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WO2023071994A1 - Use of nad+ precursor in preparation of drug for treating lens sclerosis-related diseases - Google Patents

Use of nad+ precursor in preparation of drug for treating lens sclerosis-related diseases Download PDF

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Publication number
WO2023071994A1
WO2023071994A1 PCT/CN2022/127077 CN2022127077W WO2023071994A1 WO 2023071994 A1 WO2023071994 A1 WO 2023071994A1 CN 2022127077 W CN2022127077 W CN 2022127077W WO 2023071994 A1 WO2023071994 A1 WO 2023071994A1
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Prior art keywords
lens
nad
precursor
pharmaceutical composition
administration
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PCT/CN2022/127077
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French (fr)
Chinese (zh)
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刘泉
魏来
冯绮婷
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刘泉
魏来
冯绮婷
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Publication of WO2023071994A1 publication Critical patent/WO2023071994A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/10—Ophthalmic agents for accommodation disorders, e.g. myopia
    • 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
    • A61P27/12—Ophthalmic agents for cataracts
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
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    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/40—Nucleotides, nucleosides or bases

Definitions

  • the invention relates to the technical field of biomedicine, in particular to the application of an NAD + precursor in the preparation of medicines for treating diseases related to lens sclerosis.
  • Lens sclerosis is a common sign of age-related eye diseases such as presbyopia and senile cataract, but the mechanism of lens sclerosis has not yet been fully elucidated. Many studies have pointed out that the increase in the proportion of water-insoluble protein in the lens protein may be the key factor.
  • Water-insoluble proteins are mainly composed of various post-translationally modified macromolecular proteins. Among them, oxidation modification is the more common one. Oxidized crystallins, especially ⁇ -crystallins, can form disulfide bonds or glycosylation cross-links between molecules and within molecules, leading to abnormal aggregation of proteins and the formation of high molecular weight protein.
  • ⁇ -crystallin is a small molecular chaperone, which can prevent abnormal protein folding and aggregation. Its loss will easily cause other proteins to bind abnormally with the cell membrane and affect the deformation ability of cell tissue. Furthermore, this change will promote the formation of intercellular barriers, resulting in the inability of antioxidant substances to be transported into the nucleus of the lens, and the metabolites in the nucleus cannot be smoothly discharged, which will continuously aggravate the degree of oxidative stress in the lens.
  • Presbyopia is an age-related physiological change in the eye that is closely related to a significant decline in the ability to accommodate within the eye.
  • the Helmholtz adjustment mechanism hypothesis believes that a series of shape changes such as increased thickness, decreased diameter, and increased curvature of the front and rear surfaces caused by the contraction of the ciliary muscle during near vision can enhance the refractive power of the lens and make the object image accurately focused on the retina.
  • the lens gradually hardens and the elasticity of the capsule decreases, resulting in partial or even complete loss of its deformation ability, resulting in a continuous decline in the adjustment range, so that close vision is not enough to meet the daily needs of individuals.
  • Presbyopia manifests as difficulty in near vision and inability to sustain near vision, seriously affecting the quality of life and work of middle-aged and elderly people. Although you can choose to wear glasses or surgical treatment to correct presbyopia, these methods have certain limitations, such as not simple, traumatic, etc., and more importantly, they cannot fundamentally treat presbyopia.
  • Senile cataract is one of the main eye diseases that cause blindness in the world.
  • the clouding and hardening of the lens accompanied by aging is its typical manifestation.
  • Age and environmental exposure are the main risk factors for the onset of senile cataract, which can cause metabolic abnormalities in the lens, and oxidative stress is the currently recognized pathogenesis.
  • Oxidative stress of proteins in the lens will change the morphology of lens fiber cells, destroy their highly ordered structure, and then affect the transparency.
  • oxidative stress may also be the underlying reason for the decrease in the elastic deformation capacity of the lens, and the increase in lens hardness not only further threatens the vision of the middle-aged and elderly people, but also limits the choice of cataract surgery and postoperative visual acuity. quality recovery.
  • many compounds with antioxidant properties have been found to be effective in improving cataract opacity, but their effects on hardness have not yet been studied.
  • Nicotinamide adenine dinucleotide also known as coenzyme I, participates in multiple processes of energy synthesis and metabolism in cells. At the same time, it can also activate a variety of enzymes in the form of coenzyme substrates, and has multiple functions such as anti-oxidative stress and anti-apoptosis.
  • Nicotinamide mononucleotide (nicotinamide mononucleotide, NMN) is the precursor substance of NAD + synthesis, many studies have confirmed that its exogenous supplementation can significantly increase the level of NAD + in the body, and has shown to improve the level of energy metabolism, mitochondrial function and Positive reactions such as oxidative stress. However, there is still no research on the effect of NMN and related compounds on lens hardness (presbyopia, senile cataract).
  • Patent WO 2018052019A1 discloses a visual function improving agent and a visual function improving method, but only generally points out that NMN can improve visual function, and does not mention changing the hardness of the lens, and the usage method and effective usage amount are different from the present application.
  • the first aspect of the present invention provides the application of NAD + precursor in the preparation of medicines for treating diseases related to lens sclerosis, and the medicines are administered locally.
  • the diseases related to lens sclerosis include presbyopia and cataract.
  • the local administration is ocular administration, and more preferably, the local administration includes lens culture in vitro or subconjunctival injection.
  • the drug includes 10-500 mmol/L NAD + precursor.
  • the concentration of the NAD + precursor is 10-40 (eg, 10, 20, 30, 40) mmol/L.
  • the concentration of the NAD + precursor is 500mmol/L.
  • the concentration of the NAD + precursor is 20-50 (eg, 20, 30, 40, 50) mmol/L.
  • the NAD + precursors include tryptophan, niacin, nicotinamide, reduced form of nicotinamide riboside (NRH), nicotinamide mononucleotide (NMN), trigonelline, nicotinamide mononucleotide
  • NAD + precursor is nicotinamide mononucleotide.
  • the medicine also contains other vision-improving ingredients, further preferably, the other vision-improving ingredients include anthocyanins, lutein, docosahexaenoic acid, astaxanthin, lycopene, Taurine, Panthenol, Potassium Aspartate, Chondroitin Sulfate, Zinc, Calcium or Magnesium etc.
  • the other vision-improving ingredients include anthocyanins, lutein, docosahexaenoic acid, astaxanthin, lycopene, Taurine, Panthenol, Potassium Aspartate, Chondroitin Sulfate, Zinc, Calcium or Magnesium etc.
  • the second aspect of the present invention provides a pharmaceutical composition, which includes 10-500mmol/L NAD + precursor, and pharmaceutically acceptable auxiliary materials.
  • the concentration of the NAD + precursor is 10-40 (eg, 10, 20, 30, 40) mmol/L.
  • the concentration of the NAD + precursor is 500mmol/L.
  • the concentration of the NAD + precursor is 20-50 (eg, 20, 30, 40, 50) mmol/L.
  • the NAD + precursors include tryptophan, niacin, nicotinamide, reduced form of nicotinamide riboside (NRH), nicotinamide mononucleotide (NMN), trigonelline, nicotinamide mononucleotide
  • NAD + precursor is nicotinamide mononucleotide.
  • the pharmaceutical composition is administered locally to improve visual function in the pharmaceutical field.
  • the pharmaceutical dosage forms include powder, tablet, sustained-release tablet, chewable tablet, effervescent tablet, lozenge, buccal tablet, sublingual tablet, capsule, fine granule, granule, such as oral preparation, such as pill, dry syrup , solutions, suspensions, syrups and elixirs, as well as eye drops, eye drops, eye ointments, injections, infusions, external preparations, etc.
  • the pharmaceutical composition also includes pharmaceutically acceptable excipients and other pharmaceutically known pharmaceutical additives in the dosage form, which can be properly mixed into the drug according to their physical and chemical properties, biological characteristics, etc. composition.
  • excipients lactose, starch, crystalline cellulose, sodium phosphate, etc.
  • solvents water, soybean oil, physiological saline, non-aqueous solvents for injection, etc.
  • binders starch, gelatin, gum arabic, sodium alginate, Cameron sodium, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, etc.
  • disintegrants starch, sodium carboxymethylcellulose, etc.
  • lubricants talc, magnesium stearate, calcium stearate, polyethylene glycol, sucrose fatty acid ester, etc.
  • coating agents white sugar, hydroxypropyl cellulose (HPC), shellac, gelatin, glycerin, Hydroxy
  • a method of altering lens hardness comprising the use of NAD + precursors.
  • the method includes using the above-mentioned pharmaceutical composition.
  • the concentration of the NAD + precursor is 10-500 mmol/L.
  • the concentration of the NAD + precursor is 10-40 (eg, 10, 20, 30, 40) mmol/L.
  • the concentration of the NAD + precursor is 500mmol/L.
  • the concentration of the NAD + precursor is 20-50 (eg, 20, 30, 40, 50) mmol/L.
  • the NAD + precursors include tryptophan, niacin, nicotinamide, reduced form of nicotinamide riboside (NRH), nicotinamide mononucleotide (NMN), trigonelline, nicotinamide mononucleotide
  • NAD + precursor is nicotinamide mononucleotide.
  • the method includes topical administration.
  • the method comprises: culturing the lens in NAD + precursor solution in vitro.
  • the culture time is 12 hours.
  • the method comprises: administering NAD + precursor for subconjunctival injection.
  • the injection volume is 5 ⁇ L/time.
  • the fourth aspect of the present invention provides a method for preventing and/or treating eye diseases, the method comprising using NAD + precursor.
  • the concentration of the NAD + precursor is 10-500 mmol/L.
  • the concentration of the NAD + precursor is 10-40 (eg, 10, 20, 30, 40) mmol/L.
  • the concentration of the NAD + precursor is 500mmol/L.
  • the concentration of the NAD + precursor is 20-50 (eg, 20, 30, 40, 50) mmol/L.
  • the NAD + precursors include tryptophan, niacin, nicotinamide, reduced form of nicotinamide riboside (NRH), nicotinamide mononucleotide (NMN), trigonelline, nicotinamide mononucleotide
  • NAD + precursor is nicotinamide mononucleotide.
  • the eye diseases include presbyopia and cataract.
  • the method comprises: culturing the lens in NAD + precursor solution in vitro.
  • the culture time is 12 hours.
  • the method comprises: administering NAD + precursor for subconjunctival injection.
  • the injection volume is 5 ⁇ L/time.
  • the subject of the agent for changing the hardness of the lens, the food composition, and the pharmaceutical composition of the present invention can be mammals, such as humans, monkeys, dogs, rabbits, mice, and rats.
  • treating means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but not necessarily The complete elimination of all disease-related signs, symptoms, conditions, or disorders is contemplated.
  • the NAD + precursor in the present invention acts directly on the eye through local administration at a specific concentration, which can effectively increase the axial strain of the aging lens, improve the hardness of the lens, and partially restore elasticity. Therefore, the compound can restore the adjustment ability and treat presbyopia and senile cataract by regulating the hardness of the lens.
  • Figure 1 Changes in lens hardness in vitro, where * is P ⁇ 0.05, ** is P ⁇ 0.01;
  • Figure 2 Changes in lens hardness in vivo experiments (subconjunctival injection), where * is P ⁇ 0.05;
  • Figure 3 Changes in the degree of lens opacity in senile cataract in vitro; the arrows indicate ring-shaped cataracts;
  • Figure 4 Changes in lens hardness of senile cataract in vitro; where * is P ⁇ 0.05;
  • Figure 5 Changes in lens hardness in vivo experiments (gastric administration).
  • mice Eight-month-old mice (C57BL/6J) were randomly divided into control and treatment groups, and the lenses were incubated in complete MEM medium (containing 10% fetal bovine serum and 1% penicillin/streptomycin) for 2 hours to recover metabolic activity. Subsequently, the medium in the treatment group was replaced with serum-free MEM medium containing different concentrations (10-40mmol/L, specifically 10, 20, 30, 40mmol/L) of nicotinamide mononucleotide solution, and the control group was only serum-free MEM medium and incubate for 12 hours at 37°C in a 5% CO 2 incubator. After 12 hours, wash the lens 3 times with light buffer of HBSS solution. Lens elasticity was measured using the known coverslip indentation method.
  • the specific operation is: in the acrylic square vessel containing 65-70mL HBSS solution, the lens is stably fixed in the special small hole in the vessel in the axial direction, and the rectangular prism at a certain position from the lens can reflect the overall outline of the lens.
  • the lens was deformed by the weight of the cover glass, and the deformation was counted to evaluate the hardness.
  • mice Eight-month-old mice (C57BL/6J) were randomly divided into a control group and a treatment group. After the mice were anesthetized by intraperitoneal injection of pentobarbital sodium, the conjunctiva of the right eye was fully exposed.
  • Nicotinamide mononucleotide solution improves lens turbidity and hardness in senile cataract mice
  • mice 14-month-old C57BL/6J mice were selected and randomly divided into control group and treatment group. After the materials were collected, a stereomicroscope (SteREO Discovery V8 stereomicroscope, Zeiss, Germany) was used to record the opacity degree of the mouse lens under sterile conditions to ensure the successful preparation of the senile cataract mouse model. The lenses were incubated for 2 hours in complete MEM medium (containing 10% fetal calf serum and 1% penicillin/streptomycin) to restore metabolic activity.
  • complete MEM medium containing 10% fetal calf serum and 1% penicillin/streptomycin
  • the medium in the treatment group was replaced with serum-free MEM medium containing different concentrations (20-50mmol/L, specifically 20, 30, 40, 50mmol/L) of nicotinamide mononucleotide solution, and the control group was only serum-free MEM medium and incubate for 12 hours at 37°C in a 5% CO 2 incubator. After 12 hours, wash the lens 3 times with light buffer of HBSS solution. Lens opacity was recorded again using a stereomicroscope prior to hardness testing. Lens hardness was then measured using the coverslip indentation method described above.
  • Example 3 Nicotinamide mononucleotide solution gavage administration method has a general effect on improving the hardness of the lens of mice
  • mice Eight-month-old C57BL/6J mice were randomly divided into control group and treatment group. After the mouse is weighed, hold the mouse with the left hand so that its head, neck and body are in a straight line, use a 1mL syringe and a No. 8 gavage needle, enter the gavage needle from the corner of the mouse's mouth, touch the palate and push it inward gently , after entering the stomach, inject the drug solution at 100mg/kg/day, once a day, for 1 month. One month after gavage, the eyeballs were removed to obtain the lens, and the hardness of the lens was measured by the above-mentioned cover glass indentation method.
  • nicotinamide mononucleotide solution can enhance the adjustment ability and treat presbyopia and senile cataract by increasing the axial strain of the lens, improving the hardness of the lens, and reducing the opacity of the lens in cataract. .

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Abstract

Provided is a use of a NAD+ precursor in the preparation of a drug for treating lens sclerosis-related diseases. In particular, the concentration of the NAD+ precursor is 10-500 mmol/L, and a lens ex vivo is cultured in a NAD+ precursor solution by using a local administration means, or, the NAD+ precursor solution is used for subconjunctival injection, so as to treat lens sclerosis-related diseases, which has an important value for preparing medicines for treating lens sclerosis-related diseases.

Description

NAD +前体在制备治疗晶状体硬化相关疾病的药物中的应用 NAD +Application of the precursor in the preparation of drugs for the treatment of lens sclerosis related diseases 技术领域technical field
本发明涉及生物医药技术领域，具体地说，涉及一种NAD +前体在制备治疗晶状体硬化相关疾病的药物中的应用。 The invention relates to the technical field of biomedicine, in particular to the application of an NAD + precursor in the preparation of medicines for treating diseases related to lens sclerosis.
背景技术Background technique
晶状体硬化是老花眼、老年性白内障等年龄相关性眼病的常见体征，而关于晶状体硬化的机理现仍未能完全阐明。较多研究指出，晶状体蛋白质中水不溶性蛋白质比例增加可能是关键因素。水不溶性蛋白质主要由各种翻译后修饰的大分子蛋白质组成。其中，氧化修饰是较常见的一种，氧化后的晶状体蛋白，尤其是α-晶状体蛋白，可在分子间和分子内形成二硫键或糖基化交联，导致蛋白质异常聚集进而形成高分子量蛋白质。α-晶状体蛋白是小分子伴侣，具有阻止蛋白质异常折叠聚集的作用，它的丢失容易引起其他蛋白质与细胞膜异常结合，影响细胞组织形变能力。再者，这种改变会促使细胞间屏障形成，导致抗氧化物质无法运输入晶状体核区、核内代谢产物不能顺利排出，不断加重晶状体内氧化应激程度。Lens sclerosis is a common sign of age-related eye diseases such as presbyopia and senile cataract, but the mechanism of lens sclerosis has not yet been fully elucidated. Many studies have pointed out that the increase in the proportion of water-insoluble protein in the lens protein may be the key factor. Water-insoluble proteins are mainly composed of various post-translationally modified macromolecular proteins. Among them, oxidation modification is the more common one. Oxidized crystallins, especially α-crystallins, can form disulfide bonds or glycosylation cross-links between molecules and within molecules, leading to abnormal aggregation of proteins and the formation of high molecular weight protein. α-crystallin is a small molecular chaperone, which can prevent abnormal protein folding and aggregation. Its loss will easily cause other proteins to bind abnormally with the cell membrane and affect the deformation ability of cell tissue. Furthermore, this change will promote the formation of intercellular barriers, resulting in the inability of antioxidant substances to be transported into the nucleus of the lens, and the metabolites in the nucleus cannot be smoothly discharged, which will continuously aggravate the degree of oxidative stress in the lens.
老视是一种与年龄相关的眼部生理性改变，与眼内调节能力的显著下降密切相关。Helmholtz调节机制假说认为，近距离视物时因睫状肌收缩引起的晶状体厚度增加、直径减少，前后表面曲率增加等一系列形状变化可增强晶状体屈光力，使物像能准确聚焦于视网膜上。然而随着年龄的持续增长，晶状体逐渐出现变硬、囊膜弹性下降等情况，导致其形变能力部分甚至全部丧失，使得调节幅度不断下降以致近距离视力不足以满足个人日常生活需求。老视所表现的视近困难、视近不能持久严重影响中老龄人群的生活工作质量。尽管可以选择配戴眼镜或手术治疗对老视进行矫治，但这些方式都具有一定的限制性，如简便性不强、具有创伤性等，更重要的是它们不能从根本上治疗老视。Presbyopia is an age-related physiological change in the eye that is closely related to a significant decline in the ability to accommodate within the eye. The Helmholtz adjustment mechanism hypothesis believes that a series of shape changes such as increased thickness, decreased diameter, and increased curvature of the front and rear surfaces caused by the contraction of the ciliary muscle during near vision can enhance the refractive power of the lens and make the object image accurately focused on the retina. However, as the age continues to grow, the lens gradually hardens and the elasticity of the capsule decreases, resulting in partial or even complete loss of its deformation ability, resulting in a continuous decline in the adjustment range, so that close vision is not enough to meet the daily needs of individuals. Presbyopia manifests as difficulty in near vision and inability to sustain near vision, seriously affecting the quality of life and work of middle-aged and elderly people. Although you can choose to wear glasses or surgical treatment to correct presbyopia, these methods have certain limitations, such as not simple, traumatic, etc., and more importantly, they cannot fundamentally treat presbyopia.
老年性白内障是全球致盲的主要眼病之一，伴随着年龄增长出现的晶状体混浊、变硬是其典型表现。年龄和环境暴露是老年性白内障发病的主要危险因素，可引起晶状体 内代谢异常，其中氧化应激是目前公认的发病机理。晶状体内蛋白质氧化应激会改变晶状体纤维细胞形态、破坏其高度有序的排列结构，进而影响透明度。除此以外，如上述提及，氧化应激还可能是晶状体弹性形变能力下降的潜在原因，而晶状体硬度增加不仅进一步威胁中老龄人群视力情况，更是限制了白内障手术方式的选择和术后视觉质量的恢复。目前已出现较多具有抗氧化特性的化合物能有效改善白内障混浊程度，但其对硬度的影响暂仍无研究。Senile cataract is one of the main eye diseases that cause blindness in the world. The clouding and hardening of the lens accompanied by aging is its typical manifestation. Age and environmental exposure are the main risk factors for the onset of senile cataract, which can cause metabolic abnormalities in the lens, and oxidative stress is the currently recognized pathogenesis. Oxidative stress of proteins in the lens will change the morphology of lens fiber cells, destroy their highly ordered structure, and then affect the transparency. In addition, as mentioned above, oxidative stress may also be the underlying reason for the decrease in the elastic deformation capacity of the lens, and the increase in lens hardness not only further threatens the vision of the middle-aged and elderly people, but also limits the choice of cataract surgery and postoperative visual acuity. quality recovery. At present, many compounds with antioxidant properties have been found to be effective in improving cataract opacity, but their effects on hardness have not yet been studied.
烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD +)，又称为辅酶I，参与细胞内能量合成代谢的多个过程。与此同时，它还可以以辅酶底物的形式激活多种酶类，具有抗氧化应激、抗凋亡等多重作用。烟酰胺单核苷酸(nicotinamide mononucleotide,NMN)是NAD +合成的前体物质，多项研究证实其外源性补充可显著增加体内NAD +水平，并表现出改善体内能量代谢水平、线粒体功能以及氧化应激状态等正向反应。然而目前仍未有关于NMN及相关化合物对晶状体硬度(老视、老年性白内障)影响的探究。 Nicotinamide adenine dinucleotide (nicotinamide adenine dinucleotide, NAD + ), also known as coenzyme I, participates in multiple processes of energy synthesis and metabolism in cells. At the same time, it can also activate a variety of enzymes in the form of coenzyme substrates, and has multiple functions such as anti-oxidative stress and anti-apoptosis. Nicotinamide mononucleotide (nicotinamide mononucleotide, NMN) is the precursor substance of NAD + synthesis, many studies have confirmed that its exogenous supplementation can significantly increase the level of NAD + in the body, and has shown to improve the level of energy metabolism, mitochondrial function and Positive reactions such as oxidative stress. However, there is still no research on the effect of NMN and related compounds on lens hardness (presbyopia, senile cataract).
专利WO 2018052019A1公开了一种视功能改善剂及视功能改善方法，但仅泛泛指出NMN可改善视功能，并未提及改变晶状体硬度，且使用方法及有效使用量与本申请不同。Patent WO 2018052019A1 discloses a visual function improving agent and a visual function improving method, but only generally points out that NMN can improve visual function, and does not mention changing the hardness of the lens, and the usage method and effective usage amount are different from the present application.
发明内容Contents of the invention
本发明的第一方面，提供了NAD +前体在制备治疗晶状体硬化相关疾病的药物中的应用，所述的药物为局部给药。 The first aspect of the present invention provides the application of NAD + precursor in the preparation of medicines for treating diseases related to lens sclerosis, and the medicines are administered locally.
优选的，所述的晶状体硬化相关疾病包括老花眼和白内障。Preferably, the diseases related to lens sclerosis include presbyopia and cataract.
优选的，所述的局部给药为眼部给药，进一步优选的，所述的局部给药包括晶状体离体给药培养或结膜下注射给药。Preferably, the local administration is ocular administration, and more preferably, the local administration includes lens culture in vitro or subconjunctival injection.
优选的，所述的药物包括10～500mmol/L的NAD +前体。 Preferably, the drug includes 10-500 mmol/L NAD + precursor.
在本发明的一个实施方式中，所述的NAD +前体的浓度为10～40(例如，10、 20、30、40)mmol/L。 In one embodiment of the present invention, the concentration of the NAD + precursor is 10-40 (eg, 10, 20, 30, 40) mmol/L.
在本发明的另一个实施方式中，所述的NAD +前体的浓度为500mmol/L。 In another embodiment of the present invention, the concentration of the NAD + precursor is 500mmol/L.
在本发明的另一个实施方式中，所述的NAD +前体的浓度为20～50(例如，20、30、40、50)mmol/L。 In another embodiment of the present invention, the concentration of the NAD + precursor is 20-50 (eg, 20, 30, 40, 50) mmol/L.
优选的，所述的NAD +前体包括色氨酸、烟酸、烟酰胺、还原形式的烟酰胺核糖核苷(NRH)、烟酰胺单核苷酸(NMN)、葫芦巴碱、烟酸单核苷酸或烟酸核糖核苷中的一种或两种及以上，进一步优选的，所述的NAD +前体为烟酰胺单核苷酸。 Preferably, the NAD + precursors include tryptophan, niacin, nicotinamide, reduced form of nicotinamide riboside (NRH), nicotinamide mononucleotide (NMN), trigonelline, nicotinamide mononucleotide One or two or more of nucleotides or nicotinic acid ribonucleoside, further preferably, the NAD + precursor is nicotinamide mononucleotide.
优选的，所述的药物还包含其他视觉改善成分，进一步优选的，所述的其他视觉改善成分包括花青素、叶黄素、二十二碳六烯酸、虾青素、番茄红素、牛磺酸、泛酚、天冬氨酸钾、硫酸软骨素、锌、钙或镁等。Preferably, the medicine also contains other vision-improving ingredients, further preferably, the other vision-improving ingredients include anthocyanins, lutein, docosahexaenoic acid, astaxanthin, lycopene, Taurine, Panthenol, Potassium Aspartate, Chondroitin Sulfate, Zinc, Calcium or Magnesium etc.
本发明的第二方面，提供了一种药物组合物，所述的药物组合物包括10～500mmol/L的NAD +前体，以及药学上可接受的辅料。 The second aspect of the present invention provides a pharmaceutical composition, which includes 10-500mmol/L NAD + precursor, and pharmaceutically acceptable auxiliary materials.
在本发明的一个实施方式中，所述的NAD +前体的浓度为10～40(例如，10、20、30、40)mmol/L。 In one embodiment of the present invention, the concentration of the NAD + precursor is 10-40 (eg, 10, 20, 30, 40) mmol/L.
在本发明的另一个实施方式中，所述的NAD +前体的浓度为500mmol/L。 In another embodiment of the present invention, the concentration of the NAD + precursor is 500mmol/L.
在本发明的另一个实施方式中，所述的NAD +前体的浓度为20～50(例如，20、30、40、50)mmol/L。 In another embodiment of the present invention, the concentration of the NAD + precursor is 20-50 (eg, 20, 30, 40, 50) mmol/L.
优选的，所述的NAD +前体包括色氨酸、烟酸、烟酰胺、还原形式的烟酰胺核糖核苷(NRH)、烟酰胺单核苷酸(NMN)、葫芦巴碱、烟酸单核苷酸或烟酸核糖核苷中的一种或两种及以上，进一步优选的，所述的NAD +前体为烟酰胺单核苷酸。 Preferably, the NAD + precursors include tryptophan, niacin, nicotinamide, reduced form of nicotinamide riboside (NRH), nicotinamide mononucleotide (NMN), trigonelline, nicotinamide mononucleotide One or two or more of nucleotides or nicotinic acid ribonucleoside, further preferably, the NAD + precursor is nicotinamide mononucleotide.
优选的，所述的药物组合物为局部给药，以改善药物领域中的视觉功能。所述的药物剂型包括粉末、片剂、缓释片、咀嚼片、泡腾片、锭剂、含服片、舌下片、胶囊、细颗粒、颗粒剂，例如口服制剂，如丸、干糖浆、溶液、悬浮液、糖浆和酏剂，以及滴眼液、滴眼剂、眼膏、注射剂、输液、外部制剂等。Preferably, the pharmaceutical composition is administered locally to improve visual function in the pharmaceutical field. The pharmaceutical dosage forms include powder, tablet, sustained-release tablet, chewable tablet, effervescent tablet, lozenge, buccal tablet, sublingual tablet, capsule, fine granule, granule, such as oral preparation, such as pill, dry syrup , solutions, suspensions, syrups and elixirs, as well as eye drops, eye drops, eye ointments, injections, infusions, external preparations, etc.
优选的，所述的药物组合物还包括药物可接受的辅料适以及其他所述剂型的可药用已知的药物添加剂，可根据其理化性质、生物学特性等，适当地混合到所述药物组合物中。例如，辅料(乳糖、淀粉、结晶纤维素、磷酸钠等)、溶剂(水、大豆油、生理盐水、注射用非水溶剂等)、粘合剂(淀粉、明胶、阿拉伯胶、海藻酸钠、卡梅隆钠、甲基纤维素、乙基纤维素、羟丙基甲基纤维素、羟丙基纤维素、聚乙烯醇吡咯烷酮等)、崩解剂(淀粉、羧甲基纤维素钠等)、润滑剂(滑石粉、硬脂酸镁、硬脂酸钙、聚乙二醇、蔗糖脂肪酸酯等)、包衣剂(白糖、羟丙纤维素(HPC)、虫胶、明胶、甘油、羟丙基甲基纤维素、邻苯二甲酸羟丙基甲基纤维素、邻苯二甲酸醋酸纤维素等)、稳定剂(亚硫酸氢钠、硫代硫酸钠、依地酸钠、柠檬酸钠、抗坏血酸、二丁基羟基甲苯等)、防腐剂(对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯、苄醇、苯酚、氯丁醇、苯扎氯铵、苄索氯铵、脱氢乙酸钠、硫柳汞等，增稠剂(甲基纤维素、羧甲基纤维素钠、软骨素硫酸钠、海藻酸钠等)、悬浮剂(各种非离子表面活性剂、甲基纤维素、羧甲基纤维素钠等)、乳化剂(阿拉伯胶、胆固醇、山梨糖醇倍半油酸酯、聚山梨酯80、十二烷基硫酸钠等)、缓冲液(柠檬酸、乙酸、磷酸钠、硼酸)、表面活性剂(氢化蓖麻油、聚山梨酯80等)、着色剂(水溶性食用色素、色淀色素等)，调味剂(乳糖、白糖、葡萄糖、甘露醇等)、香味剂(芳香精油等)、增塑剂(邻苯二甲酸酯、植物油、聚乙二醇等)。Preferably, the pharmaceutical composition also includes pharmaceutically acceptable excipients and other pharmaceutically known pharmaceutical additives in the dosage form, which can be properly mixed into the drug according to their physical and chemical properties, biological characteristics, etc. composition. For example, excipients (lactose, starch, crystalline cellulose, sodium phosphate, etc.), solvents (water, soybean oil, physiological saline, non-aqueous solvents for injection, etc.), binders (starch, gelatin, gum arabic, sodium alginate, Cameron sodium, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, etc.), disintegrants (starch, sodium carboxymethylcellulose, etc.) , lubricants (talc, magnesium stearate, calcium stearate, polyethylene glycol, sucrose fatty acid ester, etc.), coating agents (white sugar, hydroxypropyl cellulose (HPC), shellac, gelatin, glycerin, Hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, cellulose acetate phthalate, etc.), stabilizers (sodium bisulfite, sodium thiosulfate, sodium edetate, citric acid sodium, ascorbic acid, dibutyl hydroxytoluene, etc.), preservatives (methylparaben, ethylparaben, propylparaben, benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, Benzethonium chloride, sodium dehydroacetate, thimerosal, etc., thickeners (methylcellulose, sodium carboxymethylcellulose, sodium chondroitin sulfate, sodium alginate, etc.), suspending agents (various nonionic surfactants , methylcellulose, sodium carboxymethylcellulose, etc.), emulsifiers (gum arabic, cholesterol, sorbitol sesquioleate, polysorbate 80, sodium lauryl sulfate, etc.), buffer (lemon acid, acetic acid, sodium phosphate, boric acid), surfactant (hydrogenated castor oil, polysorbate 80, etc.), coloring agent (water-soluble food coloring, lake coloring, etc.), flavoring agent (lactose, white sugar, glucose, mannitol etc.), flavoring agents (aromatic essential oils, etc.), plasticizers (phthalates, vegetable oils, polyethylene glycol, etc.).
本发明的第三方面，提供了一种改变晶状体硬度的方法，所述的方法包括使用NAD +前体。 In a third aspect of the present invention, there is provided a method of altering lens hardness, said method comprising the use of NAD + precursors.
任选的，所述的方法包括使用上述的药物组合物。Optionally, the method includes using the above-mentioned pharmaceutical composition.
优选的，所述的NAD +前体的浓度为10～500mmol/L。 Preferably, the concentration of the NAD + precursor is 10-500 mmol/L.
在本发明的一个实施方式中，所述的NAD +前体的浓度为10～40(例如，10、20、30、40)mmol/L。 In one embodiment of the present invention, the concentration of the NAD + precursor is 10-40 (eg, 10, 20, 30, 40) mmol/L.
在本发明的另一个实施方式中，所述的NAD +前体的浓度为500mmol/L。 In another embodiment of the present invention, the concentration of the NAD + precursor is 500mmol/L.
在本发明的另一个实施方式中，所述的NAD +前体的浓度为20～50(例如，20、30、40、50)mmol/L。 In another embodiment of the present invention, the concentration of the NAD + precursor is 20-50 (eg, 20, 30, 40, 50) mmol/L.
优选的，所述的NAD +前体包括色氨酸、烟酸、烟酰胺、还原形式的烟酰胺核糖核苷(NRH)、烟酰胺单核苷酸(NMN)、葫芦巴碱、烟酸单核苷酸或烟酸核糖核苷中的一种或两种及以上，进一步优选的，所述的NAD +前体为烟酰胺单核苷酸。 Preferably, the NAD + precursors include tryptophan, niacin, nicotinamide, reduced form of nicotinamide riboside (NRH), nicotinamide mononucleotide (NMN), trigonelline, nicotinamide mononucleotide One or two or more of nucleotides or nicotinic acid ribonucleoside, further preferably, the NAD + precursor is nicotinamide mononucleotide.
优选的，所述的方法包括局部给药。Preferably, the method includes topical administration.
在本发明的一个实施方式中，所述的方法包括：将晶状体离体培养于NAD +前体溶液中。 In one embodiment of the present invention, the method comprises: culturing the lens in NAD + precursor solution in vitro.
优选的，所述的培养时间为12小时。Preferably, the culture time is 12 hours.
在本发明的另一个实施方式中，所述的方法包括：将NAD +前体用于结膜下注射。 In another embodiment of the invention, the method comprises: administering NAD + precursor for subconjunctival injection.
优选的，所述的注射量为5μL/次。Preferably, the injection volume is 5 μL/time.
本发明的第四方面，提供了一种预防和/或治疗眼部疾病的方法，所述的方法包括使用NAD +前体。 The fourth aspect of the present invention provides a method for preventing and/or treating eye diseases, the method comprising using NAD + precursor.
优选的，所述的NAD +前体的浓度为10～500mmol/L。 Preferably, the concentration of the NAD + precursor is 10-500 mmol/L.
在本发明的一个实施方式中，所述的NAD +前体的浓度为10～40(例如，10、20、30、40)mmol/L。 In one embodiment of the present invention, the concentration of the NAD + precursor is 10-40 (eg, 10, 20, 30, 40) mmol/L.
在本发明的另一个实施方式中，所述的NAD +前体的浓度为500mmol/L。 In another embodiment of the present invention, the concentration of the NAD + precursor is 500mmol/L.
在本发明的另一个实施方式中，所述的NAD +前体的浓度为20～50(例如，20、30、40、50)mmol/L。 In another embodiment of the present invention, the concentration of the NAD + precursor is 20-50 (eg, 20, 30, 40, 50) mmol/L.
优选的，所述的NAD +前体包括色氨酸、烟酸、烟酰胺、还原形式的烟酰胺核糖核苷(NRH)、烟酰胺单核苷酸(NMN)、葫芦巴碱、烟酸单核苷酸或烟酸核糖核苷中的一种或两种及以上，进一步优选的，所述的NAD +前体为烟酰胺单核苷酸。 Preferably, the NAD + precursors include tryptophan, niacin, nicotinamide, reduced form of nicotinamide riboside (NRH), nicotinamide mononucleotide (NMN), trigonelline, nicotinamide mononucleotide One or two or more of nucleotides or nicotinic acid ribonucleoside, further preferably, the NAD + precursor is nicotinamide mononucleotide.
优选的，所述的眼部疾病包括老花眼和白内障。Preferably, the eye diseases include presbyopia and cataract.
在本发明的一个实施方式中，所述的方法包括：将晶状体离体培养于NAD +前体溶液中。 In one embodiment of the present invention, the method comprises: culturing the lens in NAD + precursor solution in vitro.
优选的，所述的培养时间为12小时。Preferably, the culture time is 12 hours.
在本发明的另一个实施方式中，所述的方法包括：将NAD +前体用于结膜下注射。 In another embodiment of the invention, the method comprises: administering NAD + precursor for subconjunctival injection.
优选的，所述的注射量为5μL/次。Preferably, the injection volume is 5 μL/time.
本发明中改变晶状体硬度的试剂、食品组合物、药物组合物的受试者可以为哺乳动物，如，人类、猴、狗、兔、小鼠、大鼠等。The subject of the agent for changing the hardness of the lens, the food composition, and the pharmaceutical composition of the present invention can be mammals, such as humans, monkeys, dogs, rabbits, mice, and rats.
本发明所述的“治疗”表示在疾病已开始发展后减缓、中断、阻止、控制、停止、减轻、或逆转一种体征、症状、失调、病症、或疾病的进展或严重性，但不一定涉及所有疾病相关体征、症状、病症、或失调的完全消除。As used herein, "treating" means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but not necessarily The complete elimination of all disease-related signs, symptoms, conditions, or disorders is contemplated.
本发明中的NAD +前体在特定浓度下通过局部给药的方式，直接作用于眼部，能够有效增加衰老晶状体的轴向应变程度，改善晶状体硬度，部分恢复弹性。因此，该化合物可通过调节晶状体硬度从而起到恢复调节能力、治疗老视和老年性白内障的作用。 The NAD + precursor in the present invention acts directly on the eye through local administration at a specific concentration, which can effectively increase the axial strain of the aging lens, improve the hardness of the lens, and partially restore elasticity. Therefore, the compound can restore the adjustment ability and treat presbyopia and senile cataract by regulating the hardness of the lens.
附图说明Description of drawings
以下，结合附图来详细说明本发明的实施例，其中：Hereinafter, embodiments of the present invention will be described in detail in conjunction with the accompanying drawings, wherein:
图1：体外实验晶状体硬度变化图，其中*为P<0.05，**为P<0.01；Figure 1: Changes in lens hardness in vitro, where * is P<0.05, ** is P<0.01;
图2：体内实验(结膜下注射给药)晶状体硬度变化图，其中*为P<0.05；Figure 2: Changes in lens hardness in vivo experiments (subconjunctival injection), where * is P<0.05;
图3：体外实验老年性白内障晶状体混浊程度变化图；其中箭头为环形白内障；Figure 3: Changes in the degree of lens opacity in senile cataract in vitro; the arrows indicate ring-shaped cataracts;
图4：体外实验老年性白内障晶状体硬度变化图；其中*为P<0.05；Figure 4: Changes in lens hardness of senile cataract in vitro; where * is P<0.05;
图5：体内实验(灌胃给药)晶状体硬度变化图。Figure 5: Changes in lens hardness in vivo experiments (gastric administration).
具体实施方式Detailed ways
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实 施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
实施例1烟酰胺单核苷酸溶液降低小鼠晶状体硬度Example 1 Niacinamide mononucleotide solution reduces lens hardness in mice
1、体外实验：1. In vitro experiment:
将8月龄小鼠(C57BL/6J)随机分为对照组和处理组，将晶状体先于完全MEM培养基(含10％胎牛血清和1％青霉素/链霉素)中孵育2小时以恢复代谢活性。随后，处理组培养基更换为含有不同浓度(10～40mmol/L，具体为10、20、30、40mmol/L)烟酰胺单核苷酸溶液的无血清MEM培养基，对照组仅为无血清MEM培养基，并在37℃，5％CO 2培养箱中培养12小时。12小时后，用HBSS溶液轻缓冲洗晶状体3遍。使用已知的盖玻片压痕法测量晶状体弹性。具体操作是：在盛有65～70mL HBSS溶液的亚克力方型器皿中，晶状体以轴向方向稳定固定于器皿中特制的小孔内，距离晶状体一定位置的直角棱镜可反射出晶状体整体轮廓。利用盖玻片重量使晶状体产生形变，统计形变大小以评估硬度情况。 Eight-month-old mice (C57BL/6J) were randomly divided into control and treatment groups, and the lenses were incubated in complete MEM medium (containing 10% fetal bovine serum and 1% penicillin/streptomycin) for 2 hours to recover metabolic activity. Subsequently, the medium in the treatment group was replaced with serum-free MEM medium containing different concentrations (10-40mmol/L, specifically 10, 20, 30, 40mmol/L) of nicotinamide mononucleotide solution, and the control group was only serum-free MEM medium and incubate for 12 hours at 37°C in a 5% CO 2 incubator. After 12 hours, wash the lens 3 times with light buffer of HBSS solution. Lens elasticity was measured using the known coverslip indentation method. The specific operation is: in the acrylic square vessel containing 65-70mL HBSS solution, the lens is stably fixed in the special small hole in the vessel in the axial direction, and the rectangular prism at a certain position from the lens can reflect the overall outline of the lens. The lens was deformed by the weight of the cover glass, and the deformation was counted to evaluate the hardness.
2、体内实验：2. In vivo experiment:
将8月龄小鼠(C57BL/6J)随机分为对照组和处理组，使用戊巴比妥钠腹腔注射将小鼠麻醉后，充分暴露右眼球结膜。先使用30G针头在距离角膜缘1～2mm颞侧球结膜处，以水平方向与眼球成15°角将针头刺入，并轻挑起球结膜，随后用Hamilton 701RN TLC注射器(10μL，针头26s G)经穿刺口进针2mm，缓慢将5μL注射液(对照组：生理盐水注射液；处理组：500mmol/L烟酰胺单核苷酸溶液)注入，可见该处球结膜形成鱼泡样隆起。术后给予左氧氟沙星滴眼液点眼。右眼结膜下注射每天进行1次，连续6天。6天后摘取眼球以获得晶状体，使用上述盖玻片压痕法测量晶状体硬度。Eight-month-old mice (C57BL/6J) were randomly divided into a control group and a treatment group. After the mice were anesthetized by intraperitoneal injection of pentobarbital sodium, the conjunctiva of the right eye was fully exposed. First use a 30G needle to penetrate the temporal bulbar conjunctiva 1-2mm away from the corneal limbus at an angle of 15° to the eyeball in the horizontal direction, and gently lift the bulbar conjunctiva, then use a Hamilton 701RN TLC syringe (10μL, needle 26s G ) into the needle 2 mm through the puncture port, slowly inject 5 μL of injection (control group: saline injection; treatment group: 500 mmol/L nicotinamide mononucleotide solution), and it can be seen that the bulbar conjunctiva forms a fish bubble-like bulge. Levofloxacin eye drops were given after the operation. The subconjunctival injection of the right eye was performed once a day for 6 consecutive days. Eyeballs were enucleated 6 days later to obtain lenses, and lens hardness was measured using the coverslip indentation method described above.
最终结果如图1和2所示，不论是体外实验和体内实验，与对照组相比较，处理组均能增加中老龄小鼠晶状体的轴向应变(P<0.05)，体外实验呈现出剂量依赖性。The final results are shown in Figures 1 and 2. Both in vitro and in vivo experiments, compared with the control group, the treatment group can increase the axial strain of the lens of middle-aged and aged mice (P<0.05), and the in vitro experiments showed a dose-dependent sex.
实施例2烟酰胺单核苷酸溶液改善老年性白内障小鼠晶状体混浊程度和硬度Example 2 Nicotinamide mononucleotide solution improves lens turbidity and hardness in senile cataract mice
选择14月龄C57BL/6J小鼠，随机分为对照组和处理组。取材后，先使用体视显微镜(SteREO Discovery V8体视显微镜，德国蔡司)在无菌条件下记录小鼠晶状体混浊程度，以确保成功制备老年性白内障小鼠模型。晶状体于完全MEM培养基(含10％胎牛血清和1％青霉素/链霉素)中孵育2小时以恢复代谢活性。随后，处理组培养基更换为含有不同浓度(20～50mmol/L，具体为20、30、40、50mmol/L)烟酰胺单核苷酸溶液的无血清MEM培养基，对照组仅为无血清MEM培养基，并在37℃，5％CO 2培养箱中培养12小时。12小时后，用HBSS溶液轻缓冲洗晶状体3遍。在硬度检测之前，再次使用体视显微镜记录晶状体混浊程度。随后使用上述盖玻片压痕法测量晶状体硬度。 14-month-old C57BL/6J mice were selected and randomly divided into control group and treatment group. After the materials were collected, a stereomicroscope (SteREO Discovery V8 stereomicroscope, Zeiss, Germany) was used to record the opacity degree of the mouse lens under sterile conditions to ensure the successful preparation of the senile cataract mouse model. The lenses were incubated for 2 hours in complete MEM medium (containing 10% fetal calf serum and 1% penicillin/streptomycin) to restore metabolic activity. Subsequently, the medium in the treatment group was replaced with serum-free MEM medium containing different concentrations (20-50mmol/L, specifically 20, 30, 40, 50mmol/L) of nicotinamide mononucleotide solution, and the control group was only serum-free MEM medium and incubate for 12 hours at 37°C in a 5% CO 2 incubator. After 12 hours, wash the lens 3 times with light buffer of HBSS solution. Lens opacity was recorded again using a stereomicroscope prior to hardness testing. Lens hardness was then measured using the coverslip indentation method described above.
结果如图3和4所示，处理前小鼠晶状体已出现环状、边界清晰、中央区域稍混浊等老年性白内障表现，处理后两组晶状体透明度下降，但处理组混浊程度明显较对照组轻；同时，处理组的轴向应变程度较对照组显著增加(P<0.05)。The results are shown in Figures 3 and 4. Before the treatment, the lens of the mouse had senile cataract symptoms such as ring shape, clear boundary, and slight turbidity in the central area. After treatment, the lens transparency of the two groups decreased, but the degree of turbidity in the treatment group was significantly lighter than that in the control group. ; At the same time, the degree of axial strain in the treatment group was significantly higher than that in the control group (P<0.05).
实施例3烟酰胺单核苷酸溶液灌胃给药方式对改善小鼠晶状体硬度效果一般Example 3 Nicotinamide mononucleotide solution gavage administration method has a general effect on improving the hardness of the lens of mice
将8月龄C57BL/6J小鼠随机分为对照组和处理组。小鼠称重后，左手抓住小鼠，使其头、颈和身体呈一直线，使用1mL注射器和8号灌胃针，灌胃针头从小鼠嘴角进入，抵住上颚后轻轻往内推进，进入胃部后按照100mg/kg/天推注药液，每天1次，持续1个月。灌胃1个月后摘取眼球以获得晶状体，使用上述盖玻片压痕法测量晶状体硬度。Eight-month-old C57BL/6J mice were randomly divided into control group and treatment group. After the mouse is weighed, hold the mouse with the left hand so that its head, neck and body are in a straight line, use a 1mL syringe and a No. 8 gavage needle, enter the gavage needle from the corner of the mouse's mouth, touch the palate and push it inward gently , after entering the stomach, inject the drug solution at 100mg/kg/day, once a day, for 1 month. One month after gavage, the eyeballs were removed to obtain the lens, and the hardness of the lens was measured by the above-mentioned cover glass indentation method.
结果如图5所示，灌胃给药后晶状体轴向应变程度较对照组增加，但差异不具有统计学意义(P＝0.09)。The results are shown in Figure 5, the degree of axial strain of the lens after intragastric administration was higher than that of the control group, but the difference was not statistically significant (P=0.09).
综上所述，以上结果表明，烟酰胺单核苷酸溶液通过增加晶状体轴向应变程度、改善晶状体硬度、降低白内障晶状体混浊程度，从而起到增强调节能力、治疗老视、老年性白内障的作用。In summary, the above results show that nicotinamide mononucleotide solution can enhance the adjustment ability and treat presbyopia and senile cataract by increasing the axial strain of the lens, improving the hardness of the lens, and reducing the opacity of the lens in cataract. .
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

Claims (10)

  1. NAD +前体在制备治疗晶状体硬化相关疾病的药物中的应用，其特征在于，所述的药物为局部给药。 The application of NAD + precursor in the preparation of medicines for treating lens sclerosis related diseases is characterized in that the medicines are administered locally.
  2. 根据权利要求1所述的应用，其特征在于，所述的晶状体硬化相关疾病包括老花眼和白内障。The application according to claim 1, characterized in that the diseases related to lens sclerosis include presbyopia and cataract.
  3. 根据权利要求1或2所述的应用，其特征在于，所述的局部给药为眼部给药。The application according to claim 1 or 2, characterized in that the local administration is eye administration.
  4. 根据权利要求3所述的应用，其特征在于，所述的局部给药包括晶状体离体给药培养或结膜下注射给药。The application according to claim 3, characterized in that, the local administration includes lens ex vivo administration and culture or subconjunctival injection administration.
  5. 根据权利要求1-4任一所述的应用，其特征在于，所述的NAD +前体包括色氨酸、烟酸、烟酰胺、还原形式的烟酰胺核糖核苷、烟酰胺单核苷酸、葫芦巴碱、烟酸单核苷酸或烟酸核糖核苷中的一种或两种及以上。 The application according to any one of claims 1-4, characterized in that the NAD + precursors include tryptophan, nicotinic acid, nicotinamide, nicotinamide riboside in reduced form, nicotinamide mononucleotide , trigonelline, nicotinic acid mononucleotide or nicotinic acid ribonucleoside or two or more.
  6. 根据权利要求1-5任一所述的应用，其特征在于，所述的药物包括10～500mmol/L的NAD +前体。 The use according to any one of claims 1-5, characterized in that the drug comprises 10-500 mmol/L NAD + precursor.
  7. 一种药物组合物，其特征在于，所述的药物组合物包括10～500mmol/L的NAD +前体，以及药学上可接受的辅料。 A pharmaceutical composition, characterized in that the pharmaceutical composition includes 10-500 mmol/L of NAD + precursor, and pharmaceutically acceptable auxiliary materials.
  8. 一种改变晶状体硬度的方法，所述的方法包括使用权利要求7所述的药物组合物。A method for changing the hardness of a lens, said method comprising using the pharmaceutical composition according to claim 7.
  9. 根据权利要求8所述的方法，其特征在于，所述的方法包括局部给药。The method according to claim 8, characterized in that said method comprises topical administration.
  10. 根据权利要求8或9所述的方法，其特征在于，所述的方法包括将晶状体离体培养于药物组合物中，或，将药物组合物用于结膜下注射。The method according to claim 8 or 9, characterized in that the method comprises culturing the lens in vitro in the pharmaceutical composition, or using the pharmaceutical composition for subconjunctival injection.
PCT/CN2022/127077 2021-10-27 2022-10-24 Use of nad+ precursor in preparation of drug for treating lens sclerosis-related diseases WO2023071994A1 (en)

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