CN115006404A

CN115006404A - Pharmaceutical composition with optic nerve protection effect and application thereof

Info

Publication number: CN115006404A
Application number: CN202210947006.4A
Authority: CN
Inventors: 王尧龙; 袁华; 张黎; 朱吉; 牟婕; 林燕
Original assignee: Kunming Pharmaceutical Group Biotechnology Lufeng County Co ltd; KPC Pharmaceuticals Inc
Current assignee: Kunming Pharmaceutical Group Biotechnology Lufeng County Co ltd; KPC Pharmaceuticals Inc
Priority date: 2022-08-09
Filing date: 2022-08-09
Publication date: 2022-09-06

Abstract

The invention discloses a pharmaceutical composition with an optic nerve protection effect and application thereof, wherein active ingredients of the pharmaceutical composition with the optic nerve protection effect comprise cannabidiol, tetrahydrocannabinol and WIN-55, 212-2. The external preparation for eyes using the pharmaceutical composition as an active ingredient is eye drops, eye gel, eye ointment, external wet dressing for eyes or external atomized liquid for eyes. The pharmaceutical composition provided by the invention is composed of Cannabidiol (CBD), Tetrahydrocannabinol (THC), WIN-55 and 212-2, has wide composition source and reasonable proportion of the components, can play a synergistic effect, can reduce the intraocular pressure of a model mouse with damaged optic nerves to a normal level, simultaneously reduce the concentration of vitreous glutamic acid of the model mouse, can influence the expression of relevant optic nerve cell genes of the mouse on a gene level, and has the obvious effects of reducing the intraocular pressure and protecting the optic nerves.

Description

Pharmaceutical composition with optic nerve protection effect and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a pharmaceutical composition with an optic nerve protection effect and application thereof.

Background

Retinal neuropathies are a group of diseases involving optic nerve atrophy and retinal ganglion cell depletion that can be classified by etiology into various diseases including traumatic optic neuropathy, ischemic optic neuropathy, glaucomatous optic neuropathy, inflammatory optic neuropathy, toxic/trophic optic neuropathy, and hereditary optic neuropathy. Apoptosis of Retinal Ganglion Cells (RGCs) and formation of high intraocular pressure are common characteristics of the onset of these optic nerve diseases, generally speaking, if the intraocular pressure is reduced by 30-40%, the risk of developing glaucomatous optic neuropathy is reduced by more than 65%, and timolol and betaxolol are clinically used to control intraocular pressure, but the effect response is not clear. In recent years, with considerable progress in the research of the apoptosis pathway of RGCs after optic nerve injury, not only the neurobiological understanding of RGCs is improved, but also the exploration of novel neuroprotective therapies is stimulated, and the optic nerve protection strategy of protecting and rescuing the injured RGCs, reducing the intraocular pressure and preventing the injured RGCs from cytotoxic stimulation is a key objective in the clinical treatment of optic nerve diseases.

The ideal medicine for treating retinal neurodegenerative diseases has the following conditions: (1) has exact curative effect on reducing intraocular pressure; (2) can continuously generate the effect of reducing intraocular pressure; (3) can improve the blood circulation of ocular papilla without adverse effect; (4) can produce superposition effect with other medicine compositions; (5) no obvious side effect on the whole body and the local part; (6) the price is appropriate and the use is convenient. Cannabis, a drug with potential medical value, has started various researches on cannabinoids in the treatment of retinal neurodegenerative diseases after Helper and Frank discovered that cannabinoids can reduce intraocular pressure by 25% to 30% in 1971, and the treatment of ocular hypertension is mostly limited to adrenergic agonists, and such drugs can reduce intraocular pressure but have short duration of action. Cannabinoid compounds can regulate ocular hypotension by activating the CB receptor system and have effects lasting for more than 2 hours, and CB1 cannabinoid receptors have been found throughout the eye, especially in the ciliary body and iris, indicating the physiological role played by the endocannabinoid system in neurotransmitter regulation, ocular tension regulation, classical cannabinoid levo- Δ ⁹ Tetrahydrocannabinol [ 9-tetra-hydrocanabinol, THC ], can inhibit N-methyl-D-aspartic acid (NMDA) -induced Ca ^2＋ The composition can be taken to effectively eliminate peroxy and inhibit the synthesis of tumor necrosis factor alpha, thereby playing a role in neuroprotection. Aminoalkyl indoles WIN-55,212-2, can cause intracellular Ca2 through CB1 receptor ^＋ And further has a modulating effect on the synapse, inner nuclear layer, ganglion cell layer and photoreceptors of the retina, Cannabidiol (CBD) may also block NMDA-induced retinal neurotoxicity. The person who uses the pen carries out the dose proportioning test on the cannabinoidThe three cannabinoids achieve synergistic effect through dosage adjustment, generate curative effect of 1+1+1 > 3, remarkably achieve the effect of reducing intraocular pressure, remarkably reduce the concentration of mouse vitreous glutamic acid, remarkably reduce the damage and apoptosis of mouse RGCs, and remarkably generate the effect of protecting optic nerve by regulating the nerve repair mechanism mediated by the affinity of NGF/Ntrk 1/NGFR receptors at the gene level.

At present, there is no research on reduction of vitronectin concentration, reduction of damage and apoptosis of RGCs and optic nerve protection using a cannabinoid efficacy composition, and no drug of cannabinoid formulation for external use for ocular use having an optic nerve protection efficacy is on the market. In view of this, the invention aims to provide a pharmaceutical composition with reasonable formula, wide and safe sources and good neuroprotective effect.

Disclosure of Invention

The first purpose of the invention is to provide a pharmaceutical composition with optic nerve protection function, and the second purpose of the invention is to provide the application of the pharmaceutical composition.

The first object of the present invention is achieved in that the active ingredients of the pharmaceutical composition having an optic nerve protection effect consist of cannabidiol, tetrahydrocannabinol and WIN-55,212-2.

The second object of the present invention is achieved by the use for producing an ophthalmic external preparation for protecting optic nerve.

The ophthalmic preparation is eye drops, ophthalmic gel, eye ointment, wet dressing for external use of eyes or atomized liquid for external use of eyes.

The invention takes an optic nerve damaged mouse as an animal model, a blank group, a control group and an experimental group are arranged, the experimental group adopts the pharmaceutical composition, the blank group and the control group of the invention are used for detecting relevant indexes of the optic nerve of the mouse, and the influence of the blank group, the control group and the experimental group on the intraocular pressure, the vitreous glutamic acid concentration and the expression of relevant genes of the optic nerve of the mouse is observed, so that the pharmaceutical composition can obviously reduce the intraocular pressure and the vitreous glutamic acid concentration of the mouse and reduce the damage and the apoptosis of RGCs of the mouse. Meanwhile, the test is carried out at the gene level, and the protective function of the optic nerve of the mice in an experimental group is found to be possibly related to the promotion of the NGF gene expression in the retina tissue. The composition remarkably generates the effect of protecting optic nerves by regulating the nerve repair mechanism mediated by the affinity of NGF/Ntrk 1/NGFR receptors.

The invention has the beneficial effects that:

the pharmaceutical composition comprises Cannabidiol (CBD), Tetrahydrocannabinol (THC), WIN-55 and 212-2, has wide composition sources, is reasonable in proportion of the components, can play a synergistic effect, can reduce the intraocular pressure of a mouse with an optic nerve injury model to a normal level, simultaneously reduce the concentration of vitreous glutamic acid of the mouse with the model, can influence the expression of relevant optic nerve cell genes of the mouse on a gene level, and has the effects of reducing the intraocular pressure and protecting the optic nerve.

Drawings

FIG. 1 is a graph showing the effect of intraocular pressure in mice with damaged optic nerves on the blank group, the control group and Experimental example 1;

FIG. 2 is a graph showing the effect of glutamate concentration in vitreous body of mice model with damaged optic nerve in blank group, control group and Experimental example 1;

FIG. 3 is a graph of the effect of blanks, controls and Experimental example 1 on the number of RGCs;

FIG. 4 is a graph showing the effect of blank, control and Experimental example 1 on optic nerve-related apoptosis gene expression in mice;

FIGS. 5 (A) to (F) are the effects of drugs on intraocular pressure in mouse models with damaged optic nerves in blank group, control group and Experimental example 1 and Experimental examples 2 to 7, respectively;

FIG. 6 is a graph showing the effect of intraocular pressure in a mouse model with damaged optic nerve of blank group, Experimental example 1 and comparative example 2; (B) the effect on intraocular pressure in a mouse model with damaged optic nerve was found in the blank group, Experimental example 1 and comparative example 1.

Detailed Description

The invention is described in further detail below with reference to the drawings and examples, but the invention is not limited in any way and any variations or modifications based on the teachings of the invention are within the scope of the invention.

The invention relates to a pharmaceutical composition with an optic nerve protection effect, which comprises active ingredients of cannabidiol, tetrahydrocannabinol and WIN-55, 212-2.

Preferably, the mass ratio of the cannabidiol to the tetrahydrocannabinol to the WIN-55,212-2 is 5-3: 3-1: 1.

further preferably, the mass ratio of the cannabidiol to the tetrahydrocannabinol to the WIN-55,212-2 is in the range of 5-4: 2-1: 1.

still further preferably, the mass ratio of the cannabidiol to the tetrahydrocannabinol to WIN-55,212-2 is in the range of 5:1: 1.

the invention also provides application of the pharmaceutical composition with the effect of protecting the optic nerve in preparing an external preparation for eyes for protecting the optic nerve.

The external preparation for eyes is eye drops, eye ointment, eye gel, external wet dressing for eyes or external atomized liquid for eyes.

The external preparation for the eyes comprises 7-9 wt% of the pharmaceutical composition and the balance of pharmaceutically acceptable carriers.

The pharmaceutically acceptable carrier is Tocrisolve ^TM 100 solution, eye ointment matrix, thickening agent, acid-base regulator, isotonic regulator, electrolyte agent and preservative, wherein the dosage of the pharmaceutically acceptable carrier is as follows according to the weight percentage: 80-95% of an eye ointment matrix, 0.1-10.0% of a thickening agent, 0.01-5.0% of an acid-base regulator, 7.0-9.0% of a pH value, 0-8.0% of an isotonic regulator, 0.7-1.5% of an osmotic pressure or an osmotic pressure ratio of 280-320 mmol/L, 0.001-0.5% of an electrolyte agent and 0-0.5% of a preservative.

The thickening agent is selected from one or more of carbomer, chondroitin sulfate, methylcellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, polyacrylamide, glycerol, ethyl cellulose, hydroxypropyl methylcellulose, hydrogenated hard castor oil, polyallyl alcohol, poloxamer, sodium carboxymethyl cellulose, dextran, propylene glycol, tween, polyvinylpyrrolidone, chitosan, sodium hyaluronate, cross-linked polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol and cross-linked polyacrylamide;

the acid-base regulator is selected from one or more of boric acid, borax, glacial acetic acid, triethanolamine, citric acid, sodium citrate, hydrochloric acid, acetic acid, sodium acetate, tartaric acid, sodium tartrate, sodium hydroxide, sodium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate and phosphoric acid;

the isotonic regulator is one or more selected from glycerol, propylene glycol, glycerol, sodium chloride, glucose, mannitol and sorbitol;

the electrolyte agent is selected from one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, zinc chloride and glycine;

the antiseptic is selected from one or more of methyl hydroxybenzoate, ethylparaben, propyl hydroxybenzoate, chlorobutanol, thimerosal, mercury cyanamide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

The eye external preparation also comprises one or more pharmaceutically active substances of purslane, water lily, bergamot, dog rose and borneol.

The preparation method of the external preparation for eyes comprises the following steps: weighing effective dose of the pharmaceutical composition and Tocrisolve according to the proportion of the external preparation for eyes ^TM 100 solution, eye ointment matrix, thickening agent, acid-base regulator, isotonic regulator, electrolyte agent and preservative, and can be prepared into eye drops, eye ointment, eye gel, wet dressing for external use and atomized liquid for external use according to modern pharmaceutical technology.

The dosage and the using method of the eye drops when acting on human bodies are as follows:

generally, when dropping the medicine, the subject is required to raise his head, pull the lower eyelid with the index finger of the left hand, drop the eye drops into the conjunctival sac with the right hand (the average dosage of each drop is 0.03 ml), close the eye for about 5-10 minutes after dropping, and open the eye after the medicine is fully absorbed. In order to promote the absorption of the liquid medicine in the eyes and prevent the liquid medicine from overflowing out of the eyes, the shortest interval for repeatedly dripping the liquid medicine is five minutes, and the liquid medicine is dripped 2 to 3 times a day.

Example 1

A pharmaceutical composition with an optic nerve protection effect comprises active ingredients of cannabidiol, tetrahydrocannabinol and WIN-55,212-2, wherein the weight ratio of the cannabidiol to the tetrahydrocannabinol to the WIN-55,212-2 is 5:1: 1.

example 2

A pharmaceutical composition with an optic nerve protection effect comprises active ingredients of cannabidiol, tetrahydrocannabinol and WIN-55,212-2, wherein the weight ratio of the cannabidiol to the tetrahydrocannabinol to the WIN-55,212-2 is 4: 2: 1.

example 3

A pharmaceutical composition with an optic nerve protection effect comprises active ingredients of cannabidiol, tetrahydrocannabinol and WIN-55,212-2, wherein the weight ratio of cannabidiol, tetrahydrocannabinol and WIN-55,212-2 is 3: 3: 1.

example 4

A pharmaceutical composition with an optic nerve protection effect comprises active ingredients of cannabidiol and WIN-55,212-2, wherein the mass ratio of the cannabidiol to the WIN-55,212-2 is 2: 1.

the preparation method is as in example 1.

Example 5

A pharmaceutical composition with an optic nerve protection effect comprises active ingredients of cannabidiol and WIN-55,212-2, wherein the weight ratio of the cannabidiol to the WIN-55,212-2 is 3: 1.

example 6

A pharmaceutical composition with optic nerve protection effect comprises cannabidiol as an active ingredient.

Example 7

A pharmaceutical composition with optic nerve protection effect comprises WIN-55 and 212-2 as active ingredients.

Example 8

A pharmaceutical composition with optic nerve protection effect comprises tetrahydrocannabinol as an active ingredient.

Example 9

An eye drop preparation method comprises the following steps: dissolving 8.75mg of the pharmaceutical composition described in example 1 in 50ml of Tocri according to the mixture ratiosolve ^TM 100 parts of solution, adding 0.6% carbomer, 0.15% sodium chloride, and 0.1% methyl hydroxybenzoate, stirring, heating for dissolving completely, and adding Tocrisolve ^TM 100-100 ml, regulating osmotic pressure to 300mmol/L with isoosmotic adjusting agent, regulating pH to 7.5 with acid-base regulator, filtering with 200 mesh sieve cloth to obtain filtrate, filtering with 0.22 μm microporous membrane for sterilization, aseptically packaging in eye drop bottle, and sealing to obtain cannabinoid eye drop.

Example 10

The preparation method of the eye ointment comprises the following steps: melting vaseline 60%, liquid paraffin 15% and lanolin 15% at low temperature, adding benzalkonium chloride 0.1%, filtering with a heat-insulating filter funnel, sterilizing at 160 deg.C under hot pressure for 45min, cooling to obtain eye ointment matrix, dissolving 8.75mg of the pharmaceutical composition of example 1 in 100ml of Tocrusolve according to a certain ratio ^TM Adding 10% of the solution 100 by mass, stirring uniformly to obtain paste, gradually adding into the eye ointment matrix, stirring uniformly, packaging in eye ointment tubes under aseptic condition, and sealing to obtain cannabinoid eye ointment.

Example 11

An ophthalmic gel is prepared by the following steps: 8.75mg of the pharmaceutical composition described in example 1 is dissolved in 100ml of Tocrusolve according to the mixture ratio ^TM 100, uniformly stirring the solution with 0.3 percent of hydroxypropyl methylcellulose, 0.5 percent of hydrogenated hard castor oil, 0.1 percent of propylene glycol, 0.5 percent of sodium chloride, 0.1 percent of boric acid and 0.3 percent of ethylparaben respectively, and heating the solution to completely dissolve the mixture to form a cannabinoid solution; spreading 8% carbomer on Tocrisolve ^TM Swelling into gel solution 100, adding the above cannabinoid solution, stirring, filtering with 200 mesh sieve to obtain filtrate, autoclaving at 100 deg.C for 45min or filtering with 0.45 μm microporous membrane for sterilization, and packaging under aseptic condition to obtain cannabinoid eye gel product.

Example 12

A preparation method of the wet dressing towel for external use for eyes comprises the following steps: 8.75mg of the pharmaceutical composition described in example 1 is dissolved in 50ml of Tocrusolve according to the mixture ratio ^TM 100 solution, and purslane according to the mass ratioMixing the extract 1.5%, water lily extract 1.5%, bergamot fruit oil 0.3%, Rosa canina fruit oil 0.5%, and Borneolum 1%, stirring, heating to dissolve completely, and adding Tocrusolve ^TM 100 to 100ml of solution, regulating osmotic pressure to 300mmol/L by using an isotonic regulator, regulating pH to 7.5 by using an acid-base regulator, filtering by using a 200-mesh sieve cloth to obtain filtrate, and performing autoclaving or filter sterilization to obtain a cannabinoid solution; cutting medical non-woven fabrics into 6 × 12cm long strips, autoclaving at 100 deg.C for 45min, soaking the non-woven fabrics in hemp solution, sterilizing plastic bag and plastic sealing machine by ultraviolet irradiation, placing the non-woven fabrics soaked with medicinal liquid in the plastic bag, sealing 1 non-woven fabrics in 1 small plastic bag, sealing 12 small plastic bags in 1 large plastic bag, and sealing to obtain cannabinoid wet dressing towel for external use, or pre-packaging and sterilizing by cobalt-60 irradiation.

Example 13

The preparation method of the atomized liquid for the external use of the eye is realized according to the following steps: 8.75mg of the pharmaceutical composition described in example 1 is dissolved in 50ml of Tocrusolve according to the mixture ratio ^TM Mixing 100 solutions, respectively, with herba Portulacae extract 1.5%, water lily extract 1.5%, bergamot fruit oil 0.3%, Rosa canina fruit oil 0.5%, and Borneolum 1% by mass, stirring, heating to dissolve completely, adding Tocrisolve ^TM 100ml of the solution, regulating osmotic pressure to 300mmol/L by using an isotonic regulator, regulating the pH value to 7.5 by using an acid-base regulator, filtering by using 200-mesh sieve cloth to obtain filtrate, and sub-packaging in a sealed bottle under the aseptic condition by adopting autoclaving or filter sterilization to obtain the finished cannabinoid eye external use atomized liquid.

Experimental example 1

1. Cannabinoid eye drop preparation

1.1 Experimental animals

30 healthy adult KM male mice have no eye diseases and weight of 20-25 g, and are provided by Kunming medical university laboratory animal school. The mice were completely randomly divided into 10 mice in the blank group (normal group), 10 mice in the control group (ocular hypertension model group), and 10 mice in the experimental group (cannabinoid formulation group in example 1), each of which had 20 eyes.

Drugs and agents

4% chloral hydrate, 4% neutral formaldehyde (from Sigma-Aldrich); 1% chloramphenicol eye drops (available from bosch, shandong, lunford pharmaceuticals, ltd.); 1% lidocaine (purchased from Wuhan Bealca pharmaceuticals Co., Ltd.), Cannabidiol (CBD), Tetrahydrocannabinol (THC), WIN-55,212-2, glutamic acid standard (purchased from Beinan Biometrics testing Co., Ltd.); tocrisolvet 100 (available from hong Kong advanced technology industries, Ltd.); phthalic Aldehyde (OPA), boric acid, methanol, mercaptopropionic acid, sodium citrate, sodium nitrate, etc. (available from shanghai square field chemical limited) Trizol (available from Thermo Scientific); reverse transcription kit (purchased from promega, cat # A3500); SYBR Premix Ex Taq (TaKaRa Co., Ltd., cat # RR 420A); the primers were synthesized by Shanghai Saibance Gene technology, Inc. and quality checked.

Main instrument

Reichert 7CR tonometer (available from Yimu medical instruments, Inc., Shanghai); agilent model 100 liquid chromatograph (from tokyo agile holdings group); clean bench (Shanghai Kenyan instruments Co., Ltd.); autoclave (Shandong Boke instruments Co., Ltd.); X-22R high speed refrigerated centrifuge (BECKMAN Corp.); stereo microscopes (Shanghai point should be optical instruments); real Time-PCR (Thermo Fisher Scientific Co.).

Cannabinoid eye drop formulation

Mixing Cannabidiol (CBD) 25mg, Tetrahydrocannabinol (THC) 5mg, WIN-55, 212-215 mg (mass ratio 5:1: 1), and dissolving 8.75mg of the above composition in 100ml of Tocrusolve ^TM 100, mixing uniformly, fully shaking to obtain 40ml of cannabinoid eye drops with final concentration of 8.75%, and storing in a refrigerator at 4 deg.C in dark.

Preparation of optic nerve injury mouse model

The basal intraocular pressure was measured 7 days before molding, twice at a fixed time each day by a dedicated person. Except for the normal control group, mice were subjected to abdominal anesthesia with 4% chloral hydrate (1 ml/100 g) and eye dropping with 1% lidocaine, and a No. 5 scalp needle connected to a physiological saline bottle infusion tube was inserted into the anterior chamber to keep the vertical height of the infusion bottle and the mice at 150 cm. Retinal ischemia and pallor and edema can be seen through ophthalmoscope observation, the blood flow of the scalp and the needle bottom of the eye is blocked, the bulbar conjunctiva edema is pallor after 15-30 min, and the eyeball becomes opaque. Measuring intraocular pressure at different time intervals after injection, keeping the body temperature of the animal at 36-37 ℃, and dripping physiological saline at any time to keep the cornea moist. The intraocular pressure is determined to be stably increased to be successful in molding, and during the period, the intraocular pressure is reduced, the patients are injected once again, and 1% chloramphenicol is dripped to prevent infection.

Experimental methods

2.1 administration and intraocular pressure measurement

After the model is successfully made for 8 days, the blank group and the control group are applied with physiological saline to double eyes once a day in the morning and at night. The experimental groups were applied to both eyes once a day, morning and evening, with 8.75% cannabinoid eye drops. Intraocular pressure was measured half an hour before administration every day.

High performance liquid chromatography for detecting concentration of vitriol glutamic acid

After treatment for 16 days, the test mice were sacrificed by spondylolisthesis, 0.1ml of posterior pole vitreous body was extracted, diluted to 0.5ml with physiological saline, and placed in a 1.5ml Eppendorf tube for cryopreservation at-70 ℃.

Preparation of derivatization reagents

6.7mg of o-phthalaldehyde was dissolved in 12ml of methanol, 1ml was taken out, 5. mu.l of thiopropionic acid was added, and then the volume was adjusted to 10ml with a sodium borate buffer solution having a pH of 9.6. + -. 0.02 to a final pH of 9.0. + -. 0.05. Aging for more than 120min in dark to reduce the blank of the reagent. Storing in a refrigerator at 4 deg.C.

Derivatization reaction and high performance liquid chromatography for determining glutamic acid content

After the glass body sample is rapidly thawed, the glass body sample is centrifuged for 10min at 8000r/min, the supernatant is taken, 100 mul of derivatization reagent is respectively added, and after 15min reaction, 60 mul of an automatic sample injector is carried out. The glutamic acid content in the sample was calculated from the chromatographic peak area by measurement of a standard (10. mu.g/ml) using an external algorithm.

Retinal slice RGCs enumeration

The eyes are completely removed, the corneoscleral edge is marked by suture and treated by a fixing solution, and the eyes are dehydrated by conventional alcohol gradient, transparent by xylene and embedded by paraffin. Sections were cut with a microtome, approximately 5 μm thick, HE stained, and mounted. Three visual fields are randomly selected on the temporal side of the papilla of the posterior pole under a light mirror, the number of the RGCs of the three continuous slices is read, and the average number is taken for recording.

Real-Time fluorescence quantitative PCR (Real-Time PCR) detection of retina tissue related gene level

Blank group, control group and experimental group are selected from mouse retina tissue, total RNA is extracted by Trizol method, after RNA concentration and purity are measured, cDNA is synthesized under the catalysis of reverse transcriptase, and then real-time fluorescence quantitative PCR detection of retina tissue related gene is carried out.

The sequence of the retinal tissue-associated gene primer used is shown in table 1 below:

TABLE 1 primers for optic nerve-related genes

Quantitative real-time PCR was performed on the applied real-time PCR system using SYBR premix Ex-Taq II kit (TaKaRa, Japan), and 45 amplifications were performed using cycles of 95 ℃ 15 s and 60 ℃ 30s at 72 ℃ 30s, using beta-actin mRNA as an internal reference, using comparative 2- ^△△CT Gene expression data were analyzed by the method and repeated 3 times per group.

Statistical method

SPSS 13.0 software is adopted for statistical analysis, single-factor variance analysis is adopted for comparison among multiple groups, independent sample t test is adopted for comparison between the two groups, data are normally distributed and expressed by mean plus or minus standard deviation, and the difference P < 0.05 is considered to have statistical significance.

Results and analysis

3.1 intraocular pressure test results

Histograms of intraocular pressure were plotted for the blank, control and experimental groups at two time periods of 8d and 16 d. In 16d, the mean intraocular pressure is the mean value of the intraocular pressure of each experimental group mouse, the mean intraocular pressure of a blank group is 21.13 +/-0.97 mmHg, the mean intraocular pressure of a control group is 41.79 +/-2.32 mmHg, and the mean intraocular pressure of an experimental group is 23.74 +/-1.67 mmHg, compared with the blank group and the control group, the intraocular pressure of the experimental group is obviously reduced and close to the blank group after the treatment by the formula, and has a significant difference (P < 0.01) with the control group.

Experiments show that the experimental group can significantly reduce the intraocular pressure of mice (P < 0.01), and the results are shown in figure 1.

Vitreous glutamic acid concentration

In order to study the effect of the formulation on inhibition of protein toxin release and accumulation in mice, the vitronemic acid concentrations of the blank group, the control group and the experimental group were simultaneously evaluated. The concentration of the vitriolic glutamic acid of the experimental group treated by the formula is 1.04 +/-0.44 mu g/ml, the concentration of the vitriolic glutamic acid has no significant difference (p is more than 0.05) compared with the concentration of the glutamic acid of the normal group of 0.76 +/-0.20 mu g/ml, and the concentration of the vitriolic glutamic acid has significant difference (p is less than 0.01) compared with the concentration of the glutamic acid of the control group of 7.69 +/-1.01 mu g/ml, so that after the treatment by the formula, the local concentration of the glutamic acid can be effectively prevented from being increased, and the damage of RGC and optic nerve can be reduced.

Experiments show that the experimental group can significantly reduce the concentration of mouse vitric glutamic acid (P < 0.01), and the results are shown in figure 2.

Number observation

In order to visually evaluate the influence of the formula on the apoptosis of the RGCs, the numbers of the RGCs in a blank group, a control group and an experimental group are counted, the number of the RGCs in the experimental group treated by the formula is 51.40 +/-6.04, the number of the RGCs in the experimental group is not significantly different from the number of the RGCs in a normal group which is 56.60 +/-6.09 (p is more than 0.05), and the number of the RGCs in the control group is 20.90 +/-3.18 (p is less than 0.01).

Experiments show that the experimental group can significantly reduce the damage and apoptosis of the mouse RGCs (P < 0.01), and the results are shown in FIG. 3.

Molecular mechanism study of optic nerve

Blank group, control group and experimental group for extracting mouse retina tissue RNA and performing real-time fluorescent quantitative PCR 2 ^-△△CT Values represent the relative mRNA expression levels of the gene in mice. As shown in FIGS. 4A-4B, the experimental group expression levels of NGF mRNA 0.87 + -0.30, Ntrk1 mRNA 0.68 + -0.10, and Nfkb1 mRNA 0.89 + -0.14 were significantly up-regulated (P.sup. + -0.32 + -0.07, Ntrk1 mRNA 0.19 + -0.04, and Nfkb1 mRNA 0.47 + -0.14) relative to the control group expression levels of NGF mRNA, as analyzed with beta-actin mRNA as an internal reference (P.sup. + -4B)<0.05）(ii) a The expression quantity of MAPK8 mRNA 1.11 +/-0.21 and NGFR mRNA 1.28 +/-0.12 in the experimental group is obviously reduced relative to the expression quantity of MAPK8 mRNA 2.17 +/-0.48 and NGFR mRNA 1.77 +/-0.33 in the control group (P)<0.05); see Table for details this result further demonstrates that protection of the mouse optic nerve in the experimental group of compositions may be associated with affinity-mediated nerve repair mechanisms that modulate NGF/Ntrk 1/NGFR receptors. The result shows that the composition of the experimental group can promote the expression of NGF in optic nerve damaged mouse retina tissues and improve the expression of NGF and a high affinity receptor Ntrk1 thereof, thereby exciting a nerve repair mechanism mediated by the combination of nerve growth factors and Ntrk1 receptors; the composition of the experimental group can reduce the expression of MAPK8 and NGFR which are low-affinity receptors of NGF, thereby inhibiting the nerve cell apoptosis mechanism mediated by the combination of NGF and NGFR.

Experimental example 2

A pharmaceutical composition was formulated in accordance with the formulation of example 2, eye drops were prepared in accordance with the method of example 1, and intraocular pressure test was performed on the optic nerve-injured mice. The test results are shown in fig. 5 (a) and (B), and the results of the effect on the mean intraocular pressure of 16d in the mouse are shown in table 2 below.

Experimental example 3

A pharmaceutical composition was formulated in accordance with the formulation of example 3, eye drops were prepared in accordance with the method of experimental example 1, and intraocular pressure test was performed on the optic nerve-injured mice. The results of the test are shown in (A) and (B) in FIG. 5, and the results of the effect on the mean intraocular pressure of 16d in the mouse are shown in Table 2.

Experimental examples 4 to 8

Drugs were formulated according to the formulations of examples 4 to 8, eye drops were prepared according to the method of experimental example 1, and intraocular pressure test was performed on the optic nerve-injured mice. The results of the tests are shown in (C) to (F) of FIG. 5, respectively, and the results of the effects on the mean intraocular pressure of 16d in the mouse are shown in Table 2.

Comparative example 1

The pharmaceutical composition in the comparative example consists of the following raw materials in proportion: cannabidiol (CBD), betaxolol, WIN-55,212-2, and the mass ratio is 5:1: 1. eye drops were prepared according to the method of experimental example 1, and intraocular pressure test was performed on the optic nerve-injured mice. The results of the test are shown in (A) and (B) in FIG. 5, and the results of the effect on the mean intraocular pressure of 16d in the mouse are shown in Table 2.

Comparative example 2

The pharmaceutical composition in the comparative example consists of the following raw materials in proportion: cannabidiol (CBD), timolol, WIN-55,212-2, and the mass ratio is 5:1:1, eye drops were prepared according to the method of Experimental example 1, and intraocular pressure test was performed on the optic nerve-injured mice. The results of the test are shown in (A) and (B) in FIG. 5, and the results of the effect on the mean intraocular pressure of 16d in the mouse are shown in Table 2.

Comparative example 3

The active ingredient of the drug in this comparative example was timolol, and eye drops were prepared according to the method of experimental example 1 and subjected to intraocular pressure test on mice with impaired optic nerves. The results of their effect on the mean intraocular pressure at 16d in mice are shown in table 2.

Comparative example 4

The active ingredient of the drug in this comparative example was betaxolol, eye drops were prepared according to the method of experimental example 1, and intraocular pressure test was performed on the optic nerve-injured mice. The results of their effect on the mean intraocular pressure at 16d in mice are shown in table 2.

TABLE 2 results of the effect of the pharmaceutical compositions of the experimental examples and comparative examples on the mean intraocular pressure of the mice at 16d

As can be seen from the results of Table 2 and FIGS. 5A to 5F, the compositions of examples 2 to 3 have the same composition as that of example 1, but the compositions have different ratios and different effects on the intraocular pressure of mice, wherein the intraocular pressure reducing effect of example 1 on mice is the best, and the intraocular pressure reducing effects of the compositions of examples 2 and 3 are poor in the monomeric drugs of examples 6 to 7.

As can be seen from table 2 and the results of (a) and (B) in fig. 6, the pharmaceutical compositions of experimental examples 1 to 3 to which Tetrahydrocannabinol (THC) was added had better effect of reducing intraocular pressure in mice than comparative example 1 and comparative example 2, of which example 1 had the best effect of reducing intraocular pressure in mice.

Claims

1. A pharmaceutical composition with optic nerve protection effect is characterized in that: it is composed of cannabidiol, tetrahydrocannabinol and WIN-55, 212-2.

2. The pharmaceutical composition with the effect of protecting the optic nerve according to claim 1, wherein the weight ratio of the cannabidiol to the tetrahydrocannabinol to the WIN-55,212-2 is 5-3: 3-1: 1.

3. the pharmaceutical composition with the effect of protecting optic nerves as claimed in claim 2, wherein the mass ratio of cannabidiol, tetrahydrocannabinol, WIN-55,212-2 is 5-4: 2-1: 1.

4. the pharmaceutical composition with the effect of protecting optic nerves as claimed in claim 3, wherein the weight ratio of cannabidiol, tetrahydrocannabinol, WIN-55,212-2 is 5:1: 1.

5. use of the pharmaceutical composition having an optic nerve protective effect according to claim 1 for the preparation of an ophthalmic external preparation for protecting optic nerve.

6. The use of claim 5, wherein the external preparation for eye is eye drops, eye ointment, eye gel, external wet pad for eye or external atomized liquid for eye.

7. The use of claim 5, wherein the ophthalmic preparation for external use comprises 7% to 9% by weight of the pharmaceutical composition of claim 1, and the balance is a pharmaceutically acceptable carrier.

8. The use of claim 7, wherein the pharmaceutically acceptable carrier is Tocrisolve ^TM 100 solution, eye ointment matrix, thickener, acid-base regulator, isotonic regulator, electrolyte agent, and antiseptic agentThe dosage percentage of the pharmaceutically acceptable carrier is as follows: 80-95% of an eye ointment matrix, 0.1-10.0% of a thickening agent, 0.01-5.0% of an acid-base regulator, 7.0-9.0% of a pH value, 0-8.0% of an isoosmotic regulator, 280-320 mmol/L of osmotic pressure regulator or 0.7-1.5% of an osmotic pressure ratio, 0.001-0.5% of an electrolyte agent and 0-0.5% of a preservative.

9. The use according to claim 8,

a. the thickening agent is selected from one or more of carbomer, chondroitin sulfate, methylcellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, polyacrylamide, glycerol, ethyl cellulose, hydroxypropyl methylcellulose, hydrogenated hard castor oil, polyallyl alcohol, poloxamer, sodium carboxymethyl cellulose, dextran, propylene glycol, tween, polyvinylpyrrolidone, chitosan, sodium hyaluronate, cross-linked polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol and cross-linked polyacrylamide;

b. the acid-base regulator is selected from one or more of boric acid, borax, glacial acetic acid, triethanolamine, citric acid, sodium citrate, hydrochloric acid, acetic acid, sodium acetate, tartaric acid, sodium tartrate, sodium hydroxide, sodium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate and phosphoric acid;

c. the isotonic regulator is one or more selected from glycerol, propylene glycol, glycerol, sodium chloride, glucose, mannitol and sorbitol;

d. the electrolyte agent is selected from one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, zinc chloride and glycine;

e. the antiseptic is selected from one or more of methyl hydroxybenzoate, ethyl hydroxybenzoate, propyl hydroxybenzoate, chlorobutanol, thimerosal, mercury bicyanate, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

10. The use of claim 5, wherein the ophthalmic external preparation further comprises one or more pharmaceutically active substances selected from the group consisting of purslane, water lily, bergamot, rosa canina, and borneol.