusting the pH value to 4.7 to form working fluid. Dissolving 0.1g of STZ into 5mL of the working solution, and mixing uniformly to obtain the STZ buffer solution required by the diabetes molding, wherein the STZ buffer solution is prepared for use.

3. Diabetic foot rat wound closure rate

The wounds of the rats were observed daily, recorded by photography, and the size of the wounds was recorded.

According to wound closure% — 100 × [ (WN-W0)/W0 ]; w0 — wound area on day 0; WN is the wound area on

days

0, 3, 6, 8, and 10. The wound closure of the diabetic wound control group was negative, indicating that the wound area was large and the wound was deteriorated. The wound closures in the normal group and the doxorphine-treated group were positive, indicating that the wound area became small and the wound healed. The data are shown in table 1.

TABLE 1 wound closure Rate

From the results it can be seen that: on the sixth day, the wound surfaces of the plaster treatment groups with the three concentrations of high, medium and low are obviously reduced and begin to scab, while the wound surface area of the diabetic foot wound surface group is still larger and is in the ulcer stage; the eighth day, the scar of the plaster-treated group had already fallen off, the wound area was further reduced, wherein the wound healing rate of the high-concentration and medium-concentration doxorphine treatment had already reached about 80%, the wound of the diabetic foot wound group had just scabbed, and had a larger wound; on the tenth day, the wounds of the high-concentration and medium-concentration doxorphine groups of the plaster treatment group are basically healed, the healing rate of the low-concentration doxorphine treatment reaches about 80%, and the wounds of the diabetic foot wound group still have larger wounds.

The results of the two positive control groups are: compared with the blank group, the aspirin group has a certain effect of promoting wound healing, the wound begins to scab in the sixth day, and the wound gradually falls off in the eighth and ninth days, but the area of the aspirin group is different from the healing rate of the compound treatment group. The diclofenac group has slower wound healing speed, and the wound is healed by about 70 percent in the tenth day, so the effect of promoting the wound healing is poor.

Aspirin in combination with sodium prostacyclin treatment continued for 8 weeks due to poor wound healing within 10 days. After 8 weeks, the treatment effective rate of the treatment mode observation group is about 95%, the healing rate is about 80%, and the treatment effect is better, but the treatment time is long, the adverse reaction rate is high, and about 12.5% of mice have adverse reactions such as dizziness, nausea, gastrointestinal hemorrhage and the like.

4- [6- [ 4-Isopropoxyphenyl ] pyrazolo [1,5-a ] pyrimidin-3-yl ] quinoline has also been applied as a patch and wound treatment for diabetic feet, with the wound healing on the tenth day being about 87.

The compound of the invention has the advantages of relatively quick response and no other adverse states of experimental animals except the diabetic phenotype.

Similarly, the diclofenac group had insignificant wound healing efficacy within 10 days and long treatment times.

The structural formula of aspirin is:

the structural formula of diclofenac is:

although aspirin and diclofenac have similar structures to the compounds and have been reported to be studied as anti-inflammatory agents for treating diabetic feet, their external therapeutic effects are not good. This also suggests that conventional anti-inflammatory drugs are not effective in treating diabetic foot.

During the entire recovery period, it was seen that the high concentration group treated wounds better than the medium concentration group, the medium concentration group better than the low concentration group, and the overall group better than the blank control group.

4. Blood glucose values in diabetic foot rats

The results of testing blood glucose levels in the mice are shown in Table 2.

It can be seen from blood sugar values that the blood sugar values of rats in the diabetic wound group, the doxorphine group, the aspirin group and the diclofenac group are not obviously fluctuated in the whole treatment process and are all at high blood sugar levels, which shows that the external plaster does not influence the blood sugar value of an organism and promotes wound healing only externally in an inflammation inhibition mode.

5. Enzyme-linked immunosorbent assay for inflammatory IL-1 beta and IL-6

THP-1 (human monocyte) cells were stimulated with LPS (lipopolysaccharide) and PA (palmitic acid) to activate NLRP3 inflammasome using classical inflammatory stimulation modalities. And then treating the cells with sterile compound with 20uM concentration, aspirin and diclofenac for 2h and blank treatment respectively. After cell treatment, cell culture medium supernatants were collected and assayed for IL-1 β, IL-6 protein levels in the supernatants using an ELISA kit (R & D system) according to manufacturer's protocol.

According to analysis of ELISA detection results, the IL-1 beta and IL-6 levels in cell supernatants of the doxorphine-treated group are obviously lower than those of aspirin and diclofenac-treated groups, and the IL-1 beta and IL-6 levels of a blank group are the highest. The experimental result shows that under the same treatment condition, aspirin and diclofenac have certain anti-inflammatory effects, but doxorphine has better anti-inflammatory capability than the aspirin and the diclofenac, and certain powerful theoretical data are provided for the invention. And multiple data prove that the compound has remarkably better anti-inflammatory action than general anti-inflammatory medicaments sold on the market and common anti-inflammatory medicaments disclosed in the prior art. This may be one of the reasons why the compounds are more effective in treating diabetic feet.

The experiments show that the compound shown in the formula (I) has good inflammation inhibiting and diabetic wound recovery promoting effects in vivo and in vitro, and the plaster disclosed by the invention has a good treatment effect on diabetic foot wounds.

The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The application of the compound in preparing the external medicine for treating diabetic foot is characterized in that the molecular structural formula of the compound is shown as the formula (I):

wherein R1 includes:

r2 includes:

2. the use of claim 1, wherein the compound has the formula

3. The use of claim 1, wherein the external medicament is any one of a patch, a gel, a lotion, a cream, and a spray.

4. The use according to claim 3, wherein said patch further comprises adjuvants and absorbents commonly used in the art.

5. The use according to claim 4, wherein the auxiliary materials and the absorbent are titanium dioxide, polyvinyl alcohol, glycerol or menthol, and the dosage of the auxiliary materials and the absorbent is 5-15% of the weight of the raw material medicine of the plaster, preferably 5-10%.

6. The use of claim 3, wherein the gel is prepared from a drug and an excipient, and the excipient comprises one or more of a humectant, a solubilizer and a gel.

7. The use of claim 3, wherein the lotion comprises one or more of a surfactant, a skin care agent, a preservative, a bactericide, and a solvent in addition to a drug.

8. Use according to claim 3, wherein the cream comprises, in addition to a medicament, one or more of a moisturizer, an emollient, a solvent, a preservative and a fragrance.

9. Use according to claim 3, wherein the spray comprises, in addition to the medicament, one or more of a surfactant, a cooling component, a solvent and a stabiliser.