CN102199206B - Insulin analogue having quick response and stability under acidic condition and preparation thereof - Google Patents
Insulin analogue having quick response and stability under acidic condition and preparation thereof Download PDFInfo
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Abstract
The invention relates to an isulin analogue having quick response and stability under acidic condition, and a medicinal composition and a medicinal preparation thereof. Asparagine (Asn) at the A 21 position of the chain A of the isulin analogue is mutated into glycine (Gly), the isulin analogue can be mixed with a long-acting isulin analogue such as insulin glargine to form a premixed preparation with two functions of quick-acting sugar reduction and stable long-acting sugar reduction, and the problems that the conventional natural insulin (of pigs, cows and human) and quick-acting isulin analogues are unstable in the acidic environment, and the conventional isulin premixed preparation is not clarified and needed to be introduced with a foreign protein (such as protamine) serving as a slow release preparation so as to easily cause immune reaction, and is needed to be injected twice each day, namely in the morning and evening are solved, so that the safety of the preparation is higher.
Description
Technical field
The present invention relates to human insulin analogue, specifically, relate to a kind of can quick acting and under acidic conditions stable insulin analog, and the pharmaceutical composition and the pharmaceutical preparation that comprise this insulin analog.
Background technology
Diabetes are a kind of common incretion metabolism diseases.In recent years, the morbidity of whole world diabetes is all at rapid growth.And in China, along with the change of people's lives mode and the acceleration of aging process, the morbidity of diabetes is fast rise trend, break through 9,000 ten thousand people at diabetes mellitus in China patient sum in 2010, become the important Chronic Non-Communicable Diseases of another serious harm people ' s health after cardiovascular and cerebrovascular diseases, tumour.The acute and chronic complication of diabetes, especially the chronic disease complication is involved a plurality of organs, disables, lethality rate is high, has a strong impact on patient's physical and mental health, and brings white elephant for individual, family and society.
It is the important means for the treatment of diabetes and blood sugar being well controlled that insulinize is taken as always.Normal human's physiology insulin secretion was stimulated mutually and steadily phase composite of basis by when meal, be that people's rear periphery blood sugar of having meal increases, stimulate and store in the pancreas and newborn Regular Insulin release, and at ordinary times in order to keep the euglycemia level of body, a small amount of basal insulin of pancreatic secretion.Therefore, when making diabetic subject's meal and basic blood sugar all be well controlled, needs of patients is injected Semilente Insulin (every day three times) before the meal in every day, and in morning every day and before sleeping in each injection once or protamine zine insulin.Like this, the diabetic subject must bear 4-5 time frequency injection every day.For this reason, people have developed the solvable Regular Insulin that quick-acting effects have namely been arranged and have had again the premix insulin preparation of the crystalline insulin of retarding action (Regular Insulin-protamine crystallization) (referring to " hundred bright pharmacy science " (Remington ' s Pharmaceutical Sciences) in same preparation, the 17th edition, the 973-977 page or leaf, 1985).But still there is following problem in these premix insulin preparations: 1. contain foreign protein-protamine in the preparation, easily produce immune response; 2. drug effect time length less than 24 hours after injection, the patient still needs to inject every day twice; 3. still there is peak value in drug effect, and every day, twice drug effect peak stack easily caused ante prandium and the front hypoglycemia of sleeping; 4. existing premix insulin preparation is suspension, if mix inhomogeneously before the patient infusion, easily causes the lasting difference of solvable phase and crystallization phases ratio in the liquid, affects glycemic control.
At present, also has a kind of Recent Development of Long-acting Insulin Analogs-Lantus on the market, it is owing to can simulate the physiological foundation insulin secretion, steadily without the peak, blood sugar control easily up to standard in, almost do not have risk of hypoglycemia, and the time length is about 24 hours, meet human lives's work-rest cycle, only need inject once every day, and favored by patient and doctor.But also there are some problems in Lantus when clinical application: 1. onset slow (onset time is about 1.5 hours), and to the too high patient of mealtime blood sugar, or the more patient that has meal once in a while can't effectively reduce its postprandial blood sugar; 2. can not mix with other Regular Insulin use, this is because the iso-electric point of various Regular Insulin is different, Lantus is stable under acidic conditions, and natural insulin and existing Semilente Insulin analogue are stable under neutrallty condition, if Lantus is directly mixed with other Regular Insulin, can cause Lantus that precipitation occurs before injection and can not use, and other Regular Insulin after mixing also because the impact that changed by the mixture potential of hydrogen, stability reduces, and security also reduces immediately.Therefore, on the package insert of Lantus, the manufacturer indicates especially and Lantus is not mixed with the Regular Insulin of other any types.
In Chinese patent application 200780019450.3, propose by adding specific acid stabilizer and sequestrant, conventional human insulin preparation VIAjectTM is mixed with Lantus under the condition of low pH, to realize co-user's Regular Insulin and Lantus.But the method need to additionally be added the auxiliary materials such as organic acid, sequestrant on the one hand in preparation; On the other hand, under the condition of low pH, the Asn that the INSULIN A chain is 21 is very easy to the deamination degraded (referring to " stability of Regular Insulin " (Stability of insulin), Jens Brange, 1994), so that product is extremely unstable, and this patent application is not carried out systematic study to insulin human's stability and the stability of combined preparation under the low pH condition, and the data of safety of product is incomplete.In addition, this patent application does not also solve the problem of product stability and maintenance product clinical characteristics from the molecular structure aspect of Regular Insulin.
Summary of the invention
The objective of the invention is to solve the existing existing problem of above-mentioned Regular Insulin premix formulations, provide a kind of under acidic conditions insulin analog or its pharmacologically acceptable salt of stable and quick acting, can mix with long-acting insulin analogs such as Lantuss, make clarification preparation, it is that injectable uses that said preparation need not mixing before use, have simultaneously quick-effective hypoglycemic and two kinds of functions of steady long-acting hypoglycemic, only need inject once every day, can simulate the secretion pattern of physiology Regular Insulin, when blood sugar safety is up to standard, can reduce more risk of hypoglycemia, and do not contain foreign protein and extra auxiliary material, security is higher.
Under the condition of low pH, the Asn (l-asparagine) of INSULIN A chain A21 position is very easy to the deamination DeR occurs, so that insulin molecule is extremely unstable under acidic conditions.The inventor finds by large quantity research, the Asn of INSULIN A chain A21 position is replaced with Gly (glycine), the insulin analog of resulting brand-new sequence has beat all stability when acid ph value, and when acid and pH neutral, all have higher solubleness, thereby solved the problems referred to above.
Therefore, in order to realize purpose of the present invention, the invention provides a kind of insulin analog or its pharmacologically acceptable salt, it is stable and energy quick acting under acidic conditions, described insulin analog is for sporting the insulin human of glycine (Gly) to the l-asparagine (Asn) of its A chain of major general A21 position, wherein insulin human's structure as shown in Figure 1, insulin human A chain-ordering is the aminoacid sequence shown in the SEQ ID NO:1, and insulin human B chain-ordering is the aminoacid sequence shown in the SEQ ID NO:2.
Preferably, the insulin human that described insulin analog is replaced by Gly for the Asn on the A21 position only, i.e. Gly
A21The insulin human, its A chain-ordering is the aminoacid sequence shown in the SEQ ID NO:3, the B chain-ordering is the aminoacid sequence shown in the SEQ ID NO:4.
Preferably, described insulin analog is that the Asn on the A21 position is replaced by Gly, and the Pro on the B28 position is replaced by Lys, and the insulin human that replaced by Pro of the Lys on the B29 position, i.e. Gly
A21-Lys
B28-Pro
B29The insulin human, its A chain-ordering is the aminoacid sequence shown in the SEQ ID NO:5, the B chain-ordering is the aminoacid sequence shown in the SEQ ID NO:6.
Preferably, described insulin analog is that the Asn on the A21 position is replaced by Gly, the insulin human that the Pro on the B28 position is replaced by Asp, i.e. Gly
A21-Asp
B28The insulin human, its A chain-ordering is the aminoacid sequence shown in the SEQ ID NO:7, the B chain-ordering is the aminoacid sequence shown in the SEQ ID NO:8.
The present invention also comprises the pharmacologically acceptable salt of above-mentioned insulin analog, and described pharmacologically acceptable salt is including but not limited to zinc salt, sodium salt, sylvite, magnesium salts, calcium salt.
Insulin analog of the present invention or its pharmacologically acceptable salt can adopt any generally acknowledged peptide synthetic technology preparation, such as solution method, solid-phase synthesis (J.Stewart etc., " solid-phase peptide is synthetic ", Freeman and Co., San Francisco, 1969), semi-synthesis method, gene engineering research, DNA recombination method etc.
The insulin analog of said structure optimization or its pharmacologically acceptable salt have been eliminated Regular Insulin factor unstable under acidic condition from molecular structure, so that insulin molecule of the present invention is difficult for desamination reaction occurs, has very high stability under acidic conditions.Therefore this insulin analog or its pharmacologically acceptable salt can with Recent Development of Long-acting Insulin Analogs for example Lantus form pharmaceutical composition, or make the acid premix insulin preparation of clarification.
Therefore, the present invention provides a kind of pharmaceutical composition on the other hand, it comprises insulin analog of the present invention or its pharmacologically acceptable salt, and Recent Development of Long-acting Insulin Analogs or its pharmacologically acceptable salt, wherein said insulin analog of the present invention is for sporting at least the insulin human of glycine (Gly) in its A chain A21 position.
Preferably, described insulin analog of the present invention is selected from Gly
A21Insulin human, Gly
A21-Lys
B28-pro
B29Insulin human or Gly
A21-Asp
B28The insulin human, described Recent Development of Long-acting Insulin Analogs is selected from Lantus.
Preferably, in aforementioned pharmaceutical compositions, the percentage composition of insulin analog of the present invention and Recent Development of Long-acting Insulin Analogs is 30: 70-70: 30, and more preferably 50: 50.
The present invention provides a kind of insulin medicament preparation that comprises aforementioned pharmaceutical compositions on the other hand, and preferably, the pH of this pharmaceutical preparation is 3.5-4.5, more preferably 3.8-4.2.
The said medicine preparation also can further comprise one or more pharmaceutically acceptable auxiliaries, such as isotonic agent, buffer reagent, solubilizing agent, sanitas etc.Its isotonicity agent includes but not limited to glycerine, sodium-chlor etc.; Buffer reagent includes but not limited to phosphate buffered saline buffer, acetate buffer etc.; Solubilizing agent includes but not limited to physilogically acceptable acid example hydrochloric acid, acetic acid, citric acid etc.; Sanitas includes but not limited to phenol, meta-cresol, methyl p-hydroxybenzoate etc.
The said medicine preparation is the clarified liq preparation, and it can be used by the mode of injection, oral, oral cavity, hypogloeeis, vagina, rectum or nasal administration.Preferably, this pharmaceutical preparation is used by injection.When using, the patient can be chosen in before dinner every day or injection is once after the dinner according to self living habit.
In the acid premix insulin preparation of the present invention, two kinds of insulin analogs have good solubleness, and physical and chemical stability is constant, the clinical effect characteristics are also constant.After subcutaneous injection, protamine zine insulin can form small precipitation and play long-acting after subcutaneous injection, and Semilente Insulin analogue of the present invention still can keep higher solubleness under the neutral pH in vivo and bring into play quick-acting effects.
Premix insulin preparation of the present invention has the following advantages: 1. preparation clarification, thereby need not before use mixing and can extract accurate dosage injection; 2. clinical effect has on the time that existing Semilente Insulin is rapid-action, the advantage of the steady hypoglycemic of protamine zine insulin concurrently; The patient can according to self living habit be chosen in before dinner every day or after the dinner injection once can simulate the secretion pattern of physiology Regular Insulin; 4. can solve hyperglycemia after dinner every day; be difficult for again night particularly occurring hypoglycemia at other times; make blood sugar maintain a relatively stably state; when making blood sugar safety up to standard, at utmost reduce risk of hypoglycemia; protection diabetic subject's capillary blood vessel; prevent or the generation of delaying complications of diabetes, further improve diabetic subject's quality of life.
Insulin analog of the present invention has no the overt toxicity reaction in animal body.The pharmacodynamic evaluation result shows, premix insulin preparation of the present invention is in injection beginning in rear 10 minutes onset, stably blood sugar reducing function was arranged in 1-8 hour always, effect is still obvious in the time of 12 hours, can satisfy that the clinical diabetes patient requires that frequency injection is few, rapid-action, longer duration, hypoglycemic steadily, demand that the hypoglycemia incidence is low.
Description of drawings
Fig. 1: insulin human's structure.
Fig. 2: each the time point electrophoresis detection of expression collection of illustrative plates of sweet Insulin lispro of recombinating.
Fig. 3: the sweet Insulin lispro RP-HPLC that recombinates detects collection of illustrative plates.
Fig. 4: the retention time of recombinate sweet Insulin lispro and the Lantus high performance liquid chromatography after with 50: 50 per-cent premix is measured collection of illustrative plates.
Fig. 5: different Regular Insulin are to the hypoglycemic percentage (%) of tetraoxypyrimidine hyperglycemic rat effect.
Embodiment
Preparation and the affirmation of embodiment 1 insulin analog of the present invention
Material:
Bacterial strain:
Clone bacterial classification Escherichia coli (DH5a) is genetically engineered common tool bacterial classification,
Express bacterial classification Escherichia coli (3110) and be purchased from US mode culture collection warehousing (ATCC).Enzyme and reagent:
Molecular cloning toolenzyme and reagent are purchased from Beijing through company of HTC of section.
Plasmid extraction test kit, PCR purification kit are purchased from sky, Beijing root company (TianGen).
Rite-directed mutagenesis test kit (Fast Mutagenesis System) is purchased from full formula King Company (TransGene).
Substratum:
LB substratum, ammonia benzyl resistance LB substratum, M9 substratum and enrichment medium are genetically engineered field substratum commonly used, and the prescription of each substratum can be referring to molecular cloning third edition volume two appendix 2.
Method:
Plasmid extraction amplification, polymerase chain reaction, PCR product purification, plasmid reclaim, connect and transform intestinal bacteria, these all are the routine operation methods in the genetically engineered research field, can be referring to Sambrook J, Fristsh EF, Maniatis T.MolecularCloning; A Laboiatory Manual 2nd ed.NY:Cold Spring Harbor Laboratory Piess, 1989, pp.16-340.
1.Gly
A21-Lys
B28-Pro
B29Insulin human's's (sweet Insulin lispro) preparation:
(1) structure of the sweet Insulin lispro plasmid of restructuring
The amplification of Insulin lispro plasmid: will be according to existing Lys
B28-Pro
B29The Insulin lispro plasmid that insulin human's (Insulin lispro) sequence makes is transferred among the Escherichia coli (DH5a), then join in the ammonia benzyl resistance LB substratum, 225rpm in shaking table, cultivated 16 hours for 37 ℃, the Insulin lispro plasmid is increased, then extract plasmid with the plasmid extraction test kit.
PCR rite-directed mutagenesis: use rite-directed mutagenesis test kit (Fast Mutagenesis System), with 60 times of templates as rite-directed mutagenesis of Insulin lispro plasmid dilution of amplification, take two primers of design as the upstream and downstream primer, carry out pcr amplification.
Wherein primer sequence is as follows:
N-Gf：CTCGAGAACTACTGCGGCTAATGAAGCTTGATC
N-Gr：CCGCAGTAGTTCTCGAGCTGGTACAGGCTG
The PCR system is:
Plasmid template (5ng) 1ul,
Upstream primer N-Gf (10uM) 0.5uL,
Downstream primer N-Gr (10uM) 0.5uL,
The 5 times of quick Pfu damping fluid of Trans Start 5uL,
dNTPs(10mM)1uL，
The quick Pfu archaeal dna polymerase of TransStart 0.7uL,
Sterilized water 16.3uL,
Cumulative volume: 25uL.
Amplification condition is: 94 ℃ of 4min;
94 ℃ of 30sec, 55 ℃ of 30sec, 25 circulations of 72 ℃ of 2.5min;
72℃10min。
The purifying of PCR product and digestion: above-mentioned PCR product is carried out purifying with the PCR purification kit of TianGen company, then 1ul DMT enzyme is added in the 40ul PCR purified product, mixing is hatched for 37 ℃ and was digested in 1 hour.
The structure of sweet Insulin lispro plasmid and affirmation:
The DMT enzymic digestion product of 2ul is added in the DMT competent cell of 50ul (from rite-directed mutagenesis test kit (Fast Mutagenesis System), when competent cell just thaws, adding digestion product), flicks mixing, ice bath 30 minutes; In 42 ℃ of accurate heat shocks 45 seconds, placed immediately 2 minutes on ice; Be added in the LB substratum of 250ul room temperature, 225rpm in shaking table cultivated 1 hour for 37 ℃.Then get 200ul bacterium liquid and be laid on the ammonia benzyl resistance LB substratum overnight incubation.
Select 10 transformants that grow, access respectively in the ammonia benzyl resistance LB substratum overnight incubation.Second day extracts plasmid with the plasmid extraction test kit again from these 10 overnight culture;
10 plasmids that extract are delivered to Beijing Nuo Sai company carry out sequencing, to identify the exactness of mutator gene.Select the sweet Insulin lispro plasmid of the restructuring that is loaded with correct mutator gene.
(2) the sweet Insulin lispro of restructuring is expressed the structure of engineering bacteria
The sweet Insulin lispro plasmid of the restructuring that is loaded with correct mutator gene that 2ul is screened joins in the competent cell of 100ul Escherichia coli (3110) (adding product when competent cell just thaws), flick mixing, ice bath 30 minutes; In 42 ℃ of accurate heat shocks 90 seconds, placed immediately 2 minutes on ice; Be added in the LB substratum of 300ul room temperature, 225rpm in shaking table cultivated 1 hour, and then got 50ul bacterium liquid and be laid on the ammonia benzyl LB substratum overnight incubation for 37 ℃.
Picking list bacterium colony from the culture plate of expressing bacterial classification Escherichia coli (3110), be inoculated in the 5ml ammonia benzyl LB substratum, 225rpm in shaking table, cultivated 8 hours for 37 ℃, therefrom drawing 100ul bacterium liquid is inoculated in the M9 substratum that 1L contains 20% enrichment medium again, 225rpm in shaking table cultivated 16 hours for 37 ℃, made the expression engineering bacteria of a large amount of sweet Insulin lispros.
(3) fermentation expression of the sweet Insulin lispro engineering bacteria of restructuring
Above-mentioned qualified engineering bacteria is seeded in the 2L ammonia benzyl LB liquid nutrient medium, in shaking table 32 ℃, 250rpm cultivated about 6 hours, transferred species is in the 5L fermentor tank, when being cultured to the nectar degree and being the OD600 value for 1.5-2.0, switching enters in the 250L fermentor tank again, and carries out under the following conditions fermentation culture: pH and be controlled at about 7.0; Dissolved oxygen is controlled between the 7-65%, and temperature is controlled at 32 ℃ in earlier stage, treats to be warming up to 35 ℃ after the OD600 to 20, keeps being warming up to 37 ℃ after 2 hours, until fermentation ends.The pH value is by adding ammonia vol control in the fermenting process; Dissolved oxygen leans on feed supplement, passes into pressurized air and adjusts stirring velocity control.Fig. 2 is gained sweet Insulin lispro each the time point electrophoresis detection of expression collection of illustrative plates in the 250L fermentor tank of recombinating.Can find out sweet Insulin lispro after fermentation 15 hours by electrophoretogram, the visible significantly target protein band of expression in 13kDa place, electrophoresis scanning expression amount was 27.37% in 26 hours.
(4) former renaturation and the conversion of sweet Insulin lispro
With the engineering bacteria behind the abduction delivering through centrifugal recovery thalline, thalline is suspended in the thalline washings, and (pH 8.0,50mM Tris-Cl) in, 4-15 ℃ of agitator treating 1 hour, recentrifuge reclaims thalline, then thalline is suspended in the frozen water, in high pressure homogenizer, carry out cytoclasis (750-800 normal atmosphere), by the sweet Insulin lispro inclusion body of centrifugal collection, inclusion body is again by washing (pH8.0,50mM Tris-Cl, 0.2%Triton X-100), centrifugal recovery precipitation.Then the gained inclusion body is carried out protein denaturation under high concentration urea (8M), carry out protein renaturation under the lower concentration urea (1.6M), the sweet Insulin lispro that obtains correct conformation is former.
To transfer pH to 10.0 with 50mM Tris behind the sweet Insulin lispro original solution desalination, in mass ratio 1: 500-1: 1000 (E/S, be enzyme/precursor proinsulin) add respectively trypsinase and protaminase, 37 ℃ are reacted 30min, obtain the sweet Insulin lispro of correct conformation.
(5) purifying of sweet Insulin lispro and affirmation
Sweet Insulin lispro after the conversion is through cation-exchange chromatography (CM) purifying, and chromatography comprises pH5.0 with damping fluid, the 10mM citric acid, and gradient elution carries out with 0-0.8M NaCl gradient, and purity is more than 90% behind the sweet Insulin lispro chromatography.Behind reverse phase HPLC, detect purity through RP-HPLC and reach 99.77% (referring to Fig. 3) again, meet the standard-required under domestic and international pharmacopeia recombinant human insulin's item.Gained is recombinated sweet Insulin lispro through amino acid sequence analysis, and resulting structures is consistent with expection.
2.Gly
A21Insulin human's (sweet Regular Insulin) and Gly
A21-Asp
B28Insulin human's's (sweet insulin aspart) preparation:
The preparation process of sweet Regular Insulin and sweet insulin aspart is consistent with the preparation process of sweet Insulin lispro, just in the step of plasmid construction, replaces the Insulin lispro plasmid to carry out with existing insulin human's plasmid and insulin aspart plasmid respectively.
The stability of embodiment 2 insulin analogs of the present invention under acidic conditions
To place respectively by the insulin analog of the present invention that embodiment 1 method prepares the pH value is 4.0 ± 0.2 solution, and gained solution is clear state.With mentioned solution 2-8 ℃ store 9 months after, total increasing amount of the associated protein in the various insulin analog solution is no more than 1.26%; The increasing amount of macromolecule protein is no more than 0.55%; The reduction amount of tiring is no more than 1%, all in the scope of 95%-105%.
The above results explanation and since insulin analog of the present invention with under the acidic conditions easily the A21 position N of deamination replace with glycine, therefore under acidic conditions, shown preferably stability.
Preparation method and the composition analysis of embodiment 3 pharmaceutical preparations of the present invention
1. the preparation method of pharmaceutical preparation of the present invention
＜raw material 〉:
Sweet Insulin lispro: the method by embodiment 1 prepares.
Lantus: Gan Li pharmaceutcal corporation, Ltd provides, lot number GLGB09001.
Hydrochloric acid (lot number 20070802), sodium hydroxide (lot number 20090804) are available from Hunan Er-kang Pharmaceutical Co., Ltd.; Zinc chloride (lot number 20090522), meta-cresol (07496PK) are available from capital, Shanghai company limited; Glycerine is available from Suichang of Zhejiang Province Hui Kang pharmaceutcal corporation, Ltd, lot number 20090921.
＜preparation process 〉:
The preparation of Regular Insulin mother liquor: precision takes by weighing sweet Insulin lispro and each 500,000 unit of Lantus respectively, join in 3 liters of distilled water, the stirring suspendible is even, after dropping 3M hydrochloric acid dissolves it fully, fill into an amount of liquor zinci chloridi, make that every milliliter of zinc content is about 30ug in the final insulin mixture, then moisturizing is to 5L.
The preparation of other auxiliary material mother liquors: precision takes by weighing glycerine 160 gram, and meta-cresol 27.0 grams are put in an amount of water for injection, is stirred to after whole dissolvings moisturizing to 4 liter.
Blending means: other auxiliary material mother liquors are joined in the Regular Insulin mother liquor, transfer the pH value to 4.0 of solution ± 0.2 with 10% sodium hydroxide or 10% hydrochloric acid again, moisturizing is to 10 liters of final volume, is stored under 2-8 ℃ the condition.
The concentration of sweet Insulin lispro and Lantus respectively is 50IU/ml in the final gained settled solution, and the concentration of gross activity Regular Insulin composition is 100IU/ml.
2. the composition analysis of pharmaceutical preparation of the present invention
Analytical procedure: high performance liquid chromatography
Analysis condition: use the alkyl silane bonded silica gel to be C4 post (the 3.5 μ m of weighting material, 15cm * 4.6mm I.D.), (precision takes by weighing AMSP 5.9998g with the sodium perchlorate salt buffer, add water 900ml dissolving, add sodium perchlorate 14.046g dissolving again, be mixed, transferring its pH value with perchloric acid is 2.5, add water to 1L, filter) be mobile phase A; Get sodium perchlorate salt buffer-acetonitrile (50: 50) again and be Mobile phase B, flow velocity is 1.0ml/min, and column temperature is 40 ℃, and the detection wavelength is 214nm.Gradient is the 60%B balance, and original state is 60%B, is 100%B when then in 49 minutes B% being increased to 74%, 50 minute, continues 10 minutes, is 60%B in the time of 61 minutes, continues 5 minutes.
Analytical results: Fig. 4 is that the retention time of the sweet Insulin lispro of the present invention and the Lantus high performance liquid chromatography after with 50: 50 per-cent premix is measured collection of illustrative plates.As can be seen from Figure 4, when pH4.0, quick-acting and content ratio Recent Development of Long-acting Insulin Analogs is 50: 50 substantially, and the peak-to-peak resolution of two samples is greater than 2.0, illustrate that two kinds of analogues can be separated fully under this condition, namely can carry out the effective content analysis, guarantee that the content of each component in the production process is accurately controlled.
Prepared Gly in the embodiment of the invention 1
A21Insulin human, Gly
A21-Asp
B28The insulin human also can mix with Lantus as stated above, and can feed intake in varing proportions as required, and making percentage composition is 30: 70-70: 30 hybrid medicine preparation.Obtained preparation also can be separated when the pH4.0 fully by high-efficient liquid phase chromatogram technique analysis, namely can carry out the effective content analysis, guarantees that the content of each component in the production process is accurately controlled.
The safety evaluation test of embodiment 4 pharmaceutical preparations of the present invention
＜test drug 〉: the pharmaceutical preparation of the present invention of embodiment 3 preparations.
＜experimental animal 〉:
The Wistar rat, body weight 200～220g is provided credit number by Jilin University's Experimental Animal Center: SCXK-(Ji) 2003-0007.
Kunming kind small white mouse, body weight 18～22g is provided by Changchun Biological Products Institute, credit number: SCXK-(Ji) 2006-0001.
Cavy, body weight 300～350g, Jilin University's Experimental Animal Center, credit number: SCXK-(Ji) 2008-0004.
Rabbit, body weight 2.0～3.0kg, Jilin University's Experimental Animal Center, credit number: SCXK-(Ji) 2008-0004.
＜test method and result 〉:
1. studies on acute toxicity:
According to existing Acute toxicity research method (referring to Xu Shuyun, " pharmacological experimental methodology " second edition, the People's Health Publisher, the 201st page), the acute toxic reaction that subcutaneous injection pharmaceutical preparation of the present invention causes after the fasting of observation mouse, record active situation and the death toll of experimental animal in 14 days, calculate LD50.
Calculated results is:
Male mice LD50=3.346U/Kg, its credible 2.372～4.719U/kg that is limited to of 95%;
Female mice LD50=4.767U/kg, its credible 3.556～6.391U/kg that is limited to of 95%;
Male rat LD50=7.447U/kg, its credible 6.159～9.004U/kg that is limited to of 95%.
Dead animal is dissected visual inspection, and main organs is without unusually.
2. long term toxicity research:
Test method: give respectively rat skin lower injection 3.72U/kg (LD50 1/2), 2.0U/kg (rat drug effect dosage), 1.0U/kg (LD50 the 1/8) pharmaceutical preparation of the present invention of dosage, and physiological saline (control group), every day 1 time, in continuous 5 weeks, drug withdrawal observed for 2 weeks again.
Test-results: during the administration, between drug withdrawal decubation, body weight, dietary amount, amount of drinking water and the general activity etc. and the more equal no significant difference of physiological saline group (control group) of each dosage group rat of pharmaceutical preparation of the present invention.During 2 week of 5 weeks of administration, drug withdrawal, index and the more equal no significant differences of physiological saline group (control group) such as each dosage group rat serum conventional index of pharmaceutical preparation of the present invention, blood coagulation item index, blood biochemistry index, serum antibody, organ coefficient and organs and tissues pathological examination.
3. immunotoxicity test:
According to having animal immune toxicity test method now (referring to " technical director's principle of Chinese medicine, natural drug immunotoxicity (supersensitivity, photosensitized reaction) research ", Bureau of Drugs Supervision of country, 2005) carry out the Active general anaphylaxis (ASA) of cavy and the passive cutaneous anaphylaxis test (PCA) of rat.
1. Active general anaphylaxis (ASA)-cavy test:
With pharmaceutical preparation 0.87U/kg of the present invention (cavy drug effect dosage 1/2), 3.48U/kg (cavy drug effect dosage 2 times) through subcutaneous injection in cavy, the next day 1 time, totally 5 times, the 10th day 2 times of amounts by intravenous injection said medicine preparation after the last sensitization excite, observe and record booster injection after in 30 minutes cavy have or not allergic symptom: the result of two dosage groups all is negative.
2. passive cutaneous anaphylaxis test (PCA)-rat test:
With pharmaceutical preparation 1.0U/kg of the present invention (rat drug effect dosage 1/2), 4.0U/kg (rat drug effect dosage 2 times) through subcutaneous injection in rat, the next day 1 time, totally 5 times, after last sensitization the 11st day, aorta abdominalis blood sampling, Dispersal risk serum.With above-mentioned antibody serum with normal saline dilution after, at rat intradermal injection 0.1ml, after 48 hours, the intravenous injection antigen (include 0.75% Evans Blue dyestuff) identical with priming dose excites, measure the blueness reaction spot diameter of skin inboard after 30 minutes and judge yin and yang attribute: blue reaction spot diameter is judged to be the positive greater than 5mm person, and the result of two dosage groups of pharmaceutical preparation of the present invention all is negative.
4. pungency, hemolytic test:
According to existing animal pungency, hemolytic test method (referring to " technical director's principle of Chinese medicine, natural drug pungency, hemolytic research ", national Bureau of Drugs Supervision, 2005) rabbit is carried out irritation test and hemolytic test.
1. irritation test
With pharmaceutical preparation 0.5U/kg of the present invention (rabbit drug effect dosage 1/2), 2.0U/kg (rabbit drug effect dosage 2 times) single through subcutaneous injection in rabbit, from injecting up to 72h, injection site does not have the reaction symptoms such as erythema, oedema, hyperemia, and its histopathological examination result shows that also two dosage groups of pharmaceutical preparation of the present invention are to rabbit subcutis nonirritant.
2. hemolytic test
Haemolysis and coacervation did not all occur in pharmaceutical preparation of the present invention in 3 hours.
Can be found out by above safety evaluation test-results: pharmaceutical preparation of the present invention is showed no the obvious toxic-side effects impact to animal, does not have allergic phenomena, the injection site vacuum response, and pharmaceutical preparation itself is without haemolysis.Illustrate that pharmaceutical preparation of the present invention has very high security.
The pharmacodynamics test of embodiment 5 pharmaceutical preparations of the present invention
1, test drug:
The pharmaceutical preparation of the present invention of embodiment 3 preparations;
Restructuring Insulin lispro injection liquid (100IU/ml, lot number: 22080301, provided by Gan Li pharmaceutcal corporation, Ltd);
The restructuring insulin glargine injecta (100IU/ml, lot number: 12080303, provided by Gan Li pharmaceutcal corporation, Ltd).
2, experimental animal: the Wistar rat, male, 50, body weight 250～280g is provided credit number by Jilin University's Experimental Animal Center: SCXK-(Ji) 2003-0007.
3, test reagent: Reagent kit of glucose, Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd., lot number: 081251.200802.
4, test apparatus: GF-D800 type semi-automatic biochemical analyzer, Shandong Gaomi Caihong Analytical Instrument Co., Ltd..The TGL-16B desk centrifuge.
5, test design, concrete grammar be referring to Xu Shuyun, " pharmacological experimental methodology " third edition, and the People's Health Publisher, 1517 pages:
1. animal grouping and modeling:
50 Wistar rats are divided into 5 groups at random, 10 every group, are respectively:
Normal group (not setting up hyperglycemia model, administration physiological saline);
Model group (setting up hyperglycemia model, administration physiological saline);
Insulin lispro group (setting up hyperglycemia model, the administration Insulin lispro);
Lantus group (setting up hyperglycemia model, administration Lantus group);
Pharmaceutical preparation group of the present invention (setting up hyperglycemia model, administration pharmaceutical preparation of the present invention).
Wherein hyperglycemia model is set up by the following method: a shilling Wistar rat fasting be can't help water 24 hours, and tail vein injection tetraoxypyrimidine 60mg/kg forms permanent hyperglycemia model after 48 hours.
2. administration and blood sampling:
After hyperglycemia model is set up, make all Wistar rat fasting can't help water 16h, and blood sugar before the survey medicine of before administration, taking a blood sample first, then to the corresponding Regular Insulin of animal skin hemostasis of Insulin lispro group, Lantus group, pharmaceutical preparation group of the present invention, dosage is all elected 2.0IU/kg as; Animal skin hemostasis equal-volume physiological saline to model group, Normal group.Then 10min, 30min, 1h, 2h, 3h, 4h, 8h, 12h, the blood sampling of 16h eye socket after administration, the centrifugal 5min of 3000rpm/min gets serum, and end-point method is surveyed blood sugar.
3. data processing:
The hypoglycemic percentage calculates by following formula:
Hypoglycemic percentage (%)=(blood glucose value before (blood glucose value behind the front blood glucose value-medicine of medicine)/medicine) * 100%
Test-results adopts
A t check is organized in expression, same time point blood sugar and hypoglycemic percentage.Calculation result sees Table 1, table 2 and Fig. 5.
The different Regular Insulin of table 1 on the impact of tetraoxypyrimidine hyperglycemic rat blood glucose value (mmol/L) (
N=10)
| Group | Normal group | Model group | The Insulin lispro group | The Lantus group | Pharmaceutical preparation group of the present invention |
| Before the medicine | 4.25±0.31 *** | 30.59±5.44 | 31.10±3.97 | 30.49±5.11 | 30.83±5.15 |
| 10min behind the medicine | 4.38±0.39 *** | 30.52±5.32 | 21.18±3.76 *** | 30.16±4.74 | 24.58±4.56 * |
| 30min behind the medicine | 4.38±0.47 *** | 30.04±4.72 | 8.25±2.65 *** | 28.98±4.83 | 16.58±2.47 *** |
| 1h behind the medicine | 4.31±0.50 *** | 29.32±4.79 | 7.47±1.67 *** | 22.44±6.62 * | 11.88±2.06 *** |
| 2h behind the medicine | 4.39±0.48 *** | 29.67±5.66 | 12.02±2.47 *** | 17.58±3.82 *** | 11.61±2.00 *** |
| 3h behind the medicine | 4.27±0.44 *** | 29.14±5.41 | 18.41±2.60 *** | 15.40±2.97 *** | 15.14±1.87 *** |
| 4h behind the medicine | 4.12±0.29 *** | 27.83±6.49 | 23.77±2.74 | 14.70±2.44 *** | 17.74±2.52 *** |
| 8h behind the medicine | 3.90±0.32 *** | 28.32±5.78 | 28.17±3.89 | 17.10±2.90 *** | 20.79±3.23 ** |
| 12h behind the medicine | 3.84±0.35 *** | 30.79±5.62 | 32.42±3.07 | 23.52±2.69 ** | 26.23±3.60 * |
| 16h behind the medicine | 3.67±0.32 *** | 32.15±4.97 | 31.96±2.90 | 30.90±3.04 | 31.95±4.24 |
Annotate with model group and compare:
*P＜0.05,
*P＜0.01,
* *P＜0.001
The different Regular Insulin of table 2 on the impact of tetraoxypyrimidine hyperglycemic rat hypoglycemic percentage (%) (
N=10)
| Group | Normal group | Model group | The Insulin lispro group | The Lantus group | Pharmaceutical preparation group of the present invention |
| 10min behind the medicine | -3.17±6.16 | 0.14±2.08 | 32.12±6.02 *** | 0.94±1.96 | 20.38±4.73 *** |
| 30min behind the medicine | -3.07±8.84 | 1.43±5.36 | 73.47±7.74 *** | 4.92±1.49 | 46.01±3.68 *** |
| 1h behind the medicine | -1.54±10.14 | 3.65±8.76 | 75.98±4.46 *** | 27.30±12.26 *** | 61.44±2.99 *** |
| 2h behind the medicine | -3.26±8.88 | 3.05±5.66 | 61.42±5.87 *** | 42.54±6.03 *** | 62.30±2.78 *** |
| 3h behind the medicine | -0.49±7.77 | 4.62±6.22 | 40.45±7.39 *** | 49.56±3.95 *** | 50.31±6.59 *** |
| 4h behind the medicine | 2.75±6.58 | 9.41±8.32 | 22.49±12.51 * | 51.60±5.00 *** | 42.12±5.21 *** |
| 8h behind the medicine | 8.00±7.72 | 7.45±8.18 | 9.43±5.46 | 43.89±3.60 *** | 32.13±8.11 *** |
| 12h behind the medicine | 9.50±8.82 * | -1.01±9.81 | -4.72±5.80 | 21.69±11.59 *** | 14.62±9.22 ** |
| 16h behind the medicine | 13.48±6.07 ** | -6.43±16.85 | -3.33±7.15 | -2.71±12.02 | -4.20±7.06 |
Annotate with model group and compare:
*P＜0.05,
*P＜0.01,
* *P＜0.001
Can find out that from the above results 10min begins onset after the injection of Insulin lispro group, 30min～1h blood sugar reducing function peaking, the 4h effect is still obvious, and 8h is later on without blood sugar reducing function; 1h begins onset after the injection of Lantus group, and 1h～8h blood sugar reducing function is steady, and the 12h effect is still obvious; 10min begins onset after the pharmaceutical preparation group injection of the present invention, and 1h～8h has stably blood sugar reducing function always, and the 12h effect is still obvious, has simultaneously quick-effective hypoglycemic and two kinds of functions of steady long-acting hypoglycemic.
Claims (7)
1. an insulin analog or its pharmacologically acceptable salt is characterized in that, described insulin analog is that the Asn on the A21 position is replaced by Gly, and the Pro on the B28 position is replaced by Lys, and the insulin human that replaced by Pro of the Lys on the B29 position.
2. insulin medicament composition, comprise: insulin analog claimed in claim 1 or its pharmacologically acceptable salt, and Recent Development of Long-acting Insulin Analogs or its pharmacologically acceptable salt, wherein said Recent Development of Long-acting Insulin Analogs is selected from Lantus.
3. pharmaceutical composition according to claim 2, the percentage composition of wherein said insulin analog and Recent Development of Long-acting Insulin Analogs is 30:70-70:30.
4. pharmaceutical composition according to claim 3, the percentage composition of wherein said insulin analog and Recent Development of Long-acting Insulin Analogs is 50:50.
5. an insulin medicament preparation comprises each described pharmaceutical composition and pharmaceutically acceptable auxiliaries among the claim 2-4.
6. pharmaceutical preparation according to claim 5, the pH of this pharmaceutical preparation is 3.5-4.5.
7. pharmaceutical preparation according to claim 6, the pH of this pharmaceutical preparation is 3.8-4.2.
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| CN102628072B (en) * | 2012-03-20 | 2013-12-04 | 甘李药业股份有限公司 | Preparation method of insulin |
| CN104587455A (en) * | 2013-10-31 | 2015-05-06 | 江苏万邦生化医药股份有限公司 | Insulin preparation |
| CN106177917B (en) * | 2016-08-29 | 2019-11-19 | 合肥天麦生物科技发展有限公司 | A kind of insulin aspart injection and preparation method thereof |
| CN109957001B (en) * | 2017-12-26 | 2022-11-18 | 甘李药业股份有限公司 | Preparation method of insulin crystal of glargine |
| EP3915572A1 (en) | 2020-05-29 | 2021-12-01 | Adocia | Pharmaceutical composition in the form of an injectable aqueous solution including at least a rapid acting insulin analog and a glucagon suppressor with prandial action |
| EP3915571A1 (en) | 2020-05-29 | 2021-12-01 | Adocia | Pharmaceutical composition in the form of an injectable aqueous solution including at least a rapid acting insulin analog and a glucagon suppressor with prandial action |
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| WO2009067636A2 (en) * | 2007-11-20 | 2009-05-28 | Ambrx, Inc. | Modified insulin polypeptides and their uses |
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| CN1044820A (en) * | 1989-02-09 | 1990-08-22 | 伊莱利利公司 | Insulin analog |
| WO2009067636A2 (en) * | 2007-11-20 | 2009-05-28 | Ambrx, Inc. | Modified insulin polypeptides and their uses |
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