Application of pyruvate in the preparation of a drug for preventing or treating central pain after stroke

Abstract

The present invention relates to the field of medicine and health care products, and aims to provide a new medical use of pyruvate, namely, the use of pyruvate in the preparation of medicines and health care products for treating central pain after stroke. Experimental verification has shown that pyruvate can significantly reduce the mortality rate of mice after thalamic hemorrhage, and alleviate the hyperalgesia of mechanical and thermal stimulation caused by thalamic hemorrhage.

Description

Technical Field

[0001] The present invention relates to the field of medicines and health products, and aims to provide a new medical use of bacteriocin, namely, use of bacteriocin in preparing medicines and health products for treating central pain after stroke. Background Art

[0002] The information disclosed in this background technology section is only intended to increase some understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to ordinary technicians in this field.

[0003] Central post-stroke pain (CPSP), also known as thalamic pain, is a common neuropathic pain syndrome associated with stroke, manifested as abnormal pain perception in the affected limb, such as burning sensation, numbness, lightning-like spontaneous pain, and induced pain such as hyperalgesia and allodynia. In addition, patients also experience symptoms such as sensory impairment and sensory perversion in the affected limb. Long-term CPSP greatly reduces the patient's quality of life, affects mood, sleep and social function, and in severe cases even leads to depression, self-harm and suicide.

[0004] There is currently no effective drug for the treatment of CPSP. Conventional analgesics and even opioids are not recommended as first-line drugs; while anti-epileptic and antidepressant drugs, such as amitriptyline, have certain preventive and therapeutic effects, they have large individual differences in effect and strong side effects. As my country's population ages, the number of CPSP patients will continue to increase. Therefore, it is very important to develop effective treatment strategies.

[0005] Obacunone is a limonoid triterpenoid compound with anticancer, insecticidal and lipid-lowering activities. Traditional Chinese medicinal materials such as Phellodendron chinense and Dictamni cortex contain Obacunone, which is commonly used in clinical treatment of inflammatory diseases and has the effect of detoxification and sore treatment. In addition, the combination of Phellodendron chinense, Dictamni cortex and other Chinese medicines is also used for the prevention and treatment of stroke, with the functions of clearing away heat and detoxification, removing blood stasis and dredging collaterals, and promoting diuresis and reducing swelling. Summary of the invention

[0006] Currently, there is no effective drug for the treatment of CPSP, and existing treatment options have large individual differences and strong side effects. The present invention aims to clarify the role of chloroquine in alleviating CPSP and provide new treatment methods and strategies.

[0007] The technical solution adopted by the present invention is as follows:

[0008] In a first aspect of the present invention, there is provided a use of pyruvate in the preparation of a product for preventing or treating central pain after stroke.

[0009] Preferably, the product is a medicine or a health food.

[0010] The bacteriocin of the present invention can be used alone or in the form of a pharmaceutical composition or a health food composition.

[0011] In a second aspect of the present invention, a pharmaceutical composition for preventing or treating central pain after stroke is provided, wherein the active ingredient of the pharmaceutical composition is obacillus ketone.

[0012] Preferably, the pharmaceutical composition contains 0.1 to 99.9% by weight of the bacterium ketone of the present invention as an active ingredient.

[0013] Preferably, the pharmaceutical composition further comprises other drugs compatible with Houttuynia cordata and pharmaceutically acceptable carriers and / or excipients.

[0014] In the present invention, the pharmaceutically acceptable carrier will not destroy the pharmaceutical activity of the bacterium ketone of the present invention, and its effective amount, that is, the amount that can play the role of a drug carrier, is non-toxic to the human body.

[0015] Preferably, the pharmaceutically acceptable carrier includes, but is not limited to, lecithin, aluminum stearate, aluminum oxide, ion exchange materials, self-emulsifying drug delivery systems, Tween or other surfactants, serum proteins, buffer substances such as phosphates, aminoacetic acid, sorbic acid, water, salts, electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, magnesium silicate, a mixture of partial glycerides of saturated fatty acids, and the like.

[0016] Preferably, the excipients include binders (such as microcrystalline cellulose), fillers (such as starch, glucose, anhydrous lactose and lactose beads), disintegrants (such as cross-linked PVP, cross-linked sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose), lubricants (such as magnesium stearate), as well as absorption promoters, adsorption carriers, flavoring agents, sweeteners, excipients, diluents, wetting agents, etc.

[0017] The present invention's Houttuynia cordata and its pharmaceutical composition can be prepared according to conventional methods in the art and can be administered by intestinal, parenteral or topical routes. Oral preparations include capsules, tablets, oral liquids, granules, pills, powders, pills, ointments, etc.; parenteral preparations include injections, etc.; topical preparations include creams, patches, ointments, sprays, etc. Oral preparations are preferred.

[0018] The administration routes of the present invention's Houttuynia cordata and its pharmaceutical composition can be oral, sublingual, transdermal, intramuscular or subcutaneous, mucocutaneous, intravenous, urethral, ​​vaginal, etc.

[0019] In a third aspect of the present invention, a health food composition for relieving central pain after stroke is provided, wherein the health food composition comprises obacillus ketone.

[0020] Preferably, the health food composition further comprises food additives, for example, antioxidants, pigments, enzyme preparations, etc.

[0021] In a fourth aspect of the present invention, a product for preventing, alleviating or treating central pain after stroke is provided, wherein the product comprises the above-mentioned pharmaceutical composition or the above-mentioned health care product composition.

[0022] Compared with the related art known to the inventor, one of the technical solutions of the present invention has the following beneficial effects:

[0023] Existing CPSP treatment drugs have problems such as large individual differences in effect, strong side effects, and addiction. The present invention provides a new solution for CPSP treatment. After experimental verification, huangbai ketone can significantly reduce the mortality rate of mice after thalamic hemorrhage and alleviate the hyperalgesia of mechanical and thermal stimulation caused by thalamic hemorrhage. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 .Histological identification of thalamic hemorrhage in mice induced by local injection of type IV collagenase. The red area in the local magnified image is the hemorrhage focus.

[0026] Figure 2 .Effect of intragastric administration of pyruvate on the survival of mice with cerebral hemorrhage. Drug solvent group, N = 15; pyruvate group, N = 10.

[0027] Figure 3 .Effect of intragastric administration of pyloric spondylosis on mechanical hyperalgesia of the plantar surface of mice with cerebral hemorrhage. Drug solvent group, N = 8; pyloric spondylosis group, N = 8. Two-way ANOVA test, * P<0.05.

[0028] Figure 4 .Effect of intragastric administration of pyloric sedative on plantar temperature pain sensitivity in mice with cerebral hemorrhage. Drug solvent group, N = 8; pyloric sedative group, N = 8. Two-way ANOVA test, * P<0.05.

[0029] Figure 5 Effects of intragastric administration of pyruvate on sensory and motor neurological deficits in mice with cerebral hemorrhage. Drug solvent group, N = 8; pyruvate group, N = 8. Two-way ANOVA test. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0031] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.

[0032] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0033] 1. Experimental materials:

[0034] 1.1 Medicinal materials:

[0035] Houttuynia cordata (CAS: 751-03-1, molecular weight 454.52, HPLC purity ≥ 98%, Beijing Solaibao Technology Co., Ltd.).

[0036] 0.5% hydroxypropyl methylcellulose solution (HPMC, autoclaved, Beijing Coolable Technology Co., Ltd.) was used as the drug solvent.

[0037]

[0038] 1.2 Experimental Animals:

[0039] Twenty-six 8- to 10-week-old C57BL / 6 male mice were provided by Guangdong Yaokang Biotechnology Co., Ltd., with animal qualification certificate number: No.44824700012781. They were placed in an SPF-grade breeding environment and had free access to food and water.

[0040] 2. Experimental methods:

[0041] 2.1 Establishment of CPSP animal model

[0042] The hemorrhagic stroke model was induced by injecting type IV collagenase into the left thalamus.

[0043] Specifically, mice were anesthetized with isoflurane gas (5% induction, 1.2% to 1.5% maintenance) and fixed in a prone position on a stereotaxic instrument, with a constant temperature plate placed under the body to maintain the animal body temperature at 36.5°C. 0.25% bupivacaine was injected intradermally on both sides of the neck and abdomen 10 minutes before surgery for analgesia. The skin on the top of the head was incised along the midline of the skull to expose the skull, and then the skull was drilled and type IV collagenase (150nL, 0.05U) was injected at the following stereotaxic coordinates: bregma-1.8mm, lateral 1.8mm to the midline, cortex-3.5mm. 1mL of normal saline was injected intraperitoneally every day for 7 days after surgery and soft food was fed.

[0044] 2.2 Drug intervention

[0045] To evaluate the alleviating effect of pyruvate on CPSP in the acute and chronic stages of thalamic hemorrhage, we administered 200 μL of pyruvate solution (50 mg / kg) by oral gavage once a day on days 0-7 and 21-28 after stroke modeling. During the experiment, fresh preparations were prepared every day, pyruvate was dissolved with 0.5% HPMC, and used on the day of preparation. The solvent control group used the same volume of 0.5% HPMC.

[0046] 2.3 Experimental groups and animal use

[0047] Drug solvent group (stroke model, fed with drug solvent):

[0048] 0.5% HPMC was administered and thalamic hemorrhage was induced.

[0049] A total of 16 rats were included, of which 15 were used for survival statistics (8 of which were used for pain behavior experiments and neurological deficit assessment), and 1 was stained with hematoxylin-eosin.

[0050] Huangbaxin group (stroke model, fed with Huangbaxin solution):

[0051] The mice were fed with 50 mg / kg of chloroquine and thalamic hemorrhage was induced by surgery.

[0052] There were 10 mice in total, and the survival rate of 10 mice was counted (8 of them were used for pain behavior experiments and neurological deficit assessment).

[0053] 2.4 Hematoxylin-eosin staining

[0054] Hematoxylin-eosin staining is one of the commonly used detection methods in histology and pathology. Briefly, on the second day after stroke surgery, we deeply anesthetized the animals and used 10% formalin for cardiac perfusion. Then, we took the brain, embedded it in paraffin, and sliced ​​it along the coronal plane with a thickness of 5 μm. After that, the tissue slices were fixed on a glass plate and the conventional steps of the staining were used, which were soaked in hematoxylin and eosin dye solution in turn, then dehydrated with xylene and alcohol, sealed, and observed under a light field microscope.

[0055] 2.5 Survival rate statistics

[0056] The number of animal deaths was counted daily from 0 to 28 days after stroke modeling, and the survival curve was drawn.

[0057] 2.6 Pain index assessment (behavioral)

[0058] Behavioral assessments were performed before stroke modeling and 3, 7, 14, 21, and 28 days after modeling.

[0059] (a) Von Frey test (assessment of mechanical hyperalgesia)

[0060] The experimental animals were placed in a quiet environment for half an hour to reduce the influence of external pressure on the experimental results. The animals were contained in a plexiglass box with a fine metal mesh at the bottom to facilitate plantar stimulation. A von Frey filament with a hardness of 0.4 g was used to vertically stimulate the posterior plantar part (right plantar) of the animal on the contralateral side of the stroke brain area from the bottom for a total of 10 times, and the retraction and grasping ratio was calculated.

[0061] (b) Hot plate test

[0062] Adjust the temperature of the hot plate to 50 degrees Celsius. Place the experimental animal on the hot plate and observe the latency (in seconds) of its heat pain reaction. Heat pain reactions include: lifting the hind feet off the ground, licking the soles of the feet, and jumping. Test 3 times in total and calculate the average value.

[0063] 2.7 Assessment of neurological deficits

[0064] The Bederson scoring method was used to evaluate the sensorimotor neurological deficits of animals after stroke before stroke modeling, and 3 and 7 days after stroke modeling. The scoring criteria are: 1 point (mild): the animal twists its body to the opposite side of the stroke brain after the tail is lifted in the air; 2 points (moderate): it turns in circles to the opposite side of the stroke brain when walking on the ground; 3 points (severe): the trunk of the experimental animal is pushed laterally, and the resistance of the opposite side of the stroke brain is significantly reduced; 4 points (severe): no autonomous action ability. After the test, the final score of one indicator is selected as the result.

[0065] 2.8 Statistical analysis

[0066] All experimental data were expressed as mean ± standard error. SPSS16.0 statistical software was used to analyze the experimental data using two-way ANOVA test. P < 0.05 indicated that the statistical difference was significant.

[0067] 3. Results

[0068] 3.1 HE staining to identify thalamic hemorrhage model

[0069] The results are as follows Figure 1 As shown by hematoxylin-eosin staining, 3 days after the unilateral injection of 0.05U of type IV collagenase into the thalamus of mice, obvious bleeding was observed, indicating that this model was established.

[0070] 3.2 Houttuynia cordata reduces the mortality rate of mice after thalamic hemorrhage:

[0071] The results are as follows Figure 2 As shown, 28 days after modeling, thalamic hemorrhagic stroke caused the death of 2 mice, with a mortality rate of 13.33% (control group, N = 15). No mice died in the group fed with 50 mg / kg of pyruvate (N = 11), indicating that pyruvate can improve the survival rate after stroke.

[0072] 3.3 Houttuynia cordata relieves mechanical allodynia caused by thalamic hemorrhage:

[0073] In this part, we evaluated the effect of feeding pyruvate on mechanical hyperalgesia caused by thalamic hemorrhage by Von Frey behavioral experiment. Figure 3 As shown in Figure 2, compared with the solvent control group, feeding 50 mg / kg of pyruvate significantly alleviated the mechanical allodynia of the contralateral hind paw of mice 3 and 28 days after stroke ( * P<0.05, N=8 / group), indicating that pyruvate alleviated CPSP.

[0074] 3.4 Houttuynia cordata relieves temperature allodynia caused by thalamic hemorrhage:

[0075] In this part, we used a hot plate behavioral experiment to evaluate the effect of feeding pyruvate on the hyperalgesia of thermal stimulation caused by thalamic hemorrhage. Figure 4 As shown in the results, compared with the solvent control group, feeding 50 mg / kg of pyruvate significantly alleviated the thermal allodynia of the plantar surface of mice 7, 14 and 28 days after stroke ( * P<0.05, N=8 / group), further confirming that pyruvate alleviated CPSP.

[0076] 3.5 Houttuynia cordata has no significant effect on neurological damage after thalamic hemorrhage:

[0077] Furthermore, we chose to feed 50 mg / kg of pyruvate to verify its effect on the sensorimotor function of mice after thalamic hemorrhage. Figure 5 As shown in the results, the neurological deficit of mice significantly increased on the third day after unilateral thalamic hemorrhage (Bederson score), and gradually decreased on the seventh day. Feeding pyruvate can partially alleviate the neurological deficit on the third day after stroke, but there is no statistically significant difference, indicating that pyruvate has no obvious therapeutic effect on the sensorimotor deficit caused by thalamic hemorrhage.

[0078] 4. Conclusion

[0079] Huangbaxin can significantly reduce the mortality rate of mice after thalamic hemorrhage and alleviate the abnormal pain caused by thalamic hemorrhage.

[0080] Furthermore, the present invention provides the use of chloroquine in the preparation of medicines or health foods for preventing and treating central pain after stroke.

[0081] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A use of phellodendron in the preparation of a drug for preventing or treating central pain after stroke, wherein the stroke is hemorrhagic stroke; the pain is mechanical allodynia caused by thalamic hemorrhage and / or thermal allodynia caused by thalamic hemorrhage.

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