WO2022052454A1 - 含酯基芳香丙酰胺类化合物在制备治疗干眼症药物中的应用 - Google Patents

含酯基芳香丙酰胺类化合物在制备治疗干眼症药物中的应用 Download PDF

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WO2022052454A1
WO2022052454A1 PCT/CN2021/085266 CN2021085266W WO2022052454A1 WO 2022052454 A1 WO2022052454 A1 WO 2022052454A1 CN 2021085266 W CN2021085266 W CN 2021085266W WO 2022052454 A1 WO2022052454 A1 WO 2022052454A1
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dry eye
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朱新法
朱然
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长春金赛药业有限责任公司
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Priority to EP21865517.3A priority patent/EP4212153A1/en
Priority to US18/245,055 priority patent/US20230364047A1/en
Priority to AU2021340604A priority patent/AU2021340604A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/325—Carbamic acids; Thiocarbamic acids; Anhydrides or salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4406—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 3, e.g. zimeldine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • A61P5/26—Androgens

Definitions

  • the invention belongs to the technical field of medicine, and in particular relates to the application of an ester group-containing aromatic propionamide compound in the preparation of a medicament for treating dry eye.
  • Dry eye is a general term for a variety of diseases characterized by abnormal tear quality or quantity or abnormal dynamics caused by any reason, resulting in decreased tear film stability, accompanied by ocular discomfort and/or ocular surface tissue lesions. Keratoconjunctivopathy and can affect vision. In recent years, with the widespread use of computers and smart phones, people's eye use time has continued to increase, coupled with the influence of bad eye habits, resulting in a significant increase in the incidence of dry eye disease. Dry eye syndrome has become a global epidemic disease, and the incidence of dry eye syndrome in my country is gradually increasing and has a tendency to be younger. Studies have shown that the inflammatory response of the ocular surface tissue is the main cause of the decrease in tear secretion in dry eye.
  • the treatment of dry eye is mainly based on artificial tears, corticosteroids, and immunosuppressants such as cyclosporine A.
  • artificial tears are only a substitute for tears, and have no therapeutic effect by themselves; long-term use of corticosteroids and cyclosporine A and other drugs have certain toxic and side effects on the ocular surface. Therefore, there is an urgent need for a drug with good effect and long-term use without obvious local and systemic side effects.
  • the purpose of the present invention is to provide an application of an ester-containing aromatic propionamide compound in the preparation of a medicament for treating dry eye.
  • the technical scheme adopted in the present invention is:
  • ester-containing aromatic propionamide compound in preparation of medicament for treating dry eye.
  • the dry eye is dry eye caused by sex hormone imbalance.
  • ester-containing aromatic propionamide compound is used in combination with a pharmaceutically acceptable excipient in the preparation of a drug for the treatment of dry eye.
  • ester-containing aromatic propionamide compound and pharmaceutically acceptable auxiliary materials are prepared in the form of ointment.
  • the pharmaceutically acceptable excipients are mineral oil and petrolatum.
  • the ester-containing aromatic propionamide compound is C 22 H 19 F 3 N 4 O 4 or C 22 H 18 F4N 4 O 4 , wherein C 22 H 19 F 3 N 4 O 4 or C 22 H 18
  • F4N 4 O 4 For the preparation method and structural formula of F4N 4 O 4 , refer to the contents described in the patent document with the application number of 2014103602169.
  • the effective dose of the C 25 H 17 F 3 N 4 O 4 is 0.5-6.75 mg/kg.
  • ester-containing aromatic propionamide compounds provided by the present invention are a new class of selective non-steroidal androgen receptor modulators, and have the effect of regulating androgen receptors, so they can be used for Treatment of dry eye caused by androgen imbalance, and animal experiments show that it can increase tear secretion in castrated rabbits, reduce corneal fluorescence staining score, increase tear film breakup time, and significantly improve experimental dry eye. effect.
  • Figure 1 Schematic diagram of the results of pathological examination of Harder's glands in each group.
  • Figure 2 Schematic diagram of the pathological examination results of meibomian glands in each group.
  • Figure 3 Graph of the effect of tear film break-up time in male cynomolgus monkey model animals.
  • Figure 4 Histogram of the effect of tear film break-up time in male cynomolgus monkey model animals.
  • Figure 5 Schematic diagram of the results of fluorescent staining of the corneas of both eyes in the experiment of male cynomolgus monkeys.
  • Fig. 6 Graph of the effect of tear secretion of model animals in the experiment of male cynomolgus monkeys.
  • Figure 7 Bar graph of the effect of tear secretion of model animals in the male cynomolgus monkey experiment.
  • Figure 8 Schematic diagram of animal body weight changes in each group of male New Zealand white rabbits.
  • Figure 9 Effects of each group on tear film break-up time in male New Zealand white rabbits.
  • Figure 10 Schematic diagram of the fluorescent staining results of each group of male New Zealand white rabbits.
  • Figure 11 Schematic diagram of the effect of each group on the secretion of tears in male New Zealand white rabbits.
  • the following experiments are used to confirm the effect of the ester-containing arylpropionamide compound of the present invention in treating dry eye.
  • the EG compounds mentioned in the text are all ester-containing aromatic propionamide compounds, and EG-12, EG-312, EG-17, EG-317 are respectively C 22 H 19 F 3 N 4 O 4 , C 22
  • For the specific preparation methods and structural formulas of H 18 F4N 4 O 4 , C 25 H 17 F 3 N 4 O 4 and C 25 H 16 F 4 N 4 O 4 refer to the contents described in the patent document with the application number of 2014103602169.
  • test substance EG-12 (C 22 H 19 F 3 N 4 O 4 ), EG-312 (C 22 H 18 F4N 4 O 4 ), EG-17 (C 25 H 17 F 3 N 4 O 4 ) ), EG-317 (C 25 H 16 F4N 4 O 4 )
  • test substance airtight, room temperature, cool place
  • Preparation method of the test substance after grinding, add mineral oil and vaseline to dissolve into a paste
  • Test substance preparation frequency one preparation for three days
  • Feeding method Sufficient supply, ad libitum
  • Feed testing in line with nutritional and hygienic standards, the Agricultural Product Quality Supervision, Inspection and Testing Center of the Ministry of Agriculture (Zhengzhou), the Henan Provincial Center for Disease Control and Prevention, and the Henan Institution Laboratory Animal Quality Supervision and Testing Station provide test reports.
  • Type sterile water
  • Sterilization method automatic flushing of reverse osmosis system and online circulation disinfection
  • Water supply method Put it into a drinking bottle and ingest it freely.
  • the quarantine period is 7 days, and the general condition is observed every day during the quarantine period. Body weight was measured on the first and last days of the quarantine period.
  • Dosing frequency the positive group 1 (carbomer eye drops) was administered twice a day, once in the morning and in the afternoon, and the other groups were administered once a day, regularly in the morning.
  • Normal control group 0.9% saline, 2 drops per eye each time.
  • Model control group 0.9% saline, 2 drops per eye each time.
  • Solvent control group (1 mL of mineral oil + 0.5 g of Vaseline) for three days, about 0.05 g/eye, 0.1 g/eye.
  • Positive 1 drug group carbomer eye drops: carbomer eye drops, 1 drop per eye each time.
  • Testosterone Propionate Injection the specification is 25mg/lmL, calculated as 20 drops in 1mL, 1.25mg/drop, 2 drops per eye each time, 5.0mg/only, 2.5mg/drop Eye.
  • 11.2 Administration method start administration at the planned dose on the 35th day after surgery, and continue for 14 days.
  • Detection indicators The basal tear secretion (Schirmer I test), corneal fluorescence staining (PI) and tear break-up time (BUT) were detected in each group before modeling and after topical anesthesia on the 0, 7, and 14 days of administration.
  • Basal tear secretion volume (Schirmer I test): qualitative filter paper (35mm ⁇ 6mm), the anterior segment of 5mm is folded and placed in the temporal conjunctival sac of the lower eyelid, and the length of tear infiltration of the test paper (including the reflex segment) is detected and recorded after 5 minutes.
  • PI Corneal fluorescence staining scoring standard: 1% sodium fluorescein was instilled in both eyes of rabbits, without washing, and observed with a slit lamp microscope. No staining of corneal epithelium, 0 points; staining area less than 25%, 1 point; staining area of 25%-50%, 2 points; staining area of 50%-75%, 3 points; staining area greater than 75%, 4 points.
  • Break-up time of tear film (BUT): use a glass rod dipped in fluorescein to drop into the conjunctival sac of the lower eye of the rabbit, and record under the slit lamp after blinking from the last blink to the first black spot on the cornea time of appearance.
  • the EG-17 and EG-317 groups were compared with the model group and the solvent control group There was no difference in the staining of the cornea and conjunctiva of the rabbits; on the 14th day of administration, the two positive groups and the EG-12 and EG-312 groups significantly decreased the corneal and conjunctival staining scores of the rabbits in the model group and the solvent control group, and the difference was statistically significant.
  • the EG-17 and EG-317 groups had no significant difference in corneal staining; on the 28th day after drug withdrawal, the conjunctival staining scores of the rabbits in each administration group were increased, which were higher than those in the normal control group.
  • Control group Harderian glands and meibomian glands were neatly arranged, and there was no cell degeneration and necrosis, fibrous tissue proliferation and inflammatory cell infiltration.
  • Model group and solvent group Harderian's gland: disordered tissue arrangement, degeneration and necrosis of a large number of acinar cells, proliferation of fibrous tissue in necrotic foci, and infiltration of inflammatory cells locally.
  • Meibomian glands The duct wall is thinned, the lumen is enlarged, and the connective fibers increase and show fibrosis. Some acinars lost their normal acinar structure, showing disordered arrangement of tissue structures, smaller cell volume, significantly reduced cytoplasm, densely crowded nuclei, and atrophic acinars.
  • EG-12 group and positive 2 group (testosterone propionate) group were significantly improved compared with model group.
  • the epithelium of Harderian gland was neatly arranged, some acinar epithelial cells were mildly degenerated and fibrous connective tissue was mildly hyperplasia, and there was no obvious inflammatory cell infiltration.
  • the atrophy of the meibomian glands was significantly reduced, the cell volume became normal, the ratio of nucleus to cytoplasm was moderate, and the proliferation of fibrous connective tissue was not obvious.
  • EG-12 and EG-312 can increase the tear secretion of castrated rabbits, decrease the corneal fluorescence staining score, increase the tear film breakup time, and have obvious improvement effects on experimental dry eye.
  • EG-17 and EG-317 had no obvious effect on experimental dry eye.
  • EG017 Batch number, Q18-045; provider, Yaoyuan Pharmaceutical Chemical (Shanghai) Co., Ltd.; physical properties, off-white powder; storage conditions, airtight, room temperature.
  • CMC-Na Sodium carboxymethyl cellulose
  • Fluorescein sodium batch number
  • BCBQ5572V manufacturer
  • Sigma-Aldrich (Shanghai) Trading Co., Ltd. physical properties, orange-red powder; storage conditions, dry, airtight preservation.
  • Sutai 50 (Virbac-50); batch number, 6VUL: manufacturer, Virbac S.A., France Vic Co., Ltd.; storage conditions, airtight, room temperature.
  • Grouping All animals were randomly divided into 6 groups according to body weight, basal tear secretion volume and tear film breakup time before modeling: non-model solvent control group (sham), model group (Model), EG017 low-dose 0.5 mg/kg group, EGO17 medium dose 1.5mg/kg group, EGO17 high dose (H) 4.5mg/kg group, 7 in each group.
  • Castration surgery modeling After the animals underwent a one-week adaptation period/baseline data collection, bilateral castration surgery was performed. After overnight fasting, the monkeys were given intramuscular injection of Shutai 50 (5 mg/kg) for general anesthesia, routine disinfection, and sterile towels were applied. , then squeeze the bilateral case pills into the scrotum from the abdominal cavity and pinch it tightly, use a sterile blade to make a small incision in the scrotum, squeeze out the testis, ligate the spermatic vein and vas deferens, excise the bilateral testicles and attachment rate, suture the skin of the scrotum Apply iodine tincture and inject antibiotics for 1 week after surgery to prevent infection.
  • Shutai 50 5 mg/kg
  • sterile towels were applied.
  • squeeze the bilateral case pills into the scrotum from the abdominal cavity and pinch it tightly use a sterile blade to make a small incision in the scrotum, squeeze out the testis
  • End point Acute cold wind stimulation modeling Considering that the cynomolgus monkeys were castrated by surgery, the animals took a long time to form the model. In order to expand the model effect, the upper and lower eyelids of the animals were fixed and the eyeballs were exposed before the last test of the animals in each group (except the Sham group). , use a high-power hair dryer (cold air mode) at a distance of 10 cm from the animal's eyes, and stimulate the animal's eyes with continuous blowing for 3 minutes.
  • a high-power hair dryer cold air mode
  • the animals were given the corresponding therapeutic drugs at the corresponding time according to Table 4.
  • the non-model solvent control (Sham) group and the model (Model) group were given 0.5% CMC-Na solvent by gavage, and the low, medium and high test drug groups were given EG017 solution (0.5, 1.5, 4.5 mg/kg) by gavage. All groups were administered continuously for 14 weeks, and the administration volume was 5ml/kg, once a day. Animals were weighed weekly and clinically observed twice daily.
  • Ophthalmological examination The following ophthalmological examinations were performed after the animals were fasted and given intramuscular injection of Shutai 50 (5 mg/kg) to induce general anesthesia.
  • the detection items were tear film break-up time, corneal fluorescence staining, and basal tear secretion.
  • Tear film break-up time (BUT); tear film break-up time was measured before modeling/administration, and measured every 2 weeks after administration, once for each left and right eye, 3 times for each test, and the average was taken value.
  • Measurement method drop 50ul of 0.25% sodium fluorescein in each eye of the monkey without rinsing. After blinking for 2-3 times, use a slit lamp to observe under the diamond blue filter from the last blink to the corneal appearance. 1 dark spot at a time.
  • corneal fluorescence staining score the corneal fluorescence staining score was detected once before modeling/administration, and once every 2 weeks after administration, once for the left and right eyes.
  • Fluorescein staining was scored using a 12-point method: the cornea was divided into 4 quadrants, with 0-3 points in each quadrant; 0 points for no staining, 1 point for 1-30 punctate staining, and more than 30 punctate staining and staining 2 points for no fusion, 3 points for corneal punctate coloration fusion, filaments, and ulcers.
  • Tear secretion (Schirmer I test): The tear secretion was detected once before modeling/administration, and the measurement was performed every two weeks after administration, and the left and right eyes were measured once.
  • Measurement method After the corneal fluorescence staining score measurement is completed, gently wipe the excess saline flush (without touching the eyeball), and leave it for a period of time (about 5 minutes) to eliminate the effect of corneal fluorescence staining on tear secretion: use Commercial tear test strips were folded and placed in the conjunctival sac on the side of the lower eye, and the tear infiltration length of the test strip was recorded after 3 minutes.
  • 0.25% sodium fluorescein Precisely weigh sodium fluorescein powder and dissolve it in 0.9% sodium chloride injection. Prepared as a 0.25% sodium fluorescein solution.
  • Preparation of 0.5% CMC-Na solvent Weigh enough CMC-Na powder and dissolve it in double distillation to prepare a CMC-Na clear solution with a final concentration of 0.5% for use.
  • EGO17 solution According to the body weight of the animals in each group, an appropriate amount of EG017 powder was weighed and placed in a mortar on the day before administration; a small amount of solvent was added and ground fully; Rinse the grinding body until no drug powder adheres to the grinding body; add an appropriate amount of solvent to the graduated vessel containing the drug solution until the concentration of the drug is reached; continue stirring with a magnetic stirrer until the drug solution is completely mixed into a suspension: 2 Store at -8'°C overnight, and administer the drug the next morning; move the drug solution to room temperature at least 30 minutes in advance, and continue stirring with a magnetic stirrer until the administration is completed.
  • the original experimental protocol was adjusted and revised several times, including (1) extending the treatment period for 6 weeks, and the experimental operation and operation frequency during the extended period were consistent with the previous ones; (2) in the Before the last ophthalmic examination, the castrated model animals were subjected to acute cold wind stimulation to speed up the formation of the model and facilitate the observation of the effect of drug treatment: (3) After analyzing the entire experimental data, it was decided to cancel the serum sex hormone test, tear lipid analysis and Histopathological examination of lacrimal glands and ventricle glands.
  • the tear film breakup time of the animals in the sham group was relatively stable throughout the experimental period, with no significant change, except for a transient decrease at 2 weeks (P ⁇ 0.05).
  • Model, EG017 low dose, EG017 The tear film break-up time of the animals in the middle-dose and high-dose EG017 groups did not change significantly within 12 weeks after modeling. After 14 weeks of acute cold wind stimulation, the tear film break-up time of the animals in the 5 groups decreased significantly, suggesting that The acute model of dry eye was successful.
  • EG017 can improve the tear film break-up time of animals in a dose-dependent manner; the effect of EG017 on corneal fluorescence staining scores in both eyes
  • EG017 has a good effect on improving dry eye disease in cynomolgus monkeys.
  • test compound EG017 of Xijian Pharmaceutical Technology in the model of dry eye constructed by male New Zealand white rhizomes was studied below to demonstrate its effect.
  • EG017 batch number, Q18-045; supplier, Yaoyuan Pharmaceutical Chemical (Shanghai) Co., Ltd.; physical properties: off-white powder; storage conditions, airtight, stored at room temperature.
  • CMC-Na Sodium carboxymethyl cellulose
  • Xylazine hydrochloride injection (Lu Mianning): batch number, 170413; supplier, Jilinzhou Huamu Animal Health Products Co., Ltd.; storage conditions, airtight, dry, and stored at room temperature.
  • Sodium fluorescein batch number, BCBQ5572V; supplier, Sigma-Aldrich (Shanghai) Trading Co., Ltd.: physical properties, orange-red powder; storage conditions, dry and airtight.
  • Tear test strip batch number, 20180503 13; supplier, Tianjin Jingming New Technology Development Co., Ltd.: storage conditions, airtight, room temperature.
  • Grouping 36 animals were randomly divided into 6 groups according to tear break-up time (BUT), corneal fluorescence staining score and tear secretion (Schirmer I) before modeling: sham operation vehicle control group (Sham), model vehicle control group (Model) , EG017 0.75mg/kg low dose group (L), EG017 2.25mg/kg medium dose group (M), EG017 6.75mg/kg high dose group (H), 6 in each group.
  • BUT tear break-up time
  • Schirmer I tear secretion
  • Animals were administered the test drugs according to the table below.
  • the sham-operated vehicle control group (Sham) and the model (Model) group were given 0.5% CMC-Na solvent by gavage, and the EG017 low, medium and high dose groups were given EG017 solution (0.75, 2.25, 6.75 mg/kg) by gavage. All groups were administered continuously for 14 weeks, and the administration volume was 5ml/kg, once a day.
  • the animals in the experimental group were castrated for modeling.
  • the main steps are as follows: intramuscular injection of Lu Mianning (2.5 mg/kg, IM) for general anesthesia, routine disinfection, and sterile towel covering. Squeeze the bilateral testicles into the scrotum from the abdominal cavity and squeeze them tightly, do not let them slide, make a small incision in the scrotum with a sterile blade, squeeze out the testis, ligate the spermatic vein and vas deferens, remove the testis and epididymis, and suture the skin of the scrotum. Apply iodine tincture. Postoperative antibiotics and analgesics were injected for 1 week for nursing.
  • Acute modeling by cold air stimulation Due to the long modeling time of castration surgery, in order to expand the effect of the model, the upper and lower eyes and faces of the animals were fixed and the eyeballs were exposed before the last test of the animals in each group (except the Sham group). A high-power hair dryer was used to keep the animals away The eyes are stimulated by continuous blowing of cold air 10cm from the eyes for 3 minutes.
  • the tear film break-up time was measured once before modeling/administration, once every 2 weeks after administration, and once in the left and right eyes.
  • Measurement method After intramuscular injection of Lu Mianning (2.5mg/kg, IM) under general anesthesia, drop about 100 ⁇ l of 10.5% sodium fluorescein into each eye without rinsing. The time from eye opening after the last blink to the first dark spot on the cornea was observed under the light film.
  • Corneal fluorescence staining was scored once before modeling/administration, and once every 2 weeks after administration, once for the left and right eyes.
  • the tear secretion was measured in the awake state of the animals, once before modeling/administration, and once every 2 weeks after administration, once in each of the left and right eyes.
  • the commercial tear test strip was folded and placed in the conjunctival sac on the temporal side of the eyelid, the eyes of the animal were closed, and the length of the tear test strip infiltrated after 5 minutes was detected and recorded.
  • 0.5% sodium fluorescein Precisely weigh sodium fluorescein powder and dissolve it in 0.9% sodium chloride injection. Configured as a 0.5% sodium fluorescein solution.
  • CMC-Na solvent preparation CMC-Na powder was weighed and dissolved in distilled water to prepare a CMC-Na clear solution with a final concentration of 0.5% for use.
  • EG017 solution Preparation of EG017 solution: the doses of the animals were 0.75mg/kg, 2.25mg/kg and 6.75mg/kg, and the administration volume was 5ml/kg. According to the body weight of the animals, according to the body weight of each group of animals, the animals were weighed the day before administration.
  • the EG017 solution was prepared the day before the experiment and stored in a 4°C refrigerator overnight.
  • the experimental animals were returned to room temperature before administration, and the suspension was stirred with a magnetic stirrer during administration to keep the suspension in a state of stirring, and the drug was fully suspended.
  • the tear film break-up time value is shown in Figure 9.
  • the tear film break-up time of the animals in the sham group was relatively stable throughout the experimental period, and there was no significant change.
  • Model, EG017 low-dose, EG017 medium-dose, and EG017 high-dose groups had no significant changes in tear film break-up time within 12 weeks after modeling. A very pronounced decline in performance suggests a successful acute model of dry eye.
  • EG017 can improve the tear film break-up time of animals in a dose-dependent manner.
  • the corneal fluorescent staining scores are shown in Figure 10. There was no significant change in the corneal fluorescent staining scores of all groups of animals before modeling and 12 weeks after modeling. At the 14-week detection, the models, EG017 low-dose, and EG017 medium after acute cold wind stimulation were added. The scores of animals in the high-dose and high-dose groups of EG017 increased in different degrees, indicating that the acute modeling was successful. The corneal fluorescence staining score of the Model group was higher than that of the EG017 test group, indicating that EG017 effectively inhibited the corneal damage caused by castration modeling and acute cold wind stimulation modeling.
  • the purpose of this project is to study the pharmacodynamics of Xijian compound EG017 on the castration dry eye model of New Zealand male rabbits. All animals were given intragastric administration on the second day after castration. The weight increase in the Sham group was more obvious; after 12 weeks of castration, there were no significant changes in tear film break-up time, corneal staining and tear secretion in each group. long.
  • the tear film break-up time, tear secretion and corneal staining were all significantly improved, and there was a dose correlation.
  • EG017 can significantly improve the dry eye model of castration and cold-air-blown rabbits.

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Abstract

一种含酯基芳香丙酰胺类化合物在制备治疗干眼症药物中的应用。

Description

含酯基芳香丙酰胺类化合物在制备治疗干眼症药物中的应用 技术领域
本发明属于医药技术领域，尤其涉及一种含酯基芳香丙酰胺类化合物在制备治疗干眼症药物中的应用。
背景技术
干眼症是指任何原因造成的泪液质或量异常或动力学异常，导致泪膜稳定性下降，并伴有眼部不适和(或)眼表组织病变特征的多种疾病的总称，可造成角结膜病变，并会影响视力。近年来随着电脑、智能手机的广泛使用，人们的用眼时间不断增长，加之不良用眼习惯的影响，导致干眼症发病率明显上升。干眼症已成为全球流行性疾病，我国干眼症的发病率逐渐升高并有年轻化的趋势。研究表明，眼表组织的炎症反应是干眼症中泪液分泌减少的主要诱因，目前干眼症的治疗主要以人工泪液、皮质类固醇激素以及环孢霉素A等免疫抑制剂为主。其中，人工泪液仅是一种泪液替代品，其本身无治疗作用；皮质类固醇激素及环孢霉素A等药物长期使用对眼表均有一定的毒副作用。因此，临床上亟需一种效果好且长期使用无明显局部及全身毒副作用的药物。
发明内容
本发明的目的是提供一种含酯基芳香丙酰胺类化合物在制备治疗干眼症药物中的应用。
为达到上述目的，本发明采用的技术方案是：
含酯基芳香丙酰胺类化合物在制备治疗干眼症药物中的应用。
优选地，所述干眼症为性激素失衡引起的干眼症。
优选地，所述含酯基芳香丙酰胺类化合物在制备治疗干眼症药物中与医药学上可接受的辅料联合使用。
优选地，所述含酯基芳香丙酰胺类化合物与医药学上可接受的辅料制备成涂膏形式。
优选地，所述医药学上可接受的辅料为矿物油和凡士林。
优选地，所述含酯基芳香丙酰胺类化合物为C 22H 19F 3N 4O 4或C 22H 18F4N 4O 4，其中C 22H 19F 3N 4O 4或C 22H 18F4N 4O 4的制备方法及结构式均参考申请号为2014103602169的专利文件记载的内容。
优选地，所述C 25H 17F 3N 4O 4的有效剂量为0.5-6.75mg/kg。
本发明具有的优点是：本发明提供的含酯基芳香丙酰胺类化合物是一类新的选择性非甾 体类雄激素受体调节剂，具有调节雄激素受体的作用，因此可以用于治疗因为雄激素失衡导致的干眼症，且动物实验表明其能增加去势家兔的泪液分泌量，降低角膜荧光染色评分值，增加泪膜破裂时间，对实验性干眼症有明显的改善作用。
附图说明
图1：各组哈氏腺病理切片检查结果示意图。
图2：各组睑板腺病理切片检查结果示意图。
图3：雄性食蟹猴造模动物泪膜破裂时间的影响的曲线图。
图4：雄性食蟹猴造模动物泪膜破裂时间的影响的柱状图。
图5：雄性食蟹猴实验中双眼角膜荧光染色结果示意图。
图6：雄性食蟹猴实验中造模动物泪液分泌量的影响的曲线图。
图7：雄性食蟹猴实验中造模动物泪液分泌量的影响的柱状图。
图8：雄性新西兰大白兔各组的动物体重变化示意图。
图9：雄性新西兰大白兔各组对泪膜破裂时间的影响。
图10：雄性新西兰大白兔各组荧光染色结果示意图。
图11：雄性新西兰大白兔各组对泪液分泌量的影响示意图。
具体实施方式
采用如下实验证实本发明含酯基芳香丙酰胺类化合物治疗干眼症的作用。文中所提到的EG类化合物均为含酯基芳香丙酰胺类化合物，且EG-12、EG-312、EG-17、EG-317分别为C 22H 19F 3N 4O 4、C 22H 18F4N 4O 4、C 25H 17F 3N 4O 4和C 25H 16F 4N 4O 4，其具体制备方法和结构式参考申请号为2014103602169的专利文件记载的内容。
1实验名称及实验目的
1.1实验名称
EG类化合物对雄兔去势所致干眼症的改善作用
1.2实验目的
观察受试物EG-12、EG-312、EG-17、EG-317对雄兔去势所致干眼症的改善作用
2实验起止日期：2014年8月5日—2014年10月27日
3受试物的名称、稳定性及其他特性
3.1受试物名称：EG-12(C 22H 19F 3N 4O 4)、EG-312(C 22H 18F4N 4O 4)、EG-17(C 25H 17F 3N 4O 4)、EG-317(C 25H 16F4N 4O 4)
受试物保存条件：密闭，室温、阴凉处保存
3.2受试物配制
受试物溶媒的选择：矿物油加凡士林
受试物配制方法：研磨后加入矿物油及凡士林溶成糊状
受试物配制频率：一次配供三天使用
4实验动物的种、数量、性别、体重范围、来源、动物合格证号及签发单位和饲养条件
4.1种属 日本大耳白兔
等级 普通级
预定购入动物数量和性别：45只，雄性
预定使用动物数量和性别：45只，雄性
体重：实验开始时体重均数2.4～2.8Kg
来源：郑州康达生物公司。
实验动物生产许可证号：SCXK(豫)2011-0001
动物合格证号：41001900000233
4.2饲养条件
饲养室：普通环境
温度：20～24℃，日温差：0～4℃
相对湿度：40～66％
5动物饲料、饮水和垫料的种类、来源、批号和质量情况
5.1饲料
名称：家兔饲料
生产厂家：河南省实验动物中心提供
地址：郑州市大学路40号
许可证：SCXK(豫)2005-0001
灭菌方法：Co 60照射
给料方法：足量供给，自由采食
参考标准：GB14924.3-2001
饲料检测：符合营养和卫生标准，农业部农产品质量监督检验测试中心(郑州)、河南省疾病预防控制中心、河南省实验动物质量监督检测站提供检测报告。
5.2饮水
种类：无菌水
灭菌方法：自动对反渗透系统冲洗和在线循环消毒
使用LAWS-40T实验动物饮用水处理器供水
供水方法：装入饮水瓶中，自由摄取。
水质检测：符合卫生标准，国家城市供水水质监测网郑州监测站提供检测报告。
5.3笼具及管理
笼具：RS-12型不锈钢冲洗式实验兔笼(2000×650×1720mm 3)。
每笼动物数：1只/笼。
6动物检疫
检疫7天，检疫期内每天进行一般状况观察。检疫期首日和末日测定体重。
7实验动物的分组和识别方法
7.1动物识别方法
7.1.1检疫期单笼饲养，笼具挂牌，不标记动物。
7.1.2给药观察期用饱和苦味酸溶液体染标记，每组5只家兔依次以头、背、尾、头背、白标示。
7.2实验动物分组检疫期结束，将家兔按体重随机挑选出5只作为正常对照组，剩余家兔为造模组。经检测造模成功后，将造模家兔按体重随机分成8组，分别为模型组、溶剂对照组、阳性1药(卡波姆滴眼液)组、阳性2药(丙酸睾酮注射液)组及各受试物组。
8对照品及阳性药来源
8.1 0.9％生理盐水(厂家：河南太龙药业股份有限公司，批号：13062552)；卡波姆滴眼液(厂家：Dr.GerhardMann,Chem.-Pharm.Fabrik GmbH，批号：9113)；丙酸睾酮注射液(厂家：上海通用药业股份有限公司，批号：130403)；矿物油(厂家：Sigma批号：M5904)；凡士林(厂家：南昌白云药业有限公司，批号：140501)；戊巴比妥钠(厂家：Sigma，批号：P3761)；荧光素钠(广州白云山明兴制药有限公司，批号：130926)。
9受试物和对照品的给药途径、剂量和频率
9.1给药方法：各组均为为眼局部给药。
9.2检疫期：2014.8.5-2014.8.11
9.3分组日期：2014.8.12；2014.9.15
9.4造模日期：2014.8.12
9.5给药时间：2014.9.15
9.6动物处置时间：2014.10.27
9.2用药期限：2014年9月15日～2014年9月28日，连续给药2周。
9.3给药频率：阳性1组(卡波姆滴眼液)为每天给药两次，上下午各一次，其余各组均每 天给药一次，上午定时给药。
9.4给药剂量
正常对照组：0.9％生理盐水，每次每眼滴2滴。
模型对照组：0.9％生理盐水，每次每眼滴2滴。
溶剂对照组：(1mL矿物油+0.5g凡士林)为三天的给药量，约为0.05g/眼，0.1g/只。
阳性1药组(卡波姆滴眼液)：卡波姆滴眼液，每次每眼滴1滴。
阳性2药组(丙酸睾酮注射液)：丙酸睾酮注射液，规格为25mg/lmL，按1mL20滴计算，1.25mg/滴，每次每眼滴2滴，5.0mg/只，2.5mg/眼。
受试物组(EG-N)：(150mg+1mL矿物油+0.5g凡士林)为三天的给药量，5只家兔×3天＝15次，即10mg/只，5mg/眼。
每周称1次体重。
10所使用的仪器
电子天平(上海精密科学仪器，JA1003N)；秤(上海友声衡器有限公司，ASC-A)；裂隙灯显微镜(江西枫林光学仪器有限公司，LYL-1)。
11实验方法及检测指标
11.1造模方法：将待手术家兔分别用戊巴比妥钠(50mg·kg -1)耳缘静脉注射全身麻醉，常规消毒，铺无菌巾，先后将双侧睾丸由腹腔挤入阴囊并捏紧，勿使其滑动，用消毒刀片将阴囊切一小口，且力挤出睾丸，结扎精索静脉及输精管，切除睾丸及附睾，缝合阴囊皮肤后局部涂碘酊预防感染。
11.2给药方法：术后第35天按拟定剂量开始给药，连续14天。
11.3检测指标：每组分别于造模前、给药第0、7、14天行表面麻醉后检测基础泪液分泌量(SchirmerI实验)、角膜荧光染色(PI)、泪膜破裂时间(BUT)。
11.3.1基础泪液分泌量(SchirmerI实验)：定性滤纸(35mm×6mm)，前段5mm反折置于下眼睑颞侧结膜囊内，检测记录5min后试纸泪液浸润的长度(包括反折段)。
11.3.2角膜荧光染色(PI)评分标准：家兔双眼滴1％荧光素钠，不冲洗，用裂隙灯显微镜进行观察。角膜上皮无染色，0分；染色面积小于25％，1分；染色面积为25％-50％，2分；染色面积为50％-75％，3分；染色面积大于75％，4分。
11.3.3泪膜破裂时间(BUT)：用玻璃棒蘸荧光素滴于兔下眼结膜囊内，瞬目后于裂隙灯下记录从最后1次瞬目后睁眼至角膜第1个黑斑出现的时间。
11.4组织病理学检查取左侧哈氏腺及眼睑标本，10％甲醛溶液固定，乙醇脱水、石蜡包埋、 切片、常规HE染色后，置于光学显微镜下检查哈氏腺及睑板腺。
12数据统计处理方法
数据使用
Figure PCTCN2021085266-appb-000001
表示，数据采用单因素方差分析(one-way ANOVA法检验)，组间用LSD法两两比较，检验水准α＝0.05。
13实验结果
13.1各组家兔一般状态及体重情况在造模后的5天内，造模组家兔运动次数减少、毛色较差、粪便较稀；在给药期间，各组家兔的外观、行动、被毛、粪便等一般状况良好，未见明显与药物相关的毒性症状。由于去势手术的影响，各组家兔在给药前体重均低于正常对照组，但随着实验进行，各组家兔体重均呈上升的趋势。结果如表1所示。
表1：各组家兔体重变化情况(
Figure PCTCN2021085266-appb-000002
Kg)
Figure PCTCN2021085266-appb-000003
Figure PCTCN2021085266-appb-000004
*P<0.05，与正常对照组相比
13.2与正常对照组比较，模型对照组家兔的泪液分泌量明显减少、差异有统计学意义(P＜0.05)；角膜荧光染色评分值显著增高、差异有统计学意义(P＜0.05)泪膜破裂时间均显著降低，差异有统计学意义(P＜0.05)；说明造模成功。(2)与模型组及溶剂组比较，阳性1药(卡波姆滴眼液)组、阳性2药(丙酸睾酮注射液)组、EG-12组、EG-312组的泪液分泌量明显增加、差异有统计学意义(P＜0.05)；结果如表2所示。
表2：各组不同时间泪液分泌量的比较(
Figure PCTCN2021085266-appb-000005
mm)
Figure PCTCN2021085266-appb-000006
Figure PCTCN2021085266-appb-000007
*P<0.05，与正常对照组相比； △P<0.05，与模型对照组相比； ▲P<0.05，与溶剂对照组相比
13.3各组不同时间角膜荧光染色评分的比较术后第35天进行家兔角结膜染色检测显示：造模组较正常对照组显著增加，P＜0.05，表明造模成功；给药第7天时，两个阳性组及EG-12、EG-312组较模型组与溶剂对照组家兔角结膜染色有减少趋势，但差异无显著性，EG-17、EG-317组较模型组与溶剂对照组家兔角结膜染色无差异；给药第14天时，两个阳性组及EG-12、EG-312组较模型组与溶剂对照组家兔角结膜染色评分值显著减少，差异有统计学意义，EG-17、EG-317组较模型组与溶剂对照组家兔角膜染色无显著差异；停药后第28天，各给药组的家兔结膜染色评分值均增高，与正常对照组均有显著性差异，P＜0.05，结果如表3所示。
表3各组不同时间角膜染色评分结果比较
Figure PCTCN2021085266-appb-000008
Figure PCTCN2021085266-appb-000009
P<0.05，与正常对照组相比； △P<0.05，与模型对照组相比； ▲P<0.05，与溶剂对照组相比
13.4组织病理学检查，结果如图1-图2所示
对照组：哈氏腺及睑板腺组织排列整齐，未见细胞变性坏死、纤维组织增生及炎性细胞浸润。
模型组及溶剂组：哈氏腺：组织排列紊乱，可见大量腺泡细胞变性坏死，坏死灶纤维组织增生，局部见炎性细胞浸润。睑板腺：导管管壁变薄，管腔扩大，结缔纤维增多呈纤维化。部分腺泡失去正常腺泡结构，表现为组织结构排列紊乱，细胞体积变小，细胞质明显减少，细胞核密集拥挤在一起，腺泡呈萎缩状态。
阳性药和治疗药：EG-12组和阳性2组(丙酸睾酮)组较模型组明显改善。哈氏腺上皮排列整齐，部分腺泡上皮细胞轻度变性及纤维结缔组织轻度增生，未见明显炎性细胞浸润。睑板腺腺上皮萎缩显著减轻，细胞体积趋于正常，核质比例适中，纤维结缔组织增生不明显。其余组哈氏腺和睑板腺较模型组均有不同程度的好转，腺泡组织细胞排列较模型组整齐，细胞变性、坏死数量减少，局部炎细性胞浸润和纤维组织增生不同程度减轻。睑板腺上皮细胞萎缩程度明显减轻，腺泡及导管周围结缔组织中度增生。
结论，EG-12与EG-312能增加去势家兔的泪液分泌量，降低角膜荧光染色评分值，增加泪膜破裂时间，对实验性干眼症有明显的改善作用。EG-17与EG-317对实验性干眼症并无明显作用。
以下进一步研究特测化合物EG017在雄性食蟹猴去势所致干眼病症中的药效学，以表明其作用。
实验材料
实验药品和试剂
EG017:批号，Q18-045；提供单位，药源药物化学(上海)有限公司；物理性质，类白色粉末；储存条件，密闭、室温保存。
羧甲基纤维素钠(CMC-Na):批号，C1814029:厂家，阿拉丁；物理性质，类白色粉末；储存条件，干燥、室温保存。
荧光素钠：批号，BCBQ5572V:厂家，西格玛奥德里奇(上海)贸易有限公司：物理性质，橙红色粉末；储存条件，干燥、密闭保存。
舒泰50(Virbac-50)；批号，6VUL:厂家，法国维克有限公司Virbac S.A.；储存条件，密闭、室温。
Figure PCTCN2021085266-appb-000010
Figure PCTCN2021085266-appb-000011
实验动物
雄性食蟹猴(苏州西山中科实验动物有限公司)42只，普通级，体重4.0-9.0kg,动物合
格证号：201817192,所有食蟹猴在12小时光暗循环并且温控的恒温(22+4℃),湿度40％至70％环境下饲养，饲养间内每小时空气更换10至20次，正常饲料喂养，每周记录一次体重，
实验方法
实验周期
体内实验开始日期：2018-08-27；体内实验结束日期：2018-12-11
实验流程
分组：所有动物按体重和造模前基础泪液分泌量及泪膜破裂时间，随机分为6组：非模型溶剂对照组(sham),模型组(Model),EG017低剂量0.5mg/kg组，EGO17中剂量1.5mg/kg组、EGO17高剂量(H)4.5mg/kg组，每组7只。
去势手术造模：动物经过一周适应期/基线数据采集后，进行双侧去势手术，猴子过夜禁食后用舒泰50(5mg/kg)肌肉注射全身麻醉，常规消毒，铺无菌巾，然后将双侧案丸由腹腔挤入阴囊并捏紧，用消毒刀片将阴囊切一小口，用力挤出睾丸，结扎精索静脉及输精管，切除双侧睾丸及附率，缝合阴囊皮肤后局部涂碘酊，术后抗生素注射1周，防止感染。
终点急性冷风刺激造模：考虑到食蟹猴手术去势造模，动物成模时间较长，为了扩大模型效果，在末次检测各组动物(Sham组除外)指标前固定动物上下眼睑，暴露眼球，用大功率电吹风(冷风模式)距离动物双眼10cm处，对动物双眼连续吹风刺激3分钟。
给药：动物按照表4在相应时间给予相应治疗药物。非模型溶剂对照(Sham)组和模型(Model)组灌胃给予0.5％CMC-Na溶剂，低、中、高测试药物组灌胃给予EG017溶液(0.5、1.5、4.5mg/kg)。所有组别连续给药14周，给药体积为5ml/kg,每天一次。每周称量一次 动物体重并每天进行两次临床观察。
表4：分组给药表
Figure PCTCN2021085266-appb-000012
眼科检查：动物禁食后肌肉注射舒泰50(5mg/kg)诱导全身麻醉后做以下眼科检测。
检测项依次为泪膜破裂时间、角膜荧光染色、基础泪液分泌量。
a.泪膜破裂时间(BUT)；在造模/给药前进行一次泪膜破裂时间的检测，给药后每2周进行一次测量，左右眼各测一次，每次检测3次，取平均值。
测量方法：猴每眼滴50ul0.25％荧光素钠，不冲洗，瞬目2-3次后，用裂隙灯在钻蓝滤光片下观察从最后1次瞬目后睁眼至角膜出现第1个黑斑的时间。
b,角膜荧光染色评分：在造模/给药前进行一次角膜荧光染色评分的检测，给药后每2周进行一次评估，左右眼各一次。
评估方法：泪膜破裂时间测量完成后，紧接着使用生理盐水冲洗去除过多的荧光素溶液，用裂隙灯在钴蓝滤光片下观察。
荧光素染色评分采用12分法：将角膜分为4个象限，每个象限0-3分；无染色为0分，1-30个点状着色为1分，大于30个点状着色且染色未融合为2分，出现角膜点状着色融合、 丝状物和溃疡等为3分。
c.泪液分泌量(Schirmer I实验)：在造模/给药前进行一次泪液分泌量的检测，给药后每两周进行一次测量，左右眼各测一次。
测量方法：在角膜荧光染色评分测量完成后，轻轻沾拭多余的生理盐水冲洗液(不接触到眼球)，并间隔一段时间(约5分钟)，消除角膜荧光染色对泪液分泌的影响：使用商品化泪液检测试纸，前段反折置于下眼险顾侧结膜囊内，检测记录3min后试纸泪液浸润的长度。
制剂配制
0.25％荧光素钠：精密称取荧光素钠粉末溶解于0.9％氯化钠注射液。配置成0.25％的荧光素钠溶液。
0.5％CMC-Na溶媒配制：称取足量CMC-Na粉末溶解于双蒸馏中配制成终浓度为0.5％的CMC-Na澄清溶液待用。
EGO17溶液配制：根据各组动物体重，给药前一天分别称取适量的EG017粉末，置于研钵中；加入少量溶媒并充分研磨；研磨好的药液转移到刻度器皿中，并用适量溶媒反复冲洗研体，真至研体内无药物粉末粘附；向含有药液的刻度器皿中，添加适量溶媒直至到达给药浓度；用磁力搅拌器持续搅拌直至药液完全混匀成混悬液：2-8'℃过夜保存，次日早上给药；至少提前30分钟将药液移到室温，用磁力搅拌器持续搅拌直至给药完成。
数据处理
所有的数据以Mean士SEM的方式表示。实验数据采用单因素方差分析方法(one-way ANOVA)加t-test双尾检验将各组数据进行分析比较。
实验结果
在本实验中，根据实验进展及实验数据，对原实验方案进行了多次调整修订，包括(1)延长6周治疗期，延长期内实验操作及操作频率与前面保持一致；(2)在末次眼科检测前，对去势造模动物进行急性冷风刺激造模，加快成模及利于观察药物治疗效果：(3)对整个实验数据进行分析后，决定取消血清性激素检测、泪液脂质分析及泪腺和险板腺组织病理学检查。
动物的体重及一般情况
动物体重变化Sham组动物在整个实验期间体重较稳定，没有明显变化，Model组动物体重在整个实验周期内都呈极显著性的持续下降，说明动物去势操作会很明显地降低动物体 重。EG017低剂量、EG017中剂量、EG017高剂量、组动物在去势造模后的给药期间体重呈上升趋势，说明各组药物均有效阻止了动物去势造成的体重下降情况。
EG017对造模动物泪膜破裂时间的影响
如图3及图4所示，sham组动物在整个实验期间，泪膜破裂时间较稳定，没有明显变化，除了2周时有一过性的减少(P《0.05).Model、EG017低剂量、EG017中剂量、EG017高剂量组动物在造模后12周内泪膜破裂时间均无明显变化，在14周急性冷风刺激造模后该5组动物的泪膜破裂时间有非常明显的下降表现，提示干眼症急性模型成功。EG017有改善动物泪膜破裂时间作用，且有剂量依赖性；EG017对双眼角膜荧光染色评分的影响
如图5所示，所有组别动物在造模前及造模后12周双眼角膜荧光染色评分无明显变化，14周检测时增加急性冷风刺激后Model、EG017低剂量、EG017中剂量、EG017高剂量组动物都有不同程度的评分增加，提示急性造模成功。Model组的双眼角膜荧光染色评分高于EG017测试组，表明EG017有效抑制去势造模和急性冷风刺激造模所产生的角膜损伤。
EG017对造模动物泪液分泌量的影响
如图6及图7所示，所有组别动物在造模/给药前泪液分泌量的值比较接近没有明显的差别，在造模/给药后Sham组动物整个给药期的泪液分泌量较为平稳且有上升。趋势，说明整个实验给药期动物无其他因素影响和干扰。Model组动物在造模后直至14周实验终点泪液分泌量指标呈持续下降趋势且与其他组别有显著性差异，说明去势造模显著影响动物的泪液分泌量指标。EG017治疗组有不同程度地改善了动物泪液分泌量的减少，且有剂量相关性；
结论
在本实验条件下，雄性食蟹猴完全去势后，体重有显著性性持续性下降，EG017有很好的抑制体重下降作用，甚至轻微增加体重。去势12周后，各组动物泪膜破裂时间、角膜染色及泪液分泌量均无明显变化，除了Model组泪液分泌量从10周开始有明显下降(P《0.05),可见单纯去势，对干眼成模作用较小且周期较长。
为了加强干眼成模效果并很好的观察治疗效果，在14周检测前，给所有动物(除了Sham组)双眼进行连续吹冷风刺激3分钟，造成急性干眼模型，急性造模后，Model组动物泪膜破裂时间和泪液分泌量均有显著性减少、角膜染色评分有显著性增加，提示急性造模成功：各组EG017，泪膜破裂时间、泪液分泌量及角膜染色均有很好的改善，且有剂量相关性。
综上所述，EG017对食蟹猴干眼病均有很好的改善作用。
以下研究熙健医药科技待测化合物EG017在雄性新西兰大白免去势法构建的干眼病模型中的药效,以表明其作用。
实验材料
实验药品和试剂
EG017:批号，Q18-045；供应商，药源药物化学(上海)有限公司；物理性质：类白色粉末；储存条件，密闭、室温保存。
羧甲基纤维素钠(CMC-Na)；批号，C1814029；供应商，阿拉丁；储存条件，密闭干燥、室温保存。
盐酸赛拉嗪注射液(陆眠宁)：批号，170413；供应商，吉林省华牧动物保健品有限公司；储存条件，密闭干燥、室温保存。
0.9％氯化钠注射液：批号，1809271374；供应商，辰欣药业股份有限公司；存储条件，密闭、室温保存，
荧光素钠：批号，BCBQ5572V；供应商，西格玛奥德里奇(上海)贸易有限公司：物理性质，橙红色粉末；储存条件，干燥、密闭保存。
氟尼辛葡甲胺注射液；批号，20180510:供应商，江西博莱大药厂有限公司。
拜有利注射液；批号，1332；厂家，拜耳(中国)有限公司：存储条件，2.8℃保存。
泪液检测试纸条：批号，20180503 13；供应商，天津晶明新技术开发有限公司：存储条件，密闭、室温。
主要仪器：
Figure PCTCN2021085266-appb-000013
Figure PCTCN2021085266-appb-000014
实验动物
雄性新西兰大白兔(青岛康大生物科技有限公司提供)36只，普通级，体重3～4.5kg.动物合格证号：37003900001046。
所有新西兰大白兔在12小时光暗循环并且温控的恒温(22±4℃)、湿度为40％-70％的环境下正常饲料喂养，饲养间内每小时空气更换10至20次，每周记录一次体重，实验过程中对于动物的处置符合动物伦理学要求。
实验方法
实验操作分组及给药
分组：36只动物根据造模前泪膜破裂时间(BUT)、角膜荧光染色评分、泪液分泌量(SchirmerI)随机分为6组：假手术溶媒对照组(Sham)、模型溶媒对照组(Model)、EG0170.75mg/kg低剂量组(L)、EG017 2.25mg/kg中剂量组(M)、EG017 6.75mg/kg高剂量组(H),每组6只。
给药：动物按照下表分别给予待测药物。假手术溶媒对照组(Sham)和模型(Model)组灌胃给予0.5％CMC-Na溶剂，EG017低、中、高剂量组灌胃给予EG017溶液(0.75、2.25、6.75mg/kg)。所有组别分别连续给药14周，给药体积为5ml/kg,每天一次。
每周称量一次动物体重并进行两次临床观察。
表7、分组给药表
Figure PCTCN2021085266-appb-000015
Figure PCTCN2021085266-appb-000016
动物模型的建立
实验组动物行去势造模。主要步骤如下：肌肉注射陆眠宁(2.5mg/kg,IM)进行全身麻醉，常规消毒，铺无菌巾。先后将双侧睾丸由腹腔挤入阴囊并捏紧，勿使其滑动，用消毒刀片将阴囊切一小口，挤出睾丸，结扎精索静脉及输精管，并切除睾丸及附睾，缝合阴囊皮肤后局部涂碘酊。术后注射抗生素和镇痛剂1周进行护理。
冷风刺激急性造模：由于去势手术造模时间较长，为了扩大模型效果，在末次检测各组动物(Sham组除外)指标前固定动物上下眼脸并暴露眼球，用大功率吹风筒距离动物双眼10cm连续吹冷风刺激眼睛3分钟。
泪膜破裂时间(BUT)测定
泪膜破裂时间在造模前/给药前测量一次，给药后每2周测量一次，左右眼各测一次。
测量方法：肌肉注射陆眠宁(2.5mg/kg,IM)全身麻醉后，每眼滴大约100u10.5％荧光素钠，不冲洗，瞬目2-3次后，用裂隙灯在钴蓝滤光片下观察从最后1次瞬目后睁眼至角膜出现第1个黑斑的时间。
角膜荧光染色评分
在造模前/给药前进行一次角膜荧光染色评分，给药后每2周进行一次评分，左右眼各一次。
评估方法：泪膜破裂时间测量完成后，紧接着使用生理盐水冲洗去除过多的荧光素溶液，用裂隙灯在钴蓝滤光片下观察。荧光素染色评分采用12分法：将角膜分为4个象限，每个象限0-3分；无染色为0分，1-30个点状着色为1分，大于30个点状着色且染色未融合为2分，出现角膜点状着色融合、丝状物和溃疡等为3分。
泪液分泌量测定
角膜荧光染色完成后次日在动物清醒状况下进行泪液分泌量测定，造模前/给药前检测一次，给药后每2周检测一次，左右眼各一次。将商品化泪液检测试纸条反折置于眼睑颞侧结膜囊内，闭合动物双眼，检测记录5min后泪液试纸浸润的长度。
制剂配制及给药
0.5％荧光素钠：精密称取荧光素钠粉末溶解于0.9％氯化钠注射液。配置成0.5％的荧光素钠溶液。
0.5％CMC-Na溶媒配制：称取CMC-Na粉末溶解于蒸馏水中配制成终浓度为0.5％的CMC-Na澄清溶液待用。
EG017溶液配制：动物给药剂量分别为0.75mg/kg、2.25mg/kg和6.75mg/kg,给药体积均为5ml/kg.根据动物体重，根据各组动物体重，给药前一天分别称取适量的EG017粉末，置于研钵中；加入少量溶媒并充分研磨；研磨好的药液转移到刻度器皿中，并用适量溶媒反复冲洗研钵，直至研钵内无药物粉末粘附；向含有药液的刻度器皿中，添加适量溶媒直至到达给药浓度；用磁力搅拌器持续搅拌直至药液完全混匀成混悬液：2-8℃过夜保存，次日早上给药；至少提前30分钟将药液移到室温，用磁力搅拌器持续搅拌直至给药完成。
EG017溶液于实验前一天配制，配制好后储藏于4℃冰箱过夜。在实验动物给药前恢复至室温，给药期间用磁力搅拌仪搅拌，使混悬液一直处于搅拌状态，药物充分混悬。
数据处理
将所有数据以Mean±SEM方式表示。实验数据采用软件GraphPad Prism 5双因素方差分析和T-test对各组数据进行组间和组内前后的分析比较。统计分析结果P《0.05认为差异有统计学意义，P<0.01为显著性差异，P《0.001有极显著性差异。
实验结果
动物体重及总体情况
动物体重变化情况如图8所示。各组动物体重在整个实验期间都有显著升高，但相对于sham组其它组体重升高更为明显，说明去势会造成兔子体重增加更为明显。
EG017对泪膜破裂时间的影响
泪膜破裂时间值如图9所示，sham组动物在整个实验期间，泪膜破裂时间较稳定，没有明显变化。Model、EG017低剂量、EG017中剂量、EG017高剂量组动物在造模后12周内泪膜破裂时间均无明显变化，在14周急性冷风刺激造模后该5组动物的泪膜破裂时间有非常明显的下降表现，提示干眼症急性模型成功。EG017有改善动物泪膜破裂时间作用，且有剂量依赖性。
EG017对角膜荧光染色评分的影响
角膜荧光染色评分如图10所示，所有组别动物在造模前及造模后12周双眼角膜荧光染色评分无明显变化，14周检测时增加急性冷风刺激后Model、EG017低剂量、EG017中剂量、EG017高剂量组动物都有不同程度的评分增加，提示急性造模成功。Model组的双眼角膜荧光染色评分高于EG017测试组，表明EG017有效抑制去势造模和急性冷风刺激造模所产生的角膜损伤。
EG017对泪液分泌量的影响
泪液分泌量结果如图11所示，所有组别动物在造模给药前泪液分泌量的值比较接近没有明显的差别，在造模/给药后Sham组动物整个给药期的泪液分泌量较为平稳(左眼第8周有波动上升)。第14周急性造模后，Model组和EG017组泪液分泌量均有所下降。相对Model组，EG017治疗组有不同程度地改善了动物泪液分泌量的减少，且有剂量相关性；
结论与讨论
本项目意在研究熙健化合物EG017对新西兰雄兔去势干眼病模型的药效学研究，去势造模后第二天所有动物开始灌胃给药，去势3周后去势组动物较Sham组体重升高更为明显；去势12周后，各组动物泪膜破裂时间、角膜染色及泪液分泌量均无明显变化，可见单纯去势，对干眼成模作用较小且周期较长。
为了加强干眼成模效果并很好的观察治疗效果，在14周检测前，给所有动物(除了Sham组)双眼进行连续吹冷风刺激3分钟，造成急性干眼模型。急性造模后，Model组动物泪膜破裂时间和泪液分泌量均有显著性减少、角膜染色评分有显著性增加，提示急性造模成功；
各组EG017组，泪膜破裂时间、泪液分泌量及角膜染色均有很好的改善，且有剂量相关性。
综上所述，EG017对去势及冷风吹兔子干眼病模型有明显改善作用。

Claims (7)

  1. 含酯基芳香丙酰胺类化合物在制备治疗干眼症药物中的应用。
  2. 如权利要求1所述的应用，其特征在于：所述干眼症为性激素失衡引起的干眼症。
  3. 如权利要求2所述的应用，其特征在于：所述含酯基芳香丙酰胺类化合物在制备治疗干眼症药物中与医药学上可接受的辅料联合使用。
  4. 如权利要求3所述的应用，其特征在于：所述含酯基芳香丙酰胺类化合物与医药学上可接受的辅料制备成涂膏形式。
  5. 如权利要求4所述的应用，其特征在于：所述医药学上可接受的辅料为矿物油和凡士林。
  6. 如权利要求5所述的应用，其特征在于：所述含酯基芳香丙酰胺类化合物为C 22H 19F 3N 4O 4或C 22H 18F4N 4O 4或C 25H 17F 3N 4O 4。
  7. 如权利要求6所述的应用，其特征在于：所述C 25H 17F 3N 4O 4的有效剂量为0.5-6.75mg/kg。
PCT/CN2021/085266 2020-09-14 2021-04-02 含酯基芳香丙酰胺类化合物在制备治疗干眼症药物中的应用 WO2022052454A1 (zh)

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WO2014060216A1 (en) 2012-10-18 2014-04-24 Sanofi-Aventis Deutschland Gmbh Auto-injector
CN104151197A (zh) * 2014-07-25 2014-11-19 苏州伊莱特新药研发有限公司 芳香丙酰胺类化合物及其制备方法和应用
CN112043693A (zh) * 2020-09-14 2020-12-08 宁波熙健医药科技有限公司 含酯基芳香丙酰胺类化合物在制备治疗干眼症药物中的应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014060216A1 (en) 2012-10-18 2014-04-24 Sanofi-Aventis Deutschland Gmbh Auto-injector
CN104151197A (zh) * 2014-07-25 2014-11-19 苏州伊莱特新药研发有限公司 芳香丙酰胺类化合物及其制备方法和应用
CN112043693A (zh) * 2020-09-14 2020-12-08 宁波熙健医药科技有限公司 含酯基芳香丙酰胺类化合物在制备治疗干眼症药物中的应用

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