Ophthalmic preparation containing sivimeline as well as preparation method and application of ophthalmic preparation

Abstract

The invention provides an ophthalmic preparation of sivimeline and pharmaceutically acceptable salt or stereoisomer thereof, and a preparation method and application thereof. The ophthalmic preparation comprises 0.1-10 parts of sivemeline or a salt thereof or a stereoisomer thereof and 0-10 parts of a vitamin or a vitamin-like substance. The traditional Chinese medicine composition has a good curative effect on preventing and treating eye diseases such as presbyopia, intraocular hypertension and xerophthalmia. Test results of the ophthalmic preparation on old monkeys and young monkeys show that the ophthalmic preparation has good age selectivity and can prevent and treat presbyopia symptoms of middle-aged and elderly people.

Description

[0001] This invention is a divisional application of an invention patent with an application date of January 18, 2023, application number 202310067259.7, and invention name of an ophthalmic preparation containing cevimeline, and its preparation method and application.

[0002] The present invention claims priority to application number 2022100564047 filed on January 18, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The invention belongs to the field of pharmaceutical technology, and specifically relates to a preparation method and application of an ophthalmic preparation containing Cevimeline and its pharmaceutically acceptable salt or Cevimeline stereoisomers. Background Art

[0004] The aqueous humor of the eye is produced by the secretion of ciliary epithelial cells and the exudation of blood vessels. It flows from the posterior chamber of the eye through the pupil into the anterior chamber of the eye, reaches the anterior chamber angle gap, and mainly flows into the scleral canal sinus through the trabecular meshwork (filter curtain), and finally enters the bloodstream. M cholinergic receptor agonists can shrink the iris to the center, increase the tension of the scleral protrusion, and thin the iris root, thereby expanding the anterior chamber angle gap around the iris, making it easier for the aqueous humor to enter the circulation through the trabecular meshwork and scleral canal sinus, and then reduce the intraocular pressure. The main feature of glaucoma is increased intraocular pressure, which can cause symptoms such as headaches and decreased vision, and in severe cases can cause blindness. Patients with angle-closure glaucoma (acute or chronic congestive glaucoma) have narrow anterior chamber angles and increased intraocular pressure. M cholinergic receptor agonists can rapidly reduce intraocular pressure, thereby alleviating or eliminating glaucoma symptoms. Although patients with open-angle glaucoma (chronic simple glaucoma) do not have narrow or occluded anterior chamber angle, the trabecular meshwork and Schlemm's canal undergo degeneration or sclerosis, which hinders the circulation of aqueous humor and causes increased intraocular pressure. M-cholinergic receptor agonists can reduce intraocular pressure by dilating small blood vessels around Schlemm's canal and contracting the ciliary muscle, which changes the structure of the trabecular meshwork.

[0005] Presbyopia is a refractive state. As the age increases, the lens gradually loses the ability to adjust to close-up vision and cannot focus on objects at different close distances, resulting in presbyopia. In the Internet age, the elderly have become the main force of the Internet, and there is a great demand for close vision. With the aging of the world's population, presbyopia will be a global problem that needs to be solved urgently. At present, the treatment of presbyopia mainly relies on non-drug means such as optical lenses, refractive surgery and intraocular lens surgery. Most patients with visual impairment are willing to improve the current situation of visual difficulties through a variety of correction methods, rather than simply relying on optical lenses and other similar mechanical devices for treatment and adjustment. For a long time, presbyopia and other visual disorders have been mainly treated and adjusted with optical lenses and other similar mechanical devices. There are no drugs that can be used to treat presbyopia on the domestic market. At present, dry eye, as a chronic ocular surface disease, lacks effective treatment methods. The drugs currently used in clinical practice can only relieve the dry eyes, visual fatigue and blurred vision discomfort symptoms of dry eyes, and it is difficult to achieve the purpose of cure. Therefore, a more effective therapeutic product is urgently needed in the market.

[0006] M-cholinergic receptor agonists can cause the circular muscle to contract toward the center of the pupil, relax the ciliary suspensory ligament, make the lens convex, and increase the diopter, which is suitable for near objects and can see near objects clearly. This effect is called accommodation spasm, which focuses the lens and helps treat presbyopia and refractive errors.

[0007] M-cholinergic receptor agonists can also stimulate the secretion of glands, such as sweat glands, tear glands and salivary glands. If used as ophthalmic drugs, they can promote the secretion of natural tears by stimulating the parasympathetic nerve pathway, thereby improving or treating dry eyes. M-cholinergic receptor agonists can also be used to treat dry mouth caused by chemotherapy for head and neck cancer.

[0008] Therefore, there is great room for the development of M cholinergic receptor agonists as an ophthalmic drug for the prevention and treatment of eye diseases such as presbyopia, ocular hypertension, dry eyes, refractive errors, photophobia or angle-closure glaucoma.

[0009] M cholinergic receptor agonists are widely used as drugs for miosis and lowering intraocular pressure. For example, pilocarpine is included in the Chinese Pharmacopoeia and the United States Pharmacopoeia as a miotic drug. However, the use of M cholinergic receptor agonists in the preparation of drugs for the prevention and treatment of presbyopia and dry eye has rarely been reported. Allergan published patent WO2021209955 reporting that 1.0 to 1.5% w / v pilocarpine hydrochloride eye drops treat eye diseases such as presbyopia, hyperopia, mydriasis, unequal pupil sizes, accommodative esotropia, myopia and astigmatism. The mechanism of action of Allergan's U.S.-listed drug Vuity (1.25% pilocarpine hydrochloride) is mainly through the pinhole effect to treat or improve near vision in presbyopia. However, pilocarpine has different stability under different pH conditions. The higher the pH value, the better the efficacy and the worse the stability. The most suitable pH for the human eye is 6.5-7.7. Due to the poor stability of pilocarpine, Vuity's pH is 5.0. By optimizing the formula of pilocarpine and using Allergan's proprietary pHast technology carrier, the eye drops can quickly adapt to the physiological pH of the tear film. Vuity's instructions for use and patent WO2019209955 show that Allergan's proprietary pHast technology uses excipients boric acid, sodium citrate dihydrate, sodium chloride, hydrochloric acid or sodium hydroxide, and preservative benzalkonium chloride. Vuity's active ingredients are not stable, and preservatives are used. Preservatives themselves can cause damage to the ocular surface. Long-term use may cause dry eyes, corneal and conjunctival epithelial damage, etc.

[0010] At present, there are no drugs that can be used to treat presbyopia in the Chinese market, and most of them use technology introduction to introduce foreign technology to carry out drug research. The fastest progress is the introduction of presbyopia drugs using Microline technology from Eyenovia, a clinical-stage biopharmaceutical company in the United States, by Arctic Vision Bio. Patent WO2020180793 published by Eyenovia reports that the "MicroLine" technology delivers relatively high concentrations of pilocarpine to the eyes, thereby achieving the purpose of treating presbyopia with low concentrations of pilocarpine. However, the "MicroLine" technology is not convenient for patients to use and is costly.

[0011] Another product that has made rapid progress is BRIMOCHOL, a drug for treating presbyopia developed by Visus Therapeutics of the United States, which was introduced by Zhaoke Ophthalmology in May 2022. TMPF and CARBACHOL PF, which are drugs with carbachol as the active ingredient and their combination drugs with brimonidine. Patent WO2020252061 shows that Visus uses 0.2% brimonidine, and the purpose of adding brimonidine is to solve the possible side effects of the miotic drug carbachol. Pilocarpine and carbachol are both non-selective cholinergic receptor agonists, which stimulate the contraction of the ciliary muscles that control the shape of the lens while shrinking the pupil, which may change the shape of the lens and cause its refractive characteristics to change. However, although the drug improves near vision, distance vision will decrease. Brimonidine is an α2 receptor agonist that can inhibit the contraction of ciliary muscles. When used in combination with carbachol, it can reduce the side effects of carbachol. However, the use of brimonidine at a concentration greater than or equal to 0.05% w / v will lead to an increase in congestion rebound.

[0012] Another drug that has made rapid progress is the presbyopia drug introduced by Jixing Pharmaceuticals from LENZ Therapeutics in the United States in April 2022. The presbyopia drug is a drug with acetocrine as the active ingredient. However, acetocrine is unstable in solution and inconvenient to store. Usually, acetocrine is stored in a two-bottle system: one bottle contains freeze-dried acetocrine and the other bottle contains a diluent to dissolve acetocrine before topical instillation.

[0013] Cevimeline has been approved by the FDA to treat an autoimmune disease, Sjögren's syndrome. As an M3 cholinergic receptor agonist, the M-like effects it produces are manifested as inhibition of cardiovascular activity, contraction of smooth muscles, contraction of the pupillary sphincter, and secretion of glands such as digestive glands and salivary glands, among which the effects on the eyes and glands are the most obvious. The human pupillary sphincter is mainly composed of cholinergic receptors M3, so using Cevimeline as an active ingredient to prepare drugs for the prevention or treatment of presbyopia can reduce the stimulation of the ciliary muscle while shrinking the pupil, maintain the normal refractive power of the eye, and will not affect the distance vision. This will truly solve the problem of near vision for the middle-aged and elderly people around the world.

[0014] At present, there is still a blank in the research of drugs for treating presbyopia in China, and the drugs are mainly imported. There has not been a self-developed presbyopia drug entering the clinic. In view of the various problems existing in the above presbyopia drugs, there is an urgent need to develop a drug with good stability, good efficacy, few side effects, good safety, easy use, long-term use and no obvious local and systemic toxic side effects to treat presbyopia. Summary of the invention

[0015] In view of the shortcomings of existing ophthalmic drugs, especially presbyopia drugs, the present invention provides a natural green additive-free ophthalmic preparation with good stability, high safety and convenient use and a preparation method thereof. The ophthalmic preparation contains cevimeline as an active ingredient, has a good therapeutic effect on the prevention and treatment of presbyopia, ocular hypertension, dry eye and other eye diseases, and can be used for the prevention and treatment of presbyopia at different stages; can cause pupillary sphincter contraction, shrink the pupil and cause the gap between the anterior chamber angle to expand, increase the reflux of aqueous humor, thereby reducing intraocular pressure; can promote the secretion of tears and effectively treat dry eye.

[0016] The ophthalmic preparation of the present invention may only contain the active ingredient of Cevimeline, chloride ions and sodium ions, and does not contain any other excipients such as preservatives, buffers, chelating agents and excipients, and only needs to be taken once a day. Cevimeline used in the present invention is a selective M3 cholinergic receptor agonist, and its regulation of the lens is much weaker than that of non-selective cholinergic receptor agonists. Animal experimental results show that Cevimeline eye drops are excellent in efficacy and safety in preventing or treating presbyopia, which will fill the gap in China's independently developed presbyopia drug market, and will also bring a new solution with good safety and convenient use to the treatment of presbyopia worldwide.

[0017] In order to achieve the purpose of the present invention, the disclosed technical solution is as follows:

[0018] In one aspect, the present invention discloses the use of Cevimeline or its salt or its stereoisomer in the preparation of ophthalmic preparations.

[0019] Specifically, the ophthalmic preparation comprises, by weight, 0.1-10 parts of cevimeline or its salt or its stereoisomer and 0-10 parts of vitamins or vitamin-like substances.

[0020] Preferably, the content of cevimeline in the ophthalmic preparation is 0.1-10% w / w, and the content of cevimeline can be: 1%-4% w / w, 1%-5% w / w, 1.25%-4.5% w / w, 1.25%-4% w / w, 1.5%-2.5% w / w or 1.25%-3.75% w / w.

[0021] Preferably, the salt comprises an inorganic salt or an organic salt of Cevimeline; the isomer comprises a cis isomer, a trans isomer, a diastereomer or an enantiomer of Cevimeline.

[0022] In another aspect, the present invention provides an ophthalmic formulation.

[0023] The ophthalmic preparation comprises, by weight, 0.1-10 parts of cevimeline or its salt or its stereoisomer and 0-10 parts of vitamins or vitamin-like substances.

[0024] Specifically, the salt includes an inorganic salt or an organic salt of Cevimeline, and the stereoisomer includes a cis isomer, a trans isomer or a diastereomer of Cevimeline.

[0025] The dosage forms of the ophthalmic preparation include eye drops, eye gels, eye ointments, eye patches, eye masks, eye washes or intraocular injections.

[0026] Preferably, the ophthalmic preparation comprises 1-5 parts of cevimeline or its salt or stereoisomer thereof, and the vitamin or vitamin-like substance comprises 0-2 parts by weight.

[0027] Further preferably, the ophthalmic preparation comprises 2-5 parts of cevimeline or its salt or stereoisomer thereof, and the vitamin or vitamin-like substance comprises 0.01-0.1 parts by weight.

[0028] Preferably, the concentration of cevimeline in the cevimeline ophthalmic preparation is 0.1%-10% w / w by weight; the content of cevimeline can be: 1%-4% w / w, 1%-5% w / w, 1.25%-4.5% w / w, 1.25-4% w / w, 1.5%-2.5% w / w or 1.25%-3.75% w / w; preferably 1-5%, more preferably 1.25-4.5%, further preferably 1.25-3.75%.

[0029] Preferably, the concentration of the vitamin or vitamin-like substance is 0.01%-10% w / w, preferably 0.01%-5% w / w, more preferably 0.01%-2% w / w, most preferably 0.01%-0.1% w / w, in terms of weight percentage.

[0030] The vitamins or vitamin-like substances include, but are not limited to, at least one of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, lipoic acid, pantothenic acid, calcium pantothenate, sodium pantothenate, niacinamide and folic acid.

[0031] Preferably, the pH value of the eye drops is adjusted to 4.0-9.0.

[0032] Preferably, the osmotic pressure value of the eye drops is adjusted to 260-350 mOsmol / kg.

[0033] The pH value is adjusted by a pH adjusting agent, which includes but is not limited to one or more of hydrochloric acid, boric acid, phosphoric acid, citric acid, sodium hydroxide and potassium hydroxide.

[0034] The osmotic pressure value is adjusted by an osmotic pressure regulator, which includes but is not limited to one or more of sodium chloride, sodium sulfate, potassium chloride, magnesium chloride, mannitol, mannose and dextrose.

[0035] The Cevimeline ophthalmic preparation further comprises a solvent, and the solvent is selected from water for injection or a buffer solution.

[0036] The buffer is an aqueous solution of a buffer, and the buffer includes but is not limited to one or more of carbonates, phosphates, citrates, acetates, borates, barbiturates and 3-(N-morpholino)propanesulfonate.

[0037] The content of vitamins or vitamin-like substances in the ophthalmic preparation can be 0 parts.

[0038] Preferably, when the content of vitamins or vitamin-like substances in the ophthalmic preparation is 0 parts, the ophthalmic preparation comprises at least one of cevimeline or its salt or cevimeline isomers in an amount of 0.1-10% w / w.

[0039] Further preferably, the content of cevimeline or its salt or cevimeline isomer is 0.01-5% w / w, preferably 1-4% w / w, further preferably 1.25-4% w / w, further preferably 1.5-2.5% w / w.

[0040] Furthermore, the ophthalmic preparation consists only of the following ingredients: cevimeline or at least one of its salts or isomers, chloride ions and sodium ions.

[0041] Preferably, when the content of vitamins or vitamin-like substances in the ophthalmic preparation is 0 parts, the chloride ions are derived from the pH regulator hydrochloric acid and the osmotic pressure regulator sodium chloride; the sodium ions are derived from the pH regulator sodium hydroxide and the osmotic pressure regulator sodium chloride.

[0042] Preferably, the pH adjusting agent adjusts the pH value of the Cevimeline eye drops to 4.0-9.0. Wherein, the amount of the pH adjusting agent used in the present invention can be determined according to the target pH to be adjusted. After mixing the materials other than the water for injection, before the preparation is fixed to volume, the pH of the eye drops is adjusted with an appropriate amount of the pH adjusting agent, thereby easily determining the specific amount of the pH adjusting agent in the preparation. In certain preferred embodiments, according to the stability of the active ingredient and the irritation of the eye drops, the pH of the eye drops is 4.0-9.0, for example, pH=4.0-4.5, pH=4.5-5.0, pH=5.0-5.5, pH=5.5-6.0, pH=6.0-7.0, pH=7.0-8.0 or pH=8.0-9.0.

[0043] Preferably, the osmotic pressure regulator adjusts the osmotic pressure value of Cevimeline eye drops to 260-350mOsmol / kg. Wherein, the usage amount of the osmotic pressure regulator in the present invention can be determined according to the target osmotic pressure desired to be adjusted. In certain preferred embodiments, the eye drops are 260-350mOsmol / kg, such as 260-270mOsmol / kg, 270-280mOsmol / kg, 280-290mOsmol / kg, 290-300mOsmol / kg, 300-310mOsmol / kg, 310-320mOsmol / kg, 320-330mOsmol / kg, 330-340mOsmol / kg, 340-350mOsmol / kg.

[0044] In another aspect, the present invention also provides the use of the above-mentioned ophthalmic preparation in the preparation of a medicament for preventing or treating eye diseases.

[0045] Preferably, the eye disease comprises presbyopia, ocular hypertension, dry eye, refractive error, photophobia or angle-closure glaucoma.

[0046] The refractive errors include, but are not limited to, astigmatism and hyperopia.

[0047] Preferably, the eye disease comprises presbyopia, which is a condition in which blurriness occurs when viewing near objects due to a decrease in the accommodative ability of the lens.

[0048] Preferably, the eye diseases include eye diseases caused by increased intraocular pressure due to narrow anterior chamber angle, which may cause headaches, decreased vision, and blindness in severe cases.

[0049] Preferably, the eye diseases include diseases involving the cornea and conjunctival tissues of the eye.

[0050] Preferably, the eye disease includes a refractive error disease caused by the inability of external light to focus normally on the retina.

[0051] Preferably, the eye disease comprises a lacrimal duct disease or a lacrimal gland disease.

[0052] The pinhole principle (produced by miosis) of the composition of the present invention can prevent and treat presbyopia and photophobia;

[0053] The composition of the present invention can adjust the diopter through the adjustment of the ciliary muscle to the lens, and can prevent and treat presbyopia, astigmatism, hyperopia and other refractive errors.

[0054] The composition of the present invention can shrink the pupil to tighten the iris toward the center, increase the tension of the scleral protrusion, and thin the iris root, thereby expanding the anterior chamber angle space around the iris, allowing the aqueous humor to easily enter the circulation through the trabecular meshwork and the scleral canal sinus, thereby reducing the intraocular pressure to treat ocular hypertension and angle-closure glaucoma (acute or chronic congestive glaucoma).

[0055] In yet another aspect, an object of the present invention is to provide a method for preparing the aforementioned ophthalmic preparation.

[0056] The preparation method may include the following steps:

[0057] (1) adding cevimeline or its salt or its stereoisomer to a portion of the solvent and mixing the mixture or further adding vitamins or vitamin-like substances and mixing the mixture;

[0058] (2) Adding excipients for ophthalmic preparations to adjust pH and osmotic pressure;

[0059] (3) Add the remaining solvent to make up the volume.

[0060] Preferably, the mixing speed in step (1) or step (2) is 50-70 rpm.

[0061] The preparation method may also include the following steps:

[0062] (1) Add water for injection into the preparation tank, add the prescribed amount of sodium chloride in sequence, and stir to dissolve;

[0063] (2) adding a prescribed amount of cevimeline or its salt or stereoisomer, stirring to dissolve, rinsing the container with water for injection, and pouring the rinse into the liquid preparation tank;

[0064] (3) After complete dissolution, sample the solution to detect pH. According to the pH value of the solution, continuously add hydrochloric acid or sodium hydroxide solution to adjust the pH internal control standard of the solution;

[0065] (4) Use water for injection to make up to the prescribed amount and stir to obtain Cevimeline pure natural green additive-free eye drops.

[0066] Preferably, the preparation method further comprises:

[0067] (5) Filter and sterilize through a microporous filter membrane, and then perform aseptic filling and sealing (Blow / Fill / Seal) under the aseptic protection of a Class A air shower.

[0068] Compared with the prior art, the present invention has the following beneficial effects:

[0069] (1) Pilocarpine (including its salts) is a toxic western medicine (nationally controlled product), and its production, purchase, use and storage are strict and complicated. Cevimeline is not a nationally controlled product and can be obtained through chemical synthesis and recrystallization purification. The starting materials are cheap and easy to obtain, the synthesis process is simple to operate, and the cost is controllable.

[0070] (2) The present invention has found that the stability of Cevimeline is better than that of pilocarpine, and the influence of temperature and pH value is less than that of pilocarpine. At the same time, when administered as a simple eye drop, there is no need to excessively add excipients to the preparation, and there is no eye irritation caused by the addition of excipients, let alone the cumulative toxic side effects of pilocarpine. Therefore, the present invention has the characteristics of convenient use, significant effect, and small side effects, and is easier to be accepted by patients. In some cases, the Cevimeline ophthalmic preparation of the present invention may not contain any other excipients such as preservatives, buffers, chelating agents and excipients, thereby reducing the irritation to the eye caused by excipients, and has high safety, few side effects, and is suitable for long-term use.

[0071] (3) The present invention is the first to discover the combination of cevimeline and vitamins, which improves the penetration of cevimeline in the eye and synergistically increases the efficacy of the drug. Animal experiments have found that cevimeline and vitamin substances have good preventive and synergistic therapeutic effects on eye diseases such as presbyopia, ocular hypertension, dry eyes, refractive errors, photophobia or angle-closure glaucoma.

[0072] (4) The stability of the cevimeline ophthalmic preparation of the present invention is better than that of the presbyopia drugs currently under development, and the effects of temperature and pH value on the drug are smaller than those of the presbyopia drugs currently under development. In addition, the present invention adopts the BFS filling technology and can be filled into single-dose packages on demand, without worrying about contamination after opening. It is easy to take and carry, and is more easily accepted by patients.

[0073] (5) The cevimeline ophthalmic preparation of the present invention only needs to be administered as eye drops once a day, and the effect can be maintained for up to 8 hours. The administration method is simple, the operation is convenient, and the effect lasts for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art.

[0075] Figure 1 : Comparison of pupil diameters before and after administration of formulation 1 and formulation 5.

[0076] Figure 2 : Comparison of lens thickness before and after administration of formulation 1 and formulation 5.

[0077] Figure 3: Comparison of pupil diameter changes before and after administration of formulations 1-4.

[0078] Figure 4 : Comparison of lens thickness changes before and after administration of formulations 1-4.

[0079] Figure 5 : Comparison of pupil diameter changes before and after administration of formulation 2 and formulations 6-9.

[0080] Figure 6 : Comparison of changes in lens thickness before and after administration of formulation 2 and formulations 6-9.

[0081] Figure 7 : Comparison of pupil diameter changes before and after administration of formulation 2 and formulations 10-13.

[0082] Figure 8 : Comparison of changes in lens thickness before and after administration of formulation 2 and formulations 10-13.

[0083] Fig. 9 : Comparison of normal intraocular pressure changes before and after administration of formulations 2-4.

[0084] Fig.10 : Comparison of changes in mydriatic intraocular pressure before and after administration of formulations 2-4.

[0085] Fig.11 : Comparison of pupil diameters before and after administration of formulations 17-23.

[0086] Fig.12 : Comparison of pupil diameter changes before and after administration of formulation 21 and formulations 25-29.

[0087] Fig.13 : Comparison of lens changes in young and middle-aged cynomolgus monkeys before and after administration of formulations 24 and 25. DETAILED DESCRIPTION

[0088] The present invention will be further described below in conjunction with specific implementation modes so that those skilled in the art can more clearly understand the solutions of the present invention, but the protection scope of the present invention is not limited thereby.

[0089] The experimental methods and technical terms used in the following examples, unless otherwise specified, are generally operated according to conventional conditions commonly understood by those of ordinary skill in the art to which the present invention belongs. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0090] Among them, the pH adjuster uses hydrochloric acid, boric acid, phosphoric acid, citric acid, sodium hydroxide or potassium hydroxide to adjust the eye drops to the required pH, and the osmotic pressure regulator uses sodium chloride, sodium sulfate, potassium chloride, magnesium chloride, mannitol, mannose or dextrose to adjust the eye drops to the required osmotic pressure.

[0091] The young cynomolgus monkeys used in the following examples were about 3-4 years old, sourced from Fangchenggang Kanglu Biotechnology Co., Ltd. and Hainan Jingang Biotechnology Co., Ltd., of ordinary grade, male or female, and administered with 50 μL / eye single eye drops in both eyes. The cynomolgus monkeys used for safety and tolerability evaluation were about 3-5 years old, of ordinary grade, sourced from Guangdong Chunsheng Biotechnology Development Co., Ltd., half male and half female, and administered with 50 μL / eye eye drops in both eyes, tid.

[0092] The middle-aged and elderly cynomolgus monkeys used in the following examples were about 8-15 years old and were sourced from Guangdong Chunsheng Biotechnology Development Co., Ltd. They were of ordinary grade and of both sexes and were administered with a single eye drop of 50 μL / eye in both eyes.

[0093] The New Zealand white rabbits used for safety and tolerability evaluation in the following examples were sourced from Pizhou Dongfang Breeding Co., Ltd., and 50 μL / eye was administered tidly in both eyes.

[0094] After all animals arrive at the research facility, veterinary staff will conduct physical examinations on all animals and quarantine / acclimate the animals. There will be at least 7 days between the arrival of the animals at the research facility and the first day of dosing, and a comprehensive examination will be conducted again before the experiment.

[0095] Example 1: Preparation of eye drop formulation 1 (4.5% Cevimeline)

[0096] Add an appropriate amount of Cevimeline to the dispensing bottle, add some water and dissolve it with a rotator at a speed of 60rpm, observe the appearance every 15 minutes to see if it is dissolved; after it is completely dissolved, measure its pH and osmotic pressure, use pH regulator and osmotic pressure regulator to adjust its pH and osmotic pressure to the designed value pH=5.6; osmotic pressure: 323mOsmol / kg; then add the remaining injection water to the concentration of Cevimeline to 4.5%, then sterile filter, and package for subsequent use. The eye drop formula was tested for physical appearance, content determination, impurities, pH value and osmotic pressure. The pH regulator is sodium hydroxide and hydrochloric acid, and the osmotic pressure regulator is sodium chloride.

[0097] Example 2: Preparation of eye drop formulation 2 (2.5% Cevimeline)

[0098] According to the operating steps of Example 1, 2.5% Cevimeline eye drops were prepared, with pH=6.0 and osmotic pressure: 316 mOsmol / kg. The pH regulators were sodium hydroxide and hydrochloric acid, and the osmotic pressure regulator was sodium chloride.

[0099] Example 3: Preparation of eye drop formulation 3 (1.25% Cevimeline)

[0100] According to the operating steps of Example 1, 1.25% Cevimeline eye drops were prepared, with pH = 5.9 and osmotic pressure: 310 mOsmol / kg. The pH regulators were sodium hydroxide and hydrochloric acid, and the osmotic pressure regulator was sodium chloride.

[0101] Comparative Example 1: Eye Drops Formula 4 (1.25% Pilocarpine Hydrochloride)

[0102] According to the operating steps of Example 1, 1.25% pilocarpine hydrochloride eye drops were prepared, with pH = 5.3 and osmotic pressure: 300 mOsmol / kg. The pH regulators were sodium hydroxide and hydrochloric acid, and the osmotic pressure regulator was sodium chloride.

[0103] Comparative Example 2: Preparation of eye drop formula 5 (1% pilocarpine nitrate)

[0104] According to the operating steps of Example 1, 1% pilocarpine nitrate eye drops were prepared, with pH = 5.3 and osmotic pressure of 300 mOsmol / kg. The pH regulators were sodium hydroxide and hydrochloric acid, and the osmotic pressure regulator was sodium chloride.

[0105] Example 4: Preparation of eye drop formula 6 (2.5% Cevimeline + 0.01% Vitamin B12)

[0106] Add Cevimeline to the dispensing bottle, add some water and dissolve it with a rotator at a speed of 60rpm, observe the appearance every 15min to see if it is dissolved; add vitamin B12 after it is dissolved, continue to dissolve it with a rotator at a speed of 60rpm, observe the appearance every 15min to see if it is dissolved; measure its pH and osmotic pressure after it is completely dissolved, and use pH regulator and osmotic pressure regulator to adjust its pH and osmotic pressure to the designed value pH=6.0; osmotic pressure: 320mOsmol / kg; then add the remaining water to a concentration of Cevimeline of 2.5%, then sterile filter, and package for subsequent use. The eye drop formula was tested for physical appearance, content determination, impurities, pH value and osmotic pressure. The pH regulator is sodium hydroxide and hydrochloric acid, and the osmotic pressure regulator is sodium chloride.

[0107] Example 5: Preparation of eye drop formula 7 (2.5% Cevimeline + 0.05% Lipoic Acid)

[0108] According to the operation steps of Example 4, 0.05% lipoic acid was used instead of 0.01% vitamin B12 to prepare a 2.5% cevimeline and 0.05% lipoic acid eye drop composition, with a pH of 5.9 and an osmotic pressure of 320 mOsmol / kg. The pH regulators were sodium hydroxide and boric acid, and the osmotic pressure regulator was sodium sulfate.

[0109] Example 6: Preparation of eye drop formula 8 (2.5% Cevimeline + 0.05% sodium pantothenate)

[0110] According to the operation steps of Example 4, 0.05% sodium pantothenate was substituted for 0.01% vitamin B12 to prepare a 2.5% cevimeline and 0.05% sodium pantothenate eye drops composition, with pH = 5.9 and osmotic pressure: 322 mOsmol / kg. The pH regulators were potassium hydroxide and phosphoric acid, and the osmotic pressure regulator was potassium chloride.

[0111] Example 7: Preparation of eye drop formula 9 (2.5% Cevimeline + 0.1% niacinamide)

[0112] According to the operation steps of Example 4, 0.1% nicotinamide was used instead of 0.01% vitamin B12 to prepare a 2.5% cevimeline and 0.1% nicotinamide eye drops composition, with pH=6.0 and osmotic pressure: 323 mOsmol / kg. The pH regulators were sodium hydroxide and hydrochloric acid, and the osmotic pressure regulator was mannose.

[0113] Comparative Example 3: Preparation of eye drop formula 10 (0.01% vitamin B12)

[0114] Add an appropriate amount of vitamin B12 to the ingredient bottle, then add some water and dissolve it with a rotator at a speed of 60rpm. Observe the appearance every 15 minutes to see if it is dissolved; after it is completely dissolved, measure its pH and osmotic pressure, and use pH regulator and osmotic pressure regulator to adjust its pH and osmotic pressure to the designed value pH=6.0; osmotic pressure: 321mOsmol / kg; then add the remaining water to a concentration of 0.01% of vitamin B12, then sterile filter, and package for subsequent use. The eye drop formula was tested for physical appearance, content determination, impurities, pH value and osmotic pressure. The pH regulator is sodium hydroxide and hydrochloric acid, and the osmotic pressure regulator is sodium chloride.

[0115] Comparative Example 4: Preparation of Eye Drops Formula 11 (0.05% Lipoic Acid)

[0116] According to the operation steps of comparative example 3, lipoic acid was used instead of vitamin B12 to prepare 0.05% lipoic acid eye drops, with pH=5.9 and osmotic pressure: 318mOsmol / kg. The pH regulators were sodium hydroxide and boric acid, and the osmotic pressure regulator was sodium sulfate.

[0117] Comparative Example 5: Preparation of eye drop formula 12 (0.05% sodium pantothenate)

[0118] According to the operation steps of Comparative Example 3, sodium pantothenate was used instead of vitamin B12 to prepare 0.05% sodium pantothenate eye drops, with pH=6.0 and osmotic pressure: 320mOsmol / kg. The pH regulators were potassium hydroxide and phosphoric acid, and the osmotic pressure regulator was potassium chloride.

[0119] Comparative Example 6: Preparation of eye drop formula 13 (0.1% niacinamide)

[0120] According to the operation steps of Comparative Example 3, nicotinamide was used instead of vitamin B12 to prepare 0.1% nicotinamide composition eye drops, with pH=6.0 and osmotic pressure: 323mOsmol / kg. The pH regulators were sodium hydroxide and hydrochloric acid, and the osmotic pressure regulator was mannose.

[0121] Example 8: Preparation of eye drop formulation 14 (0.1% Cevimeline + 4% niacinamide)

[0122] According to the operation steps of Example 4, 4% nicotinamide was used instead of 0.01% vitamin B12 to prepare a 0.1% cevimeline and 4% nicotinamide eye drops composition, the pH of which was 4.0. The pH regulators were sodium hydroxide and hydrochloric acid, and the osmotic pressure regulator was sodium chloride.

[0123] Example 9: Preparation of eye drop formulation 15 (4.5% Cevimeline + 0.1% niacinamide)

[0124] According to the operation steps of Example 4, 0.1% nicotinamide was used instead of 0.01% vitamin B12 to prepare a 4.5% cevimeline and 0.1% nicotinamide eye drops composition, the pH of which was 9.0. The pH regulators were sodium hydroxide and hydrochloric acid, and the osmotic pressure regulator was sodium chloride.

[0125] Example 10: Preparation of eye drop formulation 16 (2.5% Cevimeline + 2% niacinamide)

[0126] According to the operation steps of Example 4, nicotinamide was used instead of vitamin B12 to prepare a 2.5% cevimeline and 2% nicotinamide eye drops composition, pH = 6.5. The pH regulators were sodium hydroxide and hydrochloric acid, and the osmotic pressure regulator was sodium chloride.

[0127] Experiment 1: Stability study of eye drop formula 3 (Example 3):

[0128] An appropriate amount of freshly prepared 1.25% Cevimeline eye drops formulation 3 (different pH) solution was taken for HPLC analysis, and then the subpackaged samples were stored at 5° C., 25° C. and 40° C. for 90 days to measure their contents. The results are shown in Table 1.

[0129] Table 1 Changes in the content of Cevimeline eye drops of the present invention at different temperatures (based on the content at zero time as 100%)

[0130]

[0131]

[0132] Result analysis:

[0133] As can be seen from Table 1, the Cevimeline solutions with pH values ​​of 5 and 6.5 are very stable when stored at 5° C., 25° C. and 40° C. for 90 days, and the content is substantially unchanged. Therefore, the Cevimeline eye drops prepared by the present invention are considered to be stable when stored at room temperature, and can be stored at room temperature for easy carrying.

[0134] Experiment 2: Stability study of eye drop formula 5 (Comparative Example 2)

[0135] An appropriate amount of freshly prepared 1% pilocarpine nitrate eye drops solution of Formulation 5 (different pH values) was taken for HPLC analysis, and then small samples stored at room temperature were taken and their contents were determined regularly. The results are shown in Table 2.

[0136] Table 2 Changes in pilocarpine content at different pH values ​​(with its zero-time content as 100%)

[0137]

[0138] Result analysis:

[0139] As can be seen from Table 2, the pilocarpine nitrate solution with a pH of 4.2 is more stable in chemical properties than the pilocarpine nitrate solution with a pH of 8.0, but its therapeutic effect is weaker than the latter, and pH 4.2 exceeds the pH requirement range for eye drops specified in the pharmacopoeia.

[0140] Experiment 3: Study on the efficacy of eye drops formula 1 and formula 5:

[0141] Test animals: cynomolgus monkeys, 2 monkeys / group, both eyes / monkey, i.e. 4 eyes / group, 1 group of cynomolgus monkeys was selected for each formulation.

[0142] Preparation before the experiment: Freshly prepared eye drops formula 1 (4.5% cevimeline) and formula 5 (1% pilocarpine nitrate, pH = 4.5); all animals were checked for abnormalities before the experiment.

[0143] Test method: 50 μl / eye of each formulation was administered as a single eye drop to both eyes, and pupil size (diameter) and lens thickness were measured before administration (0h), 0.5h, 1h, and 2h after administration, as well as observation of eye irritation.

[0144] The comparison results of pupil diameter before and after administration of formulation 1 and formulation 5 are shown in Figure 1 , according to the analysis of this figure: the miotic effect of Formula 1 and Formula 5 groups was obvious after the animals were administered, and the strongest time point and miotic intensity of the two formula groups were basically the same, and the miotic effect was most significant at 1 hour. The pupil diameter of Formula 5 group began to gradually recover around 1 hour; the time for Formula 1 in Example 1 of the present invention to start miotic effect was shorter than that of Formula 5 in Comparative Example 2, and the miotic duration was longer.

[0145] The comparison results of lens thickness before and after administration of formulation 1 and formulation 5 are shown in Figure 2 , from this figure, it is analyzed that the lens thickness of the animals in the Formula 5 group increased significantly after administration, but the increase was not as large as that in the Formula 1 group. The change trend of the lens thickness of the animals in the two formula groups was basically the same. The lens thickness was the thickest at 1 hour, and the lens thickness began to gradually recover around 1 hour. The Formula 1 group of Example 1 of the present invention began to play an earlier role in regulating the lens thickness than the Formula 5 group of Comparative Example 2, and the lens thickness was maintained for a longer time.

[0146] During the administration period, no obvious abnormalities were observed in both eyes of the animals, and no irritation symptoms such as redness and swelling of the eyelids, conjunctival congestion, dryness of the ocular surface, and increased ocular secretions occurred.

[0147] Experiment 4: Study on the efficacy of eye drop formulas 1-4 and 6-13:

[0148] Test animals: cynomolgus monkeys, 2 monkeys / group, both eyes / monkey, i.e. 4 eyes / group, 1 group of cynomolgus monkeys was selected for each formulation.

[0149] Preparation before the experiment: Freshly prepared eye drops formula 1; formula 2; formula 3; formula 4; formula 6; formula 7; formula 8; formula 9; formula 10; formula 11; formula 12; formula 13; all animals were checked for abnormalities before the experiment.

[0150] Test method: 50 μl / eye of each formulation was administered as a single eye drop to both eyes, and pupil size (diameter) and lens thickness were measured before administration (0h), 0.5h, 1h, 2h, 3h, 5h, and 7h after administration, as well as observation of eye irritation.

[0151] The comparison results of pupil diameter changes before and after administration of formulations 1-4 are shown in Figure 3, from this figure analysis, we can see that: Formula 1 and Formula 4 groups have obvious miotic effects on animals after administration, and the strongest time point and miotic intensity of the two formula groups are basically the same, and the miotic effect is most significant at 1 hour. The pupil diameter of Formula 4 group began to gradually recover around 1 hour; the duration of miosis in Formula 1 group was longer than that in Formula 4 group. Formula 2 and Formula 3 groups have obvious miotic effects on animals after administration, and the miotic effect is most significant at 0.5h to 1h, and the duration of miosis is basically the same. From this result analysis, it can be concluded that the higher the concentration of Cevimeline, the more significant the miotic effect, and the duration of miosis does not change significantly with the change of concentration.

[0152] The comparison results of the lens thickness changes before and after administration of formulations 1-4 are shown in Figure 4 , from this figure analysis: the lens thickness of the animals in the formula 1 and formula 4 groups increased significantly after administration. The lens thickness of the formula 1 increased the most at 1 hour, and then gradually recovered; the highest point of the formula 4 appeared at 0.5 hours, and then gradually recovered. The lens thickness of the formula 2 and formula 3 groups also increased significantly after administration. From this result analysis, it can be concluded that the higher the concentration of Cevimeline, the more obvious the increase in lens thickness, and the effect of the formula 1 group is more prominent than that of the formula 4 group.

[0153] The comparison results of pupil diameter changes before and after administration of formulation 2 and formulations 6-9 are shown in Figure 5 , from this figure, it can be analyzed that adding different vitamins or vitamin-like compounds on the basis of Formula 2 can improve the miotic effect, among which the effect of Formula 6 is obvious. Therefore, it can be seen that vitamins or vitamin-like compounds can promote and synergistically improve the efficacy of Cevimeline in treating presbyopia.

[0154] The comparison results of the changes in lens thickness before and after administration of formulation 2 and formulations 6-9 are shown in Figure 6 Adding different vitamins or vitamin-like compounds to Formula 2 can increase the thickness of the lens, among which Formula 6 has the most obvious effect, which is consistent with the result of pupil constriction.

[0155] The comparison results of pupil diameter changes before and after administration of formulation 2 and formulations 10-13 are shown in Figure 7 , from this figure, it can be analyzed that vitamins or vitamin-like compounds alone cannot have a miotic effect. Figure 5 The analysis showed that vitamins or vitamin-like compounds and active ingredients act synergistically to enhance and prolong the efficacy of cevimeline in treating presbyopia.

[0156] The comparison results of the changes in lens thickness before and after administration of formulation 2 and formulations 10-13 are shown in Figure 8 , from this figure analysis: vitamins or vitamin-like compounds alone can not cause changes in the lens, combined with Figure 6The analysis showed that vitamins or vitamin-like compounds and active ingredients act synergistically to enhance and prolong the efficacy of cevimeline in treating presbyopia.

[0157] During the administration period, no obvious abnormalities were observed in both eyes of the animals, and no irritation symptoms such as redness and swelling of the eyelids, conjunctival congestion, dryness of the ocular surface, and increased ocular secretions occurred.

[0158] Experiment 5: Intraocular pressure test study of eye drop formula 2-4 (study under normal conditions):

[0159] Test animals: healthy New Zealand rabbits, 3 rabbits / group, two eyes / rabbit, i.e. 6 eyes / group, 1 group of New Zealand rabbits was selected for each formulation.

[0160] Preparation before the experiment: freshly prepared eye drops formulation 2-4; check both eyes of each animal within 24 hours before the experiment to ensure that the rabbit eyes have no irritation symptoms such as redness, swelling, congestion, and no corneal defects and conjunctival damage; all animals are checked for abnormalities before the experiment.

[0161] Test method: 50 μl / eye of each preparation formula was administered as a single eye drop to both eyes, and intraocular pressure was tested before administration (0h), and 0.25h, 0.5h, 1h, 2h, 3h, 4h, and 5h after administration, as well as eye irritation observation.

[0162] The intraocular pressure of formulation 2-4 fluctuated slightly from 0h before administration to 5h after administration. The comparison results are shown in Fig. 9 Overall, there was no significant difference compared with before administration, which was within the normal fluctuation range.

[0163] During the administration period, no obvious abnormalities were observed in both eyes of the animals, and no irritation symptoms such as eyelid redness and swelling, conjunctival congestion, ocular dryness, increased ocular secretions, etc. were observed. During the administration period, tear secretion was observed in one animal in each of the experimental groups of formula 2 and 3.

[0164] Experiment 6: Intraocular pressure test study of eye drop formula 2-4 (study after mydriasis):

[0165] Test animals: healthy New Zealand rabbits, 3 rabbits / group, two eyes / rabbit, i.e. 6 eyes / group, 1 group of New Zealand rabbits was selected for each formulation.

[0166] Preparation before the experiment: freshly prepared eye drops formulation 2-4; check both eyes of each animal within 24 hours before the experiment to ensure that the rabbit eyes have no irritation symptoms such as redness, swelling, congestion, and no corneal defects and conjunctival damage; all animals are checked for abnormalities before the experiment.

[0167] Test method: Use compound tropicamide eye drops to dilate the animals' pupils and increase the intraocular pressure. Then, administer sodium chloride eye drops (as a blank control) and 50 μl / eye of each formulation to both eyes once. Test the intraocular pressure before mydriasis, before administration (0h), and 0.25h, 0.5h, 1h, 2h, 3h, 4h, and 5h after administration, as well as observe for eye irritation.

[0168] The intraocular pressure of each group of animals before mydriasis was slightly different. After mydriasis with compound tropicamide eye drops, the intraocular pressure increased, which is consistent with the slight increase in intraocular pressure after clinical use of mydriatic drugs. The overall trend after administration of formulations 2-4 was basically the same, and the comparison results of the changes are shown in Fig.10 After administration, intraocular pressure gradually began to return to its pre-mydriasis state.

[0169] During the administration period, no obvious abnormalities were observed in both eyes of the animals, and no irritation symptoms such as redness and swelling of the eyelids, conjunctival congestion, dryness of the ocular surface, and increased ocular secretions occurred.

[0170] In the following examples, the pH regulator selected is hydrochloric acid or sodium hydroxide to adjust the eye drops to a desired pH, and the osmotic pressure regulator is sodium chloride to adjust the eye drops to a desired osmotic pressure.

[0171] Example 11: Preparation of eye drop formulation 17 (1.75% Cevimeline, pH 6.0)

[0172] The preparation method is as follows:

[0173] Add an appropriate amount of water for injection to the dispensing bottle, add the prescribed amount of sodium chloride in order, and stir to dissolve; after it is completely dissolved, add the prescribed amount of cevimeline and stir to dissolve; after it is completely dissolved, measure its pH, and adjust pH to 6.0 with sodium hydroxide; use water for injection to make up to the prescribed amount, and measure its osmotic pressure and record the measured value, osmotic pressure: 296mOsmol / kg. Then sterile filter and package for subsequent use. The eye drop formula was tested for physical appearance, content determination, impurities, pH value and osmotic pressure.

[0174] Example 12: Preparation of eye drop formulation 18 (2.0% Cevimeline, pH 6.0)

[0175] Referring to the operating steps of Example 11, 2.0% Cevimeline eye drops were prepared, with pH = 6.0 and osmotic pressure: 297 mOsmol / kg.

[0176] Example 13: Preparation of eye drop formulation 19 (2.25% Cevimeline, pH 5.0)

[0177] Referring to the operating steps of Example 11, 2.25% Cevimeline eye drops were prepared, with pH = 5.0 and osmotic pressure: 289 mOsmol / kg.

[0178] Example 14: Preparation of eye drop formulation 20: 2.25% Cevimeline, pH 6.0

[0179] Referring to the operating steps of Example 11, 2.25% Cevimeline eye drops were prepared, with pH = 6.0 and osmotic pressure: 280 mOsmol / kg.

[0180] Example 15: Preparation of eye drop formulation 21: 2.25% Cevimeline, pH 6.5

[0181] Referring to the operating steps of Example 11, 2.25% Cevimeline eye drops were prepared, with pH = 6.5 and osmotic pressure: 279 mOsmol / kg.

[0182] Example 16: Preparation of eye drop formulation 22: 2.25% Cevimeline, pH 7.0

[0183] Referring to the operating steps of Example 11, 2.25% Cevimeline eye drops were prepared, with pH = 7.0 and osmotic pressure: 287 mOsmol / kg.

[0184] Example 17: Preparation of eye drop formulation 23: 2.25% Cevimeline, pH 7.5

[0185] Referring to the operating steps of Example 11, 2.25% Cevimeline eye drops were prepared, with pH = 7.5 and osmotic pressure: 286 mOsmol / kg.

[0186] Comparative Example 7: Preparation of eye drop formulation 24 (1.25% pilocarpine hydrochloride, boric acid / sodium citrate system)

[0187] Add an appropriate amount of water for injection to the ingredient bottle, add the prescribed amount of sodium chloride, boric acid and sodium citrate in order, and stir to dissolve; after all of them are dissolved, add the prescribed amount of pilocarpine hydrochloride, and stir to dissolve; after all of them are dissolved, measure the pH, and adjust the pH to 5.0 with sodium hydroxide; use water for injection to make up to the prescribed amount, and measure the osmotic pressure and record the measured value, osmotic pressure: 280mOsmol / kg. Then sterile filter and package for subsequent use. The eye drop formula was tested for physical appearance, content determination, impurities, pH value and osmotic pressure.

[0188] Comparative Example 8: Preparation of eye drop formula 25 (1.25% cevimeline, boric acid / sodium citrate system)

[0189] Referring to the operating steps of Comparative Example 7, 1.25% Cevimeline eye drops were prepared, with pH = 6.5 and osmotic pressure: 279 mOsmol / kg.

[0190] Comparative Example 9: Preparation of eye drop formula 26 (3% Cevimeline, boric acid / sodium citrate system)

[0191] Referring to the operating steps of Comparative Example 7, 3% Cevimeline eye drops were prepared, with pH = 6.5 and osmotic pressure: 286 mOsmol / kg.

[0192] Comparative Example 10: Preparation of eye drop formula 27 (2.25% cevimeline, sodium citrate system)

[0193] Referring to the operating steps of Comparative Example 7, 2.25% Cevimeline eye drops were prepared without adding boric acid, and the pH value was 6.5; the osmotic pressure was 296 mOsmol / kg.

[0194] Comparative Example 11: Preparation of eye drop formula 28 (2.25% Cevimeline, phosphoric acid system)

[0195] Referring to the operating steps of Comparative Example 7, phosphoric acid was used instead of boric acid and sodium citrate to prepare 2.25% Cevimeline eye drops, with pH = 7.0 and osmotic pressure: 287 mOsmol / kg.

[0196] Comparative Example 12: Preparation of eye drop formula 29 (2.25% cevimeline, citric acid system)

[0197] Referring to the operating steps of Comparative Example 7, citric acid and disodium hydrogen phosphate were used instead of boric acid and sodium citrate to prepare 2.25% cevimeline eye drops, with pH = 7.0 and osmotic pressure: 281 mOsmol / kg.

[0198] Example 18: Preparation of eye drop formulation 30 (4% Cevimeline, pH 6.5)

[0199] Referring to the operating steps of Example 11, 4% Cevimeline eye drops were prepared, with pH = 6.5 and osmotic pressure: 292 smol / kg.

[0200] Example 19: Preparation of eye drop formulation 31 (0.9% saline)

[0201] Add an appropriate amount of water for injection to the dispensing bottle, add the prescribed amount of sodium chloride in order, and stir to dissolve; after it is completely dissolved, use water for injection to make up to the prescribed amount, and measure its pH and osmotic pressure and record the measured values, pH = 7.1, osmotic pressure 288mOsmol / kg. Then sterile filter and package for subsequent use. The eye drop formula was tested for physical appearance, content determination, impurities, pH value and osmotic pressure.

[0202] Experiment 7: Study on the efficacy of eye drop formula 17-23:

[0203] Test animals: middle-aged and elderly cynomolgus monkeys (8-15 years old), 2 monkeys / group with both eyes, i.e. 4 eyes / group, 1 group of cynomolgus monkeys was selected for each formulation.

[0204] Preparation before the experiment: Freshly prepared eye drops formula 17-23; all animals were checked for abnormalities before the experiment.

[0205] Test method: 50 μL / eye of each formulation was administered once to both eyes, and the blood pressure before administration (0 h) was measured.

[0206] The pupil size (diameter) and lens thickness were measured at 0.25 h, 1 h, 2 h, 4 h, 6 h, and 8 h after administration, and ocular irritation was observed.

[0207] The comparison results of pupil diameter before and after administration of formulations 17-23 are shown in Fig.11 , from this figure analysis: the strongest time point of mydriasis in animals of the 7 formula groups is basically the same, all of which take effect 15 minutes after administration, and the mydriasis is most significant at 1 hour, and the mydriasis lasts up to 8 hours. When the pH of the eye drops is 6, the constriction effect of the pupils at concentrations of 1.75%, 2% and 2.25% is not much different, and the concentration of 2.25% has a slightly higher effect intensity. The fixed concentration of 2.25% was used to compare the constriction effect of different pH on the pupil. The results showed that the mydriasis effect of pH 6-7 was similar. Therefore, Cevimeline eye drops can choose a pH value similar to that of tears to improve the comfort of patients. From this result analysis, it can be concluded that Cevimeline eye drops have good tolerance, fast onset, and long duration of action. Only one eye drop is needed per day to maintain the effect for up to 8 hours. The administration method is simple, the operation is convenient, and it is more easily accepted by patients.

[0208] The change in lens thickness before and after administration of formulation 17-23 was not obvious in middle-aged and elderly monkeys, with a change of about 5%. Based on the results, it can be concluded that Cevimeline eye drops have a mild regulation on the lens, will not cause spasms, have little effect on lens thickness, and will not affect long-distance vision.

[0209] During the administration period, no obvious abnormalities were observed in both eyes of the animals, and no irritation symptoms such as redness and swelling of the eyelids, animal irritability, conjunctival congestion, dry eye surface, and increased eye secretions occurred.

[0210] Experiment 8: Study on the efficacy of eye drops formula 21 and formulas 25-29:

[0211] Test animals: middle-aged and elderly cynomolgus monkeys (8-15 years old), 2 monkeys / group with both eyes, i.e. 4 eyes / group, 1 group of cynomolgus monkeys was selected for each formulation.

[0212] Preparation before the experiment: Freshly prepared eye drops formula 21 and formula 25-29; all animals were checked for abnormalities before the experiment.

[0213] Test method: 50 μL / eye of each formulation was administered as a single eye drop to both eyes, and the pupil size (diameter) and lens thickness were measured before administration (0h), 0.25h, 1h, 2h, 4h, 6h and 8h after administration, and the eye irritation was observed.

[0214] The comparison results of pupil diameter changes before and after administration of formulation 21 and formulations 25-29 are shown in Fig.12 From this figure, we can see that no matter which buffer system is used, the miotic effect of the six groups of animals in the formulations 21 and 25-29 is similar to the results of Experiment 1: the strongest time point is basically the same, all taking effect 15 minutes after administration, and the miotic effect is most significant at 1 hour, and the miotic time is as long as 8 hours. Different buffer systems have different effects on miotic effect. The most comfortable pH 6.5-7 eye drops for human eyes are selected, among which green no-additive drops are

[0215] The miotic effect of eye drops formula 21 was the most obvious. Based on the results, it was concluded that Cevimeline eye drops did not contain any preservatives, buffers, chelating agents, excipients or other excipients, which reduced the irritation to the eyes caused by excipients, had high safety, few side effects, and was suitable for long-term use.

[0216] The changes in lens thickness before and after administration of formulation 25-29 were similar to the results of experiment 1.

[0217] During the administration period, no obvious abnormalities were observed in both eyes of the animals, and no irritation symptoms such as redness and swelling of the eyelids, animal irritability, conjunctival congestion, dry eye surface, and increased eye secretions occurred.

[0218] Experiment 9: Study on the lens accommodation effect of eye drop formula 24 and 25 in young and middle-aged cynomolgus monkeys:

[0219] Test animals: Young cynomolgus monkeys (3-4 years old), 2 monkeys / group with both eyes, i.e. 4 eyes / group; middle-aged and elderly cynomolgus monkeys (8-15 years old), 2 monkeys / group with both eyes, i.e. 4 eyes / group; 1 group of young cynomolgus monkeys and 1 group of middle-aged and elderly cynomolgus monkeys were selected for each formulation.

[0220] Preparation before experiment: Freshly prepared eye drops formula 24 and formula 25; all animals were checked for abnormalities before experiment.

[0221] Test method: 50 μL / eye of each formulation was administered as a single eye drop to both eyes, and the lens thickness was measured before administration (0h), 0.25h, 1h, 2h, 3h, 5h and 7h after administration in young monkeys, and 0.25h, 1h, 2h, 4h, 6h and 8h after administration in middle-aged and elderly monkeys, and the eye irritation was observed.

[0222] The results of the comparison of lens changes in young and middle-aged cynomolgus monkeys before and after administration of formulations 24 and 25 are shown in Fig.13 From this figure, we can see that: whether it is young monkeys or middle-aged and elderly monkeys, the ability of pilocarpine to regulate the lens is greater than that of cevimeline; and the intensity of pilocarpine's regulation on the lens of young monkeys and middle-aged and elderly monkeys is basically the same, and even the duration of action on the lens of middle-aged and elderly monkeys is longer. There is a slight difference in the intensity of regulation of cevimeline on the lens of young monkeys and middle-aged and elderly monkeys. The ability of cevimeline to regulate the lens of middle-aged and elderly monkeys is weaker than that of young monkeys, and the duration is shorter. From this result, it can be analyzed that cevimeline M3 receptor agonist has better selectivity for the target, hardly affects distance vision, and is more suitable for the prevention or treatment of presbyopia symptoms in the middle-aged and elderly groups.

[0223] During the administration period, no obvious abnormalities were observed in both eyes of the animals, and no irritation symptoms such as redness and swelling of the eyelids, animal irritability, conjunctival congestion, dry eye surface, and increased eye secretions occurred.

[0224] Experiment 10: Evaluation of the ocular safety and tolerability of cevimeline eye drops in a rabbit model:

[0225] Test animals: New Zealand white rabbits, ordinary grade, aged more than 140 days at the start of dosing, male: 2.4-3.0kg; female: 2.9-3.3kg; female animals are nulliparous and non-pregnant animals. 12 (6 males / 6 females) New Zealand white rabbits were divided into 3 groups, with 2 males and 2 females in each group, i.e. 8 eyes / group; 1 group of New Zealand white rabbits was selected for each formulation. The other 2 animals / sex were kept as reserve animals until 5 days after dosing.

[0226] Preparation before the experiment: Freshly prepared eye drops formula 21 (medium concentration group), formula 30 (high concentration group) and formula 31 (solvent control group); all animals were checked for abnormalities before the experiment, and no animals with poor health or obvious physical abnormalities were used for the experiment. When randomly grouped, the weight difference of animals in all experiments was within ±20% of the average weight of the same sex. The experimental plan and changes or operations involving animal management and use were reviewed and approved by the company's animal management and use committee before starting. During the experiment, veterinary technicians supervised the animal welfare issues in the experiment and communicated with the subject leader to decide whether to adopt an appropriate treatment plan.

[0227] Experimental method: 50 μL / eye of each formulation was administered tid to both eyes, and the ocular safety and tolerability of cevimeline eye drops in rabbits were recorded and analyzed.

[0228] Detailed clinical observation and cage-side observation: Before the start of the trial, a detailed clinical observation was conducted on all animals (including reserve animals) before dosing on Day 1, and then a detailed clinical observation was conducted once a week during the dosing period. A detailed clinical observation was conducted on all experimental animals on the planned dissection day. Before the start of the trial, all experimental animals were cage-side observed once a day starting from Day-2. Cage-side observation was conducted once a day during the trial. Animals that were scheduled for detailed clinical observation during the same time period were not cage-side observed. No symptoms related to the test article were observed during the entire trial. On Day 16 before dissection, it was observed that both the vehicle control group and the high-dose group had mild conjunctival congestion in the right eye on Day 16. This symptom is not considered to be related to Cevimeline Eye Drops because the symptom was also observed in the eyes of the vehicle group.

[0229] Eye irritation scoring: Before the start of dosing, eye irritation scoring was performed twice in both eyes of all animals (including reserve animals), once a day after the last dosing on days 1-7 of dosing, and once in the second week of dosing.

[0230] During the trial, no ocular irritation related to Cevimeline Ophthalmic Solution was observed. Conjunctival hyperemia / discharge observed during the ocular irritation scoring was not related to Cevimeline Ophthalmic Solution as it was observed in both treated and vehicle-treated eyes and occurred at similar rates between groups.

[0231] Body weight: Each animal (including the reserve animals) was weighed once in the early stage of the experiment, and once a week during the entire In-Life period. The body weight was weighed once on the planned dissection day (after fasting). During the experiment, no weight changes related to Cevimeline eye drops were observed.

[0232] Food intake measurement: The quantitative food intake was assessed by weighing the feed daily starting from Day-2 and throughout the experimental period (except during fasting). The animals were fasted overnight before the planned autopsy. No changes in food intake related to Cevimeline eye drops were observed during the experimental period.

[0233] Ophthalmological examination: All animals in the 3 groups underwent a complete ophthalmological examination of both eyes, including ocular irritation, before the start of dosing, 1 week, and 2 weeks after dosing. The examination included fundus examination, using an indirect ophthalmoscope and condenser, and slit lamp examination. The anterior segment of both eyes was examined with the aid of a slit lamp before mydriasis. Ocular structures were scored according to the McDonald-Shadduck scoring system. Fluorescein staining (sterile ophthalmic test strips of sodium fluorescein moistened with sterile saline) was used to evaluate the condition of the corneal surface. For the examination of the posterior segment, mydriatic drops were placed in both eyes before the examination. The posterior segment (fundus) of the eye was examined with the aid of an indirect ophthalmoscope.

[0234] During the trial, no ophthalmic symptoms related to Cevimeline eye drops were found. In the McDonald-Shadduck score examination, mild conjunctival congestion, a small amount of secretions, and local small-area fluorescence staining were observed in the three groups on Day 7, Week 1, and Week 2. The incidence in each group was low or similar to that in the vehicle group. Small anterior lens cortex opacity was observed in one eye of the animal in the medium-dose group, and small anterior lens cortex opacity was also observed in this eye before administration. Small anterior lens capsule opacity was observed in one eye of the animal in the vehicle group, and small anterior lens capsule opacity was also observed before the change of administration, and other animals that were administered with the drug were not found. Therefore, the McDonald-Shadduck score examination results were considered to be not related to Cevimeline eye drops. All other ocular findings before the trial and at the time of administration were considered to be common background or spontaneous findings consistent with the age and strain of the species.

[0235] Intraocular Pressure: The intraocular pressure of all animals in groups 1-3 was measured using a TonoVet tonometer on different 3 days during the adaptation period before the start of dosing. The measurements were taken before the start of dosing, 1 week after dosing, and within 2 weeks. The animals were fixed so that the test eye was perpendicular to the probe of the TonoVet tonometer, and the IOP was measured at least 3 times until the value stabilized. The average of the last three stable values ​​was calculated as the final IOP. No changes in intraocular pressure related to Cevimeline eye drops were observed during the trial.

[0236] Tear secretion test: Before the experiment, all animals (including reserve animals) were tested for tear secretion twice. Tear secretion was measured once in both eyes of all animals on Day 2, Day 8, and Day 15.

[0237] The tear secretion measurement method is as follows: After the animal is restrained and the mood is stable, fold one end of the tear test paper and put it into the 1 / 3 conjunctival sac of the lower eyelid. After closing the eyes for 5 minutes, take out the test paper and measure its wet length from the folded part. If the test paper falls off within 5 minutes, wait 30 minutes before taking the second measurement.

[0238] During the trial, no changes in tear secretion were observed related to cevimeline eye drops.

[0239] Visual electrophysiology (ERG): Dark-vision and photopic electroretinogram (ERG) examinations were performed on both eyes of all animals before drug administration and before dissection. Animals were adapted to the dark environment for at least 0.5 hours. The pupils were dilated by instilling mydriatics and recorded in the raw data. Animals were anesthetized by a combined intramuscular injection of ketamine (30 mg / kg) and thiazine (2 mg / kg). Benzothiozine hydrochloride injection (4.5 mg / kg) was also given to ensure that the animals woke up normally. The actual anesthesia method was recorded in the raw data. During the experiment, no retinal electrophysiological changes related to Cevimeline eye drops were observed. Changes in latency or amplitude (including statistically significant changes) were considered incidental changes because of their small amplitude and lack of dose compliance, and were not related to Cevimeline eye drops.

[0240] Toxicokinetics: After topical administration of medium and high doses of Cevimeline eye drops to male and female rabbits three times a day for 14 consecutive days, plasma concentrations of Cevimeline at each dose were measured to determine systemic exposure. On Day 1 and Day 14, blood samples were collected from all rabbits at 0 hours (pre-dose) and 0.25, 1, 2, 4, 6, and 12 hours after the last dose. For each time point, approximately 0.75-1 mL of whole blood was collected from the central ear artery of conscious rabbits. Whole blood will be placed in labeled blood collection tubes with K2EDTA as an anticoagulant. The sample tubes will be gently inverted several times to ensure mixing and immediately placed in wet ice for storage. Plasma was obtained by centrifugation at 3200×g, 4°C for 10 minutes within 1 hour after blood collection. Plasma was transferred to a clearly labeled transparent polypropylene tube, immediately placed vertically on dry ice, and stored in an ultra-low temperature freezer at <60°C until shipped to the Bioanalytical Department for analysis. The whole blood collection, plasma collection, and storage time were recorded in the experimental record. The test results showed that Cevimeline had a rapid onset of action, with the average time to peak Cevimeline levels in experimental animals in all dose groups occurring 15 minutes after the first dose; there was no significant gender difference in systemic exposure in all dose groups; systemic exposure on the first and 14th days in all dose groups increased with increasing doses; and no significant drug accumulation was observed in systemic exposure in both sexes in all dose groups.

[0241] Histopathology: All experimental animals were dissected on day 16. During the autopsy, the gross lesions of all organs were recorded, and the body and musculoskeletal system; the animal's body surface and natural orifices; the outer surface of the cranial cavity and brain; the thoracic cavity, abdominal cavity and pelvic cavity, as well as the intracavitary tissues and organs and gross lesions were observed with the naked eye.

[0242] At planned necropsy, organ weights of adrenal glands (bilateral), brain, heart, kidneys (bilateral), liver and gallbladder, ovaries (bilateral), spleen, testes (bilateral), and thymus were collected from all experimental animals. Paired organs were weighed together, and relative organ weights (brain weight to body weight ratio) were calculated based on brain weight and the final weight of the animal. In addition, all masses and gross ocular lesions were collected. Eyeballs and optic nerves, testes and epididymis were fixed in modified Davidson's solution, and other tissues were fixed in 10% neutral buffered formalin solution. Eyeballs and optic nerves, testes and epididymis were fixed in the relevant fixative for 24 to 72 hours and then transferred to 10% formalin neutral buffered solution. All ocular tissues (eyeballs and optic nerves, upper and lower eyelids, lacrimal glands, Harderian glands, eye muscles) were divided into left and right. Before being placed in the fixative, sutures were made at approximately 12 o'clock in the bulbar conjunctiva of each eyeball as a histological positioning mark.

[0243] For all experimental animals, limited samples such as eye tissues (eyeballs and optic nerves, upper and lower eyelids, lacrimal glands, Harderian glands, eye muscles), nasal conchas and all gross lesions and masses were removed from the fixative, sampled, dehydrated, paraffin-embedded, sectioned, HE-stained, and microscopically examined, and analyzed to produce a pathology report.

[0244] The results showed that Cevimeline eye drops were highly safe for New Zealand rabbits. The rabbits tolerated low and high concentrations of the drug well when given eye drops three times a day for 14 consecutive days. There were no Cevimeline-related animal deaths or changes in organ weights, macroscopic changes, or histopathological changes.

[0245] Experiment 11: Evaluation of the ocular safety and tolerability of Cevimeline eye drops in monkey models:

[0246] Test animals: Cynomolgus monkeys, normal grade, age range of about 3-5 years, weight range of 2.0 to 3.6 kg. Female animals are nulliparous and non-pregnant animals. 6 (3 males / 3 females) cynomolgus monkeys were divided into 3 groups, 1 male and 1 female in each group, i.e. 4 eyes / group; 1 group of cynomolgus monkeys was selected for each formulation. Another 1 animal / sex was kept as a reserve animal until 5 days after administration.

[0247] For pre-experimental preparation, experimental methods, and experimental content, please refer to Experiment 10.

[0248] The results showed that cevimeline eye drops were highly safe for crab-eating macaques. The monkeys were given low and high concentrations of eye drops three times a day for 14 consecutive days and tolerated them well. There were no cevimeline-related animal deaths or changes in organ weights. No changes were observed with the naked eye. No cevimeline-related eye irritation was observed. There were no changes in body weight, food intake, ophthalmic examination, body temperature, intraocular pressure, tear secretion, visual electrophysiological examination, fundus photography, and histopathology during clinical observation.

[0249] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. Use of Cevimeline or its salt or its stereoisomer in the preparation of ophthalmic preparations, characterized in that: The ophthalmic preparation comprises the active ingredient of cevimeline and vitamins or vitamin-like substances, wherein the vitamins or vitamin-like substances are lipoic acid, vitamin B12, sodium pantothenate or niacinamide; When the vitamin or vitamin-like substance is lipoic acid, the added amount of cevimeline is 2.5% w / w, and the added amount of lipoic acid is 0.05% w / w.

2. The use according to claim 1, characterized in that: The ophthalmic preparation comprises, by weight, 0.1-10 parts of cevimeline or its salt or its stereoisomer and 0-10 parts of vitamins or vitamin-like substances.

3. An ophthalmic preparation, characterized in that: Calculated by weight, it contains 0.1-10 parts of cevimeline or its salt or stereoisomer thereof and 0-10 parts of vitamins or vitamin-like substances; The vitamin or vitamin-like substance is lipoic acid, vitamin B12, sodium pantothenate or niacinamide; When the vitamin or vitamin-like substance is lipoic acid, the added amount of cevimeline is 2.5% w / w, and the added amount of lipoic acid is 0.05% w / w.

4. The ophthalmic preparation according to claim 3, characterized in that By weight, the composition comprises 1-5 parts of cevimeline or its salt or its stereoisomer, and the vitamin or vitamin-like substance comprises 0-2 parts.

5. The ophthalmic preparation according to claim 4, characterized in that By weight, it contains 2-5 parts of cevimeline or its salt or its stereoisomer, and the vitamin or vitamin-like substance contains 0.01-0.1 parts.

6. The ophthalmic preparation according to claim 2, characterized in that The dosage forms of the ophthalmic preparation include eye drops, eye gels, eye ointments, eye patches, eye masks, eye washes or intraocular injections.

7. The ophthalmic preparation according to claim 6, characterized in that The pH value of the ophthalmic preparation is adjusted to 4.0-9.

0.

8. The ophthalmic preparation according to claim 7, characterized in that The osmotic pressure value of the ophthalmic preparation is adjusted to 260-350 mOsmol / kg.

9. The ophthalmic preparation according to any one of claims 7 to 8, characterized in that The pH value is adjusted by a pH adjusting agent, which includes one or more of hydrochloric acid, boric acid, phosphoric acid, citric acid, sodium hydroxide and potassium hydroxide; the osmotic pressure value is adjusted by an osmotic pressure adjusting agent, which includes one or more of sodium chloride, sodium sulfate, potassium chloride, magnesium chloride, mannitol, mannose and dextrose.

10. The ophthalmic preparation according to claim 9, characterized in that The cevimeline ophthalmic preparation further comprises a solvent, and the solvent is selected from water for injection or a buffer; wherein the buffer is an aqueous solution of a buffer, and the buffer comprises one or more of carbonates, phosphates, citrates, acetates, borates, barbiturates and 3-(N-morpholine)propanesulfonates.

11. The ophthalmic preparation according to claim 3, characterized in that The salt includes an inorganic salt or an organic salt of Cevimeline, and the stereoisomer includes a cis isomer, a trans isomer or a diastereomer of Cevimeline.

12. The ophthalmic preparation according to claim 3, characterized in that The invention comprises at least one of cevimeline or its salt or cevimeline isomers in an amount of 0.1-10% w / w.

13. Use of the ophthalmic preparation according to any one of claims 3 to 12 in the preparation of a medicament for preventing or treating eye diseases.

14. The use according to claim 13, characterized in that The eye disease includes at least one of presbyopia, ocular hypertension, dry eye, refractive error, photophobia or angle-closure glaucoma.

15. The use according to claim 14, characterized in that: The eye diseases include: (1) Presbyopia, a condition in which blurriness of near objects is caused by a decrease in the accommodative ability of the lens; (2) Eye diseases that cause headaches, decreased vision, and even blindness due to the narrow anterior chamber angle, and also include diseases involving the cornea and conjunctiva of the eye; (3) A refractive error caused by the inability of external light to focus normally on the retina; (4) Tear duct disease or tear gland disease.

16. The method for preparing the ophthalmic preparation according to any one of claims 3 to 12, characterized in that: The steps include: (1) adding cevimeline or its salt or its stereoisomer to a portion of the solvent and mixing the mixture or further adding vitamins or vitamin-like substances and mixing the mixture; (2) Adding excipients for ophthalmic preparations to adjust pH and osmotic pressure; (3) Add the remaining solvent to make up the volume.

17. The preparation method according to claim 16, characterized in that: Also includes: (4) Filter and sterilize through a microporous filter membrane, and then perform blow-fill-seal under the aseptic protection of a Class A air shower to aseptically fill the finished product.

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