WO2022268167A1 - 叶酸类衍生物在制备治疗隐形眼镜不适及干眼症药物的应用 - Google Patents

叶酸类衍生物在制备治疗隐形眼镜不适及干眼症药物的应用 Download PDF

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WO2022268167A1
WO2022268167A1 PCT/CN2022/100814 CN2022100814W WO2022268167A1 WO 2022268167 A1 WO2022268167 A1 WO 2022268167A1 CN 2022100814 W CN2022100814 W CN 2022100814W WO 2022268167 A1 WO2022268167 A1 WO 2022268167A1
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eye
dry eye
discomfort
contact lens
expression
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PCT/CN2022/100814
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French (fr)
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成永之
顾睿
连增林
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连云港金康和信药业有限公司
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Priority to AU2022300058A priority Critical patent/AU2022300058A1/en
Priority to EP22827659.8A priority patent/EP4360635A1/en
Priority to CN202280045156.4A priority patent/CN117561065A/zh
Priority to JP2023579360A priority patent/JP2024524276A/ja
Publication of WO2022268167A1 publication Critical patent/WO2022268167A1/zh

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    • 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
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D475/00—Heterocyclic compounds containing pteridine ring systems
    • C07D475/02—Heterocyclic compounds containing pteridine ring systems with an oxygen atom directly attached in position 4
    • C07D475/04—Heterocyclic compounds containing pteridine ring systems with an oxygen atom directly attached in position 4 with a nitrogen atom directly attached in position 2

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  • the present invention relates to medicines or health foods for the treatment of dry eye, the present invention relates to a method for improving the experience of wearing contact lenses for wearers who are uncomfortable wearing contact lenses, and further the present invention relates to eye drops and contact lens discomfort symptoms for the treatment of dry eye eye drops.
  • contact lens discomfort is characterized by episodic or persistent ocular sensations associated with contact lens wear.
  • symptomatic Symptom descriptions eg, eye irritation, burning, stinging, and pain
  • contact lens wearers are highly similar to those associated with dry eye disease, which involves inflammatory responses and neuropathic pain.
  • Dry eye is a chronic inflammatory disease in which tear instability is accompanied by increased tear osmolarity, which activates stress signals in resident immune cells of the ocular surface epithelium and triggers the activation of innate inflammatory molecules.
  • Th1 cytokines namely interferon gamma (IFN- ⁇ )
  • IFN- ⁇ interferon gamma
  • the term "dry eye” is accurately described as a feeling of the eyes.
  • Ocular Surface Disease Index (OSDI) is the most commonly used questionnaire for clinical diagnosis of dry eye. It is mainly used to diagnose dry eye and evaluate the severity of the disease. OSDI mainly asks about the sensation of the eyes, including the stimulation of light and wind on the eyes, the influence of reading, writing and watching TV on the comfort of the eyes, etc.
  • the above-mentioned problems are all aimed at the unpleasant feeling and emotional experience of the eyes. Without the diagnosis of biomarkers such as inflammatory factors, dry eye syndrome can actually be attributed to a painful disease. Painful disorders can be broadly divided into two categories: nociceptive pain and neuropathic pain, both of which may be associated with dry eye.
  • Nociceptive pain is usually transient and the result of tissue damage and inflammation, and neuropathic pain is Pain caused by a disease or disease directly affecting the somatosensory system tends to be chronic.
  • nociceptive and neuropathic pain triggers may be the same, but these trigger stimuli can lead to transient or persistent changes in sensory neuronal phenotype [Galor A, Levitt RC, Felix ER, Martin ER, Sarantopoulos CD.
  • Neuropathic ocular pain an important yet underevaluated feature of dry eye. Eye(Lond). 2015; 29(3):301 ⁇ 312.].
  • dry eye syndrome has a wide range, but in reality, patients who go to the ophthalmology clinic for medical treatment often belong to dry eye symptoms with more severe symptoms, that is, dry eye syndrome with aqueous tear deficiency, including patients with lack of lacrimal glands or blocked lacrimal ducts. Some dry eye patients do not have tear deficiency and belong to evaporative dry eye, but these patients are more sensitive to wind and light [Chhadva P, Lee T, Sarantopoulos CD, et al. Human Tear Serotonin Levels Correlate with Symptoms and Signs of Dry Eye. Ophthalmology. 2015; 122(8):1675 ⁇ 1680.].
  • Lifitegrast is a small molecule that inhibits the binding of leukocyte-associated antigen 1 (LFA-1) on T cells to its ligand intercellular adhesion molecule 1 (ICAM1) on antigen-presenting, epithelial and vascular endothelial cells, which are present in Clinical trials have improved the symptoms of dry eye, but these treatments cannot address the acute impact of dry eye on the ocular surface, especially under the acute stimulation of light and wind, they cannot effectively relieve eye irritation. Corticosteroids have shown efficacy in response to acute irritation, however, long-term use of corticosteroids carries risks of cataracts and glaucoma.
  • LFA-1 leukocyte-associated antigen 1
  • IAM1 intercellular adhesion molecule 1
  • Nerve growth factor is a potent stimulator of axonal growth and regeneration, and NGF has been shown to aid in the healing of persistent epithelial defects, but during nerve injury, NGF release increases neuronal excitability and reduces pain threshold. Substantial evidence has shown that NGF is important in the mediation and enhancement of pain, leading to the development of NGF antagonists as potential analgesics and antihyperalgesics. Therefore, whether the use of NGF antagonists or NGF treatment is beneficial to patients with dry eye syndrome still needs further research.
  • 5-Methyltetrahydrofolate a form of folic acid in the body, has been shown to reduce levels of homocysteine, an important risk factor for macular degeneration in older eyes.
  • folic acid can promote the secretion of nerve growth factor to promote the repair of nerve damage.
  • NGF non-selective cation channel
  • TRPV1 non-selective cation channel
  • TRPV1 expression changes and thermal hyperalgesia and Associated with the development of hyperalgesia.
  • Folinic acid is also a derivative of folic acid, which can be converted into 5-methyltetrahydrofolate in the body. There are no studies reporting a beneficial effect of folic acid on patients with dry eye or contact lens discomfort.
  • the difficulty in animal models of contact lens discomfort is that there is a lack of contact lens designs suitable for animals, the manufacture of animal contact lenses is challenging, and animal compliance is difficult to guarantee.
  • the inventors have found that the symptoms of contact lens discomfort are very similar to those of some dry eye syndromes, especially patients with normal tear secretion who feel dry eyes and discomfort, usually have the same discomfort with contact lenses.
  • Recent studies have identified a significant association between contact lenses and dry eye [Tan L L, Morgan P, Cai Z Q, et al. Prevalence of and risk factors for symptomatic dry eye disease in Singapore [J]. Clinical and Experimental Optometry,2015,98(1):45-53.], so it is feasible to try to use the dry eye model to replace the animal model of contact lens discomfort.
  • the technical scheme of the present invention is to provide a new use of folic acid derivatives.
  • Another technical scheme of the present invention is to provide medicine or health food containing the compound for treating dry eye and contact lens discomfort symptoms. Eye drops for symptoms of dry eye and contact lens discomfort.
  • the R is selected from methyl or formyl
  • the X can form a pharmaceutically acceptable salt with 5-methyltetrahydrofolate or folinic acid
  • the X includes calcium ions, sodium ions, Organic amines, glucosamine, etc.
  • the dry eye syndrome mentioned therein refers to evaporative dry eye syndrome, that is, lacrimal gland secretion is normal but sensitive to external stimuli, especially symptoms of fear of wind, photophobia, dry eyes, and burning sensation.
  • Symptoms of the discomfort of the contact lens include symptoms such as dry eyes, pain, foreign body sensation, and itching.
  • the present invention also provides an eye drop for dry eye treatment, comprising 5-methyltetrahydrofolate or a pharmaceutically acceptable salt thereof, and used for suppressing eye discomfort caused by light or wind.
  • the eye drops for treating dry eye syndrome provided by the present invention also contain arginine.
  • 5-methyltetrahydrofolate or a pharmaceutically acceptable salt thereof is contained at a concentration of 0.01 to 10% by weight/volume.
  • the present invention also provides a medicine or health food for patients with contact lens discomfort, which contains 5-methyltetrahydrofolate or a pharmaceutically acceptable salt thereof, and is used for relieving dryness, gritty feeling, itching and other symptoms.
  • the medicine or health food for patients with contact lens discomfort includes oral preparations and eye drops.
  • the present invention is based on the discovery that 5-methyltetrahydrofolate can improve the contact lens wearing experience in symptomatic contact lens wearers, thereby providing a proactive approach to improving contact lenses in these patients Wearing experience.
  • the pathological mechanism of symptomatic contact lens wearers is similar to that of some dry eye patients, especially those with dry eye syndrome but normal lacrimal gland secretion.
  • 5-Methyltetrahydrofolate can also improve the eye experience of this type of patients .
  • 5-Methyltetrahydrofolate can reduce the expression of some inflammatory factors in the ocular surface tissue, especially IL-17, and can inhibit the expression of MMP-2 related to the trigeminal ganglion on the same side of the eye.
  • the research of the present invention shows that chronic dry eye, especially lacrimal gland secretion is normal, but the mechanism of dry eye that shows dry eye symptoms may be different from severe dry eye.
  • chronic dry eye involves persistent ocular surface inflammation associated with Th17 responses, which may be primarily or exclusively mediated by Th17 cell responses alone.
  • Th17 shows the properties of significant long-term effect memory cells.
  • the present invention finds that chronic dry eye not only involves ocular surface inflammation but also chronic neuroinflammation.
  • the present invention reveals for the first time some specific effects of chronic dry eye on the ophthalmic nerve, especially MMP-2.
  • tear film urea may be a potential diagnostic marker for dry eye syndrome, and that tear film urea level was significantly lower in dry eye patients compared with healthy people.
  • Urea is formed during the metabolism of some amino acids, especially arginine.
  • the present invention finds that the use of arginine alone cannot significantly improve the ocular surface damage of model mice, but it has a significant improvement effect when used in combination with folinic acid.
  • Fig. 1 The content of IL-17 in tears of different contact lens wearers (*, compared with group C, p is less than 0.05; #, compared with group B, p is less than 0.05).
  • Fig. 2 Tear secretion test in dry eye model mice (*, p ⁇ 0.05 compared with the control group).
  • Figure 5 investigates the effect of 5-methyltetrahydrofolate eye drops on the ocular surface damage of mice (*, p ⁇ 0.05 compared with the control group).
  • Figure 6 investigates the effect of 5-methyltetrahydrofolate eye drops on the inflammatory factors of the ocular surface of mice (*, compared with the control group, p is less than 0.05).
  • Figure 7 investigates the effects of 5-methyltetrahydrofolate eye drops on the expression of metalloproteinases and inflammatory factors in the trigeminal ganglion of mice (*, p is less than 0.05 compared with the control group).
  • Figure 8 investigates the effect of folinic acid, the composition of folinic acid and arginine, and 5-methyltetrahydrofolate eye drops on the secretion of tears in mice
  • Figure 9 investigates the effects of folinic acid, the composition of folinic acid and arginine, and 5-methyltetrahydrofolate eye drops on the ocular surface damage of mice (*, compared with the control group, p is less than 0.05).
  • Dry eye is divided into dehydration dry eye and evaporative dry eye [Bron AJ, Yokoi N, Gafney E, Tiffany JM. Predicted phenotypes of dry eye: proposed consequences of its natural history. Ocul Surf. 2009; 7 (2):78 ⁇ 92.] point out the different development of typical dry eye, in the history of evaporative dry eye, the integrity of the tear film lipid layer is reduced, tear evaporation is increased, resulting in increased osmotic pressure, corneal irritation Nerve endings, dry eye symptoms occur, leading to increased blinking and a compensatory increase in tear secretion. The exact mechanism by which changes in the ocular surface affect tear function is unclear, but in some dry eye patients, tear secretion not only does not decrease but increases, similar apparently contradictory results have been demonstrated in human clinical observations .
  • the mice in the dry and windy environment were taken out, and tested with phenol red cotton thread to measure the production of tears, and compared with mice raised in a standard cage in a normal environment.
  • corneal fluorescein staining was performed on mice, and the cornea was checked to record punctate staining.
  • mice in a dry environment were transferred to a standard breeding box (temperature about 20°C, humidity: 40-60%), and continued to be raised for 3 months. The mice were taken out for measurement of tear secretion and corneal fluorescence staining test. The results showed that small The lacrimal secretion of the mice returned to normal or even supernormal levels (see Figure 2).
  • the corneal staining test showed that the corneal damage of the mice did not disappear due to the restoration of tear secretion, and the mice showed persistent low-level corneal damage (see FIG. 3 ).
  • the mice in group A were instilled 5 ⁇ L of 5-methyltetrahydrofolate eye drops in each eye every day
  • the mice in group B were instilled with 5 ⁇ L of 0.5% arginine eye drops in each eye every day
  • the mice in group C were instilled with phosphate buffered saline in each eye every day. Solution 5 ⁇ L.
  • the tear fluid was collected on the 3rd, 5th, 7th and 10th day after treatment, and the secretion of tear fluid was detected. On the 10th day after treatment, the mice in each group were stained with corneal fluorescence.
  • mice were euthanized and ocular tissues such as cornea, lacrimal gland and meibomian gland were removed and washed in PBS, cornea, conjunctiva and draining lymph nodes were collected and stored in cold sterile PBS at -80 °C, The samples were homogenized and centrifuged on ice, and the levels of IFN- ⁇ , IL-17, IL-6 in the supernatant were determined using ELISA kits (Raybiotech).
  • the ipsilateral trigeminal ganglion and trigeminal nerve trunk of the mouse eye were quickly dissected on ice, and immediately frozen in liquid nitrogen, stored at -80°C, and obtained from the same side using the NucleoSpin RNA purification II kit (NucleoSpin RNA S, Germany).
  • RNA was extracted from the lateral trigeminal ganglion and trigeminal nerve trunk, and PCR amplification was performed with mouse-specific cDNA for matrix metalloproteinases MMP-9, MMP-2, and TNF- ⁇ , and each primer was 24, 28, 32, 36 and The reaction was terminated after an interval of 40 cycles to ensure that the PCR product was within the linear range of the amplification curve and to achieve semi-quantitative RT-PCR for the expression of the relevant gene.
  • the present invention also investigated the expression of inflammatory genes related to the trigeminal nerve of dry eye (see Figure 7), and found that 5-methyltetrahydrofolate eye drops can significantly inhibit the gene expression of MMP-2, and also have a certain effect on TNF- ⁇ . Inhibitory effect, but not significant on MMP-9 inhibition. It has been reported that the expression of metalloproteinases in nerve cells can mediate neural pain. MMP-9 mediates early pain during injury, while MMP-2 is more inclined to chronic pain. The results are unexpected. It may be that 5-methyltetrahydrofolate has the ability to penetrate the aqueous humor circulation barrier and act directly on the nervous system.
  • the tear fluid was collected on the 3rd, 5th, 7th and 10th day after treatment, and the secretion of tear fluid was detected. On the 10th day after treatment, the mice in each group were stained with corneal fluorescence. The results showed that in the eyes of mice in each group, there was no significant difference in tear secretion (see Figure 8). Corneal damage was improved, and the difference was significant compared with the baseline state (see Figure 9).
  • Our data suggest that the combination of folinic acid and arginine may be a novel approach to relieve dry eye syndrome.

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Abstract

一种干眼症治疗用药品，及一种改善佩戴隐形眼镜不适的人群改善佩戴体验的方法，进一步的涉及一种治疗滴眼液。

Description

叶酸类衍生物在制备治疗隐形眼镜不适及干眼症药物的应用 技术领域
本发明涉及干眼症治疗用药品或保健食品，本发明涉及改善佩戴隐形眼镜不适的佩戴者佩戴隐形眼镜的体验的方法，进一步的本发明涉及治疗干眼症的滴眼液和隐形眼镜不适症状的滴眼液。
背景技术
佩戴隐形眼睛时，隐形眼镜会直接与眼表以及上下眼睑的内侧区域相互作用，三叉神经的感觉纤维密集的分布这些区域，每年均有隐形眼镜佩戴者放弃继续佩戴隐形眼镜，佩戴中产生的不适是主要原因。隐形眼镜的不适的特征在于与隐形眼镜佩戴有关的发作性或持续不适的眼部感觉，目前，尽管这种不适反应的病因的机制的研究证据有限，但是值得注意的是，经历过有症状的隐形眼镜的佩戴者的症状描述(例如，眼睛刺激、灼热感、刺痛和疼痛)与干眼病有关的症状高度相似，后者涉及炎症反应与神经性疼痛。
干眼症是一种慢性炎性疾病，泪液的不稳定性伴随着泪液渗透压的增加，从而激活了眼表上皮的驻留免疫细胞中的应激信号，并触发了先天性炎性分子的产生，进一步表达的Th1细胞因子即干扰素γ(IFN-γ)会促进结膜杯状细胞功能障碍和死亡，加剧泪膜的不稳定，加剧炎症并形成恶性循环[Pflugfelder SC,de Paiva CS.The Pathophysiology of Dry Eye Disease:What We Know and Future Directions for Research.Ophthalmology.2017；124(11S):S4‐S13.]。虽然人们对于干眼症的病理生理学的知识有了长足的进步，但现实是干眼疾病的病理机制不能简单归结于单一的原因，现有的治疗方法并不能有效改善所有患者的不适感和角膜上皮疾病。
“干眼”这一词准确来说是形容眼睛的一种感觉，眼表疾病指数量表(OSDI)为临床上诊断干眼最常用的问卷，主要用于诊断干眼及评估病情严重程度，OSDI中主要是询问眼睛的感觉包括光和风对眼睛的刺激、读书写字和看电视对眼睛舒适感的影响等等。上述的问题均是针对眼睛的不愉快的感觉和情感体验，缺乏炎症因子等生物标志物的诊断下，干眼症其实可以归结为一种疼痛疾病。疼痛性疾病大致可分为两类：伤害性疼痛和神经性疼痛，两者都可能与干眼症有关，伤害性疼痛通常是短暂的，是组织损伤和炎症的结果，神经性疼痛是由于病变或疾病直接影响躯体感觉系统而引起的疼痛，更倾向于慢性。在干眼症中，伤害性和神经性疼痛的诱因可能相同，但是这些诱因刺激可导致感觉神经元表型的短暂或持续的变化[Galor A,Levitt RC,Felix ER,Martin ER,Sarantopoulos CD.Neuropathic ocular pain:an  important yet underevaluated feature of dry eye.Eye(Lond).2015；29(3):301‐312.]。
干眼症的诊断定义范围较大，而现实中去眼科门诊就医的患者往往属于症状较严重的干眼症状，即水性泪液缺乏性干眼症，包含泪腺缺乏或泪腺管阻塞的患者。部分干眼症患者并不存在泪液缺乏的现象属于蒸发性干眼症，而这类患者却显示对风和光更为敏感[Chhadva P,Lee T,Sarantopoulos CD,et al.Human Tear Serotonin Levels Correlate with Symptoms and Signs of Dry Eye.Ophthalmology.2015；122(8):1675‐1680.]。上述泪液分泌正常的干眼症患者与有症状的隐形眼睛佩戴者具有高度相似的症状，其病理机制也可能非常类似，二者均对冷热刺激非常敏感[Situ P,Simpson T,Begley C.Hypersensitivity to Cold Stimuli in Symptomatic Contact Lens Wearers.Optom Vis Sci.2016；93(8):909‐916.]，存在一定的“季节效益”。公认的观点是隐形眼镜相关的结膜炎发作受一年中的时间影响，冬天发生角膜浸润的风险增加了2到4倍。
泪液分泌正常的干眼症患者与有症状的隐形眼睛佩戴者其眼睛不是由感染或组织伤害引起的，介于炎症和正常状态之间。Ruslan Medzhitov引入了“副炎症”的概念，隐形眼镜引起的细微组织扰动会导致组织压力或故障引起接近基础状态的副炎症状态，在这种条件下，需要驻留的免疫细胞根据问题的严重程度小范围递送其他白细胞和血浆蛋白，但不显示炎症的典型特征[Medzhitov R.Origin and physiological roles of inflammation[J].Nature,2008,454(7203):428-435.]。虽然有症状的隐形眼镜患者和泪液分泌正常的干眼症患者在终止佩戴隐形眼镜或脱离光/风的环境，不适感会减缓或消除，但是依然会给患者带来非常大的痛苦。因为眼部的神经密度非常大，且次级神经位于三叉神经节，产生有关疼痛的患者可能会伴随偏头痛或抑郁，会对社交，身体和心理功能产生负面影响。
当前针对干眼症的治疗方法主要是针对炎症，例如环孢素和Lifitegrast，靶向T细胞，这是导致慢性干眼症的病理生理学关键因素。Lifitegrast是一种小分子，可抑制T细胞上的白细胞相关抗原1(LFA-1)与其在抗原呈递，上皮和血管内皮细胞上的配体细胞间粘附分子1(ICAM1)结合，这些分子在临床试验中改善了干眼症的症状，但是这些疗法不能解决干眼症对眼表的急性影响，特别是在光和风的急性刺激下无法有效缓解眼睛刺激。皮质类固醇则在应对急性刺激中显示出功效，然而，长期使用皮质类固醇会带来白内障和青光眼的风险。
针对炎症的疗法不能有效改善所有患者的不适感和角膜上皮疾病，这部分患者具有神经性疼痛和中枢敏化迹象，特别是泪液分泌正常的干眼症患者和隐形眼镜不适者。针对神经的疗法目前也存在很大争议，γ-氨基丁酸类似物加巴喷丁和喷他巴林已用于抑制异常的眼 觉，但目前仅用于减轻严重的眼部疼痛的疾病，尽管这些抗癫痫药可能对中度眼部不适的患者有所帮助，但是缺乏临床数据，且可能存在明显的不良影响。神经生长因子(NGF)是轴突生长和再生的有效刺激剂，NGF已经被证明在持续性上皮缺损的愈合中提供帮助，但是在神经损伤期间，NGF的释放会增加神经元兴奋性并降低疼痛阈值。大量证据表明，NGF在疼痛的介导和增强中很重要，这导致了NGF拮抗剂作为潜在的止痛药和抗痛觉过敏药的发展。因此使用NGF拮抗剂还是NGF治疗是否对干眼症患者有益，依然需要进一步的研究。
5-甲基四氢叶酸是叶酸在人体内的一种形式，有研究表明叶酸能够降低同型半胱氨酸水平，而同型半胱氨酸又是老年眼睛黄斑病变的重要风险因素。有研究表明叶酸，维生素B6，维生素B12联合治疗能够降低老年眼睛黄斑病变的风险。另一方面叶酸又能够促进神经生长因子的分泌来促进神经损伤的修复。对干眼症患者来言，NGF的分泌可能会带来疼痛的增强，并且促进神经的敏化，特别是NGF会促进非选择性阳离子通道TRPV1在损伤的感觉纤维的致敏[Stratiievska A,Nelson S,Senning EN,Lautz JD,Smith SE,Gordon SE.Reciprocal regulation among TRPV1 channels and phosphoinositide 3-kinase in response to nerve growth factor.Elife.2018；7:e38869.]，TRPV1的表达变化与热痛觉过敏和痛觉过敏发展相关。亚叶酸同样也是叶酸的衍生物，在体内可以转化为5-甲基四氢叶酸。目前尚无叶酸对干眼症或是隐形眼镜不适患者有益作用的研究报道。
尽管目前人们开发了许多针对干眼症的新治疗方法，但是被药品管理机构批准的治疗药物依然匮乏，治疗对干眼症干预试验的失败的其中主要原因是缺乏适当的疾病指标和合适的临床前病理模型。
对于隐形眼镜不适症状的动物模型的难点在于，缺乏适合动物的隐形眼镜设计，动物隐形眼镜的制造具有挑战性，且动物依从性难以保证。发明人发现隐形眼镜不适的症状表现与部分干眼症的表现非常类似，特别是泪液分泌正常却有眼干不适感觉的患者，通常对隐形眼镜也同样不适。最近的研究已经确定了隐形眼镜与干眼之间的显著关联[Tan L L,Morgan P,Cai Z Q,et al.Prevalence of and risk factors for symptomatic dry eye disease in S ingapore[J].Clinical and Experimental Optometry,2015,98(1):45-53.]，因此尝试使用干眼症模型来替代隐形眼镜不适的动物模型是可行的。
发明内容
本发明的技术方案是提供一种叶酸类衍生物的新用途，本发明的另一技术方案是提供含有该化合物治疗干眼症和隐形眼镜不适症状的药物或保健食品，提供一种用于治疗干眼症 和隐形眼镜不适症状的滴眼剂。
本发明通式I所述的化合物在制备治疗干眼症或缓解佩戴隐形眼镜不适的药物或保健食品的用途。
Figure PCTCN2022100814-appb-000001
其中，所述的R选自甲基或甲酰基，所述的X能与5-甲基四氢叶酸或亚叶酸形成药学上可接受的盐的物质，所述X包括钙离子、钠离子、有机胺、氨基葡萄糖等。
其中所述的干眼症是指蒸发性干眼症，即泪腺分泌正常但对外界刺激敏感，特别是具有怕风、畏光、眼睛干涩、痛灼热感的症状。所述隐形眼镜不适的症状包括眼睛干涩、疼痛、有异物感、及刺痒等症状。
本发明还提供一种干眼症治疗用的滴眼剂，其包含5-甲基四氢叶酸或其药学上可接受的盐，且用于抑制由光或风导致的眼部不适。优选的，本发明提供的干眼症治疗用滴眼剂中还含有精氨酸。
优选的是，以0.01～10％重量/容量％的浓度包含5-甲基四氢叶酸或其药学上可接受的盐。
本发明还提供一种用于隐形眼镜不适患者的药物或保健食品，其包含5-甲基四氢叶酸或其药学上可接受的盐，用于缓解干燥，砂砾感，发痒等症状。所述用于隐形眼镜不适患者的药物或保健食品包含口服制剂，滴眼剂。
如本文本所公开的，本发明基于以下发现：5-甲基四氢叶酸可以改善有症状隐形眼镜佩戴者的隐形眼镜佩戴体验，从而提供一种采取主动措施的方法来改善这些患者的隐形眼镜佩戴体验。此外有症状的隐形眼镜佩戴者与部分干眼症患者的病理机制接近，特别是有干眼症却泪腺分泌正常的患者，有关5-甲基四氢叶酸同样可以改善该类型患者的眼部体验。5-甲基四氢叶酸能够降低眼表组织的部分炎性因子的表达，特别是IL-17，并且能够抑制眼同侧三叉神经节有关MMP-2的表达。
本发明的研究表明慢性干眼症，特别是泪腺分泌正常，却表现出干眼症状的干眼症的机制可能与重度干眼症不同，本发明发现急性干燥的风刺激后小鼠，在正常环境下培养3个 月，小鼠眼部角膜依然存在损伤，但是有关眼表组织中炎性因子的表达却与很多前人研究不同，未观察到高水平的炎性因子和MMP-9的表达，慢性干眼症涉及Th17反应相关的持续性眼表炎症，可能主要或仅是有Th17细胞相应单独介导的。炎症发生后有关T细胞进一步的表型，Th17显示了显著的长效效应记忆细胞的性质，本发明发现慢性干眼症不仅涉及眼表的炎症还涉及慢性神经炎症。本发明首次揭示了慢性干眼症对眼神经的某些特定影响，特别是MMP-2。
最近的一项研究表明泪膜尿素的水平可能是干眼症潜在的诊断标志物，在干眼症的人群中，泪膜尿素水平与健康人群相比显著降低。尿素是一些氨基酸代谢过程中形成的，尤其是精氨酸。本发明发现单独使用精氨酸并未能够显著改善模型小鼠的眼表损伤，但是与亚叶酸联合使用后，具有显著的改善效果。
附图说明
图1不同隐形眼镜佩戴者眼泪中IL-17的含量(*，与C组比p小于0.05；#，与B组相比，p小于0.05)。
图2干眼症模型小鼠眼泪分泌试验(*，与对照组相比p小于0.05)。
图3干眼症模型小鼠角膜染色试验(*，与对照组相比p小于0.05)。
图4考察5-甲基四氢叶酸滴眼剂对小鼠泪液分泌影响
图5考察5-甲基四氢叶酸滴眼剂对小鼠眼表损伤的影响(*，与对照组相比p小于0.05)。
图6考察5-甲基四氢叶酸滴眼剂对小鼠眼表炎性因子的影响(*，与对照组相比p小于0.05)。
图7考察5-甲基四氢叶酸滴眼剂对小鼠眼三叉神经节的表达金属蛋白酶、炎性因子的影响(*，与对照组相比p小于0.05)。
图8考察亚叶酸，亚叶酸与精氨酸组合物，5-甲基四氢叶酸滴眼液对小鼠泪液分泌影响
图9考察亚叶酸，亚叶酸与精氨酸组合物，5-甲基四氢叶酸滴眼液对小鼠眼表损伤的影响(*，与对照组相比p小于0.05)。
具体实施方式
实施例1改善隐形眼镜不适症状的初步考察
共找到10例对佩戴隐形眼镜不适的成人参与研究，其中8人声明其对于隐形眼镜佩戴 有不适感，2人为无症状隐形眼镜佩戴者，参与者佩戴的隐形眼镜均为硅水凝胶型隐形眼镜。所有参与者健康状况良好，通过眼表疾病指数(OSDI)评估眼部的不适症状和严重程度，并接受验光师检查眼部是否有发炎等症状。
参与研究人员平均年龄33岁，标准差SD为11岁，3名男性，7名女性，结果发现所有参与者均无典型的眼部炎症状态。与无症状隐形眼镜佩戴者相比，有症状的佩戴者反馈对光和风敏感(n＝6，75％)。
将有症状的隐形眼镜佩戴者分为2组，A组(n＝4)，B组(n＝4)，将无症状的隐形眼镜佩戴者分为C组，各组24小时内未佩戴隐形眼镜，之后收集眼泪后，开始A组每天服用含有6S-5-甲基四氢叶酸钙5mg/片的膳食补充剂，3日后各组开始佩戴隐形眼镜，6小时后收集各组眼泪，检测IL-2、IL-6、IL-17、TNF-α的含量(ELISA法)。并对各组进行访视，回答佩戴眼镜的不适程度。
统计分析，A组中有3人表示隐形眼镜不适感几乎消失，仅有1例认为效果不显著。炎性因子方面难以检测与量化不同组之间的差异，但是IL-17却显示出隐形眼镜不适者与正常者存在差异，而5-甲基四氢叶酸能够降低泪液中IL-17的水平，见图1。
实施例2慢性干眼病的小鼠造模
干眼症分为缺水性干眼症和蒸发性干眼症，[Bron AJ,Yokoi N,Gafney E,Tiffany JM.Predicted phenotypes of dry eye:proposed consequences of its natural history.Ocul Surf.2009；7(2):78‐92.]指出了典型干眼症的不同发展，在蒸发性干眼的发展史中，泪膜脂质层的完整性降低，泪液蒸发增加，导致渗透压增加，刺激角膜神经末梢，发生干眼症状，导致眨眼增加和泪液分泌代偿性增加。有关眼表变化影响泪液功能的确切机制尚不清楚，但是在部分干眼症患者发展过程中不仅泪液分泌不仅不降低反而会升高，在人类的临床观察中也证明了类似的明显矛盾的结果。
由此可见，合适干眼症的动物模型非常重要，仅以泪腺破坏，或高渗盐水造模无法体现真实干眼或眼镜不适的状态。利用特定的环境条件制作干眼症的小鼠模型最为贴近真实情况。
120只8至12周大的雌性BALB/c小鼠，安置在隔离密封的笼子中，笼子上方开孔，使用空气泵，将经无水CaSO 4干燥后的空气泵入笼子，空气湿度RH＝19.5％±5％，流量为15L/min，温度为21-23℃。分别于第5天和第15天及30天将干燥，有风环境下的小鼠取出，用酚红棉线试验，测量泪液的产生，并与在正常环境的标准笼中饲养小鼠进行比较。同时对小鼠进行角膜荧光素染色，检查角膜记录点状染色。
结果表明，小鼠在干燥环境第五天中泪液分泌下降，而在第30天泪液分泌却上升，显示可能出现代偿性的泪液增加。将30天后干燥环境的小鼠转移至标准饲养箱(温度20℃左右，湿度：40～60％)，继续饲养3个月，取出小鼠进行泪液分泌的测量和角膜荧光染色试验，结果显示小鼠泪液分泌恢复正常甚至超正常水平(见图2)。
角膜染色试验表明，小鼠角膜损伤并未因泪液分泌恢复而消失，小鼠表现出持续的低水平的角膜损伤(见图3)。
实施例3 5-甲基四氢叶酸对慢性干眼症模型鼠的影响
5-甲基四氢叶酸滴眼液配置，取6S-5-甲基四氢叶酸精氨酸盐冻干粉，与纯化水配置成0.5％浓度的5-甲基四氢叶酸滴眼液。精氨酸盐酸盐与纯化水配置成0.5％浓度。
实验分3组，取实施例2中慢性干眼症模型小鼠20只，分为5-甲基四氢叶酸组(A组，n＝10)，精氨酸组(B组，n＝10)，取正常小鼠4只作为对照组。A组小鼠每天双眼各滴注5-甲基四氢叶酸滴眼液5μL，B组小鼠每天双眼各滴注0.5％精氨酸滴眼液5μL，C组每天双眼各滴注磷酸盐缓冲液5μL。治疗后第3、5、7和10天分别收集泪液，检测泪液分泌，治疗后第10天对各组小鼠进行角膜荧光染色。
治疗结束时，对动物实施安乐死并取出角膜，泪腺和睑板腺等眼部组织在PBS中洗涤，收集角膜，结膜和引流淋巴结并将其存储在-80℃的冷的无菌PBS中，将样品在冰上均质并离心，使用ELISA试剂盒(Raybiotech)测定上清液中IFN-γ、IL-17、IL-6的水平。
取小鼠的眼同侧三叉神经节及三叉神经干在冰上快速解剖，并立即使用液氮冷冻，储存在-80℃下，使用NucleoSpin RNA纯化Ⅱ试剂盒(NucleoSpin RNA S，德国)从同侧三叉神经节及三叉神经干中提取RNA，用针对基质金属蛋白酶MMP-9，MMP-2，TNF-α的鼠特异性cDNA进行PCR扩增，每个引物以24、28、32、36和40个循环的间隔后终止反应，以确保PCR产物在扩增曲线的线性范围内，对有关基因表达实现半定量RT-PCR。
结果显示各组小鼠眼类中，泪液分泌无明显差异(见图4)，荧光色素染色却显示出，5-甲基四氢叶酸能够改善角膜的损伤，与基线状态相比差异性显著(见图5)。眼表及淋巴结的结果表明，该模型的慢性干眼症的炎性因子并未诱导表达，可能是眼表驻留的免疫细胞在接收刺激后，才能显示出显著性的差异，但是IL-17则显示较大差异(见图6)，可能是表达IL-17的Th17细胞常驻在慢性干眼的眼表中。本发明发现5-甲基四氢叶酸滴眼液能够显著降低IL-17炎性因子，这与前述的实验结果相吻合。
本发明还考察了干眼症三叉神经有关炎性基因表达(见图7)，结果发现5-甲基四氢叶酸滴眼液能够显著抑制MMP-2的基因表达，并且对TNF-α也有一定的抑制作用，但是对 MMP-9抑制不显著。有关金属蛋白酶在神经细胞的表达会介导神经疼痛已经有所报道，MMP-9介导了损伤期间，前期的疼痛，而MMP-2更倾向于慢性疼痛，有关结果出乎人的意料。可能是5-甲基四氢叶酸具有穿透房水循环屏障直接作用于神经系统的缘故。
实施例4亚叶酸对慢性干眼症模型鼠的影响
5-甲基四氢叶酸精氨酸盐在水溶液中的稳定性非常差。因此有必要验证左旋亚叶酸对于干眼症模型小鼠的治疗可能性。取左旋亚叶酸钙100mg溶于20mL纯化水，配置成亚叶酸钙滴眼液。取6S-5-甲基四氢叶酸精氨酸盐冻干粉与纯化水配置成0.5％的滴眼液。取左旋亚叶酸钙100mg与精氨酸盐酸盐80mg与20ml纯化水配置成组合滴眼液。
实验分4组，取实施例2中慢性干眼症模型小鼠40只，分为左旋亚叶酸组(A组，n＝10)，5-甲基四氢叶酸组(B组，n＝10)，左旋亚叶酸钙精氨酸组合组(C组，n＝10)，磷酸盐缓冲液组(D组，n＝10)作为对照组。治疗后第3、5、7和10天分别收集泪液，检测泪液分泌，治疗后第10天对各组小鼠进行角膜荧光染色。结果表明各组小鼠眼类中，泪液分泌无明显差异(见图8)，荧光色素染色显示左旋亚叶酸钙与精氨酸组合物，以及5-甲基四氢叶酸精氨酸盐能够显著改善角膜的损伤，与基线状态相比差异性显著(见图9)。我们的数据表明左旋亚叶酸与精氨酸的配合使用可能是缓解干眼症的一种新方法。

Claims (8)

  1. 通式I所述的化合物在制备治疗干眼症或佩戴隐形眼镜不适的药物或保健食品的用途：
    Figure PCTCN2022100814-appb-100001
    其中，所述的R选自甲基或甲酰基，所述的X为能与5-甲基四氢叶酸或亚叶酸形成药学上可接受的盐的物质，包括钙离子、钠离子、有机胺、氨基葡萄糖等。
  2. 根据权利要求1所述的用途，所述干眼症是蒸发性干眼症。
  3. 根据权利要求1-2所述的用途，所述药品包含干眼症治疗用滴眼剂。
  4. 根据权利要求3，所述滴眼剂中还包含精氨酸。
  5. 根据权利要求4所述用途，所述滴眼剂能够用于抑制眼表炎性因子表达导致的炎症，抑制眼三叉神经节金属蛋白酶MMP-2，TNF-α的表达导致的神经炎及神经痛。
  6. 根据权利要求1所述的用途，所述佩戴隐形眼镜不适的症状包括眼睛干涩、疼痛、有异物感、及刺痒。
  7. 根据权利要求6所述的用途，所述药品或保健食品能够抑制眼表炎性因子表达导致的炎症，抑制眼三叉神经节金属蛋白酶MMP-2，TNF-α的表达导致的神经炎及神经痛。
  8. 根据权利要求4～7中任一项所述的用途，其中，所述治疗用滴眼剂还包含药学上可用的辅料。
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