CN103520706A - Oral recombined human serum albumin fusion protein preparation formula, products of the protein preparation, and applications of the protein preparation - Google Patents

Abstract

The invention provides a universal, simple and extremely effective oral preparation which is manufactured from one or more recombined human serum albumin fusion proteins with biological functions, a proper amount of an excipient, a proper amount of an adhesion agent, a proper amount of a nutrition agent and a proper amount of a buffer salt. The preparation technology, formula compositions and production technology of the oral preparation are simple, unique, high-quality and prone to industrialization. Products with different forms manufactured from the oral preparation, which comprises spray directly adsorbed through an oral cavity, and tablets directly adsorbed through hypogloeeis or adsorbed after enteric dissolution, can be used directly for human health care, and physical function improvement, or can be used as oral medicines with clinic treating significance.

Description

Pharmaceutical formulation for recombined human serum albumin fusion proteins for oral use, product and purposes

Technical field

The present invention is the continuation application of Chinese invention patent ZL200910210379.8, is also the continuation application of Chinese invention patent ZL021428816, ZL2004100428148, ZL200710057571.9 and ZL200710059770.3.The formula that the present invention relates to recombined human serum albumin fusion proteins oral formulations forms and preparation method, and relates to single fusion rotein or the method that be used in combination of different fusion rotein in pharmaceutical formulation.Be particularly related to by the oral formulations formula of human serum albumin and the formed recombination fusion protein of the human cell growth factor, preparing product and in health care and application medically.

Technical background

Human serum albumin (HSA) is a kind of soluble and monomeric protein, forms half of Tot Prot in blood.Albumin, as a kind of underlying carrier, carries and transmits fatty acid, steroid and hormone molecule etc., and its stable inert nature is a key factor that maintains blood pressure.Serum albumin is that a kind of spherical nonglycosylated, molecular weight is 65 kilodaltons, has 585 amino acid whose serum proteins.This albumen (albumin precursor) is processed by the conversion of Golgi body again, removes guiding polypeptide, and is secreted into extracellular.Serum albumin has 35 cysteine, and in blood, albumin is a monomer (referring to Brown JR, " albuminous structure, function and application " Pergamum, New York, 1977) that has 17 disulfide bond.When there is no secrete polypeptide, the albumin product in yeast cells is mispairing state, by the antigenicity (comparing with naturalness albumin in blood plasma) that loses 90%, and forms insoluble albumin polymer, does not have albuminous biological activity.Now for clinical human serum albumin, all from human plasma, extract.The production that utilizes the recombinant expressed albumin of microorganism (rHSA) is open in patent of invention ZL200410057313.7 " structure of high efficient expression human serum albumin yeast strain and fermentation purifying process " authorizes in the China of patent EP330451, EP361991 and Yu Zailin, Fu Yan.

Albumin is the main component in blood, at people's in-vivo content, is that every liter of blood contains 40 grams, and its life-span half-life is 14-20 days.In sum, therapeutic protein and albumin form after fusion rotein, and fusion rotein will have great advantage makes it to resist zymolysis in organism, and can make therapeutic protein in vivo, the external life-span improves greatly, reach prolonged drug, can also higher dosage use simultaneously.In pharmaceutical preparation forms, albumin is also often used as the stabilizing agent of medicine, especially biological medicament.Utilize technique for gene engineering, human serum albumin and therapeutic protein gene fusion are expressed as to fusion rotein in yeast, the stability that can increase therapeutic protein in serum and while storing, and longer half-life, reach long-actingization of protein drug.This protein modified prolonged drug common techniques platform, open in Yu Zailin, rich rock China mandate patent of invention ZL02142881.6, ZL200410042814.8 and ZL200710057571.9, in this as foundation and document incorporated by reference.

Therapeutic protein and recombined human serum albumin fusion proteins are all clinically with injection system administration.Yu Zailin, Fu Yan etc. disclose general recombined human serum albumin fusion proteins injection preparation formula and preparation technology and medical application in Chinese invention patent ZL200910210379.8 " general pharmaceutical formulation for recombined human serum albumin fusion proteins for injection ".The present invention continues to disclose a kind of oral formulations formula, preparation method, product and medical application of recombined human serum albumin fusion proteins.

Human serum albumin fusion proteins refers to that human serum albumin's mature peptide complete amino acid sequence and therapeutic protein mature peptide complete amino acid sequence link by different way, the brand-new aminoacid peptide chain of formation.Between two protein molecules in fusion rotein, can be provided with or not be provided with connection peptides.Therapeutic protein mature peptide can link with the N-end of human serum albumin's mature peptide aminoacid sequence by its C-end, or the N-of therapeutic protein mature peptide aminoacid sequence holds C-with human serum albumin's aminoacid sequence to hold to link to form.Therapeutic protein can be any protein with biological function.Recombined human serum albumin fusion proteins refer to human serum albumin fusion proteins utilize technique for gene engineering in external source expression system through gene expressions such as yeast, vertebrate cells (such as CHO), antibacterial, fungus, animal, insecticide, plants, through scale separation and purification be prepared from.Recombined human serum albumin fusion proteins includes, but are not limited to, all hemocytees stimulate generate the factors, human growth hormone, insulin, human glucagon, people's insulin secretion accelerating peptide, interleukin, interferon, Skin Cell somatomedin,

Vivo and vitro experiment inventor by animal and cell is verified human serum albumin fusion proteins can irritation cell hypertrophy, and have than the cell growth factor of monomer time longer with external stop (long half-lift and persistence) in vivo, cell also finds that with zoopery fusion rotein has mole biological activity identical with human cytokines monomer.During the combination of the fusion rotein (HSA/GF) being formed by HSA and different somatomedin when compound use, can have and stimulate the reparation of various skin cell and tool synergistic function simultaneously, and be applicable to the treatment of beauty treatment, wound and disease,

Oral administration of drugs is because convenient drug administration, degree of complying with are high, and becomes a kind of optimal administering mode.But the bioavailability of pharmaceutical grade protein oral administration administration is not high, therefore, this approach is not generally suitable for the administration of the such biomacromolecule of protein.Recombined human serum albumin fusion proteins has the long-lasting and remarkable result of disease treatment clinically.Inventor continues to find the oral formulations formula that is suitable for recombined human serum albumin fusion proteins, to carry out expansion, grind high, reach more effectively and convenient with the preparation of low cost very, obtain the useful new pharmaceutical formulation that health state forms new health promoting product and has clinical efficacy that improves.Particularly, in the situation of not desired to treat clinically " will strictly confirm to enter the drug dose of blood system ", oral fusion rotein is to arrive to improve condition, and arriving fine setting physical function is one of main purpose of the present invention.

Mucosal drug delivery system is to use suitable carrier in the position administration of human body mucosa, as nasal mucosa, oral mucosa, eye mucosa, rectal mucosa, uterus and vaginal mucosa, medicine sees through mucosa and enters the administering mode that body blood circulation produces whole body or local therapeutic effects.Mucosal drug delivery dosage form need to have the adjuvant of bioadhesive.The adjuvant that sticks that can be used as medicine-releasing system comprises, natural polymer, there are gelatin, chitosan, agglutinin, sodium alginate etc., also have synthetic polymer carbomer (CP), polyvinylpyrrolidone (PVP), hydroxypropyl emthylcellulose (HPMC), hydroxypropyl cellulose (HPC) and Polyethylene Glycol (PEG) etc.These polymer can attach on aqueous biomembrane well, thereby make medicine can bring into play rapidly drug effect at lesions position, can maintain for a long time drug effect again.Mucosal drug delivery has been widened the route of administration of many medicines greatly, particularly some polypeptide classes, macromole class medicine.Cheek mucosal drug delivery and the administration of oral administration oral mucosa have following characteristics: the blood vessel at the cheek mucosa in (1) oral mucosa and mucosa position, Sublingual is intensive, and blood flow is abundant, and the permeability of mucous membrane tissue is only second to nasal mucosa; (2) compare with percutaneous dosing with nasal-cavity administration, in oral mucosa, the activity of enzyme is lower, therefore can effectively avoid degraded and the metabolism of medicine; (3) oral mucosa has the strong toleration stimulating to external world, and some compositions that are subject in preparation when mucous membrane tissue produce zest and damage, after discontinuing medication, can recover preferably; (4) dosage form is easily located, medication is convenient, can remove at any time medicine, easily by patient, accepted (S Gupta, et al.Oral delivery of therapeutic proteins and peptides:a review on recent developments.Drug Delivery, 2013, Vol.20, No.6, Pages237-246).Oral vaccine is method newer in protein vaccine, and two kinds of oral vaccine Sa guest's oral polio vaccine and Ty21 antityphoid vaccine are put into vaccination plan on the U.S. and other places.At present, the vaccine that comprises the diseases such as acquired immune deficiency syndrome (AIDS), anthrax, cervical cancer, cholera, gastroenteritis, diarrhoea and malaria is all being attempted oral immunity treatment (Aziz MA et al, Oral vaccines:new needs, new possibilities.Bioessays, 2007,29 (6): 591-604).Soluble protein antigen can stimulate generation immunity (Verdonck F et al when oral administration, Oral immunization of piglets with recombinant F4fimbrial adhesin FaeG monomers induces a mucosal and systemic F4-specific immune response, Vaccine, Volume22, Issues31-32,22October2004, Pages4291-4299).

Oral protein drug main will adopt two administration routes, the one, utilize bio-adhesive (bio-adhesion) principle, to play location " platform " effect with polymeric material, by mucosal drug delivery, make preparation there is the feature of local targeting, long-lasting, slow release.Bernkop-Schnurch and Pasta[Intestinal peptide and protein delivery:Novel bioadhesive drug-carrier matrix shielding from enzymatic attack.J Pharm Sci, 1998,87 (4): 430-4] research shows, conjugate by chitosan and derivant and enzyme inhibitor reduces the biodegradation of digestive enzyme to proteins and peptides class medicine, can greatly improve oral insulin bioavailability.The 2nd, adopt the form of protecting, by antigen coated, the destruction that protectiveness material can protect medicine not to be subject to pipe intestinal digesting enzyme, increases medicine when reaching intestinal, with the close contact of intestinal mucosa, improves bioavailability.

Protein is in gastrointestinal inherent instability, and due to the relative molecular mass compared with high and polar surfaces characteristic and to biomembranous hypotonicity, shows that protein can only avoid directly through gastrointestinal administration when for whole body therapeutic.By selecting different administering modes to reach higher bioavailability, employing comprises that protein molecular structure is modified, researched and developed novel effective enteric coated capsule, intestinal absorption promoter, adds enzyme inhibitor, colon positioning release, medicine pulsed doser and medicine carrying microballoons, nano-particle discharges and the several different methods such as receptor mediated targeted carrier, improves oral administration biaavailability, protein blood drug level and the stability of pharmaceutical grade protein in intestinal of pharmaceutical grade protein.By various salvos, allow pharmaceutical protein can resist the attack of esophagus stomach function regulating Digestive system and arrive safely small intestinal, then discharging, and with other materials, reducing the malabsorption of small intestinal, by intestinal mucosa, make pharmaceutical protein can enter blood system.

Pasting material interpolation as binder comprises natural polymer, there are gelatin, chitosan, agglutinin, sodium alginate etc., also have synthetic polymer carbomer (CP), polyvinylpyrrolidone (PVP), hydroxypropyl emthylcellulose (HPMC), hydroxypropyl cellulose (HPC) and Polyethylene Glycol (PEG) etc.These polymer can attach on aqueous biomembrane well, thereby make medicine can bring into play rapidly drug effect at lesions position, can maintain for a long time drug effect again.

Alginic acid sulfuric ester (sodium alginate) hydrophilic is strong, in cold water and warm water, can dissolve, and forms the very uniform solution of thickness.The true solution forming has other analog and is difficult to the flexibility, homogeneity and other good characteristics that obtain.There is the effect of very strong protecting colloid, strong to the emulsifying ability of oils and fats.As this characteristic of the dispersant acid labile of fusion rotein to medicine of great use, can make medicine avoid the attack of gastric acid, and gel swelling can make medicine discharge with the speed of expectation in small intestinal.

Mucosal drug delivery has been widened the route of administration of recombined human serum albumin fusion proteins greatly, particularly some growth hormone, leukocyte increasing, rise the medicine of erythrocyte, infection, raising immunity of organisms.

Solving macro-molecular protein, also can be as micromolecule polypeptide can oral administration to add blood circulation of human body system be in pharmaceutical formulation and production technology, to make effective improvement.

Summary of the invention

Present inventor discloses a kind of recombined human serum albumin fusion proteins preparations for oral administration formula, preparation technology and product.Embodiment has verified the oral formulations that contains human serum albumin fusion proteins, by oral, no matter be oral buccal tablet or enteric coated capsule, all arrives the body local position tissue of appointment.Fusion rotein antibody test result shows that recombined human serum albumin fusion proteins can enter in blood of human body.Formulation products, to human body nonirritant, is applicable to the treatment of body-care, health improvement and some disease, completes thus the present invention.

The present invention's recombined human serum albumin fusion proteins applicatory includes, but are not limited to, fusion rotein (Yu Zailin, Chinese patent ZL02142881.6 that human serum albumin and hematopoietic cell proliferation stimulating factor form; US7,244,833B2), include, but are not limited to: human serum albumin and human red cell proliferin (rHSA/EPO), with human interleukin (rHSA/ILs), with human stem cell factor (rHSA/SCF), with human blood platelets proliferin (rHSA/TPO), with Filgrastim (rHSA/GCSF), with human glucagon (rHSA/GLP-1), with people's insulin secretion accelerating peptide (rHSA/Exendin-4), with insulin human (rHSA/Insulin) or the fusion rotein that forms with the huge phasmid colony-stimulating factor of people (rHSA/GM-GSF); Human serum albumin and human interferon and analog (Fu Yan, Yu Zailin ZL200410042814.8), include, but are not limited to: with interferon-ALPHA 2a, α-2b or-1b (rHSA/IFN α-2a ,-2b or-1b), with interferon-beta (rHSA/IFN-β), with interferon-γ (rHSA/IFN γ ), interferon-' alpha ' 8 (rHSA/IFN α 8) or the fusion rotein that forms with interferon-ω (rHSA/IFN ω); Human serum albumin and human skin cell's somatomedin (Yu Zailin, rich rock Chinese patent ZL200710057571.9), comprise, but be not limited to, with human epidermal growth factor (rHSA/EGF), with human acid fibroblast growth factor (rHSA/aFGF), with human alkaline fibroblast growth factor (rHSA/bFGF), with human insulin-like cell growth factor (rHSA/IGF), with human horny cell growth factor-2 (rHSA/KGF) or the fusion rotein that forms with vectors containing human platelet-derived growth (rHSA/PDGF).

Recombined human serum albumin fusion proteins can be by, but be not limited to, yeast, vertebrate cells, as Chinese hamster ovary celI, antibacterial, insecticide, animal or plant express, through diverse ways separation and purification, obtain with stock solution form or lyophilized form and suitable adjuvant mixed preparing.

Different recombined human serum albumin fusion proteins can be separately in oral formulations disclosed by the invention formula or compound use, adjuvant in can filling a prescription with formation oral formulations, independent one or more common combinations that have in excipient, binder, thickening agent, emulsifying agent, wetting agent, buffer agent function are used, to form oral formulations, and in order to can reach reparation, renewal, the correction of human body cell, take and reach being healthy and strong, improving looks or treating as object of the mankind, and be prepared into product, use.

In pharmaceutical formulation of the present invention, can be to contain recombinant human cell growth factor, recombinant human somatropin's monomer, functional, treatment recombinant protein that to be non-warp merge with human serum albumin, comprise, but be not limited to recombinant human somatropin, recombinant human epidermal growth factor, recombinant human insulin, human glucagon.

A preparations for oral administration that contains human serum albumin fusion proteins, is characterized in that:

Described preparations for oral administration is by recombined human serum albumin fusion proteins and pharmaceutically acceptable excipient sucrose, mannitol, tween 80 and maintain the buffer salt of pH; And pharmaceutically acceptable adhesion adjuvant chitosan, sodium alginate, hyaluronic acid, hydroxypropyl emthylcellulose (HPMC) form jointly;

(1) in, contained recombined human serum albumin fusion proteins can be a kind of fusion rotein, can be also containing two or more different fusion rotein;

(2) in, contained recombined human serum albumin fusion proteins content is 0.1-10%;

(3) the pharmaceutically acceptable excipient adjuvant described in is mannitol or glycine, and acceptable salt buffer is phosphate buffer or acetate buffer or citrate buffer; Wherein the mannitol content of excipient adjuvant is 0.2-6%; The content of sucrose is 0.5-1.5%, and the content of polyoxyethylene sorbitan monoleate is 0.002-0.006%, and the concentration of salt buffer is 5-100mM, and pH value is 5.5-7.0;

(4) the pharmaceutically acceptable adhesion adjuvant described in is chitosan, sodium alginate, hyaluronic acid, hydroxypropyl emthylcellulose (HPMC); Wherein the content of adhesion adjuvant is chitosan 0.2-2%, sodium alginate 0.4-4%, hyaluronic acid 0.1-1%, hydroxypropyl emthylcellulose (HPMC) 0.2-2%.

In the oral formulations that contains recombined human serum albumin fusion proteins lyophilized powder, also can contain lecithin, cephalin, aminoacid, vitamin, trace element etc.Such as: glycine, α-GPC (L-α-choline glycerophosphatide), L-arginine, L-3,4 dihydroxyphenylalanine bean extract, GAMMA AMINOBUTRIC ACID, the acid of L-glutamy, ILE, 1B etc., vitamin C, vitamin E, biotin etc.

For reaching goal of the invention, the technical solution used in the present invention is:

Prepare a kind of preparations for oral administration that contains human serum albumin fusion proteins, the fusion rotein that the human serum albumin of containing in described preparations for oral administration and therapeutic protein form, and with medically acceptable excipient, binder and stabilizing agent adjuvant, in the pharmaceutically acceptable salt buffer of pH4-8, by lyophilization, form white loose body lyophilized powder, it meets water, can redissolve rapidly for supernatant liquid, for clinical treatment effect, the content of described recombined human serum albumin fusion proteins is 0.1-10mg/ oral buccal tablet or 0.1-10mg/ grain enteric coated capsule.

Recombined human serum albumin fusion proteins can be with various adjuvants together with aqueous solution form, start to form by lyophilization the preparation that contains fusion rotein, also first recombination human serum albumin can be formed to lyophilized powder with suitable pharmaceutical adjunct separately, and then prepare by a certain percentage with other adjuvant, after mixing, tabletting forms tablet finished product; Or pack in enteric coated capsule after preparation, form enteric coated capsule formula finished product, for oral application.

The expression of recombined human serum albumin fusion proteins and preparation method can be with reference to the mandate patent of invention (U.S. patent of invention US7 of Yu Zailin, Fu Yan, 244,833B2, US7,442,371B2, US7,572,437B2, the open CN101260145A of Chinese invention patent ZL02142881.6, Chinese invention patent ZL200410042814.8, Chinese invention patent ZL200410057313.7 and Chinese invention patent application) etc.

Obtain the described excipient and the stability adjuvant that in recombined human serum albumin fusion proteins lyophilized powder, use and can take the circumstances into consideration as required to add, as alcohols, amino acids and saccharide etc.In the present invention, preferred adjuvant excipient is mannitol, glycine, methionine, hetastarch, dextran, gelatin hydrolysate, polyvidone, alanine and other aminoacid, and stability adjuvant is lactose, sucrose, trehalose, glucose, Raffinose and arginine.For stop the tested surfactant of Protein formation polymer be medical science acceptable surfactant as Polysorbate (tween) 80, Polysorbate (tween) 20, cyclodextrin, dextran, poloxamer etc.The buffer system of preparation can be by calcium salt; Acetic acid sodium salt, phosphate, lactate, citrate, borate, phthalate, sodium chloride salt, monoammonium glycyrrhizinate, Cys hydrochlorate, anhydrous sodium sulfite or sodium hydroxide proper content form.

As the adjuvant of excipient, add and be preferably mannitol or glycine or the two and all contain, be preferably mannitol, content concn is 2-6%, is preferably 3-5%, more preferably 4.0%.The Optimization of Adjuvant with stability action is sucrose and trehalose, and content concn is 0.5-2%, is preferably sucrose 0.5-1.5%, more preferably 1.0%.Surfactant is preferably polyoxyethylene sorbitan monoleate, and content concn is 0.001-0.02%, is preferably 0.002-0.006%, and more preferably 0.004%.Buffer solution system as preparation is preferably citric acid, phosphate and acetic acid sodium salt, is preferably phosphate or citric acid, and content concn is 5-100mM, and being preferably content concn is 10-50mM, more preferably 10mM.The pH value of preparation is preferably 4.0-8.0, more preferably pH5.0-7.0.A mole osmotic pressure for pharmaceutical formulation is preferably the suitableeest scope 200-500mOsm of human body, is preferably 200-400mOsm, more preferably 220-340mOsm.Recombined human serum albumin fusion proteins is prepared into the pharmaceutical formulation of lyophilized powder and technique referring to Chinese invention patent ZL200910210379.8. such as Yu Zailins

Pasting material interpolation as binder comprises natural polymer, there are gelatin, chitosan, agglutinin, sodium alginate etc., also have synthetic polymer carbomer (CP), polyvinylpyrrolidone (PVP), hydroxypropyl emthylcellulose (HPMC), hydroxypropyl cellulose (HPC) and Polyethylene Glycol (PEG) etc.These polymer can attach on aqueous biomembrane well, thereby make medicine can bring into play rapidly drug effect at lesions position, can maintain for a long time drug effect again.Mucosal drug delivery has been widened the route of administration of recombined human serum albumin fusion proteins greatly, particularly some growth hormone, rise white, rise interferons medicine red, viral infection resisting.

Alginic acid sulfuric ester (sodium alginate) hydrophilic is strong, in cold water and warm water, can dissolve, and forms the very uniform solution of thickness.The true solution forming has other analog and is difficult to the flexibility, homogeneity and other good characteristics that obtain.There is the effect of very strong protecting colloid, strong to the emulsifying ability of oils and fats.As this characteristic of the dispersant acid labile of fusion rotein to medicine of great use, can make medicine avoid the attack of gastric acid, and gel swelling can make medicine discharge with the speed of expectation in small intestinal.

The maximum feature of preparation is can not produce any allergy, increase new toxic and side effects human body.Can add the more obvious adjuvant of the protective effect of fusion rotein, and can add any adjuvant that is conducive to product object, nutrient substance for example, as essential amino acid, vitamin etc.

In preparation optimization formula in the production process of recombined human serum albumin fusion proteins crude drug (stock solution), institute's water is without thermal source water for injection, agents useful for same is all through without thermal source treatment step, glass equipment used all passes through xeothermic baking except the processing of thermal source, and recipe ingredient used is " pharmaceutical grade " or " chemical analysis is pure " and meets the requirement of biological product to pharmaceutical formulation adjuvant.

Lyophilization can be so that human serum albumin fusion proteins preparation obtains very stable preservation state and the convenience of reprocessing.In water preparation formula, fusion rotein, when storage, along with the time, can form human serum albumin and the therapeutic protein segment of monomer free, and this is that water preparation dosage form mode is inevitable.Inventor, through comparing water preparation and freeze-dried powder preparation, demonstrates advantage of the present invention fairly obvious.The lyophilization production technology of recombined human serum albumin fusion proteins is as follows: stock solution adds through preferred adjuvant (mannitol, sucrose, polyoxyethylene sorbitan monoleate and saline solution) and is mixed with semi-finished product, after 0.22m positive press filtration, be divided in cillin bottle, or be placed in open square position, or be placed in tinfoil paper monolithic forming disk, enter freeze drying box and start lyophilizing program.Freeze-drying curve is :-50 ℃ of pre-cooling 4hr,-20 ℃ of 8hr that distil under vacuum state, then carry out gradient increased temperature and start to resolve ,-10 ℃ of 1hr, 0 ℃ of 1hr, 10 ℃ of 1hr, 29 ℃ (product temperature starts end vacuum while rising to 26 ℃), resolves about 9h altogether, through indexs such as pressure, detects rear pressing cover, go out freeze dryer, the finished product that is placed in cillin bottle is sealed up aluminium-plastic combined cover; Be placed in the aseptic plastic bag of being transferred to of open square position; Be placed in the finished product of tinfoil paper monolithic forming disk through pressing tinfoil paper cover plate.Lyophilizing the entire process have about needs 20-26h.Lyophilizing obtains the white loose body of molding, and moisture content is in 3.0%.After adding the solution of labelled amount or water or oral administration or oral cavity sublingual administration to take, all can redissolve rapidly for supernatant liquid.Freeze-drying curve can be controlled and be carried out optimization of profile by technical staff.

Preparation of the present invention and dosage form are preparation and the dosage forms that meets clinical medicine medication requirement, process for producing is simply controlled, simplified the oral medication method that can be used for producing various human serum albumin fusion proteins, be applicable to, comprise, but be not limited to, the fusion rotein that recombination human serum albumin and various therapeutic protein form directly preparation becomes oral medication, or in perfusion enteric solubility capsule, oral administration reaches drug delivery to specifying human body local location, melt Hou Bei relevant people soma and absorb, enter human recycle system.

In the study on the stability of pharmaceutical formulation for recombined human serum albumin fusion proteins medicament, the calibrating index of choosing has lyophilized powder redissolution speed, clarity detects, lipidated protein detects SDS-polyacrylamide gel electrophoresis and high performance liquid chromatography, protein content, biological activity determination at cellular level, pH value, the mensuration of various adjuvant content, moisture, sterility test or bacteria containing amount and no pathogenicity bacterium check, abnormal toxicity tests, the routines such as bacterial endotoxin inspection and the calibration method of formulating voluntarily decide pharmaceutical formulation and finished product whether to meet the requirement of oral formulations or medicinal preparation for oral administration.

In oral formulations formula, can be that recombined human serum albumin fusion proteins content is 1-5%, chitosan content is 0.5%, sodium alginate content is 1%, hyaluronic acid (hyaluronic acid) 0.3%, hydroxypropyl emthylcellulose (HPMC) content are 0.4%, mannitol content is 4.0%, cane sugar content is 1.0%, polyoxyethylene sorbitan monoleate content be 0.004% and the concentration of phosphate buffer be 10mM, pH6.5.Formulation products form can be buccal tablets, is placed in Sublingual and dissolves and absorb; Also can be manually or mechanical system perfusion enteric coated capsule in, oral administration enters oral cavity, through esophagus, stomach, finally enter capsule dissolves delivery formulations and absorption in small intestinal.

Oral formulations can be that recombination human serum albumin content is 5-20mg, lecithin, cephalin, α-GPC (L-α-choline glycerophosphatide), L-arginine, L-3,4 dihydroxyphenylalanine bean extract, GAMMA AMINOBUTRIC ACID, the acid of L-glutamy, ILE, 1B, vitamin A, vitamin B, vitamin C, vitamin E and biotin and 5-20% edible ethanol and suitable quantity of water, spray into buccal and absorption in oral cavity in spray mode.

Oral formulations oral administration gives after (medicine) animal, with the special double-antibody sandwich of functional (therapeutic) protein (ELISA) test kit in fusion rotein, the existence of functional protein molecule in blood, all can be detected, confirm that fusion rotein enters in blood.

The present invention reaches this high molecular weight protein mass-energy of recombined human serum albumin fusion proteins (molecular weight is greater than 70kD) via preparation of the present invention, as micromolecule represents polypeptide, by oral, can enter body inner blood blood circulation, demonstrate and there is corresponding bioactive functions, thereby reach beyond thought invention effect for this reason, complete the present invention.

Accompanying drawing explanation:

Accompanying drawing 1, the various recombined human serum albumin fusion proteins of being expressed by yeast engineering bacteria or the Chinese hamster ovary celI engineering strain SDS-PAGE collection of illustrative plates after separation and purification.Electrophoretic band in figure is respectively from left to right standard protein molecular weight, the functional protein monomer in rHSA/GCSF, rHSA/IFN α 2a, rHSA/IFN α 2b, rhEGF, rHSA/EGF, rHSA/EPO, rhGH, rHSA/GH and rHSA/KGF fusion rotein or fusion rotein.

Accompanying drawing 2, a kind of repressed can be oral or can be used as the sheet type product that contains recombined human serum albumin fusion proteins that in oral cavity, topical plaster is used (scale demonstrates the diameter of a kind of lyophilized powder sheet type of demonstration).

Accompanying drawing 3, a kind of freeze-dried powder preparation dosage form that contains recombined human serum albumin fusion proteins for oral cavity sublingual administration tablet form.

Accompanying drawing 4, a kind of recombined human serum albumin fusion proteins oral enteric capsule that contains.It shown in figure, is a kind of oral enteric capsule product that contains fusion rotein, it is to contain 2 kinds of recombined human serum albumin fusion proteins, is respectively the enteric coated capsule (left side) of recombination human serum albumin/erythropoietin fusion rotein (rHSA/EPO) and recombination human serum albumin/granulocyte stimulating factor fusion rotein (rHSA/GCSF) and without the empty enteric coated capsule (right side) of filling.In enteric coated capsule, can contain fusion rotein not of the same race simultaneously or contain the fusion rotein of various dose simultaneously, capsule can be that different colours, different size are shown it mutually, hereby to distinguish.

Accompanying drawing 5, the plasma concentration curve of representational fusion rotein oral formulations in mice.

Accompanying drawing 6, contain rHSA/EPO fusion rotein enteric coated capsule after mice is oral, the counting radix of the red blood cell count(RBC) in the time of 72 hours in blood during compared with 0 hour is significantly increased, and shows and provides the corresponding biological action of functional protein.

Embodiment:

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only for illustrating how the present invention implements, and particularly uses known implementation step and technology, rather than will limit interest field of the present invention.

The production and preparation of embodiment 1, recombined human serum albumin fusion proteins crude drug (stock solution)

The structure of the expressing gene Engineering Yeast strain of recombined human serum albumin fusion proteins or the strain of vertebrate cells CHO genetically engineered cell is completed by the inventor.Recombined human serum albumin fusion proteins comprises, but be not limited to, inventor gives respectively disclosed recombined human serum albumin fusion proteins in Chinese invention patent ZL021428816, ZL2004100428148, ZL200710057571.9 and ZL200710059770.3.The strain of the genetic engineering bacterium work seed bank of recombinant Human Serum Albumin Expression fusion rotein is cultivated the suitable time in suitable culture fluid, then accesses 30L seed tank.With the speed of 550 revs/min, at tank, be cultured to cell density and reach OD ₆₀₀more than=10.Utilize minimal medium increase, amplify or produce in the above fermentation tank of 200L.Fermentation process is shown in the description of the Chinese granted patent ZL200410057313.7 of Yu Zailin, Fu Yan.Growth is under 30 ℃ of conditions, and methanol induction carries out under the condition of 20 ℃-29 ℃.When 48-120hr, gather in the crops strain fermentation and express the supernatant that contains fusion rotein, proceed to the separation and purification program of fusion rotein.

Use the fusion rotein of expressing cho cell to take rHSA/hEPO as example, use suitable serum-free medium, frozen rHSA/hEPO genetic engineering Chinese hamster ovary celI strain is recovered to square vase, be expanded to Cell viability more than 95%.The cell of amplification is transferred to shaking flask (shaker flasks, 250ml), controls shaking flask inoculum density 4-5 * 10 ⁵cells/ml, volume of culture 80-100ml.The cell of propagation is increased continuously (incubation time 72h, the ratio that goes down to posterity 1:4), the cell of 250ml shake-flask culture is continued to amplification culture to 8-10 1000ml (volume of culture 400ml-500ml) shaking flask and carry out suspension culture.Control cell inoculum density and be about 4 * 10 ⁵cells/ml, Cell viability is not less than 94%, controls 37 ℃ of cultivation temperature, vibration rotating speed 120rpm, sampling observation of cell growing state (form, density, motility rate etc.).

The Chinese hamster ovary celI suspension of synchronized culture in 8-10 1000ml shaking flask is collected, shaken up sampling counting, be seeded to 120L bioreactor, control cell inoculum density 1.8-2.5 * 10 ⁵cells/ml, motility rate 95% left and right, initial incubation volume 38-45L, the condition of culture such as the pH (6.9-7.2) of control reactor, temperature (37 ℃), DO (40%-60%), rotating speed (40-60rpm); Be cultured to cell density and reach 1.5 * 10 ⁶cells/ml left and right, motility rate 95%, adds serum-free medium to 85L left and right and continues to cultivate.

Be cultured to the approximately the 5th day and start to add special abundant " fed-batch medium ".In incubation, sampling detects cell density, motility rate etc., and according to culture parameters (pH, DO etc.), determine that suitable stream adds strategy, until cell culture volumes arrives 100L, protein content accumulative total and cell density peaks and fusion rotein without obvious degradation time, add appropriate glacial acetic acid and EDTA, collecting cell suspension carries out purification process.

The separation and purification production technology that various recombined human serum albumin fusion proteins adopts is shown in the China mandate patent of invention ZL200810089645.1 of inventor's Yu Zailin, Fu Yan.Supernatant Middle St separation and purification (thalline or cell are abandoned it) for feather weight fusion rotein by Capto-MMC filler chromatographic column that summary obtains after fermentation tank (yeast) or bioreactor (CHO) scale cultivation for the engineering bacteria that contains recombined human serum albumin fusion proteins.Chromatographic column separation and purification through continuous two different fillers, acquisition meets other recombined human serum albumin fusion proteins of medicinal requirements level, can be used for the stock solution (crude drug) of the key component recombined human serum albumin fusion proteins in the preparations for oral administration formula in the present invention.

Accompanying drawing 1 is the SDS-PAGE results of yeast engineering bacteria or the Chinese hamster ovary celI engineering strain various recombined human serum albumin fusion proteins of expressing after separation and purification.Electrophoretic band in figure is respectively from left to right standard protein molecular weight, the functional protein monomer in rHSA/GCSF (85.2kD), rHSA/IFN α 2a (85.7kD), rHSA/IFN α 2b (85.7kD), rhEGF (6kD), rHSA/EGF (72.7kD), rHSA/EPO (99kD), rhGH (22.2kD), rHSA/GH (88.6kD) and rHSA/KGF (86kD) fusion rotein or fusion rotein.Fusion rotein can be different because of the molecular size range of functional protein, and vary in size.

Recombined human serum albumin fusion proteins can also obtain Expression product in antibacterial, virus, many animals cell, transgenic animals and plants, and for the preparation of the present invention's preparations for oral administration product.

The preparation of embodiment 2, recombined human serum albumin fusion proteins lyophilized powder

The universal pharmaceutical formulation (patent of invention ZL200910210379.8 " general pharmaceutical formulation for recombined human serum albumin fusion proteins for injection " authorizes in Yu Zailin, rich rock China) that the lyophilized powder of preparing recombined human serum albumin fusion proteins can be used inventor to invent.Wherein the mannitol content of excipient adjuvant can be 0.2-6%; The content of sucrose can be 0.5-1.5%, and the content of polyoxyethylene sorbitan monoleate can be 0.0002-0.006%, and the concentration of salt buffer can be 5-100mM, and pH value can be 5.5-7.0; In lyophilized powder, can contain 0.02-2% recombined human serum albumin fusion proteins.In a concrete freeze-dried powder preparation formula, excipient content is the phosphate buffer of 4.0% mannitol, 1.0% sucrose, 0.0004% polyoxyethylene sorbitan monoleate and 10mM, pH5.5.

The design of cryodesiccated production technology and freeze-drying curve has mainly considered that fusion rotein finished product is to be placed in cillin bottle or to be directly placed in opening freezing square position, and the lyophilization machine equipment monopolizing characteristic using and slightly different.Recombined human serum albumin fusion proteins is to have relatively stable on heatingly, enters after freeze dryer case after perfusion, with-50 ℃, keeps 4hr.Now freezing speed is faster, and the ice crystal of generation is less, more soluble during redissolution, can guarantee that sample freezes reality simultaneously, non-foaming during evacuation; In freeze-drying process, mainly by sublimation drying.At-20 ℃, remove most of moisture under maintaining, then by progressively heating up, make the goods temperature that raises slowly, along with the rising of products temperature further reduces moisture; Final step maintains 29 ℃.And when products temperature reaches 26 ℃, just start to start end vacuum, and can make moisture drop to lower, make products temperature can not be raised to too high simultaneously, guarantee active unaffected.Generally, the preparation of recombined human serum albumin fusion proteins lyophilized powder is that various adjuvants are mixed with fusion rotein, after filtration sterilization, with semi-finished product, divide (cillin bottle adds half plug) after sabot, put into freeze dryer freezing partition plate, according to set lyophilizing program, carry out lyophilizing.After lyophilizing EP (end of program), square position or cillin bottle shift out freeze drying box (cillin bottle needs gland and rolls aluminium lid step).Lyophilized powder is carried out the analyses such as moisture, purity, biological activity, redissolution rate, clarity by step procedure.

The recombined human serum albumin fusion proteins lyophilized powder of preparing through this production technology can be directly as the key component in preparations for oral administration formula, again with other adjuvant, as jointly formed together preparations for oral administration product after excipient, binder or nutrient mixing.

The recombined human serum albumin fusion proteins of mentioning and not mentioning in all patents of the present invention all can be prepared recombined human serum albumin fusion proteins lyophilized powder (or lyophilized injectable powder) with this general freeze-drying powder production technique.

Embodiment 3, the oral formulations formula that contains recombined human serum albumin fusion proteins lyophilized powder form

Can any ratio and be mixed into powder shape with the content of adhesion adjuvant as adjuvants such as 0.2-2% chitosan, 0.4-4% sodium alginate, 0.1-1% hyaluronic acid (hyaluronic acid), 0.02-0.5% hydroxypropyl emthylcelluloses (HPMC) by the recombined human serum albumin fusion proteins lyophilized powder of embodiment 2 manufactures, and form the key component of preparations for oral administration formula.

Particularly, the present invention has completed screening and the enforcement that the content that contains recombined human serum albumin fusion proteins lyophilized powder 0.1-1.0%, adhesion adjuvant consists of the pharmaceutical formulation of chitosan 0.5%, sodium alginate 1%, hyaluronic acid 0.5%, hydroxypropyl emthylcellulose (HPMC) 1%.

The inventor so above-mentioned preparation powder of preparation can pass through tablet machine compression molding (accompanying drawing 2), direct oral cavity buccal or be placed in sublingual administration, per mucous membrane system absorption and enter blood circulation; Also the preparation powder perfusion (filling) of so preparation can be entered in enteric coated capsule and forms capsule product for after oral, by stomach, enter intestinal dissolving and absorb by intestinal mucosa system, and enter blood circulation.

The system of embodiment 4, oral recombined human serum albumin fusion proteins lyophilized powder tablet respectively

By the recombined human serum albumin fusion proteins stock solution that contains 50-200mg, (HPMC) 1% is miscible with pre-solvent 1% trehalose, 4% mannitol, chitosan 0.5%, sodium alginate 1%, hyaluronic acid 0.5%, hydroxypropyl emthylcellulose, and then directly in freeze dryer, system respectively becomes freeze-dried powder preparation.

According to the different in kind of recombined human serum albumin fusion proteins, through sublingual administration, can improve the situation of human body.For example, while containing recombination human serum albumin/granulocyte stimulating factor fusion rotein, through sublingual administration, can improve total leukocyte and the leukocytic counting of grain; While containing recombination human serum albumin/erythropoietin fusion rotein, through sublingual administration, can improve red blood cell count(RBC); Oral application is in oral ulcer reparation, treatment;

Figure 3 shows that another kind contains human serum albumin fusion proteins lyophilized powder tablet product containing taking.Be a kind of sheet type of directly making through lyophilization mode can be oral, can be used as topical plaster in oral cavity, be directly applied to the sheet type product that contains recombined human serum albumin fusion proteins (scale demonstrates the diameter of the lyophilized powder sheet type of demonstration) of skin surface after can redissolving, its recombined human serum albumin fusion proteins containing can be rHSA/EGF, also can be prepared into the tablet product that contains any other fusion rotein.

The preparation agent technique of embodiment 5, oral recombined human serum albumin fusion proteins enteric coated capsule

The oral formulations that contains recombined human serum albumin fusion proteins of being prepared by embodiment 3 directly pours into (filling) in any enteric hollow capsule that meets Chinese Pharmacopoeia requirement (the visual volumes of formulation of capsule size is selected), enteric hollow capsule can be manufacture with plant substrates or animal substrate any commercial, should meet the national standard requirement of state-promulgated pharmacopoeia capsule class packaging material.The method of perfusion enteric hollow capsule can be Mechanical Method, can be also with artificial process perfusion, but all should in meeting aseptic and free of contamination super clean environment, pour into.Enteric coated capsule after perfusion should preferably be kept at the outer package of plastic-aluminum material, with drying condition storage and transportation under room temperature.

An embodiment is particularly, by the preparation that contains recombined human serum albumin fusion proteins of embodiment 3 preparations, complete the oral enteric capsule product that contains recombined human serum albumin fusion proteins of perfusion (filling) as shown in Figure 4, it contains 2 kinds of recombined human serum albumin fusion proteins, is respectively the enteric coated capsule of recombination human serum albumin/erythropoietin fusion rotein (rHSA/EPO) and recombination human serum albumin/granulocyte stimulating factor fusion rotein (rHSA/GCSF) and without the empty enteric coated capsule of filling.In enteric coated capsule, can contain fusion rotein not of the same race simultaneously or contain the fusion rotein of various dose simultaneously, capsule can be that different colours, different size are shown it mutually, hereby to distinguish.

The preparation of embodiment 6, a kind of oral recombined human serum albumin fusion proteins mouth sprays and technique

The recombined human serum albumin fusion proteins of being prepared by embodiment 2 can mix as lecithin, cephalin, α-GPC (L-α-choline glycerophosphatide), L-3,4 dihydroxyphenylalanine bean extract, GAMMA AMINOBUTRIC ACID, L-arginine, the acid of L-glutamy, ILE, 1B, vitamin B, vitamin C, biotin and 5-20% edible ethanol and suitable quantity of water with adjuvant (comprising excipient, binder etc.) and nutrient substance with any ratio and content, be poured in spray bottle, in spray mode, spray in oral cavity buccal and absorption.

Implement pharmaceutical formulation for concrete one and consist of in every 1000ml volume, the cephalin of the recombined human serum albumin fusion proteins that contains 500mg, the lecithin of 50mg, 100mg; The L-glutamy acid of the L-3,4 dihydroxyphenylalanine bean extract of 60mg, the γ-aminobutyric acid of 30mg, the L-aminoacid of 40mg, 50mg, the ILE of 35mg, the vitamin B of the 1B of 30mg, 50mg are, the edible ethanol of the biotin of the vitamin C of 100mg, 20mg (Biotin), 150ml finally adds sterilized water to 1000ml.During concrete preparation, be that method and program are that other component except recombinant human serum albumin fusion rotein and sterilized water is first joined in edible ethanol, then in stirring, slowly add sterilized water to 900ml volume, now again recombined human serum albumin fusion proteins stock solution is slowly added in slowly stirring, finally with sterilized water, supply cumulative volume to 1000ml.

In formula agent processed, contain the function that edible ethanol also has anti-corrosion function and fat-soluble auxiliary agent.

Recombined human serum albumin fusion proteins in formulation products can be to contain a kind of recombination human serum albumin/growth hormone fusion protein; It can be recombination human serum albumin/erythropoietin fusion rotein, can be recombination human serum albumin/granulocyte stimulating factor fusion rotein, can be recombination human serum albumin/epithelical cell growth factor fusion rotein.Recombined human serum albumin fusion proteins in formulation products can be that the mixing that contains two kinds of recombination human serum albumin/growth hormone fusion proteins and recombination human serum albumin/epithelical cell growth factor fusion rotein forms, and also can be comprised of recombination human serum albumin/erythropoietin fusion rotein and recombination human serum albumin/granulocyte stimulating factor fusion rotein.Can certainly be to contain three kinds of recombined human serum albumin fusion proteins.Recombined human serum albumin fusion proteins in formulation products addition in every 1000ml can be 50mg-500mg not etc.The content in preparation of recombined human serum albumin fusion proteins can add according to the application purpose of product, and the amount adding also can be to be applied as object, and the fusion rotein amount that design can be absorbed in body decides.

The example of the practical application of embodiment 7, oral formulations

A, buccal tablets are used for the application of dental ulcer treatment: recombined human serum albumin fusion proteins (rHSA/EGF) freeze-dried powder preparation that contains that embodiment 3 is obtained is pressed into the sheet type (seeing accompanying drawing 2) that is greater than ulcer surface area, is directly attached to ulcer place.Lyophilized powder sector-meeting is dissolved gradually.Obvious effective rate after 20 random volunteers use is 100%.In 3 days, all obtain healing completely.The ulcer time of improving does not more use quickening healing time and approximately can shift to an earlier date 3 days, and user does not generally need could obtain for 5-10 days healing completely.Lyophilized powder sheet also can be fixedly arranged in advance in a carrier film (the edible instant soluble seal of any commercialization can be saccharide film, can be protein-based film.Can be chitosan, can be pectin, can be fiber-like, can be starch, can be Ovum Gallus domesticus album or can be also each film of soybean protein system).

The application of B, oral cavity sublingual lozenge: the lyophilized powder sheet that contains recombined human serum albumin fusion proteins that embodiment 3 is obtained, be placed in Sublingual, containing changing, take it.Fusion rotein can be recombination human serum albumin/growth hormone fusion protein (rHSA/GH), can be recombination human serum albumin/granulocyte stimulating factor fusion rotein, can be recombination human serum albumin/erythropoietin fusion rotein (rHSA/EPO), can be also compound fusion rotein.For example: the combination of the rHSA/EPO lyophilized powder of the rHSA/GH that 100ml cumulative volume contains 50mg, the rHSA/GCSF of 30mg and 30mg and appropriate adjuvant, for example: in adjuvant, contain particularly 0.5% chitosan of unit of weight, 1% sodium alginate, 0.5% hyaluronic acid, 1% hydroxypropyl emthylcellulose (HPMC).

C, after oral 24 hours, experimenter is through venous blood sampling, use respectively ELISA test kit and detect blood sample, confirm that functional protein (hGH, hGCSF, hEPO) in various fusion rotein can detect and be significantly higher than the content in the blood sample of 0 hour in blood.Meanwhile, leukocyte and red blood cell count(RBC) are also higher than radix.Show that protein enters in Hematological System of Professional Workers by Sublingual is oral.

D, oral enteric capsule are fed mouse test: by the oral enteric capsule (4# of embodiment 5 preparations, 126mg) for mouse test, the oral formulations enteric coated capsule that contains 0.6mg recombined human serum albumin fusion proteins (rHSA/GCSF) in each capsule gavages mice (the about 15-20g of each Mouse Weight) with water.During administration, confirm that capsule does not break in oral cavity, entered esophagus.After oral 0,2,6,12,24,48,72 hour, get Mus tail blood (every mice in process of the test only by blood sampling 2-3 time; Same time point is got 2 mices, calculating mean value).End user's granulocyte stimulating factor (G-CSF) test kit (U.S. eBioscience company, article No.: BMS2001INST, title: Instant Human G-CSF ELISA Kit), detect the G-CSF protein content (blood drug level) in blood sample.Result shows in different period blood, can detect the existence of G-CSF protein in the mice of administration, in the time of 12-24 hour, arrive peak, in blood, continue to maintain the G-CSF protein to 72 hour higher than baseline, also demonstrate long-lasting (accompanying drawing 5) of fusion rotein.Total leukocyte in the blood sample of 24 hours, 48 hours, 72 hours and grain numeration of leukocyte, be radix compared with 0 hour, and total leukocyte and granulocyte count have significant rising, show that pharmic function is embodied.This and (Fu Yan etc. that match in clinical front zoopery blood drug level testing result, pharmacodynamics, pharmacology and the toxicologic study of recombination human serum albumin/granulocyte stimulating factor fusion rotein, Chinese Medicine biotechnology, 74 phases of volume, p241-256,2012).Result of the test shows in the blood sample of oral administration that destination protein matter content is than comparing with subcutaneous injection administering mode, enters many that G-CSF fusion rotein in blood will lack, and according to dosage oral 1% left and right of only having an appointment of estimating specifications enters blood system and suffered.Computational methods can be 60 kilograms by normal person's body weight, and total blood volume is that 5L calculates, and the functional protein content pg level in every microlitre blood sample, extrapolates the content in total blood flow volume.This also meets bibliographical information, and the utilization rate of oral way protein is lower.The object of the invention is to determine that fusion rotein (medicine) can enter blood circulation, and passes over the medication amount that enters blood by oral.By the fusion rotein content in each enteric coated capsule, be to improve in right amount, the corresponding fusion rotein that has a higher amount enters the chance of blood circulation within the specific limits simultaneously, and improves the content of moment medicine in blood.More than protein the protease hydrolysis via in intestinal is become to little peptide section, also can absorb and arrive the object that enters blood by intestinal chorion epidermis cell.

E, the application of oral enteric capsule that contains two kinds of different fusion rotein: will contain recombination human serum albumin/granulocyte stimulating factor fusion rotein (rHSA/GCSF) and recombination human serum albumin/erythropoietin fusion rotein (rHSA/EPO) lyophilized powder and adjuvant and mix to be packed into and in enteric coated capsule, give mice, by the ELISA method of listing with B item, the existence of GCSF and EPO in mouse blood, can be detected respectively with quantitative, show that two kinds of fusion rotein are all absorbed and entered into blood by intestinal cell.In the time of 72 hours, the blood sample radix Ret% of reticulocyte before than (Ret%) administration is significantly increased, and had leukocyte increasing and risen erythrocytic biological function and effect (accompanying drawing 6: reticulocyte changing trend diagram).Recombination human serum albumin/insulin fusion rotein (rHSA/Insulin) is filled in enteric coated capsule together with adjuvant, has also obtained treating diabetes and the clinical treatment of symptom improvement and the effect of long-lasting insulin action.To use object, be as the criterion, in an enteric coated capsule, can contain multiple different fusion rotein simultaneously, fusion rotein also can exist by different content jointly.Enteric coated capsule can be that different sizes, different colours are for different objects or the needs of difference.

The application of F, oral spray: the oral spray that embodiment 6 is obtained is when health product are actually used in human body and can obtain remarkable body constitution and improve, can in blood, apply corresponding ELISA test kit and the existence of corresponding functional protein be detected, show that the ingredient (peptide section) of fusion rotein or fusion rotein can enter Hematological System of Professional Workers.If oral spray, as administration human body, also can obtain corresponding clinical effectiveness.

Embodiment result shows that oral formulations formula of the present invention has good protective effect to recombined human serum albumin fusion proteins, and goods formula fitness is good, can be suitable for various measured people's Recombinant Serum Albumin fusion rotein.Existing 4 fusion rotein separately or two kinds, two or more fusion rotein combinations for prepared can practical application oral formulations formula, prepared and can be used for human health care, body constitution improvement, reparation, clinical treatment medication.Can affirm that oral formulations disclosed by the invention has stability and effectiveness.Pharmaceutical formulation is simple, with low cost simultaneously, each component of preparation is effective to human-body safety.

Claims (6)

1. a preparations for oral administration that contains recombined human serum albumin fusion proteins, is characterized in that:

(1) described preparations for oral administration is by recombined human serum albumin fusion proteins and pharmaceutically acceptable excipient sucrose, mannitol, tween 80 and maintain the buffer salt of pH; And pharmaceutically acceptable adhesion adjuvant chitosan, sodium alginate, hyaluronic acid, hydroxypropyl emthylcellulose (HPMC) form jointly;

(2) in (1), contained recombined human serum albumin fusion proteins can be a kind of fusion rotein, can be also containing two or more different fusion rotein;

(3) in (1), contained recombined human serum albumin fusion proteins content is 0.01-1.0%;

(4) the pharmaceutically acceptable excipient adjuvant described in (1) is mannitol or glycine, and acceptable salt buffer is phosphate buffer or acetate buffer or citrate buffer;

(5) the pharmaceutically acceptable adhesion adjuvant described in (1) is chitosan, sodium alginate, hyaluronic acid, hydroxypropyl emthylcellulose (HPMC);

Wherein the mannitol content of excipient adjuvant is 0.2-6%; The content of sucrose is 0.5-1.5%, and the content of polyoxyethylene sorbitan monoleate is 0.002-0.006%, and the concentration of salt buffer is 5-100mM, and pH value is 5.5-7.0;

Wherein the content of adhesion adjuvant is chitosan 0.2-2%, sodium alginate 0.4-4%, hyaluronic acid 0.1-1%, hydroxypropyl emthylcellulose (HPMC) 0.2-2%.

2. preparations for oral administration as claimed in claim 1, it is characterized in that wherein recombined human serum albumin fusion proteins content is 0.01-0.5%, chitosan content is 0.5%, sodium alginate content is 1%, hyaluronic acid 0.3%, hydroxypropyl emthylcellulose (HPMC) content are 0.5%, mannitol content is 4.0%, cane sugar content is 1.0%, polyoxyethylene sorbitan monoleate content be 0.004% and the concentration of phosphate buffer be 10mM, pH6.5.

3. oral formulations as claimed in claim 1, it is characterized in that wherein recombination human serum albumin content is 5-20mg, γ-aminobutyric acid, the acid of L-glutamy, ILE, 1B, L-arginine, lecithin, cephalin, α-GPC (L-α-choline glycerophosphatide), L-3,4 dihydroxyphenylalanine bean extract, vitamin A, vitamin B, vitamin C, vitamin E and biotin and 5-20% edible ethanol and suitable quantity of water.

4. oral formulations as claimed in claim 1 is placed in Sublingual dissolving in tablet mode.

5. oral formulations as claimed in claim 1 is oral to enteric solution in enteric coated capsule parcel mode.

6. oral formulations as claimed in claim 3 extremely absorbs with buccal in liquid mode entrance.

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