Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/5005—Wall or coating material
  • A61K9/5021—Organic macromolecular compounds
  • A61K9/5026—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/5005—Wall or coating material
  • A61K9/5021—Organic macromolecular compounds
  • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/5005—Wall or coating material
  • A61K9/5021—Organic macromolecular compounds
  • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers

Definitions

• the present invention relates to a composition for preparing a microcapsule, and more particularly to a composition for preparing a microcapsule suitable for oral administration.
• Oral administration is the most common and user-accepted route of administration, but this route will pass through the user's digestive system, so these drugs usually need to have a good embedding rate to protect the active substances from being digested. The liquid is destroyed.
• the release rate of the active material is often poor, and the efficiency of the active material is lowered. Therefore, how to strike a balance between the embedding rate and the release rate has become a difficult problem that is difficult to overcome in the field.
• the microcapsule vaccine used in the early aquaculture industry consisted of sodium alginate and calcium chloride, which had a good release rate, but the embedding rate was poor, so the active substance was easily damaged by gastric juice.
• the microcapsules most commonly used in food or medicine are composed of sodium alginate, calcium chloride and chitin. By adding chitin, the embedding rate of the vaccine is increased to protect the active substance from the destruction of gastric juice.
• aquaculture organisms usually have a shorter intestinal tract, such vaccines, despite their excellent embedding rate, sacrifice the release rate, making it impossible for the active substance to be completely released in the relatively short intestinal tract. The effect of the active substance.
• Another object of the present invention is to provide a medicament for coating activity using microcapsules prepared by the composition
• the substance prevents the active substance from being damaged by the digestive juice (gastric juice) and enables the active substance to be released quickly.
• the present invention provides a composition for preparing a microcapsule comprising: 20-50 Wt% of a hydrophilic polymer having a carboxylic acid functional group; 30-50% by weight of a hardener; 0.5- 5 wt% of chitin; and 1-20 wt% of polyamines.
• the present invention also provides a medicament comprising an active substance which is encapsulated in a microcapsule prepared by the composition.
• the polyamine compound has a molecular weight of from 100 to 2000 g/mol o
• the polyamine compound comprises tetraethylene pentamine > spermine, triethylene tetramine, spermidine > > triethylene tetramine, pentylene diamine, butylene diamine, propylene diamine, ethylene diamine, polyethylene Polyethyleneimine PEI or a combination thereof.
• the hydrophilic polymer having a carboxylic acid functional group is sodium alginate, gelatin, seaweed extract or a combination thereof.
• the hardener refers to calcium chloride, calcium carbonate, sodium chloride, calcium acetate, calcium gluconate, calcium sulphate, calcium citrate, sodium hydroxide or a combination thereof.
• the drug has an embedding rate of from 85 to 100%.
• the active substance of the drug has a release rate of 70-100% in an alkaline environment for 4 hours.
• the medicament further comprises an adjuvant.
• the microcapsules preferably have an average particle size of from 10 to 1000 ⁇ , more preferably from 20 to 250 ⁇ m.
• the medicament is an oral vaccine.
• the oral vaccine can be a first generation vaccine, a second generation vaccine or a third generation vaccine.
• the active substance in the oral vaccine is an attenuated pathogen, a non-activated pathogen, a surface antigen of a pathogen of a dead pathogen, a recombinant protein, an immunoglobulin, an antigenic determinant or a combination thereof.
• the composition of the present invention is disadvantageous in view of the fact that common drugs are difficult to achieve both the embedding rate and the release rate.
• the obtained microcapsules have excellent embedding rate and rapid release rate, so that the medicine prepared by using the microcapsule can not only prevent the gastric juice from damaging the active substance, but also can rapidly release the active substance in an appropriate environment, in particular Suitable for short water products in the digestive tract.
• FIG. 1A-1F show the capsularity of each sample of Example 1; FIG. 1A is sample 1E; FIG. 1B is sample
• Figure 1C is a comparison sample 1E
• Figure 1D is a comparison sample 2E
• Figure 1E is a comparison sample 3E
• Figure 1F is a comparison sample 4E;
• Figure 2 shows the results of the activity test of the sample 1E-1%, the sample lE-3%, and the comparative sample 4E after the gastric acid treatment;
• Figure 3 shows the results of the activity test of the sample 1S-1%, the sample lS-3%, and the comparative sample 4S after gastric acid treatment.
• the present invention relates to a composition for preparing microcapsules which can be mixed with an active material to form a medicament in the form of a microcapsule.
• the medicine provided by the invention has excellent embedding rate and rapid release rate, can protect the active substance from damage of the digestive juice, and can release the active substance quickly under a suitable environment. Therefore, the medicament of the present invention is particularly suitable for oral administration, and is also particularly suitable for use in a water product having a short digestive tract.
• drug generally refers to a product in which a microcapsule obtained by using the composition of the present invention is coated with an active material, and the active material and an additive other than the microcapsule may be further contained therein.
• the drug may be, but is not limited to, a drug, a health food, a vaccine or a combination thereof, and the active substance is an active ingredient in the drug, the health food, or the vaccine.
• the active substance contains a microorganism, a protein or other substance which has a disease prevention, a disease treatment, and a health promotion.
• a specific example of the medicament of the present invention is a vaccine comprising an attenuating pathogen, an inactivated pathogen, a dead pathogen, a surface antigen of a pathogen, a recombinant protein, an immunoglobulin, an epitope, or a combination thereof.
• the average particle diameter of the microcapsules of the present invention is preferably from 10 to 1000 ⁇ m, more preferably from 20 to 250 ⁇ m. Unless otherwise specified, the microcapsules at the time of calculating the average particle diameter may be in a state in which the active material is embedded, or may be in a state in which the active material is not embedded.
• composition for preparing a microcapsule of the present invention comprises at least the following components: sodium alginate, calcium chloride, chitin and a polyamine compound; more specifically, 20-50% by weight of a hydrophilic group having a carboxylic acid functional group Polymer; 30-50 wt% of hardener; 0.5-5 wt% of chitin; and 1-20 wt% of polyamines.
• the hydrophilic polymer having a carboxylic acid functional group is used for mixing with the active material, and by virtue of it The carboxylic acid functional group produces a crosslinking reaction with the chitin.
• the hydrophilic polymer having a carboxylic acid functional group is highly biocompatible, including, but not limited to, sodium alginate, gelatin, seaweed extract, or a combination thereof.
• the hardener is used to provide structural stability of the medicament of the present invention.
• ionic compounds which generate positively charged cations after dissociation may be used, including, but not limited to, calcium chloride, calcium carbonate, sodium chloride, calcium acetate, calcium gluconate, calcium sulfate, calcium citrate, sodium hydroxide or Its combination.
• the hydrophilic polymer having a carboxylic acid functional group is sodium alginate
• the hardener is calcium chloride.
• Sodium alginate and calcium chloride are the main components commonly used in the preparation of microcapsules.
• the microcapsules prepared by these two components have poor porosity and poor embedding rate, so that the active substance is easily destroyed by gastric juice.
• Chitin also known as chitosan, is a natural polymer that is highly biocompatible and is therefore widely used in biomedical materials. The addition of chitin to the present invention produces a crosslinking reaction with sodium alginate having a carboxylic acid functional group, thereby increasing the embedding rate of the microcapsules.
• the embedding rate is explained by a person skilled in the art; in short, it refers to the extent to which the active substance is coated with other components of the drug without being in contact with the external environment.
• the drug embedding rate of the present invention means the degree to which the active material is coated with the microcapsule of the present invention in the medicament of the present invention.
• the embedding rate of the active substance in the medicament of the present invention is 85 to 100%; among them, the components mainly providing a good embedding effect are sodium alginate, calcium chloride and chitin.
• the rate of release is explained by those well known in the art; in short, it refers to the rate at which the active substance in the drug is released from the drug structure into the environment. While increasing the embedding rate, it may significantly affect the release rate of active substances in the drug.
• the polyamine compound is added to the medicament of the present invention so that the release rate of the medicament of the present invention in the intestinal tract reaches 70-100% in 4 hours. More specifically, the polyamine compound has a property of rapidly dissolving in an alkaline environment, and therefore, it is mainly responsible for protecting the active substance from the destruction of gastric juice, and polyamine, compared with chitin, sodium alginate and calcium chloride. The compound is dissolved in the alkaline environment of the intestines, which in turn allows the active substance to be rapidly released.
• the polyamine compound is a polyamine compound having a molecular weight of 100 to 2000 g/mol, which comprises: tetraethylene pentamine > spermine, triethylene tetramine , spermamine, triethylene tetramine, pentylene diamine, butylene diamine, propylene diamine, ethylene diamine Diamine), Polyethyleneimine PEI, or a combination thereof, but the selection of the polyamine compound is not limited to the above species.
• the composition for preparing microcapsules of the present invention may further comprise 0.5-5% by weight of an emulsifier, and/or 40-60% by weight of an oil phase substance.
• the emulsifier and the oil phase material provide the function of a surfactant during the process of making the microcapsules of the compositions of the present invention.
• the emulsifier and the oil phase material are selected without limitation, and may be prepared according to the desired drug.
• the size of the material is chosen to achieve the desired HLB value (Hydrophile-Lipophile Balance Number).
• the emulsification includes: span20>span40>span60> span65 >span80> span85 >tween20> tween21 >tween40>tween60> tween61 > tween65 >tween80> tween81 > tween85 or a combination thereof, but not limited to the above categories.
• the oil phase material includes: vegetable oil such as soybean salad oil, safflower oil, sunflower oil, corn oil, olive oil, Penglai rice oil, peanut oil, animal oil such as roasted ghee, fragrant oil, refined lard or Combination, but not limited to the above categories.
• the drug is a vaccine.
• the vaccine comprises a first generation vaccine, a second generation vaccine or a third generation vaccine; and the active substance comprises: an attenuated pathogen, a dead pathogen, a surface antigen of a pathogen, an immunoglobulin, an epitope, or a combination thereof, but It is not limited to the above categories.
• the vaccine may further comprise an adjuvant.
• the adjuvant may be embedded in the microcapsules prepared by the composition as the active substance is generally, or may be formed separately from the microcapsules containing the active substance, independently of the microcapsules.
• a vaccine kit may be embedded in the microcapsules prepared by the composition as the active substance is generally, or may be formed separately from the microcapsules containing the active substance, independently of the microcapsules.
• the term "adjuvant” refers to an inducing substance used to enhance the immune response, which not only enhances the efficiency of the vaccine, but also reduces the amount of active substance used, and is of great value in terms of cost and safety considerations.
• the vaccine adjuvant of the present invention comprises: a chemical functional adjuvant and a physical functional adjuvant, for example, including an aluminum salt, an acetylated tyrosine, an acetylated saccharide, an anion-derivatized polysaccharide, or a cationic derivative.
• the polysaccharide is, but not limited to, the above species.
• One of ordinary skill in the art can select the type and amount of adjuvant as desired.
• composition for preparing microcapsules and the medicaments prepared therewith provided by the present invention in conjunction with the drawings. It should be noted that the following examples are merely illustrative of the use of the present invention. The characteristics and advantages of the composition for preparing the microcapsules and the medicaments prepared therewith are not intended to limit the scope of the invention.
• Example 1 Encapsulation, embedding rate and release rate of a drug (vaccine) in the form of a microcapsule of the present invention
• the capsularity and embedding rate of the drug prepared according to the spirit of the present invention was observed in this example.
• the medicament prepared in this embodiment is a vaccine for use in a water supply product, and the active substance is Vibrio carchariae which is treated by inactivation.
• the active substance and the aqueous sodium alginate solution are uniformly mixed, and then the oil phase material (soybean salad oil) which has been uniformly mixed with the emulsifier (Span 80) is added, and all the components are quickly mixed into the first mixture at a rotation speed of 900 rpm.
• the oil phase material sibean salad oil
• emulsifier Span 80
• chitin, a polyamine compound (DETA or PEI) and calcium chloride are uniformly mixed into a second mixture.
• the second mixture was dropped into the first mixture and stirring was continued for 10 minutes to obtain a medicament (vaccine microcapsule) of the present example.
• composition and concentration of each sample of the present embodiment (including drugs made according to the spirit of the present invention, and comparative samples) Table 1: The components of the composition for preparing microcapsules of Example 1 and their concentrations
• the prepared drug was placed on a weighing paper and observed to be cystic by the naked eye.
• the results are shown in Figs. 1A - 1F; wherein the numbers in Figures 1A - 1F correspond to the English numbers listed in Table 1.
• the absorption values of each sample and the comparative sample were measured by OD540 and converted into the embedding rate of the drug.
• the determination of the embedding rate by OD540 is based on the measurement knowledge well known in the art, and when the absorbance value is defined as 1, the number of colonies is 10 9 CFU/mL. Briefly, 15 mg of the prepared drug (vaccine microcapsule) was placed in 5 mL of NaHC0 3 (aq), and the microcapsules were cleaved to release the active substance to become a rupture solution. The OD540 value of the rupture fluid was measured and substituted into the colony number calibration line to determine the total colony concentration: X CFU/mL (CFU; colony-forming unit). Calculate the embedding rate (EE%) by taking the value obtained into the following formula:
• 'lm S represents the amount of active ingredient contained per milligram of microcapsules.
• Comparative sample 1E (Fig. 1C) showed poor cysticity, and about 50% of the drug was a sheet-shaped unformed by-product. Comparing the embedding rate of sample 1E is only 64%, presumably because the crosslinking speed of calcium ion (calcium chloride) and sodium alginate is too fast, resulting in the active substance being extruded into the microcapsule structure during the process of drug formation. outer.
• Sample 1E (Fig. 1A) and sample 2E (Fig. 1B) have excellent cysticity and the finished product is uniform and consistent.
• the entrapment rate of sample 1E and sample 2E is as high as 100%, and the drug loading per gram of drug can reach 299 mg and 287 mg, respectively.
• this example is based on the comparison of the ratios of the samples 1E, 4E and the sample 1E in the same manner, and changed to Vibrio alginolyticus (S4Y; I3 ⁇ 4n'o a/gm ⁇ ric ⁇ M ⁇ as the active substance).
• the samples 1S, 4S and 1S were compared for subsequent testing.
• the average particle sizes of the samples 1E, 2E and 1S were all 70 ⁇ .
• Example 2 Activity test of the vaccine prepared in Example 1
• the oral vaccine is estimated to remain in the stomach of the fish for about 2 hours, so the vaccine must be tolerant to gastric acid for more than 2 hours.
• the drug prepared in Example 1 was placed in gastric acid for 1 to 3 hours, and the remaining percentage of activity of the active substance was measured.
• This activity test was designed according to the United States Pharmacopoeia. Simply put, take 150 mg of the sample or compare the sample, place it in the basket, and then wrap the 250 mesh screen on the outside of the basket and the rotating shaft to avoid sample leakage and interfere with the amount of bacteria detected. .
• the device was placed in 50 ml of gastric acid for 1, 2 or 3 hours, then transferred to deionized water for washing and lyophilized.
• the dried drug is then placed in a rupture solution and treated in a turbulent manner for 2 hours to release the active substance from the drug.
• the active substance-containing rupture fluid was formulated into a suspension (with a bacterial concentration of 10 8 cfo/ml) in a coating buffer and placed in a 96-well plate (100 ⁇ /per well). The 96-well plate was then placed at 4 °C.
• bovine serum albumin (BSA) (or 5% skim milk powder) was added to each well and allowed to react at 37 ° C for 1 hour.
• BSA bovine serum albumin
• rabbit antibacterial serum was added to each well and reacted at 37 ° C for 2 hours.
• 100 ⁇ M of goat anti-rabbit serum was added to each well and reacted at 37 °C for 2 hours.
• 100 ⁇ M of the color former was added to each well and allowed to react for 30 minutes, and the absorbance at OD 405 nm was interpreted by a spectrum analyzer.
• the resulting data was interpolated into the calibration curve to calculate the percent remaining activity, which is the ability of the drug to protect the active substance for each sample.
• this experiment was carried out in addition to comparing samples 1E and 4E, and in addition to comparing the ratio of sample 4E (containing 10% by weight of chitin, containing no polyamines), respectively, adding 1 ⁇ % or 3 ⁇ % of DETA replaces it Half of the chitin, and supplemented with sodium alginate to a total concentration of 100 Wt% (ie, containing 54 wt% sodium alginate, 40 ⁇ 1% calcium chloride, 5 wt% chitin and 1 ⁇ % DETA , or 52 ⁇ % sodium alginate, 40% calcium chloride, 5wt% chitin and 3wt ° / DETA), prepared samples 1E-1% and samples 1 ⁇ -3% for testing.
• Wt% ie, containing 54 wt% sodium alginate, 40 ⁇ 1% calcium chloride, 5 wt% chitin and 1 ⁇ % DETA , or 52 ⁇ % sodium alginate, 40% calcium chloride, 5wt% chitin and 3wt
• sample 4S ratio containing 10% by weight of chitin, no polyamine compound
• 1% or 3% of DETA was added to replace half of the chitin, and sodium alginate was added.
• BP containing 54 wt% sodium alginate, 40 ⁇ 1% calcium chloride, 5 wt% chitin and lwt ° / DETA, or 52 wt% sodium alginate, 40 ⁇ % chlorine Calcium, 5 ⁇ 1% chitin and 3 ⁇ 1% of 13 ⁇ 4 butyl
• samples 1S-1% and samples 1S-3% were tested.
• samples 1S-1% and samples lS-3% reacted with gastric acid for 2 hours, and then retained 22% and 64%, respectively.
• the component mainly responsible for coating the active substance against gastric acid destruction is a polyelectrolyte membrane formed of sodium alginate, chitin and calcium chloride, and a crosslinked structure, or an egg-box structure.
• the present inventors have unexpectedly found that the addition of the polyamine compound not only increases the release rate, but more unexpectedly does not increase the extent to which the drug of the present invention is destroyed by gastric acid.
• Example 3 Release rate of the vaccine prepared in Example 1
• the release rate test of the simulated gastrointestinal tract was carried out using the comparative samples 1E, 1S, 4E, 4S and samples 1E, 1S, 2E of Example 1.
• This gastrointestinal release test was designed according to the United States Pharmacopoeia. Simply put, take 150 mg of the sample or compare the sample, place it in the basket, and then wrap the 250 mesh screen on the outside of the basket and the rotating shaft to avoid the sample seeping out and disturb the amount of bacteria detected. .
• Example 2 Experimental results of intestinal release rate of the vaccine prepared in Example 1
• Example 4 In vivo test of the efficacy of the vaccine prepared in Example 1
• This example uses a spotted grouper fry (purchased from the National Taiwan Ocean University Aquatic Animal Experimental Center, with a body length of 3 ⁇ 2 cm;).
• the fish has been successfully tamed before being purchased, and can be directly fed by feeding artificial materials.
• the fish were purchased and returned to the test after 1 week of adaptation to the environment.
• the grouper was divided into three groups: control group, experimental group 1 and experimental group 2, each group containing 30 grouper.
• each experimental grouper fed each of the experimental group 1 and the experimental group 2 per day was equivalent to an inactivated bacterial vaccine 1E (SP, sample 1E) containing 1.4 X 10 8 CFU or 2.8 X 10 7 CFU, for seven days to establish Its immunity.
• SP inactivated bacterial vaccine 1E
• each experimental grouper was injected with a pathogenic activity of 5 X 10 7 CFU/ml by intraperitoneal injection. Bacteria for the challenge test. The survival rate of fish within two weeks of the record is listed in Table 3 below.

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Citations (5)

• 2012
  • 2012-05-10 CN CN201280073082.1A patent/CN104302279A/zh active Pending
  • 2012-05-10 WO PCT/CN2012/075283 patent/WO2013166681A1/fr not_active Ceased

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