Application of fluorocitric acid Fluorocitrate in preparation of medicine for treating inflammatory pain

Abstract

The invention discloses an application of fluorocitric acid Fluorocitrate in preparation of a medicine for treating inflammatory pain, and belongs to the technical field of medicines. According to the invention, a complete Freund's adjuvant induced inflammatory pain model is constructed based on a KM mouse, an astrocyte inhibitor Fluorocitrate is given to the inflammatory pain model on the second day, and the analgesic effect of the Fluorocitrate on the inflammatory pain mouse is evaluated through a behavioral experiment. The result shows that the Flurocitrate can significantly reduce the pain sensitivity of mice with inflammatory pain, which indicates that the Flurocitrate has potential application value in treatment of inflammatory pain and is expected to be developed into a potential therapeutic drug for inflammatory pain.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of an astrocyte inhibitor in the preparation of a medicament for treating inflammatory pain. Background Art

[0002] Inflammatory pain is persistent pain caused by tissue damage or chronic inflammation, and the clinical manifestations are pain hypersensitivity and spontaneous pain, which seriously affect the quality of life of patients. At present, non-steroidal anti-inflammatory drugs (NSAIDs) and opioid drugs are the main treatment means, but the above treatment methods have side effects such as gastrointestinal damage and addiction, and there is an urgent need to develop new therapeutic drugs.

[0003] Recent studies have shown that astrocytes play a key role in the inflammatory response and pain signal transmission in the central nervous system. In an inflammatory pain model, astrocytes exacerbate neuroinflammation and pain hypersensitivity by releasing pro-inflammatory factors (such as IL-1β, TNF-α) and reactive oxygen species (ROS). Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide the application of Fluorocitrate in the preparation of a medicament for treating inflammatory pain.

[0005] The purpose of the present invention is achieved by the following technical solutions:

[0006] A medicament for treating inflammatory pain, comprising Fluorocitrate.

[0007] Further, the Fluorocitrate is sodium fluorocitrate, and the molecular formula of sodium fluorocitrate is:

[0008]

[0009] The application of Fluorocitrate in the preparation of a medicament for treating inflammatory pain

[0010] Further, the treatment of inflammatory pain includes increasing the pain threshold of inflammatory pain.

[0011] Further, the medicament further comprises a pharmaceutically acceptable lipid nanocarrier.

[0012] Further, the dosage form of the medicament is tablets, granules, capsules or injections.

[0013] Further, the Fluorocitrate is sodium fluorocitrate, and the molecular formula of sodium fluorocitrate is:

[0014]

[0015] A method for constructing a mouse model, comprising the following steps:

[0016] Step 1: Select experimental animal mice and conduct experimental grouping. The experimental grouping is as follows:

[0017] Normal saline group: Inject 40 μl of normal saline into the plantar surface of the mouse's foot, and inject 200 nl of normal saline microscopically the next day;

[0018] Inflammatory pain group: Inject 40 μl of CFA into the plantar surface of the mouse's foot, and inject 200 nl of normal saline microscopically the next day;

[0019] Inflammatory pain treatment group: Inject 40 μl of CFA into the plantar surface of the mouse's foot, and inject

[0020] Fluorocitrate 200 nl microscopically the next day;

[0021] The microscopic injection steps are as follows: Anesthetize the mouse with 2% isoflurane and fix it in a prone position on a stereotaxic apparatus. During the operation, control the rectal temperature at 37.0 ± 0.5 °C with a thermostatic blanket. The operation process includes exposing the skull, removing connective tissue, and keeping the surface dry. Zero the instrument coordinates with the bregma as the zero point. Inject normal saline or Fluorocitrate into the CA1 brain region, with an injection volume of 200 nl each, an injection speed of 40 nl / min, stop the needle for 5 minutes, and finally suture the wound and give anti-inflammatory treatment;

[0022] Step 2: Prepare for pain threshold detection of experimental animals. Conduct behavioral tests before pain threshold detection. The behavioral tests are carried out in a double-blind trial manner. Before the basic behavioral tests, let all mice adapt to the test environment for 1 hour;

[0023] Step 3: Conduct pain threshold detection on the 4th day after surgery. The pain threshold detection includes mechanical pain test and thermal pain test, and finally obtain experimental data.

[0024] Furthermore, the mechanical pain test includes using two calibrated von Frey filaments. Select the 0.07 g Von Frey filament representing low-intensity stimulus-evoked pain and the 0.4 g Von Frey filament representing high-intensity stimulus-induced hyperalgesia to apply stimuli to the central skin of the plantar surface of the left hind limb of the mouse. After the filament vertically penetrates the grid hole, apply force evenly to bend the filament into a "C" shape. The stimulation application time lasts about 1.5 seconds, repeat 10 times, with an interval of more than 5 minutes between each stimulation. Observe the mouse's paw withdrawal response and record it, and calculate the paw withdrawal frequency of the mouse under different stimulation intensities.

[0025] Further, the central skin of the hind paw of the mouse is irradiated with a heat radiation instrument for thermal pain testing, and the irradiation duration from the start to when the mouse lifts its foot or licks the sole of the foot is recorded. This is repeated 5 times, with an interval of more than 10 minutes between each irradiation. The average value of the 5 irradiation times is taken as the thermal withdrawal latency.

[0026] Advantages of the present invention:

[0027] Through the application of the astrocyte inhibitor fluorocitrate in an inflammatory pain model, experiments have shown that it can significantly reduce pain-like behaviors in diseased mice, inhibit the activation of hippocampal astrocytes, and reduce the release of pro-inflammatory factors, providing a new strategy for the treatment of inflammatory pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1 : Fluorescence image of the activation of astrocytes in the model group;

[0030] Figure 2 : Fluorescence image of the activation of astrocytes in the control group;

[0031] Figure 3 : Effect of sodium fluorocitrate on the pain threshold of inflammatory pain mice;

[0032] Figure 4 : Molecular formula of sodium fluorocitrate. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0034] Example 1

[0035] Evaluate the effect of fluorocitrate on the pain threshold of inflammatory pain.

[0036] Experimental animals: KM male mice aged 8 - 10 weeks, with a body weight of 25 - 30 g. All mice live in a breeding cage with free access to food and water, and the light is controlled for 12-hour day and night alternation.

[0037] Experimental grouping: normal saline group (control group, n = 8); CFA + solvent control (inflammatory pain group, n = 8), CFA + Fluorocitrate (inflammatory pain treatment group, n = 8). All of the above were CFA model mice.

[0038] Normal saline group: Inject 40 μl of normal saline into the plantar surface, and inject 200 nl of normal saline microscopically the next day;

[0039] Inflammatory pain group: Inject 40 μl of CFA into the plantar surface, and inject 200 nl of normal saline microscopically the next day;

[0040] Inflammatory pain treatment group: Inject 40 μl of CFA into the plantar surface, and inject 200 nl of Fluorocitrate microscopically the next day;

[0041] The steps of microscopic injection are as follows: Anesthetize the CFA model mice with 2% isoflurane and fix them in the stereotaxic apparatus in the prone position. During the operation, control the rectal temperature at 37.0 ± 0.5 °C with a thermostatic blanket. The operation process includes exposing the skull, removing connective tissue, and keeping the surface dry. Zero the instrument coordinates with the anterior fontanelle as the origin, inject normal saline or Fluorocitrate into the CA1 brain region, the injection volume is 200 nl, the injection speed is 40 nl / min, stop the needle for 5 minutes, and finally suture the wound and give anti-inflammatory treatment.

[0042] The experimental model is: an inflammatory pain model induced by complete Freund's adjuvant.

[0043] The CFA model is a pain model that uses complete Freund's adjuvant to form local inflammation in tissues to induce inflammatory pain. Expose the left hind limb of the mouse, and subcutaneously inject complete Freund's adjuvant (25 μl) into the model group and normal saline (25 μl) into the control group.

[0044] Animal treatment: Inject CFA into the left plantar surface of the mouse to obtain an inflammatory pain model induced by complete Freund's adjuvant, that is, obtain CFA model mice. Anesthetize the CFA model mice with 2% isoflurane and fix them in the stereotaxic apparatus in the prone position. During the operation, control the rectal temperature at 37.0 ± 0.5 °C with a thermostatic blanket. The operation process includes exposing the skull, removing connective tissue, and keeping the surface dry. Zero the instrument coordinates with the anterior fontanelle as the origin, inject Fluorocitrate into the CA1 brain region (ML: 1.2, AP: -1.5, DV: -1.8), the injection volume is 200 nl, and the injection speed is 40 nl / min. Finally, suture the wound and give anti-inflammatory treatment. And perform pain threshold detection on the 4th day after surgery.

[0045] Behavioral test: The behavioral test is carried out in a double-blind trial manner. Before the basic behavioral test, let all mice adapt to the test environment for 1 hour.

[0046] Mechanical pain test: Two calibrated von Frey filaments were used. The 0.07 g Von Frey filament representing low-intensity stimulus-induced pain and the 0.4 g Von Frey filament representing high-intensity stimulus-induced hyperalgesia were respectively selected to apply stimuli to the central skin of the plantar surface of the left hind limb of the mice. After the filament vertically passed through the grid holes, the filament was evenly forced to bend into a "C" shape, and the stimulation application time lasted about 1.5 seconds. It was repeated 10 times, with an interval of more than 5 minutes between each stimulation. The paw withdrawal response of the mice was observed and recorded, and the paw withdrawal frequency of the mice under different stimulation intensities was calculated. The number of paw withdrawals of the mice in 10 stimulations was recorded as PWF [(number of paw withdrawals / 10 trials) × 100 = % response frequency].

[0047] Thermal pain test: A 336-type analgesia meter (the existing model is IITC336-type analgesia meter) was used to measure the paw withdrawal latency (PWL) of noxious heat stimuli. The mice were placed in a plexiglass chamber on a glass plate, and a beam of noxious light (heat) was applied to the middle plantar surface of each hind paw. When the paw was quickly lifted, the light beam was automatically turned off. The time from the start to the stop represented the paw withdrawal latency (PWL). Five trials were carried out at 5-minute intervals. A 20-second cut-off time was used to avoid tissue damage to the paw.

[0048] The results are as Figure 3 shown. Administration of Fluorocitrate could significantly reverse the paw withdrawal time and paw withdrawal frequency of CFA-induced inflammatory pain in mice, indicating that Fluorocitrate could relieve the pain-like behaviors of inflammatory pain mice.

[0049] Figure 3 Among them, Sal+veh: normal saline group, CFA+veh: inflammatory pain group, CFA+Flu: inflammatory pain treatment group;

[0050] PWF: (number of paw withdrawals / 10 trials) × 100 = % response frequency; PWL: the time from the start of irradiating the middle of the plantar surface of the mouse paw with noxious light to paw withdrawal. Figure 3 In A: the paw withdrawal frequency of the mice after stimulating the middle of the plantar surface of the mice with a 0.07 g Von Frey filament. Figure 3 In B: the paw withdrawal frequency of the mice after stimulating the middle of the plantar surface of the mice with a 0.4 g Von Frey filament. Figure 3 In C: the time from the start of irradiating the plantar surface of the mouse paw with noxious light to paw withdrawal of the mouse.

[0051] Unless otherwise specified, the reagents or instruments used in the present invention are all commercially available.

[0052] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

[0053] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification is only to illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed.

Claims

1. A drug for treating inflammatory pain, characterized in that: Includes Fluorocitrate.

2. A drug for treating inflammatory pain according to claim 1, characterized in that: The fluorocitrate Fluorocitrate is sodium fluorocitrate, and the molecular formula of sodium fluorocitrate is:

3. Application of Fluorocitrate in the preparation of drugs for treating inflammatory pain.

4. The use of Fluorocitrate according to claim 3 in the preparation of a drug for treating inflammatory pain, characterized in that: The treating of inflammatory pain includes increasing the pain threshold of inflammatory pain.

5. The use of Fluorocitrate according to claim 3 in the preparation of a drug for treating inflammatory pain, characterized in that: The medicament also includes a pharmaceutically acceptable lipid nanocarrier.

6. The use of Fluorocitrate in the preparation of a drug for treating inflammatory pain according to claim 3, characterized in that: The dosage form of the medicine is tablet, granule, capsule or injection.

7. The use of Fluorocitrate in the preparation of a drug for treating inflammatory pain according to claim 3, characterized in that: The fluorocitrate Fluorocitrate is sodium fluorocitrate, and the molecular formula of sodium fluorocitrate is:

8. A method for constructing a mouse model, characterized in that: The following steps are involved: Step 1: Select experimental animals, mice, and divide them into experimental groups. The experimental groups are as follows: Normal saline group: mice were injected with 40 μl normal saline in the sole of their feet, and 200 nl normal saline was microinjected the next day; Inflammatory pain group: mice were injected with 40 μl CFA in the sole of their feet, and 200 nl of normal saline was microinjected the next day; Inflammatory pain treatment group: mice were injected with 40 μl CFA in the sole of their feet, and microinjected Fluorocitrate 200nl; The microinjection procedure was as follows: the mice were anesthetized with 2% isoflurane and fixed in a stereotaxic apparatus in a prone position. During the operation, the rectal temperature was controlled at 37.0±0.5℃ with a constant temperature blanket. The operation process included exposing the skull, removing connective tissue, and keeping the surface dry. The instrument coordinates were reset to zero with the bregma as the zero point. Normal saline or Fluorocitrate was injected into the CA1 brain region. The injection volume was 200nl and the injection speed was 40nl / min. The needle was stopped for 5 minutes. Finally, the wound was sutured and anti-inflammatory treatment was given. Step 2: Prepare the experimental animals for pain threshold testing and conduct behavioral tests before pain threshold testing. The behavioral tests are conducted in a double-blind manner. All mice are allowed to adapt to the test environment for 1 hour before the basic behavioral test. Step 3: Perform pain threshold test on the 4th day after surgery. The pain threshold test includes mechanical pain test and thermal pain test, and finally obtain experimental data.

9. The method for constructing a mouse model according to claim 8, characterized in that: The mechanical pain test includes using two calibrated von Frey filaments, selecting 0.07g Von Frey fiber filament representing low-intensity stimulation induced pain and 0.4g Von Frey fiber filament representing high-intensity stimulation pain hypersensitivity to stimulate the central skin of the sole of the left hind limb of the mouse. After the fiber filament passes vertically through the grid holes, the fiber filament is bent into a "C" shape with uniform force. The stimulation application time lasts for about 1.5 seconds and is repeated 10 times. The interval between each stimulation is more than 5 minutes. The mouse's paw withdrawal reaction is observed and recorded, and the mouse's paw withdrawal frequency under different stimulation intensities is calculated.

10. The method for constructing a mouse model according to claim 8, characterized in that: The heat pain test uses a thermal radiation device to irradiate the central skin of the mouse's hind foot, and records the duration of irradiation from the beginning to the time when the mouse lifts its foot or licks the sole of its foot. This is repeated 5 times, with an interval of more than 10 minutes between each irradiation. The average of the 5 irradiation times is taken as the thermal paw withdrawal latency.

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