CN102580061B - Intranasal insulin preparation and preparation method thereof - Google Patents
Intranasal insulin preparation and preparation method thereof Download PDFInfo
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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 type insulin preparation; And relate to the preparation method of this nose type insulin preparation; Also relate to the application of this nose type insulin preparation simultaneously.
Background technology
The physiology of insulin and pharmacology: insulin be a kind of by islet β cell containing 51 amino acid whose proteohormones, molecular weight is 5808 dalton, participates in regulating carbohydrate metabolism, controls blood sugar level.In body insulin definitely or relative deficiency can cause diabetes, main clinical manifestation is " three-many-one-little " (polydipsia, polyuria, polyphagia and weight loss) and glucose in urine, hyperglycemia and multiple complications etc.About there are 60,000,000 diabeticss in China at present, wherein a lot of people needs throughout one's life (as type i diabetes patient) or long term frequent (as some Type II diabetics) to utilize exogenous insulin to carry out regulating and controlling blood sugar level, in case the bad clinical complication of sugared too high or too low initiation of stopping blooding.
The use situation of insulin and problem: the most frequently used administering mode of insulin treatment is subcutaneous injection.Needs of patients is injecting Semilente Insulin 2-4 time every day before the meal, and be aided with sleep before injection appropriate in protamine zine insulin.Prolonged and repeated local injection not only easily forms scar tissue and anaphylaxis, affects the absorption of medicine, and patient can be made to feel pain, produce boredom, does not even follow the doctor's advice and causes complication, can threat to life time serious.Researcher attempts the insulin preparation developing 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 proposed at present comprise through lung, transdermal, oral, give therapeutic insulin preparation through approach such as intestinal, Sublingual and per nasal.Pulmonary's inhalation first elects the Exubera pulmonary Foradil Aerolizer formoterol fumarate of Pfizer Inc..But be namely withdrawn soon after its listing, mainly consider the latent lesion of life-time service to pulmonary function, and its relative bioavailability be only hypodermic 10-20%.Although patient more easily accepts oral enteric coated preparation, can the insulin preparation of tolerance protein hydrolytic enzyme completely not yet develop, and insulin self is difficult to through gastrointestinal mucosal epithelium.Some other substituting dosage regimen also has respective shortcoming.Such as: the bioavailability of the unhygienic inconvenience of rectally, transdermal administration is extremely low, in sublingual formulation containing potential bad foreign compound etc.
The feasibility of Giving Insulin by Pernasal Method and key point: known nasal mucosal surface is long-pending reaches 150 square centimeters, and a large amount of trickle fine hair can increase effective absorbing area.If medicine can permeate nasal membrane epithelial cell, then promptly by blood capillary abundant in nasal membrane layer and lymphatic absorption, systemic blood circulation can be entered quickly than injected s. c.Because insulin infiltration nasal membrane is epithelial limited in one's ability, therefore the relative bioavailability of regular insulin nose administration extremely low (being only hypodermic 1-2%).Therefore, the key point that the permeability of nasal membrane epithelial cell to insulin is research and development insulin intranasal formulation is increased.
The patent retrieval of various insulation administration preparation through nose: through " patent retrieval of China national Department of Intellectual Property ", finds 9 patent applications relevant to nose type insulin preparation.Apply for artificial Chinese Shanghai medical industry academy (200510028990.0; 200510028991.5; 200710173622.4; 200810035771.9), Amada Co., Ltd. DDS institute (01801146.2) and DDS Research Ltd. (02804546.7) and Nastech Pharm Co. of the U.S. (200680047851.5) and Cpex Pharmaceuticals Inc. (200780028627.6; 200980121227.9).They use porous spherical calcium carbonate, crystalline cellulose polymer, oils and fats, emulsifying agent, solubilizing agent, the large chemicals such as lopps penetrating agent and surfactant to increase the epithelial ability of insulin infiltration nasal membrane respectively.Because these foreign compounds and metabolite are scarcely present in normal body, they to the potential stimulus of nasal mucosa and after entering in body issuable chronic toxicity quite merit attention.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 the relative bioavailability obtained is very low, and result causes the safety range between dose therapeutically effective and minimum toxic dose very little.Such as, the relative bioavailability of porous spherical calcium carbonate/insulin preparation is only 5.69%.These inventions not yet can meet the requirement of diabetes clinical treatment.
Summary of the invention
The object of the invention is to be to provide a kind of nose type insulin preparation.Said preparation primarily of cell permeable peptide, insulin and liquid phase carrier three kinds of raw materials composition, for a kind of in nasal administration mode by the compound formulation in treatment insulin input patient body.Its advantage is that the insulin in preparation is easy to through nasal membrane barrier, Relative drug absorbance (blood insulin concentration) about 80%, bioavailability (hypoglycemic effect) reaches more than 60%, far above current other non-injection type insulin preparation ground.
Another object of the present invention is the preparation method being to provide a kind of nose 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.
Another object of the present invention is to provide the application of a kind of nose type insulin preparation in regulation and control blood glucose levels.The said preparation of therapeutic dose is executed the nasal mucosal surface being distributed in patient by available intranasal spray administration device, easy to use, painless, non-stimulated to nasal membrane, and capable of being fast degraded in body is aminoacid, and safety is high.
In order to realize above-mentioned object, 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 in succession as a class transmission system, they have the ability through mammalian cell membrane, and be not subject to the constraint of energy and receptor, become the new tool promoting or carry multi-medicament molecule (comprising chemical small molecule, biomacromolecule and nano-particle etc. by loading) transmembrane transport.Cell permeable peptide by several peptide molecule formed to twenties aminoacid, containing several basic amino acid and hydrophilic (polarity)/hydrophobic (nonpolar) amphiprotic group.They with non-covalent bond form and can form stable complex by loading, help the latter to enter in body in the mode of effective, harmless through tissue epithelial cell, play biological effect.In addition, the cell permeable peptide entered in body is easily degraded by proteases very soon as common amino acid molecular, and does not produce harmful metabolite.The progress of cell permeable peptide greatly accelerates the development and application of the biomacromolecule class medicines such as plasmid DNA, oligonucleotide, siRNA, peptide nucleic acid(PNA), peptides and proteins and elaioplast nanometer particle.31 kinds of cell permeable peptides and analog and various insulin react by applicant at different conditions, obtain various compositions.Then pass through a large amount of zoopery, test drug absorption rate and the bioavailability of each compositions nose administration, screening and optimizing goes out a class nose type insulin preparation.
The technical problem to be solved in the present invention is: provide a kind of and have high bioavailability, nose type insulin preparation without mucous membrane irritation and chronic toxicity.
For a nose type insulin preparation for treatment, it is made up of jointly the following raw material be dissolved in liquid phase carrier:
Cell permeable peptide 0.02-2 nanomole/microlitre liquid phase carrier;
Insulin 0.001-0.5 iu/microlitre liquid phase carrier;
This insulin preparation be a kind of with nasal administration mode by treatment insulin input patient body in compound formulation, main component is liquid phase carrier, insulin and cell permeable peptide.Specifically describe as follows:
1, in described preparation, liquid phase carrier is (but being not limited to) medical aseptic water, normal saline, phosphate buffer or artificial cerebrospinal fluid, and pH value is 6.5-7.5, and its compound method is as follows:
1) normal saline: get 9 grams, sodium chloride, add water to 1000 milliliters.
2) phosphate buffer: the sodium hydrogen phosphate aqueous solution 610 milliliters of 0.2 mol/L and the biphosphate sodium water solution 390 milliliters of 0.2 mol/L are mixed, the phosphate buffer 1000 milliliters of 0.2 mol/L, pH7.0.
3) artificial cerebrospinal fluid: get 6.279 grams, sodium chloride, 0.216 gram, potassium chloride, 0.353 gram, calcium chloride, 0.488 gram, magnesium chloride, sodium bicarbonate 1.932 grams, glucose 0.6 gram, sodium hydrogen phosphate 0.358 gram, add water to 1000 milliliters, pass into containing 95% oxygen and 5% carbon dioxide gas mixture to saturated, pH7.3-7.4.
2, in described preparation, insulin such as, for having the insulin of biological activity (energy regulating blood glucose levels), (but being not limited to) natural insulin, human insulin, insulin precurosor, insulin analog or insulin metabolism thing.
3, in described preparation, cell permeable peptide is prepared by CS936X Peptide synthesizer, and it is that a class contains 12-22 amino acid whose peptide molecule, and wherein arginine and lysine are the important component maintaining Premeabilisation of cells function.
The first grown form of this cell permeable peptide is:
Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys
When not changing arginine and lysine position, the cell permeable peptide of above-mentioned grown form can be adjusted to the cell permeable peptide of the second grown form:
Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys
The Premeabilisation of cells peptide sequence of above two kinds of grown forms can adjust as follows (but being not limited to lower example) further:
1) by after aminoterminal first half section and the location swap of c-terminus second half section, 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 converse sequencing, above-mentioned grown form 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 be formed containing 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, 1 to 6 arginine or nonspecific aminoacid is added:
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, 1 to 6 unspecific aminoacid is added:
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, 1-4 aminoacid is removed:
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) after 30% aminoacid in above-mentioned various cell permeable peptide is adjusted, the new cell permeable peptide formed.
For a nose type insulin preparation for treatment, it is made up of (better scope) jointly the following raw material be dissolved in 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
For a nose type insulin preparation for treatment, it is made up of (preferable range) jointly the following raw material be dissolved in 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
For a nose type insulin preparation for treatment, it is made up of (best scope) jointly the following raw material be dissolved in 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
For a nose type insulin preparation for treatment, it is made up of (optimum) jointly the following raw material be dissolved in liquid phase carrier:
Cell permeable peptide 0.3 nanomole/microlitre liquid phase carrier
Insulin 0.1 iu/microlitre liquid phase carrier
For a preparation method for the nose type insulin preparation for the treatment of, the steps include:
1, under room temperature (22 ± 2 DEG C, identical below), accurately take the insulin powder of 200 ius, be placed in the small test tube of 5 milliliters;
2, the hydrochloric acid solution of 200 microlitre 0.1 equivalents is added in above-mentioned small test tube lentamente, jog, dissolve insulin powder;
3, then add 1.6 milliliters of liquid phase carriers to dilute, jog mixes;
4, with the sodium hydroxide of 200 microlitre 0.1 equivalents, the above-mentioned solution containing insulin is adjusted to pH6.8-7.2;
5, accurately take the cell permeable peptide of 600 nanomoles in addition, be placed in another small test tube of 5 milliliters;
6, be transferred in the small test tube containing cell permeable peptide with micropipettor lentamente by above-mentioned insulin solutions, jog mixes 30 minutes;
7, the above-mentioned insulin solutions containing cell permeable peptide of the membrane filtration of not 0.45 micron pore size of adsorbed proteins is used;
8, the nose type insulin preparation after filtration is collected in the sterile administration device of a sealing;
9, be placed in low temperature (4 ± 1 DEG C) to save backup; Or be placed in freezing (-20 DEG C or-80 DEG C) and preserve for a long time.
For an application process for the nose type insulin preparation for the treatment of, the steps include:
1, nose type insulin preparation is placed in Intranasal sprays formula or dripping type administrator;
2, the insulin preparation of therapeutic dose is executed the nasal mucosal surface being distributed in patient;
3, the volumes of formulation 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 before and after cloth, periodic monitor blood sugar level.
Technical characteristic: containing the cell permeable peptide be made up of specific amino acids sequence in nose type insulin preparation of the present invention, it can help insulin to penetrate nasal membrane epithelial cell efficiently and enter systemic blood circulation, regulating and controlling blood sugar level.
As a kind of brand-new insulin intranasal absorption enhancer, cell permeable peptide in nose type insulin preparation of the present invention is compared with other cell permeation enhancers, there is diverse chemical constitution and the mechanism of action, the insulin in preparation can be made to produce much higher drug absorption rate and bioavailability.
This cell permeable peptide and nose when through nasal membrane epithelial cell, and after entering systemic blood circulation, have following technical characteristic with type insulin.
1, cell permeable peptide structure is relevant to the ability of transhipment insulin: the cell permeable peptide of the present invention's two kinds of grown forms used is after suitably adjusting (comprise and resequence, lengthen or shorten), and the Relative drug absorbance of its insulin preparation and relative bioavailability have to a certain degree the change of (10-30%).
2, Premeabilisation of cells peptide concentration is relevant to insulin transmembrane transport efficiency: when the concentration range of cell permeable peptide in nose type insulin preparation is 0.02-2 nanomole/microlitre, in blood, insulin level and Premeabilisation of cells peptide concentration are proportionate.Consider various composition proportion in Cost and benefit relation and preparation, the best scope of Premeabilisation of cells peptide concentration is 0.2-0.4 nanomole/microlitre.
3, cell permeable peptide can the various insulin of rapid transport: combined from different recombinant human insulin, insulin analog or natural Iletin II (Lilly) respectively by cell permeable peptide, carry out zoopery.Result shows that this several different components nose administration is after ten minutes, and in blood, insulin level obviously raises, and within 30 minutes, starts to produce significant hypoglycemic effect.
4, nose type insulin preparation can produce higher insulin Relative drug absorbance: compared with or lumbar injection subcutaneous with the laboratory animal of same dose, Premeabilisation of cells peptide concentration is the Relative drug absorbance that the nose type insulin preparation of 0.2-0.4 nanomole/microlitre can produce up to 80%.
5, nose type insulin preparation can produce higher insulin relative bioavailability: compared with or lumbar injection subcutaneous with the laboratory animal of same dose, Premeabilisation of cells peptide concentration is the relative bioavailability that the nose type insulin preparation of 0.2-0.4 nanomole/microlitre can produce up to 60%.
6, cell permeable peptide can be degraded and inactivation rapidly in vivo: after intravenously administrable, and 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, and cell permeable peptide can be dissolved in separately the isotonic normal saline or phosphate buffer that pH value is 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 can be preserved at low temperatures for a long time: the mixing of cell permeable peptide and insulin apply liquid and deposit two months under low temperature (4 DEG C), its biologic activity is without obvious change.
9, nose type insulin preparation does not produce zest to nasal membrane epithelial cell: nose type insulin preparation does not affect integrity and the motion of in vitro mucosal tissue cilium; Experiments in vivo finds that nose administration is non-stimulated to nasal membrane, does not change the epithelial Histological Study of nasal membrane.
10, cell permeable peptide does not produce acute and chronic injury to 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 the repeated multiple times use of nose type insulin preparation is stable and regular: to same animal subject once a day, continuous one week use nose type insulin, hypoglycemic effect stability, and reproducible.
12, the above-mentioned feature of cell permeable peptide and nose type insulin confirm by least two kinds of laboratory animals: histiocytic isolated experiment, and Mouse and rat experiments in vivo all confirms the above-mentioned feature of cell permeable peptide and nose type insulin preparation.
13, the Preliminary experiment results display of human body: the nose type insulin preparation giving healthy volunteer 5 iu on an empty stomach, can make blood sugar level reduce about 20%.Give the nose type insulin preparation of healthy volunteer 5 iu before dining, level of postprandial blood sugar lift-off value can be made to reduce about 70%.
According to above-mentioned technical characteristic and following concrete result of implementation, known creativeness of the present invention is:
1, first by the cell permeable peptide of suitable construction for the preparation of non-injection type insulin preparation;
2, Premeabilisation of cells peptide concentration, relation between insulin dosage and biological effect is determined;
3, the preparation method of nose type insulin preparation is established;
4, Relative drug absorbance and the relative bioavailability of insulin in preparation has been inquired into;
5, have detected the pharmacokinetics of cell permeable peptide in preparation, and to local and the acute and chronic toxic action of whole body;
6, the nose type insulin preparation with clinical practice potentiality is finally obtained.
The alternative injected s. c of nose type insulin preparation of the present invention, through Intranasal sprays formula or dripping type administration, is used for the treatment of diabetics.Each use amount is 100 microlitres (50 to 200 microlitres), interior insulin-containing 10 (5-20) iu.Concrete use is decided according to factors such as conditions of patients by doctor.
Accompanying drawing explanation
Fig. 1 is a kind of chromatography image containing 16 amino acid whose cell permeable peptides, reflects the purity of product.
Fig. 2 is a kind of mass spectral analysis image containing 16 amino acid whose cell permeable peptides, reflects the structure of product.
Fig. 3 is that a kind of cell permeable peptide promotes the experimental result that insulin intranasal absorbs, and shows Premeabilisation of cells peptide content and can promote that insulin absorbs through experimental rat nasal membrane within the specific limits, insulin concentration in the blood that increases sharply.
Fig. 4 is that a kind of cell permeable peptide helps insulin to fall hypoglycemic experimental result, shows cell permeable peptide and can promote that different insulins (as insulin human and Iletin II (Lilly)) absorbs through experiment mice nasal membrane, reduce glucose level in blood.
Fig. 5 is a kind of experimental result of nose type insulin preparation absorption efficiency, and show the drug absorption speed slightly fast (administration after 20 minute within) of nose type insulin than subcutaneous insulin injections, Relative drug absorbance is 80%.
Fig. 6 is a kind of experimental result of nose type insulin preparation blood sugar lowering effect, and show nose type insulin and can produce the blood sugar decreasing effect similar to subcutaneous insulin injections and persistent period, relative bioavailability is 60-70%.
Fig. 7 is the experimental result of a kind of cell permeable peptide metabolism in vivo: the half-life that upper drawing shows cell permeable peptide in blood is very short, and in brain, the content of cell permeable peptide is extremely low; Bottom panel show cell permeable peptide mainly accumulation and the degraded in liver, kidney entered in body.
Fig. 8 is that two kinds of cell permeable peptides help insulin to fall hypoglycemic contrast and experiment, the cell permeable peptide (16 aminoacid) showing the first basic kenel more can promote that insulin intranasal absorbs than the cell permeable peptide (18 aminoacid) after another kind of adjustment, reduce blood sugar level, and both difference is about 20%.
Fig. 9 is a kind of experimental result of nose type insulin preparation storage condition, shows after nose type insulin preparation deposits 1-60 days under low temperature (4 DEG C) and still maintains original blood sugar lowering ability (in blood glucose level) and time effect (after administration 30-120 minute).
Figure 10 is the sensitivity experiments result of a kind of animal subject to multiple dosing, shows experiment mice once a day, within continuous five days, accepts nose type insulin preparation, remains the sensitivity to insulin blood sugar lowering effect.The component (from first day to the 5th day) of every day shows similar blood sugar lowering ability (blood glucose level) and time effect (after administration 30-240 minute).
Detailed description of the invention
Further illustrate the present invention by embodiment below, but the present invention is not limited.
Embodiment 1:
With the nose of insulin human preparation experiment type insulin preparation.
The nose type insulin preparation of table one, a treated animal experiment, it is made up of the following ingredients and concentration being dissolved in phosphate buffer.
The nose type insulin preparation of table two, another treated animal experiment, it is made up of the following ingredients and concentration being dissolved in phosphate buffer.
Table one and certain cell permeable peptide described in table two are 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
Table one and certain cell permeable peptide described in table two also can be containing 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 preparation method of the above insulin preparation, the steps include:
1, accurately take the insulin of specified quantitative shown in table, be placed in a small test tube, slowly dissolve with the hydrochloric acid solution of 0.1 equivalent;
2, add liquid phase carrier to carry out being diluted to certain concentration shown in table;
3, with the sodium hydroxide of 0.1 equivalent, the above-mentioned solution containing insulin is adjusted to pH6.8-7.2 again;
4, accurately take the cell permeable peptide of specified quantitative shown in table in addition, be placed in another small test tube;
5, be transferred in the small test tube containing cell permeable peptide with micropipettor lentamente by above-mentioned insulin solutions, jog mixes 30 minutes;
6, the above-mentioned insulin solutions containing cell permeable peptide of the membrane filtration of not 0.45 micron pore size of adsorbed proteins is used;
7, the nose type insulin preparation after filtration is collected in the sterile administration device of a sealing;
8, be placed in cryopreservation, or directly use.
Embodiment 2:
With the nose of Iletin II (Lilly) preparation experiment type insulin preparation
The nose type insulin preparation of table three, a treated animal experiment, it is made up of the following ingredients and concentration being dissolved in medical aseptic water.
The nose type insulin preparation of table four, another treated animal experiment, it is made up of the following ingredients and concentration being dissolved in medical aseptic water.
Table three and certain cell permeable peptide described in table four are 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
Table three and certain cell permeable peptide described in table four also can be containing 14 amino acid whose cell permeable peptides:
Arg Trp Phe Lys Ile Gln Gln Arg Arg Trp Lys Asn Lys Lys
The preparation method of the above insulin preparation, its step is with embodiment 1.
Embodiment 3:
With the nose of insulin analog preparation experiment type insulin preparation
The nose type insulin preparation of table five, a treated animal experiment, it is made up of the following ingredients and concentration being dissolved in phosphate buffer.
The nose type insulin preparation of table six, another treated animal experiment, it is made up of the following ingredients and concentration being dissolved in medical aseptic water.
Table five and the insulin analog described in table six are insulin aspart.
Table five and certain cell permeable peptide described in table six are 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
Table five and certain cell permeable peptide described in table six also can be containing 14 amino acid whose cell permeable peptides:
Arg Gln Ile Lys Ile Trp GlnArgArg Met Lys Trp Lys Lys
The preparation method of the above insulin preparation, its step is with embodiment 1.
Embodiment 4:
The nose type insulin preparation of preparation volunteer
The nose type insulin preparation of table seven, lineup's body volunteer, it is made up of the following ingredients and concentration being dissolved in medical water or phosphate buffer.
Certain cell permeable peptide described in 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 preparation method of the above insulin preparation, its step is with embodiment 1.
Embodiment 5:
The structure of cell permeable peptide and quality analysis
Solid phase method (CS936X Peptide synthesizer) is adopted to prepare cell permeable peptide.Carry out purification product by high performance liquid chromatography, characterize purified product with mass spectrography.Now introduce structure and quality analysis (figure mono-and figure bis-) that an example contains 16 amino acid whose cell permeable peptides.
Sequence: Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys
Chemical modification: aminoterminal and c-terminus are all without modification
Molecular formula: C
104h
168n
34o
20s
1
Molecular weight: 2246.78
Outward appearance: white powder
Purity (chromatography): be greater than 95%
Structure (mass spectral analysis): stable
Store: dry cold preservation
The measurement result of cell permeable peptide purity is in table eight.
Table eight, efficient liquid phase chromatographic analysis data
Embodiment 6:
The assay method of insulin in blood
Promote that at cell permeable peptide in the experiment of insulin transmembrane transport, in blood, insulin level reflects the drug absorption ability of nose type insulin preparation.
Hypersensitive insulin human radioimmunoassay kit (LINCO RES) is adopted to measure insulin concentration in blood plasma.This radioimmunology analysis system comprises four elements: specificity anti-insulin antibody; The insulin antigen of labelled with radioisotope; By the method that conjunction type labelled antigen separates with sequestered labelled antigen; And radiosiotope measuring instrument.Concrete determination step is:
1, at particular point in time blood-sample withdrawal, be separated 50 microlitre blood plasma, cryopreservation is for subsequent use;
2, by a certain amount of
125iodine labeling insulin antigen and the common incubation of specificity insulin antibody, make labelled antigen and the antibodies of about 50%;
3, in this incubation system, unmarked insulin antigen (test plasma) is added, competitively with antibodies;
4, along with unlabelled antigen in system increases, the binding capacity of labelled antigen and antibody reduces;
5, conjunction type labelled antigen and sequestered labelled antigen are separated, measure respective radioactivity;
6, insulin concentration in test plasma (micro-iu/milliliter blood plasma) is calculated according to standard curve (making of unmarked insulin standards).
Embodiment 7:
The assay method of glucose in blood
In the blood sugar lowering experiment of insulin, in blood, glucose level reflects the biological activity of nose type insulin preparation.
Adopt the glucose level (milligram/decilitre) in ReliOn Ultima system for detecting blood sugar mensuration whole blood.The advantage of this system is: convenient (animal Tail blood samples), quick (5 seconds went out result), sensitive (being less than 1 microliters of sample), stable (measurement result has good repeatability).
Embodiment 8:
The administration of laboratory animal and sampling method
Experimental animal feeding used 22 ± 2 DEG C, 12 little time/Animal House of dark circulation, drinking-water of freely ingesting.
1, experiment mice (20 ± 4 grams, male and female half and half) is freely drunk water, fasting is after one day, lumbar injection sodium phenobarbital anesthetis (60 mgs/kg of body weight).
Under anatomic microscope, with micro sample adding appliance to every side nasal cavity slowly drip 4 microlitres by test preparation.Matched group experiment mice accepts dorsal sc or lumbar injection is subject to test preparation.At particular point in time, docking measuring blood sugar of blood extracting.
In addition, in order to the metabolism distribution of observation of cell infiltration peptide, by 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 grams, male and female half and half) is freely drunk water, fasting is after one day, lumbar injection sodium phenobarbital anesthetis (50 mgs/kg of body weight).
Supine, directly inserts nasal cavity by micro sample adding appliance, every side instil 20 microlitres by test preparation.Matched group experimental rat accepts dorsal sc or lumbar injection is subject to test preparation.At particular point in time, measure blood glucose with the syringe containing anticoagulant through taking blood from jugular vein; After centrifuging separated plasma, measure blood insulin.
Embodiment 9:
The relation of Premeabilisation of cells peptide content and Insulin transport efficiency
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 blood after 20 minutes, promote to understand cell permeable peptide the optimum efficiency that insulin intranasal absorbs.Figure tri-shows certain and contains 16 amino acid whose cell permeable peptides (Arg Trp Phe Lys Ile Gln Met Gln IleArg Arg Trp Lys Asn Lys Lys) and, when 0.1 nanomole/microlitre, can promote that insulin human (0.02 iu/microlitre) enters blood through nasal membrane epithelium; Along with Premeabilisation of cells peptide content in preparation increases, in blood, insulin human concentration also raises; When in preparation, Premeabilisation of cells peptide content is increased to 2 nanomoles/microlitre, in blood, insulin human concentration is close to peak value.This result shows, in preparation, Premeabilisation of cells peptide content has obvious dose-effect relationship with insulin intranasal transport efficacy within the specific limits.
The result display of control experiment, per nasal gives recombinant human insulin separately, does not detect insulin human in blood.
Embodiment 10:
Cell permeable peptide is to the transhipment effect of different insulin
The cell permeable peptide (0.1 nanomole/microlitre) of certain content and the insulin (0.0025 iu/microlitre) of separate sources are formed multiple formulations, and per nasal gives experiment mice.Respectively before administration and after administration when 30 and 60 minutes, measure glucose level in blood, to understand the facilitation that cell permeable peptide absorbs different insulin intranasal.
Figure tetra-show certain contain 18 amino acid whose cell permeable peptides (Arg Gln Ile Lys Ile Trp Phe GlnAsn Arg Arg Met Lys Trp Lys Lys Gly Gly) to recombinant human insulin and natural Iletin II (Lilly) via intranasal application mucous epithelium transhipment all have obvious facilitation.This result shows, under the help of cell permeable peptide, after the insulin intranasal absorption of these two kinds of separate sources, creates similar hypoglycemic effect.
The result display of control experiment, independent per nasal gives the one of cell permeable peptide, insulin or liquid phase carrier, does not all produce obvious hypoglycemic activity.
Embodiment 11:
The nose Relative drug absorbance (in blood insulin concentration) of type insulin
One group of experimental rat is given by nose type insulin preparation (including cell permeable peptide 0.8 nanomole/microlitre and recombinant human insulin's 0.02 iu/microlitre) per nasal, separately give control rats by the recombinant human insulin of isodose through subcutaneous, before administration with administration after 5,15,30,45,60,90,120,150 minutes time measure insulin human concentration in blood, calculate blood Chinese medicine Cmax, reach Cmax required time and Relative drug absorbance.
The blood Chinese medicine Cmax that figure five shows recombinant human insulin in certain nose type insulin preparation (inside containing 18 amino acid whose cell permeable peptides: Arg Gln IleLys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys Gly Gly) is 188.6 ± 125.2 micro-iu/milliliter blood plasma, and reaching Cmax required time is 18.3 ± 4.5 minutes.
Compared with subcutaneous injection, the Relative drug absorbance of Giving Insulin by Pernasal Method is 83.5 ± 21.2%.
This result shows, the drug absorption rate of nose type insulin preparation of the present invention is far above the insulation administration preparation through nose of other types.
Embodiment 12:
The nose relative bioavailability (in blood glucose level) of type insulin
One group of experiment mice is given by nose type insulin preparation (including cell permeable peptide 0.2 nanomole/microlitre and recombinant human insulin's 0.005 iu/microlitre) per nasal, separately give matched group experiment mice by the recombinant human insulin of isodose through subcutaneous, before administration with administration after 30,60,90,120,150,180,240,300,360,720 minutes time measure glucose level in blood, calculate relative bioavailability.
Figure six shows certain nose type insulin preparation (inside containing 16 amino acid whose cell permeable peptides: Arg TrpPhe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys) can make blood sugar level be down to 70% in administration after 30 minutes, be down to 50% after 90 minutes; Level before blood sugar level returns to administration gradually when 180-240 minute.
Compared with subcutaneous injection, the relative bioavailability of Giving Insulin by Pernasal Method is 66.8 ± 15.4%.
This result shows, the bioavailability of nose type insulin preparation of the present invention is far above the insulation administration preparation through nose of other types.
Embodiment 13:
Cell permeable peptide degraded in vivo and distribution (in body method)
Measure blood and each content of cell permeable peptide in internal organs of organizing can understand its metabolic rate in vivo and distribution pattern.
By 100 microlitres
111the Premeabilisation of cells peptide solution of indium (1 milli Becquerel) labelling is in tail vein injection experiments Mice Body.In injection after 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 dry, weigh.Measure radioactive intensity with gamma counter, converse radioactivity cell permeable peptide in every gram of tissue sample and account for the percentage contents of gross activity cell permeable peptide.
Figure seven shows certain and contains the Time dynamic distribution of 16 amino acid whose radioactivity cell permeable peptides (Arg Trp Phe Lys Ile GlnMet Gln Ile Arg Arg Trp Lys Asn Lys Lys) in blood, brain, liver and kidney.
Premeabilisation of cells peptide concentration in blood and brain reaches peak in 10 minutes after injection, declines rapidly subsequently; Premeabilisation of cells peptide concentration in liver reaches peak in 30 minutes after injection, declines gradually subsequently; Premeabilisation of cells peptide concentration in kidney reaches peak in 50 minutes after injection, slowly declines subsequently.
In liver and kidney, the peak value of Premeabilisation of cells peptide concentration is close, and apparently higher than the maximum concentration of cell permeable peptide in blood; In brain, the concentration of cell permeable peptide is minimum.
These results show:
1, the half checkout time about 30 minutes of cell permeable peptide in blood;
2, As time goes on, cells in vivo infiltration peptide is gradually to liver and kidney transfer;
3, liver and kidney are main accumulation and the metabolic organ of cell permeable peptide;
4, may due to the effect of blood brain barrier, cell permeable peptide is difficult to enter cerebral tissue through blood.
Embodiment 14:
Cell permeable peptide Stability Determination in blood (vitro method)
Will
111indium (the 2 milli Becquerel) cell permeable peptide of labelling and the common incubation of human serum (37 DEG C) 0,5,10,20,30,45,60,90,120,180 of 400 microlitres, 240 minutes.After mixing with the acetonitrile of 2 times of volumes, put 4 DEG C one hour, to precipitate serum albumin.The supernatant of cell permeable peptide is contained with radiation counting-high-efficient liquid phase chromatogram technique analysis.Calculate radioactivity cell permeable peptide by the speed of serum albumin hydrolase.
Result shows, certain contains 16 amino acid whose cell permeable peptides (Arg Trp Phe Lys Ile Gln MetGln Ile Arg Arg Trp Lys Asn Lys Lys) the half degradation time 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 ArgMet Lys Trp Lys Lys Gly Gly) in test tube 58.2 ± 7.1 minutes.
Embodiment 15:
The nose preservation condition of type insulin
Two parts of identical nose type insulin preparations (containing 16 amino acid whose cell permeable peptides: Arg TrpPhe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys) are placed 0 at 4 DEG C or 22 DEG C of lower seals respectively, 2,10,20,30,60, after 90 days, per nasal gives experiment mice, and the blood sugar level after mensuration administration when 90 minutes, to understand its preservation condition and time.Result shows, nose type insulin preparation should not at room temperature be preserved (see table nine) for a long time.
The insulin preparation blood sugar lowering ability (relative to the percentage ratio of Fresh preparations, %) of preserving under table nine, different condition
Figure eight show two kinds of nose type insulin preparations (be respectively: containing 16 amino acid whose cell permeable peptides:
Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys; Containing 18 amino acid whose cell permeable peptides: Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys LysGly Gly) after 4 DEG C of lower seals are placed one month, all there is good blood sugar lowering ability.
Figure nine further display after 4 DEG C of lower seals are placed two months, still can keep good blood sugar lowering ability containing 18 amino acid whose cell permeable peptides (Arg Gln Ile Lys Ile Trp PheGln Asn Arg Arg Met Lys Trp Lys Lys Gly Gly).
Embodiment 16:
Nose type insulin preparation damages the stimulation of nasal membrane
Acid phosphatase is present in normal cell.When damaged membrane, acid phosphatase will spill in cell.Therefore, detect extracellular acid phosphatase content, the integrity of cell membrane can be understood.
Whether stimulation and damage are produced to nasal membrane to observe nose type insulin preparation, cell permeable peptide, insulin, nose type insulin preparation, phosphate buffer (contrast) are instiled at the nasal mucosal surface of experimental rat respectively, with phosphate buffer (37 DEG C) the lavation nasal cavity lentamente that 5-15 milliliter is heated after 20 minutes.Then the content of acid phosphatase in biochemical reaction development process (test kit of Promega company) and spectrophotometric determination nasal lavage fluid is used.Result shows, cell permeable peptide, insulin and nose type insulin preparation all do not damage nasal membrane epithelial cell (see table ten).
The content (iu/liter) of acid phosphatase in table ten, nasal lavage fluid
In table containing 16 amino acid whose cell permeable peptides be: Arg Trp Phe Lys Ile Gln Met Gln Ile ArgArg Trp LysAsn Lys Lys.
In table containing 18 amino acid whose cell permeable peptides be: Arg Gln Ile Lys Ile Trp Phe Gln Asn ArgArg Met Lys Trp Lys Lys Gly Gly.
Embodiment 17:
Cell permeable peptide is to the toxic action of histoorgan
By cell permeable peptide (0.1 nanomole/microlitre) or the every twice-daily of phosphate buffer, within continuous two weeks, drip 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 do conventional histopathological inspection.Result shows, cell permeable peptide does not cause damage (see table ten one) to the organizational structure of main organs.
The check result of table ten one, several main organs
(-unchanged; May there be slight change in-/+; + slightly change; ++ moderate changes; +++ severe changes)
In table containing 16 amino acid whose cell permeable peptides be: Arg Trp Phe Lys Ile Gln Met Gln Ile ArgArg Trp LysAsn Lys Lys.
In table containing 18 amino acid whose cell permeable peptides be: Arg Gln Ile Lys Ile Trp Phe Gln Asn ArgArg Met Lys Trp Lys Lys Gly Gly.
Embodiment 18:
The continuous result of use of nose type insulin preparation
By nose type insulin preparation (0.005 iu/microlitre), a couple of days gives same group of experiment mice (h fast at night 12) continuously, measure the blood sugar content after administration every day, the blood sugar decreasing effect after observation nose type insulin preparation uses continuously.In other words, body is observed to the lasting sensitivity of nose type insulin preparation.
Figure ten shows containing the continuous use of 16 amino acid whose cell permeable peptides (Arg Trp Phe Lys Ile Gln Met Gln IleArg Arg Trp Lys Asn Lys Lys) five days, and the hypoglycemic effect (curve) between every day is similar.This result shows, nose type insulin preparation can give same individual test subjects in continuous many days, produces stable hypoglycemic effect.
Embodiment 19:
The nose human body preliminary test of type insulin preparation
Experimenter is adult healthy volunteers 2.
Under fasted conditions or before formal dining, give experimenter by the mode that 100 microlitre nose type insulin preparations (including 5 iu recombinant human insulin) instil with nasal cavity.
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 administration, detect the glucose level (milligram/decilitre) in the nameless end whole blood of experimenter.
Table ten two display gives the nose type insulin preparation of experimenter 5 iu on an empty stomach, and blood sugar level can be made to reduce about 20%.Give the nose type insulin preparation of experimenter 5 iu before dining, level of postprandial blood sugar lift-off value can be made to reduce about 70%.
Table ten two, experimenter's blood sugar level (milligram/decilitre)
Claims (1)
1. the cell permeable peptide in insulin preparation, is characterized in that, the aminoacid sequence of described cell permeable peptide is: Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys Gly Gly.
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