CN105878177A - Mesalazine temperature-sensitive gel enema and preparation method thereof - Google Patents

Abstract

The invention discloses a temperature-sensitive gel-type enema liquid capable of prolonging the residence time of mesalazine in the colon and a preparation method thereof. Composed of materials, stabilizers and appropriate amount of water. The temperature-sensitive gel enema for mesalazine colon provided by the invention is a free-flowing liquid at room temperature, which is injected into the colon in liquid form, and has the advantages of uniform drug distribution and good spreadability; Transformation to form a non-chemically cross-linked semi-solid gel, coupled with the mucoadhesive effect of the bioadhesive material, thereby prolonging the residence time of the drug in the colon. Compared with the conventional mesalamine enema solution, the preparation can effectively reduce the leakage of the drug solution and improve the curative effect, and has the advantages of simple preparation and little irritation.

Description

Mesalamine thermosensitive gel enema and preparation method thereof

technical field

The invention belongs to the field of pharmaceutical preparations, and relates to a mesalamine temperature-sensitive gel enema and a preparation method thereof.

Background technique

Ulcerative colitis (Ulcerative colitis, UC) is a chronic non-specific intestinal inflammation whose etiology is not very clear. The lesions are mainly in the colorectal mucosa and submucosa, and can form visible erosions and ulcers. The range of lesions mostly starts from the distal colon and develops reversibly to the proximal end, even involving the entire colon, and occasionally involving the terminal ileum, showing a continuous distribution. The incidence of UC is high in Europe and America, and relatively low in Asian and African countries. In recent years, the number of reported cases in China has gradually increased, and the incidence of UC in my country has shown an upward trend. Since the etiology and pathogenesis of the disease have not yet been elucidated, and the treatment lacks specificity, the disease is persistent and repeated, and even cancerous, seriously affecting the physical and mental health of patients, and is listed as a difficult disease by the World Health Organization.

Drug therapy is the main treatment for mild-to-moderate UC. At present, the clinical drugs used for the treatment of UC mainly include aminosalicylates, steroid hormones, immunosuppressants, and antibiotics. Mesalamine (mesalamine), also known as 5-aminosalicylic acid (5-ASA), is currently the first-line drug for the treatment of mild to moderate UC due to its definite curative effect. It exerts an anti-inflammatory effect by inhibiting the formation of prostaglandin E and leukotrienes, and its effect is local, that is, when it contacts and complexes with the intestinal mucosa, its therapeutic effect is related to the concentration of the drug in contact with the inflammatory intestinal mucosa and It is related to the duration and has little to do with the blood concentration.

Mesalamine chemical structure

Mesalamine is mainly administered orally and rectally in the clinical treatment of UC. Oral preparations are mostly sustained-release preparations that can be released in the intestinal tract, and are mainly used for the treatment of proximal colitis. Rectal topical formulations are preferred for treatment of the distal and left colon. Some data show that for mild to moderate proctitis and sigmoid colitis, the curative effect of topical application of mesalamine is better than that of oral preparations. In China, rectosigmoiditis or proctitis accounts for about 70%, and the main lesions of UC are rectum, recto-sigmoid colon, and left colon, and rectal administration has become a very important way of administering mesalamine.

Currently clinically used dosage forms for rectal administration include suppositories and enemas. The suppository enters the depth of the body and the contact area with the lesion is limited, can only act on the rectum, and is usually only used for the treatment of rectal ulcerative colitis. The commercially available mesalamine enema is mainly composed of carbomer, which is a free-flowing liquid that can reach the sigmoid colon, but it has almost no bioadhesiveness, is easily affected by intestinal peristalsis and leaks, and stays in the lesion. time is limited. Therefore, it has good clinical application value to develop mesalamine transrectal administration preparation with uniform drug distribution and strong bioadhesion for the treatment of terminal and left colitis.

In situ gel provides a good solution to the above problems. In situ gel (in situ gel) is a kind of non-chemically cross-linked semi-solid gel preparation that responds to changes in the environment and undergoes a phase transition at the site of administration after administration in a solution state. It combines the advantages of solution and gel, and also has the advantages of simple preparation process, convenient use, strong affinity for mucosal tissues, and can be used as a drug storage to prolong the residence time at the administration site. According to different formation mechanisms, in situ gels can be divided into temperature-sensitive, pH-sensitive, ion-sensitive and so on. Due to the lack of tissue fluid in the colon and the limited buffering capacity of the intestinal tract, it is not suitable to make the enema solution into a pH-sensitive or ion-sensitive gel, but it is a good choice to make the enema solution into a temperature-sensitive type.

Bostan et al. crosslinked chitosan and poly-N-isopropylacrylamide, and used the crosslinked product as the gel material to prepare a mesalazine temperature and pH dual-sensitive gel (Bostan MS, et al. Int J Biol Macromol,2013,52:177-83.), this study has not yet involved pharmacodynamics and irritation evaluation, etc. From the perspective of preparation technology, the cross-linking reaction process is complicated, and the organic solvent introduced affects to a certain extent the safety of the preparation. Ramadass et al. used collagen as the gel matrix to prepare mesalamine rectal administration in situ gel, which was also pH and temperature sensitive (RamadassSK, et al. Eur J Pharm Sci, 2013, 48(1-2) :104-10.), due to the poor stability of the carrier material collagen, the gel system must be preserved below 10°C, which brings inconvenience to the storage of the preparation. The pH of the above two dual-sensitivity gel systems are both designed at 4~5, and the pH at which the phase transition occurs is 7~8. Due to the very limited buffering capacity of the intestinal tract and the large volume of an enema, the pH of the preparation in actual medication It is difficult to reach the pH required for phase transition in a short time, so the pH sensitivity may be difficult to reflect.

Contents of the invention

Aiming at the deficiency of the existing mesalamine rectal administration preparation, the purpose of the present invention is to provide a novel mesalamine temperature-sensitive gel enema. Another object of the present invention is to provide a preparation method of the preparation.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

The temperature-sensitive gel for mesalazine colon provided by the invention comprises: main drug, temperature-sensitive gel matrix, bioadhesive material, stabilizer and appropriate amount of water, wherein the main drug is mesalazine, and the quality in the prescription is The percentage is 6.7%.

The temperature-sensitive gel matrix is poloxamer 407, poloxamer 188 and mixtures thereof. Poloxamer (poloxamer) is a non-ionic surfactant, which is an ABA-type block copolymer composed of polyoxyethylene (PEO) and polyoxypropylene (PPO). Type gel polymer materials, among which poloxamer 407 and poloxamer 188 are the most commonly used. Poloxamer has a large relative molecular mass, stable chemical properties, no metabolic transformation in the body, and good biocompatibility. By adjusting the ratio of poloxamer 407 and poloxamer 188 in the formulation, the gelation temperature of the system can be controlled around body temperature. In terms of mass percentage, the content of poloxamer 407 is 16%-19%; the content of poloxamer 188 is 3%-12%.

The mesalamine temperature-sensitive gel enema is to suspend mesalamine in the temperature-sensitive gel solution, and the gel matrix poloxamer itself has suspending properties, which is beneficial to the stability of the system.

In the described mesalamine thermosensitive gel enema, the chemical structural formula of mesalamine is as follows:

From the structural point of view, -OH and -NH ₂ are in the para position, which is unstable and easy to be oxidized and degraded. After testing, it is found that the stability of mesalamine is related to pH, and the stability is better in the solution of pH 4~5. Therefore, the pH of the gel system should be controlled between 4 and 5.

The bioadhesive material is one of chitosan, sodium hyaluronate, hydroxypropylmethyl cellulose, xanthan gum, konjac gum, bletilla striata gum or polycarbophil or a combination thereof.

The bioadhesive material is calculated by mass percentage, and its content (mass percentage) in the mesalamine thermosensitive gel enema is 0.1%-0.4%, preferably 0.2%. Preferably, the bioadhesive material is composed of polycarbophil and sodium hyaluronate, and the mass ratio of polycarbophil and sodium hyaluronate is 1:3-3:1, most preferably 3:1.

Polycarbophil (PCP) is a cross-linked polyacrylic acid polymer, which can interact with mucosal glycoproteins to form physical connections, produce a strong mucus gel network structure, and maintain the mucoadhesive system for a long time. Adhesion time without irritation to the mucous membrane, good bioadhesion and biocompatibility. In addition, PCP is a weakly acidic substance with good buffering capacity, and the addition of PCP can provide a weakly acidic environment for the gel system. Sodium hyaluronate is an acidic mucopolysaccharide. With its unique molecular structure and physical and chemical properties, it exhibits a variety of important physiological functions in the body, such as regulating the permeability of blood vessel walls and promoting wound healing. There are also a small amount of literature reporting that sodium hyaluronate has certain bioadhesive properties. The invention uses polycarbophil and sodium hyaluronate as bioadhesives, which can exert a synergistic effect to increase the adhesive force of the preparation, and also have the effect of promoting wound healing.

Described mesalamine thermosensitive gel enema is characterized in that described stabilizer is mainly made up of water-soluble antioxidant, metal complexing agent and preservative, wherein water-soluble antioxidant is preferably metabisulfite, its The mass percentage in the prescription is 0.8%~1.2%; the metal complexing agent is preferably disodium edetate, and the mass percentage in the prescription is 0.1%~0.2%.

In the described mesalamine thermosensitive gel enema, the solvent is water. The weakly acidic environment of pH 4-5 in the gel system can be provided by polycarbophil and potassium metabisulfite, so water is directly used as the solvent, which simplifies the preparation process.

The dynamic viscosity of the mesalazine thermosensitive gel enema at 25°C±0.5°C is less than 500mPa·s, which can meet the requirement that the preparation is a free-flowing liquid at room temperature, has good injectability, and the drug is easy to enter. Spread in the tract; the gelation temperature is controlled at 30~38°C, which can ensure the rapid gelation of the preparation in the body and reduce leakage; the adhesion force is greater than 70mNcm ^-2 , which can further prolong the residence time of the preparation in the lesion.

The mesalazine temperature-sensitive gel enema of the present invention uses the temperature-sensitive gel as the carrier of the mesalazine local administration preparation through the rectum, and the gel is a free-flowing liquid at room temperature, and is pushed into the colon in liquid form, and has uniform drug distribution 1. The advantages of good spreadability; rapid phase transition at body temperature to form a non-chemically cross-linked semi-solid gel, coupled with the mucoadhesive effect of bioadhesive materials, can prolong the residence time of drugs in the colon. The poloxamer in the prescription is used not only as a temperature-sensitive gel material, but also as a suspending agent to maintain the stability of the system. Compatibility of polycarbophil and sodium hyaluronate, which can promote wound healing, as bioadhesive material can further improve the bioadhesive property of the preparation. Preliminary pharmacodynamics proves that the present invention is superior to common mesalamine enema. In addition, the mesalamine thermosensitive gel enema described in the present invention also has the following advantages compared with the existing related research:

(1) Compared with the reported mesalazine temperature-sensitive liquid suppositories, the present invention has the advantages of large drug loading capacity, suitable gelation temperature, good bioadhesion and stability. ① The main drug of the temperature-sensitive gel mesalazine enema of the present invention is dispersed in the gel system in the form of suspension. Compared with completely dissolving the drug, the required amount of gel base is less, which can ensure that the preparation can be prepared in an appropriate volume. Carry out enema administration and maintain a relatively high drug concentration locally; ②The gelation temperature of the thermosensitive gel mesalazine enema of the present invention is 30-38°C, which is slightly lower than body temperature, which can ensure that the enema is kept at body temperature Rapidly gelling; 3. in the preparation of the present invention except adding more commonly used bioadhesive material polycarbophil, also added sodium hyaluronate, sodium hyaluronate is a kind of adhesive material that can promote wound healing, its Compatible with polycarbophil, while ensuring the good fluidity of the preparation at room temperature, it further improves the adhesion performance of the preparation. The external adhesion of the mesalazine temperature-sensitive gel enema liquid described in the present invention can be greater than 70 mN cm ^-2 , sustainable drug release for 8 hours; ④ good stability

(3) Compared with the temperature/pH dual sensitive gels prepared by Bostan et al. and Ramadass et al., the preparation process of the present invention is simple and does not use any organic solvent. At the same time, it also avoids the problem that the pH sensitivity due to the limited buffering capacity of the intestinal tract is difficult to achieve and affects the phase transition process.

(4) Both the temperature-sensitive gel material poloxamer and the bioadhesive material polycarbophil selected in the present invention have good biocompatibility and are almost non-irritating to mucous membranes, and are listed by the US FDA as safe pharmaceutical excipients. In the first category, the safety and availability of excipients also provide an important guarantee for the future promotion and application of this project.

Description of drawings

Figure 1: Process flow chart for the preparation of mesalamine thermosensitive gel enema.

Figure 2: In vitro release profile of mesalamine from a thermosensitive gel for colon use.

Figure 3: Body weight change curves of mice in each group during the experiment.

Figure 4: Scores of stool properties of mice in each group during the experiment.

Figure 5: Fecal occult blood scores of mice in each group during the experiment.

Figure 6: Pathological observation of the colon of rats in each group after 7 days of administration.

A: normal saline group; B: blank gel group; C: Example 1.

detailed description

The present invention will be further described below in conjunction with specific examples, so that those skilled in the art can better understand the present invention and implement it, but the given examples are not intended to limit the present invention.

In the following examples of the present invention, the composition of the mesalazine temperature-sensitive gel enema (the balance is water) and its mass percentage in the mesalazine temperature-sensitive gel enema are as follows: Example Mesalamine Poloxamer 407 Poloxamer 188 bioadhesive material metal complexing agent preservative Water Soluble Antioxidant 1 6.7% 17.6% 8.0% 0.20% (the mass ratio of polycarbophil to sodium hyaluronate is 3:1) 0.1% (EDTA) 0.1% (sodium benzoate) 0.8% potassium metabisulfite 2 6.7% 16% 3% 0.20% (the mass ratio of polycarbophil to sodium hyaluronate is 3:1) 0.1% (EDTA) 0.1% (sodium benzoate) 0.8% potassium metabisulfite 3 6.7% 19% 12% 0.20% (the mass ratio of polycarbophil to sodium hyaluronate is 3:1) 0.1% (EDTA) 0.1% (sodium benzoate) 0.8% potassium metabisulfite 4 6.7% 17.6% 8.0% 0.1% (the mass ratio of polycarbophil and sodium hyaluronate is 3:1) 0.1% (EDTA) 0.1% (sodium benzoate) 0.8% potassium metabisulfite 5 6.7% 17.6% 8.0% 0.4% (the mass ratio of polycarbophil and sodium hyaluronate is 3:1) 0.1% (EDTA) 0.1% (sodium benzoate) 0.8% potassium metabisulfite 6 6.7% 17.6% 8.0% 0.20% (the mass ratio of polycarbophil and sodium hyaluronate is 1:1) 0.1% (EDTA) 0.1% (sodium benzoate) 0.8% potassium metabisulfite 7 6.7% 17.6% 8.0% 0.20% (the mass ratio of polycarbophil and sodium hyaluronate is 1:3) 0.1% (EDTA) 0.1% (sodium benzoate) 0.8% potassium metabisulfite 8 6.7% 17.6% 8.0% 0.20% sodium hyaluronate 0.1% (EDTA) 0.1% (sodium benzoate) 0.8% potassium metabisulfite 9 6.7% 17.6% 8.0% 0.20% Polycarbophil 0.1% (EDTA) 0.1% (sodium benzoate) 0.8% potassium metabisulfite 10 6.7% 17.6% 8.0% 0.20% (the mass ratio of polycarbophil to sodium hyaluronate is 3:1) 0.1% (EDTA) 0.1% (sodium benzoate) 1.0 % potassium metabisulfite 11 6.7% 17.6% 8.0% 0.20% (the mass ratio of polycarbophil to sodium hyaluronate is 3:1) 0.1% (EDTA) 0.1% (sodium benzoate) 1.2% potassium metabisulfite 12 6.7% 17.6% 8.0% 0.20% (the mass ratio of polycarbophil to sodium hyaluronate is 3:1) — 0.1% (sodium benzoate) 0.8% potassium metabisulfite 13 6.7% 17.6% 8.0% 0.20% (the mass ratio of polycarbophil to sodium hyaluronate is 3:1) — — — Comparative example 1 6.7% 17.6% 8% 0.6% Polycarbophil 0.1% (EDTA) 0.1% (sodium benzoate) 0.8% potassium metabisulfite Comparative example 2 6.7% 17.6% 8% — 0.1% (EDTA) 0.1% (sodium benzoate) 0.8% potassium metabisulfite

As shown in Figure 1, the preparation method of the mesalamine temperature-sensitive gel enema of the above-mentioned embodiment, the steps are as follows:

(1) placing the bioadhesive material in water and stirring until uniformly dispersed to obtain the first mixture;

(2) Slowly add the temperature-sensitive gel matrix to the first mixture under stirring to make it fully wet, and place it at 4°C for 24 hours until the temperature-sensitive gel matrix is completely swollen and becomes clear without lumps solution, stirred to make it homogeneous, to obtain the second mixture;

(3) Slowly add the stabilizer to the second mixture under stirring, stir until completely dissolved, finally add the main drug mesalazine and add water to a sufficient amount, keep stirring in the dark until the drug is evenly suspended.

The character of the mesalazine temperature-sensitive gel enema that above-mentioned embodiment makes is measured, and result is as follows:

1. Evaluation indicators

Determination of gelation temperature (T _gel ): Measure T _gel by inverting the test tube method, take 2 mL of the gel solution in a test tube, place it in a constant temperature water bath and slowly raise the temperature at a rate of about 0.2°C·min ^-1 , with each increase of 0.2 °C, quickly invert the test tube, observe the flow of the solution, define the temperature when the solution in the test tube no longer flows as the gelling temperature, measure each sample 3 times, and take the average value.

Determination of viscosity before gelation: use a rotational viscometer to measure the viscosity of the sample before gelation, measure each sample 3 times, and take the average value.

Determination of sedimentation volume ratio: Take 25mL of the drug-containing gel solution with a stoppered measuring cylinder, seal it tightly, shake vigorously for 1 minute, record the initial height H ₀ of the suspension, let it stand for 3 hours, and record the final height H of the suspension , calculated according to the formula: sedimentation volume ratio = H/H ₀ .

Determination of adhesion force: refer to the method for determination of adhesion force in the literature and slightly improve the adhesion performance of the preparation. Fill a glass watch glass with a diameter of 6 cm with water, cover it and place it in a 37°C water bath with the top about 0.5 cm above the water surface, and let it balance for a period of time. Add an appropriate amount of sample to the top of the watch glass. After complete gelation, place a preheated glass slide (2.5cm×2.5cm) on the sample, and press it with a 50g weight for 3 minutes to make the slide and the gel fit tightly. Hook, thin wire, small cup and drip device, open the drip control valve and add water (5mLmin-1) to the small cup at a constant speed until the slide starts to move, take off the small cup and weigh. Adhesion calculation formula per unit area: f=9.8m/s, m is the mass of the cuvette, s is the area of the slide. ^-1 Example Adhesion (mN.cm ^-2 ) Gelation temperature (°C) Viscosity before gelling (mPa·s) Sedimentation volume ratio (%) pH 1 76.5±2.2 33.73±0.25 395.3±2.5 99.2 4.35 2 75.3±2.1 37.57±0.15 264.3±1.0 98.6 4.53 3 75.2±2.2 30.14±0.15 635.3±1.5 99.2 4.55 4 49.3±1.1 33.17±0.15 290.6±0.7 98.4 4.46 5 92.1±2.2 35.20±0.20 1021.0±3.0 99.1 4.20 6 68.2±2.1 34.65±0.05 488.1±3.1 98.8 4.35 7 56.8±2.0 33.17±0.12 432.2±2.0 99.6 4.38 8 53.6±2.8 34.27±0.12 378.3±8.4 99.7 4.49 9 68.4±2.1 34.73±0.06 431.3±3.1 98.8 4.38 10 75.1±3.2 33.95±0.67 391.2±2.8 99.4 4.32 11 75.5±1.2 33.56±0.29 397.5±1.5 98.9 4.20 12 75.1±1.6 33.75±0.35 397.4±2.5 98.3 5.52 13 74.9±1.9 33.77±0.15 395.3±3.1 97.9 5.53 Comparative example 1 92.8±2.8 / Not free flowing at room temperature / / Comparative example 2 39.1±3.0 35.03±0.21 179.0±0.5 98.0 4.57

From the table above, comprehensively analyze data such as adhesion force, gel temperature and viscosity before gel of each embodiment:

1. Comparing Examples 1 to 3, it can be seen that the mass percentage of poloxamer 407 in the mesalazine thermosensitive gel enema is the best 17.6%, and poloxamer 188 in the mesalamine The optimum mass percentage in the thermosensitive gel enema is 8.0%.

2. Comparing Examples 1, 4, and 5, it can be seen that the addition of bioadhesive materials can improve the adhesive force of the system. As the concentration of bioadhesive materials increases, the adhesive force increases gradually. With the increase of the concentration of bioadhesive materials, the gelation temperature increased, but the increase was not large; the viscosity and sedimentation volume ratio before gelation increased with the increase of the concentration of bioadhesive materials; the effect of bioadhesive materials on gelation The pH of the gel system was slightly affected, and the pH of the system decreased gradually with the increase of the concentration of the bioadhesive material. When the proportion of bioadhesive material is increased to 0.4%, the adhesive force is the largest, but at this time the viscosity has exceeded 1000mPa.s before the transaction, and it cannot flow freely at room temperature.

3, comparative examples 1, 6 and 7 can find out, when the mass ratio of bioadhesive material is polycarbophil and sodium hyaluronate is the mixture of 1:3～3:1, mesalazine thermosensitive gel enema The adhesive force, gel temperature and viscosity before gel are all suitable, and the best bioadhesive material is the mixture of polycarbophil and hyaluronic acid with a mass ratio of 3:1.

4, comparative examples 1, 8 and 9, the bioadhesive material of embodiment 1 is the mixture that the mass ratio of polycarbophil and sodium hyaluronate is 3:1, the mixture of embodiment 1 mesalazine thermosensitive gel enema The adhesion performance is significantly better than Examples 9 and 10 in which sodium hyaluronate or polycarbophil is selected as the bioadhesive material.

5. Comparative Examples 1 and 10-13, the addition of antioxidants, metal complexing agents and preservatives is almost equal to the adhesion, gel temperature, viscosity and sedimentation volume ratio of mesalazine thermosensitive gel enema. No effect, with the increase in the amount of antioxidant potassium metabisulfite, the pH decreased slightly.

6. Comparing Example 1 and Comparative Examples 1 and 2, the addition of bioadhesive materials can increase the adhesive force of the system, but too much addition will cause the viscosity of the system to be too high, which is not suitable for the use of mesalazine thermosensitive gel enema. application.

Two in vitro release

Determination of the release of mesalamine from thermosensitive gels by dynamic dialysis. Take 2 g of the mesalazine temperature-sensitive gel enema prepared in Example 1 and place it in a pretreated dialysis bag. The two ends of the dialysis bag are fastened with threads to prevent leakage. After fully gelling at 37°C, put the dialysis bag into the In 100mL 37℃ pH7.4 phosphate buffer solution, shake at 37.0℃±0.5℃ in a constant temperature shaker at a rate of 100 rmin 10.0, 12.0h sample 5mL, and immediately add an equal amount of 37 ℃ pH7.4 phosphate buffer solution. After the sample was diluted reasonably, the absorbance was measured at 316nm, and then the cumulative release was calculated. Each sample was measured 3 times, and the average value was taken. The external release rate of commercially available mesalamine enema liquid was determined by the same method, and the results are shown in Figure 2.

It can be seen from Figure 2 that the commercially available mesalamine enema drug releases about 79.0% in 2 hours, and releases completely within 4 hours; while the accumulation of mesalamine (5-ASA) in the mesalamine thermosensitive gel enema of Example 1 in 2 hours The release rate was 42.8%, the release reached 74.2% in 4 hours, and the release was complete within 8 hours, indicating that the 5-ASA thermosensitive gel enema of Example 1 has a certain sustained release effect.

3. Stability test

1. Influencing factor test

The stability data of the mesalamine thermosensitive gel enema in Examples 1 and 10-13 placed at high temperature (60°C) for 5 days and 10 days are as follows. The results showed that compared with the initial state (0 o'clock), the content of the main drug on the 5th day had a very obvious decrease.

Comparative example 1, 10～12 and embodiment 13, mesalazine stability has improved substantially, illustrates that it is very necessary to add potassium metabisulfite in the prescription.

Comparative examples 1, 10 and 11, along with the consumption of antioxidant potassium metabisulfite in prescription increases, stability improves slightly, but after the consumption of potassium metabisulfite in prescription reaches 0.8%, continue to increase again The amount of sodium metabisulfite has little contribution to the stability of mesalamine.

Comparing Example 1 and Example 12, it is illustrated that adding the metal complexing agent EDTA in the prescription helps to improve the stability.

stability test

Get three batches of the 5-ASA thermosensitive gel enema test product of Example 1, simulate the commercially available packaging, place it under the conditions of temperature 40°C ± 2°C and relative humidity 25% ± 5% for 6 months, during the test period respectively at Sampling once every 0, 1, 2, 3, and 6 months, observe and record the appearance of the test product, and measure the gelation temperature, pH, viscosity, sedimentation volume ratio, 5-ASA content and related substances.

The results are shown in the table below:

Compared with the initial state (at 0 o'clock), the color of the suspension gradually changed from milky white to light brown, and the pH was controlled within the range of 4.0~5.0 without significant change; the content of the main drug was slightly reduced, but the reduction was within 3%. Still within the labeled range, the relevant substances meet the requirements; gelation temperature, viscosity and sedimentation volume ratio have no obvious changes. Therefore, the indicators are stable in nature.

4. Animal experiments

Select 40 healthy female BALB/c mice, aged 6-8 weeks, weighing 20-22g, and randomly divide them into 4 groups: A: normal group; B: normal saline group; C: mesalazine enema group ( Abbreviation: commercially available group); D: 5-ASA thermosensitive gel enema group of Example 1 (abbreviation: Example 1).

Preparation and administration method of acute ulcerative colitis model

The model of acute ulcerative colitis in mice was prepared by drinking DSS aqueous solution freely. Except group A, the other three groups were all modeled. They drank 3.5% DSS aqueous solution freely for 5 days, and replaced fresh DSS solution every day. Group A was given normal drinking. water for 5 days. The successful establishment of the model is marked by any of the symptoms of semi-loose stool, diarrhea, positive fecal occult blood, and gross blood in the stool. The corresponding drugs were given by enema on the next day after successful modeling. The doses of the two groups C and D were 520 mg/kg (according to 5-ASA), which was converted into the equivalent dose of mice per daily enema dose for humans, for 7 consecutive days; the dose of group B was Relative to the dose of group D, the same amount of normal saline as the drug-containing gel was given for 7 consecutive days; group A had a normal diet without any treatment.

Disease activity index (DAI) assessment

From the beginning of the experiment, observe the general activity of the mice every day, record the body weight change, stool properties and fecal occult blood, refer to the literature standards and make appropriate modifications for DAI scoring, see the table below. scoring Stool properties fecal occult blood 0 normal No color after 2min 1 ╲ Within 1~2min, the purple color will gradually appear 2 loose Produce purple within 1min 3 ╲ Violet blue within 10s 4 loose stool Immediately produces purple blue

*Normal stool: shaped stool; loose stool: mushy, semi-formed stool that does not adhere to the anus; loose stool: thin, watery stool that adheres to the anus.

As can be seen from Figure 3, during the administration period, the weight loss trend of the mice in the normal saline group gradually slowed down, and the mice in the commercially available group and Example 1 group began to lose weight no longer on the 8th day, and as the number of days of administration increased, some The body weight of the mice rebounded; after the administration, the body weights of the mice in the commercially available group and Example 1 group were significantly higher than those in the normal saline group (P<0.05), but there was no significant difference between the two groups (P>0.05). By observing the stool properties and fecal occult blood of the mice in each group every day, it was found that the normal group had normal stools, and the score was always maintained at zero level; the three groups of mice after modeling gradually began to have loose stools and loose stools on the third day. In severe cases, blood in the stool can be seen with the naked eye, and blood stains can be seen on the litter, and the occult blood in the feces is positive to varying degrees; scores were significantly lower than those of the other two groups. The results are shown in Figures 4~5.

Evaluation of appearance, morphology and histological damage of the colon

After the last administration, the mice were fasted for 24 hours, killed under anesthesia, dissected, and the entire intestinal segment from the anus end to the end of the cecum was taken out, and the appearance and morphology of the colon in each group were observed, and scores were scored according to the standards in the table below. scoring form 0 no damage 1 Mucosa hyperemia, edema, no ulceration 2 Mucosa hyperemia, edema, mild erosion, no ulcer 3 Mucosa hyperemia, edema, moderate erosion, single ulcer 4 Mucosa hyperemia, edema, high degree of erosion, multiple ulcers 5 Mucosa hyperemia, edema, severe erosion, and ulcers larger than 1 cm

The mouse colon was cut longitudinally along the edge of the mesentery, and the contents of the intestines were washed with ice-cold saline. Tissue samples were taken from places with severe inflammation or ulcers, fixed with 4% paraformaldehyde, routinely dehydrated and embedded in paraffin, and sectioned at 5 μm. Routine HE staining, colonic histopathological examination under an optical microscope, and scoring according to the criteria in the table below. scoring degree of inflammation Lesion depth Crypt disruption Lesion extent (%) 0 none none none none 1 mild submucosa Basal 1/3 crypts are destroyed 1~25 2 Moderate muscle layer Basal 2/3 crypts are disrupted 26~50 3 severe serosal layer Intact surface epithelium only 51~75 4 ╲ ╲ All crypts and epithelium are destroyed 76~100

SPSS19.0 software was used for statistical analysis of the results, and the measurement data were expressed as mean ± SD. After the homogeneity of variance test, the comparison of the differences among the groups was performed by one-way ANOVA, with P<0.05 as the standard. The difference is statistically significant.

The results are shown in the table below: group Colon Appearance Morphological Damage Score Colon Histological Lesion Score normal group 0.00±0.00 0.00±0.00 saline group 3.10±0.88 ^a 9.50±1.96 ^a Commercial group 2.00± ^0.67ab 6.80± ^2.10ab Example 1 1.20± ^0.42abc 4.40± ^1.35abc

^a Compared with normal group, P<0.05; ^b Compared with normal saline group, P<0.05; ^c Compared with commercial group, P<0.05

The appearance of the colon of the mice in each group was observed with the naked eye. The colonic mucosa of the mice in the normal group was smooth, the intestinal wall was thin, and the submucosal blood vessel texture was clear, without congestion, edema, erosion and ulcer; the surface of the colon of the mice in the normal saline group was rough and granular. Intestinal wall thickening, distortion, intestinal mucosal hyperemia, edema, mild to severe erosion and ulcers can be seen; commercially available mice have thickened intestinal wall, intestinal mucosal hyperemia, edema, mild erosion, and a small number of small ulcers can be seen; implementation Example: The colon wall of the mice in group 1 was thickened, and the intestinal mucosa was slightly congested and edematous. Only 2 mice showed mild erosion, but no ulcer focus. The colon appearance and morphology damage scores of the mice in the commercial group and Example 1 group were significantly lower than those in the normal saline group (P<0.05); compared with the commercial group, the Example 1 group had a lower colon appearance and morphology damage score, and The difference between the two was statistically significant (P<0.05). The results of mouse colon histological injury scores showed that the scores of the commercially available group and Example 1 group were significantly lower than those of the normal saline group (P<0.05), and the score of Example 1 group was the lowest. Compared with the commercially available group, the difference between the two was significant Statistically significant (P<0.05).

In summary, compared with the normal saline group and the commercially available group, the curative effect of the example 1 group (the mesalamine thermosensitive gel enema of example 1) is better. At the same time, it was also found during the administration period that almost all of the mice in the normal saline group and the commercially available group leaked to varying degrees after enema administration, while the mice in the Example 1 group occasionally leaked, indicating that the use of thermosensitive gel as a mesala The oxazine carrier, supplemented with bioadhesive materials, can effectively prolong the residence time of mesalamine in the colon, thereby improving the curative effect.

5. Pathology

Select 9 female SD rats, weighing 200-250g, and randomly divide them into 3 groups, namely A: normal saline group; B: blank example 1 group; C: mesalazine colon temperature-sensitive example 1 group. Group C is given mesalamine colonic thermosensitive gel (prepared according to Example 3) by enema every day, and the dose is 360 mg/kg (based on mesalamine), for 7 consecutive days; For the blank gel of salazine, the dose is the corresponding drug equivalent to the drug-containing gel calculated relative to the dose of group C, for 7 consecutive days. The body weight, mental state and activity of rats in each group were observed every day. After the last administration, the rats were fasted for 24 hours and sacrificed under anesthesia. After dissecting, the entire intestinal segment from the anus end to the end of the cecum was taken out, and the changes in the appearance and shape of the colon in each group were observed with the naked eye. The colon was cut along the edge of the mesentery, and the feces were washed with ice saline. In order to avoid the inflammatory interference caused by the mechanical stimulation during the enema administration, the tissue samples were taken about 1 cm from the upper end of the enema depth and fixed with 4% paraformaldehyde. Afterwards, they were dehydrated and embedded in paraffin, sectioned (5 μm), stained with conventional HE, and examined for colon histopathology under an optical microscope.

The results showed that the body weight, food intake, and stool properties of the rats in each group were normal during the medication period, and the appearance of the colon in each group was observed with the naked eye after dissection, and no obvious congestion, edema, or submucosal hemorrhage was found. The pathological sections of the colonic tissue were observed under an optical microscope, as shown in Figure 6. The epithelial cells of the colonic mucosa in the three groups had no degeneration and necrosis, the distribution of glands in the lamina propria was normal, no congestion, edema, and inflammatory cell infiltration were seen. The membrane tissue structure was intact, and no abnormal changes were seen. The experimental results show that mesalamine thermosensitive gel for colon has good biocompatibility in vivo.

The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be determined by the claims.

Claims (10)

1. a mesalazine thermosensitive hydrogel enema, it is characterised in that its composition includes: principal agent, temperature sensitive hydrogel substrate, life Thing adhesion material and water,

Wherein said principal agent is mesalazine, and its mass percent shared in described mesalazine thermosensitive hydrogel enema is 6.7%；

Described temperature sensitive hydrogel substrate is made up of poloxamer188 and PLURONICS F87, and poloxamer188 is at described U.S. salad Mass percent shared in piperazine thermosensitive hydrogel enema is 16%～19%, and PLURONICS F87 is in described mesalazine temperature Mass percent shared in quick gel enema is 3%～12%；

Described bioadhesive material of stating is chitosan, hyaluronate sodium, hydroxypropyl methylcellulose, xanthan gum, Konjac glucomannan, Bletilla glucomannan Or the one in polycarbophil or a combination thereof, described bioadhesion material is shared in described mesalazine thermosensitive hydrogel enema Mass percent is 0.1%～0.4%, preferably 0.2%.

Mesalazine thermosensitive hydrogel enema the most according to claim 1, it is characterised in that described bioadhesive material by Polycarbophil and hyaluronate sodium composition, the mass ratio of polycarbophil and hyaluronate sodium is 1:3～3:1.

Mesalazine thermosensitive hydrogel enema the most according to claim 1, it is characterised in that described bioadhesive material by Polycarbophil and hyaluronate sodium composition, the mass ratio of polycarbophil and hyaluronate sodium is 3:1.

Mesalazine thermosensitive hydrogel enema the most according to claim 1, it is characterised in that poloxamer188 is described Mass percent shared in mesalazine thermosensitive hydrogel enema is 17.6%；PLURONICS F87 is in described mesalazine temperature Mass percent shared in quick gel enema is 8.0%.

Mesalazine thermosensitive hydrogel enema the most according to claim 1, it is characterised in that also include stabilizer, described Stabilizer include: metal chelating agent, preservative and antioxidant.

Mesalazine thermosensitive hydrogel enema the most according to claim 5, it is characterised in that described antioxidant is burnt sulfurous Acid potassium.

Mesalazine thermosensitive hydrogel enema the most according to claim 5, it is characterised in that described metal chelating agent is for depending on Ground acid disodium.

Mesalazine thermosensitive hydrogel enema the most according to claim 5, it is characterised in that described preservative is benzoic acid Sodium.

Mesalazine thermosensitive hydrogel enema the most according to claim 1, it is characterised in that described mesalazine is temperature sensitive solidifying The pH value of glue enema is 4.0～5.0.

10. the preparation method of the mesalazine colon thermosensitive hydrogel described in any one of claim 1～9, it is characterised in that bag Include following steps:

(1) bioadhesive material being placed in water, stirring, to being uniformly dispersed, prepares the first mixture；

(2) in the first mixture, under agitation add temperature sensitive hydrogel substrate so that it is fully moistening, transfer in 0～5 DEG C Putting, be the clear solution without agglomerate to temperature sensitive hydrogel substrate complete swelling, stirring is allowed to uniform, prepares the second mixture；

(3) taking stabilizer and under agitation join in the second mixture, stirring, to being completely dissolved, is eventually adding principal agent and adds Water is to the most enough, and lucifuge continues stirring until medicaments uniformity suspendible and get final product.