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(11) EP 2 543 357 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 09.01.2013 Bulletin 2013/02 A61K 9/00 (2006.01) A61K 47/36 (2006.01)

(21) Application number: 11173000.8

(22) Date of filing: 07.07.2011

(84) Designated Contracting States: (72) Inventor: Lin, Shyh-Shyan AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Taipei (TW) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (74) Representative: Becker Kurig Straus Designated Extension States: Bavariastrasse 7 BA ME 80336 München (DE)

(71) Applicant: Holy Stone Healthcare Co.,Ltd. Taipei City (TW)

(54) Composition for use in treating and preventing related disorder

(57) The presentinvention isrelated to ause fortreat- ease, coeliac disease, conjunctivitis, otitis, allergic rhin- ing and preventing inflammation related disorder of a itis, gingivitis, aphthous ulcer, bronchitis, gastroesopha- composition containing a and hyaluronic acid (HA) geal reflux disease (GERD), esophagitis, gastritis, en- or HA mixture, whereas the HA or the HA mixture as a teritis, peptic ulcer, inflammatory bowel disease (IBD), delivery vehicle can be a formulation including at least Crohn’s Disease, irritable bowel syndrome (IBS), intes- two HAs having different average molecular weights. The tinal inflammation or allergy, urethritis, cystitis, vaginitis, composition has been demonstrated to be capable of proctitis, eosinophilic gastroenteritis, or rheumatoid ar- reducing the therapeutic dose of a drug on the treatment thritis. and prevention of inflammation related disorders is acute inflammatory disease, chronic obstructed pulmonary dis- EP 2 543 357 A1

Printed by Jouve, 75001 PARIS (FR) 1 EP 2 543 357 A1 2

Description alleviate by counteracting the (COX) . On its own, COX enzyme synthe- [0001] The present invention provides a composition sizes , creating inflammation. In of hyaluronic acids in combination with a drug for treating whole, the NSAIDs prevent the prostaglandins from and preventing an inflammation related disorder, espe- 5 ever being synthesized, which reduces or eliminates cially where the onset of the disorder mainly occurs in the pain. Some common examples of NSAIDs are: mucosal tissue. , , and . The newer specific [0002] Hyaluronic acid, also known as hyaluronan, hy- COX-inhibitors, although it is presumed, sharing a aluronate and sodium hyaluronate, is generally referred similar mode of action, are not classified together to as HA, which is a natural macro- 10 with the traditional NSAIDs. molecule including disaccharides composed of the alter- 3. ImSAIDs were discovered to have diverse biolog- native N-acetyl-D- and D-glucuronic acid ical properties, including anti-inflammatory proper- linked via alternative β -1,3 and β -1,4 glycosidic bonds. ties. ImSAIDs work by altering the activation and mi- HA found in nature with a molecular weight (Mw) between gration of inflammatory cells, which are immune cells 50,000 Dalton (Da) and a few millions Dalton usually has 15 responsible for amplifying the inflammatory re- high viscosity. sponse (Bao F, John SM, Chen Y, Mathison RD, [0003] HA found in nature is the extra- cellular material Weaver LC. The tripeptide phenylalanine-(D) gluta- with elasticity, filling between the cells and the collagen- mate-(D)glycine modulates leukocyte infiltration and ous fibers and covering onto some epidermal tissues, oxidative damage in rat injured spinal cord. Neuro- mainly for protecting and lubricanting cells, for providing 20 science., 2006, 140(3):1011-22; Epub on 2006 Apr a platform for transporting the regulatory T cell, and also 3). The ImSAIDs represent a new category of anti- for stabilizing collagen network and protecting collagen inflammatory and are unrelated to steroid hor- network from the mechanical damage. HA is also a major mones or non-steroidal anti-inflammatory drugs. lubricant in the tendon and the tendon sheath and on the surface of the synovial membrane due to the lubricant 25 [0006] US patent 6,159,955 discloses a method of feature and the high shock absorber, and HA is helpful treating aphthous ulcers comprising administration of an for the tissue rheological mechanics, motion and the cell effective amount of a composition comprising a NSAID proliferation (referring to Delpech, B. et al., 1997. Hy- and a formulation of hyaluronic acid selected from hy- aluronan: fundamental principles and applications in can- aluronic acid, pharmaceutically acceptable salts thereof, cer. J. Intern. Med. 242, 41-48), and participates in the 30 fragments thereof and/or subunits thereof. However, it interaction on the surface of some cells, partic- did not emphasize the less dose of the NSAID being ularly to be the major receptor of CD44. CD44 is widely used, whereas mesalamine does not belong to NSAID. accepted as a marker of the activated lymphocyte (refer- [0007] The mucous membranes (or mucosae; singular ring to Teder P, et al., 2002, Resolution of lung inflam- mucosa) are linings of mostly endodermal origin, covered mation by CD44. Science). 35 in epithelium, and involved in absorption (gastrointestinal [0004] Recently, HA is applied in clinical treatment in tract) and secretion (gastrointestinal and respiratory the sodium salt form mainly in eye, skin, orthopedics, tract). They line cavities that are exposed to the external surgery, , artery treatment and in cosmetic fields. environment and internal organs and contiguous with The HA with alkali metal ion, alkaline earth metal ion (for skin at several body areas: at the nostrils, the mouth, the example, the magnesium ion), aluminum ion, ammonium 40 lips, the eyelids, the ears, the genital area, and the anus. ion, and salt form of the replacement of the ammonium The sticky, thick fluid secreted by the mucous mem- ion can be the carrier for assisting drug absorption (re- branes and glands is termed mucus. The term mucous ferring to Belgium Patent 904,547). The silver salt is used membrane refers to where they are found in the body as the mycocide and the gold salt is used for treating the and not every mucous membrane secretes mucus. among the heavy metal salt of the 45 [0008] Conjunctivitis refers to inflammation of the con- HA (referring to WO 87/05517). junctiva (the outermost layer of the eye and the inner [0005] Anti-inflammatory refers to the property of a surface of the eyelids). substance or treatment that reduces inflammation. Anti- [0009] Otitis is a general term for inflammation or in- inflammatory drugs make up about half of , fection of the ear, in both humans and other animals. remedying pain by reducing inflammation as opposed to 50 [0010] Rhinitis is defined as inflammation of the nasal , which affect the central nervous system. The membranes and is characterized by a symptom complex related drugs are described as follows: that consists of any combination of the following: sneez- ing, nasal congestion, nasal itching, and rhinorrhea. The 1. Steroids, specifically glucocorticoids, reduce in- eyes, ears, sinuses, and throat can also be involved. US flammation or swelling by binding to glucocorticoid 55 patentapplication 20050107330 discloseda pharmaceu- receptors. These drugs are often referred to as cor- tical composition for curative topical treatment of rhinitis ticosteroids. comprising at least one acidic glycosaminoglycan. But 2. Non-steroidal anti-inflammatory drugs (NSAIDs) this invention also contains at least one sympathomimet-

2 3 EP 2 543 357 A1 4 ic suitable for topical application and having vasocon- to 6 weeks (Colasa®). For the treatment of mildly to mod- strictor action or detumescent action on the mucous erately active ulcerative colitis: The usual dosage in membrane or its physiologically acceptable salts or de- adults is two 400-mg tablets to be taken three times a rivatives. It did not disclose the technical concept that day for a total daily dose of 2.4 grams for a duration of 6 less dose of drug can be used in need by combining with 5 weeks (Asacol®). The recommended dosage for the in- HA. duction of remission and the symptomatic treatment of [0011] Oral mucosa is the mucous membrane epithe- mildly to moderately active ulcerative colitis is 1 g (4 PEN- lium of the mouth. An oral ulcer is an open sore inside TASA 250 mg capsules or 2 PENTASA 500 mg capsules) the mouth, or rarely a break in the mucous membrane or 4 times a day for a total daily dosage of 4 g. Treatment the epithelium on the lips or surrounding the mouth. Once 10 duration in controlled trials was up to 8 weeks. (PENTA- formed, the ulcer may be maintained by inflammation SA®). In a preferred embodiment, the drug is a steroid, and/or secondary infection. prednisolone. The initial dose of prednisone varies de- [0012] Bronchitis is inflammation of the mucous mem- pending on the condition being treated and the age of branes of the bronchi, the airways that carry airflow from the patient. The starting dose may be from 5 to 60 mg the trachea into the lungs. Bronchitis can be divided into 15 per day and often is adjusted based on the response of two categories, acute and chronic, each of which has the condition being treated (Prednesol q). In a preferred unique etiologies, pathologies, and therapies. US patent embodiment, the drug is a NSAID, Naproxen. For Rheu- application 20030171332 discloses a method of treating matoid arthritis, , and ankylosing spondyli- respiratory conditions by a polysaccharide capable of tis: 250-500 mg Naproxen is administered orally twice binding CD44. However, only one single species of HA 20 daily and may be increased to 1.5 g/day of naproxen could be involved and the prior art did not disclose a base for limited time period. (Naprosyn q). combination of HA and a drug, not to mention merely a [0016] With regards to interaction between molecules, less dose of drug being used. intermolecular forces are first categorized into hydrogen [0013] The mucosa is the innermost layer of the gas- bond and non-covalent bond. Hydrogen bonds are es- trointestinal wall that is surrounding the lumen, or open 25 sentially electrostatic in nature, although the energy of space within the tube. This layer comes in direct contact hydrogen bond can be decomposed into additional con- with food bolus, and is responsible for absorption, diges- tributions from polarization, exchange repulsion, charge tion, and secretion which are the important processes in transfer, and mixing. A hydrogen bond is the attractive digestion. force between a hydrogen atom and an electronegative [0014] Generally known as peptic ulcer, and also30 atom, such as nitrogen, oxygen, or . The hydro- known as PUD or peptic ulcer disease, peptic ulcer is an gen bond is often described as a strong electrostatic di- ulcer (defined as mucosal erosions equal to or greater pole-dipole interaction. The most common hydrogen than 0.5 cm) of an area of the gastrointestinal tract that bonds in biological systems involve oxygen and nitrogen is usually acidic and thus causes extreme pain. Ulcers atoms. can also be caused or worsened by drugs such as aspirin, 35 [0017] As another kind of interaction between mole- Plavix (clopidogrel), ibuprofen, and other NSAIDs (non- cules, the noncovalent bond is the dominant type of in- steroid anti-inflammatory drugs). teraction between supermolecules in supermolecular [0015] Mesalamine, also known as or 5- chemistry. Noncovalent bonds are critical in maintaining aminosalicylic acid (5- ASA), is an anti- inflammatory drug the three-dimensional structure of large molecules and used to treat inflammation of the digestive tract ulcerative 40 are involved in many biological processes in which large colitis and mild-to-moderate Crohn’s disease. Mesala- molecules bind specifically but transiently to one another. mine is a bowel-specific aminosalicylate drug that acts The forces consist of four types: (1) dipole- dipole forces, locally in the gut and has its predominant actions there, (2) ion-dipole forces, (3) dipole-induced dipole force or thereby having few systemic side effects. As a derivative Debye forces, and (4) instantaneous dipole-induced di- of , mesalamine is also thought to be an45 pole forces or London dispersion forces. Molecular inter- antioxidant that traps free radicals, which are potentially actions are fundamentally electrostatic in nature and can damaging byproducts of metabolism. Mesalamine is con- be described by Coulomb’s Law. Coulomb’s law correctly sidered the active moiety of sulfasalazine, which is me- describes forces that bind (1) electrons to nuclei in atoms, tabolized to sulfapyridine and mesalamine (Lippencott’s (2) atoms to atoms in molecules, and (3) molecules to Illustrated Reviews: Pharmacology, 4th Ed. Finkel,50 molecules in liquids and solids. The detailed descriptions Cubeddu and Clark). Mesalamine is formulated for oral of such forces are common scientific knowledge which ingestion as tablets or granules, and for rectal adminis- are omitted herein. tration as a rectal suppository, suspension or enemas. [0018] The objective of the present invention is to pro- The normal therapeutic dose of three commercial drugs vide a composition for use in treating or preventing in- of mesalamine is introduced herein. Retention enema 55 flammation and/or allergy related disorder, which re- rectally for adult with ulcerative proctitis (active, chronic) quires a lower dose of anti-inflammatory drug and thus and proctosigmoiditis (active ulcerative): 4 gm retention fewer side-effects of said drug will occur once the com- enema rectally QD at bedtime (retain for 8 hours) for 3 position is administrated to a subject suffering from in-

3 5 EP 2 543 357 A1 6 flammation and/or allergy related disorder. [0023] HA based on its repeated disaccharide units [0019] Accordingly, the present invention provides a forms a linear structure (Fig. 1). However, in the high composition for use in treating or preventing an inflam- average Mw HA, by the reaction of HA with an amine, mation and/or allergy related disorder in a mammal or the carboxylic groups of the linear HA macromolecules human comprising: 5 react with the di/tri/poly amines in the same or other HA macromolecules to form an amide linkage and form an a drug suitable for the treatment of an inflammation inter- or intra-molecular bridge. Due to this reaction, the and/or allergy in a dose less than the normal thera- starting coiled HA structure is transformed into a globular peutic dose thereof, and spherical nanoparticle (referring to U.S. Patent No. hyaluronic acid (HA) or HA mixture, 10 7,879,818). In a preferred embodiment of the present whereby the HA or the HA mixture used as a delivery invention, a HA mixture has been set up comprising at vehicle delivers the drug specially to the inflamma- least two species of HAs of different average molecular tory tissue while the composition is administrated to weights including low average molecular weight hy- the mammal or human suffering from the inflamma- aluronic acid (LMWHA) and high average molecular tion and/or allergy related disorder. 15 weight hyaluronic acid (HMWHA). HAs of different mo- lecular weights have different rheology, adhesion, func- [0020] Other objectives, advantages and novel fea- tions as tissue scaffold and degradation properties in the tures of the invention will become more apparent from solution. the following detailed description when taken in conjunc- [0024] HA hydrolysis catalyzed by HAase is believed tion with the accompanying drawings. 20 to be involved in the control of the balance between long- er and shorter HA chains. Shorter HA chains seem to be IN THE DRAWINGS too short to form a stable complex and longer HA chains encounter difficulties in forming a complex, probably be- [0021] cause of steric hindrance. 25 [0025] Pursuant to the present invention, on contrary Fig. 1 shows the general chemical structure of the to conventional techniques that one single species of HA hyaluronic acid; is used for simple and regular situation, in a preferred Fig. 2 shows the affinity of HAs by fluorescent index embodiment the specific HA mixture provided by the in- in normal and injured colon tissues (*p<0.05); ventors is also involved. The specific HA mixture com- Fig. 3 shows colon tissue biopsies time, mesalamine 30 prises at least LMWHA and HMWHA. The HA with an concentration profile after Colasaq (a brand of en- average molecular weight lower than 1.5 million Da is ema preparation with 20 mg of mesalamine in 1 ml categorized as LMWHA, the preferred range of the av- solution) or HA-mesalamine dosing; erage molecular weight is between 50 kilo Da and 1.5 Fig. 4 shows comparison of the effect of mesalamine million Da, the more preferred range of the average mo- and HA-mesalamine with control group in treating 35 lecular weight is between 0.1 million and 1.5 million Da, IBD indicated by average body weight of rats and the most preferred range of the average molecular (mean6SD) through test days 1 to 14 (*p<0.05) vs. weight is between 0.1 million and 0.5 million Da. The HA group A); and with an average molecular weight higher than 1.5 million Fig. 5 shows comparison of the effect of mesalamine Da is categorized as HMWHA, the preferred range of the and HA-mesalamine with control group in treating 40 average molecular weight is between 1.5 million and 5 IBD indicated by recovery rate of rats (mean6SD) million Da, and the most preferred range of the average between test day 9 (D9) and day 14 (D14) (* p<0.05 molecular weight is between 1.5 million and 3.5 million vs. group A). Da. In a preferred embodiment, the average molecular weight of LMWHA is apart from the average molecular [0022] The scenario of the present invention is based 45 weight of HMWHA at least 0.5 million Da. The general on the previous test results of the inventors, wherein the chemical structure of the HA is illustrated as in Fig. 1. amount of hyaluronic acid (HA) binding onto the inflam- [0026] Another preferred embodiment of the composi- matory surface was higher than in the non-inflammatory tion in accordance with the present invention includes, area. Further, the results of the present invention indicate but is not limited to, a 1: 1 (w/w) mixture of LMWHA and that HA can be taken as a delivery vehicle to carry a drug 50 HMWHA by weight in a salt form of HA, and a more pre- suitable for the treatment of an inflammation and/or al- ferred embodiment of the ratio of LMWHA to HMWHA, lergy, in a preferred embodiment, mesalamine. Notwith- may be adjusted depending on the clinical purpose to be standing the detailed mechanism between HA and me- between 20:80 and 80: 20 by weight. The HA mixture with salamine is still not so clear, the results of the present a higher ratio of LMWHA to HMWHA can be more helpful invention prove a composition comprising HA and me- 55 in speeding up the treatment; on the contrary, a higher salamine can sustain the concentration of mesalamine ratio of HMWHA to HMWHA can provide a longer deg- in the colon. Hereinafter will be the detailed description radation rate to prolong the treatment effect. of the scenario. [0027] The term mucosa used herein includes, but is

4 7 EP 2 543 357 A1 8 not limited to, the mucosa of an eye, the mucosa of an flammatory drug (NSAID), mesalamine and derivatives ear, the mucosa of a nose, the mucosa of a mouth, the thereof. mucosa of a respiratory tract, the mucosa of a gastroin- [0031] The preferred embodiment of treating allergic testinal tract, the mucosa of a urinary tract and the mu- rhinitis or bronchitis can be carried out by administrating cosa of a genital tract. The preferred embodiment of the 5 the composition of the present invention involving a drug inflammation related disorder includes, but is not limited of antihistamine, anti-allergies, anticongestives, steroid to, acute inflammatory disease, chronic obstructed pul- or antiasthma to a subject in need thereof. The preferred monary disease, coeliac disease, conjunctivitis, otitis, al- embodiment of treating enteritis can be carried out by lergic rhinitis, gingivitis, aphthous ulcer, bronchitis, gas- administrating the composition of the present invention troesophageal reflux disease (GERD), esophagitis, gas- 10 involving a drug of antibiotic or antispasmodic to a subject tritis, enteritis, peptic ulcer, inflammatory bowel disease in need thereof. The preferred embodiment of treating (IBD), Crohn’s Disease, irritable bowel syndrome (IBS), peptic ulcer can be carried out by administrating the com- intestinal inflammation or allergy, urethritis, cystitis, position of the present invention involving a drug of co- vaginitis, proctitis, eosinophilic gastroenteritis, or rheu- agulant, antibiotics, antacid, H2 blocker, potassium hy- matoid arthritis. The more preferred embodiment of pep- 15 drogen ion pump blocker (PPI), cytoprotectives or mu- tic ulcer includes, but is not limited to, gastric ulcer, du- cosa protector to a subject in need thereof. The preferred odenal ulcer, esophageal ulcer and Meckel’s Diverticu- embodiment of treating IBD can be carried out by admin- lum ulcer. The more preferred embodiment of IBD in- istrating the composition of the present invention involv- cludes, but is not limited to, Crohn’s disease and ulcer- ing a drug of steroid, immunosuppressive agent, antibi- ative colitis. The more preferred embodiment of IBS in- 20 otic, 5-aminosalicylic acid (5- ASA) and derivatives, or an- cludes, but is not limited to, coeliac disease, fructose mal- ti-inflammatory to a subject in need thereof. absorption, mild infections, parasitic infections like giar- [0032] The preferred embodiment of treating IBS can diasis, functional chronic constipation and chronic be carried out by administrating the composition of the functional abdominal pain. present invention involving a drug of antiallergic, anti- [0028] The result of the present invention shows that 25 spasmodic, antidiarrheal, neurolytic, tranquilizer, narcot- HA is absorbed in the injured colon tissues obviously ic , antidepressant or serotonin antagonist to a higher than in the normal colon tissues (P<0.01, Fig. 2). subject in need thereof. In a more preferred embodiment, Comparing the differences among HAs of three average the drug in the composition of the present invention in- molecular weights absorbed in the injured colon tissues, cludes the antihistamine, anti- allergy, anti-inflammatory, the fluorescent index of absorption of 350 K Da HA by 30 immuno-suppressant, or combination thereof. the injured colon tissues was obviously higher than HAs [0033] In the preferred embodiment of oral formulation of the other two average molecular weights (2 M Da and (for example, enteric coated tablet), the enteric coating 1 M Da). Further, the fluorescent index of absorption of provides more resistance dissolution and digestion in the 1 M Da HA by even normal or injured colon tissues was stomach, and after reaching the intestine and colon, the higher than 2 M Da HA. This result explains fast dispersed 35 enteric coating will be dissolved and the HA herein will and covered effect of low Mw HA which supplies instant be released to form a protection membrane at the inflam- wound healing and protection of the tissue from being matory colon (the region of ascending or transverse co- injured further. lon). [0029] Another preferred embodiment of the composi- [0034] In the preferred embodiment of suppository for- tion ofthe present inventionincludes a HA ora HA mixture 40 mulation, the suppository containing the composition including an excipient to formulate an administrating dos- herein may be inserted into the anus and the composition age form for eye, ear, oral, nose, respiratory tract, gas- will dissolve in the rectum and spread to other regions of trointestinal tract or topical use. The more preferred em- the colon (for example the descending region) to form a bodiment of the oral dosage form is selected from the protection membrane at the inflammatory colon. group consisting of solid dosage form, solution including, 45 [0035] In the preferred embodiment of perfusion for- but not limited to suspension, tablet including, but not mulation (for example, enema), the HA or mixture of HAs limited to controlled-release tablet, and capsule includ- in the composition herein is the major active ingredient ing, but not limited to enteric-coated capsule. The more mixed with the excipient (for example, phosphate buff- preferred embodiment of the gastrointestinal tract admin- ered saline (PBS solution or suspension formulation)) istration form is selected from the group consisting of 50 directly used or in a soft tube to inject the above compo- solid dosage form, perfusion, enema, suppository, and sition into the colon. The composition will be charged into solution including, but not limited to, suspension. The the colon and spread to other regions of colon (for ex- more preferred embodiment of the topical administration ample, the descending region) to form a protection mem- form is selected from the group consisting of perfusion, brane at the inflammatory colon and to treat inflammation enema, suppository, spray, inhalation, and drop. 55 by the drug. [0030] According to the present invention, the drug [0036] The more preferred embodiment of the subject suitable for the treatment of an inflammation of the being treated by the composition used herein is mammal. present invention includes a steroid, non-steroid anti-in- The most preferred embodiment of the subject is human.

5 9 EP 2 543 357 A1 10

[0037] The composition of the present invention is the effect of the drug on inflammatory region. A preferred made by natural physical or chemical force. The inter- embodiment of the present invention is acting on the mu- molecular force between the HA and a drug of the present cosal tissue because of the characteristic of HA. Prefer- invention most probably is chelation, electrostatic inter- ably, the present invention reduces the dose and/or action or adhesion. Chelation is the formation or pres- 5 amount required of the drug and thence decreases the ence of two or more separate coordinate bonds between cost on drug. a polydentate (multiple bonded) and a single cen- [0040] Another preferred embodiment of the present tral atom. Usually these ligands are organic compounds. invention is providing a method for treating or preventing Though all molecular interactions are inherently electro- inflammation and/or allergy related disorder comprising static in nature, electrostatic interaction usually specifi- 10 administering to a mammal or human in need thereof a cally represents any of the attractive or repulsive forces composition of a drug suitable for the treatment of an between atoms and/or groups of atoms and/or molecules inflammation and/or allergy in a dose less than normal that are due to the presence of ionized chemical entities therapeutic dose and hyaluronic acid (HA), wherein the and to the electronegative and electropositive properties HA used as a delivery vehicle to deliver the drug specially of these atoms, groups, or molecules. Adhesion is any 15 to the inflammatory tissue. attraction process between dissimilar molecular species [0041] According to the present invention, the drug that can potentially bring them into "direct contact". Ad- suitable for the treatment of an inflammation and/or al- hesion is the tendency of dissimilar particles and/or sur- lergy is a steroid, non-steroidal anti-inflammatory drugs faces to cling to one another. (NSAIDs), immune selective anti-inflammatory deriva- [0038] In a preferred embodiment, the drug may be 20 tives (ImSAIDs) or mesalamine. enclosed or coated by HA or HA mixture aforementioned [0042] More preferably, the drug suitable for the treat- to form a polymer or a globular enclosure. Under this ment of an inflammation and/or allergy is, for example, situation, either HA itself or an excipient added can keep but not limited to, mesalamine, 5-aminosalicylate, sul- a certain amount of the drug and make the drug work fasalazine and olsalazine, , , efficiently on the inflammatory region. 25 acetaminophen, acetaminosalol, acetyl-salicylic acid, [0039] The biggest contribution of the present inven- acetyl-salicylic-2-amino-4-picoline-acid, 5-aminoacetyl- tion is providing a composition of a HA formulation with salicylic acid, , aminoprofen, , ampy- a drug which can significantly decrease the therapeutic rone, , , , benoxapro- dose of the drug. Because the doses of different drugs fen, bermoprofen, alpha.-bisabolol, , 5-bro- depend upon various situations of different patients, the 30 mosalicylic acid acetate, bromosaligenin, bucloxic acid, present invention only provides preferred embodiments butibufen, , celexocib, chromoglycate, cinmet- to be a representative. The preferred embodiment of the acin, clindanac, clopirac, sodium , , drug of the present invention is mesalamine, a drug ditazol, , enfenamic acid, , etofena- known for treating IBD. The result of the present invention mate, , , , fendosal, feno- shows that only one- fourth of the normal therapeutic dose 35 profen, , fepradinol, flufenac, , was used, but having almost the same efficacy to amel- , , , glutametacin, glycol iorate the inflammation of artificially induced IBD in rats. salicylate, ibufenac, ibuprofen, , indometh- In a more preferred embodiment, one-eighth of the nor- acin, , isofezolac, isoxepac, , keto- mal therapeutic dose was used in the present invention. profen, , , , meclofenamic Another preferred embodiment is that HA prefers to rec- 40 acid, , , mesalamine, me- ognize and attach to the inflammatory region (Fig. 2). In tiazinic acid, , montelukast, , a preferred embodiment, when only one species of HA naproxen, , , olsalazine, oxace- is utilized instead of a HA mixture aforementioned con- prol, , , , taining at least two species of HAs, its average molecular parsalmide, perisoxal, phenyl-acethyl-salicylate, phe- weight is between 50 kilo Dalton and 5 million Dalton. 45 nylbutazone, phenylsalicylate, pyrazolac, , pir- Though the actual mechanism of intermolecular force be- profen, , prednisolone, protizinic acid, re- tween HA and drug is still not so clear, the present in- serveratol, salacetamide, , salicylamide-O- vention has provided several evidences that mixing HA acetyl acid, salicylsulphuric acid, , salicylamide, and mesalamine can contribute to the combination of , , , suxibutazone, tamoxifen, each advantage of both, confirmed by the results of Ex- 50 , , tiaramide, ticlopridine, tinor- ample 2 below as shown in Fig. 3 and those of Example idine, , , tropesin, xenbucin, ximo- 3 below as shown in Figs. 4 and 5. The technical concept profen, , , tomoxiprol, zafirlukast combining HA with a special guidance ability (as a deliv- and cyclosporin. ery vehicle) to inflammatory region and a drug with less [0043] A preferred embodiment of the composition in dose has never been disclosed till the present invention, 55 accordance with the present invention comprises one or especially HA by itself also has ability to treat inflamma- more discrete dose units, each comprising the drug suit- tion. Therefore, the present invention provides a clever, able for the treatment of an inflammation in a dose less easy, simple way to save the drug dose and concentrate than 250 mg.

6 11 EP 2 543 357 A1 12

[0044] According to the present invention, the normal the solution of step (4) and then stirred for 10 minutes therapeutic dose of the drug suitable for the treatment of under a condition that light was prohibited. (6) 0.02 an inflammation is as known in the field of the art. For M MES buffer was slowly added into the solution of example, the normal therapeutic dose of mesalamine is step (5) until the volume reached 100 ml and then 4 g per day. Therefore, the drug suitable for the treatment 5 stirred for 24 hours at room temperature under a con- of an inflammation of the composition in accordance with dition that light was prohibited. (7) The product after the present invention is in a dose preferably less than 4 reaction was poured into a dialysis tubing (MW: g per day; more preferably, from 250 mg to 4 g per day; 12000∼14000) in 5 L dd water as a dialysis solution most preferably, from 250 mg to 1 g per day. and then stirred for 5 days at 4 °C under a condition [0045] While the invention has been described in con- 10 that light was prohibited with dialysis solution being junction with a specific best mode, it is to be understood changed every 12 hours until the dialysis solution that many alternatives, modifications, and variations will had no fluorescence. (8) The liquid after dialysis was be apparent to those skilled in the art in light of the fore- allocated into 50 cc plastic centrifuge tubes and then going description. Accordingly, it is intended to embrace reserved at -20 °C refrigerator overnight followed by all such alternatives, modifications, and variations which 15 drying in a freeze-drying machine under a condition fall within the spirit and scope of the included claims. All that light was prohibited. (9) The dried HA- f powder matters set forth herein or shown in the accompanying was reserved at -20 °C refrigerator. (10) 50 mg HA- drawings are to be interpreted in an illustrative and non- f powder was slowly added into 10 ml PBS buffer limiting sense. and then stirred for 6 hours until the powder was 20 totally dissolved. EXAMPLES 3. Colon tissue of SD-rat (Sprague-Dawley Rat) aged 7-8 weeks was cut by scalpel and then washed Example 1: The adhesion of HA in colon tissue (IVIS by PBS buffer followed by being cut to 3-4 cm long image system-vision 3) with soaking in PBS buffer finally. 25 4. Injured colon tissue was prepared by brushing by Procedure: toothbrush for 20 times longitudinally and then soak- ing in PBS buffer. [0046] 5. Normal and injured colon tissues were put into 12- well plates and then 1 ml 0.5 % HA-f solution was 1. 0.25 g High molecular weight sodium hyaluronate 30 added into each well and shaken for 2 hours at room powder (HHA; Mw: 2 MDa; Freda) and 0.25 g low temperature. Surplus HA-f solution was sucked by molecular weight sodium hyaluronate powder (LHA; tip 2 hours later, and then soaked into PBS buffer Mw: 0.35 MDa; Freda) were added into 50 ml PBS for 10 minutes followed by removing PBS buffer re- buffer (Phosphate buffered saline) respectively to peatedly for 3 times. form a 0.5 % solution, and then stirred for 6 hours 35 6. Cleaned colon tissue was placed in a 12- well plate until the powder was totally dissolved. with lining tissue upwards and then placed onto the 0.05 g LHA powder and 0.2 g HHA powder (ratio 2: dock of the IVIS (in vivo image system, XENOGEN). 8; MHA, medium molecular weight sodium hyaluro- The default parameter was set up as GFP (green nate powder) were added into 50 ml PBS buffer, and fluorescent protein) whereas the excitation was 465 then stirred for 6 hours until the powder was totally 40 nm and the emission was 500 nm and then the image dissolved. was captured by software. 2. Fluorescent HA (HA-f) was prepared by (1) 0.39 7. All values in the table are expressed as means of g MES free acid (2-(N-morpholino) ethanesulfonic n observations. The histological index was analyzed acid, Calbiochem) and was dissolved in 100 ml dd by Student’s t-test. water. (2) Solution A: 65 mg fluroresceinamine pow- 45 der, (isomer I, Fluka) was dissolved in 9 ml 95% Result: EtOH solution and then stirred for 10 minutes under a condition that light was prohibited. (3) Solution B: [0047] The fluorescent index was quantified and ar- 359 mg EDC powder (N-(3-Dimethylamino pro- ranged as in Fig. 2. The fluorescent index of normal colon pyl)-N-ethyl carbodiimide hydrochloride, Sigma)50 tissue was defined as 1. The other colon tissues tests was dissolved in 9 ml MES buffer and then stirred were calibrated by the defined value. The result showed for 10 minutes. (3) Solution C: 216 mg NHS powder that the HAs with the same average Mw were absorbed (N-Hydroxysuccinimde, Sigma) was dissolved in 9 in the injured colon tissues obviously higher than in the ml MES buffer and then stirred for 10 minutes. (4) 3 normal colon tissues (P<0.01). In comparing the differ- ml Solution A was slowly dropped into 50 ml 0.5% 55 ence between HAs of three different average molecular HA solution and then stirred for 10 minutes under a weights absorbed in the injured colon tissues, the fluo- condition that light was prohibited. (5) 3 ml Solution rescent index of absorption of 350 KDa HA by the injured B and 5 ml Solution C were separately dropped into colon tissues was obviously higher than those of the other

7 13 EP 2 543 357 A1 14 two HAs of the other two average molecular weights (2 cence characteristics of mesalamine. Triethylamine MDa and 1 MDa). Further, the fluorescent index of ab- was used as an ion-pairing agent to improve peak sorption of 1 MDa HA by even normal or injured colon symmetry. The UPLC method of mesalamine anal- tissues was higher than that of 2MDa HA. ysis was validated for measuring mesalamine over 5 a nominal linear range of 10 to 1000 ng/ml. The linear Example 2: Comparative study of colon tissue con- correlation coefficient (R2) of the method used in this centration of mesalamine after intraluminal instilla- study is 1.00. tion of different mesalamine preparations Results: Procedure: 10 [0049] The (median) concentration of mesalamine in [0048] colon tissue biopsies after instillation of Colasa q peaked after one hour instillation. After that, the concentration of 1. Experimental animals: 8-week-old male SPF- mesalamine dropped rapidly. On the contrary, HA- M con- grade Sprague-Dawley rats (280∼330 g) were sup- 15 tinuously released mesalamine during the two-hour pe- plied by BioLASCO Taiwan Co. Ltd. riod and the concentration of mesalamine was rising and 2. Test samples: A : Colasa® 9 enema (20 mg/ml, much higher than that of Colasa q after two-hour instilla- United Biomedical, Inc. Asia), B: 0.25% (w/w) HA tion (Fig. 3). The result of the present invention disclosed mixture (8:2=2000 KDa HA: 350 KDa HA) in PBS that HA-M sustained the release of mesalamine much (pH7.4) containing 5 mg/mL mesalamine (HA-M). 20 longer as compared to the commercial mesalamine en- 3. Intraluminal instillation of test samples: after lightly ema (Colasaq); however, it contained only one-fourth anesthetized by Zoletil 50, rat ventral incision was the concentration of mesalamine in Colasaq. made by surgical scissors, colon was identified, 2 segments of colon (2 cm each) were tied by cotton Example 3: The comparative effect of administering threads, 0.5 ml of test samples were injected into the 25 HA and HA-mesalamine (HA-M) on lowering down lumen of isolated colon segments, after 0.5, 1, 1.5, the inflammation of inflammatory bowel disease or 2 hours, rats were sacrificed and colon segments (IBD) were removed. For each time point of intraluminal instillation, three rats were used. Procedure: 4. Preparation of specimens: tissue biopsies were 30 washed with PBS to remove the surface contamina- [0050] tion, weighed and immediately frozen in liquid nitro- gen and stored at -80 °C until use. Biopsies were 1. Test purpose: to induce the IBD in the SPF grade crushed and 50 mM KH2PO4 solution (pH 7.4) was SD (Sprague-Dawley) rats with the TNBS in order added. Tissue cells were disrupted ultrasonically us- 35 to evaluate the comparative effect of administering ing a microprobe inserted into the suspension for 10 HA and HA-M on lowering down the inflammation. seconds, and then ultrasonic disruption was stopped 2. Test objective: HA mixture, comprising LMWHA for 20 seconds at 25 W for a total of 10 minutes. After and HMWHA, whereas the HMWHA was 2 million mixing by Vortex, samples stood for 30 minutes at Da and the LMWHA was 1 million Da and 350 kilo room temperature, to permit protein precipitation, 40 Da, mixed in the mixing ratio of 8:2 by weight which and were then centrifuged at 13000 g for 30 minutes. were categorized into group C and group D, respec- 5. Analysis of mesalamine concentration in colon tis- tively, and dissolved in PBS solution to produce a sue biopsies: mesalamine was measured by ultra concentration of 0.0625% (w/v). performance liquid chromatography (UPLC). The 3. Method: (1) Test target: Rats aged 8 weeks were method has been validated. Waters (UK) ACQUITY 45 selected, and classified into four groups: group A system and fluorescence detector (excitation 315 wastreated by PBS, group Bwas treatedby Colasa q nm, emission 430 nm) were employed and the data (20 mg mesalamine/mL), group C represented were analyzed using Empower 2. An ACQUITY col- HMWHA: LMWHA of 2 M Da : 1 M DA in the mixing umn (C18, 100x2.1 mm internal diameter, 1.7 um ratio of 8: 2, group D represented HMWHA : LMWHA particle) purchased from Waters (UK) was protected 50 of 2 M Da: 350 k Da in the mixing ratio of 8: 2. Each by a Van (Waters) guard column (C18, 5X2.1 mm group consisted of 10 rats. (2) Animal test: all rats internal diameter, 1.7 um particles). The mobile of the treating group were fasted for 2 days; in test phase consisted of 0.1 M with triethyl- day 1, the rats were anesthetized for administrating amine at pH 4.3 and acetonitrile (850: 150). The flow- 1 ml of TNBS (50 mg/mL) via the rectum; through rate was 0.2 mL/min, with a resulting pressure of 55 test days 5 to 8 and days 10 to 13, administering 1 5400 psi, and the analysis was performed at 40 °C. ml of different test agents of mesalamine (M) and Injection volume was 5 ul. Samples were derivatized two categories of HA- Ms via the rectum in groups B, using propionic anhydride to enhance the fluores- C and D everyday; in test day 9, half rats of each

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group were sacrificed to observe and record chang- thereof. es of body weight and the inflammation area. And the other half of the rats were sacrificed to observe 4. The composition of claim 1, wherein the dose of the and record changes of body weight and the inflam- drug can be less than the normal therapeutic dose mation area in day 14. All rats of the control group 5 till one-eighth thereof at least. were fasted for 2 days; in test day 1, the rats were anesthetized for administrating 1 ml of TNBS (50 5. The composition of claim 1, wherein the HA is of an mg/mL) via the rectum; in test days 5 to 8 and days average molecular weight (Mw) between 50 kilo Dal- 10 to 13, administering 1 ml of PBS via the rectum ton and 5 million Dalton. in group A everyday; in test day 9, half the rats were 10 sacrificed to observe and record changes of body 6. The composition of claim 1, wherein the HA mixture weight and the inflammation area. And the other half comprises at least two hyaluronic acids including low of the rats were sacrificed to observe and record average molecular weight HA (LMWHA) and high changes of body weight and the inflammation area average molecular weight HA (HMWHA), wherein in day 14. 15 the average Mw of the LMWHA is between 50 kilo Da and 1.5 million Da, and the average Mw of the Result: HMWHA is between 1.5 million Da and 5 million Da, wherein the LMWHA and the HMWHA are apart from [0051] at least 0.5 million Da, and a mixing ratio of the 20 LMWHA and the HMWHA is in a range from 20:80 1. Inflammatory index: the present invention used to 80:20 by weight. changes of body weights and average inflammation area as an index to view the amelioration of IBD. 7. Thecomposition ofclaim 1, which is madeby forming 2. The trend of body weight changes showed that natural physical or chemical force between the HA groups B, C and D had a better relief effect on IBD 25 and the drug, wherein the physical or chemical force than group A through day 3 to day 14 (Fig. 4). Es- is made by chelation, electrostatic interaction, adhe- pecially through days 12 to 14, the body weight sion to form a polymer or a globular enclosure for changes in all three treating groups were statistically accommodating the drug. more significant than that in group A. Also, the re- covery rate of the inflammation was better in three 30 8. The composition of claim 1, wherein the inflamma- treating groups than that in the control group in test tion related disorder is inflammation occurring in mu- day 14 (D14) (Fig. 5). Because the amount of me- cosal tissue. salamine of the HA-M formulation of the present in- vention was 4 times less than normal therapeutic 9. The composition of claim 8, wherein the inflamma- dose of Colasaq, the result of the present invention 35 tion related disorder is acute inflammatory disease, indicated that only one- fourth of the normal dose (i.e. chronic obstructed pulmonary disease, coeliac dis- the routine administrating dose of the referenced ease, conjunctivitis, otitis, allergic rhinitis, gingivitis, drug) could achieve almost the same treating effect aphthous ulcer, bronchitis, gastroesophageal reflux as the referenced drug. disease (GERD), esophagitis, gastritis, enteritis, 40 peptic ulcer, inflammatory bowel disease (IBD), Crohn’s Disease, irritable bowel syndrome (IBS), in- Claims testinal inflammation or allergy, urethritis, cystitis, vaginitis, proctitis, eosinophilic gastroenteritis, or 1. A composition for use in treating or preventing an rheumatoid arthritis. inflammation and/or allergy related disorder in a45 mammal or human comprising a drug suitable for 10. The composition of claim 8, wherein the mucosal the treatment of an inflammation and/or allergy in a tissue is the mucosa of an eye, the mucosa of an dose less than the normal therapeutic dose thereof ear, the mucosa of a nose, the mucosa of a mouth, and hyaluronic acid (HA) or HA mixture. the mucosa of a respiratory tract, the mucosa of a 50 gastrointestinal tract, the mucosa of a urinary tract 2. The composition of claim 1, wherein the drug is an or the mucosa of a genital tract. antihistamine, anti-allergy drug, anti-inflammatory drug, immuno-suppressant, or a combination there- 11. The composition of claim 1, further comprising an of. excipient to formulate the composition in an admin- 55 istrating dosage form for eye, ear, oral, nose, respi- 3. The composition of claim 2, wherein the anti- inflam- ratory tract, gastrointestinal tract or topical use. matory drug is a steroid, a non-steroid anti-inflam- matory drug (NSAID), mesalamine or derivatives 12. The composition of claim 11, wherein the adminis-

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trating dosage form for oral use is selected from the group consisting of solid dosage form, solution in- cluding suspension, tablet including eontrolled-re- lease tablet, and capsule including enteric-coated capsule. 5

13. The composition of claim 11, wherein the adminis- trating dosage form for gastrointestinal tract use is selected from the group consisting of solid dosage form, perfusion, enema, suppository, and solution 10 including suspension.

14. The composition of claim 11, wherein the adminis- trating dosage form for topical use is selected from the group consisting of perfusion, enema, supposi- 15 tory, spray, inhalation, and drop.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• BE 904547 [0004] • US 20050107330 A [0010] • WO 8705517 A [0004] • US 20030171332 A [0012] • US 6159955 A [0006] • US 7879818 B [0023]

Non-patent literature cited in the description

• DELPECH, B. et al. Hyaluronan: fundamental prin- • BAO F ; JOHN SM ; CHEN Y ; MATHISON RD ; ciples and applications in cancer.J. Intern. Med., WEAVER LC. The tripeptide phenylalanine-(D) 1997, vol. 242, 41-48 [0003] glutamate-(D) glycine modulates leukocyte infiltra- • TEDER P et al. Resolution of lung inflammation by tion and oxidative damage in rat injured spinal cord. CD44. Science, 2002 [0003] Neuroscience, 03 April 2006, vol. 140 (3), 1011-22 [0005] • FINKEL ; CUBEDDU ; CLARK. Lippencott’s Illus- trated Reviews: Pharmacology [0015]

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