TWI446915B - Composition for use in treating and preventing mucosa related disease - Google Patents

Description

Use of a hyaluronic acid mixture for the manufacture of a composition for treating or preventing mucosal related diseases or diseases

The present invention is a use of a hyaluronic acid mixture for the manufacture of a composition for treating and preventing mucosal-related diseases or diseases, wherein the symptoms of mucosal-related diseases or diseases include ulcers, inflammation, allergic reactions, and bleeding.

Hyaluronic acid has different English nouns (hyaluronic acid, hyaluronan, hyaluronate and sodium hyaluronate), which can be referred to as HA, is a natural glycosaminoglycan, which is composed of alternating N-acetyl-D-glucosamine ( N-acetyl-D-glucosamine) and D-glucuronic acid, which are linked by alternating β-1,3 and β-1,4 glycosidic linkages. Natural hyaluronic acid typically has a high viscosity with a molecular weight between 50,000 Daltons (Daton) and millions of Daltons.

Natural hyaluronic acid is a viscoelastic fluid that fills the gaps between cells and collagen fibers and covers certain epidermal tissues. Primarily used to protect and lubricate cells, providing a platform to transport regulatory T cells, stabilize the collagen network, and protect it from mechanical damage. Hyaluronic acid is also a major lubricant on the surface of tendons, tendon sheaths and sticky synovial membranes, which are characterized by their lubricating properties and high shock absorption. Hyaluronic acid also contributes to the organization of rheological mechanisms, motion and cell proliferation (see Delpech, B., Girard, N., Bertrand, P., Courel, M.-N., Chauzy, C., Delpech, A). , 1997. J. Intern. Med . 242, 41-48). And participate in some cell surface receptor interactions, especially the main receptor - CD44. CD44 has been widely accepted as a marker for lymphocyte activation (see Teder P, Vandivier RW, Jiang D, Liang J, Cohn L, Pure E, Henson PM, Noble PW. Science 2002; 296: 155-158).

Recently, hyaluronic acid is often used in clinical treatment in the form of its sodium salt, mainly used in ophthalmology, dermatology, orthopedics, general surgery, arthritis, arterial therapy and cosmetics. Hyaluronic acid containing an alkali metal ion, an alkaline earth metal ion (for example, magnesium), an aluminum ion, an ammonium ion, and a salt of a substituted ammonium ion can be used as a carrier for assisting drug absorption (refer to Belgian Patent No. 904,547). Among the heavy metal salts of hyaluronic acid, silver salts are used as fungicides; and gold salts can be used to treat rheumatoid arthritis (refer to PCT Patent WO 87/05517).

U.S. Patent No. 5,888,986 discloses the use of an effective concentration of hyaluronic acid for the treatment of cystitis and related structures wherein the weight average molecular weight (Mw) of hyaluronic acid exceeds 200,000 Daltons. In the examples, only a single hyaluronic acid of a specific weight average molecular weight is used. For example, hyaluronic acid having a weight average molecular weight of 650,000 Daltons or 1.9 million Daltons is used to treat cystitis, but a single hyaluronic acid having a specific weight average molecular weight cannot balance the therapeutic effect with long-lasting effects.

U.S. Patent Application No. 2005/0080037 discloses the treatment of acute and excessive sprains and strains by hyaluronic acid, wherein the weight average molecular weight of hyaluronic acid is only between 900,000 Daltons and 1.2 million Daltons, and the specific hyaluronic acid of a specific weight average molecular weight cannot be balanced. Treatment for quick and long lasting results.

U.S. Patent No. 7,354,910 discloses that hyaluronic acid having a weight average molecular weight of between 600,000 Daltons and 1.2 million Daltons can be used to treat inflammatory bowel disease (IBD). However, only a single weight average molecular weight hyaluronic acid cannot be combined with a quick-acting and long-lasting effect after it is injected into a patient's body, so it is clinically very uncomfortable for the patient. Convenience.

European Patent No. 1369119 discloses that hyaluronic acid having a weight average molecular weight of between 600,000 Daltons and 3 million Daltons can be used to treat autoimmune diseases. However, this patent utilizes only a single hyaluronic acid having a specific weight average molecular weight, and cannot achieve both a fast-acting and long-lasting effect.

The mucous membrane (mucosa) covers most of the surface of the endothelium, the surface of the epidermis, and has functions of absorption (in the gastrointestinal tract) and secretion (in the gastrointestinal tract and respiratory tract). The mucous membrane covers the exposed surface of the external environment and the concave surface of the internal organs, and is accompanied by skin covering a part of the body: nostrils, mouth, lips, eyelids, ears, reproductive tract and anus. The thick liquid that is secreted by mucous membranes and glands is called mucus, and depending on the mucosa, different parts of the body are called, and mucus does not necessarily secrete mucus.

Eyes: Conjunctivitis is inflammation of the conjunctiva (the conjunctiva is the outermost layer of the eye and the inner surface of the eyelid) most often caused by infection or allergic reactions (see Richards A, May 2010. "Conjunctivitis"). Pediatr Rev 31(5): 196-208).

Ear: Otitis is a general term for inflammation or infection of the ear of humans and other animals. Can be divided into: otitis externa, otitis media and otitis externa.

Allergic rhinitis: Rhinitis is inflammation of the nasal mucosa characterized by a combination of any of the following: sneezing, stuffy nose, itching, and runny nose. Eyes, ears, sinuses and throat are included. Allergic rhinitis is the most common cause of rhinitis and affects 20% of the population. Allergic rhinitis is not a life-threatening disease, but it can cause complications and can significantly affect the quality of life, indirectly leading to a lot of cost of living (see Bousquet J et al . "Allergic rhinitis management pocket reference" 2008 Allergy 2008 Aug; 63(8): 990-996). U.S. Patent Application Serial No. 2005/0107330 discloses a pharmaceutical composition for topical treatment of rhinitis comprising at least one acidic glycosaminoglycan. The invention also encompasses at least one sympathomimetic or physiologically acceptable salt or derivative suitable for topical administration, which has an effect on vasoconstriction or swelling of the mucosa, and which does not disclose or teach the incorporation of two different weights The average molecular weight of hyaluronic acid.

Mouth: The oral mucosa is located in the epidermis of the mouth. Oral ulcers are open ulcers inside the mouth, or in a few cases, cracks in the mucous membranes or epidermis around the lips or around the mouth. The type of ulcer is heterogeneous and has multiple related causes: physical wear, acidic fruit, infection, other medical conditions, methods of administration, and cancerous and nonspecific processes. Once formed, the ulcer may persist due to inflammation and/or a second infection. Two common forms are oral ulcers and cold sores. Lip herpes near the lips are caused by viruses (see JM Casiglia, et al ., October 2006. "Aphthous stomatitis". Emedecine ).

Bronchitis: The bronchial system is used to transport airflow from the trachea to the respiratory tract of the lungs. Bronchitis is inflammation of the mucosa of the bronchi and can be divided into two types: acute and chronic. Each has its own unique cause, symptoms and treatments. Acute bronchitis often occurs in the course of acute viral diseases such as colds or flu. Chronic bronchitis is most often developed as a recurrent injury to the trachea caused by inhalation stimulation. Smoking is the most common cause, followed by exposure to irritants (Cohen, Jonathan and William Powderly. "Infectious Diseases." 2nd ed. Mosby (Elsevier), 2004 " Chapter 3: Trachea , Bronchial, and Cystic Fibrosis " ( Chapter 33: Bronchitis , Bronchiectasis, and Cystic Fibrosis ). U.S. Patent Application Serial No. 2003/0171332 discloses a method for treating respiratory diseases by binding to a polysaccharide of CD44, but using only a single hyaluronic acid.

Gastrointestinal mucosa: The innermost layer of the gastrointestinal wall that coats the open space in the digestive tract or duct is the mucosa. This layer is in direct contact with the food group and is responsible for the important processes of digestion, digestion and secretion.

The mucosa is highly specialized in each digestive tract organ, which faces a low pH in the stomach, absorbs a variety of different substances in the small intestine, and also absorbs a certain amount of water in the large intestine. To reflect the different needs of these organs, the structure of the mucosa can be composed of nests that secrete glands (such as gastric pits) or folds to increase surface area (Abraham L. Kierszenbaum, 2002, Histology And Histology and Cell Biology: an introduction to pathology.

The digestive tract mucosa forms a barrier between the body and the digestive tract that contains both nutrients and potentially pathogenic microorganisms and toxins. The challenge of mucous membranes is to allow efficient delivery of nutrients through the epidermis into the body when it is necessary to force the harmful molecules and organisms into the body. The exclusion characteristic of the stomach and intestinal mucosa is called the "gastrointestinal barrier". In general, toxins and microorganisms can break through a single epidermal cell layer without any hindrance into the systemic circulation.

Gastroesophageal reflux disease (GERD) is a chronic symptom or mucosal damage caused by gastric acid reflux from the stomach to the esophagus. The typical symptom is heartburn. Gastroesophageal reflux disease is usually caused by changes in the barrier between the stomach and the esophagus. It includes: abnormal relaxation of the lower esophageal sphincter. Under normal circumstances, the lower esophageal sphincter keeps the top of the stomach closed. Esophageal damage begins with the expulsion of the stomach countercurrent; or hiatal hernia. These changes may be permanent or temporary (see DeVault KR, 1999. Updated guidelines for the diagnosis and treatment of gastroesophageal reflux disease) .The Practice Parameters Committee of the American College of Gastroenterology)". Am J Gastroenterol 94(6): 1434-42).

Peptic ulcer: A peptic ulcer is an ulcer in the area of the gastrointestinal tract, defined as mucosal erosion equal to or greater than 0.5 cm, which is usually acidic and thus causes particularly pain. Almost 70-90% of the ulcers are associated with Helicobacter pylori , which lives in the acidic environment of the stomach in a spiral shape. Ulcers can also be caused or worsened by drugs such as aspirin, Plavix, ibuprofen, and other non-steroidal anti-inflammatory drugs. , NSAIDs).

In contrast to general salty, peptic ulcers are more likely to occur in the duodenum than in the stomach. Approximately 4% of gastric ulcers are caused by malignant tumors and require multiple biopsy tests to rule out cancer. Duodenal ulcers are generally benign. Peptic ulcers are classified by the site of the attack: stomach (called gastric ulcer), duodenum (called duodenal ulcer) Ulcer), esophagus (called esophageal ulcer), Meckel's Diverticulum (called Meckel's diverticulum ulcer).

The above examples all represent mucosa-related lesions or diseases but are not limited to the above examples. The disclosed lesions or diseases may have been treated with a number of different drugs or treatments to date, but most of these treatments are achieved by complex ingredients made from highly specialized manufacturing methods, rather than simple and safe treatments. . In addition to the inventors of the present invention, there is no prior art teaching or prediction to combine two hyaluronic acids (LMWHA, low weight average molecular weight hyaluronic acid; HMWHA, high weight average molecular weight hyaluronic acid) having different weight average molecular weights, and the two hyaluronic acid The molecular weight is distinguished by a limit of 1.5 million Daltons. This represents a concept that would not be able to derive the application of the mixture of the present invention by any of the prior art in connection with any of the prior art.

In order to overcome the disadvantages of the prior art, the present invention provides a use of a hyaluronic acid mixture for the manufacture of a composition for treating and preventing a mucosal-related disease or disease, wherein the composition comprises a therapeutically effective amount of a hyaluronic acid mixture, and the mixture comprises At least two different weight average molecular weight hyaluronic acids are used to alleviate or eliminate the previously mentioned problems.

The overall concept of the present invention first understands that hyaluronic acid is decomposed by hyaluronidase (HAase). Linear hyaluronic acid (linear HA) (LMWHA) and globular HA (HMWHA) are broken down into small fragments, but globular hyaluronic acid can replenish linear hyaluronic acid after degradation due to spatial stereostructure. It helps the therapeutic effect while prolonging the duration of action. Reduce the need to constantly replenish linear hyaluronic acid. A known The activity of hyaluronic acid hydrolase can be used as a marker of inflammation. Briefly described as follows: the combination of low and high weight average molecular weight hyaluronic acid can be combined with the low weight average molecular weight hyaluronic acid to adhere to the wound and rapid treatment effect, and the high weight average molecular weight hyaluronic acid can continuously supplement the low weight average molecular weight hyaluronic acid due to degradation. Concept, so the mixture is both therapeutic and long-lasting. Thus, after a decrease in hyaluronic acid, it can be continuously replenished for sustained wound treatment or provide a soft barrier to protect the tissue from damage or further damage. The concept of the present invention will be described in detail below.

The composition for treating and preventing mucosal related diseases or diseases of the present invention comprises a therapeutically effective amount of a hyaluronic acid mixture comprising a low weight average molecular weight hyaluronic acid (LMWHA) and a high weight average molecular weight hyaluronic acid (HMWHA). As shown in Fig. 1, the low weight average molecular weight hyaluronic acid is a repeating disaccharide unit to form a linear structure. However, the high weight average molecular weight hyaluronic acid, the carboxylic group of the generalized linear hyaluronic acid macromolecule, forms an amide linkage by reaction with a di/tri/polyamine, and simultaneously forms an intermolecular bond bridge ( Intramolecular bridge). Based on this reaction, the hyaluronic acid structure that begins to wrap is transformed into spherical nanoparticle (refer to U.S. Patent No. 7,879,818). Different molecular weights have different rheology, adhesion, tissue scaffold function and degradability in aqueous solution, so hyaluronic acid mixture can balance drug efficacy and degradation rate to facilitate the treatment and prevention of mucosal related diseases or diseases, and at the same time achieve appropriate Prolonged treatment effect.

Tranchepain F. et al. revealed that hyaluronic acid has different biological functions based on its molecular weight. From a physicochemical point of view, studies on the hydrolysis of hyaluronic acid by calf hydrazide hyaluronan hydrolase indicate that the kinetic parameters depend on the length of the hyaluronic acid chain. A new process for obtaining hyaluronic acid of different molecular weights catalyzes the hydrolysis of hyaluronic acid by hyaluronan hydrolase (cf. Tranchepain F et al ., "A complete series of hyaluronic acid fragments hydrolyzed by hyaluronic acid hydrolase: by simple high performance liquid chromatography A complete set of hyaluronan fragments obtained from hydrolysis catalyzed by hyaluronidase: Application to studies of hyaluronan mass distribution by simple HPLC devices.) Anal Biochem .2006 Jan 15;348(2):232-42.). Similarly, the same team of researchers also revealed that hyaluronic acid has different biological functions depending on its chain length. It is generally believed that hyaluronic acid hydrolase catalyzes the hydrolysis of hyaluronic acid to control the balance between longer chain and shorter chain hyaluronic acid. The shorter chain hyaluronic acid appears to be too short to form a stable complex, while the longer chain hyaluronic acid also encounters difficulties in forming complexes, possibly due to steric hindrance.

Several reports have stated that hyaluronan hydrolase is involved in the inflammatory response. The activity of synovial fluid (SF) hyaluronic acid hydrolase can be used as a marker for Synovial inflammation (see Nagaya H et al ., "Assessing rheumatoid arthritis and osteoarthritis patients as joint marker molecules""Examination of synovial fluid and serum HAase activity as a joint marker in rheumatoid arthritis and osteoarthritis patients." Ann Rheum Dis ., 1999, 58(3): 186-8). Hyaluronic acid is the main component of the extracellular matrix of the periodontal ligament (PDL), which promotes the integrity of its structure and function. Hyaluronic acid contributes to the cushioning effect of the periodontal ligament during chewing, and also plays an important role in inflammation and wound healing. Studies have shown that periodontal ligament fibroblasts can release hyaluronan hydrolase, and under physiological and inflammatory conditions, can produce the activity of hyaluronan hydrolase required for extracellular hyaluronic acid metabolism (Ohno S et al ., "Hyaluronic acid hydrolase Expression and activity of hyaluronidase in human periodontal ligament fibroblasts.) J Periodontol ., 2002, 73(11): 1331-7). The shorter hyaluronic acid fragment produced by the action of hyaluronic acid hydrolase is involved in the inflammatory response and angiogenesis, whereas the native hyaluronic acid does not (Tranchepain F et al ., supra).

According to the definition of the present invention, hyaluronic acid having a weight average molecular weight of less than 1.5 million Daltons is classified as low weight average molecular weight hyaluronic acid (LMWHA). Preferably, the low weight average molecular weight hyaluronic acid has an average molecular weight ranging from 50,000 to 1.5 million Daltons. More preferably, the low weight average molecular weight hyaluronic acid has an average molecular weight ranging from 100,000 to 1.5 million Daltons. Most preferably, the low weight average molecular weight hyaluronic acid has an average molecular weight ranging from 100,000 to 500,000 Daltons. Hyaluronic acid having a weight average molecular weight of more than 1.5 million Daltons is classified as a high weight average molecular weight hyaluronic acid (HMWHA). Preferably, the high weight average molecular weight hyaluronic acid has an average molecular weight ranging from 1.5 million to 5 million Daltons. Preferably, the high weight average molecular weight hyaluronic acid has an average molecular weight ranging from 1.5 million to 2.5 million Daltons. When a mixture of low weight average molecular weight hyaluronic acid and high weight average molecular weight hyaluronic acid is administered to a therapeutic target, the low weight average molecular weight hyaluronic acid can rapidly cover the inflamed site to treat mucosal related diseases or diseases, and the high weight average molecular weight hyaluronic acid Can extend the treatment effect. because Thus, the treatment can be carried out quickly and in a long-term manner. The general chemical structure of hyaluronic acid is shown in Figure 1.

The quantification of hyaluronic acid is not only expressed by molecular weight, but also by intrinsic viscosity (η). This is directly related to the molecular weight of the polymer. Expressed by the Mark-Houwink-Sakurada (MHS) equation: [η] = KM ^α . For hyaluronic acid, in 0.15 M sodium chloride (NaCl) phosphate buffer (phosphate buffcr), pH 7.5, 20 ° C, K is 0.00057, index α is 0.75 ("for biomedical and tissue engineering pharmaceutical products "Standard Guide for Characterization and Testing of Hyaluronan as Starting Materials Intended for Use in Biomedical and Tissue Engineered Medical Product Applications", ASTM Designation : F 2347-03).

The results of the present invention show that the high weight average molecular weight hyaluronic acid can continuously replenish the low weight average molecular weight hyaluronic acid to maintain its long-lasting effect in hyaluronic acid hydrolase or artificial gastric juice. As shown in Figure 2, in the presence of hyaluronic acid hydrolase, all three hyaluronic acid retention times increased significantly with increasing degradation. As shown in Figure 3, in an acidic environment, the residence time of all three hyaluronic acids increases slowly as the degradation increases. The experimental parameters simulate the physiological environment, but the real physiological environment may be slightly different from the experimental results. However, the tendency of hyaluronic acid degradation does not change, because its natural molecular characteristics are high weight average molecular weight hyaluronic acid can supplement low weight average molecular weight hyaluronic acid.

Another preferred embodiment of the mixture of the present invention comprises, but is not limited to, mixing high and low weight average molecular weight hyaluronic acid according to a weight ratio of 1:1 (w/w), The preferred embodiment of the mixing ratio can be adjusted to a weight ratio of between 20:80 and 80:20 depending on clinical requirements. Hyaluronic acid mixtures with a higher proportion of low weight average molecular weight are more helpful for accelerating treatment. Conversely, a higher proportion of high weight average molecular weight hyaluronic acid mixture can provide longer degradation times and thus prolong the therapeutic effect.

As used herein, the mucosa includes, but is not limited to, the mucosa of the eye, the mucosa of the ear, the mucosa of the nose, the mucosa of the mouth, the mucosa of the respiratory tract, the mucosa of the gastrointestinal tract, the mucosa of the urethra, and the mucosa of the genital tract. The symptoms of the mucosa-related disease or disease of the present invention include ulcers, inflammatory reactions, allergic reactions, or bleeding. Preferred examples of mucosal related lesions or diseases include, but are not limited to, conjunctivitis, otitis, allergic rhinitis, gingivitis, oral ulcers, bronchitis, gastroesophageal reflux disease (GERD), esophagitis, gastritis, Enteritis, peptic ulcer (PU), inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), urethritis, cystitis and vaginitis. More preferred embodiments of enteritis include, but are not limited to, acute enteritis, chronic enteritis, infectious enteritis, ischemic enteritis, radioactive enteritis, and drug-induced enteritis. More preferred embodiments of peptic ulcer include, but are not limited to, gastric ulcer, duodenal ulcer, esophageal ulcer, and Meckel's Diverticulum ulcer. More preferred examples of inflammatory bowel disease (IBD) include, but are not limited to, Crohn's disease and ulcerative colitis. More preferred examples of stimulating bowel syndrome (IBS) include, but are not limited to, coeliac disease, fructose malabsorption, mild infection, such as pear-shaped flagella. Parasitic infection of giardiasis, chronic functional constipation and chronic functional abdominal pain.

The results of the present invention show that, as shown in Fig. 4, the degree of hyaluronic acid of the same weight average molecular weight was significantly absorbed in the injured colon tissue than in the normal colon tissue (P < 0.01). Comparison of the extent to which three different weight average molecular weight hyaluronic acids were absorbed in injured colon tissue, where the amount of 350,000 dalton hyaluronic acid shown by the fluorescence index was absorbed by the injured colon tissue, significantly higher than the other two weight average molecular weights. Hyaluronic acid (1 million and 2 million Daltons). Furthermore, the fluorescence index of 1 million Dalton hyaluronic acid absorbed by normal or injured colon tissue is higher than 2 million Dalton hyaluronic acid. This result explains the effect of rapid dispersion and coverage of low weight average molecular weight hyaluronic acid, which provides rapid wound healing and protects tissue from further injury. In conjunction with the above data, the present invention provides a rapid and sustained method of treating or preventing mucosal related diseases or diseases.

Another preferred embodiment of the composition of the present invention comprises the addition of an excipient to form the following dosage forms suitable for use in various parts or forms: eye, ear, mouth, nose, respiratory, gastrointestinal or topical use. More preferred embodiments of the oral dosage form of the present invention are selected from the following solid dosage forms, solutions including, but not limited to, suspensions, lozenges including, but not limited to, sustained release tablets, and capsules including, but not limited to, enteric-coated capsules (enteric-coated) Group of capsules. The preferred embodiment of the oral solid dosage form is a daily dose of between 50 mg and 1000 mg. A more preferred embodiment of the gastrointestinal dosage form is selected from the group consisting of solid dosage forms, perfusions, enemas, suppositories, and solutions including suspensions. A more preferred embodiment of the topical dosage form is selected from the group consisting of a perfusate, an enema, a spray, and an inhalant. And the group consisting of drip.

A preferred embodiment of the present invention for treating allergic rhinitis or bronchitis is the use of the composition of the present invention as a drug-binding adjuvant, wherein the drug comprises an antihistamine, an antiallergic agent, an anti-congestant, Steroids or anti-asthmatic drugs. A preferred embodiment of the present invention for treating enteritis is the use of the composition of the present invention as a drug-binding adjuvant, wherein the drug comprises an antibiotic or an antispasmodic. A preferred embodiment of the present invention for treating peptic ulcers uses the composition of the present invention as a drug-binding adjuvant, wherein the drug includes a coagulant, an antibiotic, an antacid, and an H2 blocker. (H2 blocker), potassium hydrogen ion pump blocker (PPI), cell or mucosal protective agent. A preferred embodiment of the present invention for treating inflammatory bowel disease is the use of the composition of the present invention as a drug-binding adjuvant, wherein the drug comprises a steroid, an immunosuppressive agent, an antibiotic, 5-amine salicylic acid (5) -aminosalicylic acid, 5-ASA) and its derivatives or anti-inflammatory drugs.

The present invention describes the treatment of inflammatory bowel disease by hyaluronic acid in different weight mixing ratios, which is measured by the average body weight change of the rat. As shown in Figures 5A and 5B, Group A represents a high weight average molecular weight (HMWHA): low weight average molecular weight (LMWHA) weight mixing ratio of 8:2. Group B represents HMWHA: LMWHA has a weight mixing ratio of 1:1. Group C represents the control group in which hyaluronic acid is replaced by phosphate buffer saline (PBS). Although statistical significance occurs only on days 5 and 6, the focus of this diagram is on the trend change from day 1 to day 14 for each group. As the number of experimental days increases, the experimental trend shows that both groups A and B have The effect of colitis (a type of inflammatory bowel disease). As shown in Fig. 5A, compared with the control group and the average body weight of the rats in groups A and B, after the whole experimental procedure until the 14th day, it can be seen that the administration of hyaluronic acid to colitis causes the rats of group A and B. The average weight continued to be higher than the control group. Significantly, this trend also shows that group B generally has a better therapeutic effect than group A, and represents a higher proportion of low-weight average molecular weight hyaluronic acid with better therapeutic effect. This result supports the concept of the present invention utilizing a high weight average molecular weight hyaluronic acid to supplement the low weight average molecular weight hyaluronic acid to prolong the therapeutic effect.

The present invention utilizes a rat rubbing nose as an indicator of allergy symptoms and a reduction in the number of rubbing noses as an indicator for improving allergy. As shown in Fig. 6, the results showed that on the 28th day, the results of the control group (only PBS administration) and the hyaluronic acid group were significantly different, indicating that the allergy symptoms have been successfully induced. Although there was no statistically significant difference on the 30th day, there was still a significant reduction in allergy symptoms between the control group and the A group. Therefore, the tendency to improve allergy symptoms can be highly confirmed by the experimental results of administering the hyaluronic acid mixture of the present invention to the target.

Perhaps the results of the various experiments of the present invention are somewhat different, however, it is to be noted that the appendix of the present invention can still deduce that the mixture of the present invention has the ability to improve mucosal-related lesions or diseases including allergies. The present invention has been confirmed by several related experiments, and the present invention takes a shorter experimental time course for proper display of results. Therefore, when the concept of the present invention is actually applied to an animal or a human body, a longer time course than the experiment should be required. Based on the theory and the results of the present invention, the high weight average molecular weight hyaluronic acid should be sufficient to supplement long-term assistance and/or compensate for the rapid therapeutic effects of low weight average molecular weight hyaluronic acid in the body.

A preferred embodiment of the present invention for treating irritation of intestinal tract syndrome (IBS) is the use of a composition of the present invention as a drug-binding adjuvant, wherein the drug comprises an antiallergic agent, an antispasmodic agent, an antidiarrheal inhibitor, and a nervous tissue. A neurolytic, a transquilizer, an narcotic analgesic, an antidepressant or a serotonin antagonist.

In a preferred embodiment of an oral dosage form (e.g., enteric coating tablet), the enteric coating provides better resistance to dissolution and digestion in the stomach. Until the intestines and colon are reached, the enteric coating will dissolve and the hyaluronic acid mixture will be released to form a protective film on the site of the colonic inflammation (where the ascending colon or transverse colon is located). The aim is to speed up the healing of the site of inflammation and to extend the treatment time by a long rate of degradation (long-term degradation of long-chain hyaluronic acid into short-chain hyaluronic acid).

In a preferred embodiment of the suppository, the suppository contains a hyaluronic acid mixture which can be administered to the anus such that the mixture will dissolve in the rectum and spread to other parts of the colon (e.g., descending colon) to form a protective film on the inflamed site of the colon. It can achieve accelerated treatment and sustained release.

In a preferred embodiment of the perfusate (e.g., enema), the hyaluronic acid mixture is herein the primary active ingredient and is mixed with excipients (e.g., PBS buffer), either directly or in a hose. And injected into the colon. The hyaluronic acid mixture will be infused into the colon and spread to other parts of the colon (such as the descending colon) to form a protective film on the inflamed area of the colon, which in turn accelerates the treatment of the inflamed area and achieves a sustained release effect.

A more preferred embodiment of the invention for receiving a therapeutic target is a mammal, most The preferred embodiment is labeled as human.

According to the above description, after mixing at least two pharmaceutically acceptable hyaluronic acid salts of different weight average molecular weights, rapid coverage of the injured or inflamed surface can be achieved while shortening the treatment period and lengthening the degradation time to prolong the coverage period. Therefore, rapid treatment and sustained release effects can be achieved simultaneously. The present invention provides a relatively simple and safe method of treating the aforementioned lesions or diseases and can be cost effective to provide a better choice to assist the patient in question.

The present invention is intended to be only a preferred embodiment, and it is obvious to those skilled in the art that the present invention can be modified, modified, and changed. However, the core concepts of these actions should have been disclosed by the present invention. Therefore, these conversions, modifications and variations fall within the scope of the patent application scope of this application. The descriptions of the present invention and accompanying drawings are used as illustrations and not limitation.

Example 1: Degradation of hyaluronic acid in 1 U (U/ml) hyaluronic acid hydrolase (HAase) per ml

step:

1. 0.25 g of high weight average molecular weight sodium hyaluronate powder (labeled HHA, high weight average molecular weight, molecular weight: 2 million Daltons, available from Freda) and 0.25 grams of low weight average molecular weight sodium hyaluronate powder (labeled LHA, The low weight average molecular weight, molecular weight: 350,000 Daltons, purchased from Freda, was separately added to 50 ml of phosphate buffer to form a 0.5% solution, followed by stirring for 6 hours until the powder was completely dissolved.

2. Add 0.05 g of LHA powder and 0.2 g of HHA powder (ratio 2:8, intermediate weight average molecular weight, MHA) to 50 ml PBS buffer The mixture was stirred for 6 hours until the powder was completely dissolved.

3. The mobile phase solution of the gel permeation chromatograph (GPC) system is prepared by (1) adding 35.49 grams of disodium hydrogen phosphate (Na ₂ HPO ₄ ) powder to 450 ml of deionized distilled water ( Deionized-distilled water, dd water), stirred at room temperature for 30 minutes to form a 0.5 volume molar concentration (M) Na ₂ HPO ₄ solution; (2) 18 grams of NaH ₂ PO ₄ powder was added to 250 ml of deionized The water was distilled and stirred at room temperature for 30 minutes to form a 0.5 M NaH ₂ PO ₄ solution.

4. The hyaluronic acid hydrolase powder was added to the phosphate buffer at 4 ° C to prepare a 1 U/ml hyaluronic acid hydrolase.

5. 2 ml of hyaluronic acid sample, 1 ml of 10 U/ml hyaluronic acid hydrolase and 7 ml of phosphate buffer were mixed and added to a 15 ml glass tube, and shaken vigorously for 3 minutes.

6. Place the glass tube in a water bath and shake at 50 rpm. 1 ml of each solution was taken at 15, 30, 45, 60, 75, 90, 105 and 120 minutes, respectively, and then 1 ml of hyaluronic acid hydrolase was added at the above time. Each of the 1 ml solutions was filtered through a 0.45 micron (μm) pore size filter. A 20 microliter (μl) solution was injected into the GPC system and the map was recorded.

7. The state of the GPC system is set to: (1) column: 2X GMPWx1 (TSK-gel); (2) mobile phase flow rate: 1 ml per minute (ml/min); (3) temperature: 30 °C.

8. All values shown refer to the average observed. The histological index was analyzed using Student's t-test.

result:

Figure 2 shows the hyaluronic acid retention time of the GPC map. The vertical axis represents the residence time in GPC, and the horizontal axis represents the residence time of hyaluronic acid in the solution containing hyaluronic acid hydrolase. The horizontal dashed line represents the hyaluronic acid retention time of the molecular weights of 2 million, 1 million, 350,000 and 17,000 Daltons from top to bottom. As the three hyaluronic acid degradation increases, the residence time also increases significantly. After 15 minutes of degradation, the weight average molecular weight of HHA and MHA degraded to approximately 1 million Daltons. After 37 minutes of degradation, the weight average molecular weight of HHA and MHA degraded to approximately 350,000 Daltons. After 90 minutes of degradation, the weight average molecular weight of HHA, MHA and LHA degraded to no significant difference between the three, and both were greater than 17,000 Daltons.

Example 2: Degradation of hyaluronic acid in 0.1 equivalent (N) hydrochloric acid (HCl)

step:

1. The preparation of LHA, MHA and HHA is the same as in Example 1.

2. The mobile phase solution of the GPC system was prepared in the same manner as in Example 1.

3. 5.72 ml of 17.5 N HCl and 90 ml of deionized distilled water were mixed and stirred for 10 minutes to prepare an artificial gastric juice (0.1 N HCl) as a stock solution.

4. In a 15 ml glass tube, mix 2 ml of HHA, MHA and LHA with 8 ml of artificial gastric juice and vigorously stir for 3 minutes.

5. The glass tube was placed in a water bath and shaken at 37 ° C at 50 rpm. 1 ml of each solution was taken at 6, 12, 24 and 48 hours, respectively, and then 1 ml of artificial gastric juice was added at the above time points. Each 1 ml solution was filtered through a 0.45 micron pore size filter. A 20 microliter solution was injected into the GPC system and the map was recorded.

6. All values shown refer to the average observed. The histological index was analyzed using Student's t-test.

result:

Figure 3 shows the hyaluronic acid retention time of the GPC map. The vertical axis represents the residence time in GPC, and the horizontal axis represents the residence time of hyaluronic acid in the artificial gastric juice. The horizontal dashed line represents the hyaluronic acid retention time of the molecular weights of 2 million, 1 million, 350,000 and 170,000 Daltons from top to bottom. As the three hyaluronic acid degradation increases, the residence time also slowly increases. After 7 hours of degradation, the weight average molecular weight of HHA and MHA degraded to approximately 1 million Daltons. After 31 hours of degradation, the weight average molecular weight of the MHA degraded to approximately 350,000 Daltons. After 35 hours of degradation, the weight average molecular weight of HHA also degraded to approximately 350,000 Daltons. The above data indicate that hyaluronic acid is slowly degraded in artificial gastric juice, and the weight average molecular weight of HHA and MHA is greater than 1 million Dalton after 6 hours of degradation, while the weight average molecular weight of HHA and MHA is degraded after 24 hours. Both are more than 350,000 Daltons.

Example 3: Adhesion of hyaluronic acid in colon tissue (through the third edition of the IVIS image system)

step:

1. Preparation of LHA and HHA is as in Example 1. MHA (molecular weight: 1 million Daltons, purchased from Freda) was added to 50 ml of phosphate buffer, and then stirred for 6 hours until the powder was completely dissolved for use in the next step.

2. Fluorescent hyaluronic acid (HA-f) was prepared in the following manner: (1) 0.39 g of MES free acid [2-(N-morpholine)-ethanesulfonic acid (2-(N-morpholino)) Ethylsulfonic acid (purchased from Calbiochem)] was dissolved in 100 ml of deionized distilled water. (2) Solution A: 65 mg of fluoresceinamine powder (isomer I, available from Fluka) was dissolved in 9 ml of a 95% alcohol solution and stirred for 10 minutes in the dark. (3) Solution B: 359 mg of [N-(3-dimethylpropylpropyl)-N-ethylcarbodiimide hydrochloride] [N-(3-Dimethylamino propyl)-N-ethyl carbodiimide hydrochloride, EDC] (purchased from Sigma) powder was dissolved in 9 ml of MES buffer and stirred for 10 minutes. (4) Solution C: 216 mg of N-Hydroxysuccinimde (NHS) powder (purchased from Sigma) was dissolved in 9 ml of MES buffer and stirred for 10 minutes. (5) 3 ml of the solution A was slowly dropped into 50 ml of a 0.5% hyaluronic acid solution, and stirred for 10 minutes in the dark. (6) 3 ml of the B solution and 5 ml of the C solution were respectively dropped into the solution of the step (5), and stirred for 10 minutes in the dark. (7) The 0.02 M MES buffer was slowly dropped into the solution of the step (6) until the volume reached 100 ml, and stirred at room temperature for 24 hours in the dark. (8) Pour the reacted product into a dialysis tube (molecular weight: 12000-14000), use 5 liters of deionized distilled water as dialysate, and stir at 4 ° C for 5 days in the dark, during which dialysis is changed every 12 hours. Liquid until there is no fluorescence in the dialysate. (9) The dialysis liquid was dispensed in a 50 ml plastic centrifuge tube and then stored in a -20 ° C refrigerator overnight. It was then dried in a freeze dryer under light. (10) The dried HA-f powder was stored in a refrigerator at -20 °C. (11) 50 mg of HA-f powder was slowly added to 10 ml of phosphate buffer and stirred for 6 hours until the powder was completely dissolved.

3. Cut the colon tissue of 7-8 weeks old Shih Tzu's rats (Sprague-Dawley, SD), rinse with phosphate buffer, and cut into 3-4 cm. Long, finally soaked in phosphate buffer.

4. The injured colon tissue was prepared by brushing 20 times in the longitudinal direction of the toothbrush and then immersed in phosphate buffer.

5. Place normal and injured colon tissue into a 12-well culture dish, add 1 ml of 0.5% HA-f solution to each well, and then shake at room temperature for 2 hours. Excessive HA-f solution was aspirated by a micropipette after 2 hours, and after soaking in phosphate buffer for 10 minutes, the phosphate buffer was removed. Repeat the above steps three times.

6. Place the clean colon tissue lining tissue upside in a 12-well culture plate and place it on a dock of IVIS ( in vivo image system, in vivo image system, XENOGEN). The factory parameters were set to green fluorescent protein (GFP), the excitation wavelength was set to 465 nm (nm), and the emission wavelength was set to 500 nm, and the image was captured by the software.

7. All values shown refer to the average observed. The histological index was analyzed using Student's t-test.

result:

Figure 4 shows the quantification and arrangement results of the fluorescence index. The fluorescence index of normal colon tissue is defined as 1. Other colon tissues are then calibrated against defined values. The results showed that hyaluronic acid of the same weight average molecular weight was significantly more adherent to the injured colon tissue than in normal colon tissue (P < 0.01). Comparing the results of three different weight average molecular weight hyaluronic acid adhesion to injured colon tissue, the fluorescence index of the absorbed 350,000 Dalton hyaluronic acid was significantly higher than the other two hyaluronic acids (2 million and 1 million Daltons). Even if it is normal In the injured colon tissue, the absorbed 1 million Dalton hyaluronic acid has a fluorescence index higher than 2 million Dalton hyaluronic acid.

Example 4: Changes in body weight of rats given or not administered with hyaluronic acid for colitis

step:

1. Test purpose: In order to evaluate the effect of mixing two different weight average molecular weight hyaluronic acid to treat or prevent colitis by weight ratio, trinitrobenzenesulfonic acid (TNBS) in specific pathogen-free Colitis was induced in the history of the SPF class of rats.

2. Test sample: IBD98, which contains low weight average molecular weight (LMWHA) and high weight average molecular weight (HMWHA), of which HMWHA is 2 million Dalton hyaluronic acid, LMWHA is 350,000 Dalton hyaluronic acid, the mixing ratio is 8:2 (Classified as Group A) and 1:1 (Classified as Group B). Dissolved in phosphate buffer to produce a 0.125% (w/v) concentration of hyaluronic acid solution.

3. Methods: (1) Test target: Eight-week-old rats were selected and divided into three groups: group A represents hyaluronic acid mixing ratio of 8:2; group B represents hyaluronic acid mixing ratio of 1:1; group C represents phosphoric acid Salt buffer instead of hyaluronic acid control group. (2) Animal experiments: Rats in all experimental groups (groups A and B) were given an empty stomach for two days. On the first day of the experiment, rats were anesthetized with a concentration of 50 mg of 1 ml of trinitrobenzenesulfonic acid per ml via the rectum. From day 4 to day 14, rats of groups A and B were administered 1 ml of two IBD98 via the rectum. On the ninth day, the body weight of half of the rats in group A and group B was observed. The change in body weight of the other half of the rats was observed on the 14th day. Rats in all control groups were given an empty stomach for two days. On the first day of the experiment, the rats were anesthetized via straight Intestine is administered at a concentration of 50 mg of 1 ml of trinitrobenzenesulfonic acid per ml. From day 4 to day 14, 1 ml of phosphate buffer was administered via the rectum daily. On the ninth day, the body weight of half of the rats in group C was observed. The change in body weight of the other half of the rats was observed on the 14th day.

result:

1. Inflammatory index: The present invention utilizes changes in mean body weight to observe the results of improved colitis (IBD). Changes in body weight are a convenient and straightforward indicator of the outcome of a treatment.

2. Observing the trend of group A and group B, it can be found that, as shown in Fig. 5 and Fig. 5B, colitis which represents inflammatory bowel disease (IBD) from day 1 to day 14 has a mitigating result in both groups. . Observing the average body weight change between the control group and the experimental group, as shown in Fig. 5B, the therapeutic effect of the administration of hyaluronic acid colitis caused the average body weight of the experimental group to be maintained even during the whole experiment period until the 14th day. The average weight of the control group is high.

Example 5: Therapeutic effect of hyaluronic acid on allergic diseases

step:

1. Test purpose: Allergic diseases were induced in a history-specific T. sinensis-free rat, which is allergic rhinitis.

2. Test sample: IBD98, which contains low weight average molecular weight and high weight average molecular weight, wherein HMWHA is 2 million Dalton hyaluronic acid, LMWHA is 350,000 Dalton hyaluronic acid, the mixing ratio is 1:1, soluble in phosphate buffer A 0.125% (w/v) concentration of hyaluronic acid solution was produced in the solution.

3. Methods: (1) Test criteria: 9 rats of 8 weeks old were randomly divided into two groups: a control group consisting of 4 rats (phosphate buffer) and an experiment consisting of 5 rats. Group (hyaluronic acid). (2) Animal experiment: From the first day to the seventh day of the experiment, intraperitoneally (IP) contained 1 mg of ovalbumin and 10 mg of alum physiological saline per day. Only observe from day 8 to day 13. From day 14 to day 27, egg albumin-containing saline (10 μg/10 μl per peritone) was instilled into the nostrils on both sides of the rat using a micropipette. After the allergic antigen reaction, the therapeutic effect of hyaluronic acid on the symptoms on the nose was evaluated on the 28th and 30th day, respectively. One hour before the instillation of ovalbumin, the rats were first dropped into hyaluronic acid solution or phosphate buffer (25 μl per nostril). After instillation of ovalbumin-containing saline (10 μg/10 μl per nostril) into the nostrils on both sides, the rats were placed in an observation cage (1 rat per cage) and their 30 minute course The number of times the nose is rubbed is counted. The frequency of rubbing the nose is calculated as the number of times the nose is rubbed every 30 minutes.

result:

1. Allergy improvement index: The present invention uses the number of times the rat rubs the nose as an indicator for improving allergy symptoms.

2. As shown in Figure 6, the hyaluronic acid sample has a therapeutic effect on antigen-induced nasal symptoms. The average friction nose frequency induced by ovalbumin was 56.25 ± 16.1 times / 30 minutes on day 28 and 70.0 ± 41.3 times / 30 minutes on day 30. Compared with the control group, the frequency of the experimental group rubbing the nose was statistically significantly reduced on the 28th day, but on the 30th day, there was still a significant decrease.

Figure 1 is a general chemical structural formula of hyaluronic acid.

Figure 2 is a hyaluronic acid retention time displayed by a gel permeation chromatograph; The vertical axis represents the residence time in GPC, and the horizontal axis represents the degradation time of hyaluronic acid in hyaluronic acid hydrolase (HAase) solution.

Figure 3 shows the hyaluronic acid retention time by GPC, where the vertical axis represents the residence time in GPC and the horizontal axis represents the degradation time of hyaluronic acid in an artificial gastric juice solution.

Figure 4 is the adhesion of hyaluronic acid exhibited by the fluorescence coefficient in normal and injured colon tissue, wherein the fluorescence index = colon tissue treated with hyaluronic acid / normal colon tissue treated with phosphate buffer solution.

Figure 5A is an effect of a hyaluronic acid mixture in the treatment of colitis, which is shown by the average body weight of the rats on days 1 to 9 of the experiment.

Figure 5B is an effect of a hyaluronic acid mixture in the treatment of colitis, which is shown by the average body weight of the rats on days 1 to 14 of the experiment.

Figure 6 shows the effect of hyaluronic acid on rat nasal allergy induced by ovalbumin (10 μg/10 μl/nostril). On day 28, there was a significant difference between hyaluronic acid group and control group (* p <0.05).

Claims (13)

A use of a hyaluronic acid mixture for the manufacture of a composition for treating or preventing a respiratory mucosa-related disease or disease, wherein the hyaluronic acid mixture comprises a low weight average molecular weight hyaluronic acid (LMWHA) and a high weight average molecular weight hyaluronic acid (HMWHA), wherein the low weight average The weight average molecular weight of the molecular weight hyaluronic acid is between 50,000 Daltons and 1.5 million Daltons, and the weight average molecular weight of the high weight average molecular weight hyaluronic acid is between 1.5 million Daltons and 5 million Daltons, wherein The weight average molecular weight of the low weight average molecular weight hyaluronic acid and the high weight average molecular weight hyaluronic acid is at least 500,000 Daltons, and the weight ratio of the low weight average molecular weight hyaluronic acid to the high weight average molecular weight hyaluronic acid is 20:80 to 80:20. The use according to the first aspect of the patent application, wherein the low weight average molecular weight hyaluronic acid has a weight average molecular weight of between 100,000 Daltons and 1.5 million Daltons. The use of the low weight average molecular weight hyaluronic acid has a weight average molecular weight of between 100,000 Daltons and 500,000 Daltons, according to the use of claim 2. According to the use of the first aspect of the patent application, wherein the weight average molecular weight of the high weight average molecular weight hyaluronic acid is between 1.5 million Daltons and 3.5 million Daltons. According to the use of claim 4, the weight average molecular weight of the high weight average molecular weight hyaluronic acid is between 1.5 million Daltons and 2.5 million Daltons. According to the use of the first aspect of the patent application, wherein the weight ratio of the low weight average molecular weight hyaluronic acid to the high weight average molecular weight hyaluronic acid is 1:1. According to the use of the first aspect of the patent application, wherein the therapeutically effective dose is between 0.05 mg/ml and 50 mg/ml. The use according to item 7 of the scope of the patent application, wherein the therapeutically effective dose is between 0.05 mg/ml and 5 mg/ml. According to the use of the first aspect of the patent application, the symptoms of the respiratory mucosa-related lesion or disease include inflammation, allergic reaction or bleeding. According to the use of the first aspect of the patent application, the respiratory mucosa-related lesion or disease refers to allergic rhinitis or bronchitis. The use according to claim 1, wherein the composition further comprises an excipient for formulating the composition into a dosage form suitable for use in the nose or respiratory tract. According to the application of the scope of claim 11, the dosage form is spray or inhalant for use in the nose or respiratory tract. According to the use of claim 10, wherein allergic rhinitis or bronchitis is treated with the composition as an adjuvant in combination with a drug, which is an antihistamine, an antiallergic, an anti-congestant, a steroid or an antibiotic. Asthma pills.