DRUG COMBINATION FOR PREVENTING OR TREATING IRRITABLE BOWEL SYNDROME

Abstract

Disclosed is a drug combination for preventing or treating irritable bowel syndrome. The drug combination comprises dosmalfate and lysozyme.

Claims

1. A method of preventing or treating irritable bowel syndrome comprising administering a therapeutically effective amount of dosmalfate and lysozyme to a subject in need thereof.

2. The method according to claim 1, wherein a mass ratio of dosmalfate to lysozyme is (0.05-100):1, and does not comprise 0.05:1.

3. The method according to claim 1, wherein the mass ratio of dosmalfate to lysozyme is (0.1-10):1.

4. A pharmaceutical composition for preventing or treating irritable bowel syndrome, wherein the pharmaceutical composition comprising dosmalfate and lysozyme.

5. The pharmaceutical composition according to claim 4, wherein a mass ratio of dosmalfate to lysozyme in the pharmaceutical composition is (0.05-100):1, and does not comprise 0.05:1.

6. The pharmaceutical composition according to claim 5, wherein the mass ratio of dosmalfate to lysozyme in the pharmaceutical composition is (0.1-10):1.

7. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition is an oral formulation.

8. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition also comprises pharmaceutically acceptable adjuvants.

9. A method for preparing the pharmaceutical composition according to claim 4, comprising the following step: mixing dosmalfate with lysozyme.

10. The method for preparing the pharmaceutical composition according to claim 8, comprising the following steps: preparing a dosmalfate formulation subunit and a lysozyme formulation subunit with dosmalfate and lysozyme as well as the pharmaceutically acceptable adjuvants respectively, and mixing the two formulation subunits to obtain the pharmaceutical composition.

11. The method according to claim 1, wherein the irritable bowel syndrome has the characteristic of visceral hypersensitivity.

12. The method according to claim 1, wherein the daily dosage of the dosmalfate is 0.05-20 g.

13. The method according to claim 1, wherein dosmalfate or lysozyme can be respectively administrated for 1-3 times/day.

14. The method according to claim 1, wherein the dosmalfate or lysozyme can be respectively administrated in a form of separate formulations, or in a form of existing in the same formulation.

15. The method according to claim 14, wherein the dosmalfate and lysozyme are administrated in a form of pharmaceutical composition.

16. The method according to claim 15, wherein the pharmaceutical composition is an oral formulation.

17. The method according to claim 16, wherein the oral formulation is any one selected from an oral normal-release formulation, an oral sustained-release formulation and an oral controlled-release formulation.

18. The method according to claim 1, wherein the lysozyme is prepared into an enteric formulation.

19. The pharmaceutical composition according to claims 7, wherein the oral formulation is any one selected from an oral normal-release formulation, an oral sustained-release formulation and an oral controlled-release formulation.

20. The method for preparing the pharmaceutical composition according to claim 10, wherein the dosmalfate formulation subunit being in an enteric dosage form or a gastric dosage form, and the lysozyme formulation subunit being in the enteric dosage form.

Description

DETAILED DESCRIPTION

[0047] The following examples are further enumerated to illustrate the present disclosure in detail. It should also be understood that the following examples are only used to further illustrate the present invention, and should not be construed as limiting the protection scope of the present disclosure. Some non-essential improvements and adjustments made by those skilled in the art according to the principles set forth in the present disclosure belong to the scope of the present disclosure. The specific process parameters and the like in the following examples are only an example in the appropriate range, that is, those skilled in the art can make selections within the appropriate range through the descriptions herein, and are not limited to the specific data in the following examples.

[0048] Definition:

[0049] Irritable bowel syndrome (IBS): IBS is a functional intestinal disease, exhibiting abdominal pain or discomfort associated with changes in defecation or defecation habit change relevant abdominal pain or discomfort. Abdominal pain, bloating, distension and dysporia are common features.

[0050] Visceral hypersensitivity: visceral hypersensitivity refers to enhancement of perceptibility of intestinal tract to stimulation, including a reduced threshold triggering visceral pain or uncomfortable stimulation, visceral discomfort to physiological stimulation or a strong response to noxious stimulation. Not only can rectum and colon show visceral hypersensitivity but also other areas of the digestive tract such as jejunum and esophagus can also exhibit visceral hypersensitivity. Animals or patients with irritable bowel syndrome have visceral hypersensitivity. Visceral hypersensitivity is a core pathophysiological mechanism of abdominal discomfort symptoms such as abdominal pain and abdominal bloating in IBS. Visceral hypersensitivity can amplify gastrointestinal motility events to produce symptoms. Visceral hypersensitivity in animals or patients with irritable bowel syndrome includes, but is not limited to, sensitivity to colorectal distention (pressure) stimulation, high sensitivity to temperature stimulation, and high responsiveness to physiological stimulation (such as meals).

[0051] Dosmalfate: dosmalfate is a gastric mucosa protecting agent approved for treatment of gastric and duodenal ulcers. Its chemical name is a diosmin heptadiene (bisulfate) aluminum complex with a molecular formula Al.sub.7(OH).sub.14(C.sub.28H.sub.25O.sub.36S.sub.7)[Al(OH).sub.3].sub.7. Animal experiment and human body researches both show that dosmalfate can effectively treat peptic ulcer, and its adverse reactions are mild and the incidence is low.

[0052] Lysozyme: lysozyme is a lysozyme derived from animals, plants and microorganisms, or a recombinant of natural lysozyme. For example, it can be egg lysozyme, human lysozyme, recombinant human lysozyme, phage lysozyme, etc. The lysozyme in the present disclosure also comprises its medicinal salts, such as hydrochloride, chloride, sulfate or amino acid salt, etc. Egg lysozyme is the main commercial source of lysozyme, and its activity is close to that of human lysozyme. The lysozyme used in many marketed drugs is egg lysozyme. Human lysozyme is widely distributed and abundant in human body. Lysozyme has high safety.

[0053] Enteric formulation: the enteric formulation refers to a formulation that does not release or almost does not release drug in the stomach, but can release most or all of the drug in some parts of the intestine after entering the intestine. Human intestines include small intestine and large intestine, in which the small intestine is also divided into duodenum, jejunum and ileum, and the large intestine is also divided into cecum, colon and rectum. Different parts of the digestive tract have different pH values. For example, the pH value in the stomach is about 1-3, the pH value in the small intestine is about 4-7, and the pH value in the large intestine is about 7-8. By selecting pH dependent degradation materials as formulation adjuvants, the formulations that release drugs in specific parts of the digestive tract can be prepared, such as small intestinal enteric formulations or large intestinal enteric formulations. Specifically, it can comprise a duodenal enteric formulation, a jejunal enteric formulation, an ileal enteric formulation, a cecal enteric formulation, a colon enteric formulation or a rectal enteric formulation, etc.

[0054] Exemplary preparation methods of a separate formulation of dosmalfate, a separate formulation of lysozyme, and a pharmaceutical composition of dosmalfate and lysozyme are described below.

[0055] Raw materials of lysozyme can be commercially available, and has a potency of above 20000 U/mg; raw materials of dosmalfate can be prepared according to the existing technical method.

[0056] Dosmalfate tablet: the dosmalfate tablet is obtained by tableting using common tablet adjuvants based on dosmalfate as a raw material.

[0057] Lysozyme granules: the granules are prepared by using lysozyme as a raw material, adding an equal amount of starch, evenly mixing, adding an adhesive (water) and then drying.

[0058] Lysozyme colon enteric-coated granules: the lysozyme granules are prepared by using lysozyme as a raw material, adding an equal amount of starch, evenly mixing, adding an adhesive (water) to granulation and then drying to obtain the lysozyme granules. The lysozyme colon enteric-coated granules are obtained by coating the prepared lysozyme granules using commercially available colon enteric coatings according to a conventional method.

[0059] Dosmalfate-lysozyme compounded capsules: the lysozyme granules are prepared by using lysozyme as a raw material, adding an equal amount of starch, evenly mixing, adding an adhesive (water) to granulation and then drying to obtain the lysozyme granules. The lysozyme colon enteric-coated granules are obtained by coating the prepared lysozyme granules using commercially available colon enteric coatings according to a conventional method. The dosmalfate granules are prepared by using dosmalfate as a raw material, adding common granule adjuvants and granulating. The lysozyme colon enteric-coated granules and the dosmalfate granules are put into common empty capsules in a proper proportion to obtain the dosmalfate-lysozyme compounded capsules.

[0060] The present disclosure will be illustrated in detail through animal experiments in the following examples.

[0061] The used test animals, reagents and others are commercially available. Relevant testing methods and specific operation manners of related instruments used in tests are all well-known to the skilled in the art. The used dosmalfate and lysozyme colon enteric-coated granules are all provided by Xiangbei Welman Pharmaceutical Co., Ltd.

EXAMPLES

[0062] Therapeutic Effect of a Combination of Dosmalfate and Lysozyme on Irritable Bowel Syndrome Model Animals

[0063] In this example, the irritable bowel syndrome model was induced by daily applying colorectal distension as a stimulating factor to neonatal SD rats (Sprague-Dawley rats) from 8 days old to 21 days old. This model animal had visceral hypersensitivity of irritable bowel syndrome and without colonitis and mucosal injury. Colorectal distension stimulation was not given any more from 21 days old to 8 weeks old. A test drug or placebo was administered daily by gavage from 8 weeks old to 3 months old. On the 8 weeks old (before the beginning of the 1-month drug administration period) and about 3 months old (after the end of the 1-month drug administration period), behavioral and neural responses of rats to colorectal distention stimulation were tested by an abdominal wall withdrawal reflex test and an electromyography test.

[0064] 1. Animals and Grouping

[0065] 8-day-old neonatal male SD rats with normal body weight were exposed to a 12:12 hr light-dark cycle. Every 12 neonatal rats and 1 adult female rat were co-bred with rat milk until 25 days old. Then every 4 neonatal rats were fed in the same cage, and they were allowed to eat and drink freely.

[0066] The SD rats were randomly divided into a normal control group, a model group, a dosmalfate group, a lysozyme group, a first combination group (a dosage ratio of dosmalfate to lysozyme was 0.05:1), a second combination group (a dosage ratio of dosmalfate to lysozyme was 0.1:1), a third combination group (a dosage ratio of dosmalfate to lysozyme was 10:1), and a fourth combination group (a dosage ratio of dosmalfate to lysozyme was 20:1), and 12 animals were assigned to each group.

[0067] 2. Modeling

[0068] Except for normal control group, colorectal distension (CRD) stimulation was applied to the rats in other groups every day from 8 days old to 21 days old to induce irritable bowel syndrome. The concrete operation method was as follows: a flexible balloon (with a polytetrafluoroethylene catheter; the balloon should be inflated overnight before the experiment so that it has better compliance) made of the fingers of surgical gloves, was smeared with vaseline, inserted from the anus of the rat to the colon and then fixed at the tail distanced away from the anus by 2 cm. The balloon was connected with a valve via the catheter, and the valve was additionally connected with a sphygmomanometer. Rats were placed in small plastic cages to restrict their turning and acclimatized for 30 minutes. The balloon was slowly inflated to 60 mm Hg with the sphygmomanometer, and deflated after stimulation for 20 seconds; the stimulation was repeated twice within 1 hour (with an interval of 30 minutes each time).

[0069] Rats in normal control group were treated with the method similar to that in other groups, but the balloon was not inserted into the colon, and the rats were gently palpated in the perineal area every day from 8 days old to 21 days old.

[0070] When the modeling was completed, only one rat in normal control group died, and the follow-up experimental processes were completed for the rest animals.

[0071] 3. Preparation and Administration of Drugs

[0072] Except for normal control group and model group, test drugs (wetted in 0.5% sodium carboxymethyl cellulose solution) were administrated to the rats in the other groups by gavage daily from 8 weeks old to 3 months old, and the dosage was 8 ml.

[0073] Lysozyme group: 100 mg/kg (calculated by lysozyme) of lysozyme colon enteric-coated granules.

[0074] Dosmalfate group: 100 mg/kg of dosmalfate.

[0075] First combination group: 100 mg/kg (calculated by lysozyme) of lysozyme colon enteric-coated granules, and 5 mg/kg of dosmalfate.

[0076] Second combination group: 100 mg/kg (calculated by lysozyme) of lysozyme colon enteric-coated granules, and 10 mg/kg of dosmalfate.

[0077] Third combination group: 100 mg/kg (calculated by lysozyme) of lysozyme colon enteric-coated granules, and 1000 mg/kg of dosmalfate.

[0078] Fourth combination group: 100 mg/kg (calculated by lysozyme of lysozyme colon enteric-coated granules, and 2000 mg/kg of dosmalfate.

[0079] An equal amount of 0.5% sodium carboxymethyl cellulose solution was daily administrated to model group. No drugs were administrated to normal control group.

[0080] 4. Test

[0081] The colorectal distension (CRD) model has become a standard tool for assessing visceral sensitivity in animals. Abdominal wall withdrawal reflex and EMG tests are the most commonly used methods to evaluate animal responses after colorectal distension stimulation.

[0082] 4.1 Abdominal Withdrawal Reflex (AWR) Test

[0083] The abdominal wall withdrawal reflex test was used to detect behavioral responses of rats to colorectal distension stimulation. This test was performed when rats in respective groups were 8 weeks old (before the start of the 1-month drug administration period) and 3 months old (after the end of the 1-month drug administration period). The rats were fasted overnight before undergoing the abdominal wall withdrawal reflex test, so as to reduce gastrointestinal contents.

[0084] During the test, a flexible balloon (with a thin-walled polytetrafluoroethylene catheter; the balloon should be inflated overnight before the experiment so that it has better compliance) made of the fingers of surgical gloves, was smeared with vaseline and inserted from the anus of the rat by 7 cm to the colon and fixed at the caudal point by 2 cm distanced away from the anus. The balloon was connected with the valve via the catheter, and the valve was additionally connected with a sphygmomanometer. Rats were placed in small plastic cages to restrict their turning and acclimatized for 30 minutes. The balloons were inflated with the sphygmomanometer to be tested under 20, 40 and 60 mm Hg pressures with the sphygmomanometer. During each test, the pressure was maintained for 20 seconds, and the abdominal wall withdrawal reflex score of the rats during the 20 seconds of pressure maintenance was recorded by a blinded observer. The test was repeated 3 times under each pressure condition, and the average value of the 3 tests was taken as a result. Rats were allowed to rest for 10 minutes between every two tests and also between different stresses.

[0085] The abdominal wall withdrawal reflex (AWR) has a scoring rule as following: 0=no behavioral response; 1=transient movement of a head, followed by immobility; 2=abdominal muscle contraction;

[0086] 3=lifting the abdomen off the platform of cage; 4=pelvic structure arched.

[0087] 4.2 Electromyography (EMG) Test

[0088] The visceral hypersensitivity (neural response) of rats to colorectal distension was quantified by measuring the electromyographic activity of external oblique abdominal muscles. When the rats in each group were 3 months old (after the end of the 1-month drug administration period), they were subjected to abdominal wall withdrawal reflex test followed by EMG test after abdominal wall withdrawal reflex test.

[0089] On the second day when 3-month-old rats in each group were subjected to abdominal wall withdrawal reflex test, an appropriate amount of pentobarbital solution (50 mg/ml) was injected into the abdominal cavities of the rats, and the rats were fully anesthetized. The hairs on the lower abdomen and the back of the neck were cut, a needle electrode (tungsten microelectrode) was implanted into the left side of the abdomen of the rat, fixed in the muscle layer of the external oblique abdominal muscle, and the electrode wire was crossed through the subcutaneous of the left side of the rat to a small incision at the rear neck and out of the small incision. The wounds on the abdomen and neck of the rat were sutured and the rats were returned to the cage. On the day 3 after surgery, the rats were fasted overnight to reduce gastrointestinal contents. On the day 4 after surgery, electromyography test was started. The colorectal distention stimulation was performed with the method identical to that in abdominal wall withdrawal reflex test, and the discharge activity of the external oblique abdominal muscle of the rat under the stimulation of each colorectal distention pressure (20, 40 and 60 mm Hg) was recorded by using a physiological signal acquisition and processing system. The pressure was maintained for 20 seconds each time, each pressure was tested 3 times, and the average value of the 3 tests was taken as the result. Rats were allowed to rest for 10 minutes between each test and also between different stresses.

[0090] The EMG test results are represented by the area under the curve (AUC) during the 30-second period after the onset of colorectal distention, and each datum is normalized relative to the average baseline amplitude (set to 100%).

[0091] 5. Test Results and Evaluation

[0092] Abdominal Withdrawal Reflex and EMG Tests

[0093] The higher score of the abdominal wall withdrawal reflex indicates the stronger behavior response of the rat to colorectal distension stimulation and further indicates the higher visceral sensitivity, thus showing the more severe irritable bowel syndrome.

[0094] The larger AUC value (normalized relative to the average baseline amplitude) of EMG indicates the stronger neural response of the rat to colorectal distension stimulation, and further indicates higher visceral sensitivity, thus showing the more severe irritable bowel syndrome. The statistical results of this test are listed in Tables 1-3.

TABLE-US-00001 TABLE 1 Abdominal withdrawal reflex test score results of animals in each group on 8 weeks old Abdominal withdrawal reflex scores of colorectal distension at different Numbers pressures (mean + SD ) Groups of rats 20 mm Hg 40 mm Hg 60 mm Hg Normal control group 11 0.64 ± 0.10  1.21 ± 0.22  2.33 ± 0.30  Model group 12 2.39 ± 0.53** 2.67 ± 0.40** 3.58 ± 0.41** Dosmalfate group 12 2.31 ± 0.41** 2.50 ± 0.46** 3.56 ± 0.48** Lysozyme group 12 2.14 ± 0.30** 2.58 ± 0.47** 3.61 ± 0.19** First combination group 12 2.33 ± 0.53** 2.42 ± 0.35** 3.42 ± 0.38** Second combination group 12 2.28 ± 0.42** 2.50 ± 0.30** 3.50 ± 0.30** Third combination group 12 2.31 ± 0.54** 2.56 ± 0.26** 3.64 ± 0.27** Fourth combination group 12 2.25 ± 0.38** 2.42 ± 0.21** 3.42 ± 0.35** **represents that there is significant difference compared with normal control group under the corresponding pressure (P < 0.01).

TABLE-US-00002 TABLE 2 Abdominal withdrawal reflex test score results of animals in each group at 3 months old Abdominal withdrawal reflex scores of colorectal Numbers distension at different pressures (mean ± SD ) Groups of rats 20 mm Hg 40 mm Hg 60 mm Hg Normal control group 11 0.70 ± 0.23 1.12 ± 0.27 2.36 ± 0.32 Model group 12  2.45 ± 0.56**  2.72 ± 0.45**  3.67 ± 0.43** Dosmalfate group 12 2.25 ± 0.45 2.56 ± 0.30 3.64 ± 0.30 Lysozyme group 12 2.11 ± 0.33 2.69 ± 0.41 3.56 ± 0.36 First combination group 12 2.08 ± 0.32 2.39 ± 0.37 3.44 ± 0.43 Second combination group 12   .sup. 1.06 ± 0.24.sup.##&&   .sup. 1.56 ± 0.26.sup.##&&   .sup. 2.39 ± 0.34.sup.##&& Third combination group 12  .sup.  1.64 ± 0.27.sup.##&   .sup. 1.72 ± 0.31.sup.##&&   .sup. 2.42 ± 0.38.sup.##&& Fourth combination group 12 2.11 ± 0.36 2.47 ± 0.36   .sup. 2.89 ± 0.46.sup.##&& **represents that there is significant difference compared with normal control group under the corresponding pressure (P < 0.01), .sup.##represents that there is significant difference compared with model group under the corresponding pressure (P < 0.01), .sup.&&represents that there is significant difference compared with lysozyme group under the corresponding pressure (P < 0.01), .sup.&represents that there is difference compared with lysozyme group under the corresponding pressure (P < 0.05).

TABLE-US-00003 TABLE 3 EMG test score results of animals in each group at 3 months old AUC (%) of EMG of colorectal distension at Numbers different pressures (mean ± SD ) Groups of rats 20 mm Hg 40 mm Hg 60 mm Hg Normal control group 11 141 ± 35 184 ± 17 273 ± 39  .sup.  Model group 12  303 ± 37**   437 ± 104** 637 ± 104**.sup.  Dosmalfate group 12 332 ± 39 445 ± 99 644 ± 117 .sup.  Lysozyme group 12 303 ± 36 394 ± 60 601 ± 60  .sup.  First combination group 12 286 ± 28 405 ± 80 580 ± 41  .sup.  Second combination group 12   .sup. 245 ± 27.sup.##&&   .sup. 268 ± 42.sup.##&& 334 ± 37.sup.##&& Third combination group 12   .sup. 229 ± 46.sup.##&&  .sup.  299 ± 30.sup.##& 356 ± 38.sup.##&& Fourth combination group 12 272 ± 48  .sup.  310 ± 67.sup.##& 421 ± 34.sup.##&& **represents that there is significant difference compared with normal control group under the corresponding pressure (P < 0.01), .sup.##represents that there is significant difference compared with model group under the corresponding pressure (P < 0.01), .sup.&&represents that there is significant difference compared with lysozyme group under the corresponding pressure (P < 0.01), .sup.&represents that there is difference compared with lysozyme group under the corresponding pressure (P < 0.05).

[0095] In this test, colorectal distension stimulation is continuously applied to neonatal rats to induce generation of irritable bowel syndrome model, resulting in high visceral sensitivity. Colorectal distension (CRD) is a widely used and repeatable visceral sensitivity evaluation method in preclinical and clinical researches. As a harmful stimulation to the intestine, a visceral pain response can be caused by applying colorectal distention stimulation to subjects or tested animals with visceral hypersensitivity. This mechanical distention model can better simulate symptoms occurring in patients with irritable bowel syndrome, such as abdominal pain and abdominal discomfort, and this visceral stimulation form is easy to control and has good repeatability. Colorectal distension is often used to evaluate visceral sensitivity in rodents (such as rats and mice). The colorectal distension model has become a standard tool to evaluate visceral sensitivity in rodents.

[0096] It can be seen from the results of abdominal wall withdrawal reflex and electromyography tests that the irritable bowel syndrome animal model was successfully established. The animal model has visceral hypersensitivity, which can be compared with various types of irritable bowel syndromes in humans. Compared with normal control group, the behavioral responses and neural responses of animals in model group to colorectal distention stimulation are significantly strengthened under each test pressure. It can be seen from data in Tables 1-3 that compared with normal control group, the abdominal wall withdrawal reflex score of the model group is increased significantly (P<0.01), and the AUC value of the area under the curve of EMG test is also increased significantly (P<0.01), indicating that the animals in model group maintained the visceral hypersensitivity state of irritable bowel syndrome at least from 8 weeks old to 3 months old.

[0097] The following conclusions can be drawn from the test results:

[0098] 1. After administration, there is no significant difference in numeral values of the abdominal wall withdrawal reflex and EMG test result between lysozyme group and model group (P>0.05), indicating that the alone use of lysozyme basically has no curative effect on the treatment of irritable bowel syndrome. Dosemalate group is similar to lysozyme group.

[0099] 2. After administration, compared with model group, numeral values of the abdominal wall withdrawal reflex and EMG test result in first combination group tend to reduce, but there is no statistical difference between them, indicating that the ratio of dosemalate to lysozyme in the combination can affect the curative effect on irritable bowel syndrome, so combination of dosemalate and lysozyme should reach a certain ratio in order to produce a better synergistic effect.

[0100] 3. After administration, compared with model group and lysozyme group, numeral values of the abdominal wall withdrawal reflex and EMG test result in second combination group, third combination group and fourth combination group are significantly reduced (P<0.01), indicating that the combination of dosemalate and lysozyme functions that the two components do not have when they are used alone, and plays a synergistic effect, and can be used for treatment of irritable bowel syndrome.

[0101] In this test, on the day when EMG test was completed, the rats were sacrificed, and tissues such as brain, heart, liver, spleen, lung, kidney and digestive tract (including colon) etc. were subjected to histopathological examinations. Compared with normal control group and model group, there were no obvious pathological changes in the rats in first combination group, second combination group, third combination group and fourth combination group, indicating that the combination of the present disclosure has small toxic and side effects after administration, as well as high safety. In addition, in the present disclosure, there are no typical inflammations, mucosal damages or other abnormalities in colonic histopathological examination, thus the association of inflammation and mucosal damage with the visceral hypersensitivity of test animals is excluded.

[0102] The present disclosure has been described in detail above with general descriptions, specific embodiments and the like. On the basis of the present disclosure, the skilled in the art can make reasonable modifications or improvements to the present disclosure, and these modifications or improvements made without departing from the spirit of the present disclosure belong to the content of the present disclosure.