CN110575448A - Cannabidiol composition and use thereof - Google Patents

Abstract

The present application relates to cannabidiol compositions and uses thereof. In particular, the application relates to a cannabidiol composition which contains cannabidiol and a surfactant, and the composition can remarkably improve the water solubility of the cannabidiol, so that the composition can be better applied to the fields of medicines, daily chemicals, foods, health-care products and the like.

Description

Cannabidiol composition and use thereof

technical field

The application relates to the field of chemistry, in particular to a composition containing cannabidiol (Cannabidiol, CBD), the composition can significantly improve the water solubility of cannabidiol, making it better used in medicine, daily chemicals, food, Health care products and other fields.

Background technique

Cannabis (scientific name: Cannabis sativa L.) is a cannabis plant, also known as hemp, hemp, hemp, mountain silk seedling, jute, and has important agricultural and medicinal value. Cannabis contains a toxic component, tetrahydrocannabinol (THC), which can be hallucinogenic and can be used as a drug, and has been banned for a long time.

The hemp variety (referred to as "industrial hemp") specially designed for industrial use has a THC content of less than three thousandths in the mosaic leaves during the growing period, and does not have the value of extracting the toxic component THCTHC or is directly consumed as a drug. It has economic and medicinal value, and can be legally planted on a large scale and industrialized for development and utilization.

At present, more than 500 substances have been isolated from cannabis plants, of which there are at least 86 cannabidiol compounds. Cannabidiol compounds are a unique class of substances in cannabis plants and are the main active components in cannabis plants. Research on them has always been a hot spot in cannabis research. The main cannabidiol compounds in cannabis plants are THC, cannabidiol (CBN), CBD, cannabidiol (CBG), cannabidiol (CBC), etc., of which the first three account for more than 90% of cannabidiol compounds.

Cannabidiol is one of the cannabinoids, the molecular formula is C ₂₁ H ₃₀ O ₂ , it is a pale yellow resin or crystal, usually extracted from the natural plant cannabis and can also be synthesized artificially. It has various pharmacological activities. Compound, almost insoluble in water, soluble in organic solvents such as ethanol, methanol, ether, benzene, chloroform and petroleum ether. Its structural formula is shown in the following formula I:

Cannabidiol is completely different from THC, not only does it have no hallucinogenic effects, but it is also useful in the treatment of convulsions, anxiety/depression, inflammation, cancer, rheumatoid arthritis, multiple sclerosis, epilepsy, especially refractory epilepsy, and It has good pharmacological activity in antiviral, analgesic and other aspects, therefore, it has become a research hotspot in the global medical field.

However, cannabidiol has problems such as poor water solubility, low bioavailability and weak stability, which limit its application to a large extent, as follows:

GW Pharma is a company whose main business is the research and development of cannabis-related ingredients. Its compound drug Sativex containing cannabidiol and THC has been developed and marketed for the treatment of multiple sclerosis. However, due to the problems of poor stability in gastric juice and low bioavailability of cannabidiol administered through the gastrointestinal system, it can only be developed as an oral spray, which can be absorbed and administered through the oral mucosa. At the same time, due to the solubility of cannabidiol in the water system, the company had to choose organic solvents such as absolute ethanol and propylene glycol as the means of formulation molding.

However, according to the information disclosed by Sativex in the Summary of Product Characteristics, despite the choice of oral mucosal administration and the choice of an organic solvent system, Sativex still has the following problems:

1) Pharmacokinetic behavior is greatly affected by eating or not. Compared with fasting and eating conditions, the _Cmax and AUC of cannabidiol are different by 3.3 times and 5.1 times, respectively;

2) In different subjects, it showed a high difference in pharmacokinetic characteristics, with a large coefficient of variation. After a single dose, the CV% of _Cmax of cannabidiol was 64.1%, and the CV% of AUC was 72.5. %, the CV% of _Cmax of cannabidiol was 75.7% and the CV% of AUC was 46.6% after multiple consecutive administrations;

3) In the same subject, it also showed a high degree of variability in pharmacokinetic behavior.

The existence of these problems is related to the properties of cannabidiol itself, and, of course, to the particular route of administration and formulation technology chosen by Sativex. Taken together, these huge uncertainties have brought significant differences in clinical efficacy, and at the same time, inevitably brought certain safety hazards.

In patent US20160143972A1, Russell Hobart Stebbins disclosed a method for preparing solid cannabidiol, claiming that this form of cannabidiol can be dissolved in an aqueous system. Although the above-mentioned prior art has improved the problems of poor solubility and low bioavailability of cannabidiol in water to a certain extent, the effect is unsatisfactory.

In addition, it should be further pointed out that in addition to poor water solubility, the safety, compliance, economy, and stability of cannabidiol are also unavoidable:

1) The bioavailability of cannabidiol is low, and it needs to be administered in large doses to achieve the goal of effectiveness. However, the use of large doses will inevitably bring compliance problems and increase the economic burden of patients. At the same time, large doses of drugs will also aggravate the liver. Kidney burden, leading to unpredictable liver and kidney disease;

2) Cannabidiol has high fat solubility (K oil-water = 6-7), and the apparent volume of distribution is about 32L/kg, which can be rapidly distributed to the brain, adipose tissue and other organs. The plasma protein binding rate of cannabidiol is High, and about 10% can be combined with red blood cells, long-term use is likely to accumulate in patients, especially for obese patients;

3) In Identification of Psychoactive Degradants of Cannabidiol in Simulated Gastric and Physiological Fluid, John Merrick, Brian Lane, etc. disclosed the situation of cannabidiol in simulated artificial gastric fluid, and pointed out that cannabidiol was degraded in 60 minutes under the experimental conditions. 85% and 98% are degraded in 120 minutes, which is very unfavorable. What is even more terrible is that the substance converted into cannabidiol after the degradation of artificial gastric juice is the psychoactive substance tetrahydrocannabinol, which is It is very dangerous, and it will inevitably bring huge security risks in practical applications.

To sum up, finding suitable technical means to solve the problems existing in the application of cannabidiol has become an urgent problem to be solved in this field.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the present application provides a cannabidiol composition comprising cannabidiol and a surfactant.

In certain preferred embodiments, the surfactant is selected from nonionic surfactants.

In certain preferred embodiments, the surfactant is selected from the group consisting of polypropylene glycol and/or polyoxyethylene-based adducts and polyol-type ester or amide adducts.

Polypropylene glycol as used herein refers to having A structured polymer with a molecular weight of 1000-10000 Da, preferably 5000-10000 Da, more preferably 6000-9000 Da.

Polyoxyethylene as used herein refers to having Structured polymers with a molecular weight of 500-2000 Da, preferably 1000-2000 Da.

In certain preferred embodiments, the polyhydric alcohol-type ester adduct refers to the esters formed by organic substances containing multiple hydroxyl groups in the molecules such as ethylene glycol, sorbitol, and sucrose, and higher fatty acids, such as sorbitol esters, sucrose esters, etc.

In certain preferred embodiments, the polyol-type amide adduct refers to an organic compound formed by the condensation of polyolamine (such as diethanolamine) with fatty acid, wherein the fatty acid can be coconut oil, fatty acid or lauric acid .

In certain preferred embodiments, the surfactant is a poloxamer.

In certain preferred embodiments, the poloxamer has a molecular weight of 9000 to 20000 Da, such as 9000 to 16000 Da, 9000 to 12600 Da, 9900 to 12600 Da, or 10000 to 15000 Da, preferably 9950 to 12600 Da), and a polyoxyethylene block The percentage is 40% to 80%, such as 60% to 80%, 60% to 75%, 62% to 72%, 65% to 75% or 65% to 80%.

In certain preferred embodiments, the poloxamer is P407 or a poloxamer having a molecular weight of 9950-12600 Da and a percentage of polyoxyethylene blocks of 70%.

In certain preferred embodiments, the mass ratio of cannabidiol and surfactant is 1:(1-100).

In certain preferred embodiments, the mass ratio of cannabidiol and surfactant is 1:(5-50).

In certain preferred embodiments, antioxidants are also included in the composition.

In certain preferred embodiments, the antioxidant is an organic acid or a pharmaceutically acceptable salt thereof.

In certain preferred embodiments, the antioxidant is selected from the group consisting of citric acid, tartaric acid, malic acid, succinic acid, ascorbic acid, and pharmaceutically acceptable salts thereof.

In certain preferred embodiments, the antioxidant is selected from the group consisting of citric acid, tartaric acid, malic acid, succinic acid, ascorbic acid, potassium citrate, sodium citrate, and potassium hydrogen tartrate.

In certain preferred embodiments, the antioxidant is citric acid or tartaric acid.

In certain preferred embodiments, the weight ratio of cannabidiol and antioxidant is 1:(0.01-0.2); eg, 1:(0.01-0.1).

In certain preferred embodiments, the weight ratio of cannabidiol, surfactant and antioxidant is 1:(1-100):(0.01-0.2).

In certain preferred embodiments, the weight ratio of cannabidiol, surfactant and antioxidant is 1:(5-50):(0.01-0.1).

In certain preferred embodiments, the weight ratio of cannabidiol, surfactant and antioxidant is 1:50:0.01.

In certain preferred embodiments, the composition further contains a pharmaceutically acceptable carrier or excipient.

Carriers as described herein include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, saturated vegetable matter Partial glyceride mixtures of fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, fibers Base material, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax, lanolin. The excipients refer to additives other than the main drug in the pharmaceutical preparation. It is stable in nature, has no incompatibility with the main drug, does not produce side effects, and does not affect the curative effect. It does not affect the determination of the content of the main drug. Such as binders, fillers, disintegrants, lubricants in tablets; wine, vinegar, concoction, etc. in traditional Chinese medicine pills; matrix parts in semi-solid preparations ointments and creams; preservatives in liquid preparations, Antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can all be called excipients.

In certain preferred embodiments, the composition also contains 5-20 times the weight of water as solids; preferably 10-15 times; more preferably 10 times.

The solid matter herein refers to the remaining matter after the composition or product has been sufficiently dried, that is, the sum of the matter in the composition excluding water.

In certain preferred embodiments, the composition is a nanoemulsion formulation.

In certain preferred embodiments, the nanoemulsion has a particle size of 1-200 nm.

In certain preferred embodiments, the nanoemulsion has a particle size of 1-100 nm.

In certain preferred embodiments, the composition is a tablet, granule, capsule, soft capsule or injection.

In certain preferred embodiments, the composition may be administered orally, transdermally, or by injection.

In another aspect, the present application relates to the composition in the manufacture of a medicament for the prevention and/or treatment of convulsions, anxiety/depression, inflammation, cancer, rheumatoid arthritis, multiple sclerosis, epilepsy, especially refractory epilepsy , antiviral or analgesic use.

In another aspect, the present application relates to the preparation of the composition in the preparation of foods (especially health foods) for enhancing immunity, assisting in lowering blood sugar/blood lipids, assisting in improving memory, clearing throat, improving sleep, losing weight or improving chemical liver injury the use of.

In another aspect, the present application relates to the use of the composition for treating acne or improving skin moisture/oil balance for non-therapeutic purposes.

In another aspect, the present application relates to a cosmetic product comprising the aforementioned composition.

Cosmetics mentioned in this article refers to spreading, spraying or other similar methods on any part of the human body surface, such as skin, hair, fingernails, lips and teeth, etc., to achieve cleaning, maintenance, beauty, modification and change of appearance, or correction Human body odor, chemical industrial products or fine chemical products for the purpose of maintaining good condition.

In another aspect, the present application relates to a food product (eg, a health food) comprising the aforementioned composition.

The term "health food" as used herein refers to foods with additional effects (usually health promotion or disease prevention) obtained by adding new nutrients or by increasing the content of certain inherent nutrients. In addition to meeting basic nutritional needs, the food also has the function of regulating human body functions, but it is not for the purpose of disease treatment. It may be no different from ordinary food in appearance, and for certain groups of people, it can be used as part of the daily diet.

Unless otherwise specified, the "molecular weight" mentioned herein refers specifically to the "number average molecular weight Mn".

Beneficial Effects of Invention

The application provides a cannabidiol composition, which can achieve at least one of the following technical effects:

1) The composition can significantly improve the water solubility of cannabidiol, which greatly promotes its application in many fields such as medicine, daily chemicals, food, and health care products.

2) the composition has high stability, which solves the problem that cannabidiol is unstable and easily degraded in gastric juice;

3) the composition can significantly improve the relative bioavailability of cannabidiol;

4) The pharmacokinetic behavior of cannabidiol in the body is more consistent, and the coefficient of variation of the two key pharmacokinetic indicators of _Cmax and AUC among different individuals is lower, which reduces the unpredictable risk in the process of clinical application;

5) Compared with the single administration of cannabidiol, the binding rate of cannabidiol and plasma protein in the composition is greatly reduced, which solves the problem of public reports that cannabidiol has high lipid solubility, high plasma protein binding rate, and long-term use. There is an accumulation problem.

Description of drawings

Fig. 1 is the GPC determination pattern of poloxamer P-HY prepared by the application.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

Cannabidiol: Yunnan Hansu Biotechnology Co., Ltd.

Example 1: Preparation of self-made Poloxamer P-HY and determination of performance parameters

1. The synthetic process route of poloxamer (P-HY):

(1) Condensing propylene oxide on the 1,2-propylene glycol group to obtain a polyoxypropylene intercalation segment; wherein basic 1,2-propylene glycol is used as an inducer, and the molar ratio of each substance is propylene oxide: 1,2- Propylene glycol: KOH/NaOH=(220-320): 1: (2-0.5), the reaction temperature is 90-120°C, the reaction end point (time) varies with different reaction conditions, and the reference standard is the end of the reflux state.

(2) under the catalysis of potassium hydroxide (or sodium hydroxide) and EDTA, ethylene oxide is condensed to the two ends of the gained polyoxypropylene block section of step (1), and the molar ratio of controlling starting material is ethylene oxide: Polyoxypropylene: potassium hydroxide (or sodium hydroxide): EDTA=(450-650): 1: (0.5-1): (0.05-0.1), the reaction temperature is 90-120°C, and the time is 9-20h, Finally, a poloxamer (named as P-HY) with a molecular weight range of 9954-12506 Da and a polyoxyethylene segment of 62-72% is obtained, and the final total yield is 45%-50%.

2. The specific preparation method of Poloxamer P-HY

(1) Preparation of polyoxypropylene chimeric segment

Feed reaction according to the material ratio in Table 1. First, add propylene glycol dehydrated by anhydrous sodium sulfate as an inducer, and KOH/NaOH fine powder (80-100 mesh) dried under reduced pressure in the reaction kettle in turn, fill with nitrogen to exhaust air, turn on stirring, and pour hot water (95 ~ 100 ℃) for heating, when KOH/NaOH is completely dissolved, slowly add propylene oxide, the reaction starts, when the reaction kettle heat reflux phenomenon is over, stop supplying hot water, use tap water to circulate and cool down, that is, the molecular weight is about 3500Da The crude product of yellow viscous polyoxypropylene was added to the crude product with the same amount of water, extracted with 2 times the volume of n-heptane, repeatedly washed and purified with purified water, and finally concentrated under reduced pressure to obtain a yellow oily polyoxypropylene chimeric segment.

Table 1 prepares the feed intake and output table of polyoxypropylene chimeric section

(2) Preparation of Poloxamer P-HY

Taking the polyoxypropylene chimeric segment obtained in the step (1) as a raw material, adding dry inorganic alkali fine powder and EDTA according to the charging amount in Table 2, filling with nitrogen and exhausting the air, turning on stirring, and heating in a boiling water bath, slowly Add ethylene oxide from the bottom of the reactor, continue the reaction, the reaction time is as described in Table 2, stop the reaction to obtain the crude poloxamer copolymer of pale yellow paste, dissolve with purified water, add dropwise 1 mol/L of poloxamer copolymer HCl, adjusted to pH 6.5-7.0, filtered with 300-500 mesh neutral alumina to remove macromolecular polymers, the filtrate was placed on a macroporous resin D101 column, washed with 2 times the column volume of purified water to remove salt and oligomers, Rinse three times the column volume of 15% ethanol to obtain the eluate of the target product segment. The eluate of the target product segment was concentrated under reduced pressure at 60°C to a thick paste, poured out, placed at 40°C, and vacuum-dried at -0.10Mpa for 48h to obtain a poloxamer P-HY product with a moisture content of less than 0.2%.

Table 2 prepares the feed intake and output table of Poloxamer PH-Y

3. Comparison of performance parameters between Poloxamer P-HY and various types of poloxamers on the market

(1) Determination of molecular weight of poloxamer by GPC method

Take each poloxamer sample and configure it into a 5% solution, use Waters 1515GPC gel chromatograph to measure, the chromatographic column is G5000PWXL column (10μm, 7.8*300mm), with tetrahydrofuran as mobile phase, 25 ℃, flow rate 1ml/min, Detected at 256 nm wavelength (detector model: Waters 2489).

The calibration curve obtained using polyoxypropylene with known molecular weight (molecular weight: 3890Da) as the standard is:

Log M _sam = 9.03-0.19 Vsam (R ² =0.998).

M _sam is the sample molecular weight; Vsam is the sample eluent volume.

The GPC determination spectrum data of self-made poloxamer P-HY is shown in Figure 1: The poloxamer P-HY segment is in the sixth peak, the retention time is 24.948min, it is the main substance peak, and the percentage area is 88.73% , the average molecular weight is 11432Da, the molecular weight of oligomers is mainly below 8000Da, and the larger molecular weight polymers are above 15000Da.

The molecular weight determination results of each commercially available poloxamer sample are shown in Table 3 and Table 4.

(2) Determination of poloxamer polyoxyethylene chimeric segment content:

Take each poloxamer sample, dissolve it into a 10%-20% (g/ml) solution with a solution of deuterated methanol (or heavy water) containing 1% tetramethylsilane, and take 0.5-1.0ml of the above solution and put it into NMR In the tube, add 1 drop of heavy water, shake, scan from 0* ^10-6 to 5* ^10-6 in the NMR instrument, quantify by direct comparison method, calculate the PEO value according to the following formula, and obtain the polyoxyethylene in the whole The proportion of molecular composition, the results are shown in Table 3 and Table 4:

PEO%=3300a/(33a+58)

where a=(A2/A1)-1

A1 is the integral area of the double peak at 1.15*10 ^-6 , representing the methyl group of polyoxypropylene;

A2 is the integrated area of the composite peak at (3.2-3.8)* ^10-6 , representing polyoxypropylene, polyoxyethylene _CH2O , and polyoxypropylene CHO.

(3) Calculation of hydrophilic-lipophilic balance value (HLB value):

The HLB of the surfactant is expressed by the difference in the sum of the HLBs of the respective atomic groups of the hydrophilic group and the lipophilic group.

HLB=Σ(hydrophilic group)-Σ(lipophilic group)+7

The HLB value of the oxyethylene group is 0.33, and the HLB value of the oxypropylene group is 0.15. The approximate HLB value of the poloxamer is calculated by the formula, and the results are shown in Table 3 and Table 4.

(4) pH value measurement: each poloxamer sample was prepared into a 2% (W/W) dilute solution, three parallel groups, and measured with a precision pH meter (model: METTLER TOLEDO S220), and the average value was taken. pH value, the results are shown in Table 3.

(5) Determination of the solubility of cannabidiol samples with different formulations: take each poloxamer sample to be tested and self-made poloxamer P-HY, and mix with cannabidiol in a weight ratio of 25:1 to make semi-finished products, Determine the maximum dissolved amount in 100ml of purified water at room temperature (20-25°C). Shaker was shaken to assist dissolution, and the precipitation part was not dissolved within 6-8h as the end point for judgment. The results are shown in Table 3.

Table 3 Poloxamer P-HY finished product performance comparison table

Table 4 Comparison of Poloxamer Specifications and Physical and Chemical Properties

Example 2: Preparation and performance measurement of cannabidiol composition

1. Preparation process of cannabidiol composition

The cannabidiol composition described in this application can be prepared by the following process:

a) Physical grinding: Weigh the components of the recipe, place them in a mortar of suitable size or a special grinding machine, fully grind until the components in the recipe are uniformly mixed, and then mix the uniformly mixed materials Pulverize by physical or jet pulverization or pass through a pharmacopoeia sieve;

b) Heating and melting: place each component of the recipe amount in a container of suitable size, heat it to above 65°C, so that each component of the recipe is in a molten state, then fully stir at this temperature, and then Cool to room temperature, and pulverize the homogeneous mixture using the method described in a);

c) Spray drying: Weigh the components in the recipe quantity, and disperse them in purified water at a solid content of 1 to 5%. After the dispersion is uniform, pass through a spray dryer to spray dry into powdery materials, and then use the method in a) The method carries out the pulverization of the material after spray drying;

d) dry granulation: each component of the recipe quantity is placed in a dry granulator for dry granulation, and finally the method described in a) is used to pulverize the obtained particles;

e) Wet granulation: place each component of the recipe quantity in a wet granulator, add ethanol as a wetting agent, carry out wet granulation, and then dry the obtained granules below 50°C to remove ethanol, Finally, pulverization of the obtained particles is carried out using the method described in a).

2. Screening of the types of excipients: According to the specification that the weight ratio of poloxamer and cannabidiol is 50:1, the cannabidiol composition is prepared with reference to method a), and its maximum solubility, solution state, and colloidal particle size are: Comprehensive evaluation of indicators.

The cannabidiol composition can form a thermodynamically stable nanoemulsion through self-emulsification after being dissolved in water in an amount of more than about 10 times the amount (W/W). Its maximum solubility, dissolved solution state and colloidal particle size can be measured by the following methods, and the results are shown in Table 5.

1) Determination method of maximum solubility: Due to the unique dissolution characteristics of poloxamers (for example, it is easier to dissolve at low temperature, and the formed composition has a similar effect of sustained release, etc.), the method for determining the maximum solubility of poloxamers is also different from the conventional requirements of the pharmacopoeia. At room temperature (20-25°C), 100ml of purified water, gradually add the composition in 0.05g increments (faster and then slower) and shake on a shaker to help dissolve, and the precipitation part does not dissolve overnight (6-8h) as a judgment. end.

2) Dissolving solution state: Take each group of prescription samples respectively, configure them into a 2% (w/w) solution, and observe the physical form of the dissolving solution.

3) Determination of micelle particle size: use Mastersizer 2000 laser particle size analyzer (MS2000, 0.02-2000μm, 0.2% shading) to measure wet method, the sample is prepared into a 0.1mg/ml solution, filtered with a 0.45μm filter membrane, and the initial filtrate is discarded , the subsequent filtrate is the sample to be tested.

Table 5 Screening results of types of excipients

As can be seen from the results in Table 5, the solubility of the self-made P-HY (corresponding to molecular weight 9950-12500Da, the proportion of polyoxyethylene segment 62-72%) group cannabidiol composition solubility, color and luster of the solution and the particle size of the nanoemulsion formed by self-emulsification The size (<100) was significantly better than other groups.

3. Screening of adjuvant ratios: take poloxamer P-HY and P407 as preferred objects, and follow the poloxamer: cannabidiol weight ratios of 100:1, 50:1, 10:1, 5: 1 and 1:1 prepare the cannabidiol composition according to a) method in 1, and comprehensively evaluate its maximum solubility, solution state, and colloidal particle size as indicators, and the results are shown in Table 6.

Table 6 Screening results of excipient ratio

As can be seen from the results in Table 6, when the self-made poloxamer P-HY and cannabidiol weight ratio were (5-50): 1, the obtained finished product had the best performance, and the cannabidiol solubility was up to 7.08mg/mL, And the particle size of the formed colloid bundles are all below 90 nm.

4. Screening of organic acids: The screening of excipients takes poloxamer P-HY as the research object. In the mixture of cannabidiol and poloxamer P-HY (poloxamer P-HY: CBD=25:1 (W/W)), the ratio to cannabidiol is 1/10 (W/W) The organic acids or their salts are prepared according to a) method in 1, under accelerated stability evaluation conditions (temperature 40 ℃ ± 2 ℃, relative humidity 75 ± 5%, at 0, 1, 2, 3, 6 months Sampling), with cannabidiol content (measured by HPLC external standard method) and appearance properties as indicators, the impact of organic acids was comprehensively evaluated, and the results are shown in Table 7.

Wherein, the method for measuring cannabidiol content by high performance liquid chromatography (HPLC) is as follows:

a. Chromatographic conditions and system suitability test:

Use octadecylsilane-bonded silica gel as filler (C18, 4.6×150mm, 4um chromatographic column), use acetonitrile-water (70:30) as mobile phase, detection wavelength is 210nm, flow rate is 1ml/min, column temperature is 25°C. The number of theoretical plates should not be less than 2000 according to the cannabidiol peak.

b. Preparation of reference solution:

Take an appropriate amount of the cannabidiol reference substance, accurately weigh it, and use acetonitrile to make a solution containing about 0.01 mg per 1 ml.

c. Preparation of the test solution:

Take 10mg of the sample, put it in a 1000ml volumetric flask, add acetonitrile to the mark, shake well, and take the continuous filtrate.

d. Determination method:

Precisely measure 10 μl of the reference solution and the test solution and inject them into the liquid chromatograph, and record the chromatogram. According to the external standard method, the CBD content was calculated by the peak area.

In the formula, A _sam is the peak area of CBD in the test product;

V _sam is the dilution volume of the test solution, ml;

A _std is the peak area of the reference substance CBD;

_Wstd is the weighing sample size of the reference substance, mg;

_Vstd is the dilution volume of the reference solution, ml.

Table 7 Screening results of organic acid species

It can be seen from the results in Table 7 that adding an organic acid or its salt can improve the stability of the cannabidiol composition to varying degrees, and can avoid or delay the process of degradation and discoloration. Among them, citric acid and tartaric acid are the best, which can maintain the original appearance of the finished product within 6 months of acceleration, and significantly reduce the degradation rate of cannabidiol itself, and the second best is vitamin C; organic acid salts have similar effects, but not as good as organic acids , the optimal salts are acid salts of tartaric acid (potassium hydrogen tartrate), and the second best are potassium and sodium salts of citric acid.

5. Screening of organic acid ratio: add the mixture of cannabidiol and poloxamer P-HY (poloxamer P-HY: CBD=25:1 (W/W)), respectively, and the cannabidiol group. Citric acid, tartaric acid and potassium hydrogen tartrate in fractions (W/W) of 1/1, 1/5, 1/10, 1/50, 1/100 or 1/500. A 50:1 ratio of β-cyclodextrin: cannabidiol was used as a control. Prepare the composition according to a) method in 1. Under the accelerated stability evaluation conditions (temperature 40°C±2°C, relative humidity 75±5%, sampling at 0, 1, 2, 3, and 6 months), cannabis The phenol content (measured by HPLC external standard method, the specific method is as described above) and appearance properties are used as indicators to comprehensively evaluate the ratio of organic acids in the composition.

Table 8 Screening results of organic acid ratio

From the results in Table 8, it can be seen that citric acid and tartaric acid have the same trend, and have a significant effect in the range of organic acid: cannabidiol (W/W) is 1-0.01, and the optimal ratio is organic acid: cannabidiol is (0.01 -0.1): 1, wherein when the organic acid is above 0.1, the effect is similar to 0.1; the optimal ratio of organic acid salt is potassium hydrogen tartrate: cannabidiol is (0.02-0.1): 1.

Example 3: Evaluation of Biological Activity of Cannabidiol Compositions

According to the methods disclosed by GW PHARMA LTD in patent application GB2380129A and Russell Hobart Stebbins in patent application US20160143972A1, two kinds of samples for comparative study were prepared to evaluate the technical effect of the cannabidiol composition described in this application, mainly Including stability in gastric juice, bioavailability, plasma protein binding rate and so on.

Sample to be tested:

1) select nine groups of optimized formula compositions as shown below in example 2, prepare compositions with the method described in a) in 1 and carry out follow-up experiments:

a. Prescription 1: Poloxamer P-HY: CBD: Citric Acid = 100: 1: 0.01;

b. Prescription 2: Poloxamer P-HY: CBD: Citric Acid = 50: 1: 0.01;

c. Prescription 3: Poloxamer P-HY: CBD: Citric Acid = 100: 1: 0.02;

d prescription 4: Poloxamer P-HY: CBD: citric acid = 100: 1: 0.1;

e. Prescription 5: Poloxamer P-HY: CBD: Citric Acid = 10: 1: 0.01;

f. Prescription 6: Poloxamer P-HY: CBD: Citric Acid = 5: 1: 0.01;

g. Prescription 7: Poloxamer P-HY: CBD: Citric Acid = 1: 1: 0.01;

h. Formulation 8: Poloxamer P-407: CBD: Citric Acid = 50: 1: 0.01;

i. Formulation 9: Poloxamer P-188: CBD: Citric Acid = 50: 1: 0.01.

2) GW prescription: polyoxyhydrogenated castor oil RH40: ethanol: CBD=1:2:0.1.

3) Russell's prescription: beta-glucan:CBD=15:1.

1. Investigate the stability of raw cannabidiol and different cannabidiol compositions in artificial gastric juice.

experimental method:

1) Experimental method of artificial gastric juice stability: take dilute hydrochloric acid with a concentration of 1 mol/ml, dilute with water, and adjust the pH to 1.5. Add 1g of pepsin per 100ml of liquid and mix well. Take an equal amount of 100ml of artificial gastric juice and divide it into 12 groups, add 100mg of the composition simultaneously according to each prescription, take high-purity cannabidiol micropowder (100mg, 400 mesh, purity> 99%) as a control, shake at 37 ° C constant temperature shaker , at 0, 1, 4, 12, 24h time point sampling, high performance liquid chromatography (HPLC) to determine the concentration of cannabidiol. Wherein, reference substance solution and need testing solution are prepared with reference to the following steps:

Reference substance solution: Take an appropriate amount of cannabidiol reference substance, accurately weigh it, and use acetonitrile to make a solution containing about 0.01mg per 1ml, that is.

Test solution: mix the experimental sample thoroughly, accurately measure 10ml, put it in a 100ml volumetric flask, add acetonitrile to the mark, shake well, and take the continuous filtrate. Chromatographic conditions and measurement methods are as described above.

Table 9 Stability of different cannabidiol preparations in artificial gastric juice

The experimental results show that the composition has good stability in simulated gastric juice, and no obvious degradation of cannabidiol and no production of psychoactive substance THC are found.

2. To investigate the pharmacokinetic properties of raw cannabidiol and formulations of different cannabidiol compositions in rats.

After the rats were given cannabidiol to be tested by gavage, whole blood samples were collected at different times, the plasma was separated, and the drug concentration in the plasma was determined by liquid chromatography-tandem mass spectrometry.

(1) Dosing schedule

Seventy-eight healthy SPF rats were purchased from Viton Lever Laboratory Animal Center (an experimental animal company authorized by Charles River), weighing 150-200 g, and randomly divided into 13 groups with 6 rats in each group. Each group was treated by intragastric administration, fasted for 12 h before the test, and drank water freely. 2h after the administration of unified food.

The cannabidiol raw materials and the cannabidiol compositions of each prescription were dissolved in physiological saline into a solution or suspension of 2 mg/ml (calculated as CBD content) for experimental use.

Each test group was given liquid by intragastric gavage according to the administration volume of 10ml/kg. 1) The blank control group was given normal saline by gavage; 2) The cannabidiol raw material was suspended and given by gavage directly: 3) The compositions described in prescriptions 1-9, the GW prescription and the Russell prescription were all administered by intragastric administration.

(2) Blood collection time point and sample processing:

Samples were taken at 10 time points at 10 min, 20 min, 40 min, 1.0 h, 80 min, 2.0 h, 4.0 h, 8.0 h, 12.0 h and 24 h after administration.

At the time set above, 0.3 ml of venous blood was collected from the retroocular venous plexus of the rat, placed in a heparinized test tube, centrifuged at 11,000 rpm for 5 min, and the plasma was separated and frozen in a -20°C refrigerator.

(3) Sample testing and data analysis

1) The concentration of cannabidiol in rat plasma was determined by LC/MS/MS method.

The non-compartmental model analysis of WinNonlin software was used to calculate the pharmacokinetic parameters after administration.

2) Mass spectrometry conditions:

Instrument: AB SCIEX Triple QuadTM 5500 system; ESI: positive ion; monitoring mode: SRM; cannabidiol: [M ⁺ H] ⁺ m/z 315.2/123.2; m/z 494.2/169.1.

3) UPLC condition:

Mobile phase A: 0.1% formic acid & 2mM ammonium formate water-acetonitrile solution (v:v, 95:5)

Mobile phase B: 0.1% formic acid & 2 mM ammonium formate acetonitrile-water solution (v:v, 95:5)

Table 10 UPLC conditions

4) Chromatographic conditions:

Chromatographic column: ACQUITY UPLC Protein BEH C4 column, column temperature: 60°C, flow rate: 0.7ml/min, retention time: cannabidiol: 0.96min, excellent blood sugar (internal standard): 0.87min;

5) Sample preparation: Precisely take 5 μL of blood sample, add 100 μL of internal standard, vortex, centrifuge at 1300 rpm, 4° C. for 10 min, and take 10 μL of supernatant for analysis.

(4) Detection indicators

1) Detect the concentration in the blood plasma of rats after intragastric administration of 20 mg/kg of cannabidiol;

2) Detecting the pharmacokinetic parameters (peak concentration C _max , time to peak T _max , area under the curve AUC of drug time, mixed with cannabidiol raw material) after oral administration of cannabidiol and cannabidiol composition to rats. The suspension is the relative bioavailability of the reference preparation), and the results are shown in Table 11.

(5) Research results

Compared with raw cannabidiol, cannabidiol formulations 1-9 have higher _Cmax and shorter _Tmax , and can reach effective concentrations and exert their effects more quickly after oral administration. The AUC and relative bioavailability of prescriptions 1-9 were significantly better than those of GW prescription and Russell prescription, suggesting that the cannabidiol composition has better absorption effect in vivo.

Table 11 Pharmacokinetic parameters of different cannabidiol preparations after oral administration to mice

3. Investigate the therapeutic effects of raw cannabidiol and different cannabidiol compositions on the pentylenetetrazole-induced epilepsy mouse model.

(1) Experimental method

120 male C57BL/6 mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The animals were reared under natural light 12h light/12h dark conditions with free access to water and food.

The cannabidiol raw material was dissolved in physiological saline into a 7.5mg/ml suspension for experimental use; the cannabidiol compositions of each prescription were dissolved in physiological saline into an aqueous solution or suspension of 5mg/ml (based on CBD content) for use. For experimental use; carbamazepine is dissolved in physiological saline into a 6.25 mg/ml aqueous solution or suspension for experimental use; pentylenetetrazole is dissolved in physiological saline into a 6 mg/ml aqueous solution or suspension for experimental use.

After one week of adaptive feeding, the mice were randomly divided into 12 groups according to their body weight: 1) the blank control group was given normal saline by gavage; 2) the raw cannabidiol group was given raw cannabidiol suspension (150 mg/kg, Drug volume 20ml/kg); 3) Prescription 1-9 groups were respectively given the aqueous solution of prescription 1-9 by gavage (100mg/kg, administration volume 20ml/kg); 4) The positive control group was given carbamazepine solution ( 125mg/kg, administration volume 20ml/kg). After 2 hours of intragastric administration, each group was subcutaneously injected with pentylenetetrazole 60 mg/kg (administration volume 10 ml/kg), and then began to observe and count the latency period of the first forced spasm in mice and the tonic spasm in mice within 30 minutes. total duration and frequency of seizures. The behavioral Racine grading method was used to count the most severe epilepsy grades achieved by the mice within 30 minutes, and to evaluate the effects of drugs on the occurrence and development of epilepsy. The specific Racine classification method is as follows: type 1 epilepsy, lying still; type 2 epilepsy, nodding, occasional body twitching, tail rigidity; type 3 epilepsy, single forelimb raised, hind limb spasm; type 4 epilepsy, both forelimbs raised, Standing twitching; type 5 epilepsy, standing and falling, falling to the ground with violent twitching, large jumps, and death.

(2) Experimental results:

Compared with the blank control group, the time of the first tonic-clonic spasm in the raw cannabidiol group and each cannabidiol composition prescription group except prescription 7 was significantly delayed (P<0.01, P<0.05). The total duration of tonic-clonic seizures, seizure frequency and seizure grade of the raw cannabidiol group were not significantly different from those of the blank control group. Compared with the blank control group, each cannabidiol composition group had no significant difference in these three indicators compared with the blank control group. There is improvement, among which prescriptions 1-6, 8 can significantly shorten the total duration and frequency of tonic-clonic seizures in model mice, and reduce the grade of epileptic seizures (P<0.01, P<0.05). Various data show that, compared with the raw material cannabidiol, the cannabidiol composition of the present application can more effectively reduce the severity of epilepsy and delay epileptic seizures, especially the cannabidiol composition of lower doses (poloxamer). : CBD ratios greater than 5:1 for prescriptions 1-6 and 8).

Table 12 Improvement effect of different cannabidiol preparations on pentylenetetrazole-induced epilepsy in mice

#p<0.05 compared with blank control group, ##p<0.01 compared with blank control group

4. To investigate the therapeutic effect of raw cannabidiol and different cannabidiol composition prescriptions on the depressed mice in the forced swimming experiment.

(1) Experimental method

120 Kunming mice, half male and half male, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The animals were reared under natural light 12h light/12h dark conditions with free access to water and food.

The cannabidiol raw material was dissolved in physiological saline into a 7.5mg/ml suspension for experimental use; the cannabidiol compositions of each prescription were dissolved in physiological saline into an aqueous solution or suspension of 5mg/ml (based on CBD content) for use. For experimental use; Amitriptyline is dissolved in physiological saline as a 1 mg/ml aqueous solution or suspension for experimental use.

After one week of adaptive feeding, the mice were fasted for 12-16 hours with free food and water, and were randomly divided into 12 groups according to body weight: 1) the blank control group was given normal saline by gavage; 2) the raw cannabidiol group was given raw cannabis by gavage Diphenol suspension (150mg/kg); 3) Prescription 1-9 groups were given the aqueous solution (100mg/kg) of prescription 1-9 by gavage respectively; 4) The positive control group was given amitriptyline solution (20mg/kg) by gavage. kg).

Twenty-four hours before the formal test, the mice were placed in a glass cylinder with a water depth of 10 cm (25 cm in height, 10 cm in diameter), and the water temperature was 24 ± 1 °C for 15 minutes of forced swimming training. After administration of each group, the mice were placed in a glass cylinder with a water depth of 10 cm and forced to swim for 6 minutes, and the immobility time of the mice in the last 4 minutes was observed and recorded. When the mice stopped struggling, remained motionless in the water, or only made some necessary slight movements to keep their heads floating on the water surface, the time was regarded as the swimming immobility time; among them, each cannabidiol group was administered marijuana. After 2 hours of diphenol, the mice were forced to swim for 6 minutes in the glass cylinder, and the mice in the amitriptyline group were forced to swim for 6 minutes in the glass cylinder 1 hour after administration.

(2) Experimental results

In the forced swimming experiment of mice, amitriptyline, raw cannabidiol, and each combination of cannabidiol have antidepressant effects, respectively. Compared with the raw cannabidiol group, the immobility time of the mice in each cannabidiol composition prescription group except prescription 7 was further reduced (P<0.05), indicating that the lower dose of cannabidiol composition (poloxa Mu: Formulas 1-8 and 9) with a CBD ratio greater than 5:1 have better antidepressant effects than high-dose raw cannabidiol.

Table 13 Effects of different cannabidiol preparations on immobility time of mice in forced swimming experiment

group Swimming immobile time (s) Blank control group 170.4±7.1 positive control group 123.2±6.3^# Prescription 1 group 94.8±6.7^##* Prescription 2 groups 83.9±4.1^##* Prescription 3 groups 82.5±5.4^##* Prescription 4 groups 86.1±3.9^##* Prescription 5 groups 91.3±5.8^##* Prescription 6 groups 97.8±7.5^##* Prescription 7 groups 117.2±7.9^# 8 groups of prescriptions 93.7±4.3^##* Prescription 9 groups 102.1±4.7^# Raw material cannabidiol group 111.5±7.7^#

#p<0.05 compared with the blank control group, ##p<0.01 compared with the blank control group; *p<0.05 compared with the raw cannabidiol group

5. Investigate the plasma protein binding rate of raw cannabidiol and formulations of different cannabidiol compositions

The plasma protein binding rate of the raw cannabidiol and the cannabidiol composition formulations 1-9 described in this application was evaluated by a commonly used equilibrium dialysis method.

(1) Experimental method:

1) Preparation of sample mother liquor: take raw material cannabidiol or cannabidiol composition, add methanol to prepare a total of 6 gradients of (200, 100, 50, 20, 10, 5) μg/mL, and take 200 μg/mL as a high concentration sample, 100 μg/mL was used as a medium concentration sample, and 50 μg/mL was used as a low concentration sample.

2) Blank dialysate configuration: accurately weigh 14.110g K ₂ HPO ₄ , 2.592g KH ₂ PO ₄ , 1.991g NaCl and dissolve in 950ml deionized water, adjust the pH to 7.4 with 0.1mol/L HCl, and then dilute to 1000ml.

3) Dialysis inner fluid: 200 μL of fresh rabbit plasma was taken by pipette, and 600 μL of sample (cannabidiol or cannabidiol composition mother solution) was added, which was the test solution in the dialysis membrane. The molecular weight cut-off of the dialysis membrane is 5000D (model: G-RC-18-5K), and the dialysis tank is an ampoule bottle containing 20ml of blank dialysate.

4) Specificity investigation: examine the peaks of the internal and external fluids after the plasma (or dialysate), plasma (or dialysate) + samples are dialysis balanced in the dialysis tank, and observe whether there is any interference with the determination results.

5) Standard curve preparation: Take 400 μL of each sample mother solution (5-100 μg/mL), add 200 μL of dialysate (or plasma), after mixing, add 400 μL of methanol, centrifuge to take the supernatant, and send it to HPLC for testing. Coordinates, the peak area is the ordinate, and the standard curve of the dialysate outer fluid (or inner fluid) was prepared.

6) Investigate the recovery rate of sample addition: Take 200 μL of fresh rabbit plasma with a pipette, add high, medium and low concentration samples respectively, and measure the recovery rate of cannabidiol according to the steps described in 5).

7) Determination of plasma protein binding rate: After washing the dialysis bag with deionized water, one end was ligated, and three types of drug-containing plasma (200μL+600μL) with high, medium and low concentrations were added respectively. The processing method is carried out, sampling is carried out, and it is sent to HPLC for determination.

HPLC assay conditions:

Use octadecylsilane-bonded silica gel as filler (C18, 4.6×150mm, 3um chromatographic column), use acetonitrile-water (70:30) as mobile phase, detection wavelength is 210nm, flow rate is 1ml/min, column temperature is 25°C. The number of theoretical plates should not be less than 2000 according to the CBD peak.

(2) Experimental results:

The results showed that, with the exception of prescription 7, the plasma protein binding rate of the other prescriptions was significantly lower than that of the raw material cannabidiol. It is suggested that the composition of the present application helps to solve the potential accumulation problem in cannabidiol metabolism.

Table 14 Determination results of plasma protein binding rate of different cannabidiol preparations

group Plasma protein binding % Raw material cannabidiol group 95 prescription 1 79 prescription 2 74 prescription 3 75 prescription 4 72 prescription 5 78 prescription 6 83 prescription 7 91 prescription 8 81 prescription 9 89

Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details in light of all the teachings that have been disclosed, and that these changes are all within the scope of the present invention . The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (10)

1. A cannabidiol composition comprising cannabidiol and a surfactant; Preferably, the surfactant is selected from non-ionic surfactants; for example, it is selected from the adducts of polypropylene glycol and/or polyoxyethylene as the parent and the polyol-type ester or amide adducts; preferably Loxamer; more preferably, the molecular weight of the poloxamer is 9000-20000Da, such as 9000-16000Da, 9000-12600Da, 9900-12600Da or 10000-15000Da, preferably 9950-12600Da), and the percentage of polyoxyethylene blocks is 40% to 80%, such as 60% to 80%, 60% to 75%, 62% to 72%, 65% to 75% or 65% to 80%; more preferably, the poloxamer is P407 Or a poloxamer with a molecular weight of 9950 to 12600 Da and a polyoxyethylene block percentage of 70%; Preferably, the mass ratio of the cannabidiol to the surfactant is 1:(1-100), more preferably the ratio is 1:(5-50). 2. The composition of claim 1, further comprising an antioxidant; Preferably, the antioxidant is an organic acid or a pharmaceutically acceptable salt thereof; Preferably, the antioxidant is selected from the group consisting of citric acid, tartaric acid, malic acid, succinic acid, ascorbic acid and pharmaceutically acceptable salts thereof (eg sodium or potassium salts); Preferably, the antioxidant is citric acid or tartaric acid; preferably, the weight ratio of the cannabidiol to the antioxidant is 1:(0.01-0.2); for example, 1:(0.01-0.1); Preferably, the weight ratio of the cannabidiol, surfactant and antioxidant is 1:(1-100):(0.01-0.2); preferably 1:(5-50):(0.01-0.1); preferably is 1:50:0.01. 3. The composition of claim 1 or 2, further comprising a pharmaceutically acceptable carrier or excipient. 4. The composition of any one of claims 1-3, further comprising 5-20 times the weight of water in the solids in the composition; preferably 10-15 times; more preferably 10 times; Preferably, the composition is a nanoemulsion formulation; Preferably, the particle size of the nanoemulsion is 1-200 nm, preferably 1-100 nm. 5. The composition of any one of claims 1-4, which is a tablet, granule, capsule, soft capsule or injection. 6. The composition of any one of claims 1-5 in preparation for the prevention and/or treatment of convulsions, anxiety/depression, inflammation, cancer, rheumatoid arthritis, multiple sclerosis, epilepsy, especially refractory epilepsy Use in drugs, antiviral drugs, or analgesics. 7. The composition of any one of claims 1-5 is used to prepare food (especially health food) for enhancing immunity, assisting in lowering blood sugar/blood lipids, assisting in improving memory, clearing throat, improving sleep, losing weight or improving chemical liver injury use in. 8. Use of a composition according to any one of claims 1 to 5 for treating acne or improving skin moisture/oil balance for non-therapeutic purposes. 9. A cosmetic comprising the composition of any one of claims 1-5. 10. A food (eg, a health food) comprising the composition of any one of claims 1-5.