Berberine-substituted cinnamic acid cocrystal and its use in preparing drugs for treating colitis

Abstract

The present invention discloses berberine-substituted cinnamic acid eutectic and its use in the preparation of a drug for treating colitis. The present invention grinds berberine and substituted cinnamic acid according to a molar ratio of 1: (0.9-1.1) to form a eutectic, and the prepared eutectic is not hygroscopic, effectively improves the dissolution of berberine under water conditions, significantly improves the dissolution rate, and improves the solubility by about 20%. The Uss test proves that the prepared berberine-substituted cinnamic acid eutectic is nearly 20 times higher than important parameters such as berberine hydrochloride. The present invention further provides a pharmaceutical composition for treating ulcerative colitis, the pharmaceutical composition comprising an active ingredient and a carrier acceptable to a pharmaceutical preparation, the active ingredient being a berberine-substituted cinnamic acid eutectic. The present invention also prepares the pharmaceutical composition for treating ulcerative colitis into a sustained-release pellet preparation based on an oral colon-targeted drug delivery system for treating colitis.

Description

Technical Field

[0001] The present invention relates to a cocrystal, in particular to a cocrystal of berberine-substituted cinnamic acid and a preparation method thereof. The present invention further relates to use of the cocrystal of berberine-substituted cinnamic acid in preparing a drug for treating colitis, belonging to the field of berberine cocrystals and their applications. Background Art

[0002] Ulcerative colitis (UC), also known as chronic nonspecific ulcerative colitis, is a common autoimmune disease. The main clinical symptoms are recurrent diarrhea, bloody stools with mucus, abdominal pain or tenesmus. The course of the disease is protracted and difficult to cure, lasting from several months to several decades. 80% of patients will have repeated attacks and worsening symptoms. The cancer rate of patients with a 30-year course can be as high as 40%. Therefore, it is defined by the World Health Organization (WHO) as one of the 14 intractable diseases. Ulcerative colitis can occur at any age, but it is more common in people aged 20 to 40 years. The incidence rate in China is about 5-40 / 100,000.

[0003] At present, the commonly used drugs for the treatment of ulcerative colitis are: adrenocortical hormones, such as dexamethasone; aminosalicylic acids, such as sulfasalazine and 5-aminosalicylic acid; immunosuppressants, such as cyclosporine, etc. Although the above drugs have certain therapeutic effects, they have a single target and can only relieve symptoms, so they are only suitable for short-term use; if they are administered for a long time, they will also cause severe gastrointestinal reactions, suppression of bone marrow hematopoietic function, allergies and other side effects.

[0004] For the treatment of ulcerative colitis, both traditional oral and rectal administration have certain shortcomings. After taking ordinary tablets and capsules orally, the drugs are absorbed or degraded by the whole body before reaching the colon; after rectal administration of Chinese medicine external treatment preparations such as suppositories and enemas, the drugs are limited to the rectum and descending colon, and cannot reach the main ulcer lesions such as the transverse colon and ascending colon, which affects the efficacy of the drugs.

[0005] Under normal physiological conditions, the pH value of the human gastrointestinal tract increases gradually from low to high. The pH in the gastric juice before eating is between 1.0 and 2.0, and the pH during digestion after eating is about 4.0. The pH in the proximal small intestine is about 6.5, and the pH in the distal small intestine is about 7.5. The pH drops rapidly from the ileum to the ascending colon, reaching about 6.4 in the cecum and only about 5.7 in the ascending colon. The pH gradually increases from 6.6 to 7.0 from the transverse colon to the descending colon.

[0006] The biggest challenge facing colon-targeted drug delivery systems is that after oral administration, drugs are destroyed in the stomach and small intestine due to the influence of pH or enzymes, so it is crucial to promote the delivery of a certain amount of drugs to the colon. Using natural or polymer materials with bioadhesive properties as drug carriers and combining them with drugs, the drugs can adhere to the surface of the colon mucosa through the adhesion between the adhesive materials and the mucosa, thereby prolonging the retention time of the drugs in the colon, increasing the local concentration and absorption rate of the drugs, and enhancing the bioavailability of the drugs.

[0007] The oral colon targeted drug delivery system uses appropriate formulation technology to prevent the drug from being released in the stomach, duodenum, jejunum and the front end of the ileum after oral administration. Instead, it is transported to the ileocecum and released to exert local or systemic therapeutic effects. There are many preparation technologies, including pH dependence, enzyme dependence, and time and pressure control. Among them, the pH-sensitive drug delivery system (pH-dependent OCDDS, pH-OCDDS) uses the pH change law in the human gastrointestinal tract to design and select carrier materials. pH-sensitive polymers have become the first choice for carriers. These polymers are used as pharmaceutical excipients to encapsulate drugs. After oral administration, the drugs are not released in the stomach and small intestine. When the pH reaches the end of the small intestine and the colon and is close to 7.0, the coating material dissolves and releases the active drug, achieving the purpose of colon targeted drug delivery.

[0008] Berberine hydrochloride is a quinoline alkaloid hydrochloride, which is often used in the clinical setting in the form of solid preparations (mainly tablets). Due to its safety and effectiveness, it is widely used to treat diseases such as bacillary dysentery and gastroenteritis. In recent years, berberine hydrochloride has been found to have significant pharmacological effects in the treatment of various cardiovascular and metabolic diseases such as congestive heart failure, dyslipidemia and diabetes. Berberine hydrochloride is a yellow powder with a bitter taste. In addition, due to its poor water solubility, low bioavailability and humidity-induced solid-state instability, its quality control and clinical efficacy are greatly affected.

[0009] In order to improve the physicochemical properties of berberine hydrochloride and enhance its pharmaceutical and therapeutic applications, researchers have studied berberine hydrochloride. Patent literature (CN 112521385A) mentions that berberine hydrochloride is formed into salts with anserin and saccharin to solve its bitter taste, but the water solubility of these two salts is also reduced. In view of the disadvantage that berberine hydrochloride is unstable under high humidity, the prior art uses berberine hydrochloride and myricetin and dihydromyricetin to prepare two new drug-drug co-crystals. This method improves the stability of berberine hydrochloride against high humidity, but the solubility is reduced. Most of the raw materials used for co-crystallization are classified as BCS II drugs with high permeability and low solubility. Therefore, dissolution rate and dissolution are important indicators of the performance of raw materials in vivo. The drug needs to be disintegrated and dissolved before it can be absorbed by the human body. If the drug concentration is lower than its effective concentration, it will lead to poor therapeutic effect. In the market, more than 80% of drugs are sold in tablet form, and about 40% are water-soluble. Due to this shortcoming, 90% of the candidate drugs are eliminated in the drug development stage. Therefore, it is urgent to solve problems such as drug solubility and dissolution rate.

[0010] The oral colon-specific drug delivery system (OCDDS) has the advantages of obvious targeting, increasing local drug concentration, directly acting on the lesion site, reducing drug dosage, and reducing adverse reactions. It is often used to improve the treatment effect of inflammatory bowel disease and colon cancer.

[0011] There is currently no radical and specific treatment for ulcerative colitis, and there is a severe lack of drugs with significant therapeutic effects on ulcerative colitis. In the absence of compounds with significant anti-ulcerative colitis activity, new dosage forms and biological preparations of traditional therapeutic drugs have become an active direction for the current development of new anti-ulcerative colitis drugs. Therefore, there is a significant need to find new molecular entities with high efficiency and low toxicity for anti-ulcerative colitis and develop innovative drugs.

[0012] Compared with coated tablets, microcapsules disperse a dose of drug into hundreds of units. The damage of the coating film of a single microcapsule will only cause a small amount of drug to be released suddenly, and will not cause irregular drug release from the entire microcapsule. The microcapsules are not easily affected by the gastric emptying rate and can be evenly distributed in the intestine. The microcapsules have good fluidity and are suitable for industrial packaging. Therefore, coated microcapsules have better efficacy and process reproducibility than coated tablets, and can better ensure the colon targeting of drugs for the treatment of ulcerative colitis. Summary of the invention

[0013] One of the objects of the present invention is to provide a co-crystal of berberine and substituted cinnamic acid;

[0014] The second object of the present invention is to provide a method for preparing a co-crystal of berberine and substituted cinnamic acid;

[0015] A third object of the present invention is to provide a pharmaceutical composition for treating ulcerative colitis.

[0016] A fourth object of the present invention is to provide a micropellet preparation for resisting ulcerative colitis.

[0017] The above object of the present invention is achieved through the following technical solutions:

[0018] One aspect of the present invention is to provide a cocrystal of berberine (BBR) and a substituted cinnamic acid (BBR-3MCA), wherein the substituted cinnamic acid is selected from any one of 3-methoxycinnamic acid, 4-methoxycinnamic acid, 3,4,5-trimethoxycinnamic acid, 2-methoxycinnamic acid, methylcinnamic acid, fluorocinnamic acid, chlorocinnamic acid or bromocinnamic acid;

[0019] Preferably, a eutectic is formed by berberine and methylcinnamic acid in a molar ratio of 1:(0.9-1.1); more preferably, a eutectic is formed by berberine and methylcinnamic acid in a molar ratio of 1:1; the methylcinnamic acid is selected from any one of 2-methylcinnamic acid, 3-methylcinnamic acid or 4-methylcinnamic acid, more preferably 3-methylcinnamic acid.

[0020] The eutectic of berberine and 3-methylcinnamic acid (3MCA) prepared in the present invention is a monoclinic crystal with a space group of P2(1) / c and a unit cell parameter value of α=90, β=106.940(7), γ=90;

[0021] Characteristic crystalline peaks are shown at 2θ values ​​of 15.09 and 18.34, with characteristic peaks located at 10.56°, 11.64°, 15.11°, 17.93°, 19.39°, 20.53°, 21.23°, 26.48°, 31.80°, 33.98° and 52.34°.

[0022] The nuclear magnetic resonance identification parameters of the cocrystal of berberine and 3-methylcinnamic acid (3MCA) prepared by the present invention are as follows:

[0023] 1H NMR (400MHz, DMSO-d6): δ=2.28(3H,s,Ar-CH3),3.20(2H,s,CH2),4.05(3H,s,OCH3 ),4.08(3H,s,OCH3),4.96(2H,s,CH2),6.16(2H,s,O-CH2-O),6.37(1H,d,J=16.0H z,CH=),6.99-7.06(3H,m,ArH),7.17-7.23(3H,m,ArH),7.78(1H,s,ArH),8.01(1H ,d,J=8.8Hz,ArH),8.18(1H,d,J=8.8Hz,ArH),8.94(1H,s,ArH),9.93(1H,s,ArH); 13 C NMR (100MHz, DMSO-d6): δ=21.42,26.79,55.59,57.45,62.36,102.53,105.93,108.85,120.68,120.90,121.84,124.05,124.45,12 7.04,127.89,128.90,130.94,131.10,133.40,135.45,137.25,137.86,138.05,144.06,145.99,148.10,150.22,150.82,169.49.

[0024] The infrared identification of the cocrystal of berberine and 3-methylcinnamic acid (3MCA) is as follows: IR (KBr): 3487, 3410, 3057, 2940, 1639, 1598, 1553, 1506, 1480, 1397, 1036, 793, 710 cm –1 .

[0025] The berberine and 3-methylcinnamic acid (3MCA) eutectic (BBR-3MCA) prepared by the present invention was identified by scanning electron microscopy. According to the SEM micrograph, the single crystal has a block structure, a large aspect ratio, a complete crystal face and a relatively flat crystal face. The local magnified image shows that there are some defects in the crystal face, but no obvious crystal dislocation growth phenomenon is found.

[0026] The hygroscopicity identification result shows that berberine hydrochloride (BBR-HCl) is hygroscopic, and the berberine and 3-methylcinnamic acid (3MCA) eutectic prepared by the present invention is not hygroscopic.

[0027] The results of dissolution test showed that the dissolution curves of BBR-HCl and berberine and 3-methylcinnamic acid cocrystal in water were very different. The results showed that berberine and 3-methylcinnamic acid cocrystal improved the dissolution of BBR-HCl in water conditions, the dissolution rate was significantly increased, and the solubility was increased by about 20%. The cocrystal changed the connection and stacking pattern between molecules, reduced the lattice energy of the original drug, made the solute dissolution process easier, and increased the dissolution rate.

[0028] The solubility test results showed that at the same temperature and in the same solvent, the solubility of berberine and 3-methylcinnamic acid cocrystal was slightly better than that of BBR-HCl.

[0029] After berberine and 3-methylcinnamic acid were ground to form a cocrystal, the particle size and particle size distribution, pH solubility, oil-water partition coefficient, hygroscopicity and other physical and chemical properties were measured and studied. It was found that the solubility of BBR-3MCA increased and it was soluble in water, methanol, ethanol, isopropanol, etc. The oil-water partition coefficient (logP) of BBR-3MCA was 1.16, and the logP of BBR was -0.74. The intestinal absorption rate constant (Ka value) of BBR-3MCA was 2.69*10- -3 Ka / s, the apparent absorption constant (Papp) is 1.49*10 -5 , relative to the apparent absorption constant of BBR (Papp = 9.2*10 -7 ) increased by 16 times, and the absorption rate constant (Ka=2.0*10 -4 ) increased by 13 times.

[0030] Another aspect of the present invention provides a method for preparing a berberine and substituted cinnamic acid cocrystal, comprising: grinding berberine and substituted cinnamic acid in a molar ratio of 1: (0.9-1.1) in the presence of a solvent to obtain the cocrystal.

[0031] As a preferred specific embodiment of the present invention, the solvent described in the present invention is selected from any one of water, methanol, ethanol, isopropanol, ethyl acetate or acetonitrile, or a mixed solvent composed of more than one of them in any proportion; preferably, the solvent is a 30-100% ethanol aqueous solution.

[0032] As a preferred specific embodiment of the present invention, the grinding time is preferably 0.1-10h.

[0033] Another aspect of the present invention is to provide a pharmaceutical composition for treating ulcerative colitis, the pharmaceutical composition comprising an active ingredient and a carrier acceptable for pharmaceutical preparations, wherein the active ingredient is a berberine and cinnamic acid cocrystal of a substituent provided by the present invention, wherein the daily dosage of berberine is in the range of 5 to 400 mg.

[0034] The anti-ulcerative colitis pharmaceutical composition provided by the present invention can be prepared into clinically suitable preparations according to conventional preparation methods in the art, such as oral preparations, injection preparations, sustained-release or controlled-release preparations; wherein the oral preparations can be pills, tablets, granules, powders or capsules, preferably pills, and more preferably, the pills are sustained-release micropellet preparations.

[0035] The present invention grinds berberine and substituted cinnamic acid in a molar ratio of 1: (0.9-1.1) to form a cocrystal. The prepared berberine and substituted cinnamic acid cocrystal is not hygroscopic, improves the dissolution of berberine hydrochloride (BBR-HCl) under water conditions, significantly increases the dissolution rate, and increases the solubility by about 20%. The Uss test found that the berberine and substituted cinnamic acid cocrystal prepared by the present invention is nearly 20 times higher than that of berberine hydrochloride in important parameters.

[0036] The present invention further provides a pharmaceutical composition for treating ulcerative colitis, comprising an active ingredient and a carrier acceptable to pharmaceutical preparations, wherein the active ingredient is a cocrystal of berberine and substituted cinnamic acid provided by the present invention; further, the present invention prepares the pharmaceutical composition for treating ulcerative colitis into a sustained-release pellet preparation based on an oral colon-targeted drug delivery system for treating colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Molecular structure, intermolecular interactions and molecular stacking diagram of berberine and 3-methylcinnamic acid cocrystal; (a) Ellipsoid diagram, the ORTEP molecular structure is displayed as an ellipsoid probability of 30%, (b) key intermolecular interactions (hydrogen bonds are blue-green), (c) molecular stacking.

[0038] Figure 2 This is the PXRD pattern of berberine and 3-methylcinnamic acid cocrystal.

[0039] Figure 3 The carbon spectrum (a) and hydrogen spectrum (b) of the berberine and 3-methylcinnamic acid cocrystal.

[0040] Figure 4 This is the infrared spectrum of the cocrystal of berberine and 3-methylcinnamic acid.

[0041] Figure 5 This is the DSC-TGA of the eutectic of berberine and 3-methylcinnamic acid.

[0042] Figure 6 This is the SEM image of the berberine and 3-methylcinnamic acid cocrystal.

[0043] Figure 7This is the result of the hygroscopicity of the berberine and 3-methylcinnamic acid cocrystal.

[0044] Figure 8 This is the dissolution result of berberine and 3-methylcinnamic acid cocrystal.

[0045] Fig. 9 The cumulative dissolution curve of the sustained-release pellets prepared with berberine and 3-methylcinnamic acid cocrystal as active ingredients in the present invention. DETAILED DESCRIPTION

[0046] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0047] Example 1 Preparation of berberine and methylcinnamic acid eutectic

[0048] Berberine (BBR) and 3-methylcinnamic acid (3MCA) were put into a mortar at a molar ratio of 1:1, and 60% ethanol aqueous solution was added and ground for 1 h. The powder X-ray diffraction analysis was performed, and the diffraction pattern was the same as Figure 1 The results are consistent, indicating that the obtained sample is a cocrystal of berberine and 3-methylcinnamic acid.

[0049] Example 2 Preparation of berberine and methylcinnamic acid eutectic

[0050] Berberine and 3-methylcinnamic acid, wherein the molar ratio of berberine to 3-methylcinnamic acid is 1:0.9, are placed in a three-necked flask, and an appropriate amount of 30% ethanol aqueous solution is added, and heated. Filter by suction, recover the solvent under reduced pressure, cool and crystallize, and perform powder X-ray diffraction analysis on it. The diffraction pattern is the same as Figure 1 The results are consistent, indicating that the obtained sample is a cocrystal of berberine and 3-methylcinnamic acid.

[0051] Example 3 Preparation of berberine and methylcinnamic acid cocrystal

[0052] Berberine and 3-methylcinnamic acid were taken, wherein the molar ratio of berberine to 3-methylcinnamic acid was 1:1.1, put into a mortar, added with an appropriate amount of 90% ethanol aqueous solution, and manually ground for 6 hours. The powder X-ray diffraction analysis was performed, and the diffraction pattern was the same as Figure 1 The results are consistent, indicating that the obtained sample is a cocrystal of berberine and 3-methylcinnamic acid.

[0053] Example 4 Preparation of micropellets for treating colitis

[0054] (1) Preparation of pellet core particles: Weigh a certain amount of berberine and 3-methylcinnamic acid cocrystal (prepared in Example 1) and chitosan, chitosan oligosaccharide, sodium alginate, carbomer, konjac glucan, hydroxypropyl methylcellulose or microcrystalline cellulose (the specific amount of each component is shown in Table 1), mix them thoroughly, pass through a 60-mesh sieve, slowly add the binder solution, and manually make a soft material; extrude it three times, roll it into a ball, and then transfer it to a fluidized bed for drying.

[0055] Process parameters: extrusion speed 15 rpm, spheronization speed 900 rpm, spheronization time 210 s, fluidized bed drying at 45°C for 30 min. The pellet core particles were obtained.

[0056] (2) Coating:

[0057] Preparation of coating solution: Add 5% hydroxypropylmethylcellulose phthalate, 1.5% talc, 37.4% methylene chloride, 46.7% ethanol and 9.4% water to prepare coating solution.

[0058] Coating: Fluidized bed coating, fluidized bed coating parameters: air volume 90~120m 3 / h, air inlet temperature 45 ~ 65 ℃, atomization pressure 70 ~ 110KPa, preheat to material temperature about 40 ℃, start spraying enteric coating material, after coating, 40 ℃. Activate for 12h to get pellets.

[0059] Table 1 Composition and dosage of the micropellets for treating colitis of Examples 4-12

[0060]

[0061] Example 5 Preparation of micropellets for treating colitis

[0062] The components of the micropellet preparation and the specific amounts of each component are shown in Table 1. The specific preparation method is exactly the same as that of Example 4.

[0063] Example 6 Preparation of micropellets for treating colitis

[0064] The components of the micropellet preparation and the specific amounts of each component are shown in Table 1. The specific preparation method is exactly the same as that of Example 4.

[0065] Example 7 Preparation of micropellets for treating colitis

[0066] The components of the micropellet preparation and the specific amounts of each component are shown in Table 1. The specific preparation method is exactly the same as that of Example 4.

[0067] Example 8 Preparation of micropellets for treating colitis

[0068] The components of the micropellet preparation and the specific amounts of each component are shown in Table 1. The specific preparation method is exactly the same as that of Example 4.

[0069] Example 9 Preparation of micropellets for treating colitis

[0070] The components of the micropellet preparation and the specific amounts of each component are shown in Table 1. The specific preparation method is exactly the same as that of Example 4.

[0071] Example 10 Preparation of micropellets for treating colitis

[0072] The composition of the micropill preparation and the specific amount of each component are shown in Table 1. The specific preparation method is exactly the same as that of Example 4.

[0073] Example 11 Preparation of micropellets for treating colitis

[0074] The components of the micropellet preparation and the specific amounts of each component are shown in Table 1. The specific preparation method is exactly the same as that of Example 4.

[0075] Example 12 Preparation of micropellets for treating colitis

[0076] The components of the micropellet preparation and the specific amounts of each component are shown in Table 1. The specific preparation method is exactly the same as that of Example 4.

[0077] Test Example 1 Confirmation of the berberine and 3-methylcinnamic acid cocrystal

[0078] Single crystals of the berberine and 3-methylcinnamic acid cocrystals prepared in Examples 1-3 were cultured and identified.

[0079] 1. Identification of molecular structure and molecular stacking of berberine and 3-methylcinnamic acid cocrystal Figure 1 (a) is the molecular structure diagram (ORTEP) of berberine and 3-methylcinnamic acid cocrystal (BBR–3MCA), and the molecular structure is displayed as an ellipsoid probability of 30%; Figure 1 (b) shows the key intermolecular interactions of the berberine and 3-methylcinnamic acid cocrystal (hydrogen bonds are in blue-green). Figure 1 (c) Molecular stacking of berberine and 3-methylcinnamic acid cocrystal.

[0080] 2. Identification of the crystallographic characteristics of berberine and 3-methylcinnamic acid cocrystals

[0081] Table 2 shows the crystallographic characteristics, X-ray data collection and structural refinement parameters of berberine (BBR) and 3-methylcinnamic acid cocrystals. The characterization parameters of berberine and 3-methylcinnamic acid cocrystals include: monoclinic system, space group P2(1) / c, unit cell parameter value α=90, β=106.940(7), γ=90;

[0082] Table 3 shows the characterization results of the hydrogen bond distance and angle of the berberine and 3-methylcinnamic acid cocrystal.

[0083] Table 2 Crystallographic characteristics, X-ray data collection and structure refinement parameters of BBR-3MCA

[0084]

[0085]

[0086] Table 3 Hydrogen bond distances and angles of BBR–3MCA

[0087]

[0088] The samples prepared by liquid-assisted grinding showed unique PXRD patterns. New diffraction peaks appeared, and the characteristic peaks of individual components were mostly absent ( Figure 2 ). BBR exhibits characteristic crystalline peaks at 2θ values ​​of 10.26°, 11.17°, 12.32°, 15.84°, 16.84°, 18.72°, 24.36°, and 25.22°. 3MCA exhibits characteristic crystalline peaks at 2θ values ​​of 12.70°, 14.14°, 15.06°, 18.42°, 19.13°, 19.68°, 26.16°, 26.56°, 27.45°, 28.54°, 30.55°, and 37.41°. However, the physical mixture of BBR and 3MCA exhibits characteristic crystalline peaks at 2θ values ​​of 15.09 and 18.34, similar to amorphous powder. The BBR–3MCA cocrystals showed a unique PXRD pattern with characteristic peaks located at 10.56°, 11.64°, 15.11°, 17.93°, 19.39°, 20.53°, 21.23°, 26.48°, 31.80°, 33.98°, and 52.34°. The different peaks in the cocrystal PXRD pattern may mean that there is an interaction between 3MCA and BBR to form a new cocrystal phase.

[0089] 3. NMR Identification of Cocrystals of Berberine and 3-Methylcinnamic Acid

[0090] The NMR identification parameters of berberine and 3-methylcinnamic acid cocrystal are as follows:

[0091] 1H NMR (400MHz, DMSO-d6): δ=2.28(3H,s,Ar-CH3),3.20(2H,s,CH2),4.05(3H,s,OCH3 ),4.08(3H,s,OCH3),4.96(2H,s,CH2),6.16(2H,s,O-CH2-O),6.37(1H,d,J=16.0H z,CH=),6.99-7.06(3H,m,ArH),7.17-7.23(3H,m,ArH),7.78(1H,s,ArH),8.01(1H ,d,J=8.8Hz,ArH),8.18(1H,d,J=8.8Hz,ArH),8.94(1H,s,ArH),9.93(1H,s,ArH); 13 C NMR (100MHz, DMSO-d6): δ=21.42,26.79,55.59,57.45,62.36,102.53,105.93,108.85,120.68,120.90,121.84,124.05,124.45,12 7.04,127.89,128.90,130.94,131.10,133.40,135.45,137.25,137.86,138.05,144.06,145.99,148.10,150.22,150.82,169.49.

[0092] The carbon and hydrogen spectra of berberine and 3-methylcinnamic acid cocrystal are Figure 3 shown.

[0093] 4. Infrared identification of berberine and 3-methylcinnamic acid cocrystal

[0094] The infrared identification of berberine and 3-methylcinnamic acid cocrystal is as follows:

[0095] Infrared IR(KBr):3487,3410,3057,2940,1639,1598,1553,1506,1480,1397,1036,793,710cm –1 .

[0096] The infrared spectrum of berberine and 3-methylcinnamic acid cocrystal is Figure 4 shown.

[0097] 5. Scanning electron microscopy identification of berberine and 3-methylcinnamic acid cocrystals

[0098] The crystallization habit (morphology) of the API is a critical parameter in the pharmaceutical process as it affects various pharmaceutical parameters such as the flow properties and dissolution characteristics of the drug powder. SEM micrograph of berberine cocrystal with 3-methylcinnamic acid (BBR-3MC) ( Figure 6 ) respectively show that the single crystal has a blocky structure, a large aspect ratio, a complete crystal plane and a relatively flat crystal plane. The local magnified image shows that there are some defects in the crystal plane, but no obvious crystal dislocation growth phenomenon is found.

[0099] 6. Identification of the hygroscopicity of berberine and 3-methylcinnamic acid cocrystal

[0100] The sorption / desorption isotherms of BBR-HCl and BBR-3MCA are very different ( Figure 7 ). At a relative humidity of 5%, BBR-HCl quickly absorbed about 4% of water. Then, the sample weight gradually increased as the relative humidity increased to 60%, which may be due to surface water adsorption. Then, with further increase in relative humidity, the sample weight increased steadily, and finally the total weight increased to 6.9% at a relative humidity of 90%. During the desorption process, BBR-HCl lost about 6% of its weight when the relative humidity dropped from 90% to 80%. Then, about 1.4% of the weight slowly decreased when the relative humidity dropped from 80% to 0%. Surprisingly, the curve of the BBR-3MCA cocrystal is almost consistent with the adsorption curve, indicating that there is no change in the structure before and after and almost no adsorbed water. According to the guidelines for drug hygroscopicity testing, the moisture absorption weight gain is less than 15%, but not less than 2%, so BBR-HCl is hygroscopic, while the BBR-3MCA cocrystal is not hygroscopic.

[0101] 7. HPLC analysis of berberine and 3-methylcinnamic acid cocrystal

[0102] The results of HPLC analysis showed that the percentages of BBR and 3MCA in the cocrystal of berberine and 3-methylcinnamic acid were 62.85±0.32% and 30.26±0.19%, respectively. Since the theoretical mass ratios of BBR and 3MCA in the cocrystal were 63.04% and 30.39%, respectively, the molar ratio of BBR and 3MCA was determined to be 1:1, which was consistent with the results of 1H-NMR.

[0103] 8. Dissolution Determination of Berberine and 3-Methylcinnamic Acid Cocrystal

[0104] The dissolution curves of BBR-HCl and BBR-3MCA cocrystals in water are very different, e.g. Figure 8The results show that BBR-3MCA improves the dissolution of BBR-HCl in water, significantly increases the dissolution rate, and increases the solubility by about 20%. The cocrystal changes the connection and stacking pattern between molecules, reduces the lattice energy of the original drug, makes the solute dissolution process easier, and increases the dissolution rate.

[0105] 9. Solubility determination of berberine and 3-methylcinnamic acid cocrystal

[0106] Table 4 gives the results of solubility determination. The solubility of BBR-HCl and BBR-3MCA in polar solvents and non-polar solvents (including water, methanol, ethanol, and isopropanol) was studied. At 37°C, the solubility order of BBR-HCl and BBR-3MCA was water > methanol > ethanol > isopropanol > ethyl acetate. At the same temperature and in the same solvent, the solubility of BBR-3MCA was slightly better than that of BBR-HCl. This is consistent with the relatively fast dissolution rate of BBR-3MCA ( Figure 8 ).

[0107] Table 4 Solubility of BBR-HCl and BBR-3MCA (mg / mL) in different solvents at 37°C.

[0108]

[0109] 10. Identification of the physicochemical parameters of berberine and 3-methylcinnamic acid cocrystal

[0110] After berberine and 3-methylcinnamic acid were ground to form a cocrystal (BBR-3MCA), the particle size and particle size distribution, pH solubility, oil-water partition coefficient, hygroscopicity and other physical and chemical properties were measured and studied. It was found that the solubility of BBR-3MCA increased and it was soluble in water, methanol, ethanol, isopropanol, etc. The oil-water partition coefficient (logP) of BBR-3MCA was 1.16, and the logP of BBR was -0.74. The intestinal absorption rate constant (Ka value) of BBR-3MCA was 2.69*10- - 3 Ka / s, the apparent absorption constant (Papp) is 1.49*10 -5 , relative to the apparent absorption constant of BBR (Papp = 9.2*10 -7 ) increased by 16 times, and the absorption rate constant (Ka=2.0*10 -4 ) increased by 13 times

[0111] 11. Ussing Chamber experiment of berberine and 3-methylcinnamic acid eutectic

[0112] After SD rats were fasted for 12 hours, they were anesthetized. Based on the previous research of the inventor's research group, the best absorption site of berberine was the jejunal segment. Therefore, the jejunum was selected as the intestinal segment for investigation. The abdominal cavity was opened to remove the intestinal segment, and the jejunal segment was cut about 3 cm, rinsed with saline, and placed in the KR solution in an ice bath for incubation for 10 minutes. Cut 1-2 cm on the ice bath plate, fix the intestinal mucosa in the sample clip, and then fix the sample clip in the Ussing chamber. Immediately add 5 ml of KR solution to the mucosal side and serosal side of the device, and start collecting various electrophysiological indicators. Each channel collects a resistance value every 10 seconds, and the average of all resistance values ​​during the period of each sampling time point and the previous sampling time point is taken as the resistance value at the sampling time point. During the experiment, a mixed gas of 95% O2 and 5% CO2 was continuously introduced, and the water bath temperature was maintained at 37.5°C to maintain the biological activity of the intestinal mucosa. The KR solution in the device was taken out, and 5 mL of drug solution and fresh KR solution kept warm in a 37.5°C water bath were added to the mucosal side and the serosal side, respectively. At 0, 15, 30, 45, 60, 75, 90, and 120 min after drug addition, 0.5 mL of receiving solution was taken from the serosal side and 0.5 mL of fresh KR solution was immediately added. The received solution was analyzed by fluorescence spectrophotometer.

[0113] Calculation of intestinal absorption coefficient: The samples measured by the fluorescence spectrophotometer were processed to obtain the mass concentration of the drug on the receiving side of the diffusion cell (ρ n , μg·mL -1 ), using the formula The cumulative permeation per unit area (Q, unit: mmol) was calculated, where 5 and 0.5 were the added drug amount and the sampling volume (mL).

[0114] Apparent permeability coefficient (Papp, unit: cm·s -1 ) is calculated using the formula (DQ / Dt)·(1 / Aρ0). Where dQ / dt is the drug transport amount per unit time, ρ0 is the initial concentration of the test solution on the administration side, and A is the effective permeation area (0.5 cm 2 ). The absorption rate constant Ka = L / A, where L is the slope of a plot of the cumulative absorption per unit area Q versus time.

[0115] Through a series of screening of cinnamic acid derivative berberine cocrystals, it was preliminarily found that when there are F, Cl, or Br groups at the 4-position of the benzene ring, the Ka absorption coefficient is lower than that of the cinnamic acid berberine group, but the Papp coefficient is higher than that of the cinnamic acid berberine group; when there is a methoxy group at the benzene ring, the Ka absorption coefficient and the Papp coefficient are basically equivalent to those of the cinnamic acid berberine group; when there is a methyl group at the benzene ring, the Ka absorption coefficient and the Papp coefficient are significantly higher than those of the cinnamic acid berberine group.

[0116] Table 5 Cumulative transport volume, absorption rate constant and apparent permeability coefficient of different concentrations of BBR-HCl and BBR-3MCA in rat jejunum

[0117]

[0118]

[0119] Test Example 2: Adhesion Test of Micropellets

[0120] Take the small intestine of a rabbit and fix it in a longitudinally cut polyethylene tube. Take 60 pellets of 30 mesh size (prepared in Examples 4-12, respectively) (30 mesh lactose particles are used as reference), spread them evenly on the small intestinal mucosa of the rabbit, tilt the polyethylene tube (20°) and place it in a dryer filled with saturated KNO3 (RH92.5%) for 20 minutes. After the particles are fully hydrated and interact with the small intestinal mucosa, the polyethylene tube is tilted 45°, and a peristaltic pump is used to control the phosphate buffer (pH=7.8) to elute the rabbit small intestinal mucosa from the top to the bottom. The flow rate is 20 ml / min, and the elutriation lasts for 5 minutes. The retention rate is used as an indicator, and the number of particles retained in the small intestinal mucosa is counted with the naked eye. The more particles are retained, the better the adhesion (more than half of the single particles are retained and included in the calculation).

[0121] Table 6 Ball core adhesion test results (n=6)

[0122]

[0123]

[0124] In addition, through parallel tests, different coating solutions with weight gain of 10±1%, 15±1%, 20±1%, and 25±1% were investigated. It was found that when the weight gain was 10±1%, 5.1%±0.6% was released in artificial gastric juice in 2 hours; when the weight gain was 15±1%, there was basically no release in artificial gastric juice in 2 hours, but 10.9%±1.3% was released in artificial colon fluid 1 in 2 hours; when the weight gain was 20±1%, 2.1%±0.6% was released in artificial colon fluid 1 in 2 hours; 93.4%±2.8% was released in artificial colon fluid 2; when the weight gain was 25±1%, there was basically no release in artificial colon fluid 1 in 2 hours; 40.1%±3.9% was released in artificial colon fluid 2 in 2 hours, and the final weight gain was 25±1%.

[0125] Test Example 3 Release Determination Test of Micropellets

[0126] The 2015 edition of the Pharmacopoeia of the People's Republic of China, General Chapter 0931, In Vitro Release Determination Method 1 (Basket Method) was used. The temperature was (37±0.5)°C, and the release medium was 900mL of artificial gastric juice, 900mL of artificial intestinal juice, and 900mL of artificial colonic juice. According to the transit time of general food in the digestive tract, the samples to be tested were placed in artificial gastric juice for 2h, artificial small intestinal juice A, and artificial colonic juice B, respectively. After 2h, 5mL of each sample was taken (at the same time, an equal amount of dissolution medium was added), and the release was determined by HPLC.

[0127] According to the Chinese Pharmacopoeia, artificial gastric juice: take 16.4mL of dilute hydrochloric acid, add 800mL of water and 10g of pepsin, shake well, and dilute with water to 1000mL; among them, dilute hydrochloric acid is 234mL of concentrated hydrochloric acid diluted with water to 1000mL, to obtain 9.5%-10.5% dilute hydrochloric acid;

[0128] Artificial small intestinal fluid A: pH 6.8, take 6.8g of potassium dihydrogen phosphate, add 500mL of water to dissolve, adjust the pH value to 6.8 with 0.1mol / L sodium hydroxide solution, take 10g of pancreatic enzyme, add appropriate amount of water to dissolve, mix, and dilute to 1000mL with water;

[0129] Artificial colon fluid B: pH 7.8, 5.59 g of dipotassium hydrogen phosphate and 0.41 g of potassium dihydrogen phosphate, add water to dissolve into 1000 mL.

[0130] Take the microcapsule preparations of Examples 4 to 12 and measure them according to the above-mentioned release determination method. The samples are placed in a rotating basket and subjected to dissolution tests in a simulated dissolution medium for 2 hours in the stomach, 2 hours in a simulated dissolution medium for 2 hours in the small intestine, and 2 hours in a simulated dissolution medium for 2 hours in the colon according to the specified conditions. Take 5 ml of samples at 2 hours in the stomach, 1 hour and 2 hours in the small intestine, and 1 hour, 2 hours, 3 hours, 5 hours, and 7 hours in the colon, and replenish the same volume of dissolution medium at the same temperature at the same time. The sample was filtered through a 0.45um aqueous microporous membrane. Using the berberine reference substance as a control, the berberine content in the subsequent filtrate was determined by HPLC high performance liquid chromatography, the cumulative dissolution rate of the drug was calculated, and the cumulative dissolution curve of the drug was drawn.

[0131] Fig. 9 The cumulative dissolution curves of the micropellets prepared in Examples 4-12 of the present invention. Fig. 9 From the cumulative dissolution curve of the micropellets, it can be seen that the micropellets prepared in Example 4 release faster and are prone to sudden release, the micropellets prepared in Example 10 release slower and are prone to delayed release, and the micropellets prepared in Examples 7 and 8 release more gradually.

Claims

1. A eutectic of berberine and substituted cinnamic acid, characterized in that: The eutectic is formed by berberine and 3-methylcinnamic acid in a molar ratio of 1: (0.9-1.1); The eutectic is monoclinic, with a space group of P2(1) / c, unit cell parameters of a=31.213(7) Å, b=11.679(3) Å, c=15.167(3) Å, α=90, β=106.940(7), γ=90; unit cell volume = 5289(2) Å 3 ; The eutectic exhibits characteristic crystalline peaks at 2θ values ​​of 15.09 and 18.34, and its characteristic peaks are located at 10.56°, 11.64°, 15.11°, 17.93°, 19.39°, 20.53°, 21.23°, 26.48°, 31.80°, 33.98° and 52.34°.

2. The eutectic according to claim 1, characterized in that The NMR identification parameters of the cocrystal are as follows: δ =2.28 (3H, s, Ar-CH3), 3.20 (2H, s, CH2), 4.05 (3H, s, OCH3), 4.08 (3H, s,OCH3), 4.96 (2H, s, CH2), 6.16 (2H, s, O-CH2-O), 6.37 (1H, d, J = 16.0 Hz, CH=), 6.99-7.06 (3H, m, ArH), 7.17-7.23 (3H, m, ArH), 7.78 (1H, s, ArH), 8.01(1H, d, J = 8.8 Hz, ArH), 8.18 (1H, d, J = 8.8 Hz, ArH), 8.94 (1H, s, ArH), 9.93 (1H, s, ArH); 13 C NMR (100 MHz, DMSO-d6): δ = 21.42, 26.79, 55.59,57.45, 62.36, 102.53, 105.93, 108.85, 120.68, 120.90, 121.84, 124.05, 124.45,127.04, 127.89, 128.90, 130.94, 131.10, 133.40, 135.45, 137.25, 137.86,138.05, 144.06, 145.99, 148.10, 150.22, 150.82, 169.

49.

3. The eutectic according to claim 1, characterized in that: The infrared spectra of the co-crystals are as follows: 3487, 3410, 3057, 2940, 1639, 1598, 1553, 1506, 1480, 1397, 1036, 793, 710 cm –1 .

4. A method for preparing the eutectic according to any one of claims 1 to 3, characterized in that: include: Berberine and 3-methylcinnamic acid are mixed in a molar ratio of 1: (0.9-1.1), and ground in the presence of a solvent to obtain the product; the solvent is a 30-100% ethanol aqueous solution; and the grinding time is 0.1-10 hours.

5. Use of the cocrystal according to any one of claims 1 to 3 in the preparation of drugs for treating ulcerative colitis.

6. A pharmaceutical composition for treating ulcerative colitis, comprising an active ingredient and a carrier acceptable for pharmaceutical preparations, characterized in that: The active ingredient is the co-crystal according to any one of claims 1 to 3.

7. A preparation prepared from the pharmaceutical composition according to claim 6, characterized in that: The preparations include pills, tablets, granules, powders or capsules.

8. The preparation according to claim 7, characterized in that The pills are sustained-release pellet preparations.

Images