CN113116868B - Use of tetrahydrocannabinol for the prevention and/or treatment of pulmonary hypertension - Google Patents

Abstract

The invention discloses application of tetrahydrocannabivarin in prevention and/or treatment of pulmonary hypertension, and particularly relates to application of a composition containing tetrahydrocannabivarin in prevention and/or treatment of pulmonary hypertension and complications thereof. Tetrahydrocannabinol, particularly a composition containing tetrahydrocannabinol and other cannabinoids, can remarkably reduce right ventricular systolic pressure and right ventricular hypertrophy index of a pulmonary hypertension model mouse, improve pulmonary edema and hepatic edema and improve pulmonary hypertension symptoms, particularly, the improvement effect on hepatic edema can be enhanced by combined administration, and the composition has important value for development of medicaments for treating pulmonary hypertension.

Description

Use of tetrahydrocannabivarin for the prevention and/or treatment of pulmonary hypertension

Technical Field

The invention relates to the technical field of medicines, in particular to application of Tetrahydrocannabivarin (THCV) in prevention and/or treatment of pulmonary hypertension, and particularly relates to application of a composition containing the tetrahydrocannabivarin in prevention and/or treatment of the pulmonary hypertension and complications thereof.

Background

Pulmonary Arterial Hypertension (PAH) refers to a hemodynamic and pathophysiological state in which the pulmonary arterial pressure rises above a certain threshold, and the hemodynamic diagnostic criteria are: under the resting state of sea level, the average pulmonary artery pressure detected by the right heart catheter is more than or equal to 25 mmHg. Pulmonary hypertension mainly affects the pulmonary artery and the right heart, and is manifested by right ventricular hypertrophy and right atrial dilation. The pulmonary artery trunk is dilated and the peripheral pulmonary arterioles are sparse. Hyperplasia and hypertrophy of endothelial cells and smooth muscle cells of small pulmonary arteries, fibrosis and thickening of intima of blood vessels, hypertrophy of media, stenosis of lumens, occlusion, distortion and change in a cluster shape. Pulmonary venules can also present with intimal fibroplasia and luminal obstruction. Other manifestations in patients with pulmonary hypertension include hypertrophy of the pulmonary artery adventitia and veins, increased expression of TGF- β, and increased expression of matrix proteins such as elastin, fibronectin, cytochrome C, and mucopolysaccharide. In particular to right heart failure caused by strengthening pulmonary artery vascular tension, which is a disease seriously threatening the life and health of human beings.

Pulmonary hypertension is a disease that can be treated, but no specific cure is available at present. The traditional treatment methods comprise oxygen inhalation, heart strengthening, diuresis, calcium channel blocker, anticoagulant adjuvant therapy agent and the like, and mainly play a role in relieving symptoms.

In recent years, the development and popularization of targeted therapeutic drugs (mainly including prostacyclin drugs, endothelin receptor antagonists, phosphodiesterase-5 inhibitors, newly-explored soluble guanylate cyclase agonists, 5-hydroxytryptamine transporter inhibitors, growth factor inhibitors, Rho kinase inhibitors, and the like) and treatment methods such as living lung transplantation greatly improve the prognosis of patients.

Although these drugs can alleviate the symptoms of PAH to some extent, pulmonary hypertension is still an incurable disease with high mortality, median survival time of patients receiving treatment is only 2.7 years, and pulmonary hypertension still lacks a specific cure method, so that the search for new specific therapeutic drugs is urgent.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides the application of the tetrahydrocannabinol in preparing the product for preventing and/or treating pulmonary hypertension and complications thereof.

The invention also provides a composition for preventing and/or treating pulmonary hypertension and complications thereof, which comprises more than two cannabinoids.

In particular, the above composition comprises tetrahydrocannabivarin and at least one other cannabinoid, which may be selected, for example, from: cannabidiol, cannabidivarin, cannabigerol, and the like.

In one embodiment of the present invention, the above composition comprises tetrahydrocannabivarin and cannabidiol; specifically, the mass ratio of tetrahydrocannabivarin to cannabidiol is 1:10-60 (specifically 1:10, 1:20, 1:25, 1:30, 1:40, 1:50, 1: 60), especially 1: 20-40.

In one embodiment of the present invention, the above composition comprises tetrahydrocannabivarin and cannabidiol; specifically, the mass ratio of tetrahydrocannabivarin to cannabidivarin is 1:2-10 (specifically 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 0), especially 1: 4-8.

In one embodiment of the present invention, the above composition comprises tetrahydrocannabivarin and cannabigerol; specifically, the mass ratio of tetrahydrocannabivarin to cannabigerol is 1:0.5-5 (specifically 1:0.5, 1:1, 1:2, 1:3, 1:4, 1: 5), especially 1: 2-4.

In one embodiment of the present invention, the above composition comprises tetrahydrocannabivarin, cannabidiol and cannabivarin; specifically, the mass ratio of tetrahydrocannabinol, cannabidiol and cannabidiol is 1:10-60:2-10, especially 1:20-40: 4-8.

In one embodiment of the present invention, the above composition comprises tetrahydrocannabivarin, cannabidiol and cannabigerol; specifically, the mass ratio of tetrahydrocannabivarin, cannabidiol and cannabigerol is 1:10-60:0.5-5, especially 1:20-40: 2-4.

In one embodiment of the present invention, the above composition comprises tetrahydrocannabivarin, cannabidivarin and cannabigerol; specifically, the mass ratio of tetrahydrocannabivarin, cannabidivarin and cannabigerol is 1:2-10:0.5-5, especially 1:4-8: 2-4.

In one embodiment of the present invention, the above composition comprises tetrahydrocannabivarin, cannabidiol, cannabidivarin and cannabigerol; specifically, the mass ratio of tetrahydrocannabivarin, cannabidiol, cannabivarin and cannabigerol is 1:10-60:2-10:0.5-5, especially 1:20-40:4-8: 2-4.

Specifically, the tetrahydrocannabinol and the at least one other cannabinoid may be chemically synthesized, biologically synthesized, plant extracts, or prepared by other methods.

Specifically, the tetrahydrocannabinol and the at least one other cannabinoid can be pure products of each component, and the above compositions can be obtained by mixing the above components; the tetrahydrocannabinol and at least one other cannabinoids may also be present in the same product, for example a plant extract comprising both the tetrahydrocannabinol and at least one other cannabinoids, such as an industrial cannabis whole lineage oil, for example as manufactured by the applicant, comprising: CBD 60-90% (specifically 60%, 65%, 70%, 75%, 80%, 90%), CBDV 2-12% (specifically 2%, 5%, 8%, 10%, 12%), CBG 1-5% (specifically 1%, 2%, 3%, 4%, 5%), THCV 0.5-2% (specifically 0.5%, 1%, 1.5%, 2%). The above-mentioned industrial hemp whole spectrum oils contain no THC or low to undetectable levels of THC.

Specifically, the plant extract part of the plant extract may be one or more of stem core, flower, leaf, seed and seed coat of industrial hemp, especially flower and/or leaf.

In one embodiment of the present invention, the above industrial hemp full spectrum oil is prepared by the following method:

(1) extracting industrial hemp medicinal material powder with an extraction solvent to obtain an extracting solution;

(2) filtering the extracting solution obtained in the step (1), and concentrating under reduced pressure;

(3) dissolving the concentrate obtained in the step (2) in a solvent, purifying by column chromatography, collecting eluent, and concentrating under reduced pressure to obtain a crude product of the whole-spectrum oil;

optionally, (4) adding a solvent to dissolve the crude oil of the whole spectrum obtained in the step (3), purifying by column chromatography, collecting eluent, and concentrating under reduced pressure.

Specifically, the industrial hemp medicinal material in the step (1) can be one or more of stem core, flower, leaf, seed and seed coat of the industrial hemp; in one embodiment of the present invention, the industrial cannabis medicinal material is flowers and/or leaves of industrial cannabis.

Specifically, the extraction solvent in step (1) is 60-80% (specifically, 60%, 65%, 68%, 70%, 72%, 75%, 80%) ethanol; in one embodiment of the invention, the extraction solvent is 70% ethanol.

Specifically, the material-to-liquid ratio of the industrial hemp medicine powder to the extraction solvent in the step (1) is 1:5-10 (w/v, specifically 1:5, 1:6, 1:7, 1:8, 1:9, 1: 10); in one embodiment of the invention, the feed to liquid ratio is 1: 8.

In one embodiment of the present invention, the extraction in step (1) is room temperature leaching.

Specifically, the extraction times in the step (1) are 1 to 3 times, and each time lasts for 0.5 to 2 hours; in one embodiment of the present invention, the number of extractions in step (1) is 2 for 1 hour.

Specifically, the pressure condition of the reduced pressure concentration in the step (2) is-0.08 to-0.09 Mpa.

Specifically, the temperature for concentration under reduced pressure in step (2) is 60-70 deg.C (specifically 60, 62, 64, 65, 66, 68, 70 deg.C); in one embodiment of the invention, the temperature is 65 ℃.

Specifically, the density of the concentrate in the step (2) is 1.050 to 1.100.

In one embodiment of the present invention, the solvent in step (3) is water.

In one embodiment of the present invention, the chromatography column used in step (3) is a macroporous resin column.

In one embodiment of the present invention, the diameter-height ratio of the chromatography column used in step (3) is 1: 5.

specifically, column chromatography in step (3) sequentially uses eluent 1, eluent 2 and eluent 3 for elution, wherein eluent 1 is water, eluent 2 is 40-60% (specifically, 40%, 45%, 50%, 55%, 60%) ethanol, eluent 3 is 70-90% (specifically, 70%, 75%, 80%, 85%, 90%) ethanol; in one embodiment of the invention, eluent 2 is 50% ethanol and eluent 3 is 80% ethanol.

Specifically, the eluent flow rate in column chromatography in step (3) is 5-10BV/h (specifically 5, 6, 7, 8, 9, 10 BV/h); in one embodiment of the invention, the eluent flow rate is 9 BV/h.

Specifically, the eluent collected in the column chromatography in the step (3) is obtained by eluting with an eluent 3.

Specifically, the pressure condition of the reduced pressure concentration in the step (3) is-0.08 to-0.09 MPa.

Specifically, the temperature for concentration under reduced pressure in step (3) is 60-70 deg.C (specifically 60, 62, 64, 65, 66, 68, 70 deg.C); in one embodiment of the invention, the temperature is 65 ℃.

Specifically, the solvent in step (4) is 60-80% (specifically, 60%, 65%, 68%, 70%, 72%, 75%, 80%) ethanol; in one embodiment of the invention, the solvent is 70% ethanol.

In one embodiment of the present invention, the chromatography column used in step (4) is a polymeric chromatography packing column.

In one embodiment of the present invention, the chromatography column used in step (4) has a diameter to height ratio of 1: 6.

Specifically, the eluent for column chromatography in step (4) is 60-80% (specifically 60%, 65%, 68%, 70%, 72%, 75%, 80%) ethanol; in one embodiment of the invention, the eluent is 70% ethanol.

Specifically, the flow rate of the eluent in column chromatography in the step (4) is 1-4BV/h (specifically, 1, 2, 3, 4 BV/h); in one embodiment of the invention, the eluent flow rate is 2 BV/h.

Specifically, the pressure condition of the reduced pressure concentration in the step (5) is-0.08 to-0.09 MPa.

Specifically, the temperature for concentration under reduced pressure in step (4) is 60-70 deg.C (specifically 60, 62, 64, 65, 66, 68, 70 deg.C); in one embodiment of the invention, the temperature is 65 ℃.

In the present invention, the pulmonary hypertension can be primary pulmonary hypertension or secondary pulmonary hypertension, such as arterial pulmonary hypertension (e.g., idiopathic pulmonary hypertension, hereditary pulmonary hypertension, pulmonary hypertension due to drug or poison, or persistent pulmonary hypertension in newborn), pulmonary hypertension associated with left heart disease (e.g., pulmonary hypertension due to cardiac insufficiency, pulmonary hypertension due to diastolic insufficiency, pulmonary hypertension due to valvular disease), pulmonary hypertension associated with lung disease (e.g., pulmonary hypertension due to chronic obstructive pulmonary disease, pulmonary hypertension due to emphysema, or pulmonary hypertension due to pulmonary interstitial disease), pulmonary hypertension associated with hypoxemia (e.g., pulmonary hypertension due to sleep apnea syndrome, pulmonary hypertension due to chronic altitude disease such as high-grade heart disease), or chronic thromboembolic pulmonary hypertension, especially pulmonary hypertension due to hypoxemia.

In the present invention, the complications of pulmonary hypertension may be chronic obstructive emphysema, chronic pulmonary heart disease, right heart failure, liver function damage, and the like, particularly liver function damage.

In particular, the above composition may further comprise other active ingredients for preventing and/or treating pulmonary hypertension and its complications, such as prostacyclin-based drugs (e.g., one or more of beraprost (benaprost), Treprostinil (Treprostinil), Iloprost (Iloprost) and Epoprostenol (Epoprostenol)), endothelin receptor antagonists (e.g., Bosentan (Bosentan), Ambrisentan (Ambrisentan) and Macitentan (Macitentan)), phosphodiesterase type 5 inhibitors (e.g., Sildenafil, Vardenafil (Vardenafil) and Tadalafil (Tadalafil)), soluble guanylate cyclase agonists (e.g., rioceit (ricigiuat)), 5-hydroxytryptamine transporter inhibitors (e.g., Sarpogrelate), growth factor inhibitors, and Rho kinase inhibitors.

The above compositions of the invention may be formulated in a specific dosage form for administration by any suitable route, for example oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), transdermal, intracisternal, intraperitoneal, vaginal and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) routes, preferably oral routes. It will be appreciated that the preferred route will depend upon the general condition and age of the patient to be treated, the nature of the condition to be treated and the particular active ingredient or ingredients selected.

Compositions for oral administration include solid dosage forms such as capsules, tablets, dragees, pills, lozenges, powders and granules.

Compositions for oral administration also include liquid dosage forms such as solutions, emulsions, suspensions, syrups and elixirs.

Compositions for parenteral administration include sterile aqueous and non-aqueous injectable solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions prior to use.

Other suitable dosage forms for administration include suppositories, sprays, ointments, creams, gels, inhalants, dermal patches, implants and the like.

The composition of the invention or produced according to the invention may be administered by any suitable route, for example orally in the form of tablets, capsules, powders, syrups or the like, or parenterally in the form of solutions. To prepare such compositions, methods known in the art may be employed, and any pharmaceutically acceptable carrier, diluent, excipient, or other additive commonly used in the art may be employed.

For parenteral administration, sterile aqueous solutions, aqueous propylene glycol solutions, aqueous vitamin E solutions or solutions of sesame or peanut oil of one or more of the active ingredients may be employed. If necessary, such aqueous solutions should be suitably buffered and the liquid diluent first rendered isotonic with sufficient salt or glucose. Aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The sterile aqueous medium employed is readily prepared by standard techniques known to those skilled in the art.

Injectable solutions may be prepared by dissolving one or more active ingredients and possible additives in a portion of the injectable solvent (preferably sterile water), adjusting the solution to the desired volume, sterilizing the solution and filling it in suitable ampoules or vials. Any appropriate additive commonly used in the art may be added, such as tonicity agents, preservatives, antioxidants, and the like.

Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents.

Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, agar, pectin, acacia, stearic acid, lower alkyl ethers of cellulose, corn starch, potato starch, talc, magnesium stearate, gelatin, lactose, gums and the like.

Any other adjuvants or additives conventionally used for coloring, flavoring, preserving and the like may be used, provided that they are compatible with the active ingredient or ingredients already used.

Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. Similarly, the carrier or diluent may comprise any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax.

Compositions formed by admixing one or more active ingredients of the present invention with a pharmaceutically acceptable carrier may then be conveniently administered in a variety of dosage forms suitable for the disclosed route of administration. The formulations may conveniently be presented in unit dosage form by methods known in the art of pharmacy.

The active ingredients of the present invention may be formulated as similar or dissimilar pharmaceutical compositions and unit dosage forms thereof.

If solid carriers are employed for oral administration, the formulations may be in the form of tablets, powders or pellets placed in hard gelatin capsules, or may be in the form of lozenges or pastilles.

If a liquid carrier is employed, the formulation may be in the form of a syrup, emulsion, soft gelatin capsule, or sterile injectable liquid, such as an aqueous or non-aqueous liquid suspension or solution. The pharmaceutical preparations may conveniently be presented in unit dosage form according to standard procedures for pharmaceutical formulations. The amount of active compound per unit dose may vary depending on the nature of the active compound and the intended dosage regimen. Typically, this will be in the range of 0.1mg to 5000mg per unit dose.

The invention also provides a preparation method of the composition, which can comprise a step of uniformly mixing the corresponding components in proportion or a step of extracting the plant raw materials (such as industrial hemp full-spectrum oil extracted from industrial hemp).

The invention also provides the use of a combination of tetrahydrocannabivarin and at least one other cannabinoids in a product for the prevention and/or treatment of pulmonary hypertension and its complications, the other cannabinoids being selected from: cannabidiol, cannabidivarin, cannabigerol, and the like.

Specifically, in the above application, the other cannabinoids are cannabidiol; specifically, the mass ratio of tetrahydrocannabivarin to cannabidiol is 1:10-60 (specifically 1:10, 1:20, 1:25, 1:30, 1:40, 1:50, 1: 60), especially 1: 20-40.

Specifically, in the above application, the other cannabinoids are cannabidivarin; specifically, the mass ratio of tetrahydrocannabivarin to cannabidivarin is 1:2-10 (specifically 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 0), especially 1: 4-8.

Specifically, in the above application, the other cannabinoids are cannabigerol; specifically, the mass ratio of tetrahydrocannabivarin to cannabigerol is 1:0.5-5 (specifically 1:0.5, 1:1, 1:2, 1:3, 1:4, 1: 5), especially 1: 2-4.

Specifically, in the above application, the other cannabinoids are cannabidiol and cannabidivarin; specifically, the mass ratio of tetrahydrocannabinol, cannabidiol and cannabidiol is 1:10-60:2-10, especially 1:20-40: 4-8.

Specifically, in the above application, other cannabinoids are cannabidiol and cannabigerol; specifically, the mass ratio of tetrahydrocannabivarin, cannabidiol and cannabigerol is 1:10-60:0.5-5, especially 1:20-40: 2-4.

Specifically, in the above application, other cannabinoids are cannabidiol and cannabigerol; specifically, the mass ratio of tetrahydrocannabivarin, cannabidivarin and cannabigerol is 1:2-10:0.5-5, especially 1:4-8: 2-4.

Specifically, in the above application, the other cannabinoids are cannabidiol, cannabidivarin and cannabigerol; specifically, the mass ratio of tetrahydrocannabivarin, cannabidiol, cannabidivarin and cannabigerol is 1:10-60:2-10:0.5-5, especially 1:20-40:4-8: 2-4.

The invention also provides application of the industrial hemp full-spectrum oil in preparing products for preventing and/or treating pulmonary hypertension and complications thereof.

In particular, the product may be a medicament.

The present invention also provides a method for preventing and/or treating pulmonary hypertension and its complications, comprising the step of administering to a subject in need thereof a therapeutically effective amount of tetrahydrocannabivarin or the above-described composition of the invention.

Specifically, the subject is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, e.g., monkey, chimpanzee, or baboon; in particular, the subject is a human.

Specifically, the therapeutically effective amount depends on a variety of factors including the age, weight, sex, physical health, nutritional status, time of administration, metabolic rate, severity of the condition, and the subjective judgment of the treating physician, etc.

The invention provides application of tetrahydrocannabinol in prevention and/or treatment of pulmonary hypertension, in particular application of a composition containing tetrahydrocannabinol in prevention and/or treatment of pulmonary hypertension and complications thereof, wherein the tetrahydrocannabinol, in particular the composition containing the tetrahydrocannabinol and other cannabinoids compounds, can remarkably reduce right ventricular systolic pressure and right ventricular hypertrophy indexes of pulmonary hypertension model mice, improve pulmonary edema and hepatic edema and improve pulmonary hypertension symptoms, particularly, the improvement effect on hepatic edema can be enhanced by combined administration, and the application has important value in development of medicaments for treating pulmonary hypertension.

Detailed Description

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and the following abbreviations and their corresponding materials as appearing in the present invention are:

CBDV Bisabenol

CBD cannabidiol

CBG cannabigerol

THCV tetrahydrocannabinol

In the present invention, the term "effective amount" refers to a dose that achieves treatment, prevention, alleviation and/or alleviation of the diseases or disorders described herein in a subject.

In the present invention, the term "disease and/or disorder" refers to a physical condition of the subject that is associated with the disease and/or disorder of the present invention.

In the present invention, the term "treating" includes inhibiting, delaying, alleviating, attenuating, limiting, alleviating or resolving a disease, disorder, condition or state, the onset and/or progress thereof, and/or symptoms thereof.

In the present invention, the term "prevention" includes reducing the following risks: a disease, disorder, condition or state, its occurrence and/or progression, and/or its symptoms is suffered, infected or experienced.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.

Example 1

1. Laboratory animals, reagents and instruments

(1) Laboratory animal

4-6 weeks old mice, healthy, lively, shiny hair, weight (25.15 + -2.15) g, C57BL/6 (Experimental animals center of military science medical colleges, SPF grade). Experimental animals were grouped as follows:

group 1 (normoxia wild type, as control group): under the condition of normal oxygen, 10 female mice and 10 male mice are used;

group 2 (hypoxic wild type, as control group): under the anoxic condition, 10 female mice and 10 male mice;

group 3 (THCV administration group, 0.2mg/kg body weight): the administration is carried out by intragastric administration under the anoxic condition, 10 female mice and 10 male mice;

group 4 (THCV administration group, 0.4mg/kg body weight): the administration is performed by intragastric administration under the anoxic condition, 10 female mice and 10 male mice;

group 5 (THCV administration group, 0.8mg/kg body weight): the administration is performed by intragastric administration under the anoxic condition, 10 female mice and 10 male mice;

group 6 (THCV administration group, 1.6mg/kg body weight): the administration was performed by gavage under hypoxic conditions, 10 female mice and 10 male mice.

(2) Processing method

Placing the 2 nd to 6 th groups of mice in an atmospheric hypoxia animal feeding cabin for 14 days, maintaining the oxygen concentration in the cabin at 9 to 11 percent, keeping the temperature in the cabin at 22 to 26 ℃, and constructing a pulmonary artery hypertension model; mice from groups 3-6 were gavage administered beginning on day 15, and mice from group 2 were gavage given an equal dose of saline once a day for 7 days to treat mice daily for testing on day 21. Mice in group 1 were inhaled with atmospheric air, gavaged on day 15 with an equal dose of saline, once daily for 7 days, and the other conditions were the same as those in groups 2-6.

2. Experimental methods

(1) Right Ventricular Systolic Pressure (RVSP) determination

On day 21, each group of mice was anesthetized with sodium pentobarbital (35mg/kg) by intraperitoneal injection, as referenced by Song et al (Song, y., Jones, j.e., Beppu, h., Keaney, j.f., jr., loscalezo, j., and Zhang, y.y. (2005) incorporated adaptability to pulmonary hypertension in heterologous zgoos BMPR2-mutant mice 112553- "562") right ventricular systolic blood pressure was measured using a catheter fitted to a physiological apparatus. The tail end of the catheter is connected with a signal acquisition and processing system of a multi-channel physiological recorder, and the position of the tip of the catheter is judged according to the blood pressure value displayed by a monitor and the wave-shaped shift change of the pressure curve. After the catheter entered the right ventricle, the RVSP was determined and recorded.

(2) Organ index measurement

After anesthetizing each group of mice, the thoracic cavity was opened, the heart, lung and liver were removed, each organ was washed with physiological saline, excess fluid on the organ was blotted with filter paper, the weight of lung and liver was weighed, and the organ index was calculated as compared with the weight of mouse. The heart is used to detect the right ventricular hypertrophy index.

(3) Right ventricular hypertrophy index (RVH) determination

All blood vessels and ventricles of the picked heart are stripped, the right ventricle is cut off, the weight of the right ventricle and the weight of the left ventricle plus the diaphragm are respectively weighed, and the weight of the right ventricle is divided by the combined weight of the left ventricle and the diaphragm (RV/(LV + S)).

(4) HE staining

Sample preparation: paraffin sections of mouse lung tissue.

The purpose is as follows: if the lung tissue is pathological, the vessel wall is thickened.

The cut paraffin sections were placed in an oven at 55 ℃ for 10 min.

Firstly, paraffin sections are dewaxed and descend to 70% ethanol;

xylene I: 15min

Xylene II: 7min

1:1 xylene ethanol solution: 5min

Ethanol of each grade: each for 5min

② dyeing by hematoxylin solution for 10-15 min;

flushing with tap water for 2 min;

fourthly, 0.5 percent hydrochloric acid alcohol solution is differentiated for 8 s;

returning the running water to blue for 10 min;

sixthly, 70 percent alcohol → 80 percent alcohol is added for 2min respectively;

seventhly, 50-70s of 0.5 percent eosin alcohol dye liquor;

color separation is carried out on 90 percent ethanol and 95 percent ethanol for 3min respectively;

ninthly, dehydrating the anhydrous ethanol I, II for 3 min;

absolute ethanol in r: xylene (1: 1) for 3 min;

⑪ xylene I3 min;

⑫ xylene II 3 min;

sealing neutral gum into a sheet;

after staining was complete, the nuclei appeared bluish purple and the cytoplasm appeared pink.

(5) Elastic fiber dyeing (Elastic dyeing)

Sample preparation: paraffin sections of mouse lung tissue.

Preparing a dye solution: victoria blue B1 g.

1g of new fuchsin and 1g of crystal violet.

Dissolving in 200ml hot water, sequentially adding resorcinol 4g, dextrin 4g, and 30% ferric chloride 50ml (prepared at present), decocting for 5min, filtering, dissolving precipitate and filter paper with 200ml 95% ethanol, boiling for 15min-20min, filtering (water bath), supplementing 95% ethanol to 200ml, and adding 2ml concentrated hydrochloric acid. Sealing and storing in dark.

The dyeing method comprises the following steps:

xylene I: 10 min; xylene II: 10 min; 100% ethanol: 5 min; 90% ethanol: 5 min; tap water: 5 min; 0.5% potassium permanganate for 5 min; washing with tap water for 2-3 min; 1% oxalic acid solution for 2-3min (bleaching); washing with tap water for 2-3 min; 95% ethanol for 2-3 min; dyeing for 2h by using an Elastic dyeing solution; washing away the dye liquor with 95% ethanol; washing with tap water for 2-3 min; staining with Van Gieson staining solution for 1 min; and (3) quick dehydration: 80% ethanol for 1min, 90% ethanol for 1min, anhydrous ethanol I for 5min, anhydrous ethanol II for 5min, xylene I for 5min, and xylene II for 5 min.

(6) Statistical processing

The measured data are expressed by mean ± sd, and are statistically processed by SPSS 22.0, and the statistical test is based on two-tailed T test.

3. Results of the experiment

(1) Right ventricular systolic pressure of PAH mouse

The results of the mean right ventricular systolic pressure of the mice in each group are shown in table 1.

TABLE 1 Right ventricular systolic blood pressure results for each group of mice

The results in table 1 show that, compared with the normoxic control group, the average right ventricular systolic pressure of the hypoxia model control group is obviously increased after the hypoxia lasts for 21 days, and the right ventricular systolic pressure of the mice of each experimental group is reduced after the THCV is administered.

(2) Right ventricular hypertrophy index in PAH mice

The results of the mean right ventricular hypertrophy index for each group of mice are shown in table 2.

TABLE 2 Right ventricular hypertrophy index results for each group of mice

As can be seen from the results in table 2, compared with the normoxic control group, the right ventricular hypertrophy index of the mice in the hypoxic model control group is obviously increased, and after the administration of THCV, the right ventricular hypertrophy index of the mice in each experimental group is reduced, but not reduced to a normal level (similar to the normoxic control group), and the difference between the administration groups is not significant.

(3) Organ index of PAH mice

The results of the mean organ index of each group of mice are shown in table 3.

TABLE 3 organ index results for each group of mice

The results in table 2 show that, compared with the normoxic control group, the lung and liver indexes of the mice in the hypoxia model control group are both significantly increased, the mice have pulmonary edema and hepatic edema, after the THCV is administered, the lung and liver indexes of the mice in each experimental group are reduced, the pulmonary edema and the hepatic edema are improved to a certain extent, and the improvement effect of the THCV on the pulmonary edema is more obvious.

(4) Pathological changes in pulmonary arteriole

The staining section of the lung tissue of the mouse shows that the wall of the mouse in the hypoxia model group is obviously thickened and reconstructed, the pathological reconstruction after the administration is improved, and the ratio of the wall thickness of the middle layer of the artery to the cross-sectional area of the middle layer of the wall of the middle layer of the artery is reduced.

Example 2

Experimental mice were taken and grouped as follows with reference to relevant parameters of example 1:

group 1 (combination administration group THCV 0.4mg/kg body weight + CBD 10mg/kg body weight): the administration is performed by intragastric administration under the anoxic condition, 10 female mice and 10 male mice;

group 2 (combination group THCV 0.4mg/kg body weight + CBDV 2mg/kg body weight): the administration is carried out by intragastric administration under the anoxic condition, 10 female mice and 10 male mice;

group 3 (combined administration group THCV 0.4mg/kg body weight + CBG 0.5mg/kg body weight): the administration was performed by gavage under hypoxic conditions, 10 female mice and 10 male mice.

Group 4 (combination administration group THCV 0.4mg/kg body weight + CBD 10mg/kg body weight + CBDV 2mg/kg body weight): the administration is performed by intragastric administration under the anoxic condition, 10 female mice and 10 male mice;

group 5 (combined administration group THCV 0.4mg/kg body weight + CBD 10mg/kg body weight + CBG 0.5mg/kg body weight): the administration is carried out by intragastric administration under the anoxic condition, 10 female mice and 10 male mice;

group 6 (combination administration group THCV 0.4mg/kg body weight + CBDV 2mg/kg body weight + CBG 0.5mg/kg body weight): the administration was performed by gavage under hypoxic conditions, 10 female mice and 10 male mice.

With reference to the method steps of example 1, the right ventricular systolic pressure, right ventricular hypertrophy index, liver and lung index of the mouse were measured. The results are shown in tables 4 to 6, respectively.

TABLE 4 Right ventricular systolic pressure results for each group of mice

TABLE 5 Right ventricular hypertrophy index results for each group of mice

TABLE 6 results of organ index of mice in each group

As can be seen from the results in tables 4 to 6, the above-mentioned combination regimen showed a better improvement effect in group 4 with respect to right ventricular systolic pressure and right ventricular hypertrophy index in mice, and the differences between groups 1, 2 and 5 and between groups 3 and 6 were small compared with the single administration of THCV in example 1, while the combination administration enhanced the improvement effect on liver edema with respect to visceral index.

Example 3

Crushing industrial hemp leaves, sieving with a No. 1 sieve, baking at 100 ℃ for 200min, taking a certain amount of baked hemp leaf medicinal materials, adding 70% ethanol according to the material-liquid ratio of 1:8 (w/v), stirring and extracting at room temperature for two times, each time for 1 hour, filtering, combining extracting solutions, centrifugally filtering, concentrating centrifugal filtrate under reduced pressure (65 ℃, minus 0.08 to minus 0.09 Mpa) until no alcohol smell exists and the density is 1.070 (60 ℃), supplementing purified water until the weight of the solution is 8 times of the weight of the medicinal materials, and uniformly stirring to obtain a sample loading solution.

Measuring a sample loading solution, purifying by a treated macroporous resin column (the diameter-height ratio is 1: 5) at the flow rate of 4BV/h, standing for 60min after sample loading, eluting by purified water, 50% ethanol and 80% ethanol in sequence at the flow rate of 9BV/h, determining the end point of water elution by a molish reaction, eluting by 3BV by 50% ethanol and 5BV by 80% ethanol, collecting 80% ethanol eluent, and concentrating under reduced pressure (65 ℃ and-0.08 to-0.09 Mpa) until the water content is below 5% to obtain the crude oil of the whole spectrum.

Taking the crude oil of the whole spectrum system, adding 70% ethanol, stirring and dissolving, filtering, purifying the obtained filtrate by a treated polymeric chromatographic packed column (the diameter-height ratio is 1: 6) at the flow rate of 2BV/h, eluting by 70% ethanol for 5BV at the flow rate of 2BV/h after sample loading, and concentrating the 70% ethanol eluent under reduced pressure (65 ℃, minus 0.08-minus 0.09 Mpa) to the water content of less than 5% to obtain the oil of the whole spectrum system.

The obtained oil of whole lineage has total cannabinoid content of 78.41%, wherein CBDV 10.52%, CBG 1.57%, CBD 64.80%, THCV 1.52%, and THC was not detected.

Example 4

Example 3 the industrial hemp full lineage oil obtained in example 3, which contains THCV, CBD, CBDV and CBG, was administered to pulmonary hypertension model mice to evaluate the effect of the experiment.

Referring to the method steps of example 1, 10 mice were selected, molded, and gavaged with the industrial hemp full-spectrum oil obtained in example 3 at a dosage of 60mg/kg body weight. With reference to the method steps of example 1, the right ventricular systolic pressure, right ventricular hypertrophy index, lung and liver index of the mouse were measured. Wherein the average right ventricular systolic pressure and the average right ventricular hypertrophy index are respectively 17.28 + -1.24 mmHg and 27.26 + -1.31%, and the average values of the lung index and the liver index are respectively 0.73 + -0.59% and 7.54 + -0.36. Although the application of industrial hemp full-lineage oil fails to achieve the same or better effect of the group combined with the pure compound in the group 4 of the example 2, the right ventricular systolic pressure and the right ventricular hypertrophy index of the pulmonary hypertension model mice can be obviously reduced, pulmonary edema and hepatic edema are improved, and the application has important value in the development of medicaments for treating pulmonary hypertension.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.

The listing herein of method steps in a certain order does not constitute any limitation on the order of the method steps.

Claims (7)

1. Use of a composition for the prevention and/or treatment of pulmonary hypertension and its complications, said composition being:

tetrahydrocannabinol and cannabidiol, wherein the mass ratio of the tetrahydrocannabinol to the cannabidiol is 1: 2-10;

tetrahydrocannabinol and cannabigerol, wherein the mass ratio of the tetrahydrocannabinol to the cannabigerol is 1: 0.5-5;

tetrahydrocannabinol, cannabidiol and cannabidiol, wherein the mass ratio of the tetrahydrocannabinol, the cannabidiol and the cannabidiol is 1:10-60: 2-10;

tetrahydrocannabinol, cannabidiol and cannabigerol, wherein the mass ratio of the tetrahydrocannabinol, the cannabidiol and the cannabigerol is 1:10-60: 0.5-5;

tetrahydrocannabinol, cannabidiol and cannabigerol, wherein the mass ratio of the tetrahydrocannabinol to the cannabigerol is 1:2-10: 0.5-5;

tetrahydrocannabinol, cannabidiol and cannabigerol, wherein the mass ratio of the tetrahydrocannabidiol, the cannabidiol, the cannabigerol and the cannabigerol is 1:10-60:2-10: 0.5-5.

2. The use of claim 1, wherein the tetrahydrocannabivarin, the cannabidiol, the cannabidivarin, and the cannabigerol are chemically synthesized products, biologically synthesized products, or plant extracts.

3. The use as claimed in claim 2, wherein the plant extract has a plant extract part selected from the group consisting of: one or more of a stem core, a flower, a leaf, a seed, and a shell of a seed of industrial hemp.

4. The use of claim 2, wherein the plant extract is an industrial hemp full spectrum oil comprising: 60-90% of CBD, 2-12% of CBDV, 1-5% of CBG and 0.5-2% of THCV.

5. The application of industrial hemp full-spectrum oil in preparing products for preventing and/or treating pulmonary hypertension and complications thereof comprises the following components in percentage by weight: 60-90% of CBD, 2-12% of CBDV, 1-5% of CBG and 0.5-2% of THCV.

6. The use of any one of claims 1-5, wherein the pulmonary hypertension is pulmonary hypertension due to hypoxemia.

7. The use of any one of claims 1 to 5, wherein the pulmonary hypertension complication is impaired liver function.