CN112245595A

CN112245595A - Application of A-type ultrasonic wave in developing myopia-related medicines

Info

Publication number: CN112245595A
Application number: CN202011127918.4A
Authority: CN
Inventors: 李和平; 廖琴; 蔡可阳
Original assignee: Joinn Laboratories Suzhou Co Ltd
Current assignee: Joinn Laboratories Suzhou Co Ltd
Priority date: 2020-10-19
Filing date: 2020-10-19
Publication date: 2021-01-22
Also published as: WO2022083034A1

Abstract

The invention discloses a method for verifying relaxation effect of an M receptor antagonist on ciliary smooth muscle by measuring thickness change of a lens of a non-human primate (monkey) through an A-type ultrasonic technology. The method can measure the influence of the test drugs with different concentrations on the thickness of the crystalline lens, understand the dose-effect relationship between the test drugs and the thickness of the crystalline lens, speculate the influence of the drugs on the refractive adjustment, detect the effective dose, and quickly and effectively guide the preclinical research and development work of new drugs for preventing and treating the myopia of teenagers. The method of the invention is simple, stable in technology, good in repeatability, free of tissue injury to animals and harmless to the whole body health. Because experimental data obtained from monkeys (animals most similar to human beings) have incomparable guiding significance for clinical tests of other animals, important conversion medical information is provided for the research and development of innovative drugs.

Description

Application of A-type ultrasonic wave in developing myopia-related medicines

Technical Field

The invention relates to the field of biomedical research and development, in particular to application of A-type ultrasonic waves in researching myopia-related medicines.

Technical Field

Juvenile myopia is a common eye disease in China. Because of the influence of the pressure of studying and environmental factors such as the popularization of computers, the prevalence rate is increased year by year, which is an important problem affecting the health of teenagers. How to effectively prevent and cure myopia and protect the eyesight of teenagers is a big problem which needs to be solved urgently at present.

In addition to genetic factors, the main factors responsible for juvenile myopia include the heavy learning task, insufficient lighting or visual fatigue caused by long-term close-distance writing and reading. The eyeball of the teenager is in the growth and development stage, the visual accommodation capability is strong in the elasticity of the crystalline lens, and the extensibility of the eyeball wall is large. Overuse of the eye places the ciliary muscles and extraocular muscles of the eye in a highly tense state. The excessively contracted ciliary muscle causes an increase in lens thickness and refractive power, and focal point advancement, which is a direct cause of juvenile myopia. If the above condition persists, the axis of the eye is lengthened, and the short sighted eye is formed. At present, three main measures for controlling juvenile myopia include increasing outdoor activities, relieving ciliary muscle tension by using atropic eye drops with low concentration and wearing orthokeratology glasses. The addition of outdoor activities and the application of the atropine eye drops with low concentration are favorable for relieving the spasm of ciliary muscles, relieving the fatigue state of eyes and effectively relieving the progress of myopia.

Atropine is one of anticholinergics, is an M receptor blocker, is commonly used for relieving gastrointestinal smooth muscle spasm, inhibiting glandular secretion, exciting respiratory center and relieving the inhibition of vagus nerve to heart in clinic, and is mainly used for relaxing ciliary smooth muscle, relieving lens regulation and dilating pupil in ophthalmology. In recent years, relevant research at home and abroad proves that the atropine eye drops with low concentration (0.01%) can delay the progress of the myopia too fast by inhibiting the increase of the axis of eyes of teenagers caused by the myopia. However, atropine is a prescription drug with certain toxicity, and brings side effects such as mydriasis, photophobia, blurred vision, increased intraocular pressure and the like to people due to improper use, so that the atropine has not been approved to be on the market in China. For this reason, efforts are being made in the domestic ophthalmic pharmaceutical industry to develop atropine-like drugs in an attempt to alleviate the side effects such as dilated pupils while maintaining its ciliary muscle relaxing effect. Effective dose and safety are also explored and determined in animal experiments before clinical application for long term clinical application.

The traditional method for measuring ciliary muscle tension is completed by using a complete set of system for detecting tissue muscle tension, including a micro-tension transducer, an electrophysiological recorder and the like, in a highly bionic tissue culture environment to test the response of isolated tissues to muscle relaxants or tensides (Experimental Eye research.2011 Sept; 93(3): 321-327.). The experimental conditions required are high in threshold, complex in equipment, complex and time-consuming in process, high in technical difficulty and high in experimental cost, more importantly, the experiment only obtains in-vitro tissue data, and the simulated culture environment and the living environment are far away from each other and difficult to accurately reflect the actual in-vivo situation. In order to quickly determine whether a new drug will have an effect on the ciliary muscle tone, it is desirable to establish a simple, easy and effective alternative that can be implemented in live animals.

Disclosure of Invention

The invention aims to solve the problems and discloses an application scheme of A-type ultrasonic waves in the development of myopia-related medicaments. The invention adopts an A-type ultrasonic in-vivo detection technology, and verifies the relaxation effect of M receptor antagonist medicines on ciliary smooth muscle by measuring the change of the thickness of non-human primate (monkey) eye lens before and after the M receptor antagonist medicines (atropine eye drops) are locally dropped into the eye. The method can measure the influence of the test drugs with different concentrations on the thickness of the crystalline lens, understand the dose-effect relationship between the test drugs and the thickness of the crystalline lens, speculate the influence of the drugs on the refractive adjustment, detect the effective dose, and quickly and effectively guide the preclinical research and development work of new drugs for preventing and treating the myopia of teenagers.

The scheme of the invention is as follows:

the application of the A-type ultrasonic wave in the development of myopia-related medicines: the change of the thickness of the lens of the non-human primate is measured by using A-type ultrasonic waves to verify the relaxation effect of M receptor antagonist drugs on ciliary smooth muscle, and a data model is established to guide the development of myopia-related drugs.

Further, the application of the type A ultrasonic wave in the development of myopia-related medicines comprises the following steps: and (3) constructing the dose-effect relationship between the test medicine and the thickness of the crystalline lens, speculating the influence of the medicine on the refractive adjustment, and detecting the effective dose.

Further, the application of the type A ultrasonic wave in the development of myopia-related medicines comprises the following steps:

1) animal grouping: randomly grouping non-human primates;

2) ultrasonic examination: dripping a reference substance or M receptor antagonist medicines into each eye, and respectively carrying out eye A-type ultrasonic examination and pupil measurement before and after administration to obtain related data;

3) and (3) data analysis: and carrying out data analysis on the related data.

Further, the type A ultrasonic wave is applied to the development of myopia-related medicines, in the step 1, the non-human primate is a cynomolgus monkey, and the age of the non-human primate is equivalent to the adolescence of human beings.

Further, in the step 2, the ultrasonic examination includes the following specific steps:

s1: anaesthetizing the animal, fixing the animal on the examination table in a supine position, exposing the eye to be examined by using an eyelid retractor, and dripping a reference substance or M receptor antagonist medicine;

s2: arranging the A ultrasonic probe on a fixing frame which can be adjusted in three dimensions, and enabling the head of the probe to vertically approach and lightly touch the vertex of a cornea;

s3, using A-type ultrasonic waves to respectively check the anterior chamber depth, the lens thickness, the vitreous body thickness and the eye axis length of the eye, and recording data;

and S4, immediately after the step S3 is finished, visually measuring the horizontal diameter of the pupil by using a pupillometer and recording data.

Further, in the application of the type a ultrasonic wave in developing a medicine related to myopia, in the step 3, the data analysis method includes:

a. data normalization: the measured data for each eye is standardized, that is, the measured data before administration for each eye is taken as 100%, and the measured data at each time point after administration is divided by the data before administration and multiplied by 100 to obtain the standardized value at each time point after administration.

b. Multidimensional data analysis: all statistical analyses were performed using two-tailed analysis, with the statistical level set at p < 0.05. Firstly, carrying out uniformity inspection on data by using Leven Test, and carrying out one-factor variance analysis if the data is uniform (p > 0.05); if the analysis of variance is significant (p is less than or equal to 0.05), performing LSD multiple comparison; if the result of the Leven Test is significant (p is less than or equal to 0.05), performing Kruskal-Wallis nonparametric Test; if the result of Kruskal-Wallis nonparametric test is significant (p is less than or equal to 0.05), further performing pairwise comparison by using Mann-Whitney U test.

Further, the application of the type A ultrasonic wave in the development of myopia-related medicines comprises the following specific steps:

I. animal grouping: animals were randomly divided into 6 groups of two animals each;

ultrasound examination: dripping 30 mul PBS, 1%, 0.5%, 0.1%, 0.05% or 0.01% M receptor antagonist medicine into conjunctival sac of double eyes by using a pipette, respectively carrying out ocular A ultrasonography 1h, 2h,6h, 72h and 168h before and after administration, and simultaneously measuring pupil size; during examination, an anesthetized animal is fixed on an examination table in a supine position, an eye to be examined is exposed by an eyelid opener, and an A ultrasonic probe is arranged on a fixing frame capable of being adjusted in three dimensions, so that the head of the probe is vertically close to and slightly touches the vertex of a cornea; each time of examination enables ultrasonic waves to enter from the vertex of the cornea and coincide with the axis of the eye, 8 groups of data and 1 group of average values of the anterior chamber depth, the lens thickness, the vitreous body thickness and the length of the axis of the eye are measured and obtained, each time point is repeatedly measured for 3 times, the average value of 3 times is taken, and biological measurement on the two eyes is completed one by one; in a constant indoor lighting environment, the horizontal diameter of the pupil is measured visually by using a pupillometer;

data analysis: the method comprises the following 2 steps of,

a. data normalization: standardizing the measured data of each eye, namely dividing the measured data (mm) before administration of each eye by the data before administration and multiplying the divided data by 100 to obtain the standardized value of each time point after administration;

Furthermore, the A-type ultrasonic wave is applied to the development of myopia-related medicaments, and the myopia-related medicaments are medicaments for preventing or treating juvenile myopia.

Further, the application of the type A ultrasonic wave in the development of myopia-related medicines comprises the establishment of an animal model.

Further, the application of the type A ultrasonic wave in the development of myopia-related medicines comprises the following steps of converting the thickness change of the crystalline lens and diopter by using the following formula: (1/f) ═ n-1) × { (1/R1) - (1/R2) + [ (n-1) t/nR1R2] }, where f is focal length, 1/f is diopter (D), n is refractive index (refractive index) of the lens, R1 and R2 represent radii of curvature of the anterior and posterior surfaces of the lens, and t is thickness of the lens.

Further, the application of the type A ultrasonic waves in the development of myopia-related medicines, wherein the M receptor antagonist medicines include but are not limited to atropine.

According to the scheme, the invention discloses application of A-type ultrasonic waves in developing myopia-related medicaments, which at least has the following beneficial effects:

(1) the invention utilizes the coupling optical adjustment principle between ciliary muscle and crystalline lens, uses A-type ultrasonic diagnosis technology, and locally gives M receptor antagonist (atropine) eye drops with different concentrations to the eyes of non-human primates (cynomolgus monkeys) to measure the change of the thicknesses of the crystalline lenses before and after administration, thereby establishing a dose-effect relationship curve between the change of the thicknesses of the crystalline lenses of the non-human primates (cynomolgus monkeys) and the atropine. Furthermore, by using the method, the M receptor antagonist drug (atropine) is used as a positive control drug to quickly detect the drug effect and dose-effect relationship of the developed new drug to ciliary muscle; the method can also be used for measuring and calculating the diopter change caused by the tested medicine by utilizing a mathematical formula of dioptric power, and effectively guiding the research and development work of a new medicine for preventing and treating the development of the teenager myopia.

(2) The method of the invention is simple, stable in technology, good in repeatability, free of tissue injury to animals and harmless to the whole body health. Because experimental data obtained from monkeys (animals most similar to human beings) have incomparable guiding significance for clinical tests of other animals, important conversion medical information is provided for the research and development of innovative drugs. The current application of atropine eye drops in clinical and non-clinical ophthalmic experiments mainly comprises 1. treatment of non-infectious inflammation in eyes, such as uveitis; 2. performing optometry on teenagers; 3. the low-concentration eye drops delay the development of teenager myopia (still belonging to the experimental stage). The invention detects the influence and dose-effect relationship of atropine (which can be used as a standard contrast drug) on the thickness of crystalline lens by an A ultrasonic biological measurement method, and further initiates the evaluation of non-clinical drug effect of similar drugs by a novel experimental method at home and abroad.

Drawings

FIG. 1 is a sectional view of the result of the A-mode ultrasonic testing in the automatic mode of the embodiment;

FIG. 2 is a graph showing the effect of PBS and multiple doses of atropine eye drops on thickness of cynomolgus monkey lens (Mean + -SD, 4 eyes/group)

Fig. 3 is a graph of the average lens thickness versus dose for each experimental group in the examples.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Example 1

1. Experimental materials and methods:

experimental animals: 12 healthy immature cynomolgus monkeys with normal eyes were selected, aged 45.9 months. + -. 2.4 months (equivalent to adolescent age of human), mean. + -. standard deviation (same as the latter), body weight 3.8. + -. 0.6 kg, sex and sex half.

The test drugs are: 1.0% atropine sulfate eye drops (day point, famous ancient house) and 0.5%, 0.1%, 0.05% and 0.01% atropine eye drops diluted with neutral phosphate buffer.

Comparison products: neutral phosphate buffer (PBS, pH 7.4).

The administration method comprises the following steps: a single topical eye drop, 30. mu.l per eye, was administered to both eyes simultaneously.

A detection instrument: ophthalmological A/B ultrasonic diagnostic apparatus (ODM-2100S, Tianjin Mada medical science and technology)

ODM-2100S ophthalmology type A eye axis biological parameter measurement part technical index:

ultrasonic frequency 10MHz

The A-type ultrasonic biological parameter measurement precision is that the error of the length (AL) of the eye axis is less than or equal to plus or minus 0.06 mm; the error of the value of the anterior chamber depth (AC) is less than or equal to +/-0.06 mm; the error of the values of the LENs thickness (LEN) and the vitreous body thickness (VITR) is less than or equal to +/-0.12 mm. Display resolution: 0.01mm

Type a ocular axis biological parameter measurement range:

axial Length (AL): 15.0 mm-39.0 mm

Anterior chamber depth (AC) of 2.0 mm-6.5 mm

LENs thickness (LEN) of 2.0-6.5 mm

The glass body thickness (VITR) is 12.0-33.0 mm

Illuminance measuring instrument: S400B photometer (Shuncoda, Beijing)

Detection time: before administration, 1h, 2h,6h, 72h and 168h (7 days) after drug administration.

The anesthesia method comprises the following steps: animals were fasted on the day of the experiment, weighed, injected intramuscularly with Shutai 50 (R) (A)

50, Virbac, France) 8mg/kg anesthesia.

2. The experimental steps are as follows:

animals were randomly divided into 6 groups of two animals each (4 eyes). Using a pipette to drop 30 mul PBS, 1%, 0.5%, 0.1%, 0.05% or 0.01% atropine eye drops into the conjunctival sac of the double eyes at a time, and respectively carrying out ocular A ultrasonography 1h, 2h,6h, 72h and 168h before and after administration, and simultaneously measuring the pupil size. During examination, an anesthetized animal is fixed on an examination table in a supine position, an eye to be examined is exposed by an eyelid opener, and an A ultrasonic probe is arranged on a fixing frame capable of being adjusted in three dimensions, so that the head of the probe is vertically close to and slightly touches the vertex of a cornea. To ensure measurement accuracy, each examination has the ultrasound waves coming in from the corneal vertex and coinciding with the axis of the eye. The measurement adopts an automatic mode, the instrument can automatically generate 8 groups of data including the anterior chamber depth, the lens thickness, the vitreous body thickness and the axial length of the eye and 1 group of average values (shown in figure 1) in each measurement, the measurement is repeated for 3 times at each time point, the average value is taken for 3 times, and the biological measurement of the two eyes is completed one by one. In a constant room lighting environment (450 lux), the horizontal pupil diameter was measured visually using the pupillometer (tour, Ningbo) (immediately after each A-ultrasonography). After the experiment was completed, the animals were kept warm under the blanket and returned after recovering consciousness.

3 data analysis and statistical methods:

first, the measured data for each eye was normalized by dividing the measured data (mm) before administration by the measured data (mm) before administration for each eye by the measured data (mm) at each time point after administration and multiplying the divided data by 100 to obtain a normalized value (%) at each time point after administration. The specific statistical method is as follows: statistical software SPSS13.0 was used to process the data for this experiment. All statistical analyses were performed using two-tailed analysis, with the statistical level set at p < 0.05. Firstly, carrying out homogeneity Test on data by using Leven Test, and carrying out one-factor analysis of variance if the data is homogeneous (p is more than 0.05); if the analysis of variance is significant (p ≦ 0.05), then a LSD multiple comparison is performed. If the result of the Leven Test is significant (p.ltoreq.0.05), a Kruskal-Wallis nonparametric Test is performed. If the result of Kruskal-Wallis nonparametric test is significant (p is less than or equal to 0.05), further performing pairwise comparison by using Mann-Whitney U test. In comparison to the PBS group, a means p <0.05, aa means p <0.01, aaa means p < 0.001; b represents p <0.05, bb represents p <0.01, bbb represents p <0.001, compared to the 1% atropine group; c means p <0.05, cc means p <0.01, ccc means p <0.001, compared to the 0.5% atropine group; d represents p <0.05, dd represents p <0.01, ddd represents p <0.001 compared to the 0.1% atropine group; in comparison to the 0.05% atropine group, e indicates p <0.05, ee indicates p <0.01, eee indicates p < 0.001.

4. Results of the experiment

(1) Anterior chamber depth before drug treatment the mean anterior chamber depth was 2.70mm + -0.21 mm (n ═ 24 eyes), the anterior chamber depth of animals in both post-drug PBS and atropine dose groups showed non-dose related, mild variation with irregularity, no significant difference was seen in statistical analysis except that the non-dose related disorder variation appeared in the atropine dose groups at 72h after drug treatment (as shown in Table 1)

TABLE 1 Effect of PBS and atropine on anterior chamber depth of cynomolgus monkeys (% mean. + -. standard deviation, 4 eyes/group)

(2) Lens thickness: the mean thickness of the pre-drug lenses was 3.30mm ± 0.08mm (n ═ 24 eyes). The average thickness of the lens before the PBS group is 3.30mm plus or minus 0.03mm, the lens has only slight change of-0.02 mm (-0.66%) after 1h to 72h after the drug, and is 3.29mm plus or minus 0.02mm at 168h, which is 99.65% of the level before the drug. The lens thickness of the atropine dose groups exhibited a gradual decrease trend negatively correlated to the dose at 1h, 2h and 6h post-dose, 7.43% (1% group), 5.11% (0.5% group), 3.20% (0.1% group), 0.82% (0.05% group) and 0.88% (0.01% group) at 6h post-dose, respectively, and dose-related gradual recovery trends at 72h and 168h post-dose, and recovery to 95.83% (1% group), 99.07% (0.5% group), 98.05% (0.1% group), 100.03% (0.05% group) and 99.85% (0.01% group) of the pre-dose level at 168h post-dose, respectively, as compared to pre-dose.

One-way analysis of variance of lens thickness (%) (see table 2):

1) in contemporaneous comparison with the PBS group: at each time point after 1% of the composition, the significant differences exist between the 0.5% composition and the 0.1% composition after 2h,6h and 72h (p is less than or equal to 0.05 and the symbol a).

2) In a contemporary comparison with the 1% atropine group: the significant differences (p is less than or equal to 0.05 and the symbol b) are observed at each time point after the 0.1 percent group drug is treated for 6 hours and 72 hours and after the 0.05 percent group drug is treated for 0.01 percent group drug.

3) In comparison with the 0.5% group contemporaneously: the significant differences (p is less than or equal to 0.05 and the symbol c) exist at 1h, 2h,6h and 72h after 0.05 percent of the composition and at various time points after 0.01 percent of the composition.

4) In comparison with the 0.1% group contemporaneously: the 0.05% group had significant differences (p is less than or equal to 0.05, symbol d) at 2h,6h and 72h after the drug administration, and the 0.01% group had significant differences at 6h and 72h after the drug administration.

5) In a contemporary comparison with the 0.05% group: the 0.01 percent group has significant difference (p is less than or equal to 0.05 and the symbol e) after the drug is applied for 1 hour.

TABLE 2 Effect of PBS and atropine on cynomolgus monkey lens thickness (%, mean. + -. standard deviation, 4 eyes/group)

The above lens thickness variation was correlated with the atropine dose and the maximum effect observed at each time point appeared 6h after dosing (see figure 2). Taking the change (%) of the lens thickness at 6h after the drug, the mean lens thickness of each group exhibited a high linear correlation (R) with the dose (R)²0.9119) (see fig. 3).

(3) Vitreous thickness the mean vitreous thickness before dosing was 12.35mm ± 0.18mm (n ═ 24 eyes), and the vitreous thickness of animals in both the PBS and atropine dose groups after dosing was less than 2% non-dose related, erratic, subtle changes with no significant difference in statistics (one-way anova) (see table 3).

TABLE 3 Effect of PBS and atropine on cynomolgus monkey vitreous thickness (%, mean. + -. standard deviation, 4 eyes/group)

(4) Length of eye axis: the mean axial length before dosing was 18.36mm ± 0.33mm (n ═ 24 eyes), and the axial length of the eyes of the animals in each dose group of PBS and atropine after dosing showed non-dose related, irregular, mild changes in the range of less than 3%, with no significant difference in statistical analysis (one-way anova) (see table 4).

TABLE 4 Effect of PBS and atropine on cynomolgus monkey ocular axis length (%, mean. + -. standard deviation, 4 eyes/group)

(5) Diameter of pupil: under the constant illumination of 450 lux, the diameter of the pupil before medicine is 2.0 mm. The pupil diameter of the PBS group did not change before and after dosing. The pupil diameter of each atropine administration group exhibited a gradual increase tendency positively correlated with the dose at 1h, 2h and 6h after the administration, and exhibited a gradual recovery tendency correlated with the dose at 72h and 168h after the administration. Of these, the pupil diameters of the two lowest dose groups (0.05% and 0.01%) had substantially or completely returned to the pre-drug level by 168h (7d) post-drug (see table 5).

TABLE 5 Effect of PBS and atropine on cynomolgus monkey pupil diameter (mm, mean. + -. standard deviation, 4 eyes/group)

5. Discussion of Experimental results

Conversion of lens thickness change to diopter:

the lens is a biconvex lens with an anterior surface radius of curvature (R) greater than a posterior surface radius of curvature. According to the optical principle that light rays deflect to the bottom of a triangular dioptric medium (such as glass), when passing through a crystalline lens, light rays are subjected to centripetal refraction on a light path, an existing emmetropia eye focused on a retina is called a myopia eye focused in front of the retina, and a hypermetropia eye focused behind the retina is called a hypermetropia eye. The lens adjusts the focal length by a change in thickness. When the crystalline lens becomes thick, the focal length becomes short, the imaging moves forward, when the crystalline lens becomes thin, the focal length becomes long, and the imaging moves backward. Atropine enlarges ciliary muscle ring by relaxing ciliary muscle, enhances the traction force on crystalline lens by the traction of zonules, thins the crystalline lens by traction, weakens the refractive power, lengthens the focal length, moves backwards in imaging and focuses on retina, thereby achieving the effect of preventing and relieving myopia. The effect of lens thickness variation on diopter, with all other factors affecting the refraction of the eye remaining unchanged, can be calculated by the following equation:

(1/f)＝(n–1)×{(1/R1)–(1/R2)+[(n–1)t/nR1R2]}

where f is the focal length, 1/f is diopter (D), n is the refractive index of the lens, R1 and R2 represent the radii of curvature of the anterior and posterior surfaces of the lens, and t is the thickness of the lens. The two most important factors in this formula are lens thickness (t) and focal length (f). The focal length is the reciprocal of the right side of the formula. In other words, if the right side of the formula is smaller, the focal length is longer. (refractive index: a change in the direction of light traveling from one medium into another medium of different refractive index occurs, in the eye's optics, i.e. "refraction". tissues capable of refractive action in the eye are called refractive indices, such as cornea, aqueous humor, lens, vitreous humor, etc.. refractive index of refractive indices in the eye are called refractive indices. the refractive index of each refractive index in a vacuum environment is defined as 1. the refractive index of each refractive index in a human eye is defined as 1.33 for cornea, 1.43 for lens, and about 1.33 for aqueous humor and vitreous humor. normally, the refractive index does not change significantly.)

Substituting the related indexes into the formula can calculate the change of focal length (f) or diopter (1/f) caused by the change of the lens thickness, and guide the research and development of the medicine.

1. The experiment detects the effect of reducing the thickness of crystalline lens of different doses of atropine eye drops on the dose correlation of cynomolgus monkey eyes through an ophthalmic A-type ultrasonic diagnosis technology, and verifies the relaxation effect of the atropine (M receptor blocker) on ciliary smooth muscle and the visual physiological effect of generating refractive adjustment by reducing the thickness of the crystalline lens (changing the curvature radius of the crystalline lens).

2. Also observed in this experiment was a dose-related mydriatic effect of atropine on the cynomolgus monkey pupils of varying degrees and duration.

3. The method for detecting the thickness of the crystalline lens used in the experiment has the advantages of simple operation, stable technology, good repeatability, no injury to tissues and animal health, and can be carried out in a single eye or two eyes according to the needs of the experiment. The experimental data obtained from the monkeys have incomparable guidance effect on clinical tests of other animals, and provide important transformation medical information for the research and development of innovative drugs.

4. The method can be used for evaluating and verifying the relaxation effect of the novel M receptor antagonist on ciliary smooth muscle, detecting the lowest effective dose, and quickly and effectively guiding the preclinical research and development work of a new medicine for preventing and treating teenagers myopia.

5. The application discloses a use method of a mathematical formula for calculating diopter change by using lens thickness change and related indexes to effectively guide preclinical research and development work of a new medicine for preventing and treating teenager myopia.

It is to be noted that the above experiments were carried out and completed in 3 different time phases, namely: 2020.8.3 start PBS group and 05% group; 2020.8.18 starting in 0.1% and 1% groups; 2020.9.1 begin the 0.01% group and the 0.05% group. The data of the thickness change of the crystalline lens caused by detecting different doses of the atropine eye drops can present such a high linear dose-effect relationship (R) at different times for different animal individuals²0.9119) is sufficient to demonstrate the stability, ease of operation and repeatability of the process of the invention.

Claims

The application of the A-type ultrasonic wave in the development of myopia-related medicines is characterized in that the change of the thickness of the crystalline lens of a non-human primate is measured by using the A-type ultrasonic wave to verify the relaxation effect of M receptor antagonist medicines on ciliary smooth muscle, a data model is established, and the development of myopia-related medicines is guided.
2. The use of type a ultrasound according to claim 1 in the development of a medicament associated with myopia, wherein said use comprises: and (3) constructing the dose-effect relationship between the test medicine and the thickness of the crystalline lens, speculating the influence of the medicine on the refractive adjustment, and detecting the effective dose.
3. Use of type a ultrasound waves in the development of a medicament associated with myopia according to claim 2, wherein said use comprises the steps of:

1) animal grouping: randomly grouping non-human primates;

2) ultrasonic examination: dripping a reference substance or M receptor antagonist medicines into each eye, and respectively carrying out eye A-type ultrasonic examination and pupil measurement before and after administration to obtain related data;

3) and (3) data analysis: and carrying out data analysis on the related data.
4. The use of type a ultrasound in the development of a medicament associated with myopia according to claim 3, wherein in step 1 the non-human primate is a cynomolgus monkey, the age of which corresponds to human adolescence.
5. The use of type a ultrasound waves in the development of drugs associated with myopia according to claim 3, wherein in step 2, the ultrasound examination comprises the following specific steps:

s1: anaesthetizing the animal, fixing the animal on the examination table in a supine position, exposing the eye to be examined by using an eyelid retractor, and dripping a reference substance or M receptor antagonist medicine;

s2: arranging the A ultrasonic probe on a fixing frame which can be adjusted in three dimensions, and enabling the head of the probe to vertically approach and lightly touch the vertex of a cornea;

s3, using A-type ultrasonic waves to respectively check the anterior chamber depth, the lens thickness, the vitreous body thickness and the eye axis length of the eye, and recording data;

s4, upon completion of step S3, the horizontal diameter of the pupil is measured visually using the pupillometer and the data is recorded.
6. The use of type a ultrasound according to claim 3 in the development of a medicament associated with myopia, wherein in step 3 the data analysis method comprises:

a. data normalization: standardizing the measured data of each eye, namely dividing the measured data of each eye before administration by the measured data of each time point after administration by the measured data of each eye before administration and multiplying the divided data by 100 to obtain the standardized value of each time point after administration;

b. multidimensional data analysis: all statistical analyses were performed using two-tailed analysis, with statistical levels set atp≤0.05；

First, the homogeneity of the data is checked by Leven Test, if the data is homogeneous (pMore than 0.05), performing single-factor variance analysis; if the analysis of variance is significant (pLess than or equal to 0.05), performing LSD multiple comparison; if the result of Leven Test is significant (pNot more than 0.05), performing Kruskal-Wallis non-parametric test; if the result of Kruskal-Wallis nonparametric test is significant (pLess than or equal to 0.05), and further performing pairwise comparison by using Mann-Whitney U test.
7. The use of type a ultrasound waves in the development of drugs associated with myopia, according to claim 2, characterized in that said use comprises the following specific steps:

I. animal grouping: animals were randomly divided into 6 groups of two animals each;

ultrasound examination: dripping 30 mul PBS, 1%, 0.5%, 0.1%, 0.05% or 0.01% M receptor antagonist medicine into conjunctival sac of double eyes by using a pipette, respectively carrying out ocular A ultrasonography 1h, 2h,6h, 72h and 168h before and after administration, and simultaneously measuring pupil size; during examination, an anesthetized animal is fixed on an examination table in a supine position, an eye to be examined is exposed by an eyelid opener, and an A ultrasonic probe is arranged on a fixing frame capable of being adjusted in three dimensions, so that the head of the probe is vertically close to and slightly touches the vertex of a cornea; each time of examination enables ultrasonic waves to enter from the vertex of the cornea and coincide with the axis of the eye, 8 groups of data and 1 group of average values of the anterior chamber depth, the lens thickness, the vitreous body thickness and the length of the axis of the eye are measured and obtained, each time point is repeatedly measured for 3 times, the average value of 3 times is taken, and biological measurement on the two eyes is completed one by one; in a constant indoor lighting environment, the horizontal diameter of the pupil is measured visually by using a pupillometer;

data analysis: the method comprises the following 2 steps of,

a. data normalization: standardizing the measured data of each eye, namely dividing the measured data (mm) before administration of each eye by the data before administration and multiplying the divided data by 100 to obtain the standardized value of each time point after administration;

b. multidimensional data analysis: all statistical analyses were performed using two-tailed analysis, with statistical levels set atp≤0.05；

First, the homogeneity of the data is checked by Leven Test, if the data is homogeneous (pMore than 0.05), performing single-factor variance analysis; if the analysis of variance is significant (pLess than or equal to 0.05), performing LSD multiple comparison; if the result of Leven Test is significant (pNot more than 0.05), performing Kruskal-Wallis non-parametric test; if the result of Kruskal-Wallis nonparametric test is significant (pLess than or equal to 0.05), and further performing pairwise comparison by using Mann-Whitney U test.
8. Use of type a ultrasound waves according to any of claims 1 to 7 for the development of a myopia-related drug, wherein said myopia-related drug is a drug for the prevention or treatment of juvenile myopia.
9. Use of type a ultrasound waves according to any of claims 1 to 7 in the development of a medicament associated with myopia, wherein said use comprises conversion of lens thickness variation to diopters using the following formula: (1/f) = (n-1) × { (1/R1) - (1/R2) + [ (n-1) t/nR1R2] }, where f = focal length, 1/f = diopter (D), n = refractive index (refractive index) of the lens, R1 and R2 represent radii of curvature of the anterior and posterior surfaces of the lens, and t = thickness of the lens.
10. Use of type a ultrasound waves according to any of claims 1 to 7 for the development of drugs associated with myopia, wherein said M receptor antagonist class of drugs includes but is not limited to atropine.