CN116672342A - Application of berberine compound in preparation of drug of plasma kallikrein inhibitor - Google Patents

Abstract

The application provides an application of berberine in preparing a drug of a plasma kallikrein inhibitor, which is applied to the technical field of biological medicine, wherein berberine with different concentrations is mixed with rat serum, incubated for 15min (50 mM Tris-HCl,250mM NaCl,1.5 mu M BSA; pH 7.5) in a Tris-HCl buffer system at 25 ℃, then a specific fluorogenic substrate (Z-Phe-Arg 7-amido-4-methylcoumarin hydrochloride; final concentration 200 mu M) is added, and fluorescence intensity (excitation wavelength is 360 nm) is detected at 480nm by an enzyme-labeled instrument. The final concentration of berberine (μg/mL) was: 0.1,0.3,1.0,3.0,10.0,30.0,100.0,300.0,1000.0 and 3000.0. The IC50 (mug/mL) of berberine for inhibiting PK enzyme activity is 56.04 +/-1.05, hill Slope is 1.08+/-0.05, and the berberine has obvious inhibiting effect on PK.

Description

Application of berberine compound in preparation of drug of plasma kallikrein inhibitor

Technical Field

The application relates to the technical field of biological medicines, in particular to application of berberine compounds in preparation of a drug of a plasma kallikrein inhibitor.

Background

Plasma kallikrein (Plasma Kallikrein; PK) belongs to the serine protease superfamily, which is specifically expressed by hepatocytes and is mainly distributed in the blood circulation. It was found that PK acts on high molecular weight kininogen to form Bradykinin (BK), which in turn activates the signal transduction system including nitric oxide-cyclophosphaguanosine and prostacyclin-cyclophosphadenosine via the BK receptor, inducing vasodilation, participation in blood coagulation and fibrinolysis, etc. In addition to promoting BK production, PK can also promote activation of the endogenous coagulation cascade centered around the coagulation factor FXII via the contact pathway, promoting thrombosis. The above-mentioned actions of PK constitute the core of the "thrombotic inflammation hypothesis", and are closely related to the etiology and pathology of ischemic stroke.

During cerebral ischemia reperfusion, vascular endothelium is damaged, subendothelial collagen is exposed, and platelets are activated by collagen-von Willebrand factor-platelet glycoprotein Ib axis; at the same time, the negatively charged inorganic phosphate surface exposed after platelet activation promotes conversion of FXII to active FXII (FXIIa), which in turn initiates the endogenous coagulation cascade, promoting thrombosis. FXIIa formation promotes PK production, which on the one hand further promotes FXII activation by means of positive feedback, on the other hand by promoting BK production, initiating a series of signalling cascades after BK receptors, up-regulating pro-inflammatory factors, activating glial cells, ultimately leading to brain neuronal damage.

Berberine (Berberine) is an alkaloid extracted from rhizome of Coptis chinensis plant of Ranunculaceae, also called Berberine, and has various pharmacological activities and medical application. First, berberine has inhibitory effects on various bacteria, fungi and parasites. It can inhibit the growth and reproduction of bacteria, and has strong antibacterial activity to some common pathogenic bacteria such as Escherichia coli, staphylococcus aureus, etc. Second, berberine has anti-inflammatory effect, and can inhibit inflammatory reaction and release of inflammatory mediators, and relieve inflammation symptoms. This makes berberine potentially useful in the treatment of some inflammatory diseases such as inflammatory bowel disease, arthritis and the like. Thirdly, berberine has antioxidant activity, can neutralize free radical, and reduce damage of oxidative stress to cells and tissues. The antioxidant activity is helpful for protecting cells from oxidative damage, and has certain protection effects on cardiovascular diseases, neurodegenerative diseases and the like. Fourth, berberine is widely used in the field of regulating blood sugar and blood lipid. It can improve insulin sensitivity, promote glucose absorption and utilization, and inhibit hepatic glycogen synthesis. In addition, berberine can reduce blood lipid and cholesterol level, and has certain effects in preventing and treating hyperlipidemia, hypercholesterolemia, etc.

Berberine is also studied for anti-tumor, anti-arrhythmia, improving digestive function, etc., but more researches are still needed to verify the safety and effectiveness thereof. Berberine has remarkable therapeutic effect on ischemic cerebral apoplexy, and its action mechanism involves activating adenylate activated protein kinase (AMPK), regulating downstream targets such as Nrf-2 and NF _κ B. PI3K/Akt, mTOR, etc. However, it is unclear whether berberine can act directly on PK.

Based on the above, a new technical scheme is needed to prepare a drug of the plasma kallikrein inhibitor by using natural compounds so as to meet the clinical application of ischemic cerebral apoplexy.

Disclosure of Invention

In view of this, the present application uses a fluorogenic substrate specific for commercial plasma kallikrein to observe the effect of berberine on PK activity.

The application provides an application of berberine in preparing a drug of a plasma kallikrein inhibitor.

Preferably, the berberine is incubated in a Tris-HCl buffer system comprising Tris-HCl, naCl, caCl ₂ Or BSA.

In the present application, the pharmaceutically acceptable salt is prepared by pharmaceutically acceptable base addition, and the pharmaceutically acceptable base addition salt comprises: lithium, sodium, potassium, calcium, aluminum, magnesium, zinc, bismuth, ammonium or diethanolamine salts.

In the present application, the pharmaceutically acceptable salt is prepared by pharmaceutically acceptable acid addition, and the pharmaceutically acceptable acid includes inorganic acid or organic acid, and the inorganic acid includes: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid or sulfuric acid; the organic acid includes: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acidic citric acid, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, sugar acid, formic acid, ethanesulfonic acid, pamoic acid or amino acids.

In the present application, the medicament further comprises a pharmaceutically acceptable excipient.

In the present application, the compound or pharmaceutically acceptable salt thereof may be administered to a subject by any suitable route, such as orally.

In the present application, the compounds are useful for treating diseases associated with the inhibition of carotid thrombosis.

The compound or a pharmaceutically acceptable salt thereof may be formulated into various suitable dosage forms, for example, tablets, capsules, intravenous injection, intraperitoneal injection, inhalant, aerosol, lyophilized preparation, patch, gel, spray or suppository.

The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.

The term "pharmaceutically acceptable" as used herein is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present application prepared with relatively non-toxic, pharmaceutically acceptable acids or bases. When the compounds of the present application contain relatively acidic functional groups, base addition salts can be obtained by contacting a prototype of such compounds with a sufficient amount of a pharmaceutically acceptable base in pure solution or in a suitable inert solvent. When the compounds of the present application contain relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts. See specifically Berge et al, "Pharmaceutical Salts", journal of Pharmaceutical Science 66:1-19 (1977), or, handbook of Pharmaceutical Salts: properties, selection, and Use (P.Heinrich Stahl and Camille G.Wermuth, ed., wiley-VCH, 2022).

The term "treatment" refers to therapeutic therapy. When specific conditions are involved, treatment refers to: (1) alleviating a disease or one or more biological manifestations of a disorder, (2) interfering with (a) one or more points in a biological cascade that results in or causes a disorder or (b) one or more biological manifestations of a disorder, (3) ameliorating one or more symptoms, effects, or side effects associated with a disorder, or one or more symptoms, effects, or side effects associated with a disorder or treatment thereof, or (4) slowing the progression of a disorder or one or more biological manifestations of a disorder.

The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present application can be obtained.

The reagents and materials used in the present application are commercially available.

The application has the positive progress effects that: the berberine has obvious inhibition effect on PK, which has important research value and application prospect on the treatment effect on diseases (such as ischemic cerebral apoplexy).

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the inhibition curve of berberine as PK enzyme in the present application.

Detailed Description

The application is further illustrated by way of examples below, which are not to be construed as limiting the scope of the application as defined by the examples, but are selected according to conventional methods and conditions or according to the commercial specifications without specifying the particular conditions in the examples below.

Berberine used in the examples was purchased from CSNpharm, lot number: CSN12859-1001; the purity is more than or equal to 97.80 percent. Z-Phe-Arg 7-amido-4-methylcoumarin hydrochloride, lot: MKCP6580, available from Sigma.

The instrumentation used in the examples was purchased from the following manufacturers:

an electronic balance: BT25S, certolis; multifunctional enzyme-labeled instrument: spectra Max model M5, molecular Devices company.

The effect of berberine on PK activity was observed using a commercial plasma kallikrein-specific fluorogenic substrate.

Berberine with different concentrations was mixed with rat serum, incubated in Tris-HCl buffer system at 25deg.C for 15min (50 mM Tris-HCl,250mM NaCl,1.5. Mu.M BSA; pH 7.5), then specific fluorogenic substrate (Z-Phe-Arg 7-amido-4-methylcoumarin hydrochloride; final concentration 200. Mu.M) was added, and fluorescence intensity was detected at 480nm (excitation wavelength of light was 360 nm) using an enzyme-labeled instrument. The final concentration of berberine (μg/mL) was: 0.1,0.3,1.0,3.0,10.0,30.0,100.0,300.0,1000.0 and 3000.0.

Data analysis:

data are expressed as mean ± standard error. Curve fitting was performed using GraphPad Prism 8.0.1 software.

The percent inhibition activity against PK was calculated according to the following formula:

enzyme inhibition (%) = (a) _{Blank space} -A _Measurement )*100％/A _{Blank space} 。

The technical effects are as follows:

the berberine versus PK enzyme inhibition curve is shown in FIG. 1: berberine can inhibit the enzymolysis of specific substrate by pKallikrein in a dose-dependent manner. The IC50 (μg/mL) of berberine for inhibiting PK enzyme activity is 56.04 + -1.05, and Hill Slope is 1.08+ -0.05. N=3/dose.

In conclusion, berberine has obvious inhibition effect on PK, and has important research value and application prospect on the treatment effect on diseases (such as ischemic cerebral apoplexy).

Claims (3)

1. Application of berberine in preparing medicine of plasma kallikrein inhibitor is provided.

2. The use according to claim 1, wherein the berberine is incubated in a Tris-HCl buffer system comprising Tris-HCl, naCl, caCl ₂ Or BSA.

3. The use according to any one of claims 1 to 2, wherein the medicament further comprises a pharmaceutically acceptable excipient.