KR100926387B1 - A composition for treatment of conjunctivitis comprising chlorogenic acid and derivatives thereof - Google Patents

Abstract

The present invention relates to a novel use of chlorogenic acid, and more particularly to a pharmaceutical composition for treating conjunctivitis comprising chlorogenic acid and derivatives thereof and a method for treating conjunctivitis using the same.

Chlorogenic acid, conjunctivitis, infectious conjunctivitis, allergic conjunctivitis, anti-inflammatory

Description

A composition for treatment of conjunctivitis comprising chlorogenic acid and derivatives etc.

The present invention relates to a novel use of chlorogenic acid, and more particularly to a pharmaceutical composition for treating conjunctivitis comprising chlorogenic acid and derivatives thereof and a method for treating conjunctivitis using the same.

Conjunctivitis is an inflammatory condition of the conjunctiva caused by bacteria, viruses, allergies, or environmental factors. It is the most common eye disease. It usually heals well, but in some cases it is fatal and can cause blindness due to tissue damage. . Conjunctivitis is largely classified into infectious conjunctivitis and non-infectious conjunctivitis depending on the cause.

Infectious conjunctivitis includes viral conjunctivitis, bacterial conjunctivitis, fungal conjunctivitis, chlamydial conjunctivitis, rickettsial conjunctivitis and parasitic conjunctivitis (parasitic conjunctivitis). Representative pathogens causing conjunctivitis include Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Neisseria meningitidis , Herpes simplex, and picornavirus. It can range from mild hyperemia to large amounts of purulent secretions or ocular bleeding. In general, infectious conjunctivitis is transmitted from one eye to another by contact and then transmitted to another person by an object contacted by a patient, and can be commonly used by eye-related cosmetics such as mascara.

Representative allergic conjunctivitis as non-infectious conjunctivitis is a disease in which an allergic reaction occurs in the conjunctiva and is frequently accompanied by allergic fever or allergic rhinitis. It is a typical seasonal disease that is severe in spring and improves in autumn or winter. It is common in people who are allergic predominantly, and begins before puberty and recurs for 5 to 10 years, after which the incidence is reduced and the symptoms are generally lighter. However, these days, high temperatures continue to affect all ages, regardless of season or constitution, and the main causes are animal hair, dust, pollen, and house dust mites. The substance is also known to have a great effect. Common symptoms are very itchy eyes, severe redness and bleeding with tears, and aggravation if the eyes are rubbed. There may also be scratching or burning pain, foreign body sensations, blisters, and subconjunctival bleeding. Allergic conjunctivitis includes immediate hypersensitivity reactions such as Hay fever conjunctivitis, Vernal keratoconjunctivitis and Atopic keratoconjunctivitis, and Phlycactululitis and contact blepharitis. There is a delayed-type hypersensitivity reaction such as conjunctivitis caused by dermatitis induced conjuctivitis. In the treatment of allergic conjunctivitis, the method of changing the anaphylaxis (immunotherapy) is most ideal, but in reality, it is often difficult. Currently, antihistamines, steroids such as glucocorticoids, or allergy prevention and blocking agents are used.

Infectious conjunctivitis is treated with antibiotic eye drops, and sometimes in combination with steroid drugs. Allergic conjunctivitis is treated with antihistamine eye drops or steroid eye drops. Steroidal anti-inflammatory drugs of the glucocorticoid family, a typical treatment for conjunctivitis, are commonly used as anti-inflammatory drugs and have an excellent effect on rheumatoid arthritis, and inhibit various inflammatory reactions including radioactive, mechanical, chemical, infectious and immunostimulating or Prevent.

As the steroidal anti-inflammatory drugs are widely used, there are two categories of side effects, firstly, sudden discontinuation after long-term use, and second, symptom manifested by long-term overuse. Acute adrenal insufficiency during prolonged administration of corticosteroids causes symptoms such as systemic weakness, fever, myalgia, arthralgia, and loss of appetite, increases the risk of infections with bacteria, viruses, increases body weight, and changes body shape. Insomnia is well accompanied. In particular, eye drops or ointments of glucocorticoids are prone to side effects such as glaucoma, cataracts, or bacterial infections due to intraocular pressure increase when used over a long period of time, at the discretion of the patient. Due to these side effects, there are many problems in using glucocorticoids, and therefore, there is a need for anti-inflammatory agents without such problems.

Chlorogenic acid, on the other hand, is an ester of caffeic acid and quinic acid, the main phenolic component of coffee and is isolated from the leaves and fruits of the dicotyledonous plant. These chlorogenic acids are contained in higher plants, especially fresh coffee beans and roasted coffee beans, and have been reported to be easily extracted by HPLC-UV extraction with a methanol / water mixture (Bicchi et al, J. Agric). Food Chem., 43, pp 1549-1555, 1995), as well as methods for separating natural products and their systematic classification (Clifford et al, Phytochemistry, Vol. 28, No. 3, pp829-838, 1989). These chlorogenic acids are important factors for plant metabolism, antioxidants, and are known to slow the release of glucose into the bloodstream after meals.

In addition, Korean Unexamined Patent Publication No. 10-2001-87593 discloses the use of natural or synthesized chlorogenic acid as a medicament for the prevention and treatment of male reproductive function caused by endocrine disruptors, and Japanese Patent Application Laid-Open No. 2002- No. 363075 discloses the use of chlorogenic acid, which is contained in immature fruits of coffee, perennial leaves and apples, for the prevention and treatment of hypertension, and Korean Patent Publication No. 10-2006-128907 includes such chlorogenic acid and hydroxyhydroquinone. Disclosed is a coffee beverage having an excellent blood pressure lowering effect by lowering the content. However, none of these prior documents have known that chlorogenic acid is effective in the treatment of infectious or allergic conjunctivitis by inhibiting the inflammatory response.

Although the treatment and prevention of inflammation caused by conjunctivitis and other inflammation-related disorders has progressed greatly in the past few years, there remains a need for improved methods and compositions for preventing and / or treating infectious or allergic conjunctivitis.

The inventors have recently been successful in administering steroid-transfer glutaminase inhibitors in eye-induced allergy models induced by pollen in guinea pigs to achieve comparable effects to steroids (Sohn, J., Kim, T.- I., Yoon, Y.-H., and Kim, S.-Y.Transglutaminase inhibitor: A New Anti-inflammatory Approach in Allergic Coconjunctivitis.J. Clin.Invest.111, 121-8, 2003; Soo-Youl Kim Transglutaminase 2 in inflammation.Front Biosci. 11, 3026-3035, 2006). In addition, the present inventors have screened substances useful as transglutaminase inhibitors among natural substances that have already been commercialized because of their safety, and have discovered the activity as transglutaminase inhibitors from chlorogenic acid, and have applied them to Korean Patent Application No. 10. -2007-0056811.

Based on this, the present inventors have found that chlorogenic acid or a derivative thereof is very effective for the treatment of conjunctivitis, and thus the present invention has been reached.

It is an object of the present invention to provide a pharmaceutical composition for treating conjunctivitis, comprising chlorogenic acid.

Another object of the present invention is to provide a method for treating conjunctivitis using chlorogenic acid.

The first aspect of the present invention relates to a pharmaceutical composition for treating conjunctivitis comprising chlorogenic acid or a derivative thereof.

As mentioned above, chlorogenic acid is mostly present in fruits and leaves of dicotyledonous plants, and its chemical name is 3-[[3- (3,4-dihydroxyphenyl) -1-oxo-2-propenyl] oxy]. -1,4,5-trihydroxycyclohexanecarboxylic acid (3-[[3- (3,4-dihydroxyphenyl) -1-oxo-2-propenyl] oxy] -1,4,5-trihydroxycyclohexanecarboxylic acid), It is often referred to as 3-caffeoylquinic acid. In the case of chlorogenic acid obtained naturally, small amounts of isochlorogenic acid and neochlorogenic acid, which are isomers, may be contained. The molecular formula of the chlorogenic acid is C ₁₆ H ₁₈ 0 ₉ and the molecular weight is 354.30 and the structural formula is as follows.

The chlorogenic acid used in the present invention may be natural or synthesized, and natural chlorogenic acid is known from fruits (including immature fruit) of rosaceae plants such as apples, pears, or peaches, coffee beans, cacao beans, grape seeds, artichoke, and the like. Separation and purification from natural phenol and polyphenol extracts extracted by the method according to known methods (H. Li et al., J. Chromatogr. A 1098 (2005) 66-74 and V. Ossipov et al., J. Chromatogr.A 721 (1996) 59-68, etc.). The synthesized chlorogenic acid is also obtained by synthesis by known methods (M. Lepelley et al., Plant Science 172 (2007) 978-996 and J. Stockigt et al., FEBS LETTERS 42 (1974) 131-134, etc.). can do. Such natural or synthetic chlorogenic acid can be used in addition to the commercially available product.

Derivatives of chlorogenic acid are known as methyl chlorogenate and ethyl chlorogenate as ester forms that are separated from natural products. US Patent No. 6,632,459 discloses that the hydroxyl group portion of chlorogenic acid is substituted with caffeic acid. A derivative of chlorogenic acid is disclosed, and the anticancer sensitizer according to the present invention includes a derivative having the effect of such chlorogenic acid and its equivalent anticancer sensitizer.

Conjunctivitis is an inflammatory condition of the conjunctiva caused by bacteria, viruses, allergies or environmental factors. It is the most commonly developed ocular disease, and is largely caused by infectious conjunctivitis and non-infectious conjunctivitis. Common symptoms include severe pruritus of the eye, severe bleeding and bleeding of the eyelids. The pharmaceutical composition of the present invention is effective in the treatment of conjunctivitis because it showed the same or better effect than the existing anti-inflammatory agent, especially the steroidal dexamethasone in the animal model induced conjunctivitis as shown in the following examples. Existing steroids do not cause serious side effects such as Cushing syndrome, osteoporosis, infection risk, glaucoma and cataracts.

Pharmaceutical compositions comprising chlorogenic acid or derivatives thereof of the present invention may be formulated further comprising a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not significantly irritate an organism and does not inhibit the biological activity and properties of the administered compound. In the case of oral dosage forms, for example, they may be formulated as troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. It is not limited to this. To prepare into formulations such as tablets and capsules, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin and excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch and magnesium stearate Lubricants, such as calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of capsule formulation, in addition to the above-mentioned substances, it may include a liquid carrier such as fatty oil.

In order to formulate into a parenteral formulation, chlorogenic acid can be formulated for injection such as subcutaneous injection, intravenous injection or intramuscular injection, suppository infusion or aerosol for inhalation through the respiratory system. To formulate injectable formulations, chlorogenic acid is mixed in water with stabilizers or buffers to prepare solutions or suspensions, which are formulated for unit administration of ampoules or vials. To inject into suppositories, it may be formulated into a rectal composition such as suppositories or medicated enema containing conventional suppository bases such as cocoa butter or other glycerides. When formulated for sprays such as aerosols, propellants or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.

Another aspect of the invention relates to a method of treating conjunctivitis comprising administering a composition comprising chlorogenic acid or a derivative thereof.

As used herein, the term "administration" refers to introducing a pharmaceutical composition of the present invention to a patient in any suitable manner, and the route of administration of the composition of the present invention may be various oral or parenteral routes as long as the target tissue can be reached. It can be administered through. Preferably it is administered topically to the eye.

The treatment method of the present invention includes administering chlorogenic acid in a pharmaceutically effective amount. It will be apparent to those skilled in the art that a suitable total daily dose may be determined by the practitioner within the correct medical judgment. The specific therapeutically effective amount for a particular patient may be based on the specific composition, including the type and severity of the reaction to be achieved, whether or not other agents are used in some cases, the age, weight, general health, sex and diet of the patient, time of administration, Different applications are desired depending on the route of administration and the rate of release of the composition, the duration of treatment, the drug used in conjunction with or concurrently with the specific composition and similar factors well known in the medical arts.

In one preferred embodiment of the present invention, the method of treatment of the present invention may comprise administering one or more known anti-inflammatory agents together with a pharmaceutical composition comprising chlorogenic acid or derivatives thereof.

Known anti-inflammatory agents include steroidal preparations such as prednisolone and nonsteroidal preparations, and the pharmaceutically effective amounts thereof are known in the art, and the treatment may be carried out in consideration of various conditions such as the degree of symptoms and the combination with chlorogenic acid. You can adjust the amount. Thus, by combining and administering a well-known anti-inflammatory agent, not only the side effect of a well-known anti-inflammatory agent can be reduced by chlorogenic acid or its derivatives, but also a synergistic therapeutic effect can be anticipated. These known anti-inflammatory agents may optionally be administered in combination or simultaneously, or at intervals of time with the chlorogenic acid of the invention.

As can be seen from the above, the chlorogenic acid of the present invention exhibits an excellent therapeutic effect on conjunctivitis, and can be used as an anti-inflammatory agent that does not cause the side effects of the conventional steroid preparations.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are provided to help the understanding of the present invention, and the scope of the present invention is not limited thereto.

Example  1. Preparation of experimental animals Sensitization

Female Hartley guinea pigs weighing between 250 and 300 grams were fed freely with food and water and maintained under standard temperature, relative humidity and light conditions. The first sensitization started 1-2 weeks after purchase.

Short ragweed pollen (RW) was purchased from Polyscience, Inc. (Warrington, Pa.). The sensitization was carried out in two consecutive periods of five days per week.

Pollen is 10 microliters (Merayo-Lloves, J., Calonge, M, and Foster, CS 1995. Experimental model of allergic) by micropipette into 25 nostrils and inferior conjuntival fornices once a day. conjunctivitis to ragweed in guinea pig.Curr.Eye Res. 14: 487-494). On day 15, the immunized guinea pigs were divided into five groups of five each. All immunized guinea pigs were injected with 10 μl of short ragweed-pollen powder into the lower conjunctival varnish. 100 nM chlorogenic acid eye drops and dexamethasone eye drops (Maridex, 0.1% dexamethasone; Alcon-Couvreur, Puurs, Belgium), anti-allergic eye drops Patanol (0.1% ololo) 30 min and 4 times daily after pollen application Patadine Alcon Laboratories, Inc., Fort Worth, TX) and Azeran (Azelan, 0.05% azelastine, Taejun Pharmaceuticals, Korea) were applied. One group did not receive any treatment as a negative control.

Example  2. edema of the conjunctiva and Flare  Clinical grading measurement

Clinical grading of swelling and redness of the conjunctiva was performed 24 hours after infusion of pollen using a portable slit lamp (Cail Zeiss, Oberkochen, Germany). Evaluation was performed in blinded fashion 2 hours after allergen injection. Each clinical parameter is Merayo-Lloves, J., Calonge, M, and Foster, C.S. 1995. Experimental model of allergic conjunctivitis to ragweed in guinea pig. Curr. Eye Res. Records range from 0 to 4+ (0 absent, 1+ minimal, 2+ mild, 3+ moderate, 4+ severe) as described in 14: 487-494. Each clinical score was analyzed and shown in FIG. 1.

As can be seen in Figure 1, all treated groups showed less severe clinical findings than the negative control. In particular, there was almost no difference between the dexamethasone treated group and the chlorogenic acid treated group. This shows that the chlorogenic acid of the present invention exhibits an anti-inflammatory effect similar to dexamethasone.

Example  3. Measurement of eosinophil infiltration by histological observation

The lower eyelids containing the conjunctiva of the lower pony were obtained after CO ₂ asphyxiation and killing the animals, which were then fixed in 10% buffered formalin. Each tissue was prepared for light-microscopy observation. Each sample center was incised sagitally and stained with hematoxylin and eosin. The number of eosinophils in the standardized field of conjunctival epithelium, lower epithelium and strom was measured in three consecutive fields for each treatment in each experiment (200 magnification; negative control, dexamethasone, patanol, aze) Eggs (known conjunctivitis treatment) and 5 eyes in each group of chlorogenic acid). The infiltration of eosinophils by this is shown in FIG. 2.

As can be seen in FIG. 2, all treated groups showed statistically significantly lower eosinophil infiltration compared to the negative control. However, the chlorogenic acid eye drops group had a similar effect on inhibition of eosinophil chemotaxis compared to the dexamethasone eye drop group. In addition, the chlorogenic acid group did not show a significant statistical difference compared to the positive control group. As can be seen in Figure 2, the chlorogenic acid of the present invention can be seen to show a stronger effect than the existing anti-inflammatory agents, in particular, the steroid dexamethasone.

Example 4 Analysis by Immunohistochemical Staining

4-1. Anti-inflammatory Effects of Chlorogenic Acid and TGase 2 and Relationship Analysis of NF-κB

Transglutaminase (TGase) 2 is highly expressed in diseases associated with inflammation, and inhibiting the action of TGase also decreases the activity of NF-kB, which is a transcriptional agent in the inflammatory response pathway. Therefore, the present inventors performed immunohistochemical staining to determine whether the anti-inflammatory effect of chlorogenic acid is related to the inactivation of TGase 2 and NF-κB.

The tissue section slides of the conjunctiva were incubated for 30 minutes at room temperature in 1% bovine albumin fraction V, and then incubated with labeled primary antibody for 1 hour at room temperature. The slides were washed three times for 5 minutes each with phosphate buffered saline (PBS), and then treated with a secondary antibody and stored at room temperature for 30 minutes. Slides were washed with PBS and read under confocal laser microscopy (Leica, Deerfieldd, IL).

As a result, in the negative control group treated without any treatment, transglutaminase (TGase) 2 and NF-κB-expressing cells were strongly stained, and it was confirmed that TGase 2 and NF-κB were strongly expressed. In the group treated with chlorogenic eye drops, it was confirmed that the number of TGase 2 and NF-κB-expressing cells was significantly reduced compared to the dexamethasone (steroid) treated group. The expression of TGase 2 and NF-κB did not decrease in the group administered with azeran or patanol, which are commercially available anti-allergic eye drops.

Therefore, it can be seen that chlorogenic acid reduces the expression of TGase 2 and NF-κB, so that it can be effectively used as an anti-inflammatory agent.

4-2. Relationship between anti-inflammatory effects of chlorogenic acid and expression of TGase 2 and COX-2

COX (cyclooxygenase), an enzyme involved in synthesizing prostaglandins, is known to play an important role in causing inflammatory reactions due to rapid increase in cells during inflammatory reactions. Therefore, the present inventors performed immunohistochemical staining to determine whether the anti-inflammatory effect of chlorogenic acid is related to the expression of TGase 2 and COX-2. The anti-COX-2 conjugated with an anti-TGase 2 cross-link antibody and Alexafluor594 conjugated with FITC as a secondary antibody, as described in Example 3-1. An antibody (anti-COX-2 antibody) was used.

As a result, it was confirmed that TGase 2 mediated crosslinking and COX-2 were expressed at the same location (co-localization). In the negative control group, TGase 2 mediated crosslinking and COX-2 expression were increased, whereas in the azeran or patanol treated group, the expression was slightly decreased, whereas in the dexamethasone (steroid) or chlorogenic acid eyedrop group It was confirmed that their expression is significantly reduced.

Therefore, it can be seen that chlorogenic acid reduces the expression of TGase 2 and COX-2, and thus can be effectively used as an anti-inflammatory agent.

1 is a graph showing the clinical grading scores of swelling and redness of the conjunctiva after application of chlorogenic acid and known anti-inflammatory agents after injection of short ragweed pollen. (Ct, negative control; Dx, dexamethasone; Az, azelan; Pa, patanol; Ch, chlorogenic acid)

Figure 2 is a graph showing the degree of eosinophil infiltration by histological observation of chlorogenic acid and guinea pig tissue treated with each anti-inflammatory agent. (Ct, negative control; Dx, dexamethasone; Az, azelan; Pa, patanol; Ch, chlorogenic acid)

Figure 3 is a diagram showing the immunohistochemical staining results of the relationship between the anti-inflammatory effect of chlorogenic acid and TGase 2 and NF-κB (Scale bar = 500 Mm)

Figure 4 shows the results of analyzing the relationship between the anti-inflammatory effect of chlorogenic acid, TGase 2 mediated cross-linking and COX-2 expression through immunohistochemical staining. (Scale bar = 500 Mm)

Claims (7)

Pharmaceutical composition for the treatment of non-infectious conjunctivitis and infectious conjunctivitis selected from the group consisting of hyperthermic conjunctivitis, atopic keratoconjunctivitis, seasonal allergic conjunctivitis, giant papillary conjunctivitis, pricken's keratoconjunctivitis and contact ophthalmitis comprising chlorogenic acid or derivatives thereof . The composition of claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. delete delete delete delete delete

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