CN102580061A - Intranasal insulin preparation and preparation method thereof - Google Patents
Intranasal insulin preparation and preparation method thereof Download PDFInfo
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- CN102580061A CN102580061A CN2011103930935A CN201110393093A CN102580061A CN 102580061 A CN102580061 A CN 102580061A CN 2011103930935 A CN2011103930935 A CN 2011103930935A CN 201110393093 A CN201110393093 A CN 201110393093A CN 102580061 A CN102580061 A CN 102580061A
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Abstract
The invention discloses an intranasal insulin preparation and a preparation method thereof. The intranasal insulin preparation is commonly formed by cell-penetrating peptide dissolved in a liquid-phase carrier and insulin. The cell-penetrating peptide is polypeptide molecule containing specific sequences of amino acids, so that the insulin can be promoted to enter into a human body through a mucosa of a nasal cavity, and the plasma glucose level is regulated. The preparation method of the preparation comprises the main preparing steps of: dissolving the therapeutic dose of insulin by hydrochloric acid; diluting to be required concentration by the liquid-phase carrier; regulating pH to be neutral by sodium hydroxide; mixing with a specific volume of cell-penetrating peptide; filtering and degerming; and storing at low temperature. The preparation can be applied to the surface of the mucosa of the nasal cavity of a person or an animal through an intranasal feeder. Compared with a subcutaneous insulin infusion preparation with the same dosage, according to the intranasal insulin preparation, the relative drug absorption rate is about 80 percent, and the bioavailability reaches more than 60 percent. The cell-penetrating peptide and the insulin have no stimulation on the mucosa of the nasal cavity, the amino acids can be degraded in vivo, the half-life period is short, and the safety is high.
Description
Technical field
The present invention relates to non-injection-type insulin technical field.Be specifically related to a kind of nose and use the type insulin preparation; And relate to the method for preparing of this nose with the type insulin preparation; Also relate to of the application of this nose simultaneously with the type insulin preparation.
Background technology
The physiology of insulin and pharmacology: insulin is a kind ofly to contain 51 amino acid whose proteohormones by beta Cell of islet is excretory, and molecular weight is 5808 dalton, participates in regulating carbohydrate metabolism, glucose level control.Absolute or the relative deficiency of insulin can cause diabetes in the body, and main clinical manifestation is " three-many-one-little " (polydipsia, polyuria, polyphagia and weight loss) and glucose in urine, hyperglycemia and multiple complications etc.China has 60,000,000 diabeticss at present approximately; Wherein much human needs lifelong (like the type i diabetes patient) or long-term frequent (like part type ii diabetes patient) to utilize exogenous insulin to come the regulating and controlling blood sugar level, in case the serious clinical complication of the sugared too high or too low initiation of stopping blooding.
Insulin use present situation and problem: the most frequently used administering mode of insulin treatment is subcutaneous injection.Needs of patients every day 2-4 time is injected Semilente Insulin before the meal, and be aided with sleep before injection an amount of in protamine zine insulin.Prolonged and repeated local injection not only forms scar tissue and anaphylaxis easily, influences the absorption of medicine, and can make the patient feel pain, produces boredom, and even do not follow the doctor's advice and cause complication, but threat to life when serious.Researcher attempts to develop the insulin preparation of safety, convenient, effective non-injection administration mode always for a long time.
The kind of non-injection-type insulin preparation and weak point: the substituting dosage regimen that proposes at present comprise through lung, transdermal, oral, give the therapeutic insulin preparation through approach such as intestinal, Sublingual and per nasal.The Exubera pulmonary Foradil Aerolizer formoterol fumarate of the first-elected Pfizer Inc. of pulmonary's inhalation.But promptly being recalled soon after its listing, mainly be to consider the latent lesion of life-time service to pulmonary function, and its relative bioavailability is merely hypodermic 10-20%.Though the patient is prone to accept oral enteric coated preparation, can tolerate fully that the insulin preparation of proteolytic enzyme is still untapped to come out, and insulin self is difficult to see through the gastrointestinal mucosal epithelium.Some other substituting dosage regimen also has shortcoming separately.For example: the bioavailability of the unhygienic inconvenience of rectally, transdermal administration is extremely low, contain potential bad foreign compound etc. in the sublingual formulation.
The feasibility of Giving Insulin by Pernasal Method and key point: known nasal membrane surface area reaches 150 square centimeters, and a large amount of trickle fine hair can increase effective absorbing area.If medicine can permeate the nasal membrane epithelial cell, then can promptly be absorbed by blood capillary abundant in the nasal membrane layer and lymphatic vessel, get into the systemic blood circulation quickly than subcutaneous injection mode.Because insulin infiltration nasal membrane is epithelial limited in one's ability, so the relative bioavailability of regular insulin nose administration extremely low (being merely hypodermic 1-2%).Therefore, increase nasal membrane epithelial cell is the key point of research and development insulin intranasal administration preparation to the permeability of insulin.
The patent retrieval of various insulation administration preparation through nose: through " patent retrieval of China national Department of Intellectual Property ", find 9 with nose with the relevant patent application of type insulin preparation.Apply for artificial Chinese Shanghai medical industry academy (200510028990.0; 200510028991.5; 200710173622.4; 200810035771.9), the DDS of Amada Co., Ltd. institute (01801146.2) and DDS Research Ltd. (02804546.7) and U.S. Nastech Pharm Co. (200680047851.5) and Cpex Pharmaceuticals Inc. (200780028627.6; 200980121227.9).They use chemicals such as porous spherical calcium carbonate, crystalline cellulose polymer, oils and fats, emulsifying agent, solubilizing agent, big lopps penetrating agent and surfactant to increase the epithelial ability of insulin infiltration nasal membrane respectively.Because these foreign compounds and metabolite scarcely are present in the normal body, they to the potential stimulus property of nasal mucosa with get into that the issuable chronic toxicity in back quite merits attention in the body.In addition, these application for a patent for invention description or do not announce the relative bioavailability of its insulation administration preparation through nose, otherwise resulting relative bioavailability is very low, and the result causes the safety range between dose therapeutically effective and the minimum toxic dose very little.For example, the relative bioavailability of porous spherical calcium carbonate/insulin preparation is merely 5.69%.These inventions still fail to satisfy the requirement of diabetes clinical treatment.
Summary of the invention
The objective of the invention is to be to provide a kind of nose to use the type insulin preparation.Said preparation mainly is made up of cell permeable peptide, insulin and three kinds of raw materials of liquid phase carrier, will treat the intravital compound formulation with insulin input patient for a kind of with the nasal administration mode.Its advantage is that the insulin in the preparation is easy to see through the nasal membrane barrier, and drug absorption rate (blood insulin concentration) is about 80% relatively, and bioavailability (hypoglycemic effect) reaches more than 60%, far above other the non-injection-type insulin preparation that is grinding at present.
Another object of the present invention is to be to provide the method for preparing of a kind of nose with the type insulin preparation.The main preparation process of said preparation is that cell permeable peptide and insulin are dissolved in liquid phase carrier with appropriate concentration.Simple and safe operation, repeatability is very good, and quality control is easy.
A further object of the present invention is to be to provide a kind of nose with the application in the glucose level in regulation and control blood of type insulin preparation.Available intranasal spray delivery device is executed the nasal membrane surface that is distributed in the patient with the said preparation of therapeutic dose, and easy to use, painless, non-stimulated to nasal membrane, capable of being fast degraded in the body is aminoacid, safe.
In order to realize above-mentioned purpose, the present invention adopts following technical measures:
Technical conceive of the present invention is: many biomacromolecules with potential therapeutical effect are difficult to use in clinical, and tracing it to its cause is that cell membrane is very low to the permeability of these molecules.Past is over more than 10 year; Various kinds of cell infiltration peptide is developed as one type of transmission system in succession; They have the ability of passing mammalian cell membrane; And do not receive the constraint of energy and receptor, become the new tool that promotes or carry multiple drug molecule (comprising that chemical micromolecule, biomacromolecule and nano-particle etc. receive loading) transmembrane transport.Cell permeable peptide contains several basic amino acids and hydrophilic (polarity)/hydrophobic (nonpolar) amphiprotic group by several peptide molecules of forming to twenties aminoacid.They can form stabilized complex with receiving loading with the non-covalent bond form, help the latter to see through with the mode of effective, harmless and organize epithelial cell to get in the body, the performance biological effect.In addition, getting into intravital cell permeable peptide is common amino acid molecular by proteasome degradation very soon, and does not produce deleterious metabolite.The progress of cell permeable peptide has greatly been accelerated the development and application of DNA, oligonucleotide, siRNA, PNAG3 PNA, peptide and biomacromolecule class medicines such as protein and elaioplast nanometer particle.The applicant reacts 31 kinds of cell permeable peptides and analog and various insulin under different condition, obtain various compositionss.Pass through a large amount of zooperies then, test the drug absorption rate and the bioavailability of each compositions nose administration, screening and optimizing goes out one type of nose and uses the type insulin preparation.
The technical problem that the present invention will solve is: provide a kind of nose with high bioavailability, no mucous membrane irritation and chronic toxicity to use the type insulin preparation.
A kind of nose that supplies to treat usefulness is used the type insulin preparation, and it is made up of the following raw material that is dissolved in the liquid phase carrier jointly:
Cell permeable peptide 0.02-2 nanomole/microlitre liquid phase carrier;
Insulin 0.001-0.5 iu/microlitre liquid phase carrier;
This insulin preparation is that a kind of will the treatment with insulin with the nasal administration mode imported the intravital compound formulation of patient, and main component is liquid phase carrier, insulin and cell permeable peptide.Specifically describe as follows:
1, liquid phase carrier is (but being not limited to) medical aseptic water, normal saline, phosphate buffer or artificial cerebrospinal fluid in the described preparation, and pH value is 6.5-7.5, and its compound method is following:
1) normal saline: get sodium chloride 9 grams, add water to 1000 milliliters.
2) phosphate buffer: with 610 milliliters of the sodium hydrogen phosphate aqueous solutions of 0.2 mol 390 milliliters of mixings of biphosphate sodium water solution with 0.2 mol, 1000 milliliters of the phosphate buffers of 0.2 mol, pH7.0.
3) artificial cerebrospinal fluid: get sodium chloride 6.279 grams, potassium chloride 0.216 gram, calcium chloride 0.353 gram; Magnesium chloride 0.488 gram, sodium bicarbonate 1.932 grams, glucose 0.6 gram; Sodium hydrogen phosphate 0.358 gram; Add water to 1000 milliliters, feeding contains 95% oxygen and 5% carbon dioxide gas mixture is extremely saturated, pH7.3-7.4.
2, insulin is the insulin of biologically active (ability blood sugar regulation level), for example (but being not limited to) natural insulin, human insulin, insulin precurosor, insulin analog or insulin metabolism thing in the described preparation.
3, cell permeable peptide is by the preparation of CS936X Peptide synthesizer in the described preparation, and it is one type and contains 12-22 amino acid whose peptide molecule that wherein arginine and lysine are the important component of keeping the Premeabilisation of cells function.
First kind of grown form of this cell permeable peptide is:
Arg?Trp?Phe?Lys?Ile?Gln?Met?Gln?Ile?Arg?Arg?Trp?Lys?Asn?Lys?Lys
Under the situation that does not change arginine and lysine position, the cell permeable peptide of above-mentioned grown form can be adjusted to the cell permeable peptide of second kind of grown form:
Arg?Gln?Ile?Lys?Ile?Trp?Phe?Gln?Asn?Arg?Arg?Met?Lys?Trp?Lys?Lys
More than the Premeabilisation of cells peptide sequence of two kinds of grown forms can further adjust as follows (but being not limited to down example):
1) with behind half section location swap with the c-terminus second half section before the aminoterminal, the cell of above-mentioned grown form oozes peptide sequence and can be adjusted to:
Ile Arg Arg Trp Lys Asn Lys Lys Arg Trp Phe Lys Ile Gln Met Gln; Or
Asn?Arg?Arg?Met?Lys?Trp?Lys?Lys?Arg?Gln?Ile?Lys?Ile?Trp?Phe?Gln
2) through reverse ordering, above-mentioned grown form the Premeabilisation of cells peptide sequence can be adjusted to:
Lys Lys Asn Lys Trp Arg Arg Ile Gln Met Gln Ile Lys Phe Trp Arg; Or
Lys?Lys?Trp?Lys?Met?Arg?Arg?Asn?Gln?Phe?Trp?Ile?Lys?Ile?Gln?Arg
3) arginine and lysine are exchanged, the Premeabilisation of cells peptide sequence of above-mentioned grown form can be adjusted to:
Lys Trp Phe Arg Ile Gln Met Gln Ile Lys Lys Trp Arg Asn Arg Arg; Or
Lys?Gln?Ile?Arg?Ile?Trp?Phe?Gln?Asn?Lys?Lys?Met?Arg?Trp?Arg?Arg
4) at the c-terminus of the cell permeable peptide of above-mentioned grown form, add 1 to 6 glycine or nonspecific aminoacid, can form and contain 17-22 amino acid whose cell permeable peptide:
Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys adds 1-6 Gly or nonspecific aminoacid; Or
Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys adds 1～6 Gly or nonspecific aminoacid.
5) at the c-terminus of the cell permeable peptide of above-mentioned grown form, add 1 to 6 arginine or nonspecific aminoacid:
Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys adds 1-6 Arg or nonspecific aminoacid; Or
Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys adds 1-6 Arg or nonspecific aminoacid.
6) at the c-terminus of the cell permeable peptide of above-mentioned grown form, add 1 to 6 unspecific aminoacid:
Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys adds 1-6 nonspecific aminoacid; Or
Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys adds 1-6 nonspecific aminoacid.
7) from the cell permeable peptide of above-mentioned grown form, remove 1-4 aminoacid:
Arg Trp Phe Lys Ile Gln Gln Arg Arg Trp Lys Asn Lys Lys; Or
Arg?Gln?Ile?Lys?Ile?Trp?Gln?Arg?Arg?Met?Lys?Trp?Lys?Lys
8) 30% aminoacid in the above-mentioned various cell permeable peptides is adjusted after, formed new cell permeable peptide.
A kind of nose that supplies to treat usefulness is used the type insulin preparation, and it forms (better scope) jointly by the following raw material that is dissolved in the liquid phase carrier:
Cell permeable peptide 0.05-1.0 nanomole/microlitre liquid phase carrier
Insulin 0.005-0.4 iu/microlitre liquid phase carrier
A kind of nose that supplies to treat usefulness is used the type insulin preparation, and it forms (preferable range) jointly by the following raw material that is dissolved in the liquid phase carrier:
Cell permeable peptide 0.1-0.6 nanomole/microlitre liquid phase carrier
Insulin 0.015-0.3 iu/microlitre liquid phase carrier
A kind of nose that supplies to treat usefulness is used the type insulin preparation, and it forms (preferably scope) jointly by the following raw material that is dissolved in the liquid phase carrier:
Cell permeable peptide 0.2-0.4 nanomole/microlitre liquid phase carrier
Insulin 0.03-0.2 iu/microlitre liquid phase carrier
A kind of nose that supplies to treat usefulness is used the type insulin preparation, and it forms (optimum) jointly by the following raw material that is dissolved in the liquid phase carrier:
Cell permeable peptide 0.3 nanomole/microlitre liquid phase carrier
Insulin 0.1 iu/microlitre liquid phase carrier
A kind ofly supply to treat the method for preparing of the nose of usefulness, the steps include: with the type insulin preparation
1, under room temperature (22 ± 2 ℃, below identical), accurately take by weighing the insulin powder of 200 ius, place in one 5 milliliters the small test tube;
2,200 microlitres, 0.1 normal hydrochloric acid solution is added in the above-mentioned small test tube lentamente jog, dissolving insulin powder;
3, add 1.6 milliliters of liquid phase carriers then and dilute the jog mixing;
4, with 200 microlitres, 0.1 normal sodium hydroxide the above-mentioned solution that contains insulin is transferred to pH6.8-7.2;
5, accurately take by weighing the cell permeable peptide of 600 nanomoles in addition, place in another small test tube of 5 milliliters;
6, above-mentioned insulin solutions is transferred to lentamente in the small test tube that contains cell permeable peptide jog mixing 30 minutes with micropipettor;
7, use the not above-mentioned insulin solutions that contains cell permeable peptide of membrane filtration of 0.45 micron pore size of adsorbed proteins;
8, the nose after will filtering is collected in the aseptic administrator of a sealing with the type insulin preparation;
9, it is subsequent use to place low temperature (4 ± 1 ℃) to preserve; Or place freezing (20 ℃ or-80 ℃) long preservation.
A kind ofly supply to treat the application process of the nose of usefulness, the steps include: with the type insulin preparation
1, nose is placed intranasal atomizing or instillation formula administrator with the type insulin preparation;
2, the insulin preparation of therapeutic dose is executed the nasal membrane surface that is distributed in the patient;
3, the volumes of formulation of at every turn executing cloth is 100 (50-200) microlitre;
4, execute in the preparation of cloth, insulin dose is 10 (5-20) iu at every turn;
5, execute in the preparation of cloth, cell permeable peptide dosage is 30 (15-60) nanomole at every turn;
6, in executing cloth front and back, periodic monitor blood sugar level.
Technical characterictic: nose of the present invention is with containing the cell permeable peptide of being made up of the specific amino acids sequence in the type insulin preparation, it can help insulin to penetrate the nasal membrane epithelial cell efficiently and get into systemic blood circulation, regulating and controlling blood sugar level.
As a kind of brand-new insulin intranasal absorption enhancer; Nose of the present invention is compared with other Premeabilisation of cells promoter with the cell permeable peptide in the type insulin preparation; Have the diverse chemical constitution and the mechanism of action, can make the insulin in the preparation produce much higher drug absorption rate and bioavailability.
This cell permeable peptide and nose when seeing through the nasal membrane epithelial cell, and after getting into the systemic blood circulation, have following technical characterictic with the type insulin.
1, the cell permeable peptide structure is relevant with the ability of transhipment insulin: after the suitably adjustment of cell permeable peptide process (comprise rearrangement, extend or shorten) of the used two kinds of grown forms of the present invention, and the variation that the relative drug absorption rate of its insulin preparation and relative bioavailability have to a certain degree (10-30%).
2, the Premeabilisation of cells peptide concentration is relevant with insulin transmembrane transport efficient: when cell permeable peptide when nose uses concentration range in the type insulin preparation as 0.02-2 nanomole/microlitre, insulin level and Premeabilisation of cells peptide concentration are proportionate in the blood.Consider various composition proportion in cost and benefit relation and the preparation, the best scope of Premeabilisation of cells peptide concentration is 0.2-0.4 nanomole/microlitre.
But 3, the various insulins of cell permeable peptide rapid transport: cell permeable peptide is made up with different recombinant human insulin, insulin analog or natural Iletin II (Lilly) respectively, carry out zoopery.The result showed these several kinds of different components nose administrations after ten minutes, and insulin level obviously raises in the blood, began to produce significant hypoglycemic effect in 30 minutes.
4, nose can produce the relative drug absorption rate of higher insulin with the type insulin preparation: or lumbar injection subcutaneous with the laboratory animal of same dose compared, and the Premeabilisation of cells peptide concentration is that the nose of 0.2-0.4 nanomole/microlitre can produce the relative drug absorption rate up to 80% with the type insulin preparation.
5, nose can produce higher insulin relative bioavailability with the type insulin preparation: or lumbar injection subcutaneous with the laboratory animal of same dose compared, and the Premeabilisation of cells peptide concentration is that the nose of 0.2-0.4 nanomole/microlitre can produce the relative bioavailability up to 60% with the type insulin preparation.
6, cell permeable peptide can be degraded and inactivation rapidly in vivo: behind intravenously administrable, the half-life of isotope-labeled cell permeable peptide in blood circulation is less than 60 minutes, mainly through liver and kidney katabolism.
7, cell permeable peptide dissolves in multiple liquid phase carrier: at room temperature, cell permeable peptide can be dissolved in pH value separately and ooze normal saline or phosphate buffer for the grade of neutral (6.5-7.5); Also can be miscible in above-mentioned liquid phase carrier with the insulin of therapeutic dose.
8, cell permeable peptide long preservation at low temperatures: the mixing application liquid of cell permeable peptide and insulin was deposited two months under low temperature (4 ℃), and its BA does not have obvious change.
9, nose does not produce zest to the nasal membrane epithelial cell with the type insulin preparation: nose does not influence the integrity and the motion of the mucosal tissue cilium that exsomatizes with the type insulin preparation; Find that in the body experiment nose administration is non-stimulated to nasal membrane, do not change the epithelial histology's form of nasal membrane.
10, cell permeable peptide does not produce acute and chronic injury to the in-vivo tissue organ: cell permeable peptide does not produce the acute and chronic trauma in bright county to main organs 26S Proteasome Structure and Functions such as the heart, liver, spleen, lung, kidney, brain, stomach, intestinal, muscle and reproductions.
11, the biological effect after nose uses with type insulin preparation repeated multiple times is stable and regular: to same animal subject once a day, a continuous week use nose to use the type insulin, hypoglycemic effect stability, and good reproducibility.
12, cell permeable peptide and nose are confirmed by at least two kinds of laboratory animals with the above-mentioned characteristic of type insulin: histiocytic isolated experiment, and mice and rat have all confirmed cell permeable peptide and the nose above-mentioned characteristic with the type insulin preparation in the body experiment.
13, the Preliminary experiment results of human body shows: the nose that gives healthy volunteer's 5 ius is on an empty stomach used the type insulin preparation, can make blood sugar level reduce about 20%.The nose that gives healthy volunteer's 5 ius before the dining is used the type insulin preparation, can make the level of postprandial blood sugar lift-off value reduce about 70%.
According to above-mentioned technical characterictic and following practical implementation result, but the creativeness of knowledge capital invention is:
1, first the cell permeable peptide of suitable construction is used to prepare non-injection-type insulin preparation;
2, confirmed relation between Premeabilisation of cells peptide concentration, insulin dosage and the biological effect;
3, set up the method for preparing of nose with the type insulin preparation;
4, inquired into the relative drug absorption rate and the relative bioavailability of insulin in the preparation;
5, detected the pharmacokinetics of cell permeable peptide in the preparation, and to the acute and chronic toxic action of local and whole body;
6, the nose that has finally obtained to have the clinical practice potentiality is used the type insulin preparation.
Nose of the present invention is with the alternative subcutaneous injection mode of type insulin preparation, and per nasal internal spraying formula or the administration of instillation formula are used to treat diabetics.Each use amount is 100 microlitres (50 to 200 microlitres), interior insulin-containing 10 (5-20) iu.The concrete use decided according to factors such as conditions of patients by the doctor.
Description of drawings
Fig. 1 is a kind of chromatography image that contains 16 amino acid whose cell permeable peptides, has reflected degree of purity of production.
Fig. 2 is a kind of mass spectral analysis image that contains 16 amino acid whose cell permeable peptides, has reflected the structure of product.
Fig. 3 is that a kind of cell permeable peptide promotes the experimental result that insulin intranasal absorbs, and has shown that the Premeabilisation of cells peptide content can promote insulin to absorb insulin concentration in the blood that increases sharply through the experimental rat nasal membrane within the specific limits.
Fig. 4 is the experimental result that a kind of cell permeable peptide helps the insulin blood sugar lowering, has shown that cell permeable peptide can promote different insulin (like insulin human and Iletin II (Lilly)) to absorb through the experiment mice nasal membrane, reduces glucose level in the blood.
Fig. 5 is the experimental result of a kind of nose with type insulin preparation absorption efficiency, shown nose with the type insulin than the drug absorption speed of subcutaneous injection insulin fast slightly (after the administration within 20 minutes), the drug absorption rate is 80% relatively.
Fig. 6 is the experimental result of a kind of nose with type insulin preparation blood sugar lowering effect, has shown that nose can produce blood sugar decreasing effect similar with the subcutaneous injection insulin and persistent period with the type insulin, and relative bioavailability is 60-70%.
Fig. 7 is the metabolic in vivo experimental result of a kind of cell permeable peptide: last figure has shown in the blood that the half-life of cell permeable peptide is very short, and the content of cell permeable peptide is extremely low in the brain; Figure below has shown that getting into intravital cell permeable peptide mainly accumulates and degrade in liver, kidney.
Fig. 8 is the contrast and experiment that two kinds of cell permeable peptides help the insulin blood sugar lowering; The cell permeable peptide (16 aminoacid) that has shown first kind of basic kenel can promote more that than another kind of adjusted cell permeable peptide (18 aminoacid) insulin intranasal absorbs, the blood sugar lowering level, and both difference is about 20%.
Fig. 9 is the experimental result of a kind of nose with type insulin preparation storage condition, has shown still to keep original blood sugar lowering ability (glucose level in the blood) and time effect (after the administration 30-120 minute) nose is deposited 1-60 days with the type insulin preparation under low temperature (4 ℃) after.
Figure 10 is the sensitivity experimental result of a kind of animal subject to multiple dosing, has shown experiment mice once a day, accepts nose in continuous five days to use the type insulin preparation, remains the sensitivity to insulin blood sugar lowering effect.The component of every day (from first day to the 5th day) shows similar blood sugar lowering ability (glucose level the blood) and time effect (after the administration 30-240 minute).
The specific embodiment
Below further specify the present invention with embodiment, but the present invention is not limited.
Embodiment 1:
Nose with the insulin human preparation experiment is used is used the type insulin preparation.
The nose of table one, treated animal experiment usefulness is used the type insulin preparation, and it is made up of following ingredients that is dissolved in phosphate buffer and concentration.
The nose of table two, another treated animal experiment usefulness is used the type insulin preparation, and it is made up of following ingredients that is dissolved in phosphate buffer and concentration.
Described certain cell permeable peptide of table one and table two is for containing 16 amino acid whose cell permeable peptides:
Arg?Trp?Phe?Lys?Ile?Gln?Met?Gln?Ile?Arg?Arg?Trp?Lys?Asn?Lys?Lys
Described certain cell permeable peptide of table one and table two also can be and contains 18 amino acid whose cell permeable peptides:
Arg?Gln?Ile?Lys?Ile?Trp?Phe?Gln?Asn?Arg?Arg?Met?Lys?Trp?Lys?Lys?Gly?Gly
The method for preparing of the above insulin preparation the steps include:
1, accurately takes by weighing the insulin of specified quantitative shown in the table, place in the small test tube, slowly dissolve with 0.1 normal hydrochloric acid solution;
2, add liquid phase carrier and be diluted to certain concentration shown in the table;
3, reuse 0.1 normal sodium hydroxide transfers to pH6.8-7.2 with the above-mentioned solution that contains insulin;
4, accurately take by weighing the cell permeable peptide of specified quantitative shown in the table in addition, place in another small test tube;
5, above-mentioned insulin solutions is transferred to lentamente in the small test tube that contains cell permeable peptide jog mixing 30 minutes with micropipettor;
6, use the not above-mentioned insulin solutions that contains cell permeable peptide of membrane filtration of 0.45 micron pore size of adsorbed proteins;
7, the nose after will filtering is collected in the aseptic administrator of a sealing with the type insulin preparation;
8, place cryopreservation, or directly use.
Embodiment 2:
Nose with the Iletin II (Lilly) preparation experiment is used is used the type insulin preparation
The nose of table three, treated animal experiment usefulness is used the type insulin preparation, and it is made up of following ingredients that is dissolved in medical aseptic water and concentration.
The nose of table four, another treated animal experiment usefulness is used the type insulin preparation, and it is made up of following ingredients that is dissolved in medical aseptic water and concentration.
Described certain cell permeable peptide of table three and table four is for containing 16 amino acid whose cell permeable peptides:
Arg?Trp?Phe?Lys?Ile?Gln?Met?Gln?Ile?Arg?Arg?Trp?Lys?Asn?Lys?Lys
Described certain cell permeable peptide of table three and table four also can be and contains 14 amino acid whose cell permeable peptides:
Arg?Trp?Phe?Lys?Ile?Gln?Gln?Arg?Arg?Trp?Lys?Asn?Lys?Lys
The method for preparing of the above insulin preparation, its step is with embodiment 1.
Embodiment 3:
Nose with the insulin analog preparation experiment is used is used the type insulin preparation
The nose of table five, treated animal experiment usefulness is used the type insulin preparation, and it is made up of following ingredients that is dissolved in phosphate buffer and concentration.
The nose of table six, another treated animal experiment usefulness is used the type insulin preparation, and it is made up of following ingredients that is dissolved in medical aseptic water and concentration.
The described insulin analog of table five and table six is an insulin aspart.
Described certain cell permeable peptide of table five and table six is for containing 18 amino acid whose cell permeable peptides:
Arg?Trp?Phe?Lys?Ile?Gln?Met?Gln?Ile?Arg?Arg?Trp?Lys?Asn?Lys?Lys?Arg?Arg
Described certain cell permeable peptide of table five and table six also can be and contains 14 amino acid whose cell permeable peptides:
Arg?Gln?Ile?Lys?Ile?Trp?GlnArgArg?Met?Lys?Trp?Lys?Lys
The method for preparing of the above insulin preparation, its step is with embodiment 1.
Embodiment 4:
The nose that the preparation volunteer uses is used the type insulin preparation
The nose that table seven, lineup's body volunteer use is used the type insulin preparation, and it is made up of following ingredients that is dissolved in medical water or phosphate buffer and concentration.
Described certain cell permeable peptide of table seven is for containing 16 amino acid whose cell permeable peptides:
Arg?Trp?Phe?Lys?Ile?Gln?Met?Gln?Ile?Arg?Arg?Trp?Lys?Asn?Lys?Lys
The method for preparing of the above insulin preparation, its step is with embodiment 1.
Embodiment 5:
The structure of cell permeable peptide and quality analysis
Adopt solid phase method (CS936X Peptide synthesizer) preparation cell permeable peptide.Come purification to prepare product with HPLC, characterize purified product with mass spectrography.Introduce structure and quality analysis (figure one and figure two) that an example contains 16 amino acid whose cell permeable peptides at present.
Sequence: Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys
Chemical modification: aminoterminal and c-terminus all do not have modification
Molecular formula: C
104H
168N
34O
20S
1
Molecular weight: 2246.78
Outward appearance: white powder
Purity (chromatography): greater than 95%
Structure (mass spectral analysis): stable
Store: dry cold preservation
The mensuration result of cell permeable peptide purity sees table eight.
Table eight, efficient liquid phase chromatographic analysis data
Embodiment 6:
The assay method of insulin in the blood
Promote that at cell permeable peptide insulin level has reflected the drug absorption ability of nose with the type insulin preparation in the blood in the experiment of insulin transmembrane transport.
Adopt hypersensitive insulin human radioimmunoassay kit (LINCO RES) to measure insulin concentration in the blood plasma.This radioimmunology analysis system comprises four elements: the specific anti insulin antibody; The insulin antigen of labelled with radioisotope; The method that conjunction type labelled antigen and free phenotypic marker antigen are separated; And radiosiotope measuring instrument.Concrete determination step is:
1, at the particular point in time blood-sample withdrawal, separate 50 microlitre blood plasma, cryopreservation is subsequent use;
2, with a certain amount of
125Iodine labeling insulin antigen and the common incubation of specificity insulin antibody make about 50% labelled antigen and antibodies;
3, in this incubation system, add unmarked insulin antigen (blood plasma to be measured), competitive ground and antibodies;
4, along with unlabelled antigen in the system increases, the binding capacity of labelled antigen and antibody reduces;
5, conjunction type labelled antigen and free phenotypic marker antigen are separated, measure radioactivity separately;
6, calculate insulin concentration in the blood plasma to be measured (little iu/milliliter blood plasma) according to standard curve (making) with unmarked insulin standard article.
Embodiment 7:
Determination of glucose method in the blood
In the blood sugar lowering experiment of insulin, glucose level has reflected the biological activity of nose with the type insulin preparation in the blood.
Adopt ReliOn Ultima system for detecting blood sugar to measure the glucose level (milligram/decilitre) in the whole blood.The advantage of this system is: convenient (animal afterbody blood sample), quick (going out the result 5 seconds), sensitive (being less than 1 microlitre sample), stable (measure the result good repeatability is arranged).
Embodiment 8:
The administration of laboratory animal and sampling method
Used experimental animal feeding is at 22 ± 2 ℃, 12 little time/dark circulation Animal House, the drinking-water of freely ingesting.
1, experiment mice (20 ± 4 gram, male and female half and half) is freely drunk water, fasting is after one day, lumbar injection sodium phenobarbital anesthetis (60 milligrams/kg body weight).
Under anatomic microscope, slowly drip the test preparation that receives of 4 microlitres to every side nasal cavity with micro sample adding appliance.The matched group experiment mice is accepted the subcutaneous or lumbar injection in back and is received test preparation.At particular point in time, the docking measuring blood sugar of blood extracting.
In addition, for the metabolism of observation of cell infiltration peptide distributes, with isotope-labeled cell permeable peptide in tail vein injection experiments mice body.Get blood and each histoorgan at particular point in time, measure its radioactive intensity.
2, experimental rat (190 ± 30 gram, male and female half and half) is freely drunk water, fasting is after one day, lumbar injection sodium phenobarbital anesthetis (50 milligrams/kg body weight).
Animal lies on the back, and micro sample adding appliance is directly inserted nasal cavity, the instil test preparation that receives of 20 microlitres of every side.The matched group experimental rat is accepted the subcutaneous or lumbar injection in back and is received test preparation.At particular point in time, use the syringe that contains anticoagulant through the jugular vein measuring blood sugar of blood extracting; After the centrifuging separated plasma, measure blood insulin.
Embodiment 9:
The relation of Premeabilisation of cells peptide content and insulin transport efficacy
Different content cell permeable peptide (0.04-1.5 nanomole/microlitre) is formed multiple formulations with various dose recombinant human insulin (0.01-0.2 iu/microlitre); Be instilled into the nasal cavity of experimental rat respectively; Measure insulin concentration in the blood after 20 minutes, to understand the optimum efficiency that cell permeable peptide promotes that insulin intranasal absorbs.Figure three shows that certain contains 16 amino acid whose cell permeable peptides (Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys) when 0.1 nanomole/microlitre, can promote insulin human (0.02 iu/microlitre) to see through the nasal membrane epithelium and get into blood; Along with Premeabilisation of cells peptide content in the preparation increases, insulin human concentration also raises in the blood; When Premeabilisation of cells peptide content in the preparation increased to 2 nanomoles/microlitre, insulin human concentration was near peak value in the blood.This result shows that the Premeabilisation of cells peptide content has tangible dose-effect relationship with the insulin intranasal transport efficacy within the specific limits in the preparation.
The result of control experiment shows that per nasal gives the recombinant human insulin separately, does not detect insulin human in the blood.
Embodiment 10:
Cell permeable peptide is to the transhipment effect of different insulins
The cell permeable peptide (0.1 nanomole/microlitre) of certain content is formed multiple formulations with the insulin (0.0025 iu/microlitre) of separate sources, and per nasal gives experiment mice.Before administration and after the administration 30 and 60 minutes the time, measure glucose level in the blood, respectively to understand the facilitation of cell permeable peptide to different insulin intranasals absorptions.
Figure four shows that certain contains 18 amino acid whose cell permeable peptides (Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys Gly Gly) recombinant human insulin and the transhipment of natural Iletin II (Lilly) via intranasal application mucous epithelium are all had obvious facilitation.This result shows that under the help of cell permeable peptide, the insulin intranasal of these two kinds of separate sources has produced similar hypoglycemic effect after absorbing.
The result of control experiment shows that per nasal gives a kind of of cell permeable peptide, insulin or liquid phase carrier separately, does not all produce the obvious functions of blood sugar effect.
Embodiment 11:
Nose is with the relative drug absorption rate (insulin concentration in the blood) of type insulin
Give one group of experimental rat with nose with type insulin preparation (including cell permeable peptide 0.8 nanomole/microlitre and recombinant human insulin's 0.02 iu/microlitre) per nasal; In addition with the recombinant human insulin of isodose through the subcutaneous control rats that gives; Before administration with after the administration, measure insulin human concentration in the blood 5,15,30,45,60,90,120,150 minutes the time, calculate blood Chinese medicine Cmax, reach Cmax required time and drug absorption rate relatively.
Figure five shows that (contain 18 amino acid whose cell permeable peptides: recombinant human insulin's blood Chinese medicine Cmax is 188.6 ± 125.2 little iu/milliliter blood plasma to certain nose Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys Gly Gly), and reaching the Cmax required time is 18.3 ± 4.5 minutes with the type insulin preparation.
Compare with subcutaneous injection, the relative drug absorption rate of Giving Insulin by Pernasal Method is 83.5 ± 21.2%.
This result shows, nose of the present invention is with the drug absorption rate of the type insulin preparation insulation administration preparation through nose far above other types.
Embodiment 12:
Nose is with the relative bioavailability (glucose level in the blood) of type insulin
Give one group of experiment mice with nose with type insulin preparation (including cell permeable peptide 0.2 nanomole/microlitre and recombinant human insulin's 0.005 iu/microlitre) per nasal; In addition with the recombinant human insulin of isodose through the subcutaneous matched group experiment mice that gives; Before administration with after the administration, measure glucose level in the blood 30,60,90,120,150,180,240,300,360,720 minutes the time, calculate relative bioavailability.
Figure six shows that certain nose (contains 16 amino acid whose cell permeable peptides: Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys) administration can make blood sugar level reduce to 70%, 90 minute after 30 minutes after, reduce to 50% with the type insulin preparation; Level before blood sugar level returns to administration gradually in the time of 180-240 minute.
Compare with subcutaneous injection, the relative bioavailability of Giving Insulin by Pernasal Method is 66.8 ± 15.4%.
This result shows, nose of the present invention is with the bioavailability of the type insulin preparation insulation administration preparation through nose far above other types.
Embodiment 13:
Cell permeable peptide degraded and distribution (in the body method) in vivo
Measure blood and respectively organize the content of cell permeable peptide in the internal organs can understand its metabolic rate and distribution pattern in vivo.
With 100 microlitres
111The Premeabilisation of cells peptide solution of indium (1 milli Becquerel) labelling is in tail vein injection experiments mice body.In injection the last 10,30,60,120,180,240 minutes, take out blood, the heart, liver, spleen, lung, kidney, brain, intestinal and muscle etc. respectively and organize internal organs, with filter paper dip in dried, weigh.Measure radioactive intensity with gamma counter, converse the relative percentage composition that radioactivity cell permeable peptide in every gram tissue sample accounts for the gross activity cell permeable peptide.
Figure seven shows that certain contains the time DYNAMIC DISTRIBUTION of 16 amino acid whose radioactivity cell permeable peptides (Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys) in blood, brain, liver and kidney.
Premeabilisation of cells peptide concentration in blood and the brain reached the peak in back 10 minutes in injection, descended rapidly subsequently; Premeabilisation of cells peptide concentration in the liver reached the peak in back 30 minutes in injection, descended gradually subsequently; Premeabilisation of cells peptide concentration in the kidney reached the peak in back 50 minutes in injection, slowly descended subsequently.
The peak value of Premeabilisation of cells peptide concentration is close in liver and the kidney, and in the blood maximum concentration of cell permeable peptide; The concentration of cell permeable peptide is minimum in the brain.
These results show:
1, about 30 minutes of the half checkout time of cell permeable peptide in the blood;
2, As time goes on, cells in vivo infiltration peptide shifts to liver and kidney gradually;
3, liver and kidney are mainly accumulating and the metabolism organ of cell permeable peptide;
4, maybe be owing to the effect of blood brain barrier, cell permeable peptide is difficult to get into cerebral tissue through blood.
Embodiment 14:
Cell permeable peptide is measured (vitro method) in stability in blood
Will
111Indium (2 milli Becquerel) cell permeable peptide of labelling and the common incubation of human serum (37 ℃) 0,5,10,20,30,45,60,90 of 400 microlitres, 120,180,240 minutes.Behind the acetonitrile mixing of 2 times of volumes, put 4 ℃ one hour, with the deposition serum albumin.The supernatant that contains cell permeable peptide with radiation counting-high-efficient liquid phase chromatogram technique analysis.Calculate the radioactivity cell permeable peptide by the speed of serum albumin hydrolytic enzyme degraded.
The result shows that certain contains the half degradation time of 16 amino acid whose cell permeable peptides (Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys) in test tube is 52.5 ± 5.7 minutes; Certain contains the half degradation time of 18 amino acid whose cell permeable peptides (Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys Gly Gly) in test tube 58.2 ± 7.1 minutes.
Embodiment 15:
Nose is with the preservation condition of type insulin
Two parts of identical noses (are contained 16 amino acid whose cell permeable peptides: Arg TrpPhe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys) respectively 4 ℃ or 22 ℃ of lower seals placements 0,2,10 with the type insulin preparation; 20,30,60; After 90 days; Per nasal gives experiment mice, and the blood sugar level when measuring after the administration 90 minutes is to understand its preservation condition and time.The result shows, nose is with type insulin preparation long preservation (seeing table nine) at room temperature.
The insulin preparation blood sugar lowering ability of preserving under table nine, the different condition is (with respect to the percentage ratio of fresh preparation, %)
Figure eight show two kinds of noses with the type insulin preparation (be respectively: contain 16 amino acid whose cell permeable peptides:
Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys; Contain 18 amino acid whose cell permeable peptides: Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys Gly Gly) after 4 ℃ of lower seals are placed one month, good blood sugar lowering ability is arranged all.
Figure nine further demonstration contains 18 amino acid whose cell permeable peptides (Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys Gly Gly) after 4 ℃ of lower seals are placed two months, still can keep good blood sugar lowering ability.
Embodiment 16:
Nose is with the stimulation damage of type insulin preparation to nasal membrane
Acid phosphatase is present in the normal cell.When damaged membrane, acid phosphatase will spill in cell.Therefore, detect extracellular acid phosphatase content, can understand the integrity of cell membrane.
Whether nasal membrane is produced stimulation and damage in order to observe nose with the type insulin preparation; Cell permeable peptide, insulin, nose are instiled respectively on the nasal membrane surface of experimental rat with type insulin preparation, phosphate buffer (contrast), and the phosphate buffer of heating with the 5-15 milliliter after 20 minutes (37 ℃) is the lavation nasal cavity lentamente.Use the content of acid phosphatase in biochemical reaction development process (test kit of Promega company) and the spectrophotometric determination nasal cavity irrigating solution then.The result shows that cell permeable peptide, insulin and nose all do not damage nasal membrane epithelial cell (seeing table ten) with the type insulin preparation.
The content of acid phosphatase in table ten, the nasal cavity irrigating solution (iu/liter)
Containing 16 amino acid whose cell permeable peptides in the table is: Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp LysAsn Lys Lys.
Containing 18 amino acid whose cell permeable peptides in the table is: Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys Gly Gly.
Embodiment 17:
Cell permeable peptide is to the toxic action of histoorgan
Cell permeable peptide (0.1 nanomole/microlitre) or phosphate buffer secondary every day, continuous two weeks are dripped the nasal cavity at experiment mice.Taking out the heart, liver, spleen, lung, kidney, brain, stomach, intestinal, muscle, ovary, uterus or testis etc. organizes internal organs to make the conventional organization pathological examination.The result shows that cell permeable peptide does not cause damage (seeing table ten one) to the organizational structure of main organs.
The check result of table ten one, several kinds of main organs
(no change;-/+has slight variation; + slight the variation; ++ moderate changes; +++severe changes)
Containing 16 amino acid whose cell permeable peptides in the table is: Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp LysAsn Lys Lys.
Containing 18 amino acid whose cell permeable peptides in the table is: Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys Gly Gly.
Embodiment 18:
Nose is with the continuous result of use of type insulin preparation
With nose with type insulin preparation (0.005 iu/microlitre) continuously a couple of days give same group of experiment mice (fasting at 12 hours nights), measure the blood sugar content after the administration every day, observe nose with the type insulin preparation blood sugar decreasing effect after the use continuously.In other words, observe body to the lasting sensitivity of nose with the type insulin preparation.
Figure ten shows that containing 16 amino acid whose cell permeable peptides (Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys) used five days continuously, and the hypoglycemic effect between every day (curve) is similar.This result shows that nose can give the same examination individuality that receives in continuous many days with the type insulin preparation, produced stable hypoglycemic effect.
Embodiment 19:
Nose is with the human body preliminary test of type insulin preparation
The experimenter is 2 of adult healthy volunteers.
Under the empty stomach state or before formal the dining, give the experimenter with type insulin preparation (including 5 iu recombinant human insulin) with the mode that nasal cavity instils with 100 microlitre noses.
With ReliOn Ultima system for detecting blood sugar on an empty stomach, on an empty stomach one hour, administration/not administration one hour after the meal after the administration, detect the glucose level (milligram/decilitre) in the nameless terminal whole blood of experimenter.
Table ten two demonstrations give the nose of experimenter's 5 ius on an empty stomach and use the type insulin preparation, can make blood sugar level reduce about 20%.The nose that gives experimenter's 5 ius before the dining is used the type insulin preparation, can make the level of postprandial blood sugar lift-off value reduce about 70%.
Table ten two, experimenter's blood sugar level (milligram/decilitre)
Claims (10)
1. insulin preparation, it is made up of the following raw material that is dissolved in the liquid phase carrier jointly:
Cell permeable peptide 0.02-2 nanomole/microlitre liquid phase carrier;
Insulin 0.001-0.5 iu/microlitre liquid phase carrier;
1) liquid phase carrier is medical aseptic water, normal saline, phosphate buffer or artificial cerebrospinal fluid in the described preparation, and pH value is 6.5-7.5, and its compound method is following:
A) normal saline: get sodium chloride 9 grams, add water to 1000 milliliters;
B) phosphate buffer: with 610 milliliters of the sodium hydrogen phosphate aqueous solutions of 0.2 mol 390 milliliters of mixings of biphosphate sodium water solution with 0.2 mol, 1000 milliliters of the phosphate buffers of 0.2 mol, pH7.0;
C) artificial cerebrospinal fluid: get sodium chloride 6.279 grams, potassium chloride 0.216 gram, calcium chloride 0.353 gram; Magnesium chloride 0.488 gram, sodium bicarbonate 1.932 grams, glucose 0.6 gram; Sodium hydrogen phosphate 0.358 gram; Add water to 1000 milliliters, feeding contains 95% oxygen and 5% carbon dioxide gas mixture is extremely saturated, pH7.3-7.4;
2) described insulin is the insulin of biologically active, is natural insulin, human insulin, insulin precurosor, insulin analog or insulin metabolism thing;
3) described cell permeable peptide is to contain 12-22 amino acid whose peptide molecule, and wherein arginine and lysine are the important component of keeping the Premeabilisation of cells function.
2. a kind of insulin preparation according to claim 1 is characterized in that:
Cell permeable peptide 0.05-1.0 nanomole/microlitre liquid phase carrier;
Insulin 0.005-0.4 iu/microlitre liquid phase carrier.
3. a kind of insulin preparation according to claim 1 is characterized in that:
Cell permeable peptide 0.1-0.6 nanomole/microlitre liquid phase carrier;
Insulin 0.015-0.3 iu/microlitre liquid phase carrier.
4. a kind of insulin preparation according to claim 1 is characterized in that:
Cell permeable peptide 0.2-0.4 nanomole/microlitre liquid phase carrier;
Insulin 0.03-0.2 iu/microlitre liquid phase carrier.
5. a kind of insulin preparation according to claim 1 is characterized in that:
Cell permeable peptide 0.3 nanomole/microlitre liquid phase carrier;
Insulin 0.1 iu/microlitre liquid phase carrier.
6. the described a kind of insulin preparation of claim 1, it is characterized in that: the aminoacid sequence of cell permeable peptide is in the described insulin preparation:
Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys; Or
Arg?Gln?Ile?Lys?Ile?Trp?Phe?Gln?Asn?Arg?Arg?Met?Lys?Trp?Lys?Lys。
7. the described a kind of insulin preparation of claim 6, it is characterized in that: the aminoacid sequence of cell permeable peptide is adjusted in the described insulin preparation:
Ile Arg Arg Trp Lys Asn Lys Lys Arg Trp Phe Lys Ile Gln Met Gln; Or
Asn Arg Arg Met Lys Trp Lys Lys Arg Gln Ile Lys Ile Trp Phe Gln; Or
Lys Lys Asn Lys Trp Arg Arg Ile Gln Met Gln Ile Lys Phe Trp Arg; Or
Lys Lys Trp Lys Met Arg Arg Asn Gln Phe Trp Ile Lys Ile Gln Arg; Or
Lys Trp Phe Arg Ile Gln Met Gln Ile Lys Lys Trp Arg Asn Arg Arg; Or
Lys?Gln?Ile?Arg?Ile?Trp?Phe?Gln?Asn?Lys?Lys?Met?Arg?Trp?Arg?Arg。
8. the described a kind of insulin preparation of claim 7, it is characterized in that: the aminoacid sequence of cell permeable peptide is extended in the described insulin preparation:
Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys adds 1-6 Gly or nonspecific aminoacid; Or
Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys adds 1-6 Gly or nonspecific aminoacid; Or
Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys adds 1～6 Arg or nonspecific aminoacid; Or
Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys adds 1～6 Arg or nonspecific aminoacid; Or
Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys adds 1-6 nonspecific aminoacid; Or
Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys adds 1-6 nonspecific aminoacid.
9. the described a kind of insulin preparation of claim 7 is characterized in that: the aminoacid sequence of cell permeable peptide shortens 1-4 aminoacid in the described insulin preparation:
Arg Trp Phe Lys Ile Gln Gln Arg Arg Trp Lys Asn Lys Lys; Or
Arg?Gln?Ile?Lys?Ile?Trp?Gln?Arg?Arg?Met?Lys?Trp?Lys?Lys。
10. the method for preparing of the described a kind of insulin preparation of claim 1 the steps include:
1) takes by weighing the insulin powder of therapeutic dose, place in the small test tube;
2) 0.1 normal hydrochloric acid solution is added in the small test tube lentamente jog, dissolving insulin powder;
3) add liquid phase carrier and be diluted to desired concn, the jog mixing;
The solution that 4) will contain insulin with 0.1 normal sodium hydroxide transfers to pH6.5-7.5;
5) accurately take by weighing the cell permeable peptide of given dose, place in another small test tube;
6) insulin solutions is transferred to lentamente in the small test tube that contains cell permeable peptide jog mixing 30 minutes with micropipettor;
7) use the not above-mentioned insulin solutions that contains cell permeable peptide of 0.45 micron pore size membrane filtration of adsorbed proteins;
8) insulin preparation after will filtering is collected in the aseptic administrator;
9) directly be used for regulating and control the blood glucose level, or place cryopreservation subsequent use.
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