CN106924723B - Pharmaceutical composition containing human glucagon-like peptide-1 analogue administered through oral mucosa - Google Patents
Pharmaceutical composition containing human glucagon-like peptide-1 analogue administered through oral mucosa Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/26—Glucagons
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
- A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
Abstract
The invention relates to a pharmaceutical composition containing human glucagon-like peptide-1 analogue administered through oral mucosa, which comprises high-concentration human glucagon-like peptide-1 analogue, hyaluronidase, more than one mucosa adsorbent, or further contains or does not contain more than one thickening agent. The invention also provides a preparation method of the pharmaceutical composition and application of the pharmaceutical composition in preparing a medicament for treating hyperglycemia and/or diabetes, the pharmaceutical composition is convenient to use, is nontoxic and non-irritant in the using process, has quick response after being used, and can remarkably improve the clinical medication compliance of patients.
Description
Technical Field
The invention relates to the field of medicines, in particular to a pharmaceutical composition containing a human glucagon-like peptide-1 analogue administered through oral mucosa, a preparation method thereof and application of the pharmaceutical composition in treating hyperglycemia and/or diabetes.
Background
Diabetes mellitus is a chronic disease which causes hyperglycemia, urine glucose and even a series of metabolic disorders due to insufficient insulin secretion or insulin dysfunction. If diabetes cannot be treated effectively in a timely manner, complications such as ketoacidosis, cardiovascular disease, renal failure, retinopathy, etc. may also occur. Diabetes has become a chronic disease which seriously jeopardizes the life health of human beings after tumor and cardiovascular diseases. At present, the number of diabetes patients in China reaches 1 hundred million, which accounts for about one third of the total number of diabetes patients in the world, wherein the number of diabetes patients of type 2 accounts for more than 90%.
At present, medicaments for treating type 2 diabetes mellitus need to be taken for a long time, and mainly comprise compound medicaments such as metformin, α glycosidase inhibitors, sulfonylureas, glinides, thiazolidinediones and the like, and biological medicaments such as insulin, human glucagon-like peptide-1 analogues and the like, wherein the compound medicaments have good curative effect on reducing blood sugar level in a short time, but have large side effect, and can cause islet cell function failure due to excessive insulin secretion after long-term use.
Exendin-4 is a 39 amino acid polypeptide isolated from saliva of the Swrague (Hederarmasuspenum) in the southwest of the United states and northern Mexico, has 53% amino acid homology with Glucagon-like polypeptide-1 (GLP-1) of mammals and has a molecular weight of 4186.57 various animal models indicate that pancreatic GLP-1 receptor has the same affinity for Exendin-4 and GLP-1, and the two polypeptides have the same function of stimulating GLP-1 receptor, therefore, Exendin-4 is an antagonist of GLP-1 receptor and has the functions of promoting pancreatic β cell proliferation, improving the function, promoting insulin secretion, increasing the sensitivity of the body to insulin, delaying gastric emptying, reducing appetite, and being not easily degraded by DPP-IV, and can remarkably improve fasting and postprandial blood glucose levels of type 2 diabetic patients.
Exenatide is an artificial composition of Exendin-4, and Exenatide injection (Byetta) is injected subcutaneously twice in 1 day; exenatide sustained release microspheres (Bydureon) were injected once a week. However, exenatide has a half-life of only 2.4h in vivo, needs frequent injection, and is painful in injection, resulting in poor patient compliance; in addition, the injection is inconvenient to use and may cause side effects of drug injection (such as gastrointestinal reaction, increased risk of pancreatitis and thyroid cancer), and the manufacturing process is complicated and the production cost is high. At present, Exendin-4 can also be prepared by the existing known genetic engineering recombination technology, has the characteristics of lower production cost and capability of meeting large-scale production compared with artificially synthesized
Protein and polypeptide drugs administered orally are desirable to alter the route of administration. However, after oral administration of protein polypeptide drugs, problems such as liver first pass effect and degradation of proteases in gastrointestinal tract are encountered, resulting in low bioavailability.
The oral mucosa is used as a common administration mode, the administration is convenient, and the compliance of patients is high; the oral mucosa area is about 200cm2The medicine has abundant blood vessels, can directly enter the systemic circulation through the capillary vessels, and is easier to penetrate and absorb than skin medicines; low enzyme activity, and can avoid degradation of gastrointestinal tract protease and acid and liver first pass effect, so that the medicine can be better absorbedHarvesting; compared with nasal mucosa, oral mucosa is not easy to be damaged.
Although oral mucosa drug delivery systems have been widely studied, there are no commercially available protein or polypeptide drugs absorbed through oral mucosa, which are caused by the specific biological properties of oral mucosa, such as epithelial barrier, intraepithelial barrier, basement membrane barrier, etc., resulting in low permeability coefficient of oral mucosa drug delivery, thereby preventing macromolecular drugs from entering blood circulation; meanwhile, the relative molecular weight, dissociation degree and the like of the medicine also influence the effect of the medicine in permeation and absorption through oral mucosa, so that the bioavailability of the protein medicine is low. In addition, mucus secreted from the epithelial surface of the oral cavity and physiological environments such as oral saliva, enzymes, and immune proteins constitute enzyme barriers and diffusion barriers for proteinaceous drugs. Because Exendin-4 is a macromolecular substance, is influenced by the structure of oral mucosa and the physiological environment of the oral cavity, and is not easy to be directly absorbed into blood circulation through the oral mucosa, for example, the prior art CN103566376A discloses an oral mucosa agent comprising exenatide, protein hydrolysate and a plurality of auxiliary materials, but in experiments, the ideal clinical utilization effect is still difficult to achieve, so that the development of a new dosage form of Exendin-4 for oral mucosa administration is urgently needed to achieve the ideal treatment effect, and the Exendin-4 can be used for replacing injection administration.
Disclosure of Invention
The invention provides a pharmaceutical composition containing human glucagon-like peptide-1 analogue for oral mucosa administration and a preparation method thereof, wherein the pharmaceutical composition can be used for large-scale production and preparation of human glucagon-like peptide-1 analogue preparations for oral mucosa administration, and has the characteristics of high osmotic absorption, quick response, high bioavailability, good patient compliance and the like.
The purpose of the invention is realized by the following technical scheme:
a pharmaceutical composition for oral transmucosal administration containing a human glucagon-like peptide-1 analog, comprising:
1) human glucagon-like peptide-1 analogs;
2) (ii) a hyaluronidase enzyme;
3) more than one pharmaceutically acceptable oral mucosa administration carrier, wherein the mass percent of mucosa adsorbent in the pharmaceutical composition is 1% -99%. The oral mucosa administration carrier is preferably more than one pharmaceutical mucosa adsorbent.
The human glucagon-like peptide-1 analogue is Exendin-4, and the Exendin-4 prepared by the existing well-known genetic engineering recombination technology has the characteristics of lower production cost and capability of meeting large-scale production compared with artificially synthesized Exendin-4.
The amount of Exendin-4 in the pharmaceutical composition is 5-1000 mug, and preferably, the amount of Exendin-4 in the pharmaceutical composition is 25-500 mug. The hyaluronidase in the pharmaceutical composition is hPH20, the enzymatic activity is 500-20000U, preferably, the PH20 is human PH20(hPH20), and the enzymatic activity is 1000-20000U. Namely hPH20 the ratio of enzyme activity U to Exendin-4 in the unit of mu g is 0.2-4000, preferably 2-800.
The pharmaceutical composition is preferably prepared as a solution system, such as a spray, and the solvent includes water, alcohol-containing solvent, etc.; or oral mucosa patch. In a solvent system, the concentration of the Exendin-4 in the pharmaceutical composition is 5-1000 mug/ml, preferably 25-500 mug/ml. The activity of hPH20 enzyme in the pharmaceutical composition is 500-20000U/ml, and preferably, the activity of hPH20 enzyme is 1000-20000U/ml.
Because Exendin-4 belongs to hydrophilic polypeptide, the molecular weight is about 4.2KD, and the Exendin-4 is influenced by the structure of oral mucosa and the physiological environment of the oral cavity and is difficult to directly permeate into blood to be absorbed through the epithelial cells of the oral mucosa. In order to enable the Exendin-4 to permeate into capillary vessels through oral mucosa, the technical problem to be overcome at present is to increase the osmotic absorption amount of the Exendin-4 so as to achieve the effective treatment concentration of the medicine.
According to a great amount of experimental surprise, the hyaluronidase hPH20 can form extracellular matrix pore canals smaller than 200nm in a short time when being used in the oral mucosa agent, so that the viscosity of the oral mucosa is reduced, the permeability is increased, the Exendin-4 is promoted to be rapidly diffused and permeated in the mucosa, the absorption effect of capillary vessels on the Exendin-4 is enhanced, and the bioavailability of the Exendin-4 is improved. Meanwhile, hPH20 can be rapidly cleared by plasma, has short plasma half-life and local effect, hyaluronic acid in mucosa interstitium can be completely rebuilt within 24-48h, and the integrity of mucosa structure can be rapidly recovered.
Hyaluronidase hPH20 known in the art belongs to mucopolysaccharidase, and is capable of hydrolyzing hyaluronic acid in intercellular matrix after subcutaneous injection, and helps provide bioavailability of the administered biological agent by subcutaneous injection. Because the oral mucosa administration route is different from subcutaneous injection, the effect of the hyaluronidase hPH20 in the composition of the invention is not equal to the effect of the hyaluronidase hPH20 in the subcutaneous injection, and simultaneously, as a single composition, experiments show that the hyaluronidase and the drug of the invention play a certain synergistic effect to bring the excellent effect. In experiments, compared with other common absorption penetration enhancers such as cholate, fatty acid, alcohol and surfactant, hPH20 is adopted as the oral mucosa penetration enhancer of Exendin-4, so that the injury or even the degradation effect of the conventional penetration enhancers on mucosa after long-term use can be solved, and the adverse reactions such as inflammation of the mucosa can be effectively avoided.
According to the invention, hPH20 is added, on one hand, the barrier effect of the stratum corneum of the oral mucosa can be reversibly changed to promote the permeation and absorption of the mucosa of Exendin-4, on the other hand, the oral mucosa can not be seriously stimulated and damaged, and the mucosa can be quickly recovered after being damaged. hPH20, as described, can be prepared by techniques known in the art. Specifically, hPH20 is soluble hPH20(rhPH20) prepared by gene recombination technology, and rhPH20 is mature PH20 polypeptide which lacks all or part of Glycosyl Phosphatidylinositol (GPI) attachment sites at the C-terminal, so that the polypeptide is not combined with CHO cell membrane after being recombined and expressed in Chinese Hamster Ovary (CHO) cells, thereby ensuring that rhPH20 is secreted and dissolved in a cell culture medium.
The mucosa adsorbent comprises: povidone, polyvinyl alcohol (PVA), Polycarbophil (PC), Carbomer (CP) and carboxymethyl chitosan, preferably, the mucosa adsorbent in the pharmaceutical composition is one or a combination of povidone, carboxymethyl chitosan and carbomer. In order to enable the pharmaceutical composition to effectively permeate through the oral mucosa, firstly, the composition is required to be ensured to be in contact with the oral mucosa, and the influence of the oral environment on the active ingredients of the pharmaceutical composition is reduced, so that the mucosal adsorbent is added into the pharmaceutical composition to ensure the effective utilization rate of the active ingredients of the pharmaceutical composition in the oral environment. Since mucous membrane of oral cavity secretes mucus, the main component of mucus is water, when mucous membrane adsorbent contacts with mucous membrane, the water in mucus can be absorbed, and the mucus is contacted with mucous membrane and adhered together through physical action, which is helpful to improve the utilization rate of rhPH20 and the osmotic absorption of Exendin-4 through mucous membrane. The povidone is the abbreviation of polyvinylpyrrolidone (PVP), is soluble in water and high in viscosity, can be used as a viscosity regulator, a crystallization inhibitor and a drug solubilizer in a liquid preparation, so that the drug composition can be adhered to oral mucosa, the degradation rate of rhPH20 on the mucosa interstitium is enhanced, and the permeability of Exendin-4 is improved; the carboxymethyl chitosan has good biocompatibility, biodegradability and biological adhesion, has no biotoxicity and good water solubility, can be used as a good biological material to deliver protein polypeptide drugs, can effectively maintain active ingredients in the pharmaceutical composition, and keeps mucin in epithelial tissues moist, thereby prolonging the absorption time of the active ingredients in the pharmaceutical composition on the surface of a mucous membrane, increasing the ability of the Exendin-4 to penetrate the oral mucosa and promoting the absorption of the oral mucosa on the Exendin-4; carbomer is used as a carboxyl high molecular polymer, has strong biological adhesion, improves the degradation effect of rhPH20 on mucous membrane interstitium through the mutual permeation generated between macromolecular chains on the surface of the polymer, promotes the membrane penetrating effect of Exendin-4, and can maintain the effective concentration of active substances of medicaments while releasing medicaments quickly. Specifically, the mass percentage of the mucosa adsorbent in the pharmaceutical composition is 1% -99%, preferably 2% -85%, for example, in each separately packaged liquid pharmaceutical composition, the concentration is 0.2-50 mg/ml, preferably 0.2-15 mg/ml.
The composition further contains or does not contain more than one auxiliary material with other properties, more than one thickening agent and the like. The thickening agent is selected from: carboxymethyl cellulose, sodium carboxymethyl cellulose (CMC-Na), hydroxypropyl cellulose, hydroxypropyl methyl cellulose and hydroxyethyl cellulose, and preferably, the thickening agent is CMC-Na. The CMC-Na can increase the viscosity of the pharmaceutical composition, reduce the viscosity of oral mucus to medicaments, and reduce the degradation effect of protease to active ingredients of the medicaments; after being combined with moisture in mucus, the composition is not only favorable for increasing the adhesiveness to oral mucosa, but also can enhance the adhesion effect of mucosa adsorbent to mucosa, is easy for releasing rhPH20 and Exendin-4, and promotes Exendin-4 to rapidly penetrate the mucosa to enter blood circulation to increase the serum concentration. Specifically, the mass percentage of the thickening agent in the pharmaceutical composition is 1% -99%, preferably 1% -35%, for example, in each separately packaged liquid pharmaceutical composition, the concentration of CMC-Na is 0.1-5 mg/ml, preferably, the concentration of CMC-Na is 0.2-4 mg/ml.
The active ingredients and the auxiliary materials in the prescription of the pharmaceutical composition containing the Exendin-4 administrated by the oral mucosa have better compatibility, and the type and the dosage of the contained auxiliary materials ensure that the prescription integrally maintains the effective pharmaceutical activity of the Exendin-4 and the good activity of rhPH20 on the oral mucosa; in addition, the mucosa adsorbent, the thickening agent and rhPH20 have synergistic effect on degradation of oral mucosa interstitium and osmotic absorption of Exendin-4 mucosa. The formula of the pharmaceutical composition is beneficial to the development of the subsequent preparation production process and the preparation product which is in line with clinical use is prepared by the subsequent process.
The invention also aims to provide a preparation method of the medicinal composition containing Exendin-4 for oral mucosa administration, which is characterized by comprising the following steps:
1) preparing a mixed solution containing a mucous membrane adsorbent (or further containing a thickening agent);
2) adding rhPH20 protein stock solution obtained by recombinant preparation into the mixed solution prepared in the step 1), and uniformly mixing;
3) adding the Exendin-4 protein stock solution obtained by recombinant preparation into the mixed solution prepared in the step 2), and uniformly mixing;
4) sterilizing to obtain the pharmaceutical composition containing Exendin-4 administrated through oral mucosa.
The third purpose of the invention is to provide application of a medicinal composition containing Exendin-4 administrated through oral mucosa in preparation of a medicament for treating hyperglycemia and/or diabetes, wherein the usage amount of Exendin-4 in the medicinal composition is 5-1000 mu g, preferably, the usage amount of Exendin-4 in the medicinal composition is 25-500 mu g. Because the pharmaceutical composition contains the mucosa adsorbent, the thickening agent and the high-activity rhPH20, the pharmaceutical active substance Exendin-4 can quickly permeate and absorb through oral mucosa and enter blood circulation, effective blood concentration can be achieved, the administration frequency can be reduced, and no administration pain exists, so that the clinical medication compliance of patients is obviously improved, and the Exendin-4 and rhPH20 are produced in a large scale by adopting a genetic engineering recombination technology, so that the clinical requirement for large-scale medication can be met, and the pharmaceutical composition has a good clinical application prospect.
Compared with the prior art, the invention has the following advantages and beneficial effects, including but not limited to:
1. the pharmaceutical composition provided by the invention is used for oral mucosa administration through the Exendin-4, the hyaluronidase PH20 and the mucosa adsorbent composition, on one hand, the barrier effect of the stratum corneum of the oral mucosa can be reversibly changed to promote the mucosa permeation and absorption of the Exendin-4, on the other hand, the oral mucosa can not be seriously stimulated and damaged, and can be quickly recovered after the mucosa is damaged, the mucosa adsorbent can enable the composition to be quickly adhered to the surface of the mucosa, and active substances in the pharmaceutical composition can be quickly released, so that the utilization rate of the active substances is improved.
2. The pharmaceutical composition containing the Exendin-4 for oral mucosal administration contains the thickening agent, increases the viscosity of active substances of the pharmaceutical composition, and reduces the viscosity of oral mucus on the pharmaceutical composition and the degradation effect of protease in the mucus on the active substances of the pharmaceutical composition.
3. The medicinal composition containing the Exendin-4 administered through the oral mucosa improves the conductivity of the oral mucosa to hydrophilic polypeptide, so that the Exendin-4 rapidly permeates the oral mucosa at high concentration to enter blood circulation, the effective serum concentration of the medicine is reached, the systemic bioavailability of the Exendin-4 is improved, and the medicinal composition can be used as an alternative use mode of Exendin-4 injection administration.
4. The pharmaceutical composition containing Exendin-4 administrated through oral mucosa is safe and non-toxic in formula, convenient to use, non-toxic and non-irritant in the using process, quick in drug effect taking and capable of remarkably improving clinical drug compliance of patients.
5. Experiments show that the composition of the invention has a certain synergistic effect between hyaluronidase and the drug of the invention to bring the excellent effect, and compared with the combination of hyaluronidase and CD20 antibody, the composition has difficulty in meeting the clinical effect of oral administration.
Drawings
FIG. 1 shows the effect of rhPH20 with different enzyme activities on the osmotic absorbability of Exendin-4 through TR146 oral mucosal epithelial cells, wherein ●, ■ and ▲ indicate different concentrations of Exendin-4, respectively 48, 280 and 500. mu.g/ml, and PcRepresents the permeation coefficient of Exendin-4 through TR146 cells.
FIG. 2 shows the effect of mucosal adsorbents at different concentrations on the osmotic absorption of Exendin-4 by oral mucosal epithelial cells TR146, where ●, ■ and ▲ represent different concentrations of Exendin-4, respectively 48, 280 and 500. mu.g/ml, and PcRepresents the permeation coefficient of Exendin-4 through TR146 cells.
FIG. 3 shows different concentrations of pharmaceutical composition W administered to idiopathic type II diabetes KK via single oral mucosaAyEffect of blood glucose (mmol/L) in mice, wherein ● represents blank control, ■ represents subcutaneous injection of 0.6 μ g/kg of exitin-4, ◆, ▲ and the corresponding concentrations of pharmaceutical composition W are 56, 168 and 504 μ g/kg, respectively.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the invention are not limited thereto.
Exendin-4-containing protein stock solutions used in the examples were as follows: CN200410062650.5, the concentration of the obtained protein stock solution containing Exendin-4 is 1.5mg/ml, and the purity is 98.0%; the protein stock containing rhPH20 used was the literature: the method for preparing the protein is CN200680013224.X, and the obtained protein stock solution contains rhPH20 enzyme activity of 500-20000U/ml and has the purity of 98.0%.
Example 1 pharmaceutical composition containing Exendin-4 administered through oral mucosa and method for preparing the same
Respectively unfreezing the recombinantly prepared Exendin-4 protein stock solution (with the purity of 98 percent and the concentration of 1.5mg/ml) and the rhPH20 protein stock solution (with the purity of 98 percent and the activity of 500-20000U/ml) at room temperature or 2-8 ℃, and storing at 2-8 ℃ after unfreezing.
Accurately weighing mucosa adsorbent and/or thickener according to the ratio of the components of the pharmaceutical composition A-X in the following table 1, dissolving with water for injection to prepare a mixed solution, adding unfrozen rhPH20 protein stock solution, mixing, finally adding Exendin-4 protein stock solution, stirring, mixing, filtering and sterilizing with a filter membrane with the pore diameter of 0.22 mu m under aseptic condition to obtain the pharmaceutical composition A-X respectively.
TABLE 1 pharmaceutical composition A-X with various component ratios
EXAMPLE 2 detection of active ingredients in pharmaceutical composition containing Exendin-4 administered through oral mucosa
1) Detecting the biological activity of Exendin-4 in the pharmaceutical composition A-X:
the biological activity of Exendin-4 in the above pharmaceutical compositions A-X was measured by cAMP assay, and the purity and concentration of Exendin-4 were measured by high performance liquid chromatography (wherein the column used was Source 5RPC prepacked column (4.6 × 150mm, Amersham Biosciences)).
TABLE 2 Exendin-4 biological Activity, purity and concentration in pharmaceutical compositions A-X
The experimental results are shown in Table 2, the purity of Exendin-4 in the pharmaceutical compositions A-X is above 98%, the biological activity is kept at a high level, and the concentration is basically maintained unchanged.
2) Activity test of rhPH20 in pharmaceutical composition containing Exendin-4 administered through oral mucosa:
the enzymatic activity of rhPH20 in the above-mentioned pharmaceutical compositions a-X was examined according to the method for measuring the activity of hyaluronidase (nephelometry) provided in appendix XII C of the second part of the chinese pharmacopoeia, 2010 edition. The determination method is based on the fact that hyaluronic acid reacts with serum to form stable jelly under acidic conditions, the jelly formed by reaction is reduced and becomes clear after being degraded by hyaluronidase, the clarity is measured by the wavelength of 640nm, and the content of hyaluronidase and the clarity are in direct proportion. The enzyme activity of rhPH20 was calculated from a standard curve generated by measuring dilutions of the reference standard with rhPH 20.
TABLE 3 enzymatic Activity of rhPH20 in pharmaceutical compositions A-X
The results of the experiments are shown in Table 3, the rhPH20 enzyme activity in the pharmaceutical compositions A-X is kept at a high level, and the purity is more than 98%.
Therefore, the active ingredients in the pharmaceutical composition containing Exendin-4 administrated through the oral mucosa have higher biological activity and high purity, and the biological activity and purity of Exendin-4 and the enzyme activity and purity of rhPH20 can be further improved after the thickening agent is added.
Comparative example 1 pharmaceutical composition containing Exendin-4 and preparation method thereof
The mixture ratio of the components in A1-A5 in Table 4 is shown, wherein the composition A1 does not contain rhPH20, thickening agent CMC-Na and mucous membrane adsorbent, and the other components and the mixture ratio are the same as those of the composition E; the composition A1+ contains the same components and proportions as the composition E except that the composition does not contain Exendin-4, a thickening agent CMC-Na and a mucous membrane adsorbent; the composition A2 contains the same components and proportions as those of the composition L except that the composition A2 does not contain rhPH 20; the composition A3 contains the same components and proportions as those of the composition R except that the thickening agent CMC-Na and the mucous membrane adsorbent are not contained; the composition A4 contains the same components and proportions as those of the composition U except that the composition A4 does not contain rhPH 20; composition A5A 1-A5 was prepared in the same manner as in example 1, except that the thickener CMC-Na and the mucous membrane adsorbent were not included, and the other components and their proportions were the same as those of composition X.
TABLE 4 formulation of pharmaceutical composition A1-A5
Comparative example 2 pharmaceutical composition using conventional osmotic agent instead of rhPH20
(1) Sodium deoxycholate was used as an absorption enhancer for pharmaceutical compositions, and compositions B1 to B4 were prepared in the same manner as in example 1, according to the blending ratios of the components in table 5.
TABLE 5 formulation of pharmaceutical composition B1-B4
(2) Lactoferrin hydrolysate was used as an absorption penetration enhancer of the pharmaceutical composition at a concentration of 5mg/ml, and the same method as in example 1 was used to prepare composition B5 in accordance with the ingredient ratio of B1 in table 5, wherein the lactoferrin hydrolysate was obtained by the following literature: CN 103566376A.
Comparative example 3 pharmaceutical composition containing anti-CD 20 monoclonal antibody for oral transmucosal administration
TABLE 6 pharmaceutical composition C containing anti-CD 20 monoclonal antibody
anti-CD 20 mabs, such as rituximab, can be prepared according to the ratios of the components in composition C in table 6 by the same method as in example 1, in order to achieve oromucosal administration. However, in order to prepare a composition containing rituximab at such a high concentration, concentration of the antibody is required, but the concentrated antibody is liable to aggregate, the antibody activity cannot be ensured, and after absorption through the oral mucosa, the immunogenic reaction of the therapeutic antibody is liable to occur in vivo, and the neutralizing antibody produced in this process may render the therapeutic antibody ineffective. Therefore, the preparation of the pharmaceutical composition containing the anti-CD 20 monoclonal antibody for oral mucosal administration and the use thereof in treating related diseases cannot be realized technically.
Example 3Exendin-4 osmotic absorption in vitro assay on TR146 oral mucosal epithelial cells
TR146 esophageal squamous carcinoma cells (from ATCC) are used as an oral mucosa epithelial cell osmotic transfer model to test the osmotic absorbability of Exendin-4. The method described in the documents Porter A.pharm Res,2002,19(2): 169-:
DMEM culture solution in each chamber above and below the plug-in culture dish is sucked off, and TR146 cells are washed by HBSS buffer solution. Then adding HBSS solution into upper and lower chambers respectively, after balancing for 1h, sucking out HBSS solution in upper chamber, and adding 2mL test sample (pharmaceutical composition A-X, pharmaceutical composition B1-B4). 100 μ L of sample was taken from the lower chamber at the set time and the same amount of HBSS solution was immediately replenished. And (3) analyzing the sample by adopting a chromatographic method, recording the obtained peak area (A), and calculating the accumulated permeation quantity of the Exendin-4 at each time point. The following indices were calculated from the data obtained: (1) exendin-4 apparent permeability coefficient: papp＝(dQ/dt)/(A×C0) (ii) a (2) Permeation coefficient of Exendin-4 through plug-in culture dish (without cells): pE-4＝(dQ/dt)/(A×C0) (ii) a (3) Cell permeability coefficient: 1/Papp＝1/Pc+1/PE-4(ii) a (4) Osmotic absorption enhancement ratio: ER ═ Pc (composition)/Pc (control). Where dQ/dt represents the steady state permeation rate and A is the permeation area (cm)2)，C0Refers to the initial concentration, P, of Exendin-4 in the upper chambercRefers to the permeability coefficient of Exendin-4 through cells, PE-4Meaning the osmotic coefficient of the inserted culture dish in the absence of cells, Pc (composition)Refers to the cell permeability coefficient of Exendin-4 in the pharmaceutical composition A-X through TR146, Pc (control)Refer to the cell permeability coefficient of Exendin-4 passing TR146 in composition A1-A5, wherein the control group of pharmaceutical composition E, L, R, U, X is composition A1 and A1 respectively+A2, A3, a4 and a 5.
Composition A1+The method for detecting the permeability of the oral mucosa epithelial cells of Exendin-4 through TR146 comprises the following steps: DMEM culture solution in each chamber above and below the plug-in culture dish is sucked off, and TR146 cells are washed by HBSS buffer solution. Adding HBSS solution into upper and lower chambers respectively, balancing for 1 hr, sucking out HBSS solution from upper chamber, and adding 1mL composition A1+Then, 1ml of composition A (Exendin-4 at a concentration of 48. mu.8/ml) was added. 100 μ L of sample was taken from the lower chamber at the set time and the same amount of HBSS solution was immediately replenished. And (3) analyzing the sample by adopting a chromatographic method, recording the obtained peak area (A), and calculating the accumulated permeation quantity of the Exendin-4 at each time point.
(1) Example 1 and comparative example 1 Effect of each pharmaceutical composition on the osmotic absorbability of Exendin-4 through TR146 oral mucosal epithelial cells
TABLE 7 influence of each of the pharmaceutical compositions of example 1 and comparative example 1 on the osmotic absorbability of Exendin-4 through TR146 oral mucosal epithelial cells (x. + -.s, n ═ 3)
The experimental results are shown in Table 7, Exendin-4 in the pharmaceutical composition A-X has good permeation and absorption effects through TR146 oral mucosa epithelial cells; composition A1 lacks rhPH20 and mucosal adsorbents, so that the osmotic absorption of Exendin-4 is significantly lower than that of pharmaceutical composition A; composition A1+(only contains rhPH20), when the composition A1 (only contains Exendin-4) is respectively placed in an upper chamber and a lower chamber, the osmotic absorption of the Exendin-4 through TR146 oral mucosa epithelial cells is higher than that of the composition A1, but is still significantly lower than that of the composition A, which indicates that when the rhPH20 and the effective component Exendin-4 are respectively used, the osmotic absorption of the Exendin-4 through TR146 oral mucosa epithelial cells can be promoted to a certain extent, but the osmotic absorption of the pharmaceutical composition of the invention to the Exendin-4 is still significantly lower; compositions a2, a4 contained mucosal adsorbents, so that the osmotic absorption of Exendin-4 by TR146 oral mucosal epithelial cells was slightly higher than composition a1, but due to the absence of rhPTH20, the osmotic absorption of Exendin-4 was significantly lower than composition L, U; the compositions A3 and A5 contain rhPH20, improve the permeation and absorption of Exendin-4 through TR146 oral mucosa epithelial cells, are higher than the compositions A1, A2 and A4, but are difficult to form uniform and stable substances in the compositions, and are still obviously lower than the composition R, X. Therefore, rhPH20, a mucous membrane adsorbent and a thickening agent have synergistic effect on the permeation and absorption of Exendin-4 by TR146 oral mucosal epithelial cells.
(2) Comparative example 2 Effect of conventional absorption penetration enhancer on the penetration and absorption of Exendin-4 into epithelial cells of oral mucosa of TR146
When sodium deoxycholate is used as an absorption penetration enhancer of the pharmaceutical composition, although the ordered structure of the TR146 oral mucosal epithelial cell lipid bilayer can be changed, so that the order of phospholipid molecules is reduced, the viscosity between the TR146 oral mucosal epithelial cells is reduced, and the permeability between the mucosal cells is improved, the pore size formed between the TR146 oral mucosal epithelial cells is limited, and is not larger than that formed after rhPH20 degrades hyaluronic acid between the mucosal cells, so that the penetration absorption of Exendin-4 through the TR146 oral mucosal epithelial cells is lower than that of Exendin-4 in example 1.
When the lactoferrin hydrolysate is used as an absorption penetration enhancer of the pharmaceutical composition, although the lactoferrin-4 can be embedded into the surface of the TR146 oral mucosal epithelial cells to enable the Exendin-4 to penetrate the mucosa, the capacity of reducing the viscosity of the TR146 oral mucosal epithelial cells is limited, and the force of changing the tight junction between the mucosal cells is not as strong as that of rhPH20, so that the penetration and absorption of the Exendin-4 through the TR146 oral mucosal epithelial cells are lower than that of the Exendin-4 in example 1.
Example 4 Effect of different enzyme activities of rhPH20 on osmotic absorption of Exendin-4 by TR146 oral mucosal epithelial cells
According to the mixture ratio of the compositions D1-D3 in Table 8, wherein the rhPH20 enzyme activities are respectively 300, 600, 1000, 2000, 5000, 8000, 12000 and 20000U/ml, the pharmaceutical compositions containing rhPH20 with different enzyme activities are prepared by the same method as the example 1, and the influence of the rhPH20 with different enzyme activities on the penetration and absorption of Exendin-4 oral mucosa epithelial cells through TR146 is detected by the same method as the example 3.
TABLE 8 formulation of pharmaceutical composition D1-D3
The experimental result is shown in figure 1, when the activity of the rhPH20 enzyme is 1000-20000U/ml, the osmotic absorption effect of Exendin-4 through TR146 oral mucosa epithelial cells can be effectively promoted; when the activity of the rhPH20 enzyme is less than 1000U/ml, the osmotic absorption effect of Exendin-4 through the oral mucosa epithelial cells of TR146 is lower than that of rhPH20 enzyme when the activity of the rhPH20 enzyme is 1000-20000U/ml, and when the activity of the rhPH20 enzyme is less than 500U/ml, the osmotic absorption performance of Exendin-4 through the oral mucosa epithelial cells of TR146 is extremely low, so that the bioavailability of Exendin-4 cannot be obviously increased; when the activity of the rhPH20 enzyme is higher than 20000U/ml, the osmotic absorption effect of Exendin-4 is not increased significantly any more. Therefore, when the activity of the rhPH20 enzyme is 1000-20000U/ml, the osmotic absorption of Exendin-4 through TR146 oral mucosal epithelial cells has dose dependence.
Example 5 Effect of different concentrations of mucosal adsorbents on the osmotic absorbability of Exendin-4 through TR146 oral mucosal epithelial cells
According to the mixture ratio of the compositions E1-E3 in Table 9, wherein the concentrations of the mucosal adsorbent povidone K30 are 0.2, 0.8, 1, 5, 8, 12, 15, 18 and 25mg/ml, the compositions containing mucosal adsorbents with different concentrations are prepared by the same method as in example 1, and the influence of the mucosal adsorbents with different concentrations on the osmotic absorbability of Exendin-4 oral mucosal epithelial cells through TR146 is detected by the same method as in example 3.
TABLE 9 formulation of pharmaceutical composition E1-E3
The experimental result is shown in fig. 2, when the concentration of the mucosa adsorbent povidone K30 is 1-15 mg/ml, the composition can be effectively promoted to be adhered to the oral mucosa, the osmotic absorption effect of Exendin-4 on the epithelial cells of the oral mucosa of TR146 is facilitated, and the bioavailability of the Exendin-4 can be improved; when the concentration of the povidone K30 is less than 1mg/ml, the osmotic absorption effect of the Exendin-4 through TR146 oral mucosa epithelial cells is very low, which indicates that the concentration of the povidone K30 is too low, which is not beneficial to the adhesion effect of the composition, so that the osmotic absorption effect of the Exendin-4 is obviously reduced; when the concentration of the povidone K30 is more than 15mg/ml, the osmotic absorption of the Exendin-4 through TR146 oral mucosa epithelial cells is not remarkably increased, which shows that when the concentration of the povidone K30 is more than 15mg/ml, the viscosity of the composition is remarkably increased, so that the adhesive force is high when the composition is contacted with the oral mucosa, and the diffusion absorption effect of the Exendin-4 through the TR146 oral mucosa epithelial cells is influenced. Therefore, the concentration of the povidone K30 is 1-15 mg/ml, and the osmotic absorption of the Exendin-4 through TR146 oral mucosa epithelial cells has dose dependence.
In addition, when the concentration of the mucosa adsorbent carbomer is 0.2-1 mg/ml and the concentration of the carboxymethyl chitosan is 1-5 mg/ml, the tendency chart of the osmotic absorption effect of Exendin-4 through TR146 oral mucosa epithelial cells is similar to that of povidone K30 when the concentration is 1-15 mg/ml.
EXAMPLE 6 in vivo efficacy test of Exendin-4-containing composition administered through oral mucosa
Selecting 60 spontaneous II type diabetes KKAyMice, randomized into 5 groups of 12 mice each. Mice in 3 groups of drug groups to be tested are respectively administrated with the drug composition W prepared in the example 1 through single Oral mucosa, and are respectively administrated according to the body weight of 56, 168 and 504 mu g/kg (corresponding to 2, 6 and 18 mu g/kg of Exendin-4, and the dosage is set according to the prior insulin spray Oral-lyn relative to the injection administration); the positive drug control group mice are given 0.6 mu g/kg body weight Exendin-4 by single subcutaneous injection; placebo mice were given a single oral mucosal dose of an equal amount of phosphate buffer.
After the mice of the blank control group and the drug group to be tested are anesthetized by injecting 60mg/kg sodium pentobarbital solution into tail veins, the mice are kept in a supine position on an anesthesia table, and are ligated by esophagus, and each experimental animal is ensured to be at the same position on a horizontal line when oral mucosa is administrated. Loading the compositions of the blank control group and the drug group to be tested in different spray bottles respectively, spraying the compositions of different groups into oral mucosa of animals of corresponding groups, and releasing esophageal ligation 10min after administration.
The 5 groups of animals respectively take blood from eye sockets before administration (0h), 1h, 2h, 3h, 4h, 5h and 6h after the first administration, determine fasting blood glucose concentration by glucose oxidase method, and investigate the effective component Exendin-4 of the pharmaceutical composition W on spontaneous type II diabetes KKAyEffect of blood glucose concentration in mice. On the day of blood glucose measurement with each blood draw, the dose was administered 3h after the food withdrawal and food intake was resumed after the blood draw was completed.
The results show that compared with a blank control group, 1h, 2h and 3h after the drug delivery of the drug composition through single oral mucosa and single subcutaneous injection of Exendin-4 all obviously reduce spontaneous II type diabetes KKAyBlood glucose concentration (P) in mice<0.05); high dose pharmaceutical composition W (504. mu.g/kg) and Medium dose pharmaceutical composition W (168. mu.g/kg) reduced blood glucose concentration in mice at 1h, 2h and 3h after administrationThe amplitude is obviously higher than that of Exendin-4 injected in a single subcutaneous injection; at 6h after administration, the blood glucose concentration of the mice was maintained at a level lower than that of a single subcutaneous injection of Exendin-4 by the high-dose pharmaceutical composition W (504. mu.g/kg), the medium-dose pharmaceutical composition W (168. mu.g/kg) and the low-dose pharmaceutical composition W (56. mu.g/kg); spontaneous type II diabetes KK of blank control groupAyThe blood glucose concentration of the mice was always at a higher level (see table 10, fig. 3).
TABLE 10 Single oromucosal administration of pharmaceutical composition W for idiopathic type II diabetes KKAyEffect of blood glucose (mmol/L) in mice
P <0.05 compared to placebo; p <0.01
Therefore, the pharmaceutical composition containing Exendin-4 administered through the oral mucosa prepared in the embodiment 1 of the invention has the effect of remarkably reducing the blood sugar concentration of diabetic mice, and compared with the Exendin-4 injected subcutaneously, the pharmaceutical composition prepared in the embodiment 1 of the invention can maintain the blood sugar concentration of the mice at a lower level for a longer time. The oral mucosa administration route realizes the good absorption and utilization of the drug Exendin-4.
Under the same experimental conditions, the pharmaceutical compositions A-V and X show similar hypoglycemic effects to the pharmaceutical composition W, especially the pharmaceutical composition W is the best.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (4)
1. A pharmaceutical composition comprising a human glucagon-like peptide-1 (hGLP-1) analog for oromucosal administration, comprising:
1) the concentration of the human glucagon-like peptide-1 (hGLP-1) analogue is 25-500 mu g/ml, and the human glucagon-like peptide-1 (hGLP-1) analogue is Exendin-4;
2) human hyaluronidase (hPH20) with enzymatic activity of 1000-20000U/ml;
3) the concentration of the more than one pharmaceutically acceptable mucosa adsorbent is 0.2-15 mg/ml, and the mucosa adsorbent is a composition obtained by mixing one or more than two of povidone, carboxymethyl chitosan and carbomer in any proportion;
4) the thickening agent is 1-35% by mass, and the thickening agent is sodium carboxymethylcellulose.
2. Use of the pharmaceutical composition comprising human glucagon-like peptide-1 (hGLP-1) analog according to claim 1 for oromucosal administration in the manufacture of a medicament for the treatment of hyperglycemia and/or diabetes.
3. The use according to claim 2, wherein the pharmaceutical composition is prepared as a spray or as an oromucosal patch.
4. A process for preparing a pharmaceutical composition according to claim 1, comprising the steps of:
1) preparing a mixed solution containing a mucous membrane adsorbent and a thickening agent;
2) adding the human hyaluronidase solution prepared by recombination into the mixed solution prepared in the step 1),
mixing uniformly;
3) adding the human glucagon-like peptide-1 (hGLP-1) analog solution obtained by recombinant preparation into the mixed solution prepared in the step 2), wherein the human glucagon-like peptide-1 (hGLP-1) analog is Exendin-4, and uniformly mixing;
4) and (5) sterilizing to obtain the product.
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