CN114767685A

CN114767685A - Application of tetrahydrofolic acid in bacteriostasis or prevention or treatment of eye diseases

Info

Publication number: CN114767685A
Application number: CN202210442474.6A
Authority: CN
Inventors: 刘子源; 李映昱; 黄琛; 王薇
Original assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Current assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Priority date: 2022-04-25
Filing date: 2022-04-25
Publication date: 2022-07-22
Anticipated expiration: 2042-04-25
Also published as: CN114767685B

Abstract

The invention provides an application of tetrahydrofolic acid in bacteriostasis or prevention or treatment of eye diseases. The invention finds that the tetrahydrofolic acid has good inhibition effect on fusarium, and particularly in a culture medium similar to an ocular environment, so that the tetrahydrofolic acid can effectively inhibit the growth of fusarium under the condition of local ocular administration, and further prevent and treat ocular diseases, particularly corneal fungal infection.

Description

Application of tetrahydrofolic acid in bacteriostasis or prevention or treatment of eye diseases

Technical Field

The invention relates to the field of medicines, in particular to application of tetrahydrofolic acid in bacteriostasis or prevention or treatment of eye diseases.

Background

Keratomycosis (keratomyciosis) is one of the infectious diseases in the cornea due to fungi (molds), also known as fungal keratitis (Mycotic keratitis); fungal infection of the cornea can be severely blinding and is one of the most harmful ocular infections. Fungal infection of the cornea generally occurs with the causative factor, the most prominent of which is corneal trauma. In addition, ocular surface diseases, particularly abnormalities of the tear fluid, contact lens wear, and long-term use of antibiotics and glucocorticoids are also common causative factors. Among the fungal keratitis, fusarium is the most common pathogenic bacterium.

The treatment effect of the fungal infectious keratitis is not ideal at present. Different fungi have different morphological structures, growth, reproduction, variation and resistance, pathogenic and tissue growth and infiltration modes, sensibility to antifungal medicines and the like. Because of the similarity of fungi and mammals at biological and biochemical levels and the weak penetration of the medicine to the local part of the cornea, the local medicine application efficiency of eyes is low, the systemic medicine application toxicity is high, the action sites of the medicine are limited, the medicine types are few, and more medicine-resistant bacteria are frequent. This presents great difficulties in controlling infection in the early stages of fungal keratitis and in preventing recurrence of fungal infection after surgery. At present, common medicines for clinically treating fungal keratitis are polyene or imidazole medicines, but the medicines are easy to generate adverse reactions, increase the drug resistance of fungi and cause secondary damage to patients. Therefore, there is a need to find better drugs that can effectively prevent and treat corneal fungal infections.

Folic acid, also known as pteroylglutamic acid, belongs to one of the essential B vitamins of people. In recent years, the effects of folic acid have been attracting attention with the development of molecular biology and pharmacology. The reduction product of folic acid after hydrogenation, namely 5,6,7, 8-tetrahydrofolic acid (THFA), is the active form of folic acid, and the molecular structure is as follows:

the tetrahydrofolic acid is coenzyme for transferring one-carbon group enzyme system, and is carrier of methyl, methylene, formyl and methine, so that various tetrahydrofolic acid derivatives can be formed. N-5 and N-10 are reaction sites for transferring one-carbon groups. The transfer of one-carbon units is important in the participation of amino groups in acid metabolism, purine and pyrimidine synthesis.

The invention discovers that the tetrahydrofolic acid has a strong inhibiting effect on fungi, particularly fusarium, so that the tetrahydrofolic acid can be applied to the treatment of eye diseases, particularly fungal keratitis.

Disclosure of Invention

The invention aims to provide a method for inhibiting fusarium and preventing or treating fungal keratitis and other eye diseases aiming at the problems in the prior art.

In previous studies by the inventors, nitrogen metabolism of fusarium was found to play an important role in its growth and pathogenicity. Without a nitrogen source, the fungus hardly grows; but without a carbon source, the fungus can still grow. While the cornea is an amino acid-rich tissue, proline is an important constituent thereof. Previous researches find that after fusarium infects the cornea, the proline component in the cornea is in a descending trend, which indicates that proline is probably an important nitrogen nutrient source after the fusarium infects the cornea. In further studies, fusarium isolated from the ocular surface of keratitis patients was found to have significant expression of the P5C dehydrogenase (a type of proline metabolizing enzyme) gene, and more expression of these enzymes in recurrent cases. This suggests that proline metabolism plays an important role in the pathogenic process of fusarium. As shown in fig. 1, TPM expressed by the standard strain was 0, whereas clinical isolate expression was significantly activated, and the expression of the recurrent infection strain was higher, suggesting that P5C dehydrogenase was involved in the process of fusarium infection of cornea, and is likely related to its pathogenicity. Thus, it can be concluded that the growth and pathogenicity of fusarium in the cornea is related to proline. The inventor finds that the growth of fusarium is obviously inhibited by culturing fusarium in a culture medium containing proline as a nitrogen source and adding different concentrations of tetrahydrofolic acid, and the inhibition degree is related to the concentration of the tetrahydrofolic acid.

Therefore, the invention provides the application of tetrahydrofolic acid or the salt thereof in bacteriostasis or prevention or treatment of eye diseases.

One aspect of the invention provides an application of tetrahydrofolic acid or salt thereof in preparing antibacterial drugs.

In a preferred embodiment of the invention, the bacteriostatic drug is used to inhibit fungi.

In a preferred embodiment of the invention, the bacteriostatic agent is administered to the eye to inhibit fungi.

Another aspect of the present invention is to provide a use of tetrahydrofolic acid or a salt thereof for preparing a medicament for preventing or treating an ocular disease.

In a preferred embodiment of the invention, the ocular disease comprises an ocular fungal infection, for example fungal infections of the following eyes: eyelids, conjunctiva, cornea, sclera, uvea (including iris, ciliary body, choroid), retina, vitreous, orbit, etc.

In a preferred embodiment of the present invention, the ocular diseases include blepharitis, conjunctivitis, keratitis, scleritis, uveitis (including iridocyclitis, choroiditis), retinitis, vitritis, orbital inflammatory diseases, and the like, particularly the above-mentioned inflammations caused by fungi, such as fungal blepharitis, fungal conjunctivitis, fungal keratitis, fungal scleritis, fungal uveitis (including fungal iridocyclitis, fungal choroiditis), fungal retinitis, fungal vitritis, fungal orbital inflammatory diseases, and the like.

Preferably, the fungi of the invention comprise fusarium, aspergillus, candida and actinomyces pedis. More preferably, the fungus according to the invention is selected from fusarium.

Another aspect of the present invention is to provide a pharmaceutical composition for inhibiting bacteria or preventing or treating an ocular disease, the composition comprising tetrahydrofolic acid or a salt thereof.

The tetrahydrofolic acid or the salt thereof can be used alone or in a pharmaceutical composition. Therefore, the bacteriostatic agent, the agent for preventing or treating eye diseases, and the pharmaceutical composition according to the present invention may further include one or more pharmaceutically acceptable carriers, diluents, or excipients in addition to the tetrahydrofolic acid or the salt thereof. The carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The pharmaceutical composition of the invention can also comprise an isotonic regulator, a pH regulator, a preservative, a thickening agent, an eye ointment matrix and the like, and can be adjusted by a conventional method by selecting related varieties in pharmacopoeia, textbooks and documents.

The drug/pharmaceutical composition provided by the present invention may be in the form of a cream, emulsion, solution, etc. Specifically, the medicine/medicine composition provided by the invention can be liquid or semisolid eye preparations such as eye drops, eye washes, eye gels, eye creams, intraocular injections and the like.

In the uses and compositions of the invention, tetrahydrofolic acid or a salt thereof can also be used in combination with other therapeutic agents for ocular diseases. When combination therapy is employed, the therapeutic agents may be administered together or separately. More than one therapeutic agent in a combination therapy may be administered in the same or different modes of administration. When the therapeutic agents are administered separately, they may be administered simultaneously or in any order.

Therefore, it is another aspect of the present invention to provide a combination drug comprising tetrahydrofolic acid or a salt thereof and other drugs useful for the treatment of ocular diseases.

Preferably, the other drugs are used for treating blepharitis, conjunctivitis, keratitis, scleritis, uveitis (including iridocyclitis and choroiditis), retinitis, vitritis, orbital inflammatory diseases, and the like, particularly the above-mentioned inflammations caused by fungi.

In the present invention, the salt of tetrahydrofolic acid refers to pharmaceutically acceptable salts thereof, including acid addition salts of the free compounds thereof with conventional acids, including inorganic and organic acids, such as: hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, carbonic acid, phosphoric acid, maleic acid, fumaric acid, malonic acid, succinic acid, tartaric acid, formic acid, acetic acid, caproic acid, caprylic acid, capric acid, stearic acid, amino acids, alginic acid, ascorbic acid, benzenesulfonic acid, p-toluenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, (+) -camphoric acid, camphorsulfonic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, glycolic acid, hippuric acid, (+) -L-lactic acid, (±) -DL-lactic acid, lactobionic acid, (-) -L-malic acid, (±) -DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1, 5-disulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, p-toluenesulfonic acid, undecylenic acid, and the like. In the present invention, the salt of tetrahydrofolic acid may further include an alkali metal salt such as a sodium salt or a potassium salt; alkaline earth metal salts such as calcium or magnesium salts; and salts with suitable organic ligands, such as quaternary ammonium salts.

In the present invention, the term "prevent or treat" and other similar synonyms include alleviating, alleviating or ameliorating a symptom of a disease or disorder, preventing other symptoms, ameliorating or preventing an underlying metabolic cause of a symptom, inhibiting a disease or disorder, e.g., arresting the development of a disease or disorder, alleviating a disease or disorder, ameliorating a disease or disorder, alleviating a symptom of a disease or disorder, or halting a symptom of a disease or disorder, and, further, the term includes prophylactic purposes. The term also includes obtaining a therapeutic effect and/or a prophylactic effect. The therapeutic effect refers to curing or ameliorating the underlying disease being treated. In addition, a cure or amelioration of one or more physiological symptoms associated with the underlying disease is also a therapeutic effect, e.g., an improvement in the patient's condition is observed, although the patient may still be affected by the underlying disease. For prophylactic effect, the composition can be administered to a patient at risk of developing a particular disease, or to a patient presenting with one or more physiological symptoms of the disease, even if a diagnosis of the disease has not yet been made.

In the present invention, the term "pharmaceutically acceptable" refers to a substance (e.g., carrier or adjuvant) that does not affect the biological activity or properties of the compounds of the present application, and is relatively non-toxic, i.e., the substance can be administered to an individual without causing an adverse biological response or interacting in an adverse manner with any of the components contained in the composition.

In the invention, each preparation unit in the medicine/medicine composition contains 1-3000mg of tetrahydrofolic acid or the salt thereof; the preparation unit is as follows: each, a bottle, etc. The precise dosage and dosage regimen for the drug/pharmaceutical composition of the present invention will depend upon the biological activity itself, the age, weight and sex of the patient, the needs, affliction or extent of need of the individual to whom the drug is being administered, and the judgment of the practitioner.

Advantageous effects

The invention provides an application of tetrahydrofolic acid or salt thereof in bacteriostasis or prevention or treatment of eye diseases. The invention discovers that tetrahydrofolic acid has a good inhibition effect on fungi, particularly fusarium which is the most common pathogenic bacterium of fungal keratitis, and particularly, the growth of fusarium can be effectively inhibited by adding a small amount of tetrahydrofolic acid into a culture medium similar to an eye environment. Therefore, the tetrahydrofolic acid can effectively inhibit the growth of fusarium under the condition of local administration of the eye, thereby preventing and treating eye diseases, in particular corneal fungal infection, namely fungal keratitis.

Drawings

FIG. 1: expression of the dehydrogenase gene of different fusarium strains P5C;

FIG. 2: comparing the bacteriostatic action of THFA with different concentrations under the Pro-culture medium condition;

FIGS. 3A-3D: the distribution of amino acids from different sources of cornea;

FIG. 4: comparing the bacteriostatic action of THFA under different amino acid nitrogen source culture media;

FIG. 5 is a schematic view of: influence of THFA on Fusarium growth under different amino acid nitrogen source culture medium conditions.

Detailed Description

The following description will be clearly and completely described in conjunction with the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the scope of protection of the present invention.

Example 1: bacteriostatic effect of proline as sole nitrogen source under culture medium

Preparing a culture medium (called pro-culture medium for short) with proline as a unique nitrogen source, wherein the formula is as follows:

table 1:

¹filtering and sterilizing

Wherein the formulation of the microelement solution is

Table 2:

fusarium (commercially available source: China general microbiological culture Collection center; number: CGMCC3.5840) is cultured in solid pro-culture medium, 30mL of solid culture medium is placed in each culture dish, and 5 muL of solid culture medium with concentration of 2 x 10 is added⁶The fusarium spore suspension is inoculated in a culture medium, 0mM, 1.25mM, 2.5mM, 5mM, 10mM and 20mM THFA (commercial pure product, the purity is more than or equal to 95 percent) is respectively added, the mixture is cultured for 72 hours at 29 ℃, colony observation is carried out at different time points, and the colony morphology, the diameter and the like are recorded. The THFA with different concentrations is added to intervene in the metabolism process of the fusarium proline, and the lowest concentration with a good inhibition effect is found out. As a result, as shown in FIG. 2, the growth of Fusarium was significantly inhibited by the addition of THFA, and the degree of inhibition correlated with the concentration of THFA. As can be seen from fig. 2, the colony diameters after 5mM THFA treatment were significantly less than 2.5mM, 1.25mM and 0mM groups, and both 10mM and 20mM groups were significantly less than 5mM group.

Example 2: simulating antibacterial effect under cornea amino acid mixed environment

1. Analyzing the corneal proline proportion of various species (human, rat, pig and rabbit), finding out the nitrogen source species possibly utilized after the cornea is infected by fusarium, and screening the species close to the human corneal proline proportion.

The results are shown in FIGS. 3A-3D: proline accounts for about 1.08% in human cornea, is not the most abundant amino acid in human cornea, is the highest taurine, followed by phenylalanine, lysine, then glutamine and tyrosine (fig. 3A). The proportion of proline in the rat cornea was 2.59%, and the most abundant first sites were taurine and glutamine, respectively (fig. 3B). The proportion of proline in the porcine cornea is 2.97%, and more of the first amino acids are taurine and glutamine, respectively (fig. 3C). The proportion of proline in rabbit cornea is 4.04%, and more amino acids at the first several positions are taurine and glutamine, respectively (fig. 3D).

The results show that the proportions of amino acids in the corneas of common experimental animals such as rats, pigs, rabbits and the like are not completely coincident with those of human corneas, and for proline, the corneas of rats and pigs are closest to those of human corneas, but the proportion of glutamine is higher than that of human corneas, so that the selection of dominant nitrogen sources of fungi is greatly influenced.

2. According to the obtained amino acid types, a culture medium with different combinations of amino acids as nitrogen sources is prepared, the environment of corneal amino acid mixing is preliminarily simulated, fusarium is cultured, and the growth condition of the fusarium is observed. 4 kinds of amino acids, taurine (for short, cow), glutamine (for short, Gln), glycine (for short, sweet) and proline (for short, pro), which account for more in the cornea, were selected, and a fusarium culture medium using a mixture of amino acids as a nitrogen source was prepared according to the formula shown in table 3, in which the trace element solution was the same as in example 1:

table 3:

¹filtering and sterilizing

After 10mM THFA was added to the solid medium, the culture was performed in the same manner as in example 1, and the results are shown in FIG. 4. The addition of 10mM THFA to different mixed amino acids inhibited the growth of Fusarium and significantly reduced its pigment (FIGS. 4B-D). Compared with a culture dish with mixed taurine and proline and a culture dish with single proline, the culture dish has no obvious growth change; after 1.5 days, the culture dishes added with glutamine and glycine are enhanced compared with colony pigment of single proline, which shows that different amino acids have different effects on the growth and pathogenicity of fusarium. The THFA can obviously inhibit the growth of fusarium under the conditions of different mixed amino acid nitrogen sources, and shows that the THFA has an excellent inhibition effect on fusarium.

In addition, a liquid medium, ingredients such as a solid medium, was added to a 96-well plate at 150. mu.L per well at a concentration of 2X 10 per well⁶A50. mu.L/mL suspension of Fusarium spores was incubated at 29 ℃ for 72 hours, absorbance values (OD values) were read at different time points and growth curves were generated, the results are shown in FIG. 5. The growth curve results in fig. 5 suggest that fusarium grew best in the four amino acid mixed medium, that proline and glutamine mixed second, and that it was very close to the four amino acid mixed group, that glycine and proline mixed dishes were better than proline alone, and that taurine and proline mixed group instead grew at a lower level than the proline alone group. Fusarium growth was significantly reduced in each group by the addition of 10mM THFA (FIGS. 5B-F).

The results show that THFA has good inhibition effect on the growth of fusarium in a culture medium in which proline and proline are mixed with other amino acids, so that the growth of fusarium can be effectively inhibited under the condition of topical application to eyes, and infection caused by fungi such as fusarium can be prevented and treated.

It is to be understood that only the embodiments are illustrated and claimed, wherein one or more of the features recited in the claims can be combined with any one or more of the features recited, and such combined features are also within the scope of the present application, as if such combined features were specifically recited in the present disclosure.

Claims

1. An application of tetrahydrofolic acid or its salt in preparing antibacterial medicine is disclosed.

2. Use according to claim 1, wherein the bacteriostatic drug is for inhibiting fungi.

3. Use of tetrahydrofolic acid or a salt thereof in the preparation of a medicament for the prevention or treatment of an ocular disease.

4. Use according to claim 3, wherein the ocular disease comprises fungal infections of the eye, such as fungal infections of the following eyes: eyelid, conjunctiva, cornea, sclera, uvea (including iris, ciliary body, choroid), retina, vitreous, orbit.

5. Use according to claim 3, characterized in that the ocular diseases comprise blepharitis, conjunctivitis, keratitis, scleritis, uveitis (including iridocyclitis, choroiditis), retinitis, vitritis, orbital inflammatory diseases, in particular those caused by fungi, such as fungal blepharitis, fungal conjunctivitis, fungal keratitis, fungal scleritis, fungal uveitis (including fungal iridocyclitis, fungal choroiditis), fungal retinitis, fungal vitritis, fungal orbital inflammatory diseases.

6. Use according to any one of claims 1 to 5, wherein the fungus comprises Fusarium, Aspergillus, Candida, Rhizopus.

7. A pharmaceutical composition for bacteriostatic or preventing or treating ocular diseases, which comprises tetrahydrofolic acid or a salt thereof.

8. A combination for use in bacteriostatic or preventing or treating an ocular condition comprising tetrahydrofolic acid or a salt thereof and a further drug useful in the treatment of an ocular condition.

9. The combination according to claim 8, wherein the other drugs are used for the treatment of blepharitis, conjunctivitis, keratitis, scleritis, uveitis (including iridocyclitis, choroiditis), retinitis, vitritis, orbital inflammatory diseases, in particular those caused by fungi.

10. The use according to any one of claims 1 to 6, the pharmaceutical composition according to claim 7, the combination according to any one of claims 8 to 9, wherein the salt of tetrahydrofolic acid refers to a pharmaceutically acceptable salt thereof, including acid addition salts of the free compound thereof with conventional acids, including inorganic and organic acids such as: hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, carbonic acid, phosphoric acid, maleic acid, fumaric acid, malonic acid, succinic acid, tartaric acid, formic acid, acetic acid, hexanoic acid, octanoic acid, decanoic acid, stearic acid, amino acids, alginic acid, ascorbic acid, benzenesulfonic acid, p-toluenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, (+) -camphoric acid, camphorsulfonic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, glycolic acid, hippuric acid, (+) -L-lactic acid, (±) -DL-lactic acid, lactobionic acid, (-) -L-malic acid, maleic acid, (±) -DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1, 5-disulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, p-toluenesulfonic acid, and undecylenic acid; the salts of tetrahydrofolic acid also include alkali metal salts such as, sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; and salts with suitable organic ligands, such as quaternary ammonium salts.