CN107802827A - One kind restructuring kallikrein freeze-dried powder preparation - Google Patents
One kind restructuring kallikrein freeze-dried powder preparation Download PDFInfo
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- CN107802827A CN107802827A CN201711236771.0A CN201711236771A CN107802827A CN 107802827 A CN107802827 A CN 107802827A CN 201711236771 A CN201711236771 A CN 201711236771A CN 107802827 A CN107802827 A CN 107802827A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/4853—Kallikrein (3.4.21.34 or 3.4.21.35)
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
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- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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Abstract
The invention provides a kind of kallikrein freeze-dried powder preparation, contain restructuring kallikrein, excipient, osmotic pressure regulator, pH adjusting agent and buffer in described freeze-dried powder preparation.Contain in every 1000 bottles of described freeze-dried powder preparation：Recombinate the 200PNA units of KLK10 0, the 60g of excipient 2, the 50g of osmotic pressure regulator 9, the 20g of buffer 0；Recipe ingredient used in restructuring kallikrein freeze-dried powder produced by the invention is simple, using specific excipient and hardening agent, and controls both ratios, makes that the freeze-dried powder stability of preparation is high, impurity is few, side effect is low, safe；It is easy to use and restructuring kallikrein freeze-dried powder bioavilability produced by the invention is high, absorb fast, playing a role, rapid and agents useful for same is simple, and easy to operate, production cost is low, just with storing and using.
Description
Technical field
The present invention relates to field of pharmaceutical preparations, and in particular to one kind restructuring kallikrein freeze-dried powder preparation.
Background technology
Restructuring kallikrein is one group of serine protease being present in most tissues and body fluid, is in a kind of peptide chain
Enzyme cutting.It is specific in one of carbon tip cutting peptide substrate, the active kassinin kinin of cleavable kininogen release, by kassinin kinin performance expansion
Open capillary, relaxation vascular smooth muscle, improve microcirculation, anti-freezing, thrombus dissolving, reduction blood viscosity and blood pressure, and have
Treat the effect such as infertility.Its English entitled Kallikrein and Kininogenase, Kallidinogenase,
Described by Kininogenin is all same constituents.At present from the different tissues, organ or urine of humans and animals body
Many Kallikrein constituents are obtained, wherein by content highest, it is found that (restructuring swashs the also earliest Kallikrein-1 that is named as
Peptide discharges enzyme -1).Composition content highest in the pancreas and kidney of people or mammal, its be metabolized after further through kidney, with
Urine ejection is external.Therefore, from human or animal urinate in urinary kallidinogenase (or urine restructuring kassinin kinin release alleged by one's early years for separating
Enzyme) it is actually restructuring kallikrein (Kallikrein) in new classification.Restructuring kallikrein can be prevented effectively
Thrombus, also there is good therapeutic action to disease caused by diabetes and hypertension.
But on the one hand, restructuring kallikrein is extremely unstable under solution state, therefore prepares the stable restructuring that contains and swash
Peptide release enzyme medicine be always medical domain there is an urgent need to.On the other hand, after pharmaceutical preparation is prepared into, inevitably
Various adverse reactions can be caused.According to the regulation of WHO international drug monitoring Cooperation Centres, adverse drug reaction (Adverse
Drug Reactions, ADR) refer to normal dose medicine be used for prevent, diagnose, treat disease or adjust physiological function when occur
Harmful and unrelated with medication purpose reaction.Report at present on recombinating kallikrein parenteral solution adverse reaction is main
For：Cardio-cerebrovascular adverse reaction (such as flush, drop in blood pressure, conjunctival congestion, uncomfortable in chest, the angina pectoris that occurs together etc.), god
Through system adverse reaction (headache, perspiring, dizzy, weak), digestive system adverse reaction (nausea,vomiting,diarrhea, upper digestive tract
Bleeding), Systemic reaction (heating etc.) and other adverse reactions (violent cough, fash etc.).Also once occurred serious
Adverse reaction report, such as infarct internal haemorrhage, or even death.Said medicine adverse reaction may cause drug user
It is uncomfortable, or even cause that sb.'s illness took a turn for the worse, trigger new disease.Therefore the restructuring kallikrein system of low adverse reaction is prepared
Agent is also a focal point of this area.
Such as a kind of restructuring kallikrein-1 injection of stabilization is disclosed in Chinese patent application 200810038422,
Removed in the parenteral solution containing restructuring kallikrein-1, phosphate buffer, sodium chloride and albumen enzymatic protective reagent, in order to improve it
The activity of parenteral solution, it is also added into a certain amount of preservative.Due to existing in liquid form, its stability is substantially inferior to freeze-dried powder
Preparation, therefore in order to improve stability to a certain degree, the invention adds various albumen enzymatic protective reagents and preservative, undoubtedly greatly
Its sensitization risk, and adverse reaction rate are improved greatly.Another further aspect, because restructuring kallikrein-1 is a kind of albumen
Enzyme, its activity is sensitive, therefore whether the use that can not exclude preservative can cause to recombinate the active reduction of kallikrein-1.
In order to increase the stability and portability of restructuring fowl' IBD medicine, generally restructuring kallikrein is freezed
Preserve, be configured to lyophilized formulations again during use inject solution., but due to restructuring kallikrein in the solution activity stabilized
Property it is very poor, so prepare solution be also required to use as early as possible, to avoid loss of activity.Such as Chinese patent
A kind of pharmaceutical composition for being used to improve stability of human urine kininogenase is disclosed in CN2004100960604, said composition is used
One or more combination in mannitol, dextran, gelatin hydrolysate and sodium citrate, is configured to the aqueous solution, and clinical practice makes
For dissolving human urokinase-type peptidase freeze-dried powder.Although the invention improves recombinates kallikrein freeze-dried powder, but its focus
Pharmaceutical preparation not containing restructuring kallikrein, but for the aqueous solution of solvent medicine.Also it is not concerned with recombinating completely
The adverse reaction situation of kallikrein preparation.
The content of the invention
The technical problem to be solved in the present invention is to improve the stability of restructuring kallikrein preparation, and it is bad anti-to reduce its
Answer incidence.
Term：
1st, kallikrein is recombinated：Convention is used according to the routine of this area, the term used in the present invention " recombinates kassinin kinin
Discharge enzyme ", can be with " restructuring kallikreinogen " or " kininogenase " used interchangeably；Titled with source mode before above title
(such as restructuring restructuring kallikrein or natural extraction restructuring kallikrein), or source metabolite (such as pancreas restructuring kassinin kinin
Discharge enzyme or urine restructuring kallikrein) etc., referred to involved this term of restructuring kallikrein in the present invention
It is same composition.It is a kind of serine protease, may act on kininogen, is allowed to hydrolysis and discharges kassinin kinin, passes through kassinin kinin
Performance expansion capillary, relaxation vascular smooth muscle, improvement microcirculation, increase CBF, anti-freezing, thrombus dissolving, reduction blood glue
The effect such as degree and blood pressure.According to the international practice of this area, the English name that recombinates kallikrein can be Kallikrein,
Kininogenase, Kallidinogenase or Kininogenin, but the multi-purpose words of Kallikrein mono- at present.
2nd, buffer：The term " buffer " used in the present invention refers to a kind of compound or a variety of of its pH cushioning effect of energy
The composition of compound.
3rd, albumin：The term " albumin " used in the present invention can be with " albumin " used interchangeably, its English name
Albumin, English abbreviation Alb.
Definition：
1st, in the present invention, 1PNA units are defined as：Under conditions of 37 DEG C, pH8.0, the 1 μm of olVal- of hydrolysis in 1 minute
Leu-Arg-PNA restructuring kallikrein amount is 1PNA units.
On one side, the invention provides one kind to recombinate kallikrein freeze-dried powder preparation, and it contains active constituents of medicine
Recombinate kallikrein, excipient, osmotic pressure regulator, pH adjusting agent and buffer.
The formula of restructuring kallikrein freeze-dried powder preparation provided by the invention is every 1000 bottles：
Preferably, the formula of described restructuring kallikrein freeze-dried powder preparation is every 1000 bottles：
It is highly preferred that the formula of described restructuring kallikrein freeze-dried powder preparation is every 1000 bottles：
Described excipient is selected from mannitol, glycine, dextran, gelatin hydrolysate, sodium citrate, polyethylene glycol and sea
One or more in algae sugar.
Preferably, described excipient in mannitol, glycine, dextran, gelatin hydrolysate and trehalose one
Kind is several；
It is further preferred that one or more of the described excipient in mannitol, glycine and dextran.
Mannitol is the isomer of D-sorbite, on No. two carbon atoms of two kinds of alcohols materials hydroxyl towards different,
Molecular formula is C6H14O6, molecular weight 182.17.It is soluble in water, it is the solid of white clear, the sweet taste for having similar sucrose.It is sweet
Dew alcohol has extensive use in medicine, food and field of industrial production.For example, mannitol is good diuretics in medicine, drop
Intracranial hypotension, intraocular pressure and treatment renal drug, dehydrant, sugar substitute, the excipient for also serving as tablet and solid, liquid it is dilute
Release agent；Using mannitol as initiator pressurize and manufactured poly- mannitol-propylene oxide ether is widely used in plastic industry；Eating
In terms of product, water imbibition of the product in sugar and sugar alcohol is minimum, and has tasty and refreshing sweet taste, for maltose, chewing gum, rice cake etc.
Anti-sticking, and the anti-sticking powder as general cake of food.
Glycine also known as amion acetic acid, white monoclinic system or hexagonal crystalline, or white crystalline powder, it is odorless, have
Special sweet taste, skeleton symbol NH2CH2COOH, molecular weight 75.07, there is acid and alkaline official simultaneously in glycine molecule
It can roll into a ball, it is ionizable in water, there is very strong hydrophily, belong to polar amino acid, be dissolved in polar solvent, and be insoluble in nonpolar
Solvent；Glycine food, medicine, feed and field of industrial production are all widely used.For example, glycine can cook food addition
Agent, nutritional supplement etc.；Glycine can be used as Medical Microbiology and biochemical amino acid metabolism research medication, as cephalo bacterium
The raw material, thiocymetin etc. of element；The feed increase amino acid that can be eaten as poultry, livestock and poultry particularly pet etc. adds
Add agent and attractant etc.；In the industrial production can used as pesticides intermediate, such as primary raw material as herbicide glyphosate；Electroplate liquid
Additive；PH conditioning agents etc..
Dextran is a kind of macromolecule glucose polymer of the sucrose through the fermentation synthesis of leuconostoc mesenteroide -1226,
It is one of blood substitutes optimal at present, that clinically commonly uses has medium molecular dextran, is mainly used as blood substitutes, uses
In hemorrhagic shock, traumatic shock and burn shock etc., low, small molecule dextran, microcirculation can be improved, prevent or disappear
Except intravascular erythrocyte aggregation and thrombosis etc., the signified microcirculation disorder of various shocks, diffusivity blood vessel can be also used for
Intravascular coagulation, angina pectoris, acute myocardial infarction are other peripheral vascular diseases etc..
Gelatin hydrolysate is product of the collagen through enzyme hydrolysis, and the derivative of gelatin hydrolysis, peptide bond is broken after hydrolysis, is changed into
Polypeptide, the water that can be dissolved under normal temperature, the side chain in the chemical mechanical formula of gelatin hydrolysate is still identical with gelatin, therefore remains to keep dynamic
Other performances of thing glue, such as surface-active, colloid protectiveness, water-retaining property and caking property.Gelatin hydrolysate skin care item, food and
There is application in industrial production, for example, gelatin hydrolysate can be used as NMF, be easily absorbed by the skin, the effect of its skin-care anti-wrinkle；
Gelatin hydrolysate addition its thickening and emulsification in food；In fabrics printing and dyeing, fiber optic can be increased by adding gelatin hydrolysate
Pool, prevent chemical fibre from producing electrostatic in weaving, and have assisting-dyeing effect.
Sodium citrate also known as sodium citrate, it is a kind of organic compound, white or clear crystal are odorless, have refrigerant salty
It is stable in pungent, normal temperature and air, it is micro- in humid air to have generation weathering phenomenon in dissolubility, then hot-air.Sodium citrate exists
It is used as acidity regulator, flavouring agent, stabilizer in food, beverage industry；Be used as in medical industry anticoagulant, resolving sputum agent and
Diuretics；Auxiliary agent of the alternative sodium tripolyphosphate as non-toxic detergent in detergent industry；It can also be used to brewage, inject
The fields such as liquid, photographic chemical and plating.
Polyethylene glycol is also known as the polymer of a- hydrogen-ω-hydroxyl (oxygen -1,2- second diyl), and molecular formula is HO (CH2CH2O)
NH, nontoxic nonirritant, mildly bitter flavor, there is good water solubility, and have good compatibility with many organic constituentses.Have
Good lubricity, moisture retention, dispersiveness, caking property, antistatic behaviour and flexibility etc., cosmetics, pharmacy, chemical fibre, rubber,
There is a very wide range of application in the industries such as plastics, papermaking, paint, plating agricultural chemicals, intermetallic composite coating and food processing.
Trehalose trehalose is also known as Radix Rhapontici seu Radix Echinopsis sugar, gill fungus sugar etc., is a kind of safe and reliable natural carbohydrate.Trehalose is by two
The nonreducing sugar that individual glucose molecule is formed with 1,1- glycosidic bonds, have 3 kinds of isomers i.e. trehalose (α, α), isotrehalose (β,
β) and neotrehalose (α, β), and there is non-specific protective effect to various bioactivators.Trehalose is in medicine, cosmetic
There is good application in the fields such as product, food processing and agricultural.For example, trehalose can be widely applied to beverage, chocolate and candy,
Baked product and quick-frozen food etc.；In terms of medicine, trehalose instead plasma protein is as blood product, vaccine, lymph
The stabilizer of the bioactive substances such as cell, cell tissue.
In some preferred embodiments, the weight proportion of above-mentioned various excipient is：Mannitol: glycine: dextrorotation
Sugared acid anhydride: gelatin hydrolysate: sodium citrate: polyethylene glycol: trehalose=5: 1: 2: 2: 2: 3: 3.
Described osmotic pressure regulator can be selected from the one or more in sodium chloride, glucose or albumin；Preferably
One or both of sodium chloride or glucose.
In restructuring kallikrein freeze-dried powder preparation of the present invention, the dosage of sodium chloride is every 1000 bottles：1-
10g；It is preferably every 1000 bottles：2-9g；It is more preferably every 1000 bottles：3-9g.
In restructuring kallikrein freeze-dried powder preparation of the present invention, the dosage of glucose is every 1000 bottles：1-
50g；It is preferably every 1000 bottles：6-509；It is more preferably every 1000 bottles：15-509.
In restructuring kallikrein freeze-dried powder preparation of the present invention, the dosage of albumin is every 1000 bottles：1-
50g；It is preferably every 1000 bottles：10-50g；It is more preferably every 1000 bottles：14-50g.
Described buffer can be selected from one kind in disodium hydrogen phosphate-sodium dihydrogen phosphate or citric acid-sodium citrate.
The unexpected discovery of the present invention, strengthen when being added in the formula of described restructuring kallikrein freeze-dried powder preparation
After agent, its adverse reaction rate does not have significant change, but its stability significantly improves.
One or more of the described hardening agent in methionine, arginine, niacinamide and vitamin B6；
Preferably, one or more of the above-mentioned hardening agent in methionine, arginine and vitamin B6；
It is further preferred that one or more of the above-mentioned hardening agent in methionine and arginine.
Methionine, it is one of essential amino acid for forming human body, participates in protein synthesis.Because its can not in vivo itself
Generation, so must be obtained by outside.It is often used as nutritious supplementary pharmaceutical, flavor enhancement and is gone for protect liver, antidepression, gas defence
Poison and myocardial preservation etc..
Arginine, arginine are a kind of a-amino acids, pI (amino acid isoelectric point)=10.76, be also 20 kinds it is universal from
One of right amino acid.It has improvement male sexual disfunction, improves cardiovascular related diseases, increase insulin secretion and subtract
Few flu and the effect of respiratory tract infection, therefore be normally used for male sexual function health care product, cardiovascular health-care function product, reduce sugar
Urinate the preparation of disease related complication medicine and preventing cold medicine.
Niacinamide, also known as niacinamide, it is the amide compound of nicotinic acid.It is generally used clinically for preventing and treating rough skin parallel port
Scorching, glossitis, sick sinus syndrome, the problems such as atrioventricular block.
Vitamin B6, also known as pyridoxine, it includes pyridoxol, pyridoxal and pyridoxamine, in vivo in the form of phosphate
In the presence of being a kind of water soluble vitamin.It is often used as food additives, nutritious supplementary pharmaceutical or pharmaceutical preparation, for rescuing
Poisoning by isoniazid, treatment anaemia, treatment leukopenia, treatment intelligence development sluggishness, treatment canker sore, treatment artery are hard
Change and thrombotic disease, delaactation, treatment parkinson's syndrome, treatment lockjaw, treatment gestational diabetes mellitus, contraceptive steroid
Oxalate calculus, pyridoxine dependency caused by caused depression, strengthen immunity and anticancer, treatment vitamin C
Faint from fear, relieving asthma, treating carpal tunnel syndrome etc..
Dosage of the described hardening agent in restructuring kallikrein freeze-dried powder preparation provided by the invention is every 1000
Bottle：0.2-1.4g；It is preferably every 1000 bottles：0.2-1g；It is more preferably every 1000 bottles：0.3-0.8g.
Therefore, it is as other preferred embodiments, the formula of described restructuring kallikrein freeze-dried powder preparation,
Every 1000 bottles：
In restructuring kallikrein freeze-dried powder preparation provided by the invention, the dosage of methionine is every 1000 bottles：
0.2-4g；It is preferably every 1000 bottles：0.2-2g；It is more preferably every 1000 bottles：0.3-1g.
In restructuring kallikrein freeze-dried powder preparation provided by the invention, arginic dosage is every 1000 bottles：0.2-
4g；It is preferably every 1000 bottles：0.2-2g；It is more preferably every 1000 bottles：0.3-1g.
In restructuring kallikrein freeze-dried powder preparation provided by the invention, the dosage of niacinamide is every 1000 bottles：0.2-
4g；It is preferably every 1000 bottles：0.2-2g；It is more preferably every 1000 bottles：0.3-1g.
In restructuring kallikrein freeze-dried powder preparation provided by the invention, the dosage of vitamin B6 is every 1000 bottles：
0.2-4g；It is preferably every 1000 bottles：0.2-2g；It is more preferably every 1000 bottles：0.3-1g.
In other preferred embodiments, the weight proportion of above-mentioned various hardening agents is：Methionine: arginine:
Niacinamide: vitamin B6=2: 2: 1: 1.
Applicants have unexpectedly found that appropriate regulation excipient and the weight proportion of hardening agent can discharge restructuring kassinin kinin
The stability of enzyme freeze-dried powder preparation significantly improves, and wherein the ratio of excipient and hardening agent can be 10-30: 1；Preferably, assign
The ratio of shape agent and hardening agent is 10-20: 1；It is further preferred that the ratio of excipient and hardening agent is 15: 1.
On the other hand, the invention provides the preparation method of above-mentioned restructuring kallikrein freeze-dried powder preparation, including with
Lower step：
(1) other raw materials that is weighed in addition to recombinating kallikrein according to formula dosage are placed in matching somebody with somebody in liquid bucket, use
Temperature is that 2~4 DEG C of waters for injection are stirred to dissolving；After addition medical activated carbon stirs and evenly mixs, taken off using 0.45 μm of membrane filtration
Carbon, obtain solution A；The restructuring kallikrein of dosage is formulated with 2~4 DEG C of water for injection dissolving, and is mixed with above-mentioned solution A
Uniformly, solution B is obtained；
(2) pH value for the solution B that is obtained with phosphoric acid or sodium hydroxide regulating step (1) obtains solution C to 6-7；
(3) solution C that step (2) obtains is supplied volume to 1500mL by with 2~4 DEG C of waters for injection, is mixed, is obtained molten
Liquid D；
(4) solution D that is obtained using 0.22 μm of filter membrane to step (3) carries out filtration sterilization twice, obtains solution E；
(5) dispenses solution E, partly jumps a queue, is freeze-dried in freeze dryer, rolls lid, checks, packaging, produces.
The addition of medical activated carbon in preparation method step (1) of the present invention is 0.5-2g；Preferably 1-2g；
More preferably 1-1.2g.
The adsorption treatment time of medical activated carbon in preparation method step (1) of the present invention is 5-10 minutes；It is excellent
Elect 6-9 minutes as；More preferably 7-8 minutes.
The average grain diameter of medical activated carbon in preparation method step (1) of the present invention is 3-7 μm；Preferably 4-6 μ
m；More preferably 4.5-5.5 μm.
Compared with prior art, the invention has the advantages that：
(1) recipe ingredient used in restructuring kallikrein freeze-dried powder produced by the invention is simple, uses specific excipient
And hardening agent, and both ratios are controlled, make that the freeze-dried powder stability of preparation is high, impurity is few, side effect is low, it is safe；
(2) restructuring kallikrein freeze-dried powder bioavilability produced by the invention is high, easy to use, absorbs fast, performance
Effect is rapid；
(3) freeze-dried powder produced by the invention, agents useful for same is simple, easy to operate, and production cost is low, just with storing and making
With.
Specific embodiment
Features described above mentioned in the present invention, or the feature that embodiment is mentioned can be in any combination.This case specification is solved
All features released can be used in combination with arbitrary composition form, each feature disclosed in specification, can by it is any provide it is identical,
The substituted feature substitution of impartial or similar purpose.Therefore except there is specified otherwise, disclosed feature is only impartial or similar spy
The general example of sign.
In specific embodiments of the present invention, the source for recombinating kallikrein is restructuring kallikrein, but this is simultaneously
The restructuring kallikrein for not limiting other types source is applied to technical scheme, equally can also obtain and the present invention
Identical technique effect.
The restructuring kallikrein being related in specific embodiments of the present invention can be by ordinary skill in the art means
Obtain, can also be bought by commercial sources.
The medicine that is related in specific embodiments of the present invention, material, solution etc., unless otherwise indicated, remaining has business
Industry approach is bought.
Embodiment 1 recombinates kallikrein freeze-dried powder preparation
It is formulated and is, every 1000 bottles：
Wherein, mannitol, glycine, the mass ratio of dextran are 5: 1: 2
Preparation method：(1) other raw materials that is weighed in addition to recombinating kallikrein according to formula dosage are placed in matching somebody with somebody
It is that 2~4 DEG C of waters for injection are stirred to dissolving with temperature in liquid bucket；After addition medical activated carbon stirs and evenly mixs, 0.45 μm of filter is used
Membrane filtration decarburization, obtains solution A；With the restructuring kallikrein of 2~4 DEG C of waters for injection dissolving formula dosage, and with it is above-mentioned
Solution A is well mixed, and obtains solution B；
(2) pH value for the solution B that is obtained with phosphoric acid or sodium hydroxide regulating step (1) obtains solution C to 6-7；
(3) solution C that step (2) obtains is supplied volume to 1500mL by with 2~4 DEG C of waters for injection, is mixed, is obtained molten
Liquid D；
(4) solution D that is obtained using 0.22 μm of filter membrane to step (3) carries out filtration sterilization twice, obtains solution E；
(5) dispenses solution E, partly jumps a queue, is freeze-dried in freeze dryer, rolls lid, checks, packaging, produces.
Embodiment 2 recombinates kallikrein freeze-dried powder preparation
It is formulated and is, every 1000 bottles：
Wherein, the mass ratio 5: 1: 2 of mannitol, glycine, dextran；
Methionine, arginic mass ratio 1: 1；
Excipient, the mass ratio of hardening agent are 16: 1.
Preparation method：In addition to raw material, remaining step is the same as embodiment 1.
Embodiment 3 recombinates kallikrein freeze-dried powder preparation
It is formulated and is, every 1000 bottles：
Wherein, mannitol, the mass ratio of glycine are 5: 1；
Methionine, the mass ratio of vitamin B6 are 1: 1；
The mass ratio of excipient and hardening agent is 22: 1；
Preparation method：In addition to raw material, remaining step is the same as embodiment 1.
Embodiment 4 recombinates kallikrein freeze-dried powder preparation
It is formulated and is, every 1000 bottles：
Wherein, mannitol, the mass ratio of glycine are 5: 1；
Methionine, arginic mass ratio are 1: 1；
The mass ratio of excipient and hardening agent is 15: 1；
Preparation method：In addition to raw material, remaining step is the same as embodiment 1.
Embodiment 5 recombinates kallikrein freeze-dried powder preparation
It is formulated and is, every 1000 bottles：
Wherein, mannitol, the mass ratio of gelatin hydrolysate are 5: 1；
The mass ratio of excipient and hardening agent is 17.5: 1；
Preparation method：In addition to raw material, remaining step is the same as embodiment 1.
Embodiment 6 recombinates kallikrein freeze-dried powder preparation
It is formulated and is, every 1000 bottles：
Wherein, mannitol, gelatin hydrolysate, the mass ratio of glycine are 5: 2: 1；
The mass ratio of excipient and hardening agent is 20: 1；
Preparation method：In addition to raw material, remaining step is the same as embodiment 1.
Embodiment 7 recombinates kallikrein freeze-dried powder preparation
It is formulated and is, every 1000 bottles：
Wherein, mannitol, dextran, the mass ratio of glycine are 5: 2: 1；
Methionine, arginic mass ratio are 1: 1；
The mass ratio of excipient and hardening agent is 15: 1；
Preparation method：In addition to raw material, remaining step is the same as embodiment 1.
Comparative example 1 recombinates kallikrein freeze-dried powder preparation
The difference of formula and embodiment 7 is the ratio of the ratio for reducing excipient and hardening agent, excipient and hardening agent
For 9: 1, it is 1.35g to make arginic content, and the content of methionine is 1.35g；Other are same as Example 7.
Preparation method：In addition to raw material, other are same as Example 7.
Comparative example 2 recombinates kallikrein freeze-dried powder preparation
The difference of formula and embodiment 7 is the ratio for increasing excipient and hardening agent, makes the ratio of excipient and hardening agent
Example is 40: 1, and it is 0.31g to make arginic content, and the content of methionine is 0.31g；Other are same as Example 7.
Preparation method：In addition to raw material, other are same as Example 7.
Comparative example 3 recombinates kallikrein freeze-dried powder preparation
Formula：In addition to methionine and arginine is not contained, remaining is the same as embodiment 7.
Preparation method：In addition to raw material, other are same as Example 7.
Contain restructuring kallikrein-1 disclosed in the embodiment 12 of the Chinese patent application 200810038422 of comparative example 4
Parenteral solution XI.
1. stability test
Restructuring kallikrein freeze-dried powder preparation or parenteral solution prepared by embodiment 1-7 and comparative example 1-4, in (40 ± 2
DEG C), place 6 months under the conditions of relative humidity (75 ± 5) %, monitor humidity, in 1,2,3,6 the end of month, sampling, to freeze-dried powder system
Kallikrein progress Detection of Stability is recombinated in agent and (using relative activity as index, that is, deposits the activity after N number of moon and storage 0
Active percentage at individual month), to investigate preparation accelerated stability.Equally, sample bottle is loaded on (25 ± 2 DEG C), relative humidity
Placed 24 months under the conditions of (60 ± 10) %, monitor humidity, sampled in 0,3,6,9,12,24 the end of month, in freeze-dried powder preparation
The activity for recombinating kallikrein carries out Detection of Stability, to investigate preparation long-time stability.It the results are shown in Table 1 and table 2.
The accelerated test result of table 1
The experiment for long-term stability of table 2
Restructuring restructuring kallikrein freeze-dried powder preparation provided by the invention is can be seen that by the data of Tables 1 and 2 to have
There is good stability, when containing preferred excipient and hardening agent and in preferred proportion (embodiment 7), the freeze-dried powder of preparation
There is good stability in experimentation, still have good stabilization after being deposited 24 months in long-term stable experiment
Property, up to 95%；The ratio of excipient and hardening agent is simply have adjusted in comparative example 1-2, increases the content of hardening agent and adds deduct
Few, its stability does not increase, but is more or less the same (comparative example 3) with stability when not strengthening agent；And Chinese invention
The parenteral solution XI containing restructuring kallikrein-1 disclosed in the embodiment 12 of patent 200810038422, in stability test
24 months rear stabilities are reduced to less than 85% during measure, drug inactivation, it is impossible to normal use；So figuration provided by the invention
The ratio of agent and hardening agent can improve the stability of restructuring restructuring kallikrein freeze-dried powder.
Redissolve experiment
Particulate matter assay method after redissolution：Restructuring kallikrein prepared by embodiment 1-7 and comparative example 1-3 freezes
Each 4 of dry powder formulations are dissolved in 250ml 0.9% sodium chloride solution, and the particulate matter of solution is determined after redissolution.It the results are shown in Table
3.
Table 3
| Particulate matter (individual/ml) | 10 μm of particles | 25 μm of particles |
| Embodiment 1 | 5 | 2 |
| Embodiment 2 | 4 | 1 |
| Embodiment 3 | 3 | 1 |
| Embodiment 4 | 2 | 1 |
| Embodiment 5 | 3 | 1 |
| Embodiment 6 | 4 | 2 |
| Embodiment 7 | 1 | 0 |
| Comparative example 1 | 6 | 2 |
| Comparative example 2 | 6 | 3 |
| Comparative example 3 | 3 | 2 |
Particulate matter assay method after redissolution：Restructuring kallikrein prepared by embodiment 1-7 and comparative example 1-3 freezes
Each 4 of dry powder formulations are dissolved in 250ml 5% glucose solution, and the particulate matter of solution is determined after redissolution.It the results are shown in Table 4.
Table 4
By the restructuring kallikrein freeze-dried powder prepared by the present invention it can be seen from the data in table 3 and table 4 in glucose
In redissolution effect it is better than the redissolution effect in sodium chloride, and embodiment redissolved relative to comparative example after insoluble microparticle it is bright
It is aobvious few.Particularly from preferably excipient and hardening agent and (the embodiment 1- in the preferred proportion of offer provided by the invention
7) insoluble microparticle is many less after being redissolved relative to other ratios (comparative example 1-3).
By experimental data it can be seen that when comprising only excipient in restructuring kallikrein freeze-dried powder, restructuring restructuring kassinin kinin
The activity of release enzyme freeze-dried powder can maintain 6 months still can be with normal use, and activity reduces after six months, and drug effect disappears, institute
There is synergy excipient with hardening agent in the present invention, weight can be significantly improved from specific excipient and hardening agent
Group kallikrein freeze-dried powder stability, so as to reduce restructuring kallikrein freeze-dried powder because loss of activity and caused by wave
Take.
2. adverse reaction is tested
Experimental animal：SD rats, male and female half and half, body weight 240~300g, 50/group；
Administering mode：It is injected intravenously 0.2PNA units/kg body weight；
Monitoring project：Heart rate, blood pressure, body temperature, conjunctival hemorrhage situation, injection site situation；
Experimental group：Freeze-dried powder preparation prepared by the embodiment of the present invention 7, dissolved with 0.9% sodium chloride sodium chloride injection；
Control group 1：Freeze-dried powder preparation prepared by comparative example 3 of the present invention, dissolved with 0.9% sodium chloride sodium chloride injection；
Control group 2：Freeze-dried powder preparation prepared by comparative example 4 of the present invention, dissolved with 0.9% sodium chloride sodium chloride injection；
Blank control group：0.9% sodium chloride sodium chloride injection；
Experimental result：The number of elements that the above-mentioned each monitoring project of every group of mouse occurs is counted, as a result such as following table：
| Experimental group | Control group 1 | Control group 2 | Blank control group | |
| Heart rate improves | 1 | 2 | 3 | 0 |
| Blood pressure reduces | 1 | 1 | 2 | 1 |
| Body temperature raises | 1 | 2 | 4 | 0 |
| Conjunctival hemorrhage | 0 | 1 | 3 | 0 |
| The red pain of itching in injection site | 2 | 1 | 7 | 1 |
As seen from the experiment, addition provided by the invention and the restructuring kallikrein freeze-dried powder system for not adding hardening agent
The adverse reaction rate of agent does not have notable difference, but the situation that adverse reaction occurs is considerably less than disclosed injection at present
Liquid.Illustrate that restructuring kallikrein freeze-dried powder preparation stability provided by the invention is good, and adverse reaction rate is low.
Claims (9)
1. one kind restructuring kallikrein freeze-dried powder preparation, it is characterised in that：The freeze-dried powder preparation includes：Recombinate kassinin kinin release
Enzyme, excipient, osmotic pressure regulator, pH adjusting agent and buffer.
2. freeze-dried powder preparation according to claim 1, it is characterised in that：Contain in every 1000 bottles of described freeze-dried powder preparation
Have：
3. according to the freeze-dried powder preparation described in claim 1-2, it is characterised in that：Described excipient is selected from mannitol, sweet ammonia
One or more in acid, dextran, gelatin hydrolysate, sodium citrate, polyethylene glycol and trehalose；The weight of the excipient
Match and be：Mannitol: glycine: dextran: gelatin hydrolysate: sodium citrate: polyethylene glycol: trehalose=5: 1: 2: 2: 2: 3:
3。
4. according to the freeze-dried powder preparation described in claim 1-2, it is characterised in that：Described osmotic pressure regulator is selected from chlorination
One or more in sodium, glucose or albumin.
5. according to the freeze-dried powder preparation described in claim 1-2, it is characterised in that：Described buffer can be selected from phosphoric acid hydrogen two
One kind in sodium-sodium dihydrogen phosphate or citric acid-sodium citrate.
6. the described freeze-dried powder preparation according to claim 1-2, it is characterised in that：The freeze-dried powder preparation also includes strong
Agent, one or more of the hardening agent in methionine, arginine, niacinamide and vitamin B6；The hardening agent
Weight proportion be：Methionine: arginine: niacinamide: vitamin B6=2: 2: 1: 1.
7. according to the freeze-dried powder preparation described in claim any one of 1-6, it is characterised in that：The ratio of the excipient and hardening agent
Example is 10-30: 1；Preferably, the ratio of excipient and hardening agent is 10-20: 1；It is further preferred that excipient and hardening agent
Ratio be 15: 1.
8. the preparation method of the freeze-dried powder preparation described in claim any one of 1-7, it is characterised in that the preparation method includes
Following steps：
(1) other raw materials that is weighed in addition to recombinating kallikrein according to formula dosage are placed in matching somebody with somebody in liquid bucket, use temperature
Stirred for 2~4 DEG C of waters for injection to dissolving；After addition medical activated carbon stirs and evenly mixs, using 0.45 μm of membrane filtration decarburization, obtain
To solution A；The restructuring kallikrein of dosage is formulated with 2~4 DEG C of water for injection dissolving, and is well mixed with above-mentioned solution A,
Obtain solution B；
(2) pH value for the solution B that is obtained with phosphoric acid or sodium hydroxide regulating step (1) obtains solution C to 6-7；
(3) solution C that step (2) obtains is supplied volume to 1500mL by with 2~4 DEG C of waters for injection, is mixed, is obtained solution D；
(4) solution D that is obtained using 0.22 μm of filter membrane to step (3) carries out filtration sterilization twice, obtains solution E；
(5) dispenses solution E, partly jumps a queue, is freeze-dried in freeze dryer, rolls lid, checks, packaging, produces.
9. preparation method according to claim 8, it is characterised in that：Medical activated carbon in the step of wherein described (1)
Addition be 0.5-2g；Average grain diameter is 3-7 μm.
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| CN1634576A (en) * | 2004-11-29 | 2005-07-06 | 广东天普生化医药股份有限公司 | Medicine composition for improving stability of human urine kininogenase |
| CN1931362A (en) * | 2006-09-05 | 2007-03-21 | 广东天普生化医药股份有限公司 | Application of human urokinase-type peptidase in preparing medicine for treating diabetes combined cerebral infraction |
| CN101134953A (en) * | 2007-07-02 | 2008-03-05 | 广东天普生化医药股份有限公司 | Recombinant human pancreas kininogenase |
| CN101596311A (en) * | 2008-06-02 | 2009-12-09 | 上海万兴生物制药有限公司 | Stable kallikrein-1 injection |
| CN101897959A (en) * | 2007-07-02 | 2010-12-01 | 广东天普生化医药股份有限公司 | Recombinant human pancreatic kininogenase-containing medicine for treating and/or preventing cerebral infarction |
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2017
- 2017-11-29 CN CN201711236771.0A patent/CN107802827B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1634576A (en) * | 2004-11-29 | 2005-07-06 | 广东天普生化医药股份有限公司 | Medicine composition for improving stability of human urine kininogenase |
| CN1931362A (en) * | 2006-09-05 | 2007-03-21 | 广东天普生化医药股份有限公司 | Application of human urokinase-type peptidase in preparing medicine for treating diabetes combined cerebral infraction |
| CN101134953A (en) * | 2007-07-02 | 2008-03-05 | 广东天普生化医药股份有限公司 | Recombinant human pancreas kininogenase |
| CN101897959A (en) * | 2007-07-02 | 2010-12-01 | 广东天普生化医药股份有限公司 | Recombinant human pancreatic kininogenase-containing medicine for treating and/or preventing cerebral infarction |
| CN101596311A (en) * | 2008-06-02 | 2009-12-09 | 上海万兴生物制药有限公司 | Stable kallikrein-1 injection |
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