Use of antibiotics in preparing drugs for inhibiting cancer pain

Abstract

The present invention provides the use of antibiotics in the preparation of drugs for inhibiting cancer pain. The present invention finds that in the early stage of cancer pain caused by cancer, after the intestinal flora is removed by antibiotics, the occurrence, development and maintenance of bone cancer pain are significantly inhibited, and the occurrence and development of cancer pain are inhibited by inhibiting the activation of microglia and astrocytes in the spinal cord. The present invention reveals that the use of antibiotics to eliminate intestinal flora has the potential to be applied in the clinical treatment of cancer pain.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of antibiotics in preparing medicines for inhibiting cancer pain. Background Art

[0002] Bone cancer pain is a common symptom in patients with primary osteosarcoma and patients with advanced non-primary tumor bone metastasis. Bone metastasis mostly comes from advanced breast, lung, thyroid, bladder and other organ tumor cells. Bone metastasis of tumor cells may cause bone destruction, leading to pathological fractures and obvious bone cancer pain. Cancer pain greatly aggravates the painful emotions, impairs the patient's mobility and social function, reduces the quality of life, and leads to an increase in cancer mortality. The pathogenesis of cancer pain is complex, including neuropathic pain caused by tumor compression of the peripheral and central nervous systems, and pain caused by direct stimulation of peripheral nociceptors by inflammatory factors, acidic environment and growth factors in the tumor microenvironment. Therefore, compared with single neuropathic pain, the pathogenesis of cancer pain is more complex and the pain is more severe, and the current treatment methods and effective treatment drugs for cancer pain are very limited. Therefore, it is urgent to carry out new drug research to control cancer pain and find effective treatment drugs for cancer pain. Summary of the invention

[0003] According to the first aspect, in one embodiment, there is provided use of an antibiotic in preparing a medicament for inhibiting cancer pain.

[0004] According to the second aspect, in one embodiment, there is provided use of an antibiotic in preparing a medicament for inhibiting the occurrence and / or development of cancer pain.

[0005] According to the third aspect, in one embodiment, there is provided use of an antibiotic in preparing a medicament for inhibiting the maintenance of cancer pain.

[0006] According to the fourth aspect, in one embodiment, there is provided use of an antibiotic in the preparation of a medicament for inhibiting mechanical hyperalgesia of cancer pain.

[0007] According to the fifth aspect, in one embodiment, there is provided use of an antibiotic in preparing a medicament for inhibiting microglial proliferation.

[0008] According to the sixth aspect, in one embodiment, there is provided use of an antibiotic in preparing a medicament for inhibiting astrocyte proliferation.

[0009] According to the seventh aspect, in one embodiment, there is provided use of an antibiotic in preparing a medicament for inhibiting the activation of glial cells.

[0010] Based on the use of antibiotics in the above-mentioned embodiments in the preparation of drugs for inhibiting cancer pain, the present invention has found that in the early development and maintenance stages of cancer pain caused by cancer, the use of antibiotics to clear intestinal flora has a significant inhibitory effect on the development and maintenance of bone cancer pain, and the development of cancer pain is inhibited by inhibiting the activation of microglia and astrocytes in the spinal cord.

[0011] In one embodiment, the present invention discloses that the use of antibiotics to eliminate intestinal flora has the potential to be applied in the clinical treatment of cancer pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The results of mechanical pain inhibition in a bone cancer pain model 14 days before TCI surgery in an embodiment;

[0013] Figure 2 The mechanical allodynia inhibition results of a bone cancer pain model in an embodiment on the same day of TCI surgery;

[0014] Figure 3 The results of mechanical pain inhibition in a bone cancer pain model in an embodiment after 7 days of chronic pain onset;

[0015] Figures 4 to 6 The results of immunofluorescence staining to detect the expression of specific markers of microglia and astrocytes, IB a1 and GFAP, in one embodiment;

[0016] Figures 7 to 9 The results of Western Blot detection of the expression of IB a1 and GFAP, specific markers of microglia and astrocytes, in one embodiment. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can effortlessly recognize that some of the features can be omitted in different situations, or can be replaced by other materials and methods. In some cases, some operations related to the present application are not shown or described in the specification, in order to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and they can fully understand the related operations according to the description in the specification and the general technical knowledge in the art.

[0018] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.

[0019] The serial numbers assigned to the components in this article, such as "first", "second", etc., are only used to distinguish the objects described and do not have any order or technical meaning.

[0020] The intestinal flora refers to a large number of symbiotic bacteria that are planted in the gastrointestinal tract. It has been proven that the intestinal flora plays an important role in a variety of physiological and pathological processes. Various evidences show that the intestinal flora can regulate the function of distal organs. The "microbiome-gut-brain" axis (abbreviated as the brain-gut axis) refers to the channel of interaction between the microbiome, the intestine and the brain through the circulation of microbial metabolites or vagal neurotransmitters. It has been found to play an important role in regulating neural development, neurotransmitter release and regulating glial cell function, which reveals the important connection between the intestinal flora and the nervous system. At the same time, this interaction also regulates the progression of a variety of neurological diseases, such as depression, Alzheimer's disease and autism. Therefore, the use of mixed antibiotics (Antibiotics cocktail, referred to as ABX) to eliminate the intestinal flora can regulate the occurrence and development of a variety of neurological diseases and has the potential for clinical treatment.

[0021] Currently, there is no evidence to systematically reveal the relationship between intestinal flora and cancer pain, nor is there sufficient evidence to show whether the use of ABX can be used for the potential clinical treatment of cancer pain.

[0022] According to the first aspect, in one embodiment, there is provided use of an antibiotic in preparing a medicament for inhibiting cancer pain.

[0023] In one embodiment, the antibiotic is used to prepare a medicament for inhibiting the occurrence and / or development of cancer pain.

[0024] In one embodiment, the antibiotic is used to prepare a medicament for inhibiting the maintenance of cancer pain.

[0025] In one embodiment, the antibiotic is used to prepare a medicament for inhibiting mechanical hyperalgesia of cancer pain.

[0026] In one embodiment, the antibiotic is used to prepare a drug for inhibiting microglial proliferation.

[0027] In one embodiment, the antibiotic is used to prepare a medicament for inhibiting astrocyte proliferation.

[0028] In one embodiment, the antibiotic is used to prepare a drug for inhibiting the activation of glial cells.

[0029] In one embodiment, the glial cells include but are not limited to at least one of microglia and astrocytes.

[0030] In one embodiment, the microglia include microglia in the spinal cord of an organism.

[0031] In one embodiment, the astrocytes include astrocytes in the spinal cord of an organism.

[0032] In one embodiment, the antibiotics include but are not limited to at least one of ampicillin, neomycin, metronidazole, and vancomycin.

[0033] In one embodiment, the organism includes but is not limited to humans or animals, including all mammals such as primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits and cows. In some embodiments, the organism is a human.

[0034] In one embodiment, the antibiotics include ampicillin, neomycin, metronidazole and vancomycin.

[0035] In one embodiment, among the antibiotics, by mass, ampicillin:neomycin:metronidazole:vancomycin=(0.1-5):(0.1-5):(0.1-5):(0.1-5).

[0036] In one embodiment, the antibiotics are in the ratio of ampicillin:neomycin:metronidazole:vancomycin=1:1:1:0.5 by mass.

[0037] In one embodiment, the cancer pain includes bone cancer pain.

[0038] In one embodiment, the antibiotic is used to eliminate intestinal flora.

[0039] In one embodiment, the antibiotic is used to inhibit the growth of intestinal flora.

[0040] According to the second aspect, in one embodiment, there is provided use of an antibiotic in preparing a medicament for inhibiting the occurrence and / or development of cancer pain.

[0041] According to the third aspect, in one embodiment, there is provided use of an antibiotic in preparing a medicament for inhibiting the maintenance of cancer pain.

[0042] According to the fourth aspect, in one embodiment, there is provided use of an antibiotic in the preparation of a medicament for inhibiting mechanical hyperalgesia of cancer pain.

[0043] According to the fifth aspect, in one embodiment, there is provided use of an antibiotic in preparing a medicament for inhibiting microglial proliferation.

[0044] According to the sixth aspect, in one embodiment, there is provided use of an antibiotic in preparing a medicament for inhibiting astrocyte proliferation.

[0045] According to the seventh aspect, in one embodiment, there is provided use of an antibiotic in preparing a medicament for inhibiting the activation of glial cells.

[0046] Example

[0047] In this embodiment, mixed antibiotics (ABX) were dissolved in the drinking water of rats. In the obtained solution, the final concentration of each antibiotic was as follows: 0.5g / L ampicillin, 0.5g / L neomycin, 0.5g / L metronidazole and 0.25g / L vancomycin. The drinking water was used for feeding for two weeks, and the drinking water was changed every two days to intervene in the intestinal flora. Then, the bone cancer pain model was applied to the rats, and mechanical pain and thermal pain were measured to observe whether the bone cancer pain model was regulated after the elimination of intestinal flora using ABX at different stages of pain development and maintenance, and the changes in pain-related molecules were detected by immunofluorescence staining and Western Blot.

[0048] 1. Experimental Methods

[0049] 1.1. Rat bone cancer pain model

[0050] Before surgery, Walker 256 breast cancer cells were resuspended in PBS solution and kept on ice to dilute the cell suspension to 1×10 7 Induce and maintain anesthesia in rats with 2% isoflurane, disinfect the upper 1 / 3 skin of the right tibial tubercle with 75% alcohol; make a skin incision about 0.5 cm long, carefully expose the tibia; use a microinjector to penetrate into the bone marrow cavity and slowly inject 5 μL of 5×10 5 PBS solution of breast cancer cells was injected. After the injection, the microinjector was slowly pulled out, the puncture hole was quickly sealed with bone wax, and then the wound was sutured with needle and thread. All SPF-grade experimental animals were purchased from the Experimental Animal Center of Southern University of Science and Technology and were raised in the center under standard feeding conditions. The ambient temperature was 22±3℃, the relative humidity was 55±5%, and the light was alternating 12h / 12h day and night. Rats were free to eat and drink water. Before the experiment, all animals were adapted to the environment for at least one week.

[0051] 1.2 Preparation and Use of ABX

[0052] Mixed antibiotics (ABX) (0.5 g / L ampicillin, 0.5 g / L neomycin, 0.5 g / L metronidazole and 0.25 g / L vancomycin) were dissolved in the drinking water of the rats, and the rats were fed with this drinking water for two weeks, with the drinking water being replaced every two days.

[0053] 1.3.Western Blot

[0054] After anesthesia, rats were perfused with 0.9% saline in the heart, and the lumbar spinal cord segment was removed. After the tissue sample was ground, it was lysed in RIPA Lysis Buffer containing protease inhibitors and phosphatase inhibitors. The total protein was separated by SDS-PAGE gel and transferred to a PVDF membrane. The PVDF membrane was blocked for one hour in TBST solution containing 5% BSA and incubated with primary antibodies overnight at 4°C. After the primary antibody incubation, it was washed three times with TB S solution containing 0.1% Tween-20 (TBST solution), each for 10 minutes. Next, the secondary antibody was incubated at room temperature for 1 hour and washed three times with TBST solution, each for 10 minutes. The primary antibodies used included anti-GFAP (1:500; Millipore, Burlington, MA) and anti-Iba1 (1:500; Wako, Tokyo, Japan).

[0055] 1.4 Immunofluorescence staining

[0056] After anesthetizing rats, the lumbar spinal cord was removed after cardiac perfusion with 0.9% saline and 4% paraformaldehyde. After fixation in 4% paraformaldehyde for 24 hours, the tissue was transferred to 30% sucrose for dehydration for two days. The frozen spinal cord tissue embedded in the embedding medium was cut into 20 μm tissue slices using a cryostat. Next, the tissue slices were blocked in PBS containing 10% donkey serum and 0.3% Triton X-100 for 2 hours, and then incubated with primary antibodies at 4°C overnight. After the primary antibody incubation, the tissue slices were washed 3 times in PBS for 5 minutes each time, and then incubated with secondary antibodies at room temperature for 2 hours and washed. After sealing, the slices were observed under a confocal microscope. The antibodies used included anti-GFAP (1:500; Millipore, Burlington, MA) and anti-Iba1 (1:500; Wako, Toty o, Japan).

[0057] 1.5 Mechanical withdrawal threshold testing

[0058] Select healthy rats weighing 180-200 g and place them on a metal grid for 30 minutes to adapt to the environment. 514000-20C) Mechanical stimulation was applied to the rat's hind paws, starting with a 2g filament, using the Up and Down method for testing. If the rat showed rapid paw bounce, foot retraction or foot licking, it was recorded as positive, otherwise it was recorded as negative. If the rat showed a negative reaction, the filament with a higher strength was changed; if a positive reaction was detected, the filament with a lower strength was applied (with an interval of 10-15s between adjacent stimuli), until the first positive and negative reaction occurred, and then the stimuli were applied 4 times in the same way, and the intensity of the last stimulation was recorded.

[0059] 2. Experimental results

[0060] Figures 1 to 3 To eliminate the effect of intestinal flora on TCI-induced bone cancer pain using ABX. Figure 1 )14 days and ( Figure 2 ) On the same day, ABX was used to eliminate intestinal flora, and mechanical allodynia was significantly inhibited. Using Two-way ANOVA analysis, compared with the SPF group, **P<0.01; compared with the SPF+TCI group, #P<0.05, ##P<0.01. Figure 3 ) After 7 days, ABX was used to eliminate intestinal flora, and mechanical sensitivity was also significantly inhibited. Two-way ANOVA analysis showed that compared with the SPF+CCI group, **P<0.01.

[0061] First, a bone cancer pain model induced by tibia bone cavity tumor cell implantation (TCI) was established in rats. It was found that using ABX to eliminate intestinal flora 14 days before TCI surgery could significantly inhibit the occurrence and development of bone cancer pain ( Figure 1 ). Then, we tested whether eliminating intestinal flora had an immediate effect on the occurrence and development of pain. When the chronic pain model was established and the intestinal flora was eliminated with ABX, it was also found that mechanical pain was significantly inhibited during the occurrence and development of pain and was maintained until the last behavioral test (day 28, see Figure 2 ). These results indicate that the elimination of intestinal flora with ABX during the development of cancer pain can produce an immediate, significant, and selective inhibitory effect on the mechanical allodynia of cancer pain. Then, the effect of intestinal flora on the persistence and maintenance of bone cancer pain was tested. On the 7th day after TCI treatment, when significant mechanical pain had already occurred, the elimination of intestinal flora with ABX was found to be effective in inhibiting the mechanical allodynia that had already occurred ( Figure 3). In summary, this example found that the use of ABX to eliminate intestinal flora can produce immediate and significant analgesic effects during the generation and development of TCI-induced bone cancer pain.

[0062] Figures 4 to 9 The results show the effect of ABX depletion of intestinal flora on TCI-induced activation of astrocytes and microglia. ABX was used to deplete intestinal flora 14 days before TCI surgery, and immunofluorescence ( Figures 4 to 6 ) and Western Blot method ( Figures 7 to 9 ) Detect the activation of astrocytes and microglia 14 days after TCI surgery. Two-way ANOVA analysis showed that compared with the Sham+SPF group, ***P<0.001; compared with the SPF+TCI group, ##P<0.01.

[0063] During the process of cancer pain, there will be obvious activation of microglia and astrocytes in the spinal cord. The activated glial cells can release inflammatory factors, affecting the excitability of neurons, thereby producing pain. The intestinal flora has been shown to play an important role in regulating the immune system, and has a significant impact on the maturation of microglia in the spinal cord and the function of astrocytes in the brain. Therefore, we speculate that ABX may regulate the activation of spinal glial cells during the process of cancer pain by regulating the intestinal flora, thereby regulating the occurrence and development of cancer pain. Therefore, in this example, ABX was used to eliminate the intestinal flora 14 days before TCI surgery, and the activation of glial cells in the spinal cord was detected on the 14th day after surgery. By immunofluorescence staining ( Figures 4 to 6 ) and Western Blot( Figures 7 to 9 ) method was used to detect the expression of IBa1 and GFAP, specific markers of microglia and astrocytes, respectively. It was found that the use of ABX to eliminate intestinal flora significantly inhibited the increase in the number of microglia and astrocytes and the activation of glial cells after TCI surgery.

[0064] In summary, the present invention found that in the early stage of cancer pain and the maintenance stage of pain caused by cancer, the use of ABX to remove intestinal flora has a significant inhibitory effect on the development and maintenance of bone cancer pain, and inhibits the development of cancer pain by inhibiting the activation of microglia and astrocytes in the spinal cord. These research results reveal that the use of ABX can eliminate intestinal flora, thereby revealing the potential of ABX for clinical treatment of cancer pain.

[0065] The present invention investigates that the use of ABX to eliminate intestinal flora in the early development and maintenance stages of bone cancer pain can significantly inhibit pain, indicating that ABX can be used as a drug for treating bone cancer pain. Traditional pain medications such as opioids, although they have strong analgesic effects, also have many side effects, such as nausea, vomiting, dizziness, itchy skin and other physiological discomforts, as well as addiction, withdrawal reactions and tolerance and other nervous system reactions. After using ABX to eliminate intestinal flora, there is currently no evidence that side effects will occur, and the analgesic effect is comparable to that of traditional analgesics.

[0066] The pharmacological action of ABX is to eliminate part of the intestinal flora through a mixture of multiple antibiotics. In different studies, the formula of ABX and the concentration of various antibiotics are slightly different, but after testing, it is found that all of them can eliminate intestinal flora. Therefore, the use of different antibiotic formulas to make ABX can be used as a variation of the present invention if it can eliminate all intestinal flora, and therefore, it is within the scope of protection of the present invention.

[0067] The above specific examples are used to illustrate the present invention, which are only used to help understand the present invention and are not used to limit the present invention. For those skilled in the art to which the present invention belongs, some simple deductions, modifications or substitutions can be made according to the idea of ​​the present invention.

Claims

1. Use of antibiotics in the preparation of a drug for inhibiting cancer pain, characterized in that: The cancer pain includes bone cancer pain, and the antibiotics include ampicillin, neomycin, metronidazole and vancomycin. Among the antibiotics, by mass, ampicillin:neomycin:metronidazole:vancomycin=(0.1~5):(0.1~5):(0.1~5):(0.1~5).

2. The use according to claim 1, characterized in that The antibiotic is used for preparing a medicine for inhibiting the occurrence and / or development of cancer pain.

3. The use according to claim 1, characterized in that The antibiotic is used for preparing a medicine for inhibiting the maintenance of cancer pain.

4. The use according to claim 1, characterized in that The antibiotic is used for preparing a drug for inhibiting mechanical hyperalgesia of cancer pain.

5. The use according to claim 1, characterized in that The antibiotic is used for preparing a medicine for inhibiting microglial proliferation.

6. The use according to claim 1, characterized in that The antibiotic is used for preparing medicine for inhibiting astrocyte proliferation.

7. The use according to claim 1, characterized in that The antibiotic is used for preparing medicine for inhibiting activation of glial cells.

8. The use according to claim 7, characterized in that The glial cells include at least one of microglia and astrocytes.

9. The use according to claim 8, characterized in that The microglial cells include microglial cells in the spinal cord of an organism.

10. The use according to claim 8, characterized in that The astrocytes include astrocytes in the spinal cord of an organism.

11. The use according to claim 1, characterized in that The antibiotics are used to eliminate intestinal flora.

12. The use according to claim 1, characterized in that The antibiotics are used to inhibit the growth of intestinal flora.

13. Use of antibiotics in the preparation of a drug for inhibiting the occurrence and / or development of cancer pain, characterized in that: The cancer pain includes bone cancer pain, and the antibiotics include ampicillin, neomycin, metronidazole and vancomycin. Among the antibiotics, by mass, ampicillin:neomycin:metronidazole:vancomycin=(0.1~5):(0.1~5):(0.1~5):(0.1~5).

14. Use of antibiotics in the preparation of a drug for inhibiting the maintenance of cancer pain, characterized in that: The cancer pain includes bone cancer pain, and the antibiotics include ampicillin, neomycin, metronidazole and vancomycin. Among the antibiotics, by mass, ampicillin:neomycin:metronidazole:vancomycin=(0.1~5):(0.1~5):(0.1~5):(0.1~5).

15. Use of an antibiotic in the preparation of a medicament for inhibiting mechanical hyperalgesia of cancer pain, characterized in that: The cancer pain includes bone cancer pain, and the antibiotics include ampicillin, neomycin, metronidazole and vancomycin. Among the antibiotics, by mass, ampicillin:neomycin:metronidazole:vancomycin=(0.1~5):(0.1~5):(0.1~5):(0.1~5).

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