CN111909255A

CN111909255A - Insulin derivatives and process for preparing the same

Info

Publication number: CN111909255A
Application number: CN201910390460.2A
Authority: CN
Inventors: 查若鹏; 张振山; 吴松; 刘慧玲; 陈卫
Original assignee: Ningbo Kunpeng Biotech Co Ltd
Current assignee: Ningbo Kunpeng Biotech Co Ltd
Priority date: 2019-05-10
Filing date: 2019-05-10
Publication date: 2020-11-10

Abstract

The invention provides an insulin derivative and a preparation method thereof. Specifically, the present invention provides an insulin derivative comprising: (a) the a chain of insulin; (b) the B chain of insulin; and (c) a modifying group L attached to a lysine site of the B chain of the insulin, the modifying group L containing at least one group X or group Y. The experimental result shows that the half-life of the insulin derivative is obviously prolonged while the bioactivity is maintained. The invention also discloses a preparation method of the insulin derivative and an effect of the insulin derivative in treating or preventing diabetes.

Description

Insulin derivatives and process for preparing the same

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to an insulin derivative and a preparation method thereof.

Background

Diabetes is a common multiple metabolic disease, and the incidence rate at home and abroad is on the rise. According to incomplete statistics, more than 3000 people have diabetes patients in China, and the incidence rate of diabetes is up to 3.2%. Although many diseases can lead to death, diabetes is the third leading cause of death.

Insulin is a hormone secreted by islet cells, regulates blood sugar to be maintained at a normal level through interaction with receptors on cell membranes in vivo, and is a specific medicine for treating diabetes. However, insulin cannot be directly administered orally, needs subcutaneous administration or intramuscular injection, is unstable in vivo, is easily degraded by protease, has high immunogenicity and is easily recognized by the immune system of the organism, so that the half-life period of the insulin is generally short, the administration frequency and dosage must be increased, the insulin is frequently injected, great inconvenience and pain are brought to a diabetic patient, and the ultra-long-acting insulin with longer injection interval time is clinically needed.

Accordingly, the skilled person is working to develop new, more long-acting insulin derivatives.

Disclosure of Invention

It is an object of the present invention to provide a new, more potent insulin derivative.

In a first aspect of the present invention, there is provided an insulin derivative comprising:

(a) the a chain of insulin;

(b) the B chain of insulin; and

(c) a modifying group L attached to a lysine site of the B chain of the insulin, and selected from the group consisting of:

(i) containing at least one group X, wherein the group X is a group of the formula I, or

(ii) Containing at least one group Y, wherein the group Y is a group of formula II,

wherein the wavy line represents the position of attachment to the lysine site, and m is an integer of 0 to 8; a. b, c, d, e and f are each independently integers selected from 0 to 10; n is an integer of 14 to 16.

In another preferred embodiment, the group Y is a group selected from the group consisting of:

in another preferred embodiment, the insulin comprises human insulin or animal insulin, preferably, the insulin is human insulin.

In another preferred example, the animal insulin comprises porcine insulin and bovine insulin.

In another preferred embodiment, the insulin further comprises one or more disulfide bonds between the A chain and the B chain.

In another preferred embodiment, the insulin comprises native insulin, an insulin precursor, or a variant of insulin.

In another preferred embodiment, the insulin derivative comprises an insulin precursor and the modifying group L.

In another preferred example, the A chain of the insulin has a sequence as shown in SEQ ID No. 1 or 2.

In another preferred embodiment, the B chain of the insulin has a sequence as shown in SEQ ID No. 3, 4, 5 or 6.

In another preferred example, the A chain of the insulin has a sequence shown in SEQ ID No.:1, and the B chain of the insulin has a sequence shown in SEQ ID No.:3, 5 or 6.

In another preferred example, the A chain of the insulin has a sequence shown in SEQ ID No.:2, and the B chain of the insulin has a sequence shown in SEQ ID No.:4 or 6.

In another preferred embodiment, the modifying group L is covalently linked to the lysine (K) site.

In another preferred embodiment, the modifying group L is covalently linked to the-amino group of the lysine (K).

In another preferred embodiment, the insulin comprises a PK, DKT, PKT or KPT motif and the modifying group L is linked to a lysine (K) site in said motif.

In another preferred embodiment, the insulin comprises a TPK, TKP or TDK motif and the modifying group L is attached to the lysine (K) site in said motif.

In another preferred embodiment, the insulin comprises a YTPK, YTDKT, YTPKT or YTKPT motif and the modifying group L is linked to a lysine (K) site in said motif.

In another preferred embodiment, the modifying group L is attached to lysine (K) at position 28 or 29 of the B chain.

In another preferred embodiment, the modifying group L is covalently linked to a lysine (K) corresponding to position 29 in the sequence shown in SEQ ID No. 3, 4 or 6.

In another preferred example, the B chain of the insulin has a sequence as shown in SEQ ID No. 3, 4 or 6, and the modifying group L is linked to lysine (K) at position 29 in the sequence as shown in SEQ ID No. 3, 4 or 6.

In another preferred example, the B chain of the insulin has a sequence shown in SEQ ID No. 5, and the modifying group L is linked to lysine (K) at position 28 in the sequence shown in SEQ ID No. 5.

In another preferred embodiment, the structure of the insulin derivative is selected from the group consisting of

Is insulin:

wherein m is an integer of 0 to 8; a. b, c, d, e and f are each independently integers selected from 0 to 10; n is an integer of 14 to 16.

In another preferred embodiment, the insulin derivative is selected from the group consisting of: L0-GFA 16-insulin, L2-GFA 16-insulin, L3-GFA 16-insulin, L4-GFA 16-insulin, L5-GFA 16-insulin, or L6-GFA 16-insulin.

In another preferred embodiment, the insulin derivative L0-GFA 16-insulin has the following structure, wherein

Is insulin:

in another preferred embodiment, the insulin derivative L2-GFA 16-insulin has the following structure, wherein

Is insulin:

in another preferred embodiment, the insulin derivative L3-GFA 16-insulin has the following structure, wherein

Is insulin:

in another preferred embodiment, the insulin derivative L4-GFA 16-insulin has the following structure, wherein

Is insulin:

in another preferred embodiment, the insulin derivative L5-GFA 16-insulin has the following structure, wherein

Is insulin:

in another preferred embodiment, the insulin derivative L6-GFA 16-insulin has the following structure:

in a second aspect of the invention, there is provided a pharmaceutical composition comprising an insulin derivative according to the first aspect of the invention, and a pharmaceutically acceptable carrier.

In a third aspect of the present invention there is provided the use of an insulin derivative as described in the first aspect of the present invention for the preparation of a medicament or formulation for the prevention and/or treatment of diabetes, hyperglycemia and other diseases where lowering blood glucose would be beneficial.

In a fourth aspect of the present invention, there is provided a process for the preparation of an insulin derivative, the process comprising the steps of:

(1) culturing a strain containing an insulin-coding sequence in which the coding sequence for the lysine site in the B chain of insulin is replaced with TAG (encodes a lysine derivative) in the presence of an X group-lysine, a pyrrolysinyl-tRNA synthetase and its cognate tRNA, thereby producing an insulin derivative, wherein the insulin derivative comprises:

(a) the a chain of insulin;

(b) the B chain of insulin; and

(c) a modifying group L attached to a lysine site of the B chain of the insulin, and which is a group X, the group X being as defined in the first aspect of the invention; and optionally

(2) Isolating said insulin derivative from the fermentation product.

In another preferred embodiment, the insulin comprises a PK, DKT, PKT or KPT motif, and the coding sequence for lysine (K) in said motif is replaced by TAG (encoding a lysine derivative).

In another preferred embodiment, the insulin comprises a TPK, TKP or TDK motif, and the coding sequence for lysine (K) in the motif is replaced with TAG (encodes a lysine derivative).

In another preferred embodiment, the insulin comprises a YTPK, YTDKT, YTPKT or YTKPT motif and the coding sequence for lysine (K) in which the modifying group L is linked is replaced by TAG (encoding a lysine derivative).

In another preferred embodiment, the X group-lysine is butynyloxycarbonyl-lysine.

In a fifth aspect of the present invention, there is provided a process for the preparation of an insulin derivative, the process comprising the steps of:

(1) in the presence of a compound of formula III, a pyrrolysinyl-tRNA synthetase and its cognate tRNA,

culturing a strain containing an insulin-encoding sequence in which the coding sequence for the lysine site in the B chain of insulin is replaced with TAG (encoding a lysine derivative) to provide a compound of formula IV; and

(2) reacting a compound of formula IV with a compound of formula V in an inert solvent to obtain an insulin derivative,

in the formula V, a, b, c, d, e and f are respectively and independently integers selected from 0 to 10; n is an integer of 14 to 16.

In another preferred embodiment, the resulting insulin derivative comprises:

(a) the a chain of insulin;

(b) the B chain of insulin; and

(c) a modifying group L attached to a lysine site of the B chain of the insulin, and the modifying group L is a group Y, the group Y being as defined in the first aspect of the invention.

In another preferred embodiment, the insulin comprises a TPK, TKP or TDK motif, and the coding sequence for lysine (K) in the motif is replaced by TAG (encoding a lysine derivative).

In another preferred embodiment, the compound of formula V is selected from the group consisting of: L0-GFA, L2-GFA, L3-GFA, L4-GFA, L5-GFA, or L6-GFA, wherein n is an integer of 14 to 16.

In a sixth aspect of the present invention, there is provided a method for preparing an insulin derivative, the method comprising the steps of:

(1) in the presence of a compound of formula VI, a pyrrolysinyl-tRNA synthetase and its cognate tRNA,

culturing a strain containing an insulin-encoding sequence in which the coding sequence for the lysine site in the B chain of insulin is replaced with TAG (encoding a lysine derivative) to provide a compound of formula VII; and

(2) reacting a compound of formula VII with a compound of formula VIII in an inert solvent to obtain an insulin derivative,

in the formula VIII, a, b, c, d, e and f are respectively and independently integers selected from 0 to 10; n is an integer of 14 to 16.

In another preferred embodiment, the resulting insulin derivative comprises:

(a) the a chain of insulin;

(b) the B chain of insulin; and

In a seventh aspect of the present invention, there is provided a method for preventing and/or treating diabetes, the method comprising the steps of: administering to a subject in need thereof an insulin derivative according to the first aspect of the invention.

In another preferred embodiment, the subject is a human or non-human mammal.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The present inventors have conducted extensive and intensive studies to obtain an insulin derivative, and as a result of experiments, the insulin derivative has a remarkably prolonged half-life while maintaining biological activity. The invention also provides pharmaceutical application of the insulin derivative and an effect of the insulin derivative in treating or preventing diabetes. On this basis, the inventors have completed the present invention.

The ideal effect of long acting insulin is to reestablish basal insulin secretion in diabetic patients by injecting the insulin as few times as possible. Chemical modification is one of the ways to obtain long-acting insulins, and the chemical modifier must be structurally stable, non-toxic, non-antigenic and of a suitable size and molecular weight. Through specific chemical modification, the invention can prolong the half-life period and reduce the antigenicity of the insulin while maintaining the biological activity. The modified insulin derivative is a high molecular compound with better biocompatibility, has no toxicity to human bodies, and increases the water solubility of the medicine. And can reduce the clearance rate of glomeruli to the glomeruli and increase the half-life of the drug in circulation in vivo, thereby obtaining long-acting effect.

Term(s) for

Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Fatty acid acyl compounds

The recombinant insulin protein of the butynyloxycarbonyl-lysine is connected with the fatty acid acyl compound through click reaction to obtain a series of insulin derivatives with obviously prolonged half-life.

The fatty acid acyl compound of the invention is a fatty acid acyl compound with 14-18 carbons, and the structural formula is shown as the following formula V or a compound with formula VIII:

wherein: a. b, c, d, e and f are each independently integers selected from 0 to 10; n is an integer of 14 to 16.

In another preferred embodiment, the fatty acid acyl compound is selected from the group consisting of: L0-GFA, L2-GFA, L3-GFA, L4-GFA, L5-GFA, or L6-GFA, wherein n is an integer of 14 to 16.

In another preferred embodiment, the fatty acid acyl compound is selected from the group consisting of: L0-GFA16, L2-GFA16, L3-GFA16, L4-GFA16, L5-GFA16, or L6-GFA 16.

Insulin derivatives

As used herein, the terms "insulin analogue", "insulin derivative", "derivative of the invention (insulin)" are used interchangeably and all refer to insulin derivatives according to the first aspect of the invention.

Specifically, the insulin derivatives include:

(a) the a chain of insulin;

(b) the B chain of insulin; and

(i) at least one group X, wherein the group X is a group of formula I, or

(ii) At least one group Y, wherein the group Y is a group of formula II,

GIVEQCCTSICSLYQLENYCN(SEQ ID NO.:1)

GIVEQCCTSICSLYQLENYCG(SEQ ID NO.:2)

FVNQHLCGSHLVEALYLVCGERGFFYTPKT(SEQ ID NO.:3)

FVNQHLCGSHLVEALYLVCGERGFFYTPK(SEQ ID NO.:4)

FVNQHLCGSHLVEALYLVCGERGFFYTKPT(SEQ ID NO.:5)

FVNQHLCGSHLVEALYLVCGERGFFYTDKT(SEQ ID NO.:6)

In the present invention, insulin derivatives include insulin, insulin precursors and variants of insulin. The variants of insulin differ from any naturally occurring insulin but can nevertheless perform similar actions as human insulin in a glycemic controlled manner in humans. Through genetic engineering of the underlying DNA, the amino acid sequence of insulin can be altered, thereby altering its absorption, distribution, metabolism and secretion properties. Improvements include insulin analogs that are more readily absorbed by the injection site and therefore act more rapidly than the subcutaneous injection of native insulin, intended to supply the drug level of insulin required for meal time (prandial insulin); whereas those insulin analogues, which are slowly released between 8 hours and 24 hours, are intended to provide basal levels of insulin during the day (basal insulin), especially at night. Fast-acting insulin analogs include insulin lispro (li et al) and insulin aspart (Novo Nordisk), while long-acting insulin analogs include NPH insulin, insulin glulisine (Sanofi-Aventis), insulin detemir (Novo Nordisk), and insulin glargine (cenofil-amplat).

As used herein, the term "variant" includes any variant in which (a) one or more amino acid residues are replaced with a naturally or non-naturally occurring amino acid residue; (b) the order of two or more amino acid residues is reversed; (c) both (a) and (b) are present simultaneously; (d) a spacer group is present between any two amino acid residues; (e) one or more amino acid residues are in peptoid form; (f) (iv) the (N-C) backbone of one or more amino acid residues of the peptide is modified, or any combination of (a) to (f). Preferably, the variant is one of (a), (b) or (c).

More preferably, one or two amino acid residues are replaced by one or more other amino acid residues. Still more preferably, one amino acid residue is substituted with another amino acid residue. Preferably, the substitutions are homologous.

Homologous substitutions (both substitutions and substitutions as used herein refer to the interchange of an existing amino acid residue with an alternative residue) may occur, i.e., homologous substitutions, e.g., basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitutions may also occur, i.e., substitution of one residue for another, or alternatively including unnatural amino acids such as ornithine, diaminobutyric acid ornithine, norleucine ornithine, pyridylalanine, thienylalanine, naphthylalanine and phenylglycine, a more detailed list of which is set forth below. Also more than one amino acid residue may be modified. As used herein, amino acids are classified according to the following categories: alkalinity: H. k, R, respectively; acidity: D. e; non-polar: A. f, G, I, L, M, P, V, W, respectively; polarity: C. n, Q, S, T, Y are provided.

In addition to amino acid spacers (e.g., glycine or β -alanine residues), suitable spacers that may be inserted between any two amino acid residues of the carrier moiety include: alkyl groups such as methyl, ethyl or propyl. One skilled in the art will appreciate that in another variant form, form (e), includes one or more amino acid residues in peptoid form. For the avoidance of doubt, "peptoid form" is used herein to denote variant amino acid residues in which the alpha-C substituent is located on the N atom of the residue rather than on the alpha-C. Preparation of peptides in peptoid form is known in the art, e.g., SimonRJ et al, PNAS (1992)89(20), 9367-. (f) Type modification can be carried out by the methods described in International publication No. PCT/GB 99/01855. Amino acid variants, preferably of type (a) or (b), preferably occur independently at any position. As noted above, more than one homologous or nonhomologous substitution may occur simultaneously. Other variants may be obtained by reversing the sequence of some amino acid residues within the sequence. In one embodiment, the replacement amino acid residue is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

In a preferred embodiment, the insulin derivative of the invention is derived from human insulin.

The amino acid sequence of animal insulin in different mammals is similar to human insulin (human insulin INN). However, there is considerable activity in vertebrate species. Porcine insulin differs from human diversity by only one amino acid, and bovine insulin differs by only three amino acids. Both of which have approximately the same magnitude of activity on human receptors. Bovine insulin and porcine insulin are insulin analogs (naturally occurring, obtained by extraction from the pancreas of animals) that were first used clinically when human insulin (human insulin rDNA) could not be biosynthesized. Insulin from sharks and some fish may also be effective.

In another preferred embodiment the insulin analogue of the invention is derived from human insulin or a variant thereof. More preferably, the insulin is biosynthetic insulin (human insulin rDNA).

The insulin derivatives of the present invention may be present in the form of salts or esters, in particular pharmaceutically acceptable salts or esters.

Pharmaceutically acceptable salts of the compounds of the present invention include suitable acid addition or base salts thereof. Suitable pharmaceutically acceptable salts are described in Berge et al, review J Pharm Sci,66,1-19 (1977). With strong mineral acids, such as mineral acids (e.g., sulfuric acid, phosphoric acid, or hydrohalic acids); with strong organic carboxylic acids, such as unsubstituted or substituted (e.g., by halogen) alkanecarboxylic acids having 1 to 4 carbon atoms (e.g., acetic acid); with saturated or unsaturated dicarboxylic acids, such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid or terephthalic acid; with hydroxycarboxylic acids, such as ascorbic acid, glycolic acid, lactic acid, malic acid, tartaric acid or citric acid; with amino acids, such as aspartic acid or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as unsubstituted or substituted (e.g. by halogen), alkyl-or aryl-sulfonic acids (e.g. methanesulfonic acid or p-toluenesulfonic acid).

The invention also includes solvate forms of the derivatives of the invention. The terms used in the claims encompass these forms. The invention also relates to various crystalline forms, polymorphs and (anhydrous) aqueous forms of the analogs of the invention. The pharmaceutical industry has established the way in which chemical compounds can be isolated in any such form by slightly modifying the purification and/or isolation procedures of the solvents used in the synthetic preparation of the compounds.

The invention also includes derivatives of the invention in prodrug form. Such prodrugs are generally derivatives of the invention wherein one or more suitable groups are modified such that the modification is reversible upon administration to a human or mammalian subject. Such reversion is typically performed by an enzyme naturally present in the subject, although a second agent may also be administered with such a prodrug to perform the reversion in vivo. Examples of such modifications include esters (such as any of those described above) wherein the reversal may be by an esterase. Other such systems are well known to those skilled in the art.

In one embodiment of the present invention, the insulin derivative of the present application is prepared in the following manner.

By utilizing a protein site-directed modification technology, a recombinant plasmid of a gene coding pyrryllysyl-tRNA synthetase and a recombinant plasmid of a gene coding amber codon suppression tRNA and human proinsulin introduced with N- (butynyloxycarbonyl) -lysine at the position B29 are constructed. Transfecting the recombinant plasmids into host cells, inducing expression in the host cells, separating and purifying to obtain human proinsulin with N- (butynyloxycarbonyl) -lysine introduced into the position B29;

and (2) renaturing the human proinsulin introduced with the N- (butynyloxycarbonyl) -lysine at the position B29 by dialysis, and carrying out enzymolysis treatment on the renatured human proinsulin to obtain the human insulin with the N- (butynyloxycarbonyl) -lysine.

Because the fatty acid acyl compound has an azide group, the insulin protein with terminal alkyne is introduced, and the alkyne and the azide react to generate a1, 2, 3-triazole ring by utilizing the click chemistry reaction principle to form cross-linking.

Therapeutic uses

Another aspect of the invention relates to the use of an insulin derivative as described above in medicine.

Another aspect of the present invention relates to the use of an insulin derivative as described above for the treatment or prevention of diabetes, or for the treatment or prevention of hyperglycemia.

Another aspect of the present invention relates to the use of an insulin derivative as described above for the preparation of a medicament for the treatment of diabetes, or for the treatment or prevention of hyperglycemia. As used herein, the phrase "preparing a drug" includes the use of an analog of the invention directly as a drug, in addition to the use of an analog of the invention in any stage of screening for further therapeutic agents or preparing such a drug.

Another aspect of the invention relates to a method of treating diabetes, or treating or preventing hyperglycemia in a subject in need thereof, said method comprising administering a therapeutically effective amount of an insulin derivative according to the above.

Compared with the prior art, the invention has the beneficial effects that:

(1) compared with the prior art, the insulin derivative has more lasting and stable drug effect, can reduce blood sugar for a long time, and has obviously prolonged half-life.

(2) The insulin derivative of the invention does not cause hypoglycemia, can reduce injection times and has small side effect.

(3) The preparation method has the advantages of few byproducts, high yield, low cost and simple process, and is suitable for large-scale production. Cyanogen bromide cleavage, oxidative sulfitolysis and related purification steps are not required. Without the need to use high concentrations of mercaptans or hydrophobic adsorption resins. The purification steps are few, and the production cost is low.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specifying the detailed conditions in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Example 1: synthesis of recombinant insulin protein containing butynyloxycarbonyl-lysine

A DNA fragment encoding a recombinant insulin protein comprising butynyloxycarbonyl-lysine having the amino acid sequence of SEQ ID No. 7, in which the coding sequence of lysine (K) at position 80 is replaced with TAG (encoding a lysine derivative), was chemically synthesized. The DNA fragment encoding the complete amino acid sequence of SEQ ID No. 7 was then cloned into the modified pBAD-HisA vector. The resulting plasmid was used for expression of recombinant insulin protein with the structure of butynyloxycarbonyl-lysine. This plasmid was transformed into the E.coli strain Top10 together with the enzyme plasmid pEvol-pylRs-pyl. The transformation solution was cultured overnight at 37 ℃ on LB agar medium containing 25. mu.g/mL of kanamycin and 17. mu.g/mL of chloramphenicol. Single colonies were picked and shake-cultured overnight at 220rpm at 37 ℃ in LB liquid medium containing 25. mu.g/mL kanamycin and 17. mu.g/mL chloramphenicol. Then, the overnight culture was inoculated into 100mL TB liquid medium containing 25. mu.g/mL kanamycin and 17. mu.g/mL chloramphenicol, and cultured at 37 ℃ until OD₆₀₀Is 2-4. Then, a 25% arabinose solution was added to the medium to a final concentration of 0.25%, and a 0.1M butynyloxycarbonyl-lysine solution was added to a final concentration of 5mM to induce expression of the fusion protein. The culture solution is cultured for 16-20h, and then centrifuged (10000rpm, 5min, 4 ℃) for collection.

Amino acid sequence of SEQ ID No. 7:

MVSKGEELFTGVTYKTRAEVKFEGDDDDDKTLVNRIELKGIDFENLYFQGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRGIVEQCCTSICSLYQLENYCN, wherein K at position 80 is a lysine to which is covalently attached a butynyloxycarbonyl group.

The fusion protein is expressed as insoluble "inclusion bodies". In order to release the inclusion body, the Escherichia coli cells are crushed in a high-pressure homogenizer, cell fragments and soluble Escherichia coli host protein are removed by 5000g of centrifugation, the inclusion body is washed by a solution containing Tween 80, EDTA and NaCl, and then the inclusion body is washed by pure water for 1-2 times. After washing the inclusion bodies were dissolved in 7.5M urea pH 10.5-11.5 containing 2-10mM beta-mercaptoethanol to a total protein concentration of 10-25mg/ml after dissolution. The sample is diluted 5-10 times, maintained at 4-80 and subjected to conventional folding at pH 10.5-11.7 for 14-30 hours. At 18-25 deg.C, pH is maintained at 8.0-9.5, and the mixture is digested with trypsin and carboxypeptidase B for 10-20 hours, and then 0.45M ammonium sulfate is added to stop the digestion reaction. The reverse phase HPLC analysis showed that the yield of this cleavage step was higher than 90%. The insulin analogue obtained after cleavage of trypsin with carboxypeptidase B was named butynyloxycarbonyl-lysine-human insulin. And (3) clarifying the sample by membrane filtration, and primarily purifying by hydrophobic chromatography by using 0.45M ammonium sulfate as a buffer solution A and pure water as a buffer solution B to obtain crude extract of the butynyloxycarbonyl-lysine-human insulin, wherein the electrophoretic purity reaches 90%. Then purifying by polymer reversed-phase filler and C8 reversed-phase filler to finally obtain the butynyloxycarbonyl-lysine-human insulin with the purity higher than 99 percent.

Example 2: synthesis of L0-GFA 16-insulin (n is 14)

Because the fatty acid acyl compound has an azide group, the insulin protein with terminal alkyne is introduced, and the alkyne and the azide react to generate a1, 2, 3-triazole ring by utilizing the click chemistry reaction principle to form cross-linking. To a 1.5ml clean centrifuge tube was added 4. mu.L copper sulfate (50. mu.M), followed by 3. mu.L BTTAA (300. mu.M) and 10. mu.L of the compound IV prepared in example 1 (human insulin protein N- (butynyloxycarbonyl) -lysine) (approximately 5. mu.M) in that order. At this point, the solution may be diluted to the appropriate volume or protein concentration. To this solution was added 1. mu.L of L0-GFA16 (1mM), and 2. mu.L of sodium ascorbate (2.5mM) to initiate the reaction. After about 1 hour at room temperature, 5. mu.L of SDS-PAGE sample buffer was added, heated to 100 ℃ for 10min, and analyzed by 12% SDS-PAGE. The gel was recovered, imaged on the gel, analyzed by fluorescence, and then stained with Coomassie Brilliant blue.

Example 3: synthesis of L2-GFA 16-insulin, L3-GFA 16-insulin, L4-GFA 16-insulin, L5-GFA 16-insulin, L6-GFA 16-insulin (n is 14)

To a 1.5ml clean centrifuge tube was added 4. mu.L copper sulfate (50. mu.M), followed by 3. mu.L BTTAA (300. mu.M) and 10. mu.L of the N- (butynyloxycarbonyl) -lysine human insulin protein prepared in example 1 (ca. 5. mu.M) in that order. At this point, water may need to be added to dilute the solution to the correct volume or protein concentration. To this solution was added 1. mu.L of L0-GFA16 (1mM), and 2. mu.L of sodium ascorbate (2.5mM) to initiate the reaction. After about 1 hour at room temperature, 5. mu.L of SDS-PAGE sample buffer was added, heated to 100 ℃ for 10min, and analyzed by 12% SDS-PAGE. The gel was recovered, imaged on the gel, analyzed by fluorescence, and then stained with Coomassie Brilliant blue.

Similarly, the structures of products obtained by click-reacting the IV compound prepared in example 1 with L3-GFA16, L4-GFA16, L5-GFA16, and L6-GFA16, respectively, are as follows.

Example 4: pharmacokinetic study of insulin derivatives in rat

The half-life of the insulin produced by the reaction is prolonged while the biological activity is maintained. The experiment is divided into a control group and a test group, recombinant human insulin and L6-GFA 16-insulin are respectively injected subcutaneously, the administration dose is 0.45mg/kg, and the administration is carried out once. Blood was collected at time points of 15min, 30min, 1h, 2h, 3h, 5h, 7h, 12h, and 24h after administration for each group of animals. And detecting the content of different insulin analogues by an LC-MS/MS analysis method. Plasma concentration data were statistically analyzed using the pharmacokinetic data analysis software WinNonlin 7.0 and drug parameters were calculated using the non-compartmental model (NCA) (table 1). As can be seen from Table 1, the peak reaching time of the drug in each group of animals is close and is 0.5-1 hour, the half-life of the L6-GFA 16-insulin is 3-4 times of that of the recombinant human insulin, the exposure time of the drug in vivo is prolonged, and the exposure amount is increased.

TABLE 1 major pharmacokinetic parameters in the animals of each group

The above experiment was repeated except that L2-GFA 16-insulin, L3-GFA 16-insulin, L4-GFA 16-insulin, and L5-GFA 16-insulin according to the present invention were used. As a result, the insulin derivative provided by the invention has the advantages that the biological activity is maintained, and meanwhile, the half-life period is remarkably prolonged.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Ningbo spread Biotechnology Ltd

<120> insulin derivatives and process for producing the same

<130> P2018-1685

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Claims

1. An insulin derivative, comprising:

(a) the a chain of insulin;

(b) the B chain of insulin; and

2. The insulin derivative according to claim 1, wherein the group Y is a group selected from the group consisting of:

3. the insulin derivative according to claim 1, wherein the A chain of the insulin has a sequence as shown in SEQ ID No. 1 or 2.

4. The insulin derivative according to claim 1, wherein the B chain of the insulin has a sequence as shown in SEQ ID No. 3, 4, 5 or 6.

5. The insulin derivative according to claim 1, wherein the insulin derivative is selected from the group consisting of:

6. a pharmaceutical composition comprising the insulin derivative of claim 1, and a pharmaceutically acceptable carrier.

7. Use of insulin derivatives according to claim 1 for the preparation of a medicament or a preparation for the prophylaxis and/or treatment of diabetes, hyperglycemia and other diseases where a lowering of blood glucose would be beneficial.

8. A process for the preparation of an insulin derivative, said process comprising the steps of:

(1) culturing a strain comprising an insulin coding sequence in the presence of an X group-lysine, a pyrrolysinyl-tRNA synthetase and its cognate tRNA, wherein the coding sequence for the lysine site in the B chain of insulin is TAG, thereby producing an insulin derivative, wherein the insulin derivative comprises:

(a) the a chain of insulin;

(b) the B chain of insulin; and

(c) a modifying group L attached to a lysine site of the B chain of the insulin and being a group X as defined in claim 1; and optionally

(2) Isolating said insulin derivative from the fermentation product.

9. A process for the preparation of an insulin derivative, said process comprising the steps of:

culturing a strain containing an insulin coding sequence, wherein the coding sequence for a lysine site in the B chain of insulin is TAG, thereby obtaining a compound of formula IV; and

10. A process for the preparation of an insulin derivative, said process comprising the steps of:

culturing a strain containing an insulin-encoding sequence, wherein the encoding sequence of the lysine site in the B chain of insulin in the encoding sequence is TAG, thereby obtaining a compound of formula VII; and