WO2009045054A2

WO2009045054A2 - A composition for treatment of conjunctivitis comprising chlorogenic acid and derivatives thereof

Info

Publication number: WO2009045054A2
Application number: PCT/KR2008/005799
Authority: WO
Inventors: Soo-Youl Kim; Joon-Hong Sohn
Original assignee: Seoul Biomedical Institute
Priority date: 2007-10-01
Filing date: 2008-10-01
Publication date: 2009-04-09
Also published as: KR100926387B1; KR20090033698A; WO2009045054A3

Abstract

The present invention relates to a novel use of chlorogenic acid. More particularly, the present invention relates to a pharmaceutical composition for the treatment of conjunctivitis, comprising chlorogenic acid and derivatives thereof, and to a method for treating conjunctivitis using the same. Chlorogenic acid contained in the pharmaceutical composition of the present invention exhibits excellent therapeutic effects on conjunctivitis, and thus has potential as an anti-inflammatory drug not causing side effects, which is the problem with conventional steroidal anti-inflammatory drugs.

Description

[DESCRIPTION] [Invention Title]

A COMPOSITION FOR TREATMENT OF CONJUNCTIVITIS COMPRISING CHLOROGENIC ACID AND DERIVATIVES THEREOF

[Technical Field]

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

[Background Art]

Conjunctivitis, a common ocular disorder, is an inflammation of the conjunctiva, causedby bacterial or viral infection, allergy, or environmental factors . Conjunctivitis tends to resolve itself, but occasionally leads to loss of vision due to tissue damage. Conjunctivitis is divided 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, and is caused by pathogens such as Streptococcus pneumoniae, Haemophilus influenzae, staphylococcus aureus, Neisseria meningitidis, Herpes simplex, and picornavirus . Infectious conjunctivitis causes various symptoms including redness, copious purulent discharge, and ocular bleeding. The infection can easily spread from one eye to the other, and also from one person to another person through contaminated objects, or sharing of eye cosmetics, especially mascara.

Non-infectious conjunctivitis is represented by allergic conjunctivitis, which is an allergic reaction of the conjunctiva to allergens and associated with hay fever or allergic rhinitis. Allergic conjunctivitis is a representative seasonal disease that appears most often during the spring and less often during the fall or winter. Allergic conjunctivitis usually affects those having allergic conditions . It begins to occur before adolescence and recurs over a period of five to ten years. Thereafter, the incidence appears to decrease, and symptoms subside. However, these days, allergic conjunctivitis is seen in people of all ages andboth genders, independent of the season or allergic conditions, due to abnormally high temperatures. These allergic responses are often related to animal hair, dust, pollen, house dust mite dermatophagoides, and other allergens that are present in the environment. These days, pollutants, such as car fumes or chemical dust , can also cause allergic reactions . Common symptoms of allergic conjunctivitis include itchy eyes, red eyes, eyelid swelling, and watery eyes, and these symptoms worsen with rubbing. Also, stinging or burning of the eyes as well as a gritty feeling, mucous discharge and subconjunctival haemorrhaging may occur. Allergic conjunctivitis is due either to an immediate hypersensitivity reaction causing hay fever conjunctivitis, vernal keratoconjunctivitis, atopic keratoconjunctivitis, etc., or a delayed hypersensitivity reaction causing phlyctenulosis (phlyctenular keratoconjunctivitis) , contact dermatitis induced conjunctivitis, etc. The ideal method for the treatment of allergic conjunctivitis is to change conditions causing hypersensitivity (immunotherapy), but this is difficult to achieve in practice. At present, steroidal drugs, such as anti-histamines and glucocorticoids, or allergy-preventing or -blocking drugs are used.

For the treatment of infectious conjunctivitis, antibiotic eye drops are used, if necessary, in combination with steroids. Allergic conjunctivitis is treated with anti-histamine or steroid eye drops. Representative of therapeutic drugs for conjunctivitis, steroidal glucocorticoid compounds are usually used as an anti-inflammatory drug that is very effective in rheumatoid arthritis, and inhibits or prevents inflammatory reactions to several kinds of stimuli including radiant, mechanical, chemical, infectious and immunological stimuli.

The wide use of the steroidal anti-inflammatory drugs entails two kinds of side effects. One is caused by sudden cessation of long-term administration, and the other is caused by long-term excessive use. The sudden stop of long-term administration of adrenocortical hormone causes acute adrenal insufficiency, which leads to general weakness, fever, myalgia, arthralgia, anorexia, etc., increases the risk of bacterial or viral infection, and causes weight gain and body shape change, accompanied by insomnia . In particular, the long-term administration of glucocorticoid eye drops or ophthalmic ointments by an individual' s own decision often causes such side effects as glaucoma, cataracts andbacterial infection. Due to such unwanted side effects, the use of glucocorticoids is problematic. Thus, there is a need for anti-inflammatory drugs having no such problem.

On the other hand, chlorogenic acid is an ester of caffeic acid and quinic acid, a major phenolic compound in coffee, and isolated from the leaves and fruits of dicotyledonous plants. Especially, chlorogenic acid is present in the green or roasted coffee bean, and can be readily extracted using a mixed solution of methanol and water by HPLC-UV [Bicchi et al., J. Agric. Food Chem. , 43, ppl549-1555 (1995) ] . Inaddition, its isolationmethod and genealogical classification are disclosed by Clifford et al . in Phytochemistry, Vol.28, No.3, pp829-838 (1989) . This compound is an important factor inplant metabolism, known as anantioxidant , and also known to slow the release of glucose into the bloodstream after a meal. In addition, a natural or synthesized chlorogenic acid is used for preventing or treating the decline of male reproductive function against hormone-disrupting chemicals (Korean Patent Publication No. 10-2001-87593), and chlorogenic acid extracted from coffee, nandina leaf, or unripe apple fruit is also used for preventing and treating hypertension (Japanese Patent Publication No. 2002-363075) . Further, disclosed is a coffee drink composition having an excellent effect of reducing blood pressure, in which chlorogenic acid is contained, and the content of hydroxyhydroquinone is reduced (Korean Patent Publication No. 10-2006-128907) . However, there is no mention of its inhibitory activity against infectious or allergic conjunctivitis in the prior arts.

[Disclosure] [Technical Problem]

Although many advances have been made in the treatment and prevention of conjunctivitis and other inflammations caused by inflammation-associated disorders over the past several years, there remains a need for improved methods and compositions for preventing and/or treating infectious or allergic conjunctivitis .

The present inventors have recently found that several synthetic peptides, which are transglutaminase inhibitors, have anti-inflammatory effects comparable to those of steroidal drugs when administered to a guinea pig model of pollen-induced allergic conjunctivitis (Sohn, J., Kim, T. -I, Yoon, Y. -H., and Kim, S. -Y. Transglutaminase inhibitor: A New Anti-inflammatory Approach in Allergic Conjunctivitis. J. Clin. Invest. I l l, 121-8, 2003; Soo-Youl Kim. Transglutaminase 2 in inflammation. Front Biosci. 11, 3026-3035, 2006) . In addition, the present inventors conducted screening of natural substances already deemed safe and made commercially available in order to identify substances useful as transglutaminase inhibitors. The screening resulted in the finding that chlorogenic acid has an inhibitory effect on transglutaminase, as described in Korean Patent Application No. 10-2007-0056811.

Based on the previous findings, the present inventors found that chlorogenic acid or a derivative thereof is very effective in the treatment of conjunctivitis, thereby leading to the present invention.

[Technical Solution]

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

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

[Description of Drawings]

FIG. 1 is a graph showing clinical scores for the degree of conjunctival edema and redness after guinea pigs were injected with short ragweed pollen and then treated with chlorogenic acid and known anti-inflammatory drugs (Ct, Control group; Dx, Dexamethasone; Az, Azelan; Pa, Patanol; Ch, Chlorogenic acid) ;

FIG.2 is a graph showing the results of histological analysis of eye tissues from guinea pigs, which were sensitized and treated with chlorogenic acid and known anti-inflammatory drugs, in order to evaluate the degree of eosinophil infiltration (Ct, Control group; Dx, dexamethasone; Az, Azelan; Pa, Patanol; Ch, chlorogenic acid) ;

FIG. 3 is an image showing the results of immunohistochemical staining, in which inhibitory effect of chlorogenic acid on TGase 2 and NF-κB was analyzed (Scale bar=500 Mm) ; and

FIG. 4 is an image showing the results of immunohistochemical staining, in which inhibitory effect of chlorogenic acid on TGase 2 mediated cross-linking and COX-2 expression was analyzed (Scale bar=500 Mm) .

[Best Mode]

In accordance with an aspect, the present invention relates to a pharmaceutical composition for the treatment of conjunctivitis, comprising chlorogenic acid or a derivative thereof.

As mentioned above, chlorogenic acid is generally present in the leaves or fruits of dicotyledonous plants, and has the chemical name of

3- [ [3- (3, 4-dihydroxyphenyl) -l-oxo-2-propenyl] oxy] -1,4,5-trih ydroxycyclohexanecarboxylic acid. It can be also named as 3-caffeoylquinic acid. A small amount of isomers, such as isochlorogenic acid and neochlorogenic acid, is contained in naturally present chlorogenic acid, and caffeic acid is produced at the time of hydrolysis. Chlorogenic acid has a molecular formula of Ci_δHisOg, a molecular weight of 354.30, and the following structural formula.

In the present invention, a natural or synthesized chlorogenic acid may be used, and the natural chlorogenic acid may be obtained by purifying or isolating the natural phenol and polyphenol extracted from Rosaceae fruit such as apples, pears and peaches, coffee beans, cacao beans, the seeds of grapes, and artichokes according to the known method (H. Li et al . , J. Chromatogr. A1098 (2005) 66-74 andV. Ossipovet al. , J. Chromatogr. A721(1996) 59-68 etc.) . In addition, the synthesized chlorogenic acid may be obtained by a synthetic method according to a known method (M. Lepelley et al., Plant Science 172(2007) 978-996 and J. Stockigt et al. , FEBS LETTERS 42 ( 1974 ) 131-134) . The natural or synthesized chlorogenic acid may be prepared directly or found commercially.

The chlorogenic acid derivative is an ester form isolated from a natural source, known as methyl chlorogenate and ethyl chlorogenate, and US patent No. 6, 632, 459 discloses a chlorogenic acid derivative, in which a hydroxyl group of chlorogenic acid is substitutedwithcaffeicacid. The cancer sensitizer according to the present invention includes chlorogenic acid and a derivative thereof having the effect of a cancer sensitizer, which is equivalent to chlorogenic acid.

Conjunctivitis, a common ocular disorder, is an inflammation of the conjunctiva, causedby bacterial or viral infection, allergy, or environmental factors. Conjunctivitis is divided into infectious conjunctivitis and non-infectious conjunctivitis, depending on the cause. Common symptoms include itchy eyes, red eyes and eyelid swelling. The pharmaceutical composition of the present invention, as described in the following example, displayed effects identical to or higher than those of a conventional anti-inflammatory agent, particularly the steroidal drug dexamethasone in a conjunctivitis-induced animal model. Thus, the present composition is effective in the treatment of conjunctivitis while avoiding severe side effects of conventional steroid drugs, such as Cushing' s syndrome, osteoporosis, the risk of infection, glaucoma and cataracts.

The pharmaceutical composition comprising chlorogenic acid or a derivative thereof of the present invention may be formulated together with a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compound. Examples of oral dosage forms include troches, lozenges, aqueous or emulsive suspensions, powder, granules, emulsions, hard or soft capsules, syrups, and elixirs, but are not limited thereto. For formulation such as tablets and capsules, useful are binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose and gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch and sweet potato starch, and lubricants such as magnesium stearate, calcium stearate, sodium stearylfumarate, and polyethylene glycol wax. For capsules, a liquid carrier such as a lipid may be further used, in addition to the above-mentioned compounds.

For non-oral administration, the pharmaceutical composition of the present invention may be formulated into injections for subcutaneous, intravenous, or intramuscular routes, suppositories, or sprays inhalable via the respiratory tract, such as aerosols. Injection preparations may be obtained by dissolving or suspending chlorogenic acid, together with a stabilizer or a buffering agent, in water and packaging the solution or suspension in ampules or vial units. Suppositories are typically made of a suppository base, such as cocoa butter and another glyceride, or a therapeutic laxative. For sprays such as aerosol, a propellant for spraying a water-dispersed concentrate or wetting powder may be used in combination with an additive.

In accordance with another aspect, the present invention relates to a method for treating conjunctivitis, including the step of administering the composition comprising chlorogenic acid or a derivative thereof.

As used herein, the term "administration" is intended to refer to the introduction of the pharmaceutical composition of the present invention into patients in a suitable manner. As long as it leads the composition to a desired tissue, any administration route may be adopted. Topical ocular administration is preferred.

The method of the present invention includes the step of administering chlorogenic acid in a pharmaceutically effective amount. It will be apparent to those skilled in the art that the suitable total daily dose may be determined by an attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient may vary depending on a variety of factors, including the kind and degree of a desired reaction, the specific composition, including the use of any other agents according to the intended use, the patient' s age, weight, general health, gender, and diet, the time of administration, route of administration, and rate of the excretion of the composition, the duration of the treatment, other drugs used in combination or coincidentally with the specific composition, and like factors well known in the medical arts.

In accordance with a preferred aspect, the present method may include administering the pharmaceutical composition comprising chlorogenic acid or a derivative thereof in combination with one or more known anti-inflammatory drugs.

Known anti-inflammatory drugs include steroidal drugs such as prednisolone, and non-steroidal drugs. Pharmaceutically effective amounts of the anti-inflammatory drugs are known in the art, and may be decided by an attending physician taking into account general conditions including the degree of symptoms and whether administration is to be in combination with chlorogenic acid. The combined administration of chlorogenic acid or a derivative thereof and a known anti-inflammatory drug may alleviate side effects caused by the known anti-inflammatory drug through the action of chlorogenic acid or the derivative thereof, andmay also result in synergistic therapeutic effects . The known anti-inflammatory drug may be administered as a mixture with chlorogenic acid or the derivative thereof, or either simultaneously or at different time points.

[Mode for Invention]

Hereinafter, the present invention will be described in detail with reference to the following Examples. However, these Examples are for the illustrative purposes only, and the invention is not intended to be limited by these Examples.

Example 1. Preparation and sensitization of animals

Female Hartley guinea pigs weighing between 250 g and 300 g had free access to feed and water, and were housed under conditions of standard temperature, relative humidity and light. The first sensitization was carried out one to two weeks after the guinea pigs were purchased.

Short ragweed pollen (RW) was purchased from Polyscience, Inc. (Warrington, PA) . Guinea pigs were sensitized for two periods of five consecutive days a week for each sensitization.

10 μi of short ragweed pollen was injected once per day into the nostrils and inferior conjunctival fornices of twenty five guinea pigs using a micropipette (Merayo-Lloves, J., Calonge, M, and Foster, CS. 1995. Experimental model of allergic conjunctivitis to ragweed in guineapig. Curr. Eye Res.14:487-494) . On Day 15, the immunized guinea pigs were divided into five groups, each consisting of five guinea pigs. All immunized guinea pigs were challenged with 10 μ& of short ragweed pollen powder, which was injected into inferior conjunctival fornices. 30 min after pollen application, three groups were treated four times a day with 100 nM of chlorogenic acid eye drop, dexamethasone eye drop (Maridex, 0.1% dexamethasone; Alcon-Couvreur, Puurs, Belgium), anti-allergy eye drop (Patanol, 0.1% olopatadine, Alcon Laboratories, Inc. , Fort Worth, TX) , and Azelan (0.05% azelastine, Taejoon pharm, Korea) . As a negative control, one group was not treated with anything.

Example 2. Clinical grading of conjunctival edema and redness

24 hrs after the pollen challenge, clinical grading of conjunctival edema and redness was carried out using a portable slit lamp (Cail Zeiss, Oberkochen, Germany) . The evaluation was performed in a blind fashion 2 hrs after the allergen challenge, and each clinical parameter was scored on a scale of grades ranging from 0 to 4+ (0: absent, 1+: minimal, 2+: mild, 3+: moderate, 4+: severe) as described in Merayo-Lloves, J., Calonge, M, and Foster, CS. 1995. Experimental model of allergic conjunctivitis to ragweed in guinea pig. Curr. Eye Res. 14:487-494. Clinical scores were analyzed for each group, and the results are given in FIG. 1.

As shown in FIG. 1, all treatment groups displayed less severe clinical signs compared to the negative control. However, no difference was observed between the dexamethasone treatment group and the chlorogenic acid treatment group. These results indicate that chlorogenic acid of the present invention has anti-inflammatory activity similar to that of dexamethasone.

Example 3. Histological evaluation of eosinophil infiltration

After the animals were sacrificed by CO₂ asphyxiation, the lower eyelids, including the inferior forniceal conjunctivae, were harvested and fixed in 10% buffered formalin. Each tissue was prepared for the light-microscopic examination. The eyes were sectioned sagittally through the center and stained with hematoxylin and eosin. Infiltrating eosinophils in standardized fields of conjunctival epithelium, immediate subepithelium and stroma were counted in three non-contiguous fields for each treatment in each experiment (20Ox magnification; five eyes for each group (negative control, dexamethasone, Patanol, Azelan (known therapeutic drug for conjunctivitis) , and chlorogenic acid groups) . The results for eosinophil infiltration are given in FIG. 2.

As shown in FIG. 2, compared to a negative control, all treatment groups displayed reduced eosinophil infiltration, which was statistically significant. The chlorogenic acid eye drop group exhibited similar inhibitory effects on eosinophil chemotaxis to the dexamethasone eye drop group. In particular, there was no statistically significant difference between the chlorogenic acid group and the positive control group. As shown in FIG. 2, the chlorogenic acid of the present invention was found to have anti-inflammatory activity higher than that of conventional anti-inflammatory drugs, particularly the steroidal anti-inflammatory drug dexamethasone.

Example 4. Immunohistochemical evaluation

4-1. Inhibitory effect of chlorogenic acid on TGase 2 and NF-KB

The expression of transglutaminase 2 (TGase) increases markedly upon the occurrence of various inflammatory diseases. Inhibition of TGase activity decreases the activity of the transcriptional factor NF-κB in inflammatory response. Thus, the present inventors performed immunohistochemical analysis to examine the inhibitory effect of chlorogenic acid on TGase 2 and NF-KB.

Tissue sections of conjunctiva were incubated with 1% bovine albumin fraction V at room temperature for 30 minutes, and then incubated with labeled primary antibody at room temperature for lhr. The slides were washed with phosphate buffered saline (PBS) for 5 minutes three times, and incubated with secondary antibody at room temperature for 30 minutes. Then, the slides were washed with PBS, and examined under a laser scanning confocal microscopy (Leica, Deerfield, IL) .

As a result, strong signals were observed in the negative control group, indicating high expression of transglutaminase 2 and NF-κB. The number of TGase 2 and NF-κB expressing cells was remarkably reduced in the chlorogenic acid eye drop treatment group, as compared to the dexamethasone (steroid) treatment group. No reduction in TGase 2 and NF-κB expression was observed in the anti-allergy eye drop (Azelan or Patanol) treatment group.

Accordingly, chlorogenic acid reduces the expression of TGase 2 and NF-κB, thereby being useful as an effective anti-inflammatory agent.

4-2. Inhibitory effect of chlorogenic acid on TGase 2 and COX-2 expression

COX (cyclooxygenase) , an enzyme involved in the synthesis of prostaglandins, is highly induced in inflammatory cells and plays an important role in the inflammation process. Thus, the present inventors performed immunohistochemical analysis to examine the inhibitory effect of chlorogenic acid on TGase 2 and COX-2. The experiment was performed in the same manner as in Example 4-1, except using FITC-conjugated anti-TGase 2 cross-link antibody and AlexafIuor594-conjugated anti-COX-2 antibody as a secondary antibody.

As a result, the co-localization of TGase 2 with COX-2 expression was observed. In the negative control group, the TGase 2 mediated cross-linking and COX-2 expression increased. A slight decrease in their expression was observed in the Azelan or Patanol treatment groups, whereas a significant decrease in their expression was observed in the dexamethasone (steroid) or chlorogenic acid eye drop treatment groups.

Accordingly, chlorogenic acid reduces the expression of TGase 2 and COX-2, thereby being used as an effective anti-inflammatory agent.

[Industrial Applicability]

As described hereinbefore, chlorogenic acid contained in the pharmaceutical composition of the present invention exhibits excellent therapeutic effects on conjunctivitis, and thus has potential as an anti-inflammatory drug not causing side effects, which is the problem with conventional steroidal anti-inflammatory drugs.

Claims

[CLAIMS]

[Claim 1]

A pharmaceutical composition for the treatment of conjunctivitis, comprising chlorogenic acid or a derivative thereof.

[Claim 2]

The pharmaceutical composition according to claim 1, further comprising a pharmaceutically acceptable carrier.

[Claim 3]

The pharmaceutical composition according to claim 1, wherein the conjunctivitis is allergic conjunctivitis.

[Claim 4]

The pharmaceutical composition according to claim 1, wherein the conjunctivitis is infectious conjunctivitis.

[Claim 5]

A method for treating conjunctivitis, comprising the step of administering a composition comprising chlorogenic acid or a derivative thereof.

[Claim 6]

The method according to claim 5, wherein the conjunctivitis is allergic conjunctivitis.

[Claim 7]

The method according to claim 5, wherein the conjunctivitis is infectious conjunctivitis.