Use of an mpo enzyme inhibitor for the preparation of a medicament for the prevention and / or treatment of neuroinflammatory diseases

Abstract

This invention belongs to the field of pharmaceutical technology and discloses the application of 4-amino-1-benzylpiperidine in the preparation of MPO enzyme inhibitors and drugs for the prevention and / or treatment of neuroinflammatory diseases. This invention conducts experimental studies at the enzymatic, cellular, and animal levels to investigate the effects of 4-amino-1-benzylpiperidine on MPO enzyme activity, neutrophil activation, and the prevention and / or treatment of neuroinflammatory diseases. The results show that 4-amino-1-benzylpiperidine can directly inhibit MPO enzyme activity; inhibit neutrophil activation, reduce the release of inflammatory factors; reduce infarct volume and cerebral edema after stroke; and improve neurological function impairment. It can be used to prepare protective drugs for neuroinflammatory diseases. This invention provides a novel use of 4-amino-1-benzylpiperidine in the preparation of MPO enzyme inhibitors and drugs for the prevention and / or treatment of neuroinflammatory diseases, offering a safe, effective, and economical solution for the treatment and prevention of neuroinflammatory diseases.

Description

Technical Field

[0001] This invention relates to the discovery of a novel MPO enzyme inhibitor, 4-amino-1-benzylpiperidine; to pharmaceutical compositions containing 4-amino-1-benzylpiperidine; and to the use of 4-amino-1-benzylpiperidine and its pharmaceutical compositions in the preparation of remedies for the prevention and / or treatment of neuroinflammatory diseases, belonging to the field of pharmaceutical technology. Background Technology

[0002] Myeloperoxidase (MPO) is a heme enzyme abundant in neutrophils and monocytes. In neutrophils, it accounts for approximately 5% of total cell dry weight in primary granules, while in monocytes, its content is lower, accounting for about 1% of total cellular protein. Mature MPO is stored in the azurophilic granules of fully differentiated neutrophils. Multiple release mechanisms of MPO are initiated and activated through TLR-mediated inflammatory mediators, cytokines (such as GM-CSF and TNF), and Ig / Fc receptor-mediated signaling pathways. The most important release mechanism is the rapid release of MPO by neutrophils through degranulation and cell death pathways including apoptosis and necrosis. Recent studies show that MPO can also be released from neutrophils by the compression of the extracellular trapping network.

[0003] When phagocytes in surrounding tissues and body fluids are activated, MPO can be secreted into the extracellular environment and phagosomes, where it exerts its bactericidal effect by producing hydrogen peroxide and chloride as substrates. Studies have found that while MPO kills pathogens, it is also produced in excess, leading to inflammatory tissue damage. Targeting MPO-mediated inflammatory events has become a current research hotspot. MPO activity is closely related to inflammatory diseases of the nervous system. Increased permeability of the blood-brain barrier due to inflammation or other causes leads to the infiltration of inflammatory cells such as peripheral neutrophils, resulting in increased MPO expression and activity, thereby causing central nervous system diseases, including stroke, cerebral hemorrhage, traumatic brain injury, multiple sclerosis, amyotrophic lateral sclerosis, and neurodegenerative diseases.

[0004] Studies have shown that MPO levels in patients with hemorrhagic and ischemic stroke are higher than in controls, and MPO levels are correlated with stroke severity, hematoma volume, and 6-month mortality. In a rat model of stroke, MPO activity significantly increased in the cerebral cortex perfused by the anterior cerebral artery 24 hours after reperfusion, and intracerebral MPO activity was associated with neutrophil infiltration. Using anti-neutrophil monoclonal antibodies to reduce neutrophils can decrease MPO activity, reduce cerebral edema, and decrease infarct volume. Within days of brain injury, MPO activity in cerebrospinal fluid and peripheral blood increases, and the degree of increase is positively correlated with the severity of the injury; higher MPO activity indicates more severe injury. All this evidence suggests that MPO is involved in the pathophysiological process of stroke.

[0005] Currently, there are no reports on the activity of 4-amino-1-benzylpiperidine, and its inhibitory activity against MPO has not been studied. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide the application of 4-amino-1-benzylpiperidine as an MPO enzyme inhibitor in the preparation of drugs for the prevention and / or treatment of neuroinflammatory diseases, in order to solve the problems existing in the prior art. This invention discovers that 4-amino-1-benzylpiperidine inhibits neutrophil MPO enzyme activity, reduces neutrophil activation, decreases infarct volume and cerebral edema after stroke, and improves neurological function impairment, thus possessing significant clinical application value.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides the use of 4-amino-1-benzylpiperidine, as shown in Formula I, in the preparation of MPO enzyme inhibitors and medicaments for the prevention and / or treatment of neuroinflammatory diseases.

[0009]

[0010] The 4-amino-1-benzylpiperidine is an organic compound with the molecular formula C12H18N2 and a molecular weight of 190.29.

[0011] Furthermore, the present invention provides the use of 4-amino-1-benzylpiperidine, or a pharmaceutical salt thereof, a solvate thereof, or a solvate thereof in the preparation of MPO enzyme inhibitors and medicaments for the prevention and / or treatment of neuroinflammatory diseases.

[0012] Furthermore, 4-amino-1-benzylpiperidine is used for the prevention and / or treatment of inflammatory diseases caused by increased MPO enzyme activity or to reduce the risk of said diseases.

[0013] Furthermore, the inflammatory disease refers to a neuroinflammatory disease characterized by increased infiltration and activation of neutrophils in the brain.

[0014] Furthermore, the neuroinflammatory diseases include stroke, brain injury, multiple system atrophy, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and neurodegenerative diseases.

[0015] The present invention also provides a pharmaceutical composition in the preparation of an MPO enzyme inhibitor and its use in the preparation of a drug for the prevention and / or treatment of neuroinflammatory diseases, characterized in that the pharmaceutical composition is made of 4-amino-1-benzylpiperidine of Formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient;

[0016]

[0017] This pharmaceutical formulation can be prepared according to methods known in the art. It can be formulated into any dosage form for human or animal use by combining the 4-amino-1-benzylpiperidine described in this invention with one or more pharmaceutically acceptable solid or liquid excipients and / or excipients. The content of the compounds of this invention in its pharmaceutical composition is typically 0.1-99%.

[0018] Furthermore, the pharmaceutical preparations include oral preparations or dosage forms that are not administered via the digestive tract.

[0019] Furthermore, the oral preparations are tablets, capsules, pellets, or liquid preparations; the non-digestive tract administration dosage forms are preparations administered via intramuscular injection, intravenous injection, intravenous infusion, skin administration, or mucosal administration.

[0020] Dosage forms can be liquid, solid, or semi-solid. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including o / w, w / o, and double emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can include tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, microcapsules, pellets, suppositories, films, patches, aerosols, and sprays, etc.; semi-solid dosage forms can include ointments, gels, and pastes, etc.

[0021] The compounds of this invention can be formulated into conventional formulations, sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0022] To formulate the compounds of the present invention into tablets, a wide variety of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, carboxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, croscarmellose, croscarmellose sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0023] Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0024] To formulate the drug delivery unit into capsules, the active ingredient, the compound of the present invention, can be mixed with a diluent and a disintegrant, and the mixture can be placed directly into hard or soft capsules. Alternatively, the active ingredient, the compound of the present invention, can be first formed into granules or microspheres with a diluent, binder, and disintegrant, and then placed into hard or soft capsules. The diluents, binders, wetting agents, disintegrants, and disintegrants used to prepare tablets of the compound of the present invention can also be used to prepare capsules of the compound of the present invention.

[0025] To prepare the compounds of this invention into injectable formulations, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of commonly used solubilizers, co-solvents, pH adjusters, and osmotic pressure regulators can be added. Solubilizers or co-solvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters can be phosphates, esters, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure regulators can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing lyophilized powder injections, mannitol, glucose, etc., can also be added as supporting agents.

[0026] In addition, colorants, preservatives, flavorings, tasters, or other additives may be added to the pharmaceutical preparation if necessary. To achieve the intended therapeutic effect, the pharmaceuticals or pharmaceutical compositions of the present invention can be administered using any known method of administration. The dosage of the compound pharmaceutical compositions of the present invention can vary widely depending on the nature and severity of the disease to be prevented or treated, the individual circumstances of the patient or animal, the route of administration, and the dosage form. Generally, the appropriate daily dose range for the compounds of the present invention is 0.001-100 mg / kg body weight. The above dosage can be administered as a single dose unit or divided into several dose units, depending on the physician's clinical experience and the administration regimen, including the use of other treatment methods.

[0027] The compounds of this invention can be taken alone or in combination with other therapeutic or symptomatic drugs. When the compounds of this invention have a synergistic effect with other therapeutic drugs, their dosage should be adjusted according to the actual situation. Attached Figure Description

[0028] Figure 1 Inhibitory effect of 4-amino-1-benzylpiperidine on myeloperoxidase.

[0029] Figure 2 Inhibitory effect of 4-amino-1-benzylpiperidine on neutrophil activation.

[0030] Figure 3 Effect of 4-amino-1-benzylpiperidine on the volume of cerebral infarction after stroke.

[0031] Figure 4 Effects of 4-amino-1-benzylpiperidine on neurological function after stroke.

[0032] Figure 5 Effects of 4-amino-1-benzylpiperidine on post-stroke cerebral edema. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings.

[0034] Example 1: Inhibitory effect of 4-amino-1-benzylpiperidine on MPO enzyme.

[0035] MPO enzyme activity was detected using a Cayman Myeloperoxidase Inhibitor Screening Assay Kit (Item No. 700170). The kit utilizes MPO to catalyze the reaction of hydrogen peroxide and ADHP to form a highly fluorescent compound, halogenated amine. The fluorescence intensity is directly proportional to enzyme activity. Excitation light was set at 535 nm, and emission light at 590 nm for fluorescence intensity detection. The experiment included background wells, 100% enzyme activity wells, and compound wells. The solution composition was as follows: background wells: 15 μl buffer; 100% enzyme activity wells: 12.6 μl buffer + 2.4 μl MPO; compound wells: 9.6 μl buffer + 2.4 μl MPO + 3 μl compound. 15 μl of trigger agent was added to each well to initiate the reaction, for a total volume of 30 μl. After adding the solutions, the plate was placed at room temperature and incubated with shaking for 5 min before reading the plate. Reading conditions were: excitation light at 535 nm and emission light at 590 nm. Inhibition rate calculation: % Inhibition rate = [(100% enzyme activity - inhibitor) / 100% enzyme activity] * 100.

[0036] The results show (attached) Figure 1 ), 4-amino-1-benzylpiperidine inhibits MPO enzyme activity IC50 50 It is 623.3 nM.

[0037] Table 1. IC50 of 4-amino-1-benzylpiperidine inhibiting MPO enzyme activity 50 value

[0038] 4-Amino-1-benzylpiperidine 623.3nM

[0039] Example 2: Inhibitory effect of 4-amino-1-benzylpiperidine on neutrophil activation.

[0040] Male SD rats weighing 140–160 g were used in the experiment. Blood was collected from the abdominal aorta under chloral hydrate anesthesia. Peripheral blood was first extracted from the rats and then separated. Neutrophils were isolated using 3.8% sodium citrate solution at a ratio of 1:9 (anticoagulant:blood) for anticoagulation. The isolated neutrophils were identified by trypan blue rejection assay and Giemsa staining assay, and their cell viability and purity were both greater than 90%. The extracted cells were cultured in 96-well plates using RPMI-1640 medium. Cells were divided into a normal control group, a model group, a model + positive control group (100 μM), and a model + 4-amino-1-benzylpiperidine group (5, 15, 50 μM). Cells in the model group were administered LPS (1 μg / mL) after plating, while the drug control groups were administered both LPS and the drug. Cells were cultured in an incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, the supernatant was collected, and the concentrations of inflammatory factors IL-6 and TNF-α in the supernatant were detected using an ELISA kit.

[0041] The results show (attached) Figure 2 4-Amino-1-benzylpiperidine can significantly inhibit the release of neutrophil inflammatory factors IL-6 and TNF-α, and its effect is superior to that of positive control drugs.

[0042] Table 2. Effects of 4-amino-1-benzylpiperidine on the release of inflammatory factors from neutrophils

[0043]

[0044] Example 3: Effect of 4-amino-1-benzylpiperidine on the volume of cerebral infarction after stroke

[0045] Sprague Dawley (SD) rats, weighing 250-300g, were purchased from Vital River (Beijing Vital River Laboratory Animal Technology Co., Ltd.) and anesthetized with isoflurane. The skin of the rat's neck was incised, and the common carotid artery and external carotid artery were separated. The external carotid artery was clamped using an arterial clamp. The common carotid artery was placed in the groove of the vascular electrical stimulation clamp of the YLS-14B small animal thrombosis generation instrument, and an electric shock was performed with a current intensity of 1.00 mA and a stimulation time of 240s. After the electric shock, the distal end of the common carotid artery was clamped with an arterial clamp. The thrombus was fragmented from the distal end to the proximal end using soft-tipped forceps. The arterial clamp was released, and 1-5 seconds after the thrombus fragments were seen to be flushed away, the proximal end of the common carotid artery was clamped again for 15 minutes. All arterial clamps were then removed, and the rats were disinfected and sutured. After suturing, the rats were placed in a 37°C constant temperature system. Rats in all groups were fasted the night before modeling, and the drug was administered via tail vein injection half an hour before modeling. The normal control group and the stroke model group were given the corresponding volume of physiological saline. Experimental groups: sham surgery group, model group, positive control group (Ceviles sodium), 4-amino-1-benzylpiperidine (10, 20, 40 mg / kg).

[0046] The results show (attached) Figure 3 After 24 hours of ischemia, cerebral infarction occurred on the ischemic side of the rats in the model group. Compared with the model group, 4-amino-1-benzylpiperidine reduced the infarct volume in a dose-dependent manner, with the highest dose showing the most significant effect. The experimental results indicate that 4-amino-1-benzylpiperidine can significantly improve acute cerebral ischemia injury.

[0047] Table 34 shows the effect of 4-amino-1-benzylpiperidine on the volume of cerebral infarction after stroke.

[0048]

[0049] Example 4: Effects of 4-amino-1-benzylpiperidine on neurological function after stroke.

[0050] Animal modeling and drug administration methods were the same as in Example 3. After 24 hours of ischemia, the neurological function impairment of rats was evaluated using the mNSS score and Longa score.

[0051] The results show (attached) Figure 4 Following 24 hours of ischemia, the model group rats showed significant neurological dysfunction, with markedly elevated mNSS and Longa neurological scores. Compared to the model group, 4-amino-1-benzylpiperidine dose-dependently reduced neurological scores, with the highest dose showing the most significant effect. These results indicate that 4-amino-1-benzylpiperidine can improve neurological dysfunction caused by acute cerebral ischemia.

[0052] Table 44. Effects of 4-Amino-1-benzylpiperidine on neurological function after stroke.

[0053]

[0054] Example 5: Effect of 4-amino-1-benzylpiperidine on cerebral edema after stroke.

[0055] Animal modeling and drug administration methods were the same as in Example 3. After 24 hours of ischemia, the degree of cerebral edema in rats was evaluated by wet-dry weight of brain tissue.

[0056] The results show (attached) Figure 5 After 24 hours of ischemia, the water content of the ischemic brain tissue in the model group rats was significantly increased due to blood-brain barrier disruption. Compared with the model group, high doses of 4-amino-1-benzylpiperidine significantly reduced brain water content. The experimental results indicate that 4-amino-1-benzylpiperidine can improve cerebral edema caused by acute cerebral ischemia.

[0057] Table 54. Effects of 4-Amino-1-benzylpiperidine on post-stroke cerebral edema.

[0058]

[0059] In summary, this invention investigated the inhibitory effect of 4-amino-1-benzylpiperidine on MPO enzyme and its therapeutic effect on stroke using MPO enzyme activity inhibition assays, neutrophil activation assays, and an animal model of stroke. The results showed that 4-amino-1-benzylpiperidine directly inhibited MPO enzyme activity, with an IC50 value of [missing information]. 50 The concentration was 623.3 nM; it inhibited neutrophil activation and reduced the release of inflammatory factors; it significantly reduced the infarct volume and cerebral edema after stroke and improved neurological function. Therefore, 4-amino-1-benzylpiperidine has the effect of inhibiting MPO enzyme activity and preventing and treating neuroinflammation. Using 4-amino-1-benzylpiperidine as the active substance, alone or in combination with other pharmacologically active compounds and / or extracts, various dosage forms of anti-neuroinflammatory drugs can be prepared according to conventional pharmaceutical formulation methods, or compound preparations can be made with other anti-neuroinflammatory drugs to reduce adverse drug reactions while maintaining efficacy. This provides a safe, effective, and economical solution for the prevention and treatment of neuroinflammatory diseases.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes can be made to it in practice and in detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. The use of an MPO enzyme inhibitor in the preparation of drugs for the prevention and / or treatment of stroke, characterized in that, The MPO enzyme inhibitor is 4-amino-1-benzylpiperidine as shown in Formula I or a pharmaceutically acceptable salt thereof; I。 2. The use of a pharmaceutical composition in the preparation of a drug for the prevention and / or treatment of stroke, characterized in that, The pharmaceutical composition described herein is made of 4-amino-1-benzylpiperidine as shown in Formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient; I。 3. The application according to claim 2, characterized in that, The dosage form of the pharmaceutical composition may be formulated as an oral formulation or a dosage form that is not administered via the digestive tract.

4. The application according to claim 3, characterized in that, The oral preparations include tablets, capsules, pellets, and liquid preparations; the non-digestive tract administration dosage forms include preparations administered via intramuscular injection, intravenous injection, intravenous infusion, and mucosal administration.

5. The application according to claim 4, characterized in that, The oral formulation may be formulated as a sustained-release or controlled-release formulation.

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