Patent Application
20050171008
This invention relates to the medical and pharmaceutical fields. More particularly, this invention relates to a novel insulin B chain, a monomeric insulin molecule containing the B chain, pharmaceutical compositions containing the monomeric insulin, the preparations and the applications of the monomeric insulin.
Diabetes is a growing public health threat in the world. Hyperglycemia leads to various diabetes complications that is a major cause of mortality. Tight serum glucose control is the most important factor for preventing and postponing late complications of diabetes, see The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. N Engl. J. Med., 1993 329 (14) p. 977-86.
Ever since the discovery of insulin in 1920s it has been used for the treatment of diabetes as an irreplaceable drug. Secretion of insulin in normal human body is regulated by blood glucose concentration. Serum insulin concentration will rise to maximum 30-60 minutes after meal and will recover to base level S5 hours later, thus avoid considerable fluctuation of blood glucose concentration caused by food-taking. Regular insulin preparations or administration cannot mimic the process of natural physiological insulin secretion. These therefore can not effectively control rise of serum glucose after meals and there is as a result a risk of hypoglycemia The reason lies in that insulin exists in form of dimer and hexamer at the concentration of over 100 nM in a neutral medium and works by binding to specific receptor after being dissociated into monomer. This association between insulin molecules enhances the stability of insulin and ensures precise and flexible regulation of serum glucose. However, it is an obstacle to absorbance of insulin. Insulin preparation existing in the form of a hexamer will not be absorbed through capillaries into blood until it is transformed to dimeric insulin and subsequently to monomeric insulin after injection through dilution by 50,000 to 100,000 times. The multiple dilution in muscle or subcutaneously is a slow and long process with only 50% insulin being absorbed within 24 hours. 90-120 minutes after subcutaneous injection of insulin preparation, the insulin concentration in blood reaches a relatively low peak and fails to decrease rapidly later, see Brange, J. and A. Volund, Insulin analogs with improved pharmacokinetic profiles. Adv Drug Deliv. Rev, 1999.35(2-3) 307-335.
Insulin with low a tendency to self-association in neutral medium is named monomeric insulin. Monomeric insulin can be absorbed rapidly without multiples of dilution and functions within a shorter time after injection, Therefore monomeric insulin has become one of the major targets of developing new generation of drugs for diabetes. Lyspro, monomeric insulin developed by Eli Lilly Co., has proven to be very effective in clinical trials in Europe and America in 1996. It took effect 15 minutes after subcutaneous injection, the concentration reached a maximum in blood one hour later and recovered to normal level 24 hours later. Administration of Lyspro 15 minutes after meal is as effective as that of regular insulin 20 minutes before meal. Currently another monomeric insulin Aspart of Novo Co. is in clinical trial, see Vajo, Z. and W. C. Duckworth, Genetically engineered insulin analogs: diabetes in the new millennium. Pharmacol. Rev, 2000.52(1)1-9.
We have studied a method to prepare monomeric insulin, DPI (despentapeptide insulin) or DTI (destetrapeptide insulin) by secreting monomeric insulin precursor (MIP) out of Saccharomyces Cerevisiae and then by enzymatic transpeptidation, see Cui, D. F., Li, M. Y., Zhang, Y. S. and Feng, Y. M. Monomeric destetrapeptide human insulin from a precursor expressed in Saccharomyces cerevisiae. J Pept. Res., 2001.57(3)188-92. [as also disclosed in Chinese Patent ZL98 110912.8].
However, the monomeric insulin cannot be produced on large scale because of the following defects in the process: (1) it requires chemical peptide synthesis, for example, GFFY (But) Obut; (2) the yield is below 80% after enzymatic transpeptidation with trypsin; (3) the intermediate after enzymatic transpeptidation requires treatment with strong acid, e.g. TFA, to remove the protecting group; (4) the process is complicated and there exists chemical side reaction.
Consequently, it is an urgent task in this field to develop novel monomeric insulin that can be produced on large scale and by simple preparation.
One object of this invention is to provide novel monomeric insulin that can be produced on large scale and by simple method.
Another object of this invention is to provide a pharmaceutical composition containing the monomeric insulin.
Moreover, this invention aims to provide the preparation and application of the monomeric insulin and the pharmaceutical composition.
The first aspect of this invention provides an insulin B chain with following amino acid sequence:
wherein, X23, X24, X25 and X26 are individually independent and are selected from the group of amino acids consisting of Gly, Ala, Asp, Glu, Asn, Gln, Ser, Thr, Leu, Ile, Phe, Tyr, Trp, Pro, Met, His, Val or not present and wherein at most one of the individually independent amino acids is not present; and furthermore wherein Y27 is either Lys or Arg.
In one preferred embodiment Y27 is Lys.
In another preferred embodiment X23X24X25X26 is GFFY.
The second aspect of this invention provides an insulin molecule with the above B chain.
In one preferred embodiment the insulin is monomeric.
In another preferred embodiment the sequence of the said insulin is:
in which, X23, X24, X25, X26, and Y27 are defined as above.
The third aspect of this invention provides a pharmaceutical composition containing the monomeric insulin described in this invention and pharmaceutically acceptable carrier.
The fourth aspect of this invention provides a monomeric insulin precursor containing the B chain, a connecting peptide and an insulin A chain from an amino terminal to a carboxyl terminal, wherein the connecting peptide is Ala-Ala-Lys.
The fifth aspect of this invention provides DNA encoding the insulin B chain or the monomeric insulin precursor. It also provides the vector containing the DNA and the host cell containing the DNA or the vector.
The sixth aspect of this invention provides a method to prepare the insulin, in which following steps are included:
The precursor contains insulin B chain, a connecting peptide and an insulin A chain from the amino terminal to the carboxyl terminal, wherein the insulin B chain contains following amino acid sequence:
The addition of an alkaline amino acid to the insulin B chain C-terminal would simplify the production process of insulin analogue without affecting its activity. For example, insulin analogue B27K-DTrI, obtained by adding another alkaline amino acid in DTI (destetrapeptide-(B27-30)-insulin), not only possesses properties of monomeric insulin but has in-vivo bioactivity of 80% of that of native insulin. The process of obtaining B27K-DTrI by enzyme cleavage of monomeric insulin precursor secreted by yeast instead of enzyme transpeptidation using conventional techniques consequently increases total yield and is favorable for industrialized production.
The term “DOI” used in this invention refers to desoctapeptide-(B23-30)-insulin.
The term “B27K-DTrI” used in this invention refers to destripeptide with amino acids in 27 positions in the B chain being Lys. The base structure is as follows:
wherein, X23, X24, X25 and X26 are individually independent and are selected from the group of amino acids consisting of Gly, Ala, Asp, Glu, Asn, Gln, Ser, Thr, Leu, Ile, Phe, Tyr, Trp, Pro, Met, His, Val or are not-present (i.e. vacant). At most one not present amino acid among the amino acids X23, X24, X25 and X26 is allowed. Y27 is chosen to be either Lys or Arg. According to the research, the insulin will have certain activity provided it contains the first 25 amino acids (from the 23rd position, amino acid can be optionally changed). In this invention, the amino acid in the 23rd-26th positions can be changed. The changed amino acid preferably is any one of the amino acids except Lys, Arg and Cys, especially L-amino acid.
The peptide of this invention refers to B27K-DtrL an insulin containing B27K-DtrI and a corresponding monomeric insulin precursor (MIP). The peptide of this invention covers a recombinant peptide, a synthetic peptide or preferably recombinant polypeptide. It can be chemically synthesized or obtained from prokaryotic or eukaryotic hosts (e.g. bacteria, yeast, higher plant, insect and mammalian cells).
The polynucleotide of this invention can be either in DNA or RNA form. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or not Full length or fragments of the nucleotide sequence of B27K-DTrI of this invention can be obtained by PCR amplification, recombinant engineering or synthesis. The related sequence can be obtained initially by synthesis. Usually small fragments are synthesized first and then linked to obtain the full length.
Once the sequence is obtained, the sequence can be produced on a large scale by recombinant engineering. Usually the sequence is cloned in a vector, transferred to a host cell and then produced by conventional means. The resulting sequence is purified from the host cell after proliferation.
In this invention, the B27K-DTrI polynucleotide sequence can be inserted into the recombinant expression vector. The term “recombinant expression vector” refers to a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammal cell virus such as adenovirus, retrovirus or other vectors that are well known in this field.
In summary, any plasmid and vector that is able to replicate and remain stable can be used. One important characteristic of the expression vector is that it usually contains replication origin, promoter, marker, gene and translation control components.
The expression vector containing the B27K-DTrI coding DNA sequence and appropriate transcription/translation control signals can be constructed by methods familiar to skilled persons in this field. These methods include in-vitro recombination DNA technique, DNA synthesis, in vivo recombination etc. The DNA sequence can be effectively connected to a proper promoter in the expression vector to guide synthesis of mRNA. The typical examples of these promoters are: lac or trp promoter of E. coli, eukaryotic promoter including CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter and other promoters expressed in prokaryotic or eukaryotic cells or in viruses by some controllable genes. The expression vector also includes ribosome binding site for translation initiation and transcription terminator.
In addition, the expression vector preferentially contains one or more selective marker genes for providing phenotypic characters for selecting transformed host cells such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistivity and green fluorescin protein (GFP) or tetracin, ampicillin for E. coli.
The vector containing the proper DNA sequence and the promoter or control sequence described above can be used to transform the proper host cell so that it can express protein.
The host cells can be prokaryotic cells, e.g. bacterial cells; or lower eukaryotic cells, e.g. yeast cells; or higher eukaryotic cells, e.g. mammalian cells. The typical examples are: E. coli, Streptomyces; fungal cells, e.g., yeast; plant cells; insect cells of fruit fly S2 or S9; animal cells including CHO, COS etc.
The addition to the host cell of recombinant DNA can be carried out by conventional techniques familiar to person skilled in this field. As the host is a prokaryotic organism such as E. coli, the competent cell that can absorb DNA can be collected after exponential phase of growth and then treated with CaCl2, steps of which are well known in this field. Another method is to use MgCl2 instead of CaCL2. If required, electroporation can also be applied during transformation. As the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co precipitation, conventional methods including microinjection, electroporation, liposome entrapment, etc.
The transforming factor thus obtained can be conventionally cultured to express the peptide encoded by the gene of this invention. The culture medium used in the cultivation is selected from various conventional culture media according to the host cell used. The culture is carried out under the conditions suitable for growth of the host cell. As the host cell grows to a certain cell density, a selected promoter is induced by an appropriate method (temperature switching or chemical induction) to culture the cell further.
The recombinant peptide of the methods described above can be expressed in the cell or secreted out of the cell. If required, various separation methods can be used to purify the recombinant protein according to the physical, chemical and other characters of the peptide. These methods are well known in this field. Examples of these methods include but not limited to: conventional repatriation, protein precipitant (salting out), centrifugation, permeation, super centrifugation, size exclusion chromatography (gel electrophoresis), adsorption chromatography, ion exchange chromatography, HPLC and other various liquid phase chromatography techniques and combination thereof.
In one preferred example, B27K-DTrI is firstly expressed in the form of a monomeric insulin precursor and is then obtained after treatment of enzyme cleavage. The monomeric insulin precursor contains the insulin B chain of this invention, a connecting peptide and an insulin A chain from an amino terminal end to a carboxyl terminal end. There is no specific choice of the connecting peptide provided it has a connective function and the amino acid residue connected to A chain is Lys or Arg. An example of one kind of connecting peptide is (Ala)2-5Lys, for example Ala-Ala-Lys. Any enzyme e.g. trypsin, used to cleave Lys and/or Arg, can be used for enzyme cleavage treatment. Conditions for cleavage change according to the enzyme used. One example of the conditions is: solvent: 0.02-0.05M Tris buffer, pH: 7-8, concentration of substrate at about 5 mg/ml, weight of trypsin is about 1/50 of substrate, 4-250, 1-6 hour. After enzyme cleaving MIP, the product, B27K-DTrI of this invention, is purified by size exclusion chromatography.
This invention also provides a pharmaceutical composition that contains a safe and effective dosage of B27K-DTrI peptide of this invention and a pharmacologically acceptable carrier or excipient The kind of excipient includes (but not limited to): saline, buffer, glucose, water, glycerol, ethanol and the compound. The drug preparation and the drug delivery method must match. The pharmaceutical composition of this invention can be made into a dosage for injection, for example, prepared in conventional way in physiological saline or aqueous solutions containing glucose and other co-adjuvants. The pharmaceutical composition such as tablet and capsule can also be prepared in a conventional way. All of these forms including the dosage for injection, solution, tablet and capsule will be manufactured under sterile conditions. The delivery quantity of active components is effective quantity for treatment, for example, about 1 μg/kg (weight)-about 5 mg/kg (weight). The peptide of this invention can be used together with other drugs as well.
The pharmaceutical composition of this invention can be used to treat diabetes and its complications. The safe and effective dosage of B27K-DTrI protein into mammal is usually at least 10 μg/kg (weight) and usually not more than 10 mg/kg (weight), with the preferable dosage being about 10 μg/kg (weight)-100 μg/kg (weight). Certainly, delivery method, health condition of patient etc. must be taken into consideration when the drug is delivered, which falls upon responsibility of doctors.
Monomeric insulin of this invention can not only be solely used but used together with other drugs (e.g. other insulins) for treating diabetes.
The main advantages of this invention lie in:
The insulin of this invention has a strong monomeric property.
The monomeric insulin of this invention can be obtained by one-step enzyme cleavage from monomeric insulin precursor without enzyme transpeptidation or renaturation.
The pharmaceutical composition containing the insulin has a high bioavailability when delivered not by injection.
The following examples are quoted to further elaborate this invention, but not to limit it. Where no concrete condition is mentioned, normal requirements specified in Molecule Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2001) by Sambrook et al. or suggested by the manufacturer shall apply.
613 mg zinc free porcine insulin was dissolved in 127 ml 0.05 mol/L borax solution (containing 0.001M CaCl2), pH 8.9. The solution was stirred at 37° C. in water bath for 3 hours after which 24.5 mg crystal trypsin was added, then the major peak was pooled (
2.1 Preparation of Boc-Phe-Osu
3.18 g Boc-Phe (12 mmol) and equimolar N-hydroxysuccinimide (HOSu) were mutually dissolved in 15 ml 1 μl. The reaction mixture was kept at salt ice bath (−5° C.) for 10 minutes and then an equimolar amount of dicyclohexcylcarbodiimide (DCCI) dissolved in 2 ml anhydrous tetrahydrofuran (THF) was dripped slowly into the reaction bottle with magnetic stirring at −5° C. After stirring at −5° C. more than 2 hours, the solution was allowed to stand overnight at 4° C. The urea which formed was separated by filtration with three-layer filter paper and the filtrate became a white solid. The residue was dissolved with 15 ml isopropanol and recrystallized at room temperature to yield 2.5 g (53%) product after washing and drying. The product had a melting point of 138-140° C., was homogeneous in thin layer chromatography, 3/7 system. The developing solution was a mixture of 3 volume of solution A (pyridine/acetic acid/water 4:1:1.5 by volume) and 7 volumes of solution B (ethyl acetate/isopropanol 10:4 by volume). The chromatogram was stained by chlorine-starch-KI.
2.2 Preparation of (TFA) Phe-Tyr-OC2H5
5.97 g (16.5 mmol) Boc-Phe-Osu and equimolar HCl-Tyr-OC2H5 were mutually dissolved in 80 ml anhydrous ethyl acetate and was neutralized with approx. equimolar N-methylmorpholine to pH8. The reaction mixture was stirred overnight at room temperature and washed three times respectively with 10% citric acid, 1% potassium carbonate and saturated sodium chloride. The residue was dried with anhydrous sodium sulfate for 2 hours and again rotary evaporated to an exceedingly small volume. The product was crystallized with petroleum ether, to yield 6.1 g (81%) after washing and drying. The product had a melting point of 126-128° C., homogeneous in thin layer chromatography (3/7 system). The crystal was dissolved in 50 ml dichloromethane (DCM), 25 ml trifluoroacetic acid (TFA) was dripped into the solution. After being stirred at room temperature for 35 minutes, the solution was concentrated under vacuum to remove the solvent. The product (TFA) Phe-Tyr-OC2H5 was thus obtained after washing more than five times with DCM.
2.3 Preparation of (TFA) Phe-Phe-Tyr-OC2H5
13 mmol (TFA)-Phe-Tyr-OC2H5 was dissolved in 20 ml anhydrous THF (solvent) and neutralized to pH7 with 5.4 ml N-methylmorpholine. The solution was kept at salt ice bath. Then an equimolar amount of Boc-Phe-OSu was dissolved in 35 ml anhydrous THF and was dripped into a (TFA)-Phe-Tyr-OC2H5 solution, keeping the reaction solution at neutral pH with N-methylmorpholine. The reaction mixture was stirred overnight at room temperature. After rotary evaporation to powder, the mixture was dissolved with ethyl acetate and washed three times respectively with 10% citric acid, 1% potassium carbonate, saturated sodium chloride. The residue was dried with anhydrous sodium sulfate for 2 hours and again rotary evaporated to an exceedingly small volume. The crude product Boc-Phe-Phe-Tyr-OC2H5 was crystallized with a large amount of ether to yield 2 g (30%). The product had a melting point of 106-1080, homogeneous in thin layer chromatography (3/7 system). All the product was dissolved in 25 ml DCM and 25 ml TFA was dripped into the solution. After being stirred at room temperature for 30 minutes, the solution was concentrated under vacuum to remove the solvent The product (TFA) Phe-Phe-Tyr-OC2H5 was thus obtained after more than five times washing with DCM.
2.4 Preparation of Z-Gly-Osu
10 g (47 mmol) Z-Gly and equimolar amount of Hosu were mutually dissolved in 100 ml anhydrous THF (a solvent). The reaction mixture was kept at salt ice bath (−5° C.) for 10 minutes and then equimolar amount of DCCI dissolved in 2 ml anhydrous THF was dripped slowly into the reaction bottle with magnetic string at −5° C. After stirring at −5° C. more than 2 hours, the solution was allowed to stand overnight at 4° C. The urea formed was separated by filtration with a three-layer filter and the filtrate became slightly oily after rotary evaporation. The residue was dissolved with a small amount of DCM, then a large amount of ether was added, the filtrate was scrubbed against the bottle wall until yellow precipitate formed and the solution became clear. The clear solution was poured out and allowed to stand at room temperature. The product crystallized and was collected by filtration, yield 6.5 g (46%) of product after washing and drying. The product had a melting point of 100-102° C. and was homogeneous in thin layer chromatography are (3/7 system).
2.5 Preparation of ZGly-Phe-Phe-Tyr-OC2H5
3.3 g mmol (ITA) Phe-Phe-Tyr-OC2H5 and 5 mmol Z-Gly-Osu was mutually dissolved in 10 ml of anhydrous THF (solvent). The solution was neutralized with N-methylmorpholine pH8.0 and then stirred for 72 hours at room temperature. After rotary evaporation to powder, the powder was dissolved in ethyl acetate and washed three times in turn with 10% citric acid, 1% potassium carbonate, saturated sodium chloride. The residue was dried with anhydrous sodium sulfate for 2 hours and again rotary evaporated to an exceedingly small volume. The solution then became muddy from clear then clear again after the addition of a large amount of ether. A grey yellow precipitate formed after standing at room temperature for 72 hours. The precipitate was 2 g (87%) after washing with ether and drying, the precipitate had a melting point at 148-151□. The product was homogeneous in thin layer chromatography (3/7 system).
2.6 Preparation of ZGly-Phe-Phe-Tyr-NHNH2
15 mmol 85% hydrazine hydrate was added to a solution of 2 g (2.9 mmol) Z-Gly-Phe-Phe-Tyr-OC2H5 in 9 ml anhydrous methanol. The reaction mixture was refluxed for 2 hours and then cooled to room temperature. The crystals which formed immediately were collected and washed with 10 ml anhydrous ether, 10 ml methanol/ether (5v/5v) and water to pH7. The crude yield after drying was 1.29 g (65%) and the product was homogeneous in thin layer chromatography (3/7 system).
2.7 Preparation of Z-Gly-Phe-Phe-Tyr-Lys (Boc) Obut
37 g sodium nitrite was dissolved in 150 ml distilled water and 38 g ter-butyl alcohol was added to the solution. The reaction mixture was kept in an ice bath for 10 minutes and then 60 ml 35% sulfuric acid was dripped slowly into the reaction bottle with magnetic stirring. After stirring in the ice bath more than 30 minutes, the solution was allowed to stand for 15 minutes and then the supernatant was washed three times with 5% sodium bicarbonate. 30 ml ter-butyl nitrite was produced after being dried with anhydrous sodium sulfate. 1.25 g (1.9 mmol) Z-Gly-Phe-Phe-Tyr-NHNH2 was dissolved in 8 ml anhydrous DMF by heating. 2 ml 2N HCl/THF (4 mmol) was added to the solution and the reaction mixture was kept at −20° C., then 0.3 ml ter-butyl nitrite (2.5 mmol) was slowly dripped into the solution and shaken for 10 minutes, 10 ml anhydrous ethylacetate pre-cooled to −20° C. was added to the solution. The mixture was cooled to −30° C. Meanwhile 0.65 g (1.9 mmol) (HCl)Lys(Boc)Obut was dissolved in 7 ml anhydrous DMF, 247 μl triethylamine was added into the solution after it was cooled to −30° C., then the solution and 10 ml anhydrous ethylacetate pre-cooled to −20° C. were successively added into the mixture above. Triethylamine was added to adjust the pH to 8 and then the mixture was allowed to stand at 4° C. for 72 hours after being shaken for 30 minutes at 4° C. After rotary evaporation to powder, the residue was dissolved with ethylacetate, then washed three times in turn with 10% citric acid, 1% potassium carbonate, and saturated sodium chloride. The residue was dried with anhydrous sodium sulfate for 2 hours and again rotary evaporated to oil. The product was crystallized with triple times volume of ether, yield 1 g (56%) after washing with cool ethylacetate, cool ether and drying. The product had a melting point of 160-162° C. and was homogeneous in thin layer chromatography (3/7 system).
2.8 Preparation of Gly-Phe-Phe-Tyr-Lys(Boc)Obut 0.56 g Z-Gly-Phe-Phe-Tyr-Lys(Boc)Obut was dissolved in 90 ml anhydrous methanol and the solution was adjusted to pH 3.0 with glacial acetic acid. 0.12 g Pd carbon was added. The reaction solution was stirred and mixed with hydrogen for 6 hours. Then the solution was filtered with diatomite. (Pd carbon was discarded after immersion in chloroform) and the filtrate was dried with rotary evaporation. The product (yield 93%) was obtained after repeatedly washing with ethylacetate, homogeneous in thin layer chromatography (3/7 system) and can be stained with ninhydrin. As shown in
0.237 g (0.172 mmol) Gly-Phe-Phe-Tyr-Lys(Boc)Obut was dissolved in 0.26 ml dimethylsulfone (DMSO) by warming to 500. Then the solution was incubated at 37° C. water bath and 86 mg (0.0172 mmol) DOI was slowly added to the solution. 1.82 ml 14 butanediol and 0.52 ml water pre-heated to 37° C. was added to the reaction solution and the pH was adjusted to 6.5 with 5 μl N-methylmorpholine. 7.8 mg TPCK-Trypsin was added and reaction mixture incubated at 30° C. After 2 and 4 hours, 4.5 mg TPCK-Trypsin was added each time. After 20 hours, 1 ml glacial acetic acid was added and the reaction was ended by adjusting the pH to 3 with 1 mol/l HCl. The reaction mixture was purified by Sephadex G50 fine column chromatography with 30% acetic acid as eluent 76 mg (80% yield) crude product of B27K-DTrI (Boc) Obut was obtained (
The monomeric behavior of B27K-DTrI was determined by size exclusion chromatography using a Superdex 75 (HR 10/30) column, eluted by PBS (phosphate-buffered saline, pH7.4), with a flow rate 0.4 ml/min, loading volume 0.1 ml, detected at 280 nm, at room temperature. The molecular homogeneity was measured by Symmetry factor Fs. Fs=W0.05h/2A, wherein, W0.05 is the band width at 0.05 peak height and A is the width of the first half peak at 0.05 peak height. The change of average molecular weight was measured by plotting the distribution coefficient Kav vs. protein concentration. Kav=(Vr−V0)/(Vc−Vo), wherein Vr is the retention volume, Vo the void volume and Vc the total bed volume. The retention time on the Superdex 75 column decreased with increasing concentration of zinc free insulin control as shown in
5.1 Mouse convulsion test (Chinese Pharmacopoeia, 1985, Appendix 100 pages) Male Kunmin mice (body weight 27-30 g) were randomly divided into four groups with 24 in each group, fasting 2 hours before the experiment. The sample was dissolved in saline, pH 5.0, adjusted with hydrochloric acid, 1OD280 nm=1 mg/L. The potency of the sample was estimated to be 80% of that of standard product, high dosage was 0.09 U/ml, low dosage was 0.045 U/ml and the solvent was the psychological saline with the pH adjusted to 2.5 by hydrochloric acid. 0.3 ml was subcutaneously administered to each mouse within 15 minutes at 25° C. and then these mice were kept at 37° C. for 90 minutes' observation—all of those that were convulsive, dead and could not turn over when made to lie supine are considered to be positive reaction. The data was determined by qualitative reaction test.
5.2 Mouse plasma glucose lowering test (Chinese Pharmacopoeia, 2000, Appendix 106 pages).
Male Kunmin mice (body weight 28-30 g) were randomly divided into four groups with 10 in each group. The potency of the sample was estimated to be 80% of that of standard product, high dosage was 0.07 U/ml, low dosage was 0.035 U/ml and the solvent was the psychological saline with the pH adjusted to 2.5 by hydrochloric acid. 0.25 ml sample was subcutaneously administered to each mouse at 25□ and blood from the eye venous plexus was sampled to determine the blood sugar 40 minutes after the injection; the drug was administered to each mouse for the second time three hours later according to double-crossing (high dosage of the sample in test was administered to the group with low dosage of standard product earlier) and the blood sugar was tested after 40 minutes. The data were determined by a quantitative reaction test.
The biological activity according to mouse convulsion test is 21 U/mg and FL % is 23.5% (<30%) (Table 2); the biological activity according to mouse plasma glucose lowering test is 23 U/mg and FL % is 11.9% (<25%) (Table 3); The standard product used in the experiment is 27 U/mg insulin provided by NICPBP (The National Institute for the Control of Pharmaceutical and Biological Products). The results from the two testing methods are consistent to each other, and biological activity of B27K-DTrI is 80% of that of native insulin.
In this experiment, MIP (monomeric insulin precursor) was constructed by connecting the C terminal and the N terminal with connecting peptide (e.g. Ala-Ala-Lys, for MIP structure is helpful for insulin folding and formation of correct disulfide bond after biosynthesis. The steps are as follows:
Signal peptide sequence (α-MFL, α-mating factor leader) was added to 5′terminal of MIP gene (see SEQ ID NO:3 below) obtained from chemical synthesis, then the gene was cloned into pPIC9K plasmid to get pPIC9K/MIP by use of enzymatic cleavage site of EcoRI and NotI. pPIC9K/MIP was then transferred into yeast P. pastoris after treatment with linearization and spot hybridization was employed to screen highcopy strain.
After high-density fermentation, MIP was obtained from the product by hydrophobic chromatography. The amino acid sequence was shown in SEQ ID NO:4:
Highly pure B27K-DTrI (structure in
Discussion
B27K-DTrI can also be expressed by other yeast expression system or secretion E. coli expression system with the same technical process. This technical process compared with preparation of DTI disclosed in Chinese patent application 98 1 10912.8 overleaps overelaborate steps of chemical synthesis of peptide GFFY(But)OBut, enzymatic transpeptidation, HPLC separation etc. in conventional technique, consequently increases yield by a big margin and is more suitable for industrialized production. The comparison between the two processes is shown in
The prior art cited is incorporated herein by reference.
The examples and discussion are considered illustrative of the invention and since numerous modifications will occur to those skilled in the art, it is not intended to limit the invention to the examples described. All suitable modifications and equivalents fall within the scope of the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| CN 02136107.X | Jul 2002 | CN | national |
