RU2822512C2 - New use of nicotinamide mononucletide (nmn) and nicotinamide riboside (nr) - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: group of inventions relates to the use of nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR) for preparing a drug for improving meibomian gland function or for treating or preventing meibomian gland dysfunction.

EFFECT: use of the group of inventions enables the effective treatment of meibomian gland dysfunctions.

22 cl, 5 dwg, 2 tbl, 5 ex

Description

[Technical field]

[0001]

The present invention relates to a new use of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR). For example, the present invention relates to a composition containing NMN or NR for use in the treatment or prevention of a disease, disorder or symptom associated with Hsd3b6 or a homologue thereof, or a method using them. In particular, the present invention relates to a composition containing NMN or NR for use in improving meibomian gland function or a method of using them.

[Technique level]

[0002]

Due to the proposed possibility that oxidized forms of nicotinamide adenine dinucleotide (NAD+) intermediate metabolites such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), i.e. products of the enzymatic reaction nicotinamide phosphoribosyltransferase (NAMPT), may have a lifespan prolonging effect, and are being developed as a supplement. However, the mechanism of action of NMN and NR activity is not fully understood.

[Brief summary of the invention]

[The solution of the problem]

[0003]

The present invention provides the use of nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) or a composition containing them to improve the activity of Hsd3b6 or its homologue. Improving the activity of Hsd3b6 or a homologue thereof may provide treatment or prevention of a disease, disorder or symptom associated with the activity of Hsd3b6 or a homologue thereof.

[0004]

The present invention particularly provides the use of nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) or a composition containing them to improve meibomian gland function. The composition may be characterized in that it is introduced into the eye of a subject. Examples of improving meibomian gland function include increasing the number of meibomian gland acinar cells, increasing meibomian gland lipid secretion, and the like. In one embodiment, a composition may be provided for the treatment or prevention of a disease involving meibomian gland dysfunction. In another embodiment, the composition of the present invention exhibits the effect of improving meibomian gland tissue atrophy and can increase the activity of a steroid synthase that acts on a lipid secreted by the meibomian gland. Since the composition of the present invention can normalize the composition of lipids secreted by the meibomian gland, it is understood that the lipids in the meibomian gland can be normalized. In addition, the composition of the present invention can promote the production of testosterone, which is known to promote lipid production by the meibomian gland. Since sex hormones such as testosterone influence lipid production by the meibomian glands, it is understood that the composition of the present invention can normalize the lipid composition of meibomian gland tissue.

[0005]

For example, the following items are presented in preferred embodiments of the present invention.

(Point A1)

A composition containing nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR), for use in improving the activity of Hsd3b6 or its homolog.

(Clause A1-2)

A composition comprising nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR), for use in the treatment or prevention of a disease, disorder or symptom associated with the activity of Hsd3b6 or its homologue.

(Clause A1-3)

The composition according to any of the preceding claims, wherein the disease associated with the activity of Hsd3b6 or its homologue is selected from the group consisting of meibomian gland dysfunction, diseases caused by a decrease in the level of a steroid hormone (for example, testosterone, estrogen and the like), postmenopausal syndrome, osteoporosis , lifestyle diseases, age-related androgen deficiency in men and myocardial infarction.

(Point A2)

The composition according to any one of the preceding claims, wherein the composition is administered to the eye of the subject.

(Clause A3)

A composition containing nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) for use in improving meibomian gland function.

(Clause A4)

A composition according to any one of the preceding claims for use in increasing the number of meibomian gland acinar cells.

(Clause A5)

A composition according to any one of the preceding claims for use in increasing lipid secretion from the meibomian gland.

(Clause A6)

A composition according to any of the preceding claims for use in the treatment or prevention of a disease accompanied by dysfunction of the meibomian gland.

(Paragraph A6-1)

A composition for use in the treatment or prevention of meibomian gland dysfunction, containing nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR).

(Paragraph A6-2)

The composition according to any of the preceding claims, where dysfunction of the meibomian glands is accompanied by a decrease in the secretion of the meibomian glands.

(Paragraph A6-3)

The composition according to any of the preceding claims, wherein dysfunction of the meibomian glands is accompanied by an inflammatory disease.

(Paragraph A6-4)

The composition according to any one of the preceding claims, wherein the inflammatory disease includes at least one disease selected from the group consisting of meibomitis, superficial (punctate) keratitis and blepharitis.

(Paragraph A6-5)

The composition according to any of the preceding claims, where dysfunction of the meibomian glands is accompanied by excessive accumulation of lipids in the duct.

(Paragraph A6-6)

The composition according to any one of the preceding claims, wherein the meibomian gland dysfunction is accompanied by ocular discomfort, foreign body sensation and/or pressure sensation.

(Clause A7)

The composition according to any of the preceding claims, wherein the disease associated with meibomian gland dysfunction is meibomitis, posterior blepharitis, keratitis, conjunctivitis, keratoconjunctivitis associated with meibomitis, ocular pemphigoid, Sjögren's syndrome, Stevens-Johnson syndrome, graft-versus-host disease ( GVHD) and visual impairment.

(Point B1)

A method of improving the activity of Hsd3b6 or a homologue thereof in a subject or treating or preventing a disease, disorder or symptom associated with the activity of Hsd3b6 or a homologue thereof.

(Clause B2)

The method of claim B1, comprising administering nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) to the subject.

(Paragraph B3)

The method of claim B1 or B2, further comprising measuring the activity of Hsd3b6 or a homologue thereof in the subject.

(Paragraph B4)

The method according to any one of claims B1-B3, comprising administering NMN and/or NR when the activity of Hsd3b6 or its homologue in the subject is measured and the activity is reduced.

(Clause C1)

A method of improving meibomian gland function of a subject or treating or preventing a disease, disorder or symptom accompanied by meibomian gland dysfunction.

(Clause C2)

The method of claim C1, comprising administering nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) to the subject.

(Clause C3)

The method of claim C1 or C2, further comprising testing the function of the subject's meibomian gland.

(Clause C4)

The method of any one of claims C1 to C3, comprising administering NMN and/or NR when the subject's meibomian gland function is tested and the function is reduced.

(Clause C5)

The method of item C4, wherein the meibomian gland function is tested by slit lamp eye examination, meibography, confocal microscopy, DR-1, mucocutaneous junction, meibometer, lipid chemistry, evaporation amount, BUT or subjective symptom questionnaire.

(Clause C6)

The method according to any one of claims C1-C5, wherein the disease, disorder or symptom accompanied by meibomian gland dysfunction is selected from the group consisting of meibomitis, posterior blepharitis, keratitis, conjunctivitis, keratoconjunctivitis associated with meibomitis, ocular pemphigoid, Sjögren's syndrome, Stevens syndrome -Johnson, graft-versus-host disease (GVHD) and visual impairment.

(Clause C7)

The method according to any one of paragraphs C1-C6 for increasing the number of meibomian gland acinar cells.

(Clause C8)

The method according to any one of paragraphs C1-C6 for increasing the secretion of lipids by the meibomian gland.

(Clause C9)

Method according to any one of points C1-C6 for enhancing testosterone production in the meibomian gland.

(Clause D1)

Use of nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) to obtain a composition that improves meibomian gland function.

(Clause D2)

Application according to point D1, where the composition is intended to increase the number of acinar cells of the meibomian gland.

(Clause D3)

Application according to point D1, where the composition is intended to increase the secretion of lipids by the meibomian gland.

(Paragraph D4)

Use according to any of paragraphs D1-D3, where the composition is intended for the treatment or prevention of a disease accompanied by dysfunction of the meibomian gland.

(Paragraph D5)

Application according to point D4, where the disease accompanied by meibomian gland dysfunction is selected from the group consisting of meibomitis, posterior blepharitis, keratitis, conjunctivitis, keratoconjunctivitis associated with meibomitis, ocular pemphigoid, Sjögren's syndrome, Stevens-Johnson syndrome, graft-versus-host disease "(GVHD) and visual impairment.

(Clause D6)

Use according to any of points D1-D5, where the composition is intended to enhance the production of testosterone in the meibomian gland.

(Clause E1)

Use of nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) to obtain a composition for the treatment or prevention of meibomian gland dysfunction.

(Clause E2)

Use according to point E1, where dysfunction of the meibomian glands is accompanied by a decrease in the secretion of the meibomian glands.

(Clause E3)

Use according to point E1, where dysfunction of the meibomian glands is accompanied by an inflammatory disease.

(Clause E4)

Use according to claim E3, wherein the inflammatory disease includes at least one disease selected from the group consisting of meibomitis, superficial (punctate) keratitis and blepharitis.

(Clause E5)

Use according to point E1, where dysfunction of the meibomian glands is accompanied by excessive accumulation of lipids in the duct.

(Clause E6)

Use according to point E1, where dysfunction of the meibomian glands is accompanied by discomfort in the eyes, foreign body sensation and/or a feeling of pressure.

(Point F1)

Nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) for use in improving the activity of Hsd3b6 or a homologue thereof in a subject or treating or preventing a disease, disorder or symptom associated with the activity of Hsd3b6 or a homologue thereof.

(Clause G1)

Nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) for use in improving meibomian gland function or treating or preventing a disease, disorder or symptom associated with meibomian gland dysfunction.

(Item G2)

Nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) according to clause G1, wherein the disease, disorder or symptom accompanied by meibomian gland dysfunction is selected from the group consisting of meibomitis, posterior blepharitis, keratitis, conjunctivitis, keratoconjunctivitis associated with meibomitis, ocular pemphigoid, Sjögren's syndrome, Stevens-Johnson syndrome, graft-versus-host disease (GVHD) and visual impairment.

(Item H1)

Composition for use to enhance testosterone production in the meibomian gland, containing nicotinamide mononucleotide (NMN) and/or nicotinamide riboside.

(Clause H2)

The composition of item H1, wherein the composition is administered to the eye of the subject.

(Point I1)

Method of use to increase testosterone production in a subject's meibomian gland.

(Item I2)

The method of claim I1, comprising administering nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) to the subject.

(Clause J1)

Nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) to enhance testosterone production in the meibomian gland.

(Paragraph J2)

Nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) at J1, which is administered to the eye of the subject.

(Clause K1)

Use of nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) to produce a drug to enhance testosterone production in the meibomian gland.

(Clause K2)

Application according to point K1, characterized by introduction into the eye of the subject.

[0006]

The present invention is intended to ensure that one or more of the features described above can be provided not only in the expressly disclosed combinations, but also in other combinations thereof. Additional embodiments and advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description as appropriate.

[Beneficial Effects of the Invention]

[0007]

The present invention provides a composition for use in improving the activity of Hsd3b6 or a homologue thereof, or treating or preventing a disease, disorder or symptom associated with the activity of Hsd3b6 or a homologue thereof, or a method for doing so. In particular, the present invention may provide a composition for use in improving meibomian gland function or a method for doing so.

[Brief description of drawings]

[0008]

[Figure 1] In FIG. Figure 1 is a diagram showing the atrophy of meibomian gland tissue associated with aging. Left and center are images of stained eyelids from 6-month-old and 24-month-old mice, respectively. The right side is a histogram comparing the average painted areas for 6-month-old and 24-month-old mice.

[Figure 2] In FIG. Figure 2 is a graph showing the change in meibomian gland tissue of a 2-month-old Hsd3b6 knockout (KO) mouse (meibomian gland size and number of secretory ducts). The top row shows the results for male mice, and the bottom row shows the results for female mice.

[Figure 3] In FIG. 3 is a graph showing the change in Hsd3b6 enzymatic activity and meibomian gland basal cell proliferative activity in the meibomian gland following instillation of NMN or NR eye drops for 2 weeks in 1.5-year-old wild-type mice.

[Figure 4] In FIG. 4 is a graph showing the effect of instillation of NMN or NR eye drops into 1.75 year old wild type mice for 3 months on meibomian gland tissue. The figure shows representative images of eyelid staining of the left eye (no treatment) and the right eye (eye drop instillation), as well as a graph showing the stained area in each individual (right eye/left eye).

[Figure 5] In FIG. Figure 5 is a graph showing testosterone levels in meibomian gland tissue after castration and after sham surgery in 2-month-old male wild-type (WT) and Hsd3b6 knockout (KO) mice.

[Description of Embodiments]

[0009]

The present invention is described below. Throughout the specification, a singular term should be understood to include its plural term, unless specifically stated otherwise. Thus, singular forms should also be understood to cover its plural concept, unless specifically stated otherwise. Terms used herein should also be understood to have the meaning commonly used in the art unless specifically stated otherwise. Thus, unless otherwise indicated, all terminology and scientific and technical terms used herein have the same meaning as commonly understood by one skilled in the art to which this disclosure pertains. In the event of any conflict, this specification (including definitions) shall control.

[0010]

(Definitions)

As used herein, the term “about” refers to a range of ±10% of the numerical value described after “about” unless otherwise indicated.

[0011]

As used herein, the term “subject” refers to the target of administration (transplantation) of the drug or method of treatment and prevention of the present invention. Examples of subjects include mammals (eg, humans, mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep, monkeys and the like), but primates are preferred, and humans are especially preferred.

[0012]

As used herein, “or” is used when “at least one or more” of the items listed in the proposal may be used. Whenever “within the range” of “two values” is explicitly stated herein, the range also includes the two values themselves.

[0013]

As used herein, the term “therapy (treatment)” refers to the cure of a disease or symptom, or the suppression of a symptom. “Prevention (prevention)” refers to preventing the manifestation of a disease or symptom in advance. This concept also includes delaying the onset of a disease or symptom and minimizing the manifestation of a disease or symptom.

[0014]

As used herein, the term “derivative” refers to a compound having a core structure that is the same or similar to the structure of the parent compound, but has a chemical or physical modification, such as a different functional group or an additional functional group. The derivative has the same or similar biological activity as the parent compound.

[0015]

As used herein, the term “pharmaceutically acceptable salt” refers to an inorganic or organic acid addition salt of a compound of the present invention that is relatively non-toxic. These salts may be prepared temporarily during the final isolation and purification of the compound or by reacting the purified compound in its free base form alone with a suitable organic or inorganic salt and isolating the salt thus formed.

[0016]

Examples of pharmaceutically acceptable base salts of the compound of the present invention include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; aliphatic amine salts such as trimethylamine salts, triethylamine salts, dicyclohexylamine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, procaine salts, meglumine salts, diethanolamine salts and ethylenediamine salts; aralkylamine salts such as N,N-dibenzylethylenediamine and benetamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts and isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylammonium salts, benzyltributylammonium salts, methyltrioctylammonium salts and tetrabutylammonium salts; salts of basic amino acids such as arginine salts and lysine salts; etc.

[0017]

Examples of pharmaceutically acceptable acid salts of the compound of the present invention include salts of inorganic acids such as hydrochlorides, sulfates, nitrates, phosphates, carbonates, hydrogencarbonates and perchlorates; organic acid salts such as acetates, propionates, lactates, maleates, fumarates, tartrates, malates, citrates and ascorbates; sulfonates such as methanesulfonates, isethionates, benzenesulfonates and p-toluenesulfonates; acidic amino acids such as aspartates and glutamates; etc.

[0018]

As used herein, the term “solvate” refers to a solvate of a compound of the present invention or a pharmaceutically acceptable salt thereof, including, for example, an organic solvent (eg, alcohol (ethanol or the like)-ate) solvate, hydrate, and the like. When a hydrate is formed, this can be accommodated by any number of water molecules. Examples of hydrates include monohydrates, dihydrates and the like.

[0019]

As used herein, the term “homolog” refers to a protein or gene having an amino acid sequence or base sequence derived from the same ancestor. A homologue from speciation is called an ortholog. A homologue newly created as an organism by genetic duplication is called a paralogue.

[0020]

(Active Ingredient)

Nicotinamide mononucleotide (NMN) is a compound with the following structure

[Chemical formula 1]

,

which is a nucleotide derived from nicotinamide and ribose. It is known as the biochemical precursor of NAD+ (nicotinamide adenine dinucleotide).

[0021]

Nicotinamide riboside (NR) is a compound with the following structure

[Chemical formula 2]

which is a conjugate of ribose and nicotinamide. This can be thought of as NMN with a nucleotide substitution for ribose.

[0022]

The present invention may provide a composition containing NMN and/or NR, or a method including the use (eg, administration) of NMN and/or NR. NMN and/or NR can be used in the form of their derivatives and/or their pharmaceutically acceptable salts. The examples provided herein show that NMN and NR can improve the enzymatic activity of Hsd3b6 (or the enzymatic activity of its homologue, such as HSD3B1). The examples provided herein also show that NMN and NR can restore meibomian gland tissue. In one embodiment, the composition may contain NMN. In one embodiment, the composition may contain NR.

[0023]

NMN and/or NR can be used in combination with an additional active ingredient. Examples of the additional active ingredient include, but are not limited to, an anti-inflammatory agent, an antimicrobial drug, a nutritional supplement, an antioxidant, and the like.

[0024]

(Disease)

The present invention may provide a composition for use or a method for improving the activity of Hsd3b6 or its homolog with NMN and NR. Hsd3b6 is directly controlled by the circadian clock, and the product of this gene is an important element of the aldosterone pathway. In addition, a composition for use or a method for treating or preventing a disease, disorder or symptom associated with the activity of Hsd3b6 or a homologue thereof may be provided. Examples of diseases associated with the activity of Hsd3b6 or its homolog include, but are not limited to, meibomian gland dysfunction, diseases caused by decreased steroid hormone levels (eg, testosterone, estrogen, etc.), postmenopausal syndrome, osteoporosis, lifestyle diseases, age-related androgen deficiency in men, myocardial infarction, and the like.

[0025]

In particular, the present invention may provide a composition for use or a method for improving meibomian gland function with NMN and NR. The Meibomian gland is one of the sebaceous glands located at the edge of the eyelid. The meibomian glands prevent evaporation of the eye's tear film, prevent tears from falling onto the cheek, and secrete an oily substance (sebaceous gland secretion) that lines the inside of the closed eyelid. The Meibomian gland is also known as the tarsal gland. Meibomian gland dysfunction (MGD) is defined as chronic ocular discomfort in the presence of diffuse ocular dysfunction. The cause is thought to be denaturation or stagnation of meibomian lipids, chronic infection, or hyperkeratosis of the inflamed ductal epithelium. The examples of the present invention demonstrate that NMN and NR can restore Hsd3b6 activity in the meibomian gland and can increase the size of the meibomian gland. Examples of improving meibomian gland function include increasing the number of meibomian gland acinar cells, increasing meibomian gland lipid secretion, and treating or preventing disease associated with meibomian gland dysfunction.

[0026]

One embodiment of the present invention may provide a composition for use in or a method for treating or preventing a disease involving meibomian gland dysfunction with NMN and NR. For example, a composition for treating or preventing meibomian gland dysfunction comprising nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) may be provided. Meibomian gland dysfunction may be accompanied by decreased secretion of the meibomian glands. Meibomian gland dysfunction may be accompanied by an inflammatory disease. The inflammatory disease is, for example, at least one disease selected from the group consisting of meibomitis, superficial (punctate) keratitis and blepharitis. Meibomian gland dysfunction may be accompanied by excessive accumulation of lipids in the duct. Meibomian gland dysfunction may be accompanied by ocular discomfort, foreign body sensation, and/or pressure. For example, a disease involving meibomian gland dysfunction may be, but is not limited to, a disease selected from the group consisting of meibomitis, posterior blepharitis, keratitis, conjunctivitis, meibomitis-associated keratoconjunctivitis, visual impairment, androgen deficiency, atopic dermatitis, prostatic hyperplasia, ocular pemphigoid, discoid lupus erythematosus (erythematosis), ectodermal dysplasia syndrome (anhydrotic ectodermal dysplasia), bone marrow transplantation, hypertension, menopause, Parkinson's disease, plaque psoriasis, rosacea, Sjögren's syndrome, Stevens-Johnson syndrome, toxic epidermal necrolysis , Turner syndrome, graft-versus-host disease (GVHD) and side effects associated with drug therapy (retinoids, antiandrogens, antidepressants, antihistamines or postmenopausal estrogen therapy). It is understood that the composition of the present invention can improve the function of the meibomian gland itself, so that the composition can be effectively used in any of the diseases described above.

[0027]

Another embodiment of the present invention may provide a composition for use in or a method for normalizing the composition of lipids secreted by the meibomian gland and normalizing lipids in the meibomian gland. Since the composition of the present invention shows the effect of improving meibomian gland tissue atrophy and increasing the activity of steroid synthase, which affects the lipid secreted by the meibomian gland, it is understood that the composition helps normalize lipids in the meibomian gland. The method for measuring the lipid component in the meibomian gland is not particularly limited. Various methods can be used that are well known in the art, provided that the method allows the assessment of the lipid component in the meibomian gland.

[0028]

In one embodiment of the present invention, the composition of the present invention increases the enzymatic activity of Hsd3b6. Thus, the production of testosterone, which is known to promote lipid production by the meibomian gland, may be increased. Since sex hormones, such as testosterone, influence lipid production in the meibomian glands, it is understood that the composition of the present invention helps to normalize the lipid composition of meibomian gland tissue by stimulating the production of sex hormones, including testosterone, through increasing the enzymatic activity of Hsd3b6.

[0029]

(Dosage form)

The composition of the present invention can be prepared in a suitable dosage form. For example, the composition of the present invention, when used as an ophthalmic composition, can be presented as an ophthalmic injection, ophthalmic ointment, eye drops or ophthalmic perfusate. The composition may be prepared in any dosage form such as an aerosol, liquid, extract, elixir, capsule, granule, pill, ointment, powder, tablet, solution, suspension or emulsion. The composition may include any pharmaceutically acceptable additive and/or excipient that is known in the art. Examples of additives include, but are not limited to, a tonicity agent, a buffer, a preservative, a cosolvent, and a thickener. For example, the ophthalmic composition may be presented in the form of a liquid preparation obtained by dissolving the active ingredient in an aqueous solvent (eg, water).

[0030]

The composition of the present invention can be administered by any suitable route determined by one skilled in the art. The composition may be formulated to be suitable for administration by a route selected from, but not limited to, ocular injection, topical application (including application to the eye), eye drops, intravenous injection, intravenous drip, oral administration, parenteral administration, transdermal administration and the like.

[0031]

Examples of isotonic agents include saccharides such as glucose, trehalose, lactose, fructose, mannitol, xylitol and sorbitol, polyhydric alcohols such as glycerin, polyethylene glycol and propylene glycol, inorganic salts such as sodium chloride, potassium chloride and calcium chloride, and the like . Its amount is preferably 0-5% by weight. in relation to the total amount of the composition.

[0032]

Examples of chelating agents include edentates such as disodium edetate, sodium calcium edetate, trisodium edetate, tetrasodium edetate and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or a salt thereof, sodium hexametaphosphate, citric acid and the like. Its amount is preferably 0-0.2% by weight. in relation to the total amount of the composition.

[0033]

Examples of stabilizers include sodium hydrogen sulfite and the like. Its amount is preferably 0-1% by weight. in relation to the total amount of the composition.

[0034]

Examples of pH modifiers include acids such as hydrochloric acid, carbonic acid, acetic acid and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or bicarbonates such as sodium carbonate, alkali metal acetate, such such as sodium acetate, alkali metal citrate such as sodium citrate, bases such as trometamol and the like. Its amount is preferably 0-20% by weight. in relation to the total amount of the composition.

[0035]

Examples of preservatives include sorbic acid, potassium sorbate, parahydroxybenzoate esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate and butyl parahydroxybenzoate, quaternary ammonium salts such as chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride and cetylpyridinium chloride, alkyl polyaminoethylglycine, chlorobutane ol, polyquad, polyhexamethylene biguanide, chlorhexidine etc. Its amount is preferably 0-0.2% by weight. in relation to the total amount of the composition.

[0036]

Examples of antioxidants include sodium hydrogen sulfite, dried sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherol and the like. Its amount is preferably 0-0.4 wt%. in relation to the total amount of the composition.

[0037]

Examples of solubilizing agents include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropyl methylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol and the like. Its amount is preferably 0-3% by weight. in relation to the total amount of the composition.

[0038]

Examples of thickeners include polyethylene glycol, methylcellulose, ethylcellulose, sodium carmellose, xanthan gum, sodium chondroitin sulfate, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol and the like. Its amount is desirably 0-70 wt%. in relation to the total amount of the composition.

[0039]

Eye drops can be prepared, for example, by dissolving or suspending the desired component described above in an aqueous solvent such as sterile purified water, saline or buffer (for example, borate buffer, phosphate buffer and the like), or a non-aqueous solvent such as vegetable oil such as cottonseed oil, soybean oil, sesame oil or peanut oil, adjusting the osmotic pressure to a predetermined osmotic pressure and applying sterilization such as mechanical sterilization.

[0040]

The ophthalmic ointment may include an ointment base in addition to the various components described above upon receipt. Examples of ointment bases include, but are not limited to, oil bases such as petrolatum, liquid paraffin and polyethylene, emulsion bases obtained by emulsifying an oil phase and an aqueous phase with a surfactant or the like, water-soluble bases consisting of hydroxypropyl methylcellulose, carboxymethylcellulose, polyethylene glycol and the like.

[0041]

The composition, therapeutic agent or prophylactic agent of the present invention may be provided in the form of a kit. In a specific embodiment, the present invention provides a drug package or kit containing one or more containers filled with one or more components of the composition or drug of the present invention. Optionally, such containers may be marked with information indicating approval for manufacture, use, or sale to a person by a government agency that regulates the manufacture, use, or sale of drugs or biological products, in a form prescribed by the government agency.

[0042]

As used herein, the term “kit” refers to a package containing the parts provided (eg, a therapeutic drug, a prophylactic drug, each component thereof, a user's manual, and the like), which is typically divided into two or more segments. This kit form is preferred when providing a composition that should not be provided in a mixed state for stability or the like. and is preferably used by mixing immediately before use. Such a kit preferably contains instructions or a user's guide describing how the provided parts (eg, a therapeutic drug or a prophylactic drug) are used or how the reagents are to be handled. When the kit herein is used as a reagent kit, the kit typically contains instructions or the like describing the method of use of the therapeutic agent, prophylactic agent or the like.

[0043]

As used herein, the term “instructions” is a document explaining how to use the present invention to physicians or other users. The instructions contain a description of the detection method of the present invention, how to use a diagnostic drug, or instructions for administering a drug and the like. The instructions may describe administration to the eye as the site of administration (eg, by eye drops, eye ointment, injection, and the like). The instructions have been prepared in accordance with the format prescribed by the regulatory authority of the country in which this disclosure applies (for example, the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, or the like), with a clear description confirming the approval regulatory authority. The instruction is a so-called insert. Instructions are usually provided on paper, but are not limited to. The instructions may also be provided in forms such as electronic media (eg, Internet websites or sent electronically).

[0044]

In one embodiment, eye drops containing nicotinamide mononucleotide (NMN) may be provided, wherein nicotinamide mononucleotide (NMN) is present at a concentration of 0.001 μg/ml or higher, 0.01 μg/ml or higher, 0.1 μg/ml or higher , 1 mcg/ml or higher, 10 mcg/ml or higher, 100 mcg/ml or higher, 200 mcg/ml or higher, 500 mcg/ml or higher, 1 mg/ml or higher, 2 mg/ml or higher, 5 mg/ml or higher, 10 mg/ml or higher, or 50 mg/ml or higher.

[0045]

In one embodiment, eye drops containing nicotinamide riboside (NR) may be provided, wherein nicotinamide riboside (NR) is present at a concentration of 0.001 μg/ml or higher, 0.01 μg/ml or higher, 0.1 μg/ml or higher , 1 mcg/ml or higher, 10 mcg/ml or higher, 100 mcg/ml or higher, 200 mcg/ml or higher, 500 mcg/ml or higher, 1 mg/ml or higher, 2 mg/ml or higher, 5 mg/ml or higher, 10 mg/ml or higher, or 50 mg/ml or higher.

[0046]

In one embodiment, an ophthalmic ointment containing nicotinamide mononucleotide (NMN) may be provided, wherein nicotinamide mononucleotide (NMN) is contained at a concentration of 0.001 μg/ml or higher, 0.01 μg/ml or higher, 0.1 μg/ml or higher , 1 mcg/ml or higher, 2 mcg/ml or higher, 5 mcg/ml or higher, 10 mcg/ml or higher, 50 mcg/ml or higher, 100 mcg/ml or higher, 200 mcg/ml or higher, or 500 mcg/ml or higher.

[0047]

In one embodiment, an ophthalmic ointment containing nicotinamide riboside (NR) may be provided, wherein nicotinamide riboside (NR) is present at a concentration of 0.001 μg/ml or higher, 0.01 μg/ml or higher, 0.1 μg/ml or higher , 1 mcg/ml or higher, 2 mcg/ml or higher, 5 mcg/ml or higher, 10 mcg/ml or higher, 50 mcg/ml or higher, 100 mcg/ml or higher, 200 mcg/ml or higher, or 500 mcg/ml or higher.

[0048]

The use of a formulation at a concentration of a predetermined amount or higher according to the dosage form may be useful for delivery to the meibomian gland, which is located at the back of the eyelid, as opposed to delivery to the eyeball.

[0049]

(Dose)

In one embodiment, the methods of use of the present invention include, for example, eye drops, but the methods are not limited to this. Examples thereof include administration methods (administration methods and dosage forms) such as ophthalmic ointment, anterior chamber injection, sustained release agent impregnation, subconjunctival injection, and systemic administration (oral administration and intravenous injection).

[0050]

The concentration of NMN or NR used in the present invention is typically from about 0.001 to 1000 μM (μmol/L), preferably from about 0.01 to 300 μM, more preferably from about 0.03 to 100 μM, and even more preferably from about 0.1 to approximately 30 µM. For example, other concentration ranges are typically from 0.01 nM to 100 μM, about 0.1 nM to 100 μM, about 0.001-100 μM, about 0.01-75 μM, about 0.05-50 μM, about 1-10 µM, about 0.01-10 µM, about 0.05-10 µM, about 0.075-10 µM, about 0.1-10 µM, about 0.5-10 µM, about 0.75-10 µM , about 1.0-10 µM, about 1.25-10 µM, about 1.5-10 µM, about 1.75-10 µM, about 2.0-10 µM, about 2.5-10 µM, about 3.0-10 µM, about 4.0-10 µM, about 5.0-10 µM, about 6.0-10 µM, about 7.0-10 µM, about 8.0-10 µM, about 9, 0-10 µM, about 0.01-50 µM, about 0.05-5.0 µM, about 0.075-5.0 µM, about 0.1-5.0 µM, about 0.5-5.0 µM , about 0.75-5.0 µM, about 1.0-5.0 µM, about 1.25-5.0 µM, about 1.5-5.0 µM, about 1.75-5.0 µM , about 2.0-5.0 µM, about 2.5-5.0 µM, about 3.0-5.0 µM, about 4.0-5.0 µM, about 0.01-3.0 µM , about 0.05-3.0 µM, about 0.075-3.0 µM, about 0.1-3.0 µM, about 0.5-3.0 µM, about 0.75-3.0 µM, about 1.0-3.0 µM, about 1.25-3.0 µM, about 1.5-3.0 µM, about 1.75-3.0 µM, about 2.0-3.0 µM, about 0.01-1.0 µM, approximately 0.05-1.0 µM, approximately 0.075-1.0 µM, approximately 0.1-1.0 µM, approximately 0.5-1.0 µM, approximately 0. 75-1.0 µM, about 0.09-35 µM, or about 0.09-3.2 µM, more preferably about 0.01-10 µM, about 0.1-3 µM, or about 0.1- 1.0 µM, but the concentration range is not limited to this.

[0051]

When used as eye drops, the concentration of the formulation can be determined based on about 1-10,000 times, preferably about 100-10,000 times, for example, about 1000 times the effective concentration described above, taking into account dilution with tear fluid and the like, and also toxic. The concentration can also be set to a concentration higher than that specified. The concentration is, for example, from about 0.01 μM (μmol/L) to 1000 mM (mmol/L), from 0.03 μM to 1000 mM, from about 0.1 μM to 300 mM, from about 0.3 μM to 300 mM, about 1 μM to 100 mM, about 3 μM to 100 mM, about 10 μM to 100 mM, about 30 μM to 100 mM, about 0.1 μM to 30 mM, about 0.3 μM to 30 mM, about 1 μM to 30 mM, about 3 μM to 30 mM, about 1 μM to 10 mM, about 3 μM to 10 mM, about 10 μM to 1 mM, about 30 μM to 1 mM, about 10 µM to 10 mM, about 30 µM to 10 mM, about 100 µM to 10 mM, about 300 µM to 10 mM, about 10 µM to 100 mM, about 30 µM to 300 mM, about 100 µM to 300 mM, or about 300 μM to 300 mM, and may be from about 1 mM to 10 mM, or about 1 mM to 100 mM. The concentration range can be determined by an appropriate combination of these upper and lower limits.

[0052]

The magnitude of effect of a drug of the present invention that is effective in treating a particular disease, disorder or condition may vary depending on the properties of the disorder or condition, but can be determined using standard clinical techniques based on the descriptions provided herein by those skilled in the art. areas. In addition, determination of the optimal dose range can be further facilitated by in vitro assays. Since the exact dosage used in a compound may vary depending on the route of administration or the severity of the disease or disorder, the dosage should be determined in accordance with the judgment of the physician or the condition of each patient. The dosage is not particularly limited, but may be, for example, 0.001, 1, 5, 10, 15, 100 or 1000 mg/kg body weight per administration or up to two such values. The dosing interval is not particularly limited, but may be, for example, 1, 2, or 4 doses every 1, 7, 14, 21, or 28 days, or 1, 2, or 4 doses for every number of days within the range between any two of such values. The dose, the number of doses, the dosing interval, the dosing period, and the dosing method may be suitably selected depending on the patient's age or body weight, symptom, administration route, target organ, or the like. For example, the composition of the present invention can be used in the form of eye drops. In addition, the therapeutic drug preferably contains the active ingredient in a therapeutically effective amount or in an amount effective to produce the desired effect. The effective dose can be estimated from the dose-response curve obtained in an in vitro test system or in an animal model.

[0053]

Administration of the active ingredient in a predetermined higher amount may be preferable for delivery to the meibomian gland, which is located at the back of the eyelid, as opposed to delivery to the eyeball.

[0054]

(Therapeutic method and preventive method)

The present invention may provide a method of improving the activity of Hsd3b6 or a homologue thereof, or treating or preventing a disease, disorder or symptom associated with the activity of Hsd3b6 or a homologue thereof. The method may include administering nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) to a subject.

[0055]

The method may optionally include measuring the activity of Hsd3b6 or a homolog thereof in a subject. One embodiment may provide a method of administering NMN and/or NR when the activity of Hsd3b6 or its homolog in a subject is measured and the activity is reduced.

[0056]

The present invention may provide a method for improving meibomian gland function or treating or preventing a disease, disorder or symptom associated with meibomian gland dysfunction. The method may include administering nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) to a subject.

[0057]

The present invention may provide a method for enhancing testosterone production in the meibomian gland. The method may include administering nicotinamide mononucleotide (NMN) and/or nicotinamide riboside (NR) to a subject.

[0058]

The method may optionally include testing the function of the subject's meibomian glands. One embodiment may provide a method of administering NMN and/or NR when a subject's meibomian gland function is tested and function is reduced. Meibomian gland function can be tested, for example, by slit-lamp eye examination, meibography, confocal microscopy, DR-1, mucocutaneous junction, meibometer, lipid chemistry, evaporation amount, BUT, or subjective symptom questionnaire.

[0059]

(General technology)

The molecular biological method, biochemical method and microbiological method used herein are well known and commonly used in the art, which are described, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F. M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F. M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M. A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F. M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M.A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Gait, M. J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R.L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G. T. (I996). Bioconjugate Techniques, Academic Press, Bessatsu Jikken Igaku [Experimental Medicine, Supplemental Volume] , "Idenshi Donyu & Hatsugen Kaiseki Jikken Ho [Experimental Methods for Transgenesis & Expression Analysis]" , Yodosha, 1997, or the like. Relevant portions thereof (which may constitute the entire document) are incorporated herein by reference.

[0060]

Reference literature, such as scientific literature, patents and patent applications cited herein, are incorporated herein by reference to the same extent as if each document were described in detail.

[0061]

The present invention has been described with reference to preferred embodiments for ease of understanding. Although the present invention is described below on the basis of examples, the above descriptions and the following examples are provided solely for the purpose of illustration and not limitation of the present invention. Thus, the scope of the present invention is not limited to the embodiments and examples that are specifically described herein and is limited only by the scope of the claims.

[Examples]

[0062]

Examples of the present invention are described below. Biological samples or the like, where applicable, have been handled in accordance with standards adopted by the Ministry of Health, Labor and Welfare, Ministry of Education, Culture, Sports, Science and Technology, or the like, and, where applicable, based on the Helsinki Declaration or ethical principles prepared on its basis.

[0063]

(Example 1: Aging-Associated Meibomian Gland Tissue Atrophy/Meibomian Gland Tissue in Hsd3b6 KO Mice)

[Short description]

This example demonstrates aging-associated meibomian gland tissue atrophy and alteration of meibomian gland tissue in Hsd3b6 KO mice.

[0064]

[Materials and methods]

(1) Herxheimer staining protocol for whole meibomian glands

Mouse meibomian gland tissue was stained according to the following protocol.

[0065]

As a method for preparing 100 ml of staining solution, a vessel containing the stock solution obtained by dissolving Sudan IV to saturation in 100% ethanol was left standing in advance, and 70 ml of the same amount of supernatant, 20 ml of 10% NaOH, and 10 ml of ultrapure water were mixed , mixed by inversion for 5 minutes and centrifuged to sediment the powder. The supernatant was filtered through a 0.45 μm filter and used as a staining solution.

[0066]

The mice's cervical vertebrae were twisted and scalp samples, including the meibomian glands, were taken. The sample was immersed in 4% paraformaldehyde, keeping the sample flat using a thumbtack, and immobilized using a rotator at 4°C for 24 hours. The periphery of the meibomian glands was trimmed. Meibomian glands were immersed in 50% ethanol using a rotator at room temperature for 10 minutes, then immersed in 70% ethanol for 20 minutes and immersed in a staining solution using a rotator at room temperature for 24 hours in a Falcon tube. The meibomian glands were then immersed in 70% ethanol for 10 minutes and the washing was repeated several times to confirm that the meibomian glands were well stained. Meibomian glands were immersed for 24 hours in amino alcohol in glycerol, prepared by mixing 3.75 ml of 80% amino alcohol with 8.25 ml of glycerol. Meibomian glands were placed on a glass slide and encapsulated with glycerol jelly. Glycerin jelly was prepared by first mixing 10 g of gelatin with 60 ml of ultrapure water and 70 ml of glycerin, swelling the gelatin and adding glycerin after about an hour and mixing while heating.

[0067]

(2) Analysis of the area of the meibomian gland stained with Sudan IV

Post-staining area analysis was performed using Photoshop and ImageJ and determining a color intensity threshold when viewed and compared to a spot image based on the area of areas with intensity equal to or greater than a predetermined threshold value.

[0068]

(3) Mouse

A loxP-flanked mouse was obtained, in which the region of exon 2, including the start codon of the mouse Hsd3b6 gene, is flanked by loxP sites. The mouse was crossed with a CAG-Cre transgenic mouse to produce an Hsd3b6 KO mouse that could no longer express Hsd3b6.

[0069]

The test was carried out after rearing a two-month-old mouse for 10 days under normal conditions (normal humidity conditions) with free access to food/water on a 12-hour light/12-hour dark cycle at a humidity of 40 to 60% and a room temperature of 25°C.

[0070]

The 24-month-old mouse was a 78-week-old (18-month-old) male C57BL/6J mouse purchased from OrientalBioService, Inc., which was raised for 6 months under normal conditions (normal humidity conditions). The 6-month-old C57BL/6J mouse was a 6-week-old mouse purchased and raised to 6 months of age under normal conditions (normal humidity conditions).

[0071]

[Results]

Meibomian gland tissue was stained from a 6-month-old mouse and a 24-month-old mouse according to the protocol described above, and the stained areas were compared. The results are presented in Fig. 1. Observed atrophy of meibomian gland tissue and progression of meibomian gland dysfunction (MGD) due to aging (N=7).

[0072]

In fig. Figure 2 shows the size of meibomian gland tissue and the number of secretory ducts in a 2-month-old Hsd3b6 KO mouse and a wild-type mouse. Similar to aged mice, Hsd3b6 KO mice also showed meibomian gland atrophy. Since there is no difference in the number of secretory ducts depending on the genotype, it was established that meibomian gland atrophy is not an anomaly in the process of duct formation.

[0073]

(Example 2: Instillation experiment with NMN or NR eye drops in a 1.5 year old wild type mouse for 2 weeks)

[Short description]

This example demonstrates changes in the enzymatic activity of Hsd3b6 and the proliferative activity of basal meibomian gland acinar cells in the meibomian glands following instillation of NMN or NR eye drops for 2 weeks in 1.5-year-old wild-type mice.

[0074]

[Materials and methods]

(1) The following is a summary of the eye drop instillation experiment.

[Table 1]

Animal Compound Dose Route of administration administration period Number of animals Experiment Wild type mice (1.5 years) carrier - instillation of eye drops (right eye only) 2 weeks 5 Enzyme activity (steroid synthesis activity) 5 BrdU incorporation assay (cell proliferative activity) NMN 5% (5 µl/eye, 6 times/day) 6 Enzyme activity (steroid synthesis activity) 6 BrdU incorporation assay (cell proliferative activity) NR 5% (5 µl/eye, 6 times/day) 5 Enzyme activity (steroid synthesis activity) 6 BrdU incorporation assay (cell proliferative activity)

[0075]

(2) Meibomian gland Hsd3b6 enzymatic activity measurement

Meibomian gland Hsd3b6 enzymatic activity in mice was measured according to the following protocol.

[0076]

○Preliminary preparation

Hanks' balanced salt solution (HBSS) was bubbled with 95% CO ₂ /5% O ₂ for 1 hour or more and distributed into 2 ml tubes of 200 µl each. The lid was closed when filled with 95% CO ₂ /5% O ₂ . HBSS was heated in a water bath at 37°C. The reaction incubator was heated to 37°C, and a multivortex was installed in it.

[0077]

○Launch of measuring equipment

Turn on the main power for the flow-through liquid scintillation counter (Perkin Elmer, Radiomatic 625TR) and turn on the main power for the HPLC (Waters, 2795 Separations Module) to perform setup. Mobile phase A was replaced with water, and mobile phase B was replaced with acetonitrile. After installing the column (Kanto Chemical, LiChroCART 250-4, LiChrosphere 100RP-18, particle size: 5 μm, internal diameter 4 mm × 250 mm), 40% acetonitrile (ACN) (A: 60%, B: 40%) was passed through speed 0.7 ml/min. The temperature of the column thermostat at this time was set to 40°C.

[0078]

○Purification of ³ H-pregnenolone

³ H-DHEA was added to 1 ml of 40% ACN contained in a 2 ml tube and the mixture was discarded. The microtube was mounted on an HPLC holder and single injection was selected. The pollutant peak formed after 22 minutes was not collected. Once the contaminant peak decreased at approximately 23 minutes, peak ³ of H-DHEA then appeared, so collection into a glass centrifuge tube began after reaching 1/3 cpm of the peak value. Approximately 2.5 mL was collected in just 4 minutes of collection. The liquid inside the glass centrifuge tube was mixed by vortex. 25 μl was subjected to a liquid scintillation counter and 25 μl was added to 3 ml of clear sol. After confirming that a small amount of N ₂ was coming out of the outlet nozzle, the liquid inside the glass centrifuge tube was dried with nitrogen at 75°C using a Taitec incubator.

[0079]

○Obtaining substrate

After cooling the nitrogen-dried ³ H-DHEA to room temperature, 1 μL/4000 cpm (25 μL value measured using a liquid scintillation counter) propylene glycol was added and ³ H-DHEA was dissolved by shaking for 5 minutes.

[0080]

PBS 4 μl/4000 cpm was added. The mixture was mixed by vortex. 10 μl was measured on a liquid scintillation counter to confirm that the amount was greater than 400,000 cpm. The mixture was diluted with PBS:propylene glycol=4:1. 400,000 cpm/10 µl hot solution (1), 87 µM DHEA in 10% EtOH/90% D-PBS (2), 1 mM dutasteride in 50% DMSO/50% DW (3) and 1 mM fadrozole in 50% DMSO/50% DW (4) was prepared and mixed in the ratio (1):(2):(3):(4)=5:5:1:1 as a substrate solution. Before use, it was heated in a water bath at 37°C.

[0081]

○Enzymatic reaction and extraction

The meibomian gland from the upper eyelid of one eye was excised and placed on weighing paper with the side exposing the meibomian gland on top. The sample was ground to a thickness of 200 μm using a tissue grinder. The chopped meibomian gland was transferred to HBSS. The substrate solution was shaken gently and 24 μl was added to HBSS. The mixture was pipetted.

[0082]

The mixture was incubated for 30 minutes with shaking with a microtube mixer installed inside the incubator at 37°C. After 30 minutes, the mixture was centrifuged at room temperature for 1 minute at 700 g and the meibomian gland tissue was allowed to pellet. The entire amount of HBSS was transferred into a test tube to which 2 ml of ethyl acetate was added. The mixture was shaken for 10 seconds. When the reaction was stopped, the mixture was immediately transferred to ice.

[0083]

200 μl of HBSS, warmed to 37°C, was added to the 2 ml tube containing the remaining meibomian gland. After gently shaking the substrate solution, 24 μl was added to the HBSS and the mixture was pipetted. 2 μl of 100 mM NAD ⁺ in D-PBS was added. The mixture was incubated for 30 minutes with shaking with a microtube mixer installed inside the incubator at 37°C. The mixture was centrifuged at room temperature for 1 minute at 700 g and the meibomian gland tissue was allowed to sediment. The entire amount of HBSS was transferred into a test tube to which 2 ml of ethyl acetate was added. The mixture was shaken for 10 seconds. When the reaction was stopped, the mixture was immediately transferred to ice.

[0084]

○Steroid extraction

The mixture was centrifuged at room temperature for 10 minutes at 1500 rpm. 1.6 ml of ethyl acetate layer on the top layer was collected and dried with nitrogen at 75°C. When the liquid had completely evaporated, 500 μL of 40% ACN was added. The mixture was shaken for 3 minutes. The liquid was transferred to MILLIPORE Ultra free-MC mounted on a 2 ml tube and centrifuged at room temperature for 2 minutes at 12,000 g. The glass centrifuge tube was pre-washed with 500 μL of 40% ACN and shaken for 2 minutes. After passing through the filter of the same column, the mixture was centrifuged at room temperature for 4 minutes at 12,000 g. The eluate was mixed by tapping and 10 μl was added to 3 ml of clear sol. The mixture was measured in a liquid scintillation counter (measuring the degree of reduction).

[0085]

○Measurement

The column was equilibrated at initial conditions (until baseline and pressure stabilized). Sample sets were created in the same quantity as the number of samples to be measured. Since the analysis in the first column did not stabilize, 40% ACN was used as the sample. The cap of the 2 ml measurement tube was opened. The tube was covered with thinly stretched parafilm and prepared for HPLC. Continuous analysis began.

HPLC conditions ( ³ H-DHEA)

Flow rate: 0.7ml/min

Column temperature: 40°C

Gradient

0-30 min: 40-50% ACN (0.3%/min)

30-35 min: 70% ACN

35-40 min: 100% ACN

40-50 min: 100-40% ACN (6%/min)

50-60 min: 40% ACN

[0086]

○Analysis

The chromatograph was opened with ProFSA. Smoothing was performed by setting the SDA (smoothing function) level to 8. Peaks were displayed using Find Peaks and Locate Peaks. Report Preview copied and pasted into Word. Data were obtained to determine enzymatic activity using the following formula.

[Number 1]

[0087]

(3) Analysis of cell proliferation by BrdU incorporation staining

BrdU (100 mg/kg body weight, Sigma-Aldrich) was administered intraperitoneally 30 min before mouse eyelid tissue collection. The eyelid containing the meibomian glands was immobilized with 4% PFA and embedded in paraffin. Antigens were activated by applying pressure to a 5-μm-thick segment for 2.5 minutes in 10 mM sodium citrate buffer (pH 6.0). After incubating the segment for 24 hours at 4°C using anti-BrdU antibody (1:1000 dilution; Rockland Inc.), the immune response was visible with 3,3'-diaminobenzidine (DAB) using the Envision+ System secondary antibody -HRP Labeled Polymer Anti-Rabbit (Dako). For each tissue segment, BrdU-positive cells in the meibomian gland basal cell layer were counted in a digital microscope image at 20˟ magnification. The outer circumference length of the meibomian gland acinus was measured using ImageJ software, and the total number of BrdU-positive cells was normalized to the outer circumference length. BrdU-positive cells were counted using 16 segments per individual.

[0088]

[Results]

In fig. Figure 3 shows the change in the enzymatic activity of Hsd3b6 in the meibomian gland and the change in the number of BrdU-positive cells in the basal cells of the meibomian gland due to instillation of NMN, NR eye drops or their solvent (vehicle, phosphate buffered saline). The enzymatic activity of Hsd3b6 and the number of BrdU-positive cells were significantly increased by instillation of NMN eye drops. A similar increase in Hsd3b6 enzymatic activity was observed, and the number of BrdU-positive cells also increased significantly in the NR eye drop instillation group. In particular, it can be understood that the enzymatic activity of Hsd3b6 is increased locally in the meibomian gland, and the proliferative activity of meibomian gland basal cells is increased due to the instillation of NMN or NR eye drops.

[0089]

(Example 3: 3 month experiment of instillation of NMN or NR eye drops into wild type mice aged 1.75 years)

[Short description]

This example demonstrates the effect of instillation of NMN or NR eye drops for 3 months in a 1.75-year-old wild-type mouse on meibomian gland tissue volume.

[0090]

[Materials and methods]

(1) Below is a diagram of the eye drop instillation experiment.

[Table 2]

Animal Compound Dose Route of administration administration period Number of animals experiment Mouse WT (1.75 year old) carrier - instillation of eye drops (right eye only) 3 months 8 Observation of tissue stained with lipids (meibomian gland volume assessment) NMN 5% (5 µl/eye, 4 times/day) 8 NR 5% (5 µl/
eye, 4 times/
day) 10

[0091]

(2) Mouse meibomian gland tissues were stained by Herxheimer staining of all meibomian glands in the same manner as in Example 1.

[0092]

[Results]

In fig. Figure 4 shows the change in the volume of the meibomian glands during instillation of NMN, NR eye drops and their solvent (carrier, phosphate buffered saline). A significant increase in meibomian gland volume was observed in both the NMN eye drop instillation group and the NR eye drop instillation group.

[0093]

(Example 4: 3 month experiment of instillation of NMN or NR eye drops into wild type mice aged 1.75 years)

[Short description]

This example demonstrates the effect of 3 months of instillation of NMN or NR eye drops into a 1.75 year old wild-type mouse on lipid components in the meibomian glands.

[0094]

[Materials and methods]

(1) The experiment with instillation of eye drops is carried out in the same way as in example 3.

(2) Lipid collection from mouse meibomian glands and lipid analysis

Meibomian glands were removed from the eyelid of a euthanized mouse. Each of two meibomian glands taken from one eye of each animal was placed in a glass vial containing 1 ml of a chloroform:methanol=2:1 (v/v) lipid extraction solvent mixture, and the lipids were extracted. The extract is transferred to a new bottle. The extract can be stored for several months at -20°C or below by evaporating the solution under nitrogen. The extract is then dissolved in a suitable solvent and the lipid composition is analyzed using APCI.

[0095]

[Results]

Lipid components in the meibomian gland were normalized in the NMN or NR eye drop instillation groups compared to the vehicle (phosphate buffered saline) eye drop instillation control group.

[0096]

(Example 5: Quantitative determination of testosterone in the meibomian gland)

[Materials and methods]

After homogenizing the meibomian gland excised from the eyelid with methanol/water (75:25, v/v), the steroid contained in the homogenate was extracted with dichloromethane using an Isolute SLE+ cartridge (Biotage) and evaporated under nitrogen to obtain a residue. The residue was dissolved in 50 μl of methanol/5% acetic acid solution containing Amplifex Keto reagent (SCIEX) at a concentration of 10 mg/ml and incubated for 1 hour at room temperature. Testosterone concentration was then determined by liquid chromatography with electrospray ionization tandem mass spectrometry (QTRAP 4500 LC-MS/MS system; SCIEX) (derivative transition SRM: 403→164) and normalized to the wet weight of the extracted meibomian gland.

[0097]

[Results]

In fig. Figure 5 shows the amount of testosterone in the meibomian gland tissue of wild-type and Hsd3b6-deficient mice. Testosterone contained locally in the meibomian glands was reduced by approximately 50% in Hsd3b6-deficient mice compared to wild-type mice. In addition, testosterone found locally in the meibomian glands was reduced to 10% or less in the Hsd3b6 knockout mouse with a resected gonad (testis). From this it can be understood that not only the circulating testosterone coming from the testes, but also the same level of testosterone production as in the meibomian gland contributes to the meibomian glands.

[0098]

Since the enzymatic activity of Hsd3b6 is increased by instillation of NMN or NR eye drops, it is understood that the production of testosterone, which is known to promote lipid production by the meibomian glands, is increased and the lipid composition of the meibomian gland tissue is normalized.

[0099]

(Note)

As described above, the present invention is illustrated using its preferred embodiments. However, it should be understood that the scope of the present invention should be interpreted solely on the basis of the claims. It is also intended that any patent, any patent application and any references cited herein shall be incorporated herein by reference in the same manner as if the contents were directly described herein. The present application claims priority under Japanese Patent Application No. 2019-117642, filed June 25, 2019 with the Japan Patent Office. The entire contents of which are incorporated herein by reference in the same manner as if the contents were expressly described herein.

[Industrial applicability]

[0100]

The present invention can be used in the fields of medicine, pharmaceutical products, healthcare, biology, biochemistry and the like.

Claims (22)

1. Use of nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR) to improve meibomian gland function. 2. Use according to claim 1, wherein improving meibomian gland function includes increasing the number of meibomian gland acinar cells. 3. Use according to claim 1, wherein improving the function of the meibomian gland includes increasing the secretion of lipids by the meibomian gland. 4. Application according to any one of paragraphs. 1-3, where improving meibomian gland function includes treating or preventing a disease accompanied by meibomian gland dysfunction. 5. Use of nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR) for the treatment or prevention of meibomian gland dysfunction. 6. Application according to claim 5, where dysfunction of the meibomian gland is accompanied by a decrease in the secretion of the meibomian glands. 7. Application according to claim 5, where dysfunction of the meibomian gland is accompanied by an inflammatory disease. 8. Use according to claim 7, wherein the inflammatory disease includes at least one disease selected from the group consisting of meibomitis, superficial (punctate) keratitis and blepharitis. 9. Application according to claim 5, where dysfunction of the meibomian gland is accompanied by excessive accumulation of lipids in the duct. 10. Use according to claim 5, where dysfunction of the meibomian gland is accompanied by discomfort in the eyes, a sensation of a foreign body and/or a feeling of pressure. 11. Use according to claim 4, where the disease accompanied by meibomian gland dysfunction is meibomitis, posterior blepharitis, keratitis, conjunctivitis, keratoconjunctivitis associated with meibomitis, ocular pemphigoid, Sjogren's syndrome, Stevens-Johnson syndrome, graft-versus-host disease (GVHD) and visual impairment. 12. Use of nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR) to obtain a drug to improve meibomian gland function. 13. Use according to claim 12, wherein improving meibomian gland function includes increasing the number of meibomian gland acinar cells. 14. Use according to claim 12, wherein improving the function of the meibomian gland includes increasing the secretion of lipids by the meibomian gland. 15. Use according to claim 12, where improving the function of the meibomian gland includes the treatment or prevention of a disease accompanied by dysfunction of the meibomian gland. 16. Use of nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR) for the preparation of a drug for the treatment or prevention of meibomian gland dysfunction. 17. Use according to claim 16, where dysfunction of the meibomian gland is accompanied by a decrease in the secretion of the meibomian glands. 18. Use according to claim 16, where dysfunction of the meibomian gland is accompanied by an inflammatory disease. 19. Use according to claim 18, wherein the inflammatory disease includes at least one disease selected from the group consisting of meibomitis, superficial (punctate) keratitis and blepharitis. 20. Use according to claim 16, where dysfunction of the meibomian gland is accompanied by excessive accumulation of lipids in the duct. 21. Use according to claim 16, where dysfunction of the meibomian gland is accompanied by discomfort in the eyes, a sensation of a foreign body and/or a feeling of pressure. 22. Use according to claim 15, where the disease accompanied by meibomian gland dysfunction is meibomitis, posterior blepharitis, keratitis, conjunctivitis, keratoconjunctivitis associated with meibomitis, ocular pemphigoid, Sjogren's syndrome, Stevens-Johnson syndrome, graft-versus-host disease (GVHD) and visual impairment.

Images