CN112999212B

CN112999212B - Application of cannabidiol-3-sulfonic acid in preparation of medicines for preventing and/or treating inflammation-related diseases

Info

Publication number: CN112999212B
Application number: CN201911326445.8A
Authority: CN
Inventors: 曲桂武; 崔明
Original assignee: Yantai Hemp Biotechnology Co ltd
Current assignee: Yantai Hemp Biotechnology Co ltd
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2022-06-03
Anticipated expiration: 2039-12-20
Also published as: CN112999212A

Abstract

The invention provides an application of cannabidiol-3-sulfonic acid in preparation of a medicine for preventing and/or treating inflammation-related diseases, belonging to the technical field of anti-inflammatory medicines. The application of cannabidiol-3-sulfonic acid with a structural formula shown as a formula I in preparing a medicament for preventing and/or treating inflammation related diseases; the cannabidiol-3-sulfonic acid not only has higher water solubility, but also has stronger anti-inflammatory activity compared with cannabidiol. In-vitro inflammation model experiments show that cannabidiol-3-sulfonic acid can obviously inhibit the mRNA expression levels of inflammatory factors IL-1 beta, IL-6 and iNOS, and the compounds are proved to have obvious anti-inflammatory effects and can be used for preparing medicines for treating and preventing inflammation-related diseases.

Description

Application of cannabidiol-3-sulfonic acid in preparation of medicines for preventing and/or treating inflammation-related diseases

Technical Field

The invention belongs to the technical field of anti-inflammatory drugs, and particularly relates to an application of cannabidiol-3-sulfonic acid in preparation of drugs for preventing and/or treating inflammation-related diseases.

Background

Inflammation is a common pathological process in clinical practice, and is a complex defense reaction of a living organism having a blood vessel system to an injury factor (such as an allergic reaction generated by bacteria, viruses, antigens, nuclear antibodies, and the like), which can occur in tissues and organs of various parts of the organism. When inflammatory factors act on the body, the body eliminates inflammatory factors through an inflammatory reaction, which is a process of injury and anti-injury. Many common diseases, such as folliculitis, tonsillitis, pneumonia, hepatitis, pancreatitis, nephritis, etc., as well as autoimmune diseases, atherosclerosis, wound repair, etc., belong to the inflammation category. Anti-inflammatory drugs are second only to anti-infective drugs clinically in the 2 nd main category. The medicines capable of eliminating inflammation are collectively called as anti-inflammatory medicines, and can block the generation or release of inflammatory mediators and inhibit inflammatory reaction.

The anti-inflammatory drug mainly comprises non-steroidal anti-inflammatory drug, steroidal anti-inflammatory drug and traditional Chinese medicine. Because the traditional anti-inflammatory drugs have poor selectivity, obvious side effects and great limitation on clinical application, the research and development of novel anti-inflammatory drugs are always the key points of new drug research.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of a new compound cannabidiol-3-sulfonic acid in the preparation of a medicament for preventing and/or treating inflammation-related diseases, wherein the cannabidiol-3-sulfonic acid not only has high water solubility, but also has strong anti-inflammatory activity.

The invention provides an application of cannabidiol-3-sulfonic acid with a structural formula shown as a formula I in preparation of a medicine for preventing and/or treating inflammation related diseases;

preferably, the inflammation-related disorder comprises one or more of the following: rheumatic arthritis, pancreatitis, folliculitis, tonsillitis, pneumonia, hepatitis, pancreatitis, and nephritis.

Preferably, the dosage form of the medicament is injection, sterile powder for injection, eye drops, tablets, capsules or dropping pills.

Preferably, when the medicine is an injection or sterile powder for injection, the injection dosage range of the cannabidiol-3-sulfonic acid is 5mg to 500mg per adult per time.

Preferably, when the medicine is a tablet, a capsule or a dripping pill, the oral dosage range of the cannabidiol-3-sulfonic acid is 10mg to 1500mg per adult per time.

Preferably, when the medicament is an eye drop, the mass fraction of the cannabidiol-3-sulfonic acid is 0.5-10%.

The application of the cannabidiol-3-sulfonic acid provided by the invention in preparing a medicament for preventing and/or treating inflammation-related diseases. The cannabidiol-3-sulfonic acid is used as an active ingredient, so that the cannabidiol-3-sulfonic acid has better water solubility and higher anti-inflammatory performance compared with cannabidiol. In-vitro inflammation model experiments show that cannabidiol-3-sulfonic acid can obviously inhibit the mRNA expression levels of inflammatory factors IL-1 beta, IL-6 and iNOS, and the compounds are proved to have obvious anti-inflammatory effects and can be used for preparing medicaments for preventing and/or treating inflammation-related diseases.

Drawings

FIG. 1 is a bar graph of the quantitative PCR assay for the relative expression of inflammatory factor mRNA levels;

FIG. 2 is a bar graph of IL-1. beta. mRNA expression levels in LPS stimulated RAW264.7 cells by cannabidiol-3-sulfonic acid and cannabidiol;

FIG. 3 is a bar graph of IL-6 mRNA expression levels in LPS stimulated RAW264.7 cells by cannabidiol-3-sulfonic acid and cannabidiol;

FIG. 4 is a bar graph of the expression levels of iNOS mRNA in LPS-stimulated RAW264.7 cells by cannabidiol-3-sulfonic acid and cannabidiol.

Detailed Description

The invention provides application of cannabidiol-3-sulfonic acid with a structural formula shown as a formula I in preparation of a medicament for preventing and/or treating inflammation related diseases;

in the present invention, the cannabidiol-3-sulfonic acid is a derivative of cannabidiol, and its source is preferably prepared by chemical synthesis, which is described in the patent application No. 201910800720.9.

In the invention, the cannabidiol-3-sulfonic acid can obviously inhibit the mRNA expression levels of inflammatory factors IL-1 beta, IL-6 and iNOS, and the compounds are proved to have obvious anti-inflammatory effect and can be used for preparing medicines for treating and preventing inflammation-related diseases. The present invention is not particularly limited in the kind of inflammation-related diseases, and any inflammation-related diseases may be applicable, including, for example, one or more of the following diseases: rheumatic arthritis, pancreatitis, folliculitis, tonsillitis, pneumonia, hepatitis, pancreatitis, and nephritis.

The invention has no special limitation on the specific dosage form of the prepared medicament, and the medicament can be prepared into common dosage forms, such as injection, sterile powder for injection, eye drops, tablets, capsules or dropping pills. When the medicine is an injection or sterile powder for injection, the injection dosage range of the cannabidiol-3-sulfonic acid is preferably 5 mg-500 mg per adult per time, and more preferably 10 mg-250 mg per adult per time. The pH value of the injection is preferably 6.5-7.0. The sterile powder for injection preferably further comprises a stabilizer and a filler; the types of the stabilizer include EDTA-2 Na; the filler species include mannitol. When the medicine is a tablet, a capsule or a dropping pill, the oral dosage range of the cannabidiol-3-sulfonic acid is preferably 10mg to 1500mg per adult/time, and more preferably 25 mg to 500mg per adult/time. The tablet preferably further comprises a filler, a binder and a lubricant. The filler preferably comprises starch. The adhesive preferably comprises 10% starch slurry, dry starch. The lubricant preferably comprises magnesium stearate. When the medicament is an eye drop, the mass fraction of the cannabidiol-3-sulfonic acid is preferably 0.5-10%, and more preferably 1-5%. The eye drops preferably further comprise an osmotic pressure regulator and a bacteriostatic agent; the kind of the osmotic pressure regulator preferably includes sodium chloride; the kind of the bacteriostatic agent preferably comprises methyl paraben. The content of the auxiliary materials in the pharmaceutical dosage form and the preparation method of the medicine are not particularly limited, and the methods well known in the field can be adopted.

The following examples are provided to illustrate the application of cannabidiol-3-sulfonic acid in the preparation of a medicament for preventing and/or treating inflammation-related diseases, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparation of cannabidiol-3-sulfonic acid injection

Prescription:

the operation is as follows: adding sodium chloride and cannabidiol-3-sulfonic acid into 800mL of water for injection, stirring to dissolve, adding 0.1mol/L sodium hydroxide solution to adjust pH to 6.5-7.0, adding water to a sufficient amount, stirring, filtering, subpackaging in 1mL ampoules, sealing by nitrogen, and sterilizing with flowing steam at 100 ℃ for 30min to obtain the final product.

Example 2

Preparation of cannabidiol-3-sulfonic acid freeze-dried powder for injection

Prescription:

the operation is as follows: adding cannabidiol-3-sulfonic acid, mannitol and EDTA-2Na into 1600mL of water for injection, stirring to dissolve, adding 0.1mol/L sodium hydroxide solution to adjust pH to 6.5-7.0, adding water to a sufficient amount, stirring, filtering, packaging in ampoules 2mL each, freeze drying, sealing, and checking for gas leakage.

Example 3

Preparation of cannabidiol-3-sulfonic acid eye drops

Prescription:

the operation is as follows: adding cannabidiol-3-sulfonic acid, sodium chloride and methylparaben into about 800mL of distilled water, stirring to dissolve, adding 0.1mol/L sodium hydroxide solution to adjust the pH value to 6.5-7.0, adding water to a sufficient amount, stirring uniformly, filtering and filling, and sterilizing for 30min by flowing steam at 100 ℃ to obtain the finished product.

Example 4

Preparation of cannabidiol-3-sulfonic acid tablets

Prescription:

the operation is as follows: uniformly mixing cannabidiol-3-sulfonic acid and starch, adding starch slurry to prepare a soft material, extruding and granulating by using a 14-mesh sieve, drying at 80-85 ℃, granulating by using a 12-mesh sieve, adding dry starch and magnesium stearate, uniformly mixing and tabletting to obtain the cannabidiol-3-sulfonic acid tablet.

Experimental example 5

Cannabidiol-3-sulfonic acid (CBD-3-SO)₃H) Research on anti-inflammatory action and mechanism thereof

1 Material

1.1 lyophilized powder of cannabidiol-3-sulfonic acid for injection (prepared as described in example 2) for use as a medicament and agent; mouse mononuclear macrophage RAW264.7, purchased from Shanghai cell biology institute of Chinese academy of sciences; DMSO (fuchen chemical reagent factory, tianjin, china); MTT kit (Merck Millipore) and Griess kit (Abcam) are purchased from Guangzhou Xinjin Biotechnology Co., Ltd; fetal bovine serum and DMEM medium (Gibco, distributed by Beijing Qianjin Biotechnology Co., Ltd.); trypsin (Sigma); reverse transcription kit (TaKaRa Biotechnology); RNAioso for total RNA (TaKaRa Biotechnology); TB GreenTM Premix Ex TaqTM II kit (TaKaRa Biotechnology) other reagents are analytically pure and are produced by national drug group chemical reagent Co.

1.2 apparatus qRT-PCR apparatus (eppendorf); high speed refrigerated centrifuge (Legend Micro 21R, Thermo Fisher Scientific); CO2₂Cell culture chamber (INCO 2246); a 37 ℃ incubator (Beckman coulter); a micro oscillator (IKA MS3 digital).

2 method

2.1 preparation of the solution

Preparing an LPS solution: dissolving 1mg LPS powder in 1ml ultrapure water to obtain 1mg/ml stock solution, filtering with 0.22 μm filter membrane, sealing, and storing at-20 deg.C.

CBD and CBD-3-SO₃Preparing a solution H: weighing the CBD prototype powder 3.1446X 10^-3g, 100. mu.l DMSO was added to prepare a stock solution with a concentration of 100 mM. Weighing CBD-3-SO₃H powder 3.9546X 10^-3g, 100. mu.l DMSO was added to prepare a stock solution with a concentration of 100 mM.

2.2 establishment and detection of mouse macrophage cell line RAW264.7 cell inflammation model

RAW264.7 cells are inoculated in a 6-well plate, LPS (100ng/mL) is added when the cells grow to 50% -60% for induction, the cells are collected after 0, 2, 4 and 6h of action, total RNA extraction is carried out by using RNAiso for total RNA, the concentration of the total RNA is measured, and the mRNA expression quantity of IL-6, IL-1 beta and iNOS inflammatory factors is detected by adopting a fluorescence quantitative PCR method.

2.3 cell culture

Mouse mononuclear macrophage RAW264.7 DMEM culture solution (containing 10% fetal calf serum and 1% penicillin-streptomycin double antibody) at 37 deg.C and 5% CO₂Culturing in an incubator until the cells grow adherent to the wallWhen 80% of cells are cultured in a dish, digesting and passaging by using trypsin-EDTA solution, taking RAW264.7 cells in logarithmic phase, adjusting the concentration of cell suspension, inoculating a certain volume of the cells into a 96-hole cell culture plate, culturing until the cells are attached to the wall, and adding different final concentrations (20, 40 and 80 mu mol^-1) Cannabidiol-3-sulfonic acid sample solution. Each set of 5 parallel wells.

2.4 Effect of cannabidiol-3-sulfonic acid on RAW264.7 cell viability

The concentration of the cell suspension cultured in 2.3 cultures was adjusted to 5X 10⁴each.mL^-1Inoculating to 96-well cell culture plate, inoculating 180 μ L of the culture solution per well, setting blank control group (replacing cannabidiol-3-sulfonic acid sample solution with DMEM culture solution of the same volume), culturing for 24 hr in the same manner as 2.3, adding 20 μ L of the culture solution with mass concentration of 5 mg/mL^-1Continuously culturing the MTT solution for 4 hours; the culture medium was aspirated off the wells, 150. mu.L DMSO was added to each well, the mixture was shaken for 10min to dissolve the crystals sufficiently, and the absorbance (A) was measured at 490nm for each well_490nm) Calculating the inhibition rate of the human cannabidiol-3-sulfonic acid on the cells according to the formula (1):

the cell inhibition ratio (%) (1-dose group a490 nm/blank control group a490nm) × 100 formula (1).

2.5 cannabidiol-3-sulfonic acid (CBD-3-SO)₃H) Effect on LPS stimulated mRNA expression of RAW264.7 cytokines

Cell number per well 10 in 6 well cell culture plates⁵Inoculating, adding 100ng/mL LPS for inflammatory induction after cell adherence, and respectively adding 20 μmol/L CBD-3-SO 6h later₃H and 20. mu. mol/L Cannabidiol (CBD) were intervened and a blank control (normal, without LPS induction group) and an LPS control group (LPS, i.e. induced with LPS only, without CBD-3-SO were set up₃H or CBD intervention group); collecting cells after intervening for 2h, extracting total RNA by using an RNAioso for total RNA kit, carrying out reverse transcription to synthesize cDNA, and detecting the mRNA expression quantity of IL-6, IL-1 beta and iNOS inflammatory factors by adopting a fluorescence quantitative PCR method, wherein the specific method comprises the following steps:

(1) the extraction method of the total RNA of the cells comprises the following steps:

1) the cell pellet was collected.

2)1ml of Trizol at room temperature and static lysis of cells for 5 min.

3) Adding 1/5 volumes of chloroform, shaking and mixing, and standing and incubating for 5min on ice.

4) Centrifuge at 12000rpm4 ℃ for 15 min.

6) After centrifugation, the liquid is divided into three layers, namely colorless supernatant, a middle white protein layer and a lower organic phase with color. The supernatant was transferred to another centrifuge tube (the white middle layer was not aspirated).

7) The supernatant was collected, 0.5mL of pre-cooled isopropanol was added, mixed well, and allowed to stand on ice for 10 min.

8) Centrifuge at 12000rpm4 ℃ for 10 min.

9) The supernatant was discarded, and 1mL of 75% ethanol was added thereto, followed by centrifugation at 12000rpm at 4 ℃ for 5 min.

9) Discarding 75% ethanol, naturally drying for 5-10 min, and dissolving RNA by DEPC water.

10) Determination of OD by means of a biological spectrophotometer₂₆₀/OD₂₈₀And (4) calculating the RNA concentration.

11) Purity and quality of extracted total RNA were checked by 1% agarose gel electrophoresis.

(2) The reverse transcription method of total RNA is as follows:

1) genome DNA removal reaction

In a PCR instrument, the temperature is 42 ℃ for 2 min.

2) Reverse transcription

The total volume of 20. mu.l was placed in a PCR apparatus at 37 ℃ for 15min and 85 ℃ for 5s, and the synthesized cDNA was stored at-20 ℃.

After cDNA was synthesized, amplification was carried out using a specific primer for GAPDH, IL-6, iNOS, and IL-1. beta. using GAPDH as a template and GAPDH as an internal reference, and the expression levels of IL-6, IL-1. beta. and iNOS inflammatory factor mRNA were detected by a quantitative PCR method. The primer sequences used in the quantitative PCR are shown in Table 1, the reaction system is shown in Table 2, and the reaction conditions are as follows: annealing at 60 deg.C, denaturation at 95 deg.C for 30s, denaturation at 95 deg.C for 5s, and denaturation at 60 deg.C for 20s, and repeating 40 cycles.

TABLE 1 primer sequence information

TABLE 2 quantitative PCR reaction System as follows

2.3 statistical treatment

Data was analyzed using IBM SPSS Statistics 25 software. By adopting one-way anova, the difference is significant when P is less than 0.05, and the difference is highly significant when P is less than 0.01.

3 results

3.1 quantitative PCR detection of the expression of inflammatory factors after LPS treatment of RAW264.7 cells

After the cells of RAW264.7 were treated with LPS for 6h, the expression level of each inflammatory factor in the cells was detected by quantitative PCR, and the results showed that the expression level of each inflammatory factor mRNA in the cells of LPS-treated group was significantly increased compared with that of normal group (FIG. 1).

3.2 Effect of cannabidiol-3-sulfonic acid on RAW264.7 cell viability

MTT experiment results show that cannabidiol-3-sulfonic acid has no obvious influence on the activity of RAW264.7 cells under the experiment concentration, and the cell inhibition rate is lower than 20%, which is shown in Table 3.

Table 3 effect of cannabidiol-3-sulfonic acid on RAW264.7 cell viability (n-5,

)

note: p <0.05 compared to the blank control group.

3.3 Effect of cannabidiol-3-sulfonic acid and cannabidiol on the expression levels of LPS-stimulated RAW264.7 cytokines IL-6, IL-1. beta. and iNOS mRNA

The results are shown in fig. 2, 3 and 4. The results show that after the RAW264.7 cells are treated by LPS, the cells are respectively interfered by CBD (20 mu mol/L) and CBD-3-SO3H (20 mu mol/L), and the results show that the mRNA level expression of IL-1 beta, IL-6 and iNOS of the RAW264.7 cells can be obviously reduced by CBD (20 mu mol/L) and CBD-3-SO3H (20 mu mol/L). The results also show that CBD-3-SO₃The H (20 mu mol/L) has more obvious effect and more obvious inflammation inhibiting effect.

From the above examples, it is clear that LPS stimulates RAW264.7 cells to produce inflammatory factors, and the anti-inflammatory activity of the drug is judged by detecting the expression level of mRNA of inflammatory factors, which is a method generally used for screening anti-inflammatory active ingredients. The research results show that under the experimental dosage, the cannabidiol-3-sulfonic acid shows a remarkable anti-inflammatory effect on an RAW264.7 cell model stimulated by LPS, the growth of cells is not obviously inhibited, and the anti-inflammatory activity is stronger than that of the cannabidiol. Therefore, the cannabidiol-3-sulfonic acid has good application in preparing medicines for treating and preventing inflammation-related diseases.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Claims

1. The application of cannabidiol-3-sulfonic acid with a structural formula shown as a formula I in the preparation of medicines for preventing and/or treating inflammation related diseases;

the inflammation-related disease is rheumatic arthritis, pancreatitis, pneumonia or hepatitis.

2. The use of claim 1, wherein the medicament is in the form of injection, tablet, capsule or drop pill.

3. The use as claimed in claim 2 wherein, when the medicament is an injection, the cannabidiol-3-sulfonic acid is injected in an amount ranging from 5mg to 500mg per adult per time.

4. The use as claimed in claim 2 wherein, where the medicament is in the form of a tablet, capsule or drop pill, the cannabidiol-3-sulphonic acid is administered orally in an amount in the range 10mg to 1500mg per adult human per dose.