Panax notoginseng saponins temperature-sensitive gel eye drops as well as preparation method and application thereof

Abstract

The invention discloses a panax notoginseng saponins temperature-sensitive gel eye drop for preventing and treating eye diseases, the eye drop comprises panax notoginseng saponins and a pharmaceutically acceptable first auxiliary material, and the mass ratio of the panax notoginseng saponins to the pharmaceutically acceptable first auxiliary material is 0.15-0.4; wherein the pharmaceutically acceptable first auxiliary material is a temperature-sensitive gel matrix. Through SEM and light transmittance determination, the panax notoginseng saponins temperature-sensitive gel subjected to auxiliary material type and dosage screening is characterized, and the temperature-sensitive gel is of an internal loose porous structure; the light transmittance reaches 90% or above, and blurred vision cannot be caused after eye administration; the cumulative release amount of the temperature-sensitive gel is researched by using a dialysis bag method by taking a panax notoginseng saponins solution as a contrast, and the result shows that the temperature-sensitive gel disclosed by the invention has a better slow-release effect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a temperature-sensitive gel eye drop of notoginseng total saponin, and a preparation method and use thereof. Background Art

[0002] Diabetic retinopathy (DR) is an inflammatory disease of the nerve microvessels that damages the microvascular units of nerve cells. It is the most serious and common complication of diabetes and the main cause of blindness in adults (20 to 65 years old). According to a report released by the International Diabetes Federation (IDF) on December 21, 2018, about 425 million people worldwide suffer from diabetes among adults aged 20 to 79. By 2045, this number is expected to increase to 629 million. About one-third of people with diabetes (DM) suffer from DR, and one in ten patients have vision-damaging diseases. According to the degree of fundus lesions, DR can be divided into non-proliferative DR (NPDR) and proliferative DR (PDR). The former is characterized by microaneurysms, retinal hemorrhages, cotton-like spots, venous beads, macular edema, and blood-retinal barrier (BRB) leakage, while the latter occurs with further retinal ischemia and is characterized by the growth of new blood vessels on the posterior surface of the retina and vitreous. If the disease cannot be effectively controlled, NPDR can turn into PDR, and in severe cases, retinal detachment may occur, leading to blindness.

[0003] At present, the pathogenesis of DR is still unclear. The main recognized factors include accumulation of advanced glycation end products, abnormal polyol pathway, inflammatory response, oxidative stress, abnormal cytokine production, protein kinase C activation, and hemodynamic changes. Studies have shown that elevated blood sugar can cause apoptosis of retinal microvascular pericytes, damage BRB, activate glial cells, and cause damage to the optic nerve; then retinal hypoxia and ischemia occur, and eventually angiogenesis is triggered. BRB damage is one of the most significant pathological features of early DR, and it plays an important role in regulating retinal vascular permeability. It is reported that inflammatory response plays an important role in regulating the development of DR. Activation of NF-κB nuclear transcription factors and others can promote the expression of various proinflammatory cytokines in diabetic patients and the adhesion of leukocytes to the retinal endothelium, leading to BRB destruction and vascular leakage. By using drug inhibitors or anti-inflammatory drugs to inhibit the production of inflammatory mediators, the development of DR can be significantly reduced, especially in the early stages of DR.

[0004] At present, the treatment of DR mainly consists of drug therapy, laser photocoagulation therapy and vitrectomy. Drug therapy is the first choice for DR. It refers to the injection of vascular endothelial growth factor (VEGF) antagonists into the vitreous body. However, this method is prone to cause fibrovascular constriction and persistent increase in intraocular pressure. Laser photocoagulation uses the biothermal coagulation effect of laser to destroy abnormal tissue in the retina and cause scars, thereby controlling fundus lesions. It is an invasive treatment method that can lead to postoperative visual field defects, decreased contrast sensitivity, and some other adverse reactions, including central or quasi-central melanoma. For some patients who are ineffective with laser photocoagulation therapy and vascular endothelial growth factor antagonists, vitrectomy can help improve vision. However, vitrectomy is prone to cause intraocular infection, postoperative bleeding, retinal detachment and increased intraocular pressure. Therefore, it is necessary to find more drugs with better efficacy and fewer side effects for the treatment of DR. Summary of the invention

[0005] Based on this, the present invention provides a Panax notoginseng total saponin thermosensitive gel eye drops for preventing and treating eye diseases, the eye drops comprising Panax notoginseng total saponins and a pharmaceutically acceptable first excipient, wherein the mass ratio between the Panax notoginseng total saponins and the pharmaceutically acceptable first excipient is 0.15-0.4, wherein the pharmaceutically acceptable first excipient is a thermosensitive gel matrix.

[0006] Furthermore, the Panax notoginseng total saponins contain one or more of the following chemical structural formulas:

[0007]

[0008] Furthermore, the mass ratio between the Panax notoginseng total saponins and the pharmaceutically acceptable first excipient is 0.2-0.4, such as 0.25-0.35, such as about 0.32.

[0009] Further, the thermosensitive gel matrix is ​​poloxamer. Further, the poloxamer is poloxamer 407 and / or poloxamer 188. Further, based on the volume of the eye drops, the mass volume concentration of poloxamer 407 is 24% to 28%, such as 26% to 28%, such as about 27.414%. Further, based on the volume of the eye drops, the mass volume concentration of poloxamer 188 is 0% to 1%, such as 0.1% to 0.6%, such as about 0.513%.

[0010] Furthermore, based on the volume of the eye drops, the mass volume concentration of the notoginseng total saponins is 5% to 9.5%, such as 7% to 9.5%, such as about 9%.

[0011] Further, the eye drops further include a pharmaceutically acceptable second excipient, wherein the pharmaceutically acceptable second excipient includes one or more of the following: an antibacterial agent, a penetration enhancer, a viscosity modifier, a stabilizer, a solubilizer, and a cosolvent. Further, the pharmaceutically acceptable second excipient is an antibacterial agent, such as benzalkonium chloride. Further, based on the volume of the eye drops, the mass volume concentration of the benzalkonium chloride is 0.005% to 0.05%, for example, about 0.01%. Further, the pharmaceutically acceptable second excipient does not include a pH regulator. Further, the pharmaceutically acceptable second excipient does not include an osmotic pressure regulator.

[0012] Further, the eye disease is an internal eye disease. Further, the eye disease is a retinopathy. Further, the retinopathy is diabetic retinopathy and / or central retinal vein occlusion. Further, the diabetic retinopathy is proliferative diabetic retinopathy and / or non-proliferative diabetic retinopathy.

[0013] Furthermore, the eye drops further include one or more drugs and / or extracts for preventing and treating eye diseases. Further, the drug is a vascular endothelial growth factor antagonist. Further, the vascular endothelial growth factor antagonist is selected from one or more of the following: ranibizumab, aflibercept and conbercept.

[0014] According to another aspect of the present invention, a method for preparing the above-mentioned eye drops is provided, which is a cold dissolution method.

[0015] Furthermore, the method comprises the following steps:

[0016] (1) adding an appropriate amount of a pharmaceutically acceptable first excipient to water for swelling to obtain a swollen pharmaceutically acceptable first excipient; and

[0017] (2) Weighing an appropriate amount of Panax notoginseng total saponins, adding water to dissolve, and then adding the swollen pharmaceutically acceptable first excipient to mix for the first time, and letting it stand to obtain the Panax notoginseng total saponins thermosensitive gel eye drops, wherein the pharmaceutically acceptable first excipient is a thermosensitive gel matrix.

[0018] Further, in step (1), the thermosensitive gel matrix is ​​poloxamer. Further, the poloxamer is poloxamer 407 and / or poloxamer 188. Further, based on the volume of the eye drops, the mass volume concentration of poloxamer 407 is 24% to 28%, for example, 26% to 28%, for example, about 27.414%. Further, based on the volume of the eye drops, the mass volume concentration of poloxamer 188 is 0% to 1%, for example, 0.1% to 0.6%, for example, about 0.513%. Further, the water is purified water. Further, the swelling temperature is 3°C to 5°C, for example, about 4°C. Further, the swelling time is 8 hours to 16 hours, for example, about 12 hours.

[0019] Further, step (2) further comprises adding an appropriate amount of a pharmaceutically acceptable second excipient after the first mixing and stirring and mixing. Further, in step (2), the water is purified water. Further, the pharmaceutically acceptable second excipient comprises one or more of the following: an antibacterial agent, a penetration enhancer, a viscosity modifier, a stabilizer, a solubilizer and a cosolvent. Further, the pharmaceutically acceptable second excipient is an antibacterial agent, such as benzalkonium chloride. Further, based on the volume of the eye drops, the mass volume concentration of the benzalkonium chloride is 0.005% to 0.05%, such as about 0.01%. Further, the pharmaceutically acceptable second excipient does not include a pH regulator. Further, the pharmaceutically acceptable second excipient does not include an osmotic pressure regulator. Further, the temperature of the standing is 3°C to 5°C, such as about 4°C. Further, the standing time is 8 hours to 16 hours, such as about 12 hours. Further, the result of the standing is that the eye drops are transparent and clear. Furthermore, based on the volume of the eye drops, the mass volume concentration of the total saponins of Panax notoginseng is 5% to 9.5%, such as 7% to 9.5%, such as about 9%. Furthermore, the light transmittance of the thermosensitive gel is greater than 90%.

[0020] According to another aspect of the present invention, there is provided a use of the above-mentioned eye drops in the preparation of a medicament for preventing and treating eye diseases.

[0021] Further, the eye disease is an internal eye disease. Further, the eye disease is a retinopathy. Further, the retinopathy is diabetic retinopathy and / or central retinal vein occlusion. Further, the diabetic retinopathy is proliferative diabetic retinopathy and / or non-proliferative diabetic retinopathy.

[0022] Beneficial effects of the present invention:

[0023] In order to overcome the disadvantages of conventional eye drops that are easily removed by tears and the low distribution of drugs in eye tissues, the present invention designs a novel Panax notoginseng total saponin (PNS) thermosensitive gel eye drops, the purpose of which is to increase the precorneal retention time of PNS and increase the permeability of the cornea, so as to enhance the distribution of PNS in eye tissues and the efficacy of preventing and treating diabetic retinopathy (DR). The PNS thermosensitive gel of the present invention, which has been screened by the type and amount of excipients, is characterized by SEM and transmittance measurement. The thermosensitive gel of the present invention is a porous structure with loose interior; the transmittance is more than 90%, and it will not cause blurred vision after eye administration; the cumulative release of the thermosensitive gel is studied by the dialysis bag method using the PNS solution as a control, and the results show that the thermosensitive gel of the present invention has a better sustained release effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0025] Figure 1 Schematic diagram of the blank gel forming results of P407 at different concentrations.

[0026] Figure 2 Effect surface plot for optimizing prescription T1 using central-complex design-response surface methodology.

[0027] Figure 3 Effect surface plot for optimizing prescription T2 using central-complex design-response surface methodology.

[0028] Figure 4 This is the microstructure diagram of PNS thermosensitive gel.

[0029] Figure 5 Schematic diagram of the transmittance results of PNS thermosensitive gel under visible light.

[0030] Figure 6 Schematic diagram of the cumulative release results of each component of the PNS solution.

[0031] Figure 7 Schematic diagram of the cumulative release results of each component of PNS thermosensitive gel.

[0032] Figure 8 Schematic diagram of the comparison of the cumulative release of total saponins from PNS solution and thermosensitive gel.

[0033] Fig. 9 Schematic diagram of the comparison of the cumulative release of total saponins from PNS thermosensitive gel and PNS pH sensitive gel. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0035] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0036] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0037] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0038] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0039] As described in the background technology section, the prior art for diabetic retinopathy (DR) lacks drugs with better efficacy and fewer side effects, and conventional eye drops have problems such as being easily cleared by tears and low distribution of drugs in eye tissues. In order to solve the above problems, the present invention provides a notoginseng total saponin thermosensitive gel eye drops for preventing and treating eye diseases, the eye drops comprising notoginseng total saponins and a pharmaceutically acceptable first excipient, wherein the mass ratio between the notoginseng total saponins and the pharmaceutically acceptable first excipient is 0.15 to 0.4, wherein the pharmaceutically acceptable first excipient is a thermosensitive gel matrix.

[0040] In the present invention, when the mass ratio, mass volume concentration, temperature, time or other values ​​or parameters are expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "0.15-0.4" is disclosed, the described range should be interpreted as including the range "0.15-0.4", "0.15-0.39", "0.15-0.38", "0.15-0.37", "0.15-0.36", "0.15-0.35", "0.15-0.34", "0.15-0.34", "0.15-0.33", "0.15-0.32", "0.15-0.31", "0.15- ~ 0.3", "0.15~0.29", "0.15~0.28", "0.15~0.27", "0.15~0.26", "0.15~0.25", "0.15~0.24", "0.15~0.23", "0.15~0.22", "0.15~0.21", "0.15~0.20", "0.15~0.19", "0.15~0.18", "0.15~0.17", "0.15~0.16" and the like. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all fractions within the range (at least 2 decimal places), and the technical effects of the present invention can be achieved within the above range.

[0041] The thermosensitive gel matrix of the present invention refers to a polymer that responds to temperature stimulation, and can be divided into natural polymer materials and synthetic polymer materials according to their sources. Commonly used thermosensitive gel matrices include cellulose, chitosan, and poloxamer.

[0042] In a preferred embodiment, the Panax notoginseng total saponins comprise one or more of the following chemical structural formulas:

[0043]

[0044] In a preferred embodiment, the mass ratio between the Panax notoginseng total saponins and the first pharmaceutically acceptable excipient is 0.2-0.4, such as 0.25-0.35, such as about 0.32.

[0045] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.32" includes ±5% of 0.32, or from 0.304 to 0.336.

[0046] In a preferred embodiment, the thermosensitive gel matrix is ​​poloxamer. In a preferred embodiment, the poloxamer is poloxamer 407 and / or poloxamer 188.

[0047] In order to make the thermosensitive gel have better gel formability, a phase change can occur within a certain temperature range. In a preferred embodiment, based on the volume of the eye drops, the mass volume concentration of poloxamer 407 is 24% to 28%, such as 26% to 28%, such as about 27.414%.

[0048] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 27.414" includes ±5% of 27.414, or from 26.0433 to 28.7847.

[0049] In a preferred embodiment, based on the volume of the eye drops, the mass volume concentration of Poloxamer 188 is 0% to 1%, such as 0.1% to 0.6%, such as about 0.513%.

[0050] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.513" includes ±5% of 0.513, or from 0.48735 to 0.53865.

[0051] In order to achieve better therapeutic effects of the eye drops, in a preferred embodiment, the mass volume concentration of the notoginseng total saponins is 5% to 9.5%, such as 7% to 9.5%, such as about 9%, based on the volume of the eye drops.

[0052] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 9" includes ±5% of 9, or from 8.55 to 9.45; "about 90" includes ±5% of 90, or from 85.5 to 94.5.

[0053] In a preferred embodiment, the eye drops further include a pharmaceutically acceptable second excipient, wherein the pharmaceutically acceptable second excipient includes one or more of the following: an antibacterial agent, a penetration enhancer, a viscosity regulator, a stabilizer, a solubilizer and a co-solvent.

[0054] In the present invention, the term "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, which does not cause the biological activity or properties of the compound to disappear, and is relatively non-toxic, such as, giving an individual a substance, does not cause unwanted biological effects or interacts with any of its contained components in a harmful manner. The term "pharmaceutically acceptable first adjuvant" or "pharmaceutically acceptable second adjuvant" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (that is, can induce the desired therapeutic effect without causing any undesirable local or systemic effects), which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). The above-mentioned adjuvants are preferably pharmaceutically inert, or can have a synergistic or increasing effect to enhance the therapeutic activity of the pharmaceutical composition, and the above-mentioned adjuvants are only enumerated, and the adjuvants actually used in the present invention are not limited to the above-mentioned adjuvants, and can be adjusted according to actual conditions, and the effects of the present invention can be achieved.

[0055] In a preferred embodiment, the pharmaceutically acceptable second excipient is an antibacterial agent, such as benzalkonium chloride.

[0056] In a preferred embodiment, the mass volume concentration of the benzalkonium chloride is 0.005% to 0.05%, for example, about 0.01%, based on the volume of the eye drops.

[0057] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 0.01" includes ±5% of 0.01, or from 0.0095 to 0.0105.

[0058] In a preferred embodiment, the pharmaceutically acceptable second excipient does not include a pH regulator. Since the pH value of the thermosensitive gel measured in the present invention is between 7 and 8, the thermosensitive gel of the present invention does not need to add a pH regulator.

[0059] In a preferred embodiment, the pharmaceutically acceptable second excipient does not include an osmotic pressure regulator. Since the thermosensitive gel of the present invention changes from a solution state to a gel state on the ocular surface, the influence of osmotic pressure does not need to be considered, so there is no need to add an osmotic pressure regulator such as sodium chloride to the thermosensitive gel of the present invention.

[0060] In a preferred embodiment, the eye disease is an endocervical disease. In a preferred embodiment, the eye disease is a retinopathy. In a preferred embodiment, the retinopathy is diabetic retinopathy and / or central retinal vein occlusion. In a preferred embodiment, the diabetic retinopathy is proliferative diabetic retinopathy and / or non-proliferative diabetic retinopathy.

[0061] In a preferred embodiment, the eye drops further include one or more drugs and / or extracts for preventing and treating eye diseases. In a preferred embodiment, the drug is an endothelial growth factor antagonist. In a preferred embodiment, the endothelial growth factor antagonist is selected from one or more of the following: ranibizumab, aflibercept and conbercept.

[0062] According to another aspect of the present invention, a method for preparing the above-mentioned eye drops is provided, which is a cold dissolution method.

[0063] In a preferred embodiment, the method comprises the following steps:

[0064] (1) adding an appropriate amount of a pharmaceutically acceptable first excipient to water for swelling to obtain a swollen pharmaceutically acceptable first excipient; and

[0065] (2) Weighing an appropriate amount of Panax notoginseng total saponins, adding water to dissolve, adding the swollen pharmaceutically acceptable first excipient to perform a first mixing, and letting it stand to obtain the Panax notoginseng total saponins thermosensitive gel eye drops,

[0066] Wherein, the first pharmaceutically acceptable excipient is a thermosensitive gel matrix.

[0067] In a preferred embodiment, in step (1), the thermosensitive gel matrix is ​​poloxamer. In a preferred embodiment, the poloxamer is poloxamer 407 and / or poloxamer 188.

[0068] In a preferred embodiment, based on the volume of the eye drops, the mass volume concentration of the poloxamer 407 is 24% to 28%, such as 26% to 28%, such as about 27.414%.

[0069] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 27.414" includes ±5% of 27.414, or from 26.0433 to 28.7847.

[0070] In a preferred embodiment, based on the volume of the eye drops, the mass volume concentration of Poloxamer 188 is 0% to 1%, such as 0.1% to 0.6%, such as about 0.513%.

[0071] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.513" includes ±5% of 0.513, or from 0.48735 to 0.53865.

[0072] In a preferred embodiment, the water is purified water.

[0073] In a preferred embodiment, the swelling temperature is 3°C to 5°C, such as about 4°C.

[0074] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0075] In a preferred embodiment, the swelling time is 8 hours to 16 hours, such as about 12 hours.

[0076] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12" includes ±5% of 12, or from 11.4 to 12.6.

[0077] In a preferred embodiment, step (2) further comprises adding an appropriate amount of a pharmaceutically acceptable second excipient after the first mixing and stirring to mix.

[0078] In a preferred embodiment, in step (2), the water is purified water.

[0079] In a preferred embodiment, the pharmaceutically acceptable second excipient includes one or more of the following: an antibacterial agent, a penetration enhancer, a viscosity modifier, a stabilizer, a solubilizer, and a cosolvent. In a preferred embodiment, the pharmaceutically acceptable second excipient is an antibacterial agent, such as benzalkonium chloride. In a preferred embodiment, the mass volume concentration of the benzalkonium chloride is 0.005% to 0.05%, such as about 0.01%, based on the volume of the eye drops.

[0080] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 0.01" includes ±5% of 0.01, or from 0.0095 to 0.0105.

[0081] In a preferred embodiment, the second pharmaceutically acceptable excipient does not include a pH regulator. In a preferred embodiment, the second pharmaceutically acceptable excipient does not include an osmotic pressure regulator.

[0082] In a preferred embodiment, the standing temperature is 3°C to 5°C, such as about 4°C.

[0083] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0084] In a preferred embodiment, the standing time is 8 hours to 16 hours, such as about 12 hours.

[0085] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12" includes ±5% of 12, or from 11.4 to 12.6.

[0086] In a preferred embodiment, as a result of the standing, the eye drops are transparent and clear.

[0087] In a preferred embodiment, based on the volume of the eye drops, the mass volume concentration of the notoginseng total saponins is 5% to 9.5%, such as 7% to 9.5%, such as about 9%.

[0088] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 9" includes ±5% of 9, or from 8.55 to 9.45.

[0089] In a preferred embodiment, the light transmittance of the thermosensitive gel is greater than 90%.

[0090] According to another aspect of the present invention, there is provided a use of the above-mentioned eye drops in the preparation of a medicament for preventing and treating eye diseases.

[0091] In the present invention, the term "prevention" includes "treatment" and / or "prevention", unless there is a specific description to the contrary. The terms "treatment" and "therapeutically" should be understood accordingly.

[0092] In the present invention, the term "treatment" includes alleviating, inhibiting or improving the symptoms or conditions of a disease; inhibiting the occurrence of complications; improving or preventing potential metabolic syndrome; inhibiting the occurrence of a disease or symptom, such as controlling the development of a disease or condition; alleviating a disease or symptom; reducing a disease or symptom; alleviating complications caused by a disease or symptom, or preventing or treating signs caused by a disease or symptom. As used herein, a pharmaceutical composition or a pharmaceutical preparation, after administration, can improve a disease, symptom or condition, especially improve its severity, delay the onset, slow the progression of the disease, or reduce the duration of the disease. Whether fixed or temporary administration, continuous or intermittent administration, can be attributed to or related to the administration.

[0093] In a preferred embodiment, the eye disease is an endocervical disease. In a preferred embodiment, the eye disease is a retinopathy. In a preferred embodiment, the retinopathy is diabetic retinopathy and / or central retinal vein occlusion. In a preferred embodiment, the diabetic retinopathy is proliferative diabetic retinopathy and / or non-proliferative diabetic retinopathy.

[0094] According to another aspect of the present invention, there is provided an eye drop as described above for use in preventing and / or treating an eye disease, such as an endocervical disease, such as a retinopathy, in a subject.

[0095] According to another aspect of the present invention, there is provided a method for preventing and / or treating an ocular disease, such as an endocervical disease, such as a retinopathy, in a subject, comprising administering an effective amount of the above eye drops to the subject.

[0096] In the present invention, the term "subject" is a mammal. The mammal may be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse or a cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects representing eye disease models. Preferably, the subject is a human.

[0097] The "effective amount" of the pharmaceutical composition or preparation used in the present invention can obtain the desired treatment and / or prevention effect. The amount effective for this purpose will depend on, for example, the pharmaceutical composition, the mode of administration, the stage and severity of the disease to be treated, the individual's body weight and overall health, and the judgment of the prescribing physician. The dosage can be given once a week, or once every two days or every day, or even several times a day. The dosage unit can be given in a short period of time (e.g., weeks to months) or longer time periods (months to years).

[0098] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0099] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0100] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0101] Example

[0102] Preparation Example

[0103] 1. Instruments and Materials

[0104] 1.1 Instrument

[0105] Sartorius electronic balance (Beijing Sartorius Scientific Instrument Co., Ltd.), RT10 magnetic stirrer (Aika Co., Ltd., Germany), SK1200H ultrasonic cleaner (Shanghai Kedao Ultrasonic Instrument Co., Ltd.), ZEISS Gemini300 scanning electron microscope (SEM, Carl Zeiss AG, Germany), Agilent 1260 high performance liquid chromatograph (Agilent Technologies, Inc., USA), high-speed refrigerated centrifuge (Eppendorf, Germany), pH meter (Mettler Toledo Instrument Shanghai Co., Ltd.), thermometer (Hongchang Company), Milli-Q ultrapure water preparation instrument (Millipore Company, USA), and vials (Tuochen Packaging Materials Company).

[0106] 1.2 Materials

[0107] Total saponins of Panax notoginseng (take 1000g of crude powder of Panax notoginseng main root, use 95% ethanol as solvent, soak for 40 hours, then perform percolation at a speed of 1-2ml per minute, collect the percolation liquid until the total saponins of Panax notoginseng are completely filtered out (checked by Liberman reaction), decolorize the percolation liquid with alumina, filter, recover ethanol and concentrate it to a thick paste, degrease it after water precipitation, adsorb and elute it with a resin column, collect the eluate, recover the solvent, concentrate it to a thick paste, and dry it to obtain PNS for short), poloxamer 407 (Mei Lun Biotechnology), poloxamer 188 (Mei Lun Biotechnology), and benzalkonium chloride (Yuan Ye Biotechnology).

[0108] 2. Preparation method

[0109] 2.1 Preparation of PNS thermosensitive gel

[0110] Measure 7 mL of purified water and place it in a vial, add 0.5 g of hydroxypropyl methylcellulose (HPMC) and stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.7 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly to obtain PNS thermosensitive gel 1.

[0111] 2.2 Preparation of PNS thermosensitive gel II

[0112] Weigh 0.2g chitosan (CS) and dissolve it in 10mL 0.1mol / L glacial acetic acid; weigh 1.8g sodium β-glycerophosphate (β-GP) and dissolve it in 3mL purified water; mix the two in a ratio of 7:3 to obtain a CS / β-GP solution; weigh 0.7g PNS and add it to 3mL purified water to fully dissolve it, mix it evenly with the CS / β-GP solution, adjust the pH value to about 5 with glacial acetic acid, and obtain PNS thermosensitive gel II.

[0113] 2.3 Preparation of PNS thermosensitive gel 3

[0114] Measure 7 mL of purified water and place it in a vial, add 2.4 g of poloxamer 407 (P407) and stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.7 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly to obtain PNS thermosensitive gel three.

[0115] 2.4 Preparation of PNS thermosensitive gel IV

[0116] Measure 7 mL of purified water and place it in a vial, add 1.8 g of P407 and stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.9 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly to obtain PNS thermosensitive gel IV.

[0117] 2.5 Preparation of PNS Thermosensitive Gel 5

[0118] Measure 7 mL of purified water and place it in a vial, add 2.0 g of P407 and stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.9 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly to obtain PNS thermosensitive gel V.

[0119] 2.6 Preparation of PNS Thermosensitive Gel VI

[0120] Measure 7 mL of purified water and place it in a vial, add 2.2 g of P407 and stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.9 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly to obtain PNS thermosensitive gel VI.

[0121] 2.7 Preparation of PNS thermosensitive gel VII

[0122] Measure 7 mL of purified water and place it in a vial, add 2.4 g of P407 and stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.9 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly to obtain PNS thermosensitive gel VII.

[0123] 2.8 Preparation of PNS Thermosensitive Gel VIII

[0124] Measure 7 mL of purified water and place it in a vial, add 2.6 g of P407 and stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.9 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly to obtain PNS thermosensitive gel VIII.

[0125] 2.9 Preparation of PNS thermosensitive gel

[0126] Measure 7 mL of purified water and place it in a vial, add 2.8 g of P407 and stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.9 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly to obtain PNS thermosensitive gel nine.

[0127] 2.10 Preparation of PNS Thermosensitive Gel

[0128] Measure 7 mL of purified water and place it in a vial, add 2.6 g of P407 and 0.1 g of poloxamer 188 (P188), stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.9 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly to obtain PNS thermosensitive gel ten.

[0129] 2.11 Preparation of PNS thermosensitive gel

[0130] Measure 7 mL of purified water and place it in a vial, add 2.6 g of P407 and 0.2 g of P188, stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.9 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly to obtain PNS thermosensitive gel XI.

[0131] 2.12 Preparation of PNS Thermosensitive Gel XII

[0132] Measure 7 mL of purified water and place it in a vial, add 2.6 g of P407 and 0.3 g of P188, stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.9 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly to obtain PNS thermosensitive gel twelve.

[0133] 2.13 Preparation of PNS thermosensitive gel

[0134] Measure 7 mL of purified water and place it in a vial, add 2.6 g of P407 and 0.4 g of P188, stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.9 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly to obtain PNS thermosensitive gel 13.

[0135] 2.14 Preparation of PNS Thermosensitive Gel XIV

[0136] Measure 7 mL of purified water and place it in a vial, add 2.741 g of P407 and 0.051 g of P188, stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.9 g of PNS and add it into 3 mL of purified water to fully dissolve; mix the two evenly to obtain PNS thermosensitive gel fourteen.

[0137] 2.15 Preparation of PNS Thermosensitive Gel

[0138] Measure 7 mL of purified water and place it in a vial, add 2.741 g of P407 and 0.051 g of P188, stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.9 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly, then add 1 mg of benzalkonium chloride and stir evenly to obtain PNS thermosensitive gel fifteen.

[0139] 2.16 Preparation of PNS Thermosensitive Gel

[0140] Measure 7 mL of purified water and place it in a vial, add 2.741 g of P407 and 0.051 g of P188, stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.05 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly, then add 1 mg of benzalkonium chloride and stir evenly to obtain PNS thermosensitive gel sixteen.

[0141] 2.17 Preparation of PNS Thermosensitive Gel

[0142] Measure 7 mL of purified water and place it in a vial, add 2.741 g of P407 and 0.051 g of P188, stir evenly, and place in a 4°C refrigerator overnight to allow it to fully swell; weigh 0.2 g of PNS and add it to 3 mL of purified water to fully dissolve; mix the two evenly, then add 1 mg of benzalkonium chloride and stir evenly to obtain PNS thermosensitive gel seventeen.

[0143] 3. Experimental Methods

[0144] 3.1 Screening of thermosensitive gel matrix

[0145] Hydroxypropyl methylcellulose (HPMC), chitosan (CS) and P407 were selected as gel matrices to prepare thermosensitive gels, namely PNS thermosensitive gel 1, PNS thermosensitive gel 2 and PNS thermosensitive gel 3, respectively. The appearance, gelation temperature and PNS content were investigated to select the appropriate gel matrix.

[0146] 3.2 Screening of P407 blank gel

[0147] Thermosensitive gels need to have good gel formability and can undergo phase transition within a certain temperature range. In order to screen out the P407 concentration range that meets the requirements, the gelation temperature (T1) was used as an indicator to investigate P407 blank gels in the range of 15% to 26%.

[0148] 3.3 Screening of drug loading of PNS

[0149] Eye drops have a small dosage and require a higher drug loading to achieve better efficacy. In order to screen out the maximum drug loading of PNS in thermosensitive gels, the P407 concentration was fixed at 24%, and 5%, 7.5%, 8%, 9%, 10%, and 11% PNS thermosensitive gels were prepared, and the appearance of the thermosensitive gels was observed.

[0150] 3.4 Single Factor Investigation

[0151] 3.4.1 Screening of P407 concentration

[0152] Taking T1 and the gelation temperature after tear dilution (T2) as investigation indicators, the content of PNS was fixed, and different concentrations of P407 (18%, 20%, 22%, 24%, 26%, 28%) were investigated, namely PNS thermosensitive gel 4, PNS thermosensitive gel 5, PNS thermosensitive gel 6, PNS thermosensitive gel 7, PNS thermosensitive gel 8, and PNS thermosensitive gel 9. Each concentration of thermosensitive gel was tested in triplicate, T1 and T2 were measured, and the average value was taken.

[0153] 3.4.2 Screening of P188 concentration

[0154] Taking T1 and T2 as the investigation indexes, the contents of PNS and P407 were fixed, and different concentrations of P188 (0%, 1%, 2%, 3%, 4%) were investigated, namely PNS thermosensitive gel eight, PNS thermosensitive gel ten, PNS thermosensitive gel eleven, PNS thermosensitive gel twelve, and PNS thermosensitive gel thirteen. Each concentration of thermosensitive gel was tested in triplicate, and T1 and T2 were measured to take the average value.

[0155] 3.5 Star-point design-response surface methodology to optimize prescription

[0156] According to the results of the single-factor experiment, the concentrations of P407 (A) and P188 (B) were determined as the investigation factors. Each factor was set to 5 levels, coded as ±α (1.414), ±1 and 0. T1 and T2 were used as response values. The Design Expert software was used to conduct a 2-factor 5-level central-center design-response surface method (CCD-RSM) design experiment.

[0157] 3.6 Selection of excipients for PNS thermosensitive gel

[0158] 3.6.1 Antibacterial agents

[0159] Thermosensitive gel was prepared according to the optimal formulation, 0.01% benzalkonium chloride was added as an antibacterial agent, and T1 and T2 were measured.

[0160] 3.6.2 pH regulator

[0161] Thermosensitive gel was prepared according to the optimal prescription, 0.01% benzalkonium chloride was added, and the pH value of the thermosensitive gel tear before and after dilution was measured using a pH meter.

[0162] 3.7 Characterization of PNS thermosensitive gel

[0163] 3.7.1 SEM observation

[0164] Take an appropriate amount of PNS thermosensitive gel and freeze it at -80°C, then freeze-dry it in a freeze dryer, take an appropriate amount of freeze-dried gel powder, spray-treat it with gold, and observe the microscopic morphology of the gel using SEM.

[0165] 3.7.2 Light transmittance

[0166] Take an appropriate amount of PNS thermosensitive gel and heat it to a gel state, and use a UV-visible spectrophotometer to measure the absorbance under visible light of 390-780nm.

[0167] 3.8 In vitro release of PNS thermosensitive gel

[0168] The dialysis bag method was used to investigate the in vitro release of PNS thermosensitive gel. 5 mL of PNS thermosensitive gel was precisely transferred into the dialysis bag, the bag mouth of the dialysis bag was tied tightly and placed in a 250 mL sealed bottle, 200 mL of PBS (34°C) was added as the diffusion medium, and the sealed bottle was placed on a magnetic stirrer, the temperature was controlled at 34°C, the speed was 100 rpm, 1 mL of samples were taken at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, and 72 h, and isothermal and equal volume of PBS were added in time. The samples were centrifuged and the supernatant was taken, and the PNS content was measured by HPLC.

[0169] 3.9 Comparison of in vitro release between PNS thermosensitive gel and pH sensitive gel

[0170] The dialysis bag method was used to investigate the in vitro release of PNS thermosensitive gel and pH sensitive gel (prescription 1 and prescription 2 of patent ZL202210010949.4). 0.5%, 2%, and 9% PNS thermosensitive gel and 0.5% and 2% PNS pH sensitive gel were prepared, 5 mL of PNS gel was accurately transferred into the dialysis bag, the dialysis bag was tied tightly and placed in a 250 mL sealed bottle, 200 mL of PBS (34°C) was added as the diffusion medium, and the sealed bottle was placed on a magnetic stirrer, the temperature was controlled at 34°C, the speed was 100 rpm, 1 mL of samples were taken at 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h, and 72h, and isothermal and equal volume of PBS were added in time. The supernatant was taken after the sample was centrifuged, and the PNS content was measured by HPLC, and the in vitro release of the two gels was compared.

[0171] 4. Results

[0172] 4.1 Screening of thermosensitive gel matrix

[0173] The present invention measures the gelation temperature of the thermosensitive gel prepared by HPMC, CS and P407 as gel matrix before and after tear dilution, and finds that when HPMC is used as gel matrix, the gelation temperature of the prepared thermosensitive gel is too high and is not suitable for the gelation temperature required by the eye. In the process of preparing CS thermosensitive gel, it is found that CS can fully swell under acidic conditions. Considering that the PNS drug itself is easily degraded under acidic conditions, we have also measured the content of CS and P407 thermosensitive gels. The results show that the content of PNS in CS thermosensitive gel decreases, and it also irritates the eyes, indicating that CS is not suitable as the gel matrix of the present invention; and the thermosensitive gel prepared by P407 has a clear and transparent appearance, a suitable gelation temperature, and a small effect on the PNS content. In addition, P407 has the characteristics of good biocompatibility and good stability, so P407 is finally selected as the gel matrix of PNS ophthalmic thermosensitive gel eye drops. The results are shown in Table 1.

[0174] Table 1 Effect of different gel matrices on gelation temperature and PNS content

[0175] <![CDATA[T1(℃)]]> <![CDATA[T2(℃)]]> Dosage(mg) Measured amount (mg) HPMC — — 765.00 752.40 CS 26.0 34.3 765.00 543.83 P407 26.8 34.0 765.00 750.97

[0176] Note: — indicates the temperature is greater than 40°C.

[0177] 4.2 Screening of P407 blank gel

[0178] like Figure 1 As shown, since 15%, 16%, and 17% of P407 still do not form gel when the temperature rises to 40°C, the concentration range for screening P407 is 18% and above.

[0179] 4.3 Screening of PNS content

[0180] The appearance observation results of thermosensitive gels with different contents showed that the maximum content of PNS in the thermosensitive gel was 9%. The results are shown in Table 2.

[0181] Table 2 Effect of PNS content on the appearance of thermosensitive gel

[0182]

[0183]

[0184] 4.4 Single Factor Investigation

[0185] 4.4.1 Screening of P407 concentration

[0186] The content of PNS was fixed at 9%, and different concentrations of P407 were investigated with T1 and T2 as the investigation indexes. The results are shown in Table 3. T2 was preferably between 33 and 35°C, and the preliminary screening concentration of P407 was 24% to 28%.

[0187] Table 3 Effect of P407 concentration on gelation temperature of thermosensitive gel

[0188] P407 concentration (%) <![CDATA[T1(℃)]]> <![CDATA[T2(℃)]]> 18 — — 20 — — 22 36.8 — 24 31.2 38.7 26 26.7 33.1 28 24.2 28.9

[0189] Note: — indicates the temperature is greater than 40°C.

[0190] 4.4.2 Screening of P188 concentration

[0191] The content of fixed PNS was 9%, the concentration of P407 was 26%, and different concentrations of P188 were investigated with T1 and T2 as the investigation indexes. The measurement results are shown in Table 4. It is appropriate to take T2 between 33 and 35°C, and the preliminary screening concentration of P188 is 0% to 1%.

[0192] Table 4 Effect of P188 concentration on gelation temperature of thermosensitive gel

[0193] P188 concentration (%) <![CDATA[T1(℃)]]> <![CDATA[T2(℃)]]> 0 26.7 33.1 1 30.9 38.5 2 35.0 — 3 38.5 — 4 — —

[0194] Note: — indicates the temperature is greater than 40°C.

[0195] 4.5 Star-point design-response surface methodology to optimize prescription

[0196] Using Design Expert 10.0.7 software, according to the test results of single factor investigation, the dosage of P407 (A) and P188 (B) were selected as the main influencing factors, and their dosage ranges were determined: (A) 24%-28%, (B) 0%-1%. T1 and T2 were used as evaluation indicators for prescription optimization. According to the principle of central-point design, each factor was set to 5 levels, represented by codes ±α(1.414), ±1 and 0. The factor levels, experimental design and results of the central-composite effect surface method experiment are shown in Table 5. Figure 2 and Figure 3 shown.

[0197] Table 5 Factor levels, experimental design and results of the central composite effect surface method experiment

[0198] Test No. A / ％ B / ％ <![CDATA[T1 / ℃]]> <![CDATA[T2 / ℃]]> 1 24.59 0.15 30.9 41.5 2 27.41 0.15 26.0 31.4 3 24.59 0.85 33.9 44.3 4 27.41 0.85 28.9 35.3 5 24.00 0.50 34.5 47.4 6 28.00 0.50 26.5 32.4 7 26.00 0.00 26.9 31.6 8 26.00 1.00 32.5 40.8 9 26.00 0.50 29.5 39.1 10 26.00 0.50 30.2 39.4 11 26.00 0.50 29.4 37.0 12 26.00 0.50 29.4 37.0 13 26.00 0.50 30.3 37.9

[0199] As the eye temperature is about 33-35°C and the thermosensitive gel should be in liquid state before administration (ie at room temperature), T1>26°C and T2<35°C are appropriate, and the optimal prescription is optimized: P407 27.414%, P188 0.513%.

[0200] The optimal prescription was verified. Three gels were prepared according to PNS thermosensitive gel 14, and T1 and T2 were measured. The prescription verification results showed that the deviation values ​​were all less than ±2%, and the model had good predictive performance. The results are shown in Table 6.

[0201] Table 6 PNS thermosensitive gel prescription verification results

[0202]

[0203]

[0204] 4.6 Selection of excipients for PNS thermosensitive gel

[0205] 4.6.1 Antibacterial agents

[0206] From the measured gelation temperature, it can be seen that the addition of benzalkonium chloride has no effect on the gelation temperature. The results are shown in Table 7.

[0207] Table 7 Effect of benzalkonium chloride on gelation temperature

[0208] serial number <![CDATA[T1 / ℃]]> <![CDATA[T2 / ℃]]> 1 27.0 34.4 2 26.8 34.2 3 27.5 34.5

[0209] 4.6.2 pH regulator

[0210] From the measured pH value results, it can be seen that the pH of the thermosensitive gel meets the pH requirement of 5-9 for the eye, so there is no need to add a pH adjuster. The results are shown in Table 8.

[0211] Table 8 PNS thermosensitive gel pH measurement results

[0212] serial number pH of tear before dilution pH of diluted tears 1 7.06 7.84 2 7.12 7.70 3 7.12 7.78

[0213] 4.7 Characterization of PNS thermosensitive gel

[0214] 4.7.1 SEM observation

[0215] like Figure 4 As shown, the freeze-dried PNS thermosensitive gel 15 has a porous structure with loose pores, which is conducive to drug embedding.

[0216] 4.7.2 Light transmittance

[0217] like Figure 5 As shown, the transmittance of PNS thermosensitive gel 15 under visible light of 390-780 nm is greater than 90%.

[0218] 4.8 In vitro release of PNS thermosensitive gel

[0219] like Figure 6 As shown in Figure 2, the release of each component of the PNS solution tends to be stable within 24 hours. Figure 7 As shown in Figure 15, the release of each component of PNS thermosensitive gel 15 tended to be stable at 48h; at 72h, the release of the solution reached 90%, while the release of the thermosensitive gel was 65%, and the sustained release effect was significantly better than that of the solution group (such as Figure 8 shown).

[0220] 4.9 Comparison of in vitro release between PNS thermosensitive gel and pH sensitive gel

[0221] like Fig. 9 As shown, the release amount of 0.5% PNS pH sensitive gel in 72 hours is 91%, the release amount of 0.5% PNS thermosensitive gel sixteen in 72 hours is 87%, the release amount of 2% PNS pH sensitive gel in 72 hours is 84%, and the release amount of 2% PNS thermosensitive gel seventeen in 72 hours is 45%. The release amount of the best prescription 9% PNS thermosensitive gel fifteen of the present invention is 65% in 72 hours. The results show that when the mass-to-volume ratio of PNS is the same, PNS thermosensitive gel has better sustained-release effect than pH sensitive gel.

[0222] 5. Conclusion

[0223] According to the appearance clarity, transparency and stability observation of the thermosensitive gel of the present invention, the mass volume concentration of PNS was screened out to be 9%; the gelation temperature before and after tear dilution was used as an indicator, and the contents of P407 and P188 were investigated by single factor experiment, and the dosage range of P407 was screened out to be 24% to 28%, and the dosage range of P188 was 0% to 1%; the single factor screening prescription was optimized by selecting 2 factors and 5 levels by using the star point design-response surface method, and the gelation temperature after tear dilution was used as an indicator, and the concentration of P407 was optimized to be 27.414%, and the concentration of P188 was optimized to be 0.513%. The optimal prescription; the thermosensitive gel with 0.01% benzalkonium chloride was measured, and the addition of antibacterial agent had no effect on the gelation temperature; the pH value of the thermosensitive gel was measured to be between 7 and 8, so there was no need to add a pH adjuster; the PNS thermosensitive gel was characterized by SEM and transmittance measurement, and the thermosensitive gel had a loose porous structure inside, which was conducive to the embedding of drugs. The transmittance of the thermosensitive gel was more than 90%, and it would not cause blurred vision after ocular administration; using PNS solution as a control, the dialysis bag method was used to study the cumulative release of the thermosensitive gel, and the results showed that the thermosensitive gel had a better sustained-release effect.

[0224] Preparation efficacy example

[0225] 1. Instruments and Materials

[0226] 1.1 Instrument

[0227] Accu-Check blood glucose meter (Roche, Germany); dissecting microscope (Olympus, Japan); HL-2D timed digital display constant flow pump (Shanghai Huxi Analytical Instrument Factory Co., Ltd.); electric heating blast vacuum drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd., China); IX81 fluorescence inverted microscope (Olympus, Japan); microplate reader (BioTek, USA); S0200-230V-EU vortex mixer (Labnet, USA); low-temperature high-speed refrigerated centrifuge (Eppendorf, Germany); protein vertical electrophoresis instrument and transfer mold instrument (Bio-Rad, USA); BP211D electronic analytical balance (Sartorins, Germany); ultrapure water filter (Millipore, USA).

[0228] 1.2 Materials

[0229] PNS thermosensitive gel 15, Accu-Check blood glucose test strips (Roche, Germany); PNS (preparation method is the same as the preparation method of Panax notoginseng total saponins in "1.2 Materials" in the preparation example); citric acid anhydrous, sodium citrate dihydrate, streptozotocin (STZ), Evans blue dye, formamide (Dalian Meilun Biotechnology Co., Ltd., China); fluorescein isothiocyanate-concanavalin A lectin (FITC-ConA) (Vector Laboratories, USA); intercellular adhesion molecule (ICAM-1) and claudin-1 antibodies (Santa Cruz, USA): Occludin, vascular cell adhesion molecule (VCAM-1), calcium ion binding adapter molecule-1 (Iba-1), nuclear transcription factor (NF-κB) p65, phosphorylated NF-κB p65 (p-p65), phosphorylated IκB kinase (p-IKK), phosphorylated NF-κB inhibitor (p-IκB), β-actin and lamin B1 antibodies (Cell Signaling Technology, USA); protein marker (8-180 kDa), trypsin, BCA protein assay kit, peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) (H+L) and anti-mouse IgG (H+L) (Shanghai Yisheng Biotechnology Co., Ltd., China); cytoplasm and nuclear extraction reagent (Thermo Scientific, USA); enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Viagra Biotechnology Co., Ltd., China); other reagents were purchased from Beyotime Biotechnology Co., Ltd.

[0230] 2. Experimental Methods

[0231] 2.1 Experimental animals

[0232] Male Sprague Dawley rats (weight 200 ± 20 g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (certificate number: SCXK (Beijing) 2016-0006) and were housed in an SPF-grade environment with a temperature of 25 ± 2 °C, a humidity of 55 ± 15%, and a light / dark cycle of 12 h. All rats were housed for 7 days before the experiment. Animal experiments were performed under the institutional animal care guidelines approved and supervised by the Animal Experiment Ethics Committee of Shanghai University of Traditional Chinese Medicine (approval number: PZSHUTCM211115003).

[0233] 2.2 Establishment of STZ-induced NPDR model in rats

[0234] Mix 0.1M citric acid solution and disodium citrate solution in a ratio of 14:11, adjust the pH to 4.4, and use it to prepare STZ solution. Weigh an appropriate amount of STZ powder, dissolve it in a dark place to a certain concentration, and then immediately inject it intraperitoneally into rats that have been fasting for 12 hours. The dose of STZ is 65mg / kg. Another group of rats were intraperitoneally injected with solvent control as a normal control group. After 7 days of modeling, blood was collected from the tail vein to test the fasting blood glucose concentration (FBG). FBG ≥ 16.7mmol / L (250mg / dL) was considered a successful diabetes model.

[0235] 2.3 Grouping and Dosing

[0236] The diabetic rats with successful modeling were randomly divided into model group, PNS solution group and PNS thermosensitive gel eye drops group according to body weight and FBG. Two months after intraperitoneal injection of STZ, the PNS solution group was given 9% PNS isotonic solution (osmotic pressure is the same as tears) in both eyes of the rats at a dose of 40 mg / kg, and the PNS thermosensitive gel group was given 9% PNS thermosensitive gel fifteen in both eyes of the rats at a dose of 40 mg / kg, 3 times a day, each time with an interval of 4 hours. The normal control group and the model group were given 15 μL of normal saline in both eyes of the rats, 3 times a day, each time with an interval of 4 hours, for 1 month.

[0237] 2.4 Sampling and index testing

[0238] 2.4.1 Sampling and testing

[0239] During the experiment, the body weight and FBG of the rats were recorded. After the treatment, retinal acellular capillary detection, hematoxylin and eosin (H&E) and immunofluorescence staining were performed in 4 rats in each group, Evans blue dye leakage detection was performed in 4 rats in each group, and neutrophil adhesion was performed in 4 rats in each group. Serum and retina were collected from other rats after anesthesia and stored at -80°C.

[0240] 2.4.2 Pathological sections and immunohistochemistry experiments

[0241] Rats were dissected and PBS was perfused from the left ventricle to remove blood from the vessels. The left eyes were fixed in ophthalmic fixative, embedded in paraffin, and sectioned (5 μm). Retinal sections were stained with H&E, and images of the sections were obtained using an inverted microscope (BDS200, Nikon, Japan). Paraffin-embedded retinal sections (5 μm) were deparaffinized in xylene and rehydrated with distilled water in an ethanol gradient. After endogenous peroxidase activity was quenched, retinal sections were incubated with 5% bovine serum albumin to reduce nonspecific binding. Retinal sections were incubated with the corresponding antibodies at 4°C overnight and with species-matched fluorescent secondary antibodies for 1 h at room temperature, and cell nuclei were stained with DAPI for 10 min at room temperature. Images were captured under an inverted fluorescence microscope (IX81, Olympus, Japan).

[0242] 2.4.3 Rat retinal Evans blue leakage experiment

[0243] Rats were intraperitoneally injected with 2% Evans blue 10 μL / g. After circulating for 2 hours, the left ventricle was irrigated with PBS to remove Evans blue from the mouse systemic circulation. The eyeballs were removed, the retinal tissue was peeled off, and it was placed in a vacuum drying oven at 55°C for 5 hours. The mass of the dried retinal tissue was weighed. 120 μL of formamide was added and Evans blue was extracted in a 70°C water bath for 18 hours. The extract was centrifuged at 10000rpm for 1 hour, and 100 μL of the extracted supernatant was taken and measured with an enzyme marker at a wavelength of 620nm. The absorbance value was substituted into the standard curve to calculate the amount of Evans blue. The ratio of the amount of Evans blue to the mass of the dried retinal tissue was calculated to obtain the Evans blue leakage in the retinal tissue (ng / g).

[0244] 2.4.4 Cell-free capillary experiments

[0245] The right eye was removed and fixed with 4% paraformaldehyde (PFA) for 24 h, and then washed 4 times with PBS. The retina was peeled off from the eyeball and then digested with 3% trypsin at 37°C for 1 h to separate the retinal blood vessels from the retinal tissue. In order to quantify the acellular capillaries in the retina, the isolated retinal blood vessels were stained with H&E staining, and the retinal acellular capillaries in the field of view were observed and photographed with an inverted microscope (BDS200, Nikon, Japan), and the total number of acellular capillaries in 3 fields of view was calculated, and the average value represented the number of acellular capillaries in the retinal sample.

[0246] 2.4.5 Neutrophil adhesion assay

[0247] 600 μL FITC-ConA (5 mg / mL) was injected into the rat tail vein to label adherent leukocytes and vascular endothelial cells. After 2 h of systemic circulation, the rats were dissected and PBS was perfused from the left ventricle to remove red blood cells and non-adherent leukocytes. The left eye was separated and fixed in 4% PFA for 2 h, and then the retina was separated and placed on a slide. In order to quantify the leukocytes adhered to the retina, the number of leukocytes in the field of view was observed and photographed with an inverted fluorescence microscope (IX81, Olympus, Japan), and the total number of adherent neutrophils in 3 fields of view was calculated, and the average value represented the number of neutrophils adherent to the retinal sample.

[0248] 2.4.6 ELISA experiment

[0249] Plasma was taken out from the −80 °C freezer, thawed at room temperature, and serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were measured using ELISA kits according to the manufacturer's instructions.

[0250] 2.4.7 Western blot experiment

[0251] The retina was taken out of the -80°C freezer and thawed on ice. The retina was homogenized in ice-cold lysis buffer and then centrifuged at 10,000 rpm for 5 min at 4°C. The protein concentration in the supernatant was determined using a BCA protein assay kit and each sample was normalized to the same protein concentration. The proteins were separated by SDS-PAGE and then transferred to a PVDF membrane. After blocking with 5% BSA in TBST for 1 h, the membrane was incubated with the corresponding primary antibody overnight at 4°C. The membrane was washed with TBST and incubated with the secondary antibody for 1 h at room temperature. Finally, the expression of the protein was visualized with a chemiluminescent reagent and quantified by calculating the gray density of the target protein blot compared to the internal control.

[0252] 2.4.8 Data Analysis

[0253] The experimental data were expressed as Mean±SD and analyzed by SPSS16.0 software. One-Way ANOVA statistical method was used for variance analysis. P<0.05 was considered statistically significant.

[0254] 3. Experimental results

[0255] PNS thermosensitive gel 15 can significantly reduce the increase in retinal vascular permeability (ranging from 30% to 60%), significantly reduce the increase in the number of acellular capillaries, significantly reduce the adhesion of neutrophils, significantly downregulate the increase in serum TNF-α, IL-6 and IL-1β levels, and significantly inhibit the activation of NF-κB signaling pathway. It has a certain therapeutic effect on NPDR, and the therapeutic effect of PNS thermosensitive gel 15 is better than that of PNS solution.

[0256] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A Panax notoginseng total saponin thermosensitive gel eye drops for preventing and treating eye diseases, characterized in that: The eye drops include notoginseng total saponins and a pharmaceutically acceptable first excipient, wherein the mass ratio between the notoginseng total saponins and the pharmaceutically acceptable first excipient is 0.15-0.4, and the pharmaceutically acceptable first excipient is a thermosensitive gel matrix.

2. The eye drops according to claim 1, characterized in that The Panax notoginseng total saponins include one or more of the following chemical structural formulas:

3. The eye drops according to claim 1, characterized in that The mass ratio between the total saponins of Panax notoginseng and the first pharmaceutically acceptable excipient is 0.2 to 0.4, such as 0.25 to 0.35, such as about 0.32; Preferably, the thermosensitive gel matrix is ​​poloxamer; Preferably, the poloxamer is poloxamer 407 and / or poloxamer 188; Preferably, based on the volume of the eye drops, the mass volume concentration of the poloxamer 407 is 24% to 28%, such as 26% to 28%, such as about 27.414%; Preferably, based on the volume of the eye drops, the mass volume concentration of Poloxamer 188 is 0% to 1%, such as 0.1% to 0.6%, such as about 0.513%; Preferably, based on the volume of the eye drops, the mass volume concentration of the total saponins of Panax notoginseng is 5% to 9.5%, such as 7% to 9.5%, such as about 9%; More preferably, the eye drops further include a pharmaceutically acceptable second excipient, wherein the pharmaceutically acceptable second excipient includes one or more of the following: an antibacterial agent, a penetration enhancer, a viscosity modifier, a stabilizer, a solubilizer, and a cosolvent; Also preferably, the pharmaceutically acceptable second excipient is an antibacterial agent, such as benzalkonium chloride; Preferably, the mass volume concentration of the benzalkonium chloride is 0.005% to 0.05%, for example, about 0.01%, based on the volume of the eye drops; Also preferably, the pharmaceutically acceptable second excipient does not include a pH adjuster; Also preferably, the pharmaceutically acceptable second excipient does not include an osmotic pressure regulator.

4. The eye drops according to claim 1, characterized in that The eye disease is an internal eye disease; Preferably, the eye disease is retinopathy; Preferably, the retinopathy is diabetic retinopathy and / or central retinal vein occlusion; More preferably, the diabetic retinopathy is proliferative diabetic retinopathy and / or non-proliferative diabetic retinopathy; Still preferably, the eye drops further include one or more drugs and / or extracts for preventing and treating eye diseases; Particularly preferably, the drug is an endothelial growth factor antagonist; Particularly preferably, the vascular endothelial growth factor antagonist is selected from one or more of the following: ranibizumab, aflibercept and conbercept.

5. A method for preparing the eye drops according to any one of claims 1 to 4, characterized in that: The method is a cold dissolution method.

6. The method according to claim 5, characterized in that The method comprises the following steps: (1) adding an appropriate amount of a pharmaceutically acceptable first excipient to water for swelling to obtain a swollen pharmaceutically acceptable first excipient; and (2) Weighing an appropriate amount of Panax notoginseng total saponins, adding water to dissolve, and then adding the swollen pharmaceutically acceptable first excipient to mix for the first time, and letting it stand to obtain the Panax notoginseng total saponins thermosensitive gel eye drops, Wherein, the pharmaceutically acceptable first excipient is a thermosensitive gel matrix.

7. The method according to claim 6, characterized in that In step (1), the thermosensitive gel matrix is ​​poloxamer; Preferably, the poloxamer is poloxamer 407 and / or poloxamer 188; Preferably, based on the volume of the eye drops, the mass volume concentration of the poloxamer 407 is 24% to 28%, such as 26% to 28%, such as about 27.414%; Preferably, based on the volume of the eye drops, the mass volume concentration of Poloxamer 188 is 0% to 1%, such as 0.1% to 0.6%, such as about 0.513%; Particularly preferably, the water is purified water; Particularly preferably, the swelling temperature is 3°C to 5°C, for example about 4°C; Particularly preferably, the swelling time is 8 hours to 16 hours, such as about 12 hours.

8. The method according to claim 6, characterized in that Step (2) further comprises adding an appropriate amount of a pharmaceutically acceptable second excipient after the first mixing and stirring and mixing; Preferably, in step (2), the water is purified water; Preferably, the pharmaceutically acceptable second excipient comprises one or more of the following: an antibacterial agent, a penetration enhancer, a viscosity modifier, a stabilizer, a solubilizer and a cosolvent; More preferably, the pharmaceutically acceptable second excipient is an antibacterial agent, such as benzalkonium chloride; More preferably, the mass volume concentration of the benzalkonium chloride is 0.005% to 0.05%, for example, about 0.01%, based on the volume of the eye drops; Also preferably, the pharmaceutically acceptable second excipient does not include a pH adjuster; Still more preferably, the pharmaceutically acceptable second excipient does not include an osmotic pressure regulator; Particularly preferably, the standing temperature is 3°C to 5°C, for example, about 4°C; Particularly preferably, the standing time is 8 hours to 16 hours, for example about 12 hours; Particularly preferably, as a result of the standing, the eye drops are transparent and clear; Particularly preferably, based on the volume of the eye drops, the mass volume concentration of the total saponins of Panax notoginseng is 5% to 9.5%, such as 7% to 9.5%, such as about 9%; Particularly preferably, the light transmittance of the temperature-sensitive gel is greater than 90%.

9. Use of the eye drops according to any one of claims 1 to 4 in the preparation of a medicament for preventing and treating eye diseases; Preferably, the eye disease is an endocervical disease; Preferably, the eye disease is retinopathy; Preferably, the retinopathy is diabetic retinopathy and / or central retinal vein occlusion; More preferably, the diabetic retinopathy is proliferative diabetic retinopathy and / or non-proliferative diabetic retinopathy.

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