Patent Application
20200206141
The present invention relates to the field of medical technology, in particular to a new oral colon-targeted delivery system, preparation method and application thereof. The delivery system can obtain ideal selective targeting of colon and has a good application value for diagnosis and treatment of colonic diseases.
At present, colonic diseases, such as: colon cancer and inflammatory enteritis, are threatening human health. The statistical results of cancer mortality rates in 2015 show that: the number of colorectal cancer cases in the world is 1.02 million, ranking third among all fatal cancers. 177,000 of them are Chinese patients, ranking fourth among all the countries. The distribution statistics of the incidence of inflammatory enteritis show that there is a high incidence in Western countries, the number of cases in China is gradually increased with the changes in environment and living habits, and the incidence of inflammatory enteritis is rising year by year at an alarming rate worldwide. This will pose a great threat to the health of all mankind, so it will be of great significance to design relevant methods for treatment.
Currently, there are mainly three drug delivery systems for treatment of diseases in the colon site: single pH-dependent colon-targeted drug delivery system, time-dependent colon-targeted drug delivery system or colon-targeted drug delivery system triggered by bacterial flora/enzyme as detailed below: (1) The PH-dependent targeted drug delivery system is a more common targeted drug delivery system which uses a certain pH-sensitive polymer as a coating for drug encapsulation by taking advantage of the fact that the pH value of the colon site is higher than that of the small intestine so that the coating is dissolved under the pH condition of the colon site and a large quantity of drugs are released. For example: acrylic resin copolymers, cellulose phthalate acetate and the like, wherein EUDRAGIT®S100 is an anionic copolymer based on methacrylic acid and methyl methacrylate. EUDRAGIT®S100 is dissolved at pH≥7 so that drugs are protected in the stomach from damage by the acidic environment and do not release their active ingredients until they reach the intestinal tract. (2) The time-dependent colon-targeted drug delivery system refers to drug release after activities in the gastrointestinal tract for a longer time due to the time difference produced by the swelling capacity of biomaterials to achieve the purpose of intestinal targeting. The time lag is mainly regulated and controlled by particle size, coating thickness and permeability to the colon site, but the system is greatly affected by individual differences, food categories, eating time, pathological conditions and other factors. (3) The colon-targeted drug delivery system triggered by bacterial flora/enzyme is based on the fact that the concentration of bacterial floras in the colon site increases sharply compared with that in the stomach and small intestine. These bacterial floras can secrete many specific reductases and hydrolases, such as β-glucuronidase, β-glucosidase, cellulase, nitroreductase, azo reductase, α-decarboxylase, cholesterol dehydrogenase and the like, while azo polymers, polysaccharides and other materials can be degraded by colon-specific enzymes, and the colon targeting preparation triggered by bacterial flora is designed for this reason. For example, chitosan is a natural polysaccharide in which 1,4-glycosidic bonds make it not degradable and absorbable in the stomach and small intestine but degradable under the action of enzymes produced by specific bacteria at the colon. It is a good carrier material for colon-targeted drug delivery system since it is highly targeted, safe, stable, non-toxic and biodegradable.
However, the gastrointestinal environment is complex, the pH has a broad variation range, the length of gastrointestinal tract varies, and individual differences exist. The oral colon delivery system currently reported has the disadvantages that: Single pH-dependent or time-dependent delivery of colon drugs can not guarantee complete arrival at the colon site due to individual differences. Drugs are usually released in advance in the middle and lower ends of the small intestine so that the drugs are absorbed or decomposed in advance, resulting in fewer doses to the colon site, thus a higher initial dose or repeated administration is required, which leads to serious systemic side effects. Most of the materials used by a single bacterial flora sensitive mechanism are compounds in an azo-containing structure and polysaccharides, and the compounds in an azo-containing structure have more significant colon targeting. However, a small molecular azo compound is a strong carcinogen, while polysaccharides are extremely easy to absorb water and swell due to good water absorption so that drugs are released in advance.
The technical problem to be solved by the present invention is to provide an oral colon-targeted delivery system which combines pH sensitive and bacterial flora sensitive mechanisms, effectively enhances the selective dissolution and release at the colon site by using double-layer protection, enhances the therapeutic or diagnostic imaging effect and reduces toxic side effects.
Additionally, a preparation method and application of the oral colon-targeted delivery system also need to be provided.
In order to solve the above technical problem, the present invention is realized by the technical scheme as follows:
In one aspect of the present invention, an oral colon-targeted delivery system is provided, comprising:
A bacterial flora sensitive layer, which contains polysaccharides and covers the exterior of active ingredients;
A pH sensitive layer, which contains any polymer composition dissolved under the condition of pH≥7 and covers the exterior of the bacterial flora sensitive layer.
The polysaccharides comprise β-cyclodextrin derivative and/or plant extracts and derivatives thereof.
The polymer compositions of the pH sensitive layer comprise components of the following weight percentages: 10%˜43% of methacrylic acid-methyl methacrylate copolymer, 5%˜36% of plasticizer and 5%˜22% of talcum powder.
In the present invention, the term “cover” comprises coating for solid carriers and capsules or microcapsules which encapsulate fluids and/or solids.
In the present invention, the term “active ingredient” refers to any compound or biomaterial useful for diagnosis and/or treatment of diseases.
The outer layer of the delivery system of the present invention is a pH sensitive layer material which can resist the acid environment at the middle and upper ends of the stomach and small intestine because it is dissolved only at pH≥7; the bacterial flora sensitive material in the middle layer can only be dissolved when triggered by the bacterial flora specific to the colon site so that the selective delivery capability of the delivery system to the colon site can be effectively enhanced; the inner layer comprises the active ingredients, comprising drugs or diagnostic reagents, wherein the drugs comprise antibody drugs, protein drugs, gene drugs, nanodrugs, small molecule drugs and the like; the diagnostic reagents comprise contrast agents, imaging probes and other imaging reagents for the diagnosis of inflammatory bowel disease or colon cancer.
Preferably, the drugs are in a targeted nanoparticle dosage form.
Drugs with targeted nanoparticles enter cells under the action of cell uptake by combining modified targeting molecules on the surfaces of the nanoparticles with specific receptors on the surfaces of the cells to achieve safe and effective targeted drug therapy.
More preferably, the targeted nanoparticles are PLGA-PEG nanoparticles modified by CD-98 antibody.
The CD-98 antibody-modified targeted nanoparticles of the present invention can be specifically identified and enriched at the inflammatory site of the colon, stay for a long-lasting effect and release drugs slowly so that the duration of action of the drugs is prolonged, the therapeutic effect of the drugs is improved, thus effectively solving the technical problem that drugs cannot stay at the inflammatory site or achieve the therapeutic effect because small molecule drugs are quickly brought out of the human body with chyme or feces after reaching the colon site in the treatment of diffuse colitis.
The bacterial flora sensitive layer material β-cyclodextrin derivative of the present invention is preferably β-cyclodextrin polymer crosslinked with epichlorohydrin. Options for the bacterial flora sensitive layer material are natural plant extracts such as celery extract, konjac gum or pectin and the like, or derivatives of the natural plant extracts. Other options for the bacterial flora sensitive layer material of the present invention are β-cyclodextrin derivatives, plant extracts and mixture of derivatives thereof.
For the methacrylic acid-methyl methacrylate copolymer in the pH sensitive layer material of the present invention, the preferred ratio of methacrylic acid to methyl methacrylate is 1:2.
The plasticizer in the pH sensitive layer material of the present invention can be selected from methyl or ethyl esters, organic acid fatty esters or inorganic acid esters (such as lactic acid, citric acid, succinic acid, adipic acid, sebacic acid, phthalic acid, glutaric acid or phosphoric acid), or selected from acetate esters or fatty esters of monohydric, dihydric, trihydric or polyhydric alcohols. Specifically, the plasticizer can be selected from glycerol diacetate (glycerol diacetate), glycerin triacetate (triacetin), or triethyl citrate, and any mixture of the products. The preferred plasticizer is triethyl citrate.
The oral colon-targeted delivery system of the present invention can be made into any dosage form, comprising capsules, microcapsules, tablets, pills or granules.
The preparation method of the oral colon-targeted delivery system of the capsule dosage form comprises the following steps:
Dissolve the polysaccharides in an organic solvent, draw a proper amount of the solution, add into a capsule shell, dry at 45˜65° C. for 5˜15 hours, then cool to room temperature, and obtain the bacterial flora sensitive layer;
Prepare a coating fluid of the pH sensitive layer by mixing the polymer compositions dissolved under the condition of pH≥7, spray on the outer surface of the capsule shell containing the bacterial flora sensitive layer, and obtain the oral colon-targeted delivery system sensitive to both pH and bacterial flora after drying.
In another aspect of the present invention, the application of the oral colon-targeted delivery system in the preparation of drugs for treatment of colonic diseases is also provided.
In another aspect of the present invention, the application of the oral colon-targeted delivery system in the preparation of reagents for diagnosis of colonic diseases is also provided.
In another aspect of the present invention, the application of the oral colon-targeted delivery system in the preparation of drugs for diagnosis and treatment of colonic diseases is also provided.
In another aspect of the present invention, the application of the oral colon-targeted delivery system in the preparation of drugs absorbed orally by the colon is also provided.
The colonic diseases comprise colon cancer, inflammatory colitis, intestinal amebiasis or intestinal tuberculosis.
The oral colon-targeted delivery system of the present invention combined with the pH sensitive and bacterial flora sensitive mechanisms effectively enhances selective dissolution and release at the colon site by using double-layer protection so that the drugs at the inner layer are not released until reaching the colon site, the number of times of administration is reduced, the toxic and side effects are reduced, better therapeutic effects are achieved, and the oral colon-targeted delivery system has broad application prospects.
The present invention is described in further detail below in combination with the drawings and the description of the preferred embodiments.
In order to solve the technical problem that the current single pH mechanism for delivering colon drugs often results in too little dosage reaching the colon site and the single bacterial flora triggered colon-targeted drug delivery easily leads to swelling after long-time action in the stomach and small intestine since it makes the bacterial flora sensitive layer material highly hydrophilic, which is adverse to normal onset at the colon site, the present invention develops an oral colon-targeted delivery system, comprising: a bacterial flora sensitive layer which contains polysaccharides and covers the exterior of active ingredients; a pH sensitive layer which contains any polymer composition dissolved under the condition of pH≥7 and covers the exterior of the bacterial flora sensitive layer, wherein the active ingredients comprise drugs or diagnostic imaging reagents.
The polysaccharides comprise β-cyclodextrin derivatives and/or plant extracts and derivatives thereof.
The polymer compositions of the pH sensitive layer contain components of the following weight percentages: 10%˜43% of methacrylic acid-methyl methacrylate copolymer, 5%˜36% of plasticizer and 5%˜22% of talcum powder.
Preferably, the drugs are in a targeted nanoparticle dosage form. In one preferred embodiment of the present invention described below, the targeted nanoparticles are PLGA-PEG nanoparticles modified by CD-98 antibody. The CD-98 antibody-modified targeted nanoparticles can be specifically identified and enriched at the inflammatory site of the colon, stay for a long-lasting effect and release drugs slowly so that the duration of action of the drugs is prolonged, and the therapeutic effect of the drugs is improved. This is of great significance for the current treatment of diffuse colitis. Since the existing small molecule drugs in common dosage form are quickly brought out of the human body with chyme or feces after reaching the colon site, the drugs cannot stay at the inflammatory site or play the due therapeutic effect. The pH and bacterial flora sensitive delivery system combined with the targeted nanoparticles can achieve long-acting stay at the site of colonic disease and slow release of drugs and give full play to the efficacy of drugs because the pH and bacterial flora sensitive delivery system can ensure that the targeted nanoparticles are fully delivered to the site of colon disease, and the targeted nanoparticles can specifically identify cells at the site of colonic disease and enter the cells by endocytosis without being eliminated from the body quickly with chyme or feces peristalsis.
The present invention is elaborated below through specific embodiments.
1. Preparation of pH Sensitive Layer
Use 20%±10% Eudragit S100 (RohmGmbH) and 15%±10% triethyl citrate (Sigma-Aldrich) as plasticizers and 5%±10% talc powder (Sigma-Aldrich) as antisticking agent, add the three components into ethanol to form the coating fluid of the pH sensitive layer, stir (Ningbo Scientz Biotechnology Co., Ltd., China) and mix evenly. Spray on the capsule evenly at 45±10° C. through a BY-300A small coating pan (Shanghai Huanghai Drug Inspection Instrument Co.). After drying, it becomes the pH sensitive layer film on the outer layer of the capsule and is dissolved only at pH≥7 so that the capsule can resist the acid environment at the middle and upper ends of the stomach and small intestine.
2. Preparation of Bacterial Flora Sensitive Layer
Dissolve 1.8±1 g β-cyclodextrin polymer crosslinked with epichlorohydrin(β-CDP) (Binzhou Zhiyuan Biotechnology, China) or plant fiber extract in 8±5 ml dimethyl sulfoxide (DMSO) (Sinopharm Group, China). Draw 8±5 ul of the solution, add into a mould gelatin capsule shell made by 3D printing, then dry for 10±5 h at 55±10° C. After cooling to room temperature, the capsule intermediate layer sensitive to bacterial flora in colon is obtained. The capsule intermediate layer can only be dissolved when triggered by the bacterial flora specific to the colon site, so that the selectivity to the colon site can be effectively enhanced. After clamping the capsule shell sensitive to colon bacterial flora vertically, coat the pH sensitive layer for the outer layer of the capsule according to the method in 1, and obtain the colon-targeted acid-base and bacterial flora sensitive capsule for oral administration.
Carry out in vitro investigation on the dissolution and release of the acid-base and bacterial flora sensitive capsule by the simulated gastrointestinal juice, and the result is shown in
3. Preparation of Targeted Nanoparticles
Prepare CD-98 antibody modified PLGA-PEG nanoparticles by the double emulsion method. Dissolve drugs or imaging reagents in the aqueous phase, and dissolve 20±10 mg PLGA-PEG in dichloromethane (DCM) (J&K, China). After ultrasonic processing into emulsion, add into aqueous phase containing 1.5±1% Tween −80 for secondary phacoemulsification. After stirring and volatilization for 2.5±1 h, carry out ultrafiltration with a supercentrifugation tube, wash with water for three times, obtain the nanoparticles, and freeze dry for standby application.
Add PEG end —COOH activated by NHS and EDC, add CD-98 antibody for incubation, obtain the modified CD-98 antibody nanoparticles, and freeze dry for standby application.
4. Preparation of Oral Colon-Targeted Delivery System
Dissolve β-cyclodextrin polymer crosslinked with epichlorohydrin or plant fiber extract in dimethyl sulfoxide. Draw a proper amount of the solution into the mould gelatin capsule shell, and then dry at high temperature. After cooling to room temperature, the capsule intermediate layer sensitive to colon bacterial flora is obtained.
After clamping the capsule shell sensitive to colon bacterial flora vertically, mix Eudragit S100, triethyl citrate and talc powder to form the coating fluid of the pH sensitive layer, stir and mix evenly. Spray the coating fluid outside the capsule evenly by the coating pan, and obtain the double-sensitive oral capsule with the pH sensitive layer as the outer layer and the bacterial flora sensitive layer as the intermediate layer.
Add the drug-loaded targeted nanoparticles prepared in the above 3 into the double-sensitive oral capsule, and obtain the oral colon-targeted delivery system of the present invention. The structure of the oral colon-targeted delivery system is shown in
1. Measurement of Targeted Nanoparticle Size and Surface Zeta Potential
To measure the particle size and zeta potential of nanoparticles and targeted nanoparticles by dynamic light scattering, draw 1 mL solution sample, add into the sample cell, make sure there is no bubble generated in the container, measure the particle size and zeta potential of the samples with the MALVERN nanometer particle size measuring instrument (DLC), measure each sample repeatedly for three times, and obtain the particle size and potential. Observe the samples with an electron microscopy (TEM).
The result of dynamic light scattering shows that (see Table 1) the nanoparticle size is about 143.1 nm, and the ζ potential is about −27.9 mv. As shown in
2. Drug Release
The targeted nanoparticles are released in vitro in phosphate buffer (PBS, pH6.8). Nanoparticles with the concentration of 0.02 mg/ml are suspended in a microdialyzer (Thermo Fisher, USA) and incubated in a shaking table at the speed of 100 rpm at 37° C. Take out 100 ul from each of the three tubes, add 100 ul PBS (pH=7.4, 0.01M) (Gibio, USA) to supplement the volume and balance the system at each designated point of time (0, 1, 2, 3, 4, . . . 0.12, 24 and 48 hours). Measure the 5-ASA drug content in PBS with a NanoDrop 2000 UV-Vis spectrophotometer, and fit into a curve.
The result is shown in
1. Cell Uptake
Culture RAW 264.7 macrophages in a culture dish overnight. Replace the culture medium with a serum-free medium containing NP and NP-CD98. After incubation for 3 hours, rinse RAW264.7 macrophages with PBS (pH value=7.4) thoroughly to remove excessive nanoparticles, and then fix with 4% polyformaldehyde for 20 minutes. After dilution of 6-diamidino-2-phenylindole dihydrochloride (DAPI) by 10,000 times, dye the cell nucleus for 5 minutes, and observe the cell uptake of nanoparticles under laser confocal microscopy.
2. Quantitative Cell Uptake of Nanoparticles
Inoculate RAW 264.7 macrophages in a 6-well plate. After incubation with NP and NP-CD98 for 2 hours, to remove free nanoparticles, wash with PBS for 3 times, disperse the cells, centrifugate (1000 revolutions, 10 minutes), and redisperse to the concentration of 1×106 cells/lml. Measure the cell fluorescence uptake with Accuri C6 flow cytometer (Becton Dickinson, Calif.).
The results are shown in
1. In Vivo Imaging
Prepare the fluorescent capsules by adding Rhodamine B (30 mg/ml) into the pH and bacterial flora double-sensitive (pH+bacteria) capsules, and then give intragastric administration to rats. Divide the rats into three groups. Put them to death in 3 hours, 6 hours and 12 hours. Take the whole gastrointestinal tract, including the stomach, small intestine (SI-1, SI-2, SI-3) and colon part, observe and analyze the image by the Kodak IS ex vivo FX imaging system. EX=535 nm at EM=600 nm.
The result is shown in
2. Biodistribution
Give intragastric administration to the rats with ordinary capsules, pH-sensitive capsules and TEXAS RED pH+bacteria capsules respectively, and put them to death in 18 hours. Take the whole gastrointestinal tract and divide it into four parts, including stomach, SI-1, SI-2, SI-3 and colon. Then cut into slices with a freezing microtome, and observe the specific distribution of fluorescent light under a confocal laser scanning microscope. Homogenize tissue samples in PBS, and then extract with a mixture of ethanol/DMSO (1:1 volume/volume). Analyze the fluorescent light content in the samples with a fluorescent plate reader.
The result is shown in
Put free dyes, NP and NP-CD98 in pH+bacteria capsules respectively, give intragastric administration to rats, put them to death in 18H, 24H and 30H, and take the colon site. Dye the cell nucleus with DAPI. More details of Texas Red in the tissues can be observed by laser confocal microscopy. Homogenize the tissue samples in PBS, and then extract with the mixture of ethanol/DMSO (1:1 volume/volume). Analyze the fluorescent light content in the samples with the fluorescent plate reader.
The result is shown in
1. Ordinary Capsules Encapsulating Free Drugs, pH-Sensitive Capsules Encapsulating Free Drugs and PH+Bacteria-Sensitive Capsules Encapsulating Drug-Loaded NP-CD98 are Intragastrically Administered
Investigate the drug efficacy in vivo with a DSS-induced acute enteritis model. Divide rats (about 150 g) into 5 groups, with 10 rats in each group. Except the first group as the negative control group, provide free drinking of dextran sulfate sodium (DSS) (6.5%) for 10 days for other groups respectively. Take the second group as the positive control group, intragastrically administer other groups with the free drugs encapsulated with ordinary capsules, free drugs protected by the pH layers and capsules protected by the double sensitive layer of pH+bacteria with NP-CD98 encapsulated drugs every other day. Weigh the rats on a daily basis, and count the number of dead rats. After 10 days, kill all the rats, and take out the colon and spleen. Measure the length of the colon site, and weigh the spleen.
2. MPO Activity
Quantify the neutrophil infiltration by measuring the MPO activity. Measure with Sigma's myeloperoxidase kit. MPO activity is expressed as a protein unit per milligram, and one unit is defined as the amount of 1 millimole degraded into hydrogen peroxide per minute at 25° C.
3. Results
As shown in
The above-mentioned embodiments only express the implementation of the present invention, which are described in a more specific and detailed manner but cannot be construed as restrictions on the scope of the present patent for invention. It should be pointed out that several transformations and improvements can also be made for one of ordinary skill in the art without departing from the concept of the present invention, and all of them fall into the scope of protection intended to be provided by the present invention. Therefore, the scope of protection of the present patent for invention shall be determined based upon the appended claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2016/106175 | 11/17/2016 | WO | 00 |
