CN112891331A

CN112891331A - Application of benzoate compound in preparation of medicine for relieving or treating rheumatoid arthritis

Info

Publication number: CN112891331A
Application number: CN202110307211.XA
Authority: CN
Inventors: 阿基业; 齐琦; 黄宝连
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-03-23
Filing date: 2021-03-23
Publication date: 2021-06-04
Anticipated expiration: 2041-03-23
Also published as: CN112891331B

Abstract

The invention discloses an application of benzoate compounds in preparing a medicament for relieving or treating rheumatoid arthritis, wherein the benzoate compounds are slowly metabolized in vivo to generate benzoic acid, so that the benzoic acid has the characteristics of long duration, lasting and stable concentration and small fluctuation in vivo, has better pharmacokinetic characteristics, good drug effect on rheumatoid arthritis, and lasting and effective anti-inflammatory immune effect, and can be applied to preparing a medicament for treating rheumatoid arthritis with more lasting and stable drug effect.

Description

Application of benzoate compound in preparation of medicine for relieving or treating rheumatoid arthritis

Technical Field

The invention relates to an application of benzoate compounds, in particular to an application of benzoate compounds in treating rheumatoid arthritis diseases, and belongs to the technical field of biological medicines.

Background

Rheumatoid Arthritis (RA), a chronic progressive autoimmune disease, is characterized primarily by synovitis and symmetrical, destructive joint disease, affecting 0.5-1% of adults in industrialized countries, the most common of which is in women and the elderly. Uncontrolled active rheumatoid arthritis can lead to joint damage, disability, reduced quality of life, and cardiovascular disease and other complications. The diagnosis of the disease is a combined approach involving radiographic, hematological and serological marker assessment, the pathogenesis of which is not yet clear.

Traditional therapeutic drugs such as antirheumatic drugs, non-steroidal anti-inflammatory drugs, glucocorticoids and the like have unpredictable adverse reactions. Biological agents can be used when arthritis is uncontrolled or when antirheumatics produce toxic effects, but infection and high costs limit the use of biological agents. The history of the Chinese herbal medicine for treating RA is long, and the Chinese herbal medicine has less adverse reaction and moderate treatment effect, so that the Chinese herbal medicine can be used as a supplementary and alternative treatment means in the arthritis treatment.

Sodium benzoate has been reported to have therapeutic effects in experimental cerebrospinal meningitis (EAE). Benzoic acid derived phenolic acid substances, such as gallic acid, have anti-inflammatory effects on fibroblast-like synovial cells (RA-FLS) cells isolated from synovial tissue of RA patients; protocatechuic acid significantly reduced paw swelling in rat adjuvant-induced arthritis (AIA) by reducing lipid peroxide expression and reactive free radical nitric oxide levels and increasing antioxidant enzyme expression (SOD, catalase, GSH, etc.), with anti-arthritic activity comparable to diclofenac sodium. Paeoniflorin may be metabolized by carboxylesterase in intestinal flora to generate benzoic acid to exert its pharmacological effects such as antidepressant and immunoregulatory effects, and benzoic acid may be an active metabolite of paeoniflorin to exert its anti-arthritis effect.

However, as a chronic disease, the rheumatoid arthritis disease has ideal drug pharmacokinetics such that the chronic disease has long-lasting in vivo exposure, and the benzoic acid is fast in absorption and distribution and elimination, so that the anti-inflammatory and immune drug effect is not exerted.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide application of benzoate compounds in preparation of drugs for relieving or treating rheumatoid arthritis.

The technical scheme is as follows: the invention discloses application of benzoate compounds in preparation of drugs for relieving or treating rheumatoid arthritis, wherein the formula is shown as formula I, wherein n is₁＝1,2,3,4,5,6,7，n₂＝1,2,3,4,5,6。

Preferably, the benzoate compound is a benzoic acid-octadecenyl alcohol ester compound shown as a formula V, wherein n is₁＝7，n ₂6, molecular formula: c₂₅H₄₀O₂Molecular weight: 372.303.

preferably, the pharmaceutical dosage form is a tablet, a capsule, an oral solution, a pill, a granule or a powder.

Preferably, the pharmaceutical dosage form further comprises a gel, a liniment, a patch, a topical solution, an aerosol or an injection.

Preferably, the benzoate compound comprises a derivative thereof and a drug carrier.

The benzoate compound disclosed by the invention has the characteristics of long duration, small concentration fluctuation and better pharmacokinetics, has better effectiveness and more obvious effect in the application of preparing a medicament for relieving or treating rheumatoid arthritis, and has potential pharmaceutical application prospect; CIA mice are used as model animals, benzoic acid-octadecenyl alcohol ester and sodium benzoate are used for treatment through intragastric administration, paw swelling degree and joint pathology scores are measured, and evaluation of treatment of rheumatoid arthritis by benzoic acid-octadecenyl alcohol ester is carried out. Pharmacodynamic experiment results show that the benzoate compound has a good pharmacological effect on rheumatoid arthritis, relieves swelling degree of joints of a CIA mouse after administration, and improves histopathological changes of the joints of the CIA mouse; the in vivo metabolism research result shows that the benzoate compound can show better pharmacokinetic behavior and increase the in vivo exposure of the active product benzoic acid. The benzoic acid-octadecenyl alcohol ester compound improves the fat solubility and passive membrane permeability of the medicament, improves the oral absorption degree of the medicament, is hydrolyzed by carboxylesterase of intestinal flora in intestinal tracts after being orally taken, then releases original medicament benzoic acid, and prolongs the action time of the medicament.

Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: the benzoate compound has the characteristics of long drug duration in vivo, small concentration fluctuation and better pharmacokinetics, is applied to chronic disease rheumatoid arthritis diseases, can slowly release benzoic acid after long-term in vivo exposure, has more lasting and stable concentration in vivo and has good anti-arthritis drug effect. The benzoate compound is applied to the preparation of the medicine for relieving or treating rheumatoid arthritis, and can effectively relieve and treat the rheumatoid arthritis.

Drawings

FIG. 1 is a reaction scheme for preparing benzoic acid-octadecenyl alcohol ester compound;

FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of a benzoic acid-octadecenyl alcohol ester compound;

FIG. 3 is a nuclear magnetic resonance carbon spectrum of a benzoic acid-octadecenyl alcohol ester compound;

FIG. 4 is a graph showing the time-dependent changes of the mean values of the arthritis clinical scores, wherein the Control group is a Control group, the CIA group is a model group, the NaB group is a sodium benzoate administration group of a model animal, the Lef group is a leflunomide positive drug group of the model animal, and the OB group is a benzoic acid-octadecenyl alcohol ester administration group of the model animal;

FIG. 5 is a graph showing the time-dependent changes in the mean thickness values of the paw, wherein the Control group is a Control group, the CIA group is a model group, the NaB group is a sodium benzoate administration group of a model animal, the Lef group is a leflunomide positive drug group of a model animal, and the OB group is a benzoic acid-octadecenyl alcohol ester administration group of a model animal;

FIG. 6 is a graph showing the change of the mean weight values of mice with time, wherein the Control group is a Control group, the CIA group is a model group, the NaB group is a sodium benzoate administration group of a model animal, the Lef group is a leflunomide positive drug group of the model animal, and the OB group is a benzoic acid-octadecenyl alcohol ester administration group of the model animal;

FIG. 7 is a photograph of the left hind paw of each group of representative mice taken before sacrifice on day 48 of the initial immunization, wherein Control group is Control group, CIA is model group, NaB is sodium benzoate administration group for model animal, Lef is leflunomide positive drug group for model animal, OB is octadecyl benzoate administration group for model animal;

FIG. 8 is H & E staining of joint pathology sections of each group of representative mice, wherein Control group is a Control group, CIA group is a model group, NaB is a sodium benzoate administration group of a model animal, Lef is a leflunomide positive drug group of the model animal, and OB is a benzoic acid-octadecenyl alcohol ester administration group of the model animal;

FIG. 9 is the average value of H & E staining pathological scoring results of mice in each group, wherein the Control group is a Control group, the Model group is a Model group, NaB is a sodium benzoate administration group for a Model animal, Lef is a leflunomide positive drug group for a Model animal, and OB is a benzoic acid-octadecenyl alcohol ester administration group for a Model animal;

FIG. 10 is a plot of drug concentration of benzoate versus time in plasma after a single administration of the benzoate prodrug and benzoic acid, where NaB is the sodium benzoate dosing group and OB is the benzoic acid-octadecenyl alcohol ester dosing group.

Detailed Description

The technical scheme of the invention is further explained by combining the attached drawings.

To evaluate the therapeutic effect of benzoic acid-octadecenyl alcohol ester on rheumatoid arthritis RA. The invention takes a CIA mouse as a model animal, treats the benzoic acid and the ester prodrug thereof through gastric lavage, and determines the clinical indexes (paw swelling score, paw swelling thickness and HE pathological section of a joint part) of the arthritis to evaluate the efficacy of the benzoic acid and the ester prodrug thereof in treating the arthritis.

The benzoic acid-octadecenyl alcohol ester compound adopted by the invention is a preparation reaction formula of the benzoic acid-octadecenyl alcohol ester compound as shown in figure 1; the preparation method of the compound specifically comprises the following steps:

(1) dissolving 140.6mg of 1mmol of benzoyl chloride and 268.5mg of 1mmol of oleyl alcohol in 4ml of anhydrous dichloromethane solution at room temperature, and stirring at room temperature overnight;

(2) TLC trace until reaction was complete, 10ml of 1M NaHCO was added₃Stirring overnight to remove unreacted benzoyl chloride, dichloromethaneAnd (3) extracting twice with alkane, washing once with saturated salt, drying with anhydrous sodium sulfate, concentrating, and purifying by column chromatography to obtain the benzoic acid-octadecenyl alcohol ester compound.

As shown in fig. 2, nuclear magnetic resonance hydrogen spectrum of the prepared benzoic acid-octadecenyl alcohol ester compound; as shown in fig. 3, nuclear magnetic resonance carbon spectrum of the prepared benzoic acid-octadecenyl alcohol ester compound.

The drug effect evaluation test material is as follows, leflunomide is purchased from China institute for food and drug testing; the chemical structure of benzoic acid-octadecenyl alcohol ester is shown in figure 1, synthesized by pharmaceutical chemistry laboratory of Chinese university of pharmacy; complete Freund's adjuvant reagent purchased from Sigma-Aldrich (USA); bovine type II collagen, incomplete freund's adjuvant purchased from chondrex (usa); healthy 6-7 week old SPF-grade DBA/1J mice (male) were purchased from Calvens laboratory animals, Inc., Changzhou. Animals were fed freely for one week to acclimatize under Specific Pathogen Free (SPF) conditions (temperature 22 + -2 deg.C, humidity 40-70%, 12 hr light/dark alternating environment).

Then, the mouse model of rheumatoid arthritis is made, and the primary immunization of a CIA model mouse is as follows: taking a certain amount of complete Freund's reagent and an injector, slowly dropping equal volume of bovine type II collagen (melting at 4 ℃ in advance for 3 hours before use), mixing by using an electric homogenizer (1000-3000 rpm), paying attention to heat to denature collagen so as to prevent arthritis from being induced, cooling the emulsion on an ice water bath in the whole mixing process, continuously mixing the emulsion at a high rotation speed (3000-5000 rpm) for 2 minutes, adding a drop of emulsion into a beaker after the preparation of the emulsion is finished to test the stability of the emulsion, and if the emulsion is stable, enabling milky liquid drops to float on the water surface and not be dispersed. If the emulsion dissipates at the surface of the water, it is unstable, and a few drops of complete Freund's adjuvant are added and mixed again, and then the test is performed again. The emulsion after full emulsification should exhaust air to ensure that the correct amount of emulsion is injected, and the emulsion is injected within 1 hour after preparation for molding, and is kept at 4 ℃ until use. One side of the needle tip inclined plane faces upwards and is kept parallel to the tail of the mouse, a needle head is inserted into the tail root of the mouse at a distance of 2cm, and 0.1ml of emulsion is injected subcutaneously to perform primary immunization until the needle tip reaches a position 0.5cm away from the tail root; 21 days after the primary immunization, the bovine type II collagen and incomplete Freund's adjuvant were mixed in the same way in the same volume, and after sufficient emulsification, a needle was inserted 3cm from the tail root of the model mouse until the needle point reached 1.5cm from the tail root, and 0.1ml of the emulsion was injected (the booster should be injected at a different site from the primary immunization).

Dividing DBA/1J mice into 6 groups at random, setting normal mice as a control group, and dividing the mice after molding into a model group, a leflunomide group (positive drug group), a benzoic acid-octadecenol group, a sodium benzoate low-dose group and a sodium benzoate medium-dose group at random, wherein each group comprises 6 mice; the doses for each group are given in table 1 below.

TABLE 1 grouping and dosing of mice

The administration intervention is carried out on each administration group 21 days after the primary immunization, and the preparation method of the administration solution comprises the following steps of precisely weighing 25.0mg of leflunomide, and adding a small amount of 0.5% CMC-Na solution into a mortar for multiple times to suspend 50mL of leflunomide until the concentration of the leflunomide is 0.5 mg/mL; precisely weighing 258.5mg of benzoic acid-octadecenyl enol ester, adding 50uL of tween 80 for dissolution assistance, adding a small amount of 0.5% CMC-Na, performing ultrasonic treatment for 5min, and supplementing a solvent to 50mL to obtain a uniform suspension with the concentration of 5.17 mg/mL; respectively and precisely weighing 50.0mg and 100.0mg of sodium benzoate, and respectively adding 50mL of 0.5% CMC-Na solution for multiple times in small amount to obtain uniform suspension with the concentration of 1mg/mL and 2 mg/mL. The administration groups are all administrated by intragastric administration according to 0.1mL/10g for 4 weeks.

During the experiment, the diet, activity and fur status of each group of mice were observed daily, and the mice were weighed 27, 30, 33, 36, 39, 42, 45, 48 days after the primary immunization, and clinically scored for arthritis (Polyarthritis Index, PI) on a scale of 0-4 points according to the incidence of arthritis in the mice. 0 minute: no joint inflammation (no redness); 1 minute: mild redness and swelling of the little toe joint; and 2, dividing: swelling of the little toe joint and the plantar aspect; and 3, dividing: paw swelling below the ankle joint; and 4, dividing: all paws, joints including ankle joints, were swollen. The integral is accumulated according to the pathological changes of each joint, namely the PI of each mouse is 16 points at most. The thickness of the footpad was measured with a digital caliper (the paw of the mouse was too small to determine the swelling of the paw by a volume measurer), 48 days after the initial immunization the experimental mouse was sacrificed, the ankle joint was collected and fixed in 4% paraformaldehyde, and histopathological changes were observed under a light microscope after conventional decalcification, dehydration, paraffin embedding, sectioning and H & E staining. The examination indexes comprise the damage degree of articular cartilage, synovial hyperplasia, articular stenosis, interstitial cell infiltration and fibrous tissue hyperplasia degree, the grading is carried out according to the pathological change degree, and the average score of each group is calculated by accumulating the scores.

The results of the arthritis scores of the mice in each group are shown in table 2 and figure 4, and the results show that the joints of the mice in the normal group have no pathological change symptoms, the average number of the clinical scores of the mice in the model group is the highest, the pathological change degree of the joints is the most serious, the joint pathological change degree of the model-making mice is obviously reduced by the leflunomide positive drug group, and compared with the model group, the clinical scores of the sodium benzoate low-dose group, the sodium benzoate medium-dose group and the benzoic acid-octadecenyl alcohol ester administration group are also obviously reduced, which indicates that the mice have similar anti-arthritis treatment effects.

TABLE 2 clinical arthritis scores for each group of mice

The results of the thicknesses of the paws of the mice in each group are shown in the table 3 and the figure 5, the appearance lesion degree of the ankle joint is shown in the figure 6, the paws of the mice in the normal group are free from swelling, the ankle joint and the finger tip are free from swelling, the ankle joint, the foot sole and the knuckle part of the mice in the model group are obviously swollen, compared with the mice in the model group, the leflunomide positive drug group obviously relieves the swelling degree of the paw part of the mice, the thickness of the paws is reduced, the positive drug has an inhibiting effect on arthritis, and compared with the mice in the model group, the sodium benzoate administration group and the administration of the equimolar ester prodrug thereof also obviously improve the disease process and the arthritic pathological characteristics of the mice in the arthritis.

TABLE 3 mean paw thickness of each group of mice

The weight change of each group of mice is shown in table 4 and fig. 7, the disease degree is increased along with the increase of time after primary immunization modeling, the weight of the mice is gradually reduced, compared with the normal group of mice, the weight of the mice in the model group is obviously reduced, the weight reduction degree of the mice in each administration group is lower than that of the model group, the weight reduction condition of the leflunomide group is obviously improved compared with that of the model group, the weight reduction degree of the sodium benzoate administration group has certain dose dependence, and the weight reduction condition of the benzoic acid-octadecenyl enol ester administration group is improved to a certain extent compared with that of the sodium benzoate administration group.

TABLE 4 mean body weight of mice in each group

The pathological section results of ankle joints of each group of mice are shown in table 5, fig. 8 and fig. 9. The H & E staining result of the Control group shows that the ankle tissue structure is normal and complete, and compared with the Control group, pathological changes such as joint cartilage destruction, joint synovial hyperplasia, interstitial inflammatory cell infiltration, fibrous tissue hyperplasia and the like can be seen in the ankle of the CIA model group; compared with the model group, each administration group can be seen in the similar pathological changes of the model group, and the ankle joint pathological changes of the benzoic acid-octadecenyl alcohol ester group and the sodium benzoate middle dose group are obviously reduced compared with the model group.

TABLE 5 mean values of H & E staining histological damage degree of mice in each group

Plasma pharmacokinetic experiments on benzoate compounds:

the rheumatoid arthritis disease is a chronic disease, the ideal drug pharmacokinetics property is that the long-lasting in vivo exposure exists, the benzoic acid is absorbed and distributed rapidly, the elimination speed is fast, the anti-inflammatory immune efficacy is not exerted, and therefore, the benzoic acid-octadecenyl alcohol ester prodrug is synthesized to improve the pharmacokinetics.

The method is applied to plasma pharmacokinetics experiments of benzoic acid and benzoic acid-octadecenyl alcohol ester by establishing a GC-MS/MS detection method, the pharmacokinetic experiment materials are as follows, and a sodium benzoate standard substance is purchased from Shanghai Yan Biotech limited; d₅Benzoic acid Isotope standards were purchased from Cambridge Isotope Laboratories, Inc (Cambridge, USA), benzoic acid-octadecenol ester was synthesized by the pharmaceutical chemistry laboratory of chinese pharmaceutical university; internal standard 1,2-¹³C₂Myristic acid, derivatizing reagent (MSTFA + 1% TMCS) were purchased from Sigma-Aldrich (st. louis, USA) and chromatographically pure methanol, heptane from Merk. Healthy 6-7 week old SPF grade SD rats purchased from Witongliwa laboratory animal technology, Inc. Animals are freely fed and raised for one week in an alternating environment of 12 hours light/dark at the temperature of 22 +/-2 ℃ and the humidity of 40-70 percent to adapt to the environment.

Preparing a dosing solution, precisely weighing 258.5mg of benzoic acid-octadecenyl alcohol ester, adding 50uL of Tween 80 for assisting dissolution, adding a small amount of 0.5% CMC-Na, performing ultrasonic treatment for 5min, and supplementing a solvent to 50mL to obtain a uniform suspension with the concentration of 5.17 mg/mL; respectively and precisely weighing 100.0mg of sodium benzoate, and adding 50 mL0.5% CMC-Na solution for many times in small amount to obtain uniform suspension with the concentration of 2 mg/mL. The dose administered to each rat was 1mL/100 g.

After single administration of equimolar doses of benzoic acid and benzoic acid-octadecenol ester of SD rat, blood is collected from orbital venous plexus of rat, centrifuged at 8000rpm for 10min, and the supernatant plasma is stored at-70 deg.C. Re-dissolving the plasma sample, oscillating for 30s, taking 50uL of plasma, adding 200uL of methanol solution containing the internal standard, and vortexing and oscillating for 3 min. (internal standard is isotope labeled myristic acid, mother liquor is 5mg/mL, and the dilution is 1000-fold to 5ug/mL) at 4 ℃ for 20000g x10min, centrifuging to take 150uL of supernatant to a tube inserted in a GC sample injection vial, decompressing and volatilizing, adding 60uL of derivatization reagent for redissolution (MSTFA: heptane 1: 1), oscillating by a vortex device for 3min for redissolution, standing at room temperature for 1h for derivatization reaction, carrying out GC-MS/MS 1uL sample injection analysis, and simultaneously detecting the contents of benzoic acid-octadecenyl alcohol ester prodrug and benzoic acid in plasma by a GC-MS/MS method.

The plasma drug concentration of two groups of rats after administration is changed along with time as shown in figure 10, and the results show that sodium benzoate is absorbed quickly after administration, no drug absorption phase is monitored, the concentration of benzoic acid after administration is high, which is about twice of Cmax of benzoic acid after administration of ester prodrug, and is metabolized quickly within 30min, and the benzoic acid is released slowly after administration of prodrug, and the metabolic time is prolonged compared with that of benzoic acid direct administration, which shows that the synthesized prodrug can show better pharmacokinetic behavior and increase the in vivo exposure of benzoic acid which is an active product.

Claims

1. An application of benzoate compound shown as formula I in preparing medicine for relieving or treating rheumatoid arthritis, wherein n is₁＝1,2,3,4,5,6,7，n₂＝1,2,3,4,5,6。

2. Use according to claim 1, characterized in that: the benzoate compound is shown in a formula V.

3. Use according to claim 1, characterized in that the pharmaceutical dosage form is a tablet, capsule, oral solution, pill, granule or powder.

4. The use according to claim 1, wherein the pharmaceutical dosage form further comprises a gel, a varnish, a patch, a topical solution, an aerosol or an injection.

5. The use of claim 1, wherein the benzoate compound comprises a derivative thereof and a pharmaceutical carrier.