CN104447981B - Conjugate of hydroxyl-terminated pegylated human insulin and analogues thereof - Google Patents

Conjugate of hydroxyl-terminated pegylated human insulin and analogues thereof Download PDF

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CN104447981B
CN104447981B CN201410817586.0A CN201410817586A CN104447981B CN 104447981 B CN104447981 B CN 104447981B CN 201410817586 A CN201410817586 A CN 201410817586A CN 104447981 B CN104447981 B CN 104447981B
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CN104447981A (en
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陈勇
罗华
李红亮
李祥
蒋利敏
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Chongqing Punuowei Biotechnology Co ltd
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    • C07—ORGANIC CHEMISTRY
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

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Abstract

The invention belongs to the field of insulin (C07K14/62) and relates to a conjugate of hydroxyl-terminated pegylated human insulin and analogues thereof. The immunogenicity is significantly reduced and the conjugate has higher drug safety. In addition, the invention also relates to a preparation method of the conjugate and relates to an application of the conjugate in prevention or treatment.

Description

Conjugate of hydroxyl-terminated pegylated human insulin and analogue thereof
Technical Field
The invention belongs to the field of insulin technology (C07K14/62), and more particularly relates to a conjugate formed by modifying human insulin or an analogue thereof by hydroxyl-terminated polyethylene glycol, which has higher medication safety. In addition, the invention also relates to a preparation method and application of the conjugate.
Background
Diabetes Mellitus (DM) is a metabolic disease of pancreatic dysfunction, and various pathogenic factors such as genetic factors, immune dysfunction, microbial infection and toxins thereof, free radical toxins, mental factors and the like act on the body to cause pancreatic islet hypofunction and insulin resistance, so that a series of metabolic disorders such as sugar, protein, fat, water, electrolyte and the like are caused. Clinically, hyperglycemia is taken as a main characteristic. Among them, type 1 diabetes belongs to insulin-dependent diabetes, and its patients must rely on exogenous insulin for substitution treatment; type 2 diabetes is non-insulin dependent diabetes mellitus and patients are often eventually treated with insulin as the condition progresses due to relative insufficiency of insulin caused by development of insulin resistance or damaged islet beta cells in the body.
Human insulin (Ins), which consists of A, B two peptide chains, wherein the a chain contains 21 amino acids, and the amino acid sequence is: gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn TyrCys Asn; the B chain contains 30 amino acids, and the amino acid sequence is as follows: phe Val Asn Gln His Leu Cys Gly Ser His LeuVal Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr. Human insulin shares three disulfide bonds, with cysteine at position 6 of the A chain (A6(Cys)) forming a disulfide bond with cysteine at position 11 of the A chain (A11 (Cys)); cysteine 7 of the A chain (A7(Cys)) forms a pair of disulfide bonds with cysteine 7 of the B chain (B7 (Cys)); the cysteine at position 20 of the A chain (A20(Cys)) forms a pair of disulfide bonds with the cysteine at position 19 of the B chain (B19(Cys)), which join the two chains of A, B.
Many human insulin analogues have been developed, and among them, those approved for clinical use or clinical research mainly include DesB30 (the amino acid sequence of which is deletion of threonine (Thr) at position 30 in the B chain of human insulin based on the amino acid sequence of human insulin), insulin glargine (Lantus, the amino acid sequence of which is mutation of asparagine (Asn) at position 21 in the a chain to glycine (Gly) based on the amino acid sequence of human insulin, and two arginines (Arg) added after the amino acid sequence of B chain of human insulin), insulin Aspart (Aspart, the amino acid sequence of which is mutation of proline at position 28 in the B chain of human insulin to aspartic acid (Asp) based on the amino acid sequence of human insulin), and insulin Glulisine (Glulisine, the amino acid sequence of which is mutation of glutamic acid (Glu) at position 3 in the B chain of human insulin to lysine (Lys), and lysine 29 (Lys) of B chain is mutated into glutamic acid (Glu)), insulin Lispro (Lispro, the amino acid sequence of which is based on the amino acid sequence of human insulin, proline 28 (Pro) of B chain of human insulin is mutated into lysine (Lys), and lysine 29 of B chain is mutated into proline), insulin detemir (Levemir), insulin Degludec (Degludec), and the like.
Polyethylene glycol (PEG) modification can prolong the half-life of the protein in vivo, and is generally also thought to significantly reduce immunogenicity, thereby improving drug safety. Conjugates of pegylated insulin and analogs thereof have also been reported, for example, as described in chinese patent (application) nos. 02815261, 200410089050, 200610118923, 200710085552, 200780036662, 200810011478, 200810232828, 200980122164, 201110063821. Since the end of monomethoxy polyethylene glycol (mPEG) is a non-reactive group such as methoxy, and only coupling with the protein to be modified can be directionally depended on the coupling reaction functional group (such as ester-containing group, aldehyde-containing group, amino group, carboxyl group, even hydroxyl group, and the like) at the other end, the specificity of the reaction is improved, so that mPEG is basically selected and used in the prior art of modifying proteins (especially insulin and the like) by polyethylene glycol.
However, there is increasing evidence that polyethylene glycol itself introduces new immunogenicity, and can also induce immune response in the body, and generate anti-PEG antibodies, so that on one hand, polyethylene glycol modified drugs are neutralized, the curative effect is reduced, and more seriously, potential medication safety problems are caused, immune allergy is induced, and even the death of patients is caused. For example, a number of reports have shown the use of Pegliotidase (pegylated recombinant pig urate oxidase, trade name Krystex xxa) in patientsTM) Thereafter, 70-90% of patients detected anti-PEG antibodies, especially in cases of death during their second and third phase clinical trials, were thought to be associated with their immune allergies (cortex, j.et. et. Control of plasma acid additives at risk for tumor Lysis: efficacy and safety of rasburcasee alone and rasburcaseeflowed by allopurinol compared with allopurinol alone--results of a multicenter phase III study.JClin Oncol，2010.28(27)：p.4207-13；Sundy，J.S.et al.Reduction of plasma urate levels followingtreatment with multiple doses of Pegloticase(polyethylene glycol-conjugated uricase)in patientswith treatment-failure gout：results of a phase II randomized study.Arthritis Rheum，2008.58(9)：p.2882-91；Krystexxa Prescribing Information：http://www.krystexxa.com/pdfs/KRYSTEXXAPrescribing_Information)。
The inventor also finds that the conjugate obtained by modifying human insulin and analogues thereof with polyethylene glycol selected in the prior art can also generate an anti-PEG antibody, and because insulin needs to be used for a long time, the medication safety problem cannot be underestimated under the trend that the drug administration and the public pay more attention to the drug safety. After long and arduous research, the inventor of the invention has found that, in the case of almost abandoning polyethylene glycol modification system and changing to other modification system (such as human IgG or HSA, etc.) with possibly lower immunogenicity to human, it is quite unexpected that, although hydroxyl-terminated polyethylene glycol contains reactive hydroxyl group, interferes reaction and increases by-products, so that the prior art is almost not selected to use, the conjugate of human insulin and analogues thereof with pegylated terminal hydroxyl group can obviously reduce immunogenicity and greatly increase medication safety. In addition, the conjugate can basically follow the approval process and the specification of the polyethylene glycol modified drug, and the cost for developing the drug is reduced.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide novel conjugates of pegylated human insulin or analogues thereof. In addition, the invention also provides a preparation method and application of the conjugate.
In particular, in a first aspect, the invention provides a conjugate of insulin or an analogue thereof as shown in formula I or a pharmaceutically acceptable salt thereof,
{[HO-(CH2CH2O)n]p-L}t-Y (formula I)
Wherein,
y is insulin or an analog thereof;
l is a linking group;
n is an integer of 50 to 1850, preferably an integer of 220 to 1370, and more preferably an integer of 450 to 910;
p is an integer of 1 to 5, preferably an integer of 1 to 3, more preferably an integer of 1 to 2, and most preferably 1;
t is an integer of 1 to 12, preferably an integer of 1 to 5, more preferably an integer of 1 to 3, and most preferably 1;
-is a covalent bond.
In this context, a structural formula has the meaning understood by a person skilled in the art of biological macromolecules, which is not exactly the same as the strict chemical formula. Wherein the parenthesis (i.e., "()") has a meaning in the chemical sense, meaning that the structures in the parenthesis are covalently linked in a linear sequence, repeating n times.
The middle brackets (i.e., "[ ]") refer to the parallel covalent attachment of the structure in the middle brackets to different sites on L (or L '), the attachment of the structure to L (or L') being p in number. When p is more than 1, the compound is branched polyethylene glycol; when p is 1, the polyethylene glycol is straight-chain polyethylene glycol. The latter is preferred in the present invention.
The parenthesis (i.e. "{ }") values are for structures in parenthesis covalently attached to different sites on Y in parallel, for a total of t such structures attached to Y. When t is more than 1, the insulin is the multi-PEG modified insulin or the analogues thereof; when t ═ 1, it is mono-PEG modified insulin or an analog thereof. The latter is preferred in the present invention.
In a particular embodiment of the invention, p is 1 and t is 1, i.e. a single linear PEG modification, correspondingly,the structure of the insulin or the analogue thereof is HO- (CH)2CH2O)n-L-Y. In addition, in a specific embodiment of the present invention, HO- (CH) in formula I2CH2O)nThe molecular weight of the-L moiety is 20KD or 40 KD.
As used herein, "pharmaceutically acceptable salt" refers to salts which are suitable for contact with the tissues of humans or animals without excessive toxicity, irritation, allergic response, or the like. Pharmaceutically acceptable salts are well known in the art. Such salts may be prepared during the final isolation and purification of the conjugate of the invention, or the conjugate may be prepared separately by reaction with a suitable organic or inorganic acid or base. Representative acid addition salts include, but are not limited to, acetate, dihexanoate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, 3-phenylpropionate, propionate, succinate, tartrate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Preferred acids that can be used to form pharmaceutically acceptable salts are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, oxalic acid, maleic acid, succinic acid and citric acid. Cations in pharmaceutically acceptable base addition salts include, but are not limited to, alkali or alkaline earth metal ions such as lithium, sodium, potassium, calcium, magnesium, and aluminum, and the like, as well as non-toxic quaternary ammonium cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, diethylamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like. Preferred base addition salts include phosphate, tris and acetate salts. The conjugates of the invention may be used alone or in the form of a pharmaceutically acceptable salt.
Preferably in the conjugate of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, the insulin is human insulin. The amino acid sequence of human insulin is well known in the art and can be obtained by extraction, artificial chemical synthesis or expression by recombinant DNA techniques. Human insulin was synthesized in the 1960 s.
Also preferred in the conjugate of the first aspect of the invention or a pharmaceutically acceptable salt thereof, the insulin analogue is DesB30, insulin glargine (Lantus), insulin Aspart (Aspart), insulin detemir, insulin Glulisine (Glulisine), insulin Degludec or insulin Lispro (Lispro). The insulin analogues have been approved for clinical use or clinical research, and have the activity and function of insulin, even better than that of human insulin.
Herein, the linking group is a residue (i.e., a residual portion) of the coupling reaction functional group through the coupling reaction. Both the coupling reactive functional group (prior to coupling reaction) and the linking group (after coupling reaction) are well known in the art of PEG modification of proteins. Generally, the linking group and/or coupling reactive functional group is small and has a molecular weight of less than 300Da, preferably less than 200 Da. For example, in a particular embodiment of the invention, the coupling reactive functional group is a succinimidyl propyl ester group and correspondingly, the linking group is the residue of a succinimidyl propyl ester group coupled to an amino group.
Since the conjugates of the invention are obtained with PEG bearing a terminal hydroxyl group, the coupling reaction functional group does not contain a hydroxyl group in order to prevent this hydroxyl group from interfering with the coupling reaction. The coupling reactive functional group should be capable of coupling with an active group (e.g., amino, carboxyl, thiol, etc.) on insulin or the like, and preferably the coupling reactive functional group of the present invention comprises an acetal group, an aldehyde group, a succinimide group, a maleimide group, a vinylsulfone group, an iodoacetamide group, an ester group, a carbonate group, a carboxyl group, an amino group, an aminoxy group, a thiol group, an allyl group, a vinyl group, an ethynyl group, or an azido group.
The linking group of the present invention does not contain an acetal group, an aldehyde group, a succinimide group, a maleimide group, a vinylsulfone group, an iodoacetamide group, an ester group, a carbonate group, a carboxyl group, an amino group, an aminoxy group, a thiol group, an allyl group, a vinyl group, an ethynyl group or an azido group due to a coupling reaction.
Preferably in the conjugate of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, the conjugation is site-directed, such that the homogeneity (or purity) of the conjugate is increased. For example, the coupling site of Y is an amino group and/or an alpha amino group, preferably an amino group and/or an alpha amino group of the B chain. Wherein, amino refers to amino on lysine side chain, alpha amino refers to free amino of amino acid at N end of insulin chain. Site-directed coupling may be achieved by the method of the second aspect of the invention.
In a second aspect, the invention provides a process for the preparation of a conjugate of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, which comprises administration of [ HO- (CH)2CH2O)n]p-L' is coupled with Y, wherein,
y is insulin or an analog thereof;
l' is a coupling reaction functional group;
n is an integer of 50 to 1850, preferably an integer of 220 to 1370, and more preferably an integer of 450 to 910;
p is an integer of 1 to 5, preferably an integer of 1 to 3, more preferably an integer of 1 to 2, and most preferably 1;
-is a covalent bond.
The preparation method of the second aspect of the invention can obtain t [ HO- (CH)2CH2O)n]p-L' is a product of coupling with 1Y, wherein t is an integer from 1 to 12, preferably an integer from 1 to 5, more preferably an integer from 1 to 3, most preferably 1.
In the absence of contrary or contradictory indications, terms mentioned in connection with one part of the invention may be defined or interpreted with reference to terms in other parts of the invention. For example, Y, i.e. insulin or an analogue thereof, may be referred to Y of the first aspect of the invention and preferred features thereof. As another example, L', a coupling reactive functional group, is well known in the art of PEG modification of proteins, and reference may be made to a number of publications relating to conjugates of pegylated proteins, e.g., those listed in the background section relating to conjugates of pegylated insulin and analogs thereof.
Preferably, the preparation method of the second aspect of the present invention comprises
(1) By [ HO- (CH) under conditions suitable for coupling with amino and/or alpha-amino groups2CH2O)n]p-L' (preferably HO- (CH)2CH2O)n-L') is coupled with Y;
(2) purifying (preferably by chromatography, such as gel filtration chromatography and/or ion exchange chromatography) to obtain the conjugate of insulin or its analogue shown in formula I or its pharmaceutically acceptable salt.
Preferably wherein [ HO- (CH) is used under conditions suitable for coupling with alpha amino groups2CH2O)n]p-L' (preferably HO- (CH)2CH2O)nThe coupling of-L') to Y is in a solution at pH 5.0-8.0 [ HO- (CH)2CH2O)n]p-L' (preferably HO- (CH)2CH2O)n-L') and Y are mixed and reacted at a molar ratio of 1-4: 1-4 at 4-37 ℃.
Also preferred is the reaction of [ HO- (CH) wherein2CH2O)n]p-L' (preferably HO- (CH)2CH2O)nThe coupling of-L') to Y is carried out in a solution at pH 8.0-11.5 [ HO- (CH)2CH2O)n]p-L' (preferably HO- (CH)2CH2O)n-L') and Y are mixed and reacted at a molar ratio of 1-4: 1-4 at 4-37 ℃.
The reaction can be stopped by acidification with an acid (preferably to pH 2.5-3.5) and then can be directly purified. In a specific embodiment of the invention, the purification is carried out by sequentially passing through cation chromatography and anion exchange chromatography.
In a third aspect, the present invention provides a pharmaceutical composition comprising a conjugate of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
Herein, "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, adjuvant, encapsulating material or other formulation excipient. The pharmaceutical compositions may be formulated into various dosage forms as required for therapeutic purposes, administration routes, and the like, according to techniques well known in the art, and preferably the compositions are in unit dosage forms such as tablets, films, pills, capsules (including sustained release or delayed release forms), powders, granules, tinctures, syrups and emulsions, injections or suspensions, aerosols or liquid sprays, drops, injections or powder injections, automatic injection devices, or suppositories. In a particular embodiment of the invention, the administration is by injection in the form of an injection, so that the conjugate of the first aspect of the invention or a pharmaceutically acceptable salt thereof may be combined with a non-toxic pharmaceutically acceptable inert carrier for injection, such as isotonic glucose solution, glycerol, physiological saline or a combination thereof. The pharmaceutical composition may also contain adjuvants such as protein protectant, such as Human Serum Albumin (HSA), low molecular weight peptide, amino acids and/or metal cation.
The pharmaceutical composition of the third aspect of the present invention may be used for the treatment and/or prevention of insulineable diseases, preferably for the long-term treatment and/or prevention of insulineable diseases, such as diabetes, in particular type 1 diabetes and/or type 2 diabetes.
In a fourth aspect, the invention provides the use of a conjugate of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament of low immunogenicity.
The medicaments or pharmaceutical compositions of the invention can be administered by means of administration known to the person skilled in the art, such as parenteral, oral, rectal, sublingual, pulmonary, transdermal, iontophoretic, vaginal and intranasal administration, preferably parenteral administration, such as subcutaneous, intramuscular or intravenous injection. The dosage to be administered varies depending on the form of the preparation and the desired duration of action and the condition of the subject to be treated, and the amount required for the actual treatment can be conveniently determined by a physician according to the actual condition (e.g., the condition, body weight, etc. of the patient). Due to the excellent pharmacokinetic and pharmacodynamic properties of the conjugate of the invention, the amount of insulin or the like to be administered is greatly reduced, and the frequency of administration is also greatly reduced.
In the use according to the fourth aspect of the invention, the medicament is for the treatment and/or prophylaxis of insulineable diseases, preferably for the long-term treatment and/or prophylaxis of insulineable diseases, such as diabetes, in particular type 1 diabetes and/or type 2 diabetes.
In a fifth aspect, the invention provides hydroxy terminated polyethylene glycols or [ HO- (CH)2CH2O)n]p-L' (preferably HO- (CH)2CH2O)n-L') in the preparation of conjugates of insulin or analogues thereof of low immunogenicity. Wherein, L' is a coupling reaction functional group; n is an integer of 50 to 1850, preferably an integer of 220 to 1370, and more preferably an integer of 450 to 910; p is an integer of 1 to 5, preferably an integer of 1 to 3, more preferably an integer of 1 to 2, and most preferably 1; -is a covalent bond. Preferably wherein the conjugate of insulin or an analogue thereof is a conjugate of the first aspect of the invention or a pharmaceutically acceptable salt thereof.
The conjugate of the invention has low immunogenicity, which is reflected in two aspects, firstly, the conjugate obviously reduces the immunogenicity brought by the polyethylene glycol, even reduces the immunogenicity to a level which can not be detected after long-term (about 40 days) use; secondly, the conjugate also further reduces the immunogenicity of the insulin or the analogue itself. Such low immunogenicity is particularly beneficial for pharmaceutical safety, especially for long-term pharmaceutical safety.
For clarity, herein, low immunogenicity may be defined as a reduced immunogenicity relative to a conjugate formed after replacement of the terminal hydroxyl group with a methoxy group in a conjugate according to the first aspect of the invention, i.e. low immunogenicity relative to a conjugate of insulin or an analogue thereof of formula II:
{[CH3O-(CH2CH2O)n]p-L}t-Y (formula II)
Wherein,
y is insulin or an analog thereof;
l is a linking group;
n is an integer of 50 to 1850, preferably an integer of 220 to 1370, and more preferably an integer of 450 to 910;
p is an integer of 1 to 5, preferably an integer of 1 to 3, more preferably an integer of 1 to 2, and most preferably 1;
t is an integer of 1 to 12, preferably an integer of 1 to 5, more preferably an integer of 1 to 3, and most preferably 1;
-is a covalent bond.
The invention has the beneficial and even unexpected effects that the conjugate of the hydroxyl-terminated pegylated human insulin or the analogue thereof is obtained by modifying the human insulin or the analogue thereof by adopting the hydroxyl-terminated polyethylene glycol, and on the basis of keeping the excellent properties of pharmacokinetics and pharmacodynamics, the conjugate remarkably reduces the immunogenicity brought by the polyethylene glycol, even to the level which can not be detected by long-term (about 40 days), and further reduces the immunogenicity of the insulin or the analogue thereof, thereby improving the safety of long-term use of the drug.
For the sake of understanding, the present invention will be described in detail below by way of specific examples. It is to be expressly understood that the description is illustrative only and is not intended as a definition of the limits of the invention. Many variations and modifications of the present invention will be apparent to those skilled in the art in light of the teachings of this specification. In addition, the present invention incorporates publications which are intended to more clearly describe the invention, and which are incorporated herein by reference in their entirety as if reproduced in their entirety.
Detailed Description
The present invention will be described more fully hereinafter with reference to the following examples. Unless otherwise indicated, reference may be made to the protocols and guidelines for animal experiments and SFDA, in the books organic syntheses, molecular cloning, cellular experiments, immunodetection techniques, etc., and to the instructions for use of the reagents and equipment used, which are known to those skilled in the art. Wherein, the reagents and equipment used are commercially available, unless otherwise specified.
EXAMPLE 1 preparation of conjugates of HO-PEGylated insulin and analogs thereof site-directed conjugated to amino groups
This example describes, primarily, the preparation of a modified amino group of the B chain lysine of a threonine-removed B chain 30-position human insulin analogue (DesB30) with a hydroxy-terminated polyethylene glycol succinimidyl propyl ester (HO-PEG-SPA-20K) having a molecular weight of 20KD, to obtain the corresponding conjugate (HO-PEG-20K-DesB30), wherein HO-PEG is site-directed coupled to the amino group of the B chain lysine. Specifically, 1g of DesB30 was dissolved in 20mM boric acid buffer (pH10.5) to prepare a 500ml solution, and 6g of HO-PEG-SPA-20K was added thereto and reacted at 300rpm at room temperature (25 ℃ C.) for 60 min. Then, 2M hydrochloric acid was added to reach pH3.0 to terminate the reaction. After the reaction was terminated, the reaction solution was directly applied to an SP Sepharose FF column equilibrated with an equilibration buffer (50mM sodium acetate buffer, pH 3.5). After the loading, 6 column volumes of the equilibration buffer were passed through the column, and then eluted with 50mM Tris-HCl buffer (pH8.0), and the eluted peak having a molecular weight of single PEG modification by SDS-PAGE was collected. Adding ethanol to the elution peak until the final concentration of the ethanol is 60% (V/V), loading the elution peak on a SOURCE 15Q column well balanced by an equilibrium buffer solution (60% (V/V) ethanol, 20mM Tris-HCl, pH8.0), after loading, firstly, using the equilibrium buffer solution to flow until the ultraviolet absorption baseline of the flowing solution is stable, then using an eluent (60% (V/V) ethanol, 20mM Tris-HCl, 0-0.3M NaCl, pH8.0) to carry out linear elution with NaCl gradient concentration, and collecting the maximum eluted peak, namely HO-PEG-20K-DesB 30. The purity is more than 95 percent through RP-HPLC detection; the coupling site is on the amino group of B chain lysine by peptide mapping analysis.
Referring essentially to the above preparation except that DesB30 was replaced with human insulin or an analog thereof, respectively: ins, Aspart, Lispro and Glulisine, and obtaining products HO-PEG-20K-Ins, HO-PEG-20K-AP, HO-PEG-20K-LP and HO-PEG-20K-GL of HO-PEG coupling at B chain lysine amino group of corresponding human insulin or analogues thereof respectively.
In addition, modifications were performed with mPEG instead of HO-PEG described above to obtain the products mPEG-20K-DesB30, mPEG-20K-Ins, mPEG-20K-AP, mPEG-20K-LP, and mPEG-20K-GL, respectively, with mPEG coupled to the B chain lysine amino group of the corresponding human insulin or analog thereof.
EXAMPLE 2 preparation of conjugates of HO-PEGylated insulin and analogs thereof site-directed conjugated to the alpha amino group
This example describes, primarily, the preparation of a human insulin analogue (DesB30) modified from the N-terminal alpha amino group of the B chain of threonine-30 in the B chain of human insulin by a hydroxy-terminated polyethylene glycol succinimidyl propyl ester (HO-PEG-SPA-20K) with a molecular weight of 20KD, to obtain the corresponding conjugate (HO-PEG-20K-DesB30-B1) in which HO-PEG is site-specific conjugated to the N-terminal alpha amino group of the B chain. Specifically, 1g of DesB30 was prepared into 500ml of a solution in 20mM phosphate buffer (pH7.5), and 6g of HO-PEG-SPA-20K was added thereto at 300rpm for 60min at room temperature (25 ℃). Then, 2M hydrochloric acid was added to reach pH3.0 to terminate the reaction. After the reaction was terminated, the reaction solution was directly applied to an SP Sepharose FF column equilibrated with an equilibration buffer (50mM sodium acetate buffer, pH 3.5). After the loading, 6 column volumes of the equilibration buffer were passed through the column, and then eluted with 50mM Tris-HCl buffer (pH8.0), and the eluted peak having a molecular weight of single PEG modification by SDS-PAGE was collected. Adding ethanol to the elution peak until the final concentration of the ethanol is 60% (V/V), loading the elution peak on a SOURCE 15Q column well balanced by an equilibrium buffer solution (60% (V/V) ethanol, 20mM Tris-HCl, pH8.0), after loading, firstly, using the equilibrium buffer solution to flow until the ultraviolet absorption baseline of the flowing solution is stable, then using an eluent (60% (V/V) ethanol, 20mM Tris-HCl, 0-0.3M NaCl, pH8.0) to carry out linear elution with NaCl gradient concentration, and collecting the maximum eluted peak, namely HO-PEG-20K-DesB 30-B1. The purity is more than 95 percent through RP-HPLC detection; the coupling site is on the alpha amino group at the N-terminal of the B chain by peptide mapping analysis.
Referring essentially to the above preparation except that DesB30 was replaced with human insulin or an analog thereof, respectively: ins, Aspart, Lispro and Glulisine, products HO-PEG-20K-Ins-B1, HO-PEG-20K-AP-B1, HO-PEG-20K-LP-B1 and HO-PEG-20K-GL-B1, wherein HO-PEG is coupled with B chain lysine amino group of corresponding human insulin or analogues thereof, are respectively obtained.
EXAMPLE 340 preparation of conjugates of HO-PEGylated insulin and analogs thereof
This example describes, primarily, the preparation of a modified amino group of the B chain lysine of a threonine-removed B chain 30-position human insulin analogue (DesB30) with a hydroxyl-terminated polyethylene glycol succinimidyl propyl ester (HO-PEG-SPA-40K) having a molecular weight of 40KD, to obtain the corresponding conjugate (HO-PEG-40K-DesB30), wherein HO-PEG is site-directed coupled to the amino group of the B chain lysine. Specifically, 1g of DesB30 was dissolved in 20mM boric acid buffer (pH10.5) to prepare a 500ml solution, 12g of HO-PEG-SPA-40K was added thereto, and the mixture was reacted at 300rpm at room temperature (25 ℃ C.) for 60 min. Then, 2M hydrochloric acid was added to reach pH3.0 to terminate the reaction. After the reaction was terminated, the reaction solution was directly applied to an SP Sepharose FF column equilibrated with an equilibration buffer (50mM sodium acetate buffer, pH 3.5). After the loading, 6 column volumes of the equilibration buffer were passed through the column, and then eluted with 50mM Tris-HCl buffer (pH8.0), and the eluted peak having a molecular weight of single PEG modification by SDS-PAGE was collected. Adding ethanol to the elution peak until the final concentration of the ethanol is 60% (V/V), loading the elution peak on a SOURCE 15Q column well balanced by an equilibrium buffer solution (60% (V/V) ethanol, 20mM Tris-HCl and pH8.0), after loading, firstly enabling the equilibrium buffer solution to flow through until the ultraviolet absorption baseline of the flowing solution is stable, then using an eluent (60% (V/V) ethanol, 20mM Tris-HCl, 0-0.3M NaCl and pH8.0) to carry out linear elution with NaCl gradient concentration, and collecting the maximum eluted peak, namely HO-PEG-40K-DesB 30. The purity is more than 95 percent through RP-HPLC detection; the coupling site is on the amino group of B chain lysine by peptide mapping analysis.
Referring essentially to the above preparation except that DesB30 was replaced with human insulin or an analog thereof, respectively: ins, Aspart, Lispro and Glulisine, products HO-PEG-40K-Ins, HO-PEG-40K-AP, HO-PEG-40K-LP and HO-PEG-40K-GL coupled with HO-PEG at B chain lysine amino group of corresponding human insulin or analogues thereof are respectively obtained.
Example 4 pharmacokinetic Studies of insulin and analogs thereof before and after HO-PEG modification
72 healthy Beagle (Beagle) dogs, were randomly divided into 12 groups of 6 dogs each with half of males and females for 12 groups. The Aspart, HO-PEG-20K-AP and HO-PEG-40K-AP prepared in the above embodiment or used as raw materials are respectively administered to the neck or back of each group of beagle dogs in a single dose at the dose of 0.5 IU/kg; lispro, HO-PEG-20K-LP, HO-PEG-40K-LP; ins, HO-PEG-20K-Ins, HO-PEG-40K-Ins; DesB30, HO-PEG-20K-DesB30 and HO-PEG-40K-DesB 30. Before and after administration, blood is collected from the elbow vein of Beagle dog at different time intervals, centrifuged, and serum is taken to detect the blood concentration. The concentrations of insulin or its analogues in the blood at different times were determined by Radioimmunoassay (RIA), and the experimental data were analyzed and pharmacokinetic parameters calculated using the DAS 3.2.4 pharmacokinetic program with the results shown in table 1.
TABLE 1 pharmacokinetic parameters of insulin and its analogs before and after HO-PEG modification
As shown in Table 1, the pharmacokinetic parameters show that Aspart and Lispro accord with the characteristics of quick-acting insulin, the peak is fast to reach, the half-life period is relatively short, and the clearance rate is high; ins and DesB30 are characteristic of short acting insulin, peak reaching is relatively slow, half-life is slightly longer than that of quick acting insulin, and clearance rate is slow. After the four kinds of insulin and the analogues thereof are respectively modified by HO-PEG with different molecular weights, compared with before modification, the peak reaching time is obviously delayed, the half-life period is obviously prolonged, the clearance rate is obviously reduced, and the four kinds of insulin and the analogues thereof can be used as long-acting insulin.
EXAMPLE 5 pharmacodynamic Studies of insulin and analogs thereof before and after HO-PEG modification
Selecting male SD rats qualified for quarantine adaptability observation, fasting for 6-8h before model building, without water prohibition, preparing an IDDM model by injecting Streptozotocin (STZ) 55mg/kg through one-time tail vein, and determining the blood glucose concentration by adopting a Qiangsheng steady blood glucose meter test paper method. And after 72h of molding, continuously monitoring fasting blood glucose for three days and continuously monitoring for three times, wherein the blood glucose is more than or equal to 16.7mmol/L, and judging that the molding of the diabetes model is successful.
SD rats successfully molded were randomly divided into 10 groups of 8 rats, wherein 9 groups of rats in each group were subcutaneously injected with Ins, HO-PEG-20K-AP, HO-PEG-40K-AP, HO-PEG-20K-LP, HO-PEG-40K-LP, HO-PEG-20K-Ins, HO-PEG-40K-Ins, HO-PEG-20K-DesB30 and HO-PEG-40K-DesB30, respectively, at a dose of 4IU/kg, wherein the frequency of administration for the Ins group is three times a day, the frequency of administration for the 4 HO-PEG-SPA-20K modified groups is once every 12 hours, and the frequency of administration for the 4 HO-PEG-SPA-40K modified groups is once every 24 hours; the remaining group was a model group, and the same volume of physiological saline was injected. Meanwhile, 8 healthy SD rats in the same period are randomly selected as a normal group, and physiological saline with the same volume is injected.
After 14 days of continuous administration, all groups are administered for 1 time on day 15, and blood glucose values of each group 30min before administration on day 15 and 1, 2, 4, 6, 8, 10, 12, 15, 18, 21 and 24h after administration are respectively detected by adopting Qiangsheng Steady blood glucose meter test paper. Statistical comparison analysis between groups single factor analysis of variance was performed using SPSS13.0 statistical software.
As shown in tables 2-1 and 2-2, at each detection time point, the blood sugar values of the rats in the normal group are maintained at about 4.7mmol/L, and the blood sugar values of the rats in the model group fluctuate within 20-30 mmol/L; after the Ins group is administrated, the blood sugar is obviously reduced within 1 hour, the blood sugar is basically controlled within a normal blood sugar range within 2-4 hours, but the blood sugar is obviously increased after 6 hours, and no difference exists between the Ins group and the model group after 8 hours; after four types of insulin with different structures and analogues thereof are modified by HO-PEG, the blood sugar control is more stable, the maintenance time is longer, wherein the blood sugar values of the HO-PEG-20K-AP group, the HO-PEG-20K-LP group, the HO-PEG-20K-Ins group and the HO-PEG-20K-DesB30 group at the same detection time point have no obvious difference, and the blood sugar can be effectively controlled within a normal value range within 15 hours after administration; the blood sugar values of the HO-PEG-40K-AP group, the HO-PEG-40K-LP group, the HO-PEG-40K-Ins group and the HO-PEG-40K-DesB30 group at the same detection time point are not obviously different, and the blood sugar can be still effectively controlled within a normal value range within 24 hours after the administration.
TABLE 2-1 hypoglycemic Effect of insulin and its analogs after HO-PEG modification
TABLE 2-2 hypoglycemic Effect of insulin and its analogs after HO-PEG modification
Example 6 immunogenicity Studies of insulin and analogs thereof after HO-PEG modification
Healthy New southwest rabbits are used as experimental objects, and each group comprises 8 rabbits, and the number of the rabbits is 12. Each group was subcutaneously injected with 0.3IU/kg of HO-PEG-20K-AP, HO-PEG-40K-AP, mPEG-20K-AP, HO-PEG-20K-LP, HO-PEG-40K-LP, mPEG-20K-LP, HO-PEG-20K-Ins, HO-PEG-40K-Ins, mPEG-20K-Ins, HO-PEG-20K-DesB30, HO-PEG-40K-DesB30, and mPEG-20K-DesB30, prepared in the above examples or as a control. The administration was continued for six weeks, and venous blood was collected from each group of animals before administration, 14 days, 28 days, and 42 days after the first administration, centrifuged, and serum was taken and tested for the presence of 1: 100 titer or higher of anti-PEG antibody and/or anti-corresponding insulin or its analogue by indirect ELSA method, and the results are shown in Table 3.
TABLE 3 immunogenicity of different types of PEG-modified insulin and analogs thereof
As shown in Table 3, after multiple administrations of the conjugate of HO-PEG-ylated insulin and its analogues in New southwest rabbits, no anti-HO-PEG antibody was detected in all animals, only 1: 100 anti-Lispro antibody was detected in the serum of 1 animal in HO-PEG-40K-LP group 42 days after administration, and no anti-corresponding insulin and its analogues were detected in the remaining animals; however, after multiple administrations of mPEG-modified conjugates of insulin and its analogues in New southwest rabbits, not only was the production of antibodies against mPEG detected in a different number of animals per group, but also in the corresponding insulin and its analogues. At the same detection time point, the group of insulin and analogs thereof modified with HO-PEG produced a significantly lower number of positive animals against the antibodies against the corresponding insulin and analogs thereof than the group modified with mPEG. The results show that modification of insulin and its analogues with HO-PEG introduces no new immunogenicity, at least with respect to mPEG modification, which reduces the immunogenicity of PEG to levels undetectable for weeks; furthermore, modification of insulin and analogs thereof with HO-PEG inhibits the immunogenicity of insulin and analogs thereof, and significantly reduces the immunogenicity of insulin and analogs thereof, at least relative to mPEG modification. Therefore, the HO-PEG modified insulin and the analogues thereof have better medication safety and are particularly suitable for long-term administration.

Claims (73)

1. A conjugate of insulin or an analogue thereof as shown in formula I or a pharmaceutically acceptable salt thereof,
{[HO-(CH2CH2O)n]p-L}t-Y (formula I)
Wherein,
y is insulin or an analog thereof;
l is a linking group;
n is an integer of 50 to 1850;
p is an integer of 1-5;
t is an integer of 1-12;
-is a covalent bond.
2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein n is an integer from 220 to 1370.
3. The conjugate according to claim 2, wherein n is an integer of 450 to 910, or a pharmaceutically acceptable salt thereof.
4. The conjugate according to claim 1, wherein p is an integer of 1 to 3, or a pharmaceutically acceptable salt thereof.
5. The conjugate according to claim 4, wherein p is an integer of 1 to 2, or a pharmaceutically acceptable salt thereof.
6. The conjugate of claim 5, or a pharmaceutically acceptable salt thereof, wherein p is 1.
7. The conjugate according to claim 1, wherein t is an integer of 1 to 5, or a pharmaceutically acceptable salt thereof.
8. The conjugate according to claim 7, wherein t is an integer of 1 to 3, or a pharmaceutically acceptable salt thereof.
9. The conjugate of claim 8, or a pharmaceutically acceptable salt thereof, wherein t is 1.
10. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the insulin is human insulin.
11. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the insulin analog is DesB30, insulin glargine, insulin aspart, insulin detemir, insulin glulisine, insulin degludec, or insulin lispro.
12. The conjugate of claim 1, wherein the linking group is a residue of the coupling reaction functional group that has undergone a coupling reaction and has a molecular weight of less than 300Da, or a pharmaceutically acceptable salt thereof.
13. The conjugate of claim 12, wherein the linking group is a residue of the coupling reaction functional group that has undergone a coupling reaction and has a molecular weight of less than 200Da, or a pharmaceutically acceptable salt thereof.
14. The conjugate of claim 12, or a pharmaceutically acceptable salt thereof, wherein the coupling reaction functional group comprises an acetal group, an aldehyde group, a succinimide group, a maleimide group, a vinylsulfone group, an iodoacetamide group, an ester group, a carbonate group, a carboxyl group, an amino group, an aminoxy group, a thiol group, an allyl group, a vinyl group, an ethynyl group, or an azido group.
15. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the conjugation site of Y is an amino group.
16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the conjugation site for Y is an amino group of the B chain.
17. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the conjugation site for Y is an alpha amino group.
18. The conjugate of claim 17, or a pharmaceutically acceptable salt thereof, wherein the conjugation site for Y is the alpha amino group of the B chain.
19. A process for the preparation of a conjugate as claimed in any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, which comprises administration of [ HO- (CH)2CH2O)n]p-L' is coupled with Y, wherein,
y is insulin or an analog thereof;
l' is a coupling reaction functional group;
n is an integer of 50 to 1850;
p is an integer of 1-5;
-is a covalent bond.
20. The method according to claim 19, wherein n is an integer of 220 to 1370.
21. The method according to claim 20, wherein n is an integer of 450 to 910.
22. The method according to claim 19, wherein p is an integer of 1 to 3.
23. The method according to claim 22, wherein p is an integer of 1 to 2.
24. The method of claim 23, wherein p is 1.
25. The method of claim 19, wherein the insulin is human insulin.
26. The method of claim 19 wherein the insulin analog is DesB30, insulin glargine, insulin aspart, insulin detemir, insulin glulisine, insulin deglutamide, or insulin lispro.
27. The method of claim 19, wherein the coupling reaction functional group comprises an acetal group, an aldehyde group, a succinimide group, a maleimide group, a vinylsulfone group, an iodoacetamide group, an ester group, a carbonate group, a carboxyl group, an amino group, an aminoxy group, a thiol group, an allyl group, a vinyl group, an ethynyl group, or an azido group.
28. The method of claim 19, wherein the coupling site of Y is an amino group.
29. The method according to claim 28, wherein the coupling site of Y is an amino group of B chain.
30. The method of claim 19, wherein the coupling site of Y is an alpha amino group.
31. The method of claim 30, wherein the coupling site of Y is an alpha amino group of the B chain.
32. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
33. Use of a conjugate according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament of low immunogenicity for the treatment and/or prophylaxis of insulinotropic conditions.
34. The use of claim 33, wherein the treatment and/or prevention is long-term treatment and/or prevention.
35. The use according to claim 33, wherein the insulinotropic disease is diabetes.
36. The use of claim 35, wherein diabetes is type 1 diabetes and/or type 2 diabetes.
37. The use of claim 33, wherein low immunogenicity is reduced relative to the immunogenicity of a conjugate of insulin or an analog thereof of formula II:
{[CH3O-(CH2CH2O)n]p-L}t-Y (formula II)
Wherein,
y is insulin or an analog thereof;
l is a linking group;
n is an integer of 50 to 1850;
p is an integer of 1-5;
t is an integer of 1-12;
-is a covalent bond.
38. The use of claim 37, wherein n is an integer from 220 to 1370.
39. The use of claim 38, wherein n is an integer from 450 to 910.
40. The use of claim 37, wherein p is an integer from 1 to 3.
41. The use of claim 40, wherein p is an integer from 1 to 2.
42. The use of claim 41, wherein p is 1.
43. The use of claim 37, wherein t is an integer from 1 to 5.
44. The use of claim 43, wherein t is an integer from 1 to 3.
45. The use of claim 44, wherein t is 1.
46. The use of claim 37, wherein the insulin is human insulin.
47. The use of claim 37 wherein the insulin analogue is DesB30, insulin glargine, insulin aspart, insulin detemir, insulin glulisine, insulin deglutamide or insulin lispro.
48. Use according to claim 37, wherein the linking group is the residue of a coupling reaction of the coupling reaction functional group and has a molecular weight of less than 300 Da.
49. The use of claim 48, wherein the linking group is a residue of a coupling reaction of the coupling reaction functional group and has a molecular weight of less than 200 Da.
50. The use of claim 48, wherein the coupling reaction functional group comprises an acetal group, an aldehyde group, a succinimide group, a maleimide group, a vinylsulfone group, an iodoacetamide group, an ester group, a carbonate group, a carboxyl group, an amino group, an aminoxy group, a thiol group, an allyl group, a vinyl group, an ethynyl group, or an azide group.
51. The use of claim 37, wherein the coupling site of Y is an amino group.
52. The use of claim 51, wherein the coupling site of Y is an amino group of the B chain.
53. The use of claim 37, wherein the coupling site of Y is an alpha amino group.
54. The use of claim 53, wherein the coupling site of Y is the alpha amino group of the B chain.
55.[HO-(CH2CH2O)n]p-L' for the preparation of conjugates of insulin or analogues thereof with reduced immunogenicity, wherein,
l' is a coupling reaction functional group;
n is an integer of 50 to 1850;
p is an integer of 1-5;
-is a covalent bond.
56. The use of claim 55, wherein low immunogenicity is reduced relative to the immunogenicity of a conjugate of insulin or an analog thereof of formula II:
{[CH3O-(CH2CH2O)n]p-L}t-Y (formula II)
Wherein,
y is insulin or an analog thereof;
l is a linking group;
n is an integer of 50 to 1850;
p is an integer of 1-5;
t is an integer of 1-12;
-is a covalent bond.
57. The use of claim 55 or 56, wherein n is an integer from 220 to 1370.
58. The use of claim 57, wherein n is an integer from 450 to 910.
59. The use of claim 55 or 56, wherein p is an integer from 1 to 3.
60. The use of claim 59, wherein p is an integer from 1 to 2.
61. The use of claim 60, wherein p is 1.
62. The use of claim 56, wherein t is an integer from 1 to 5.
63. The use of claim 62, wherein t is an integer from 1 to 3.
64. The use of claim 63, wherein t is 1.
65. The use of claim 55 or 56, wherein the insulin is human insulin.
66. The use of claim 55 or 56 wherein the insulin analogue is DesB30, insulin glargine, insulin aspart, insulin detemir, insulin glulisine, insulin deglutamide or insulin lispro.
67. The use of claim 56, wherein the linking group is a residue of a coupling reaction of the coupling reaction functional group and has a molecular weight of less than 300 Da.
68. The use of claim 56, wherein the linking group is a residue of a coupling reaction of the coupling reaction functional group and has a molecular weight of less than 200 Da.
69. The use of claim 55 or 67, wherein the coupling reaction functional group comprises an acetal group, an aldehyde group, a succinimide group, a maleimide group, a vinylsulfone group, an iodoacetamide group, an ester group, a carbonate group, a carboxyl group, an amino group, an aminoxy group, a thiol group, an allyl group, a vinyl group, an ethynyl group or an azido group.
70. The use of claim 55 or 56, wherein the coupling site for insulin or an analogue thereof is an amino group.
71. The use of claim 70, wherein the coupling site for insulin or an analogue thereof is the amino group of the B chain.
72. The use of claim 55 or 56, wherein the coupling site for insulin or an analogue thereof is an alpha amino group.
73. The use of claim 72, wherein the coupling site for insulin or an analogue thereof is the alpha amino group of the B chain.
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CN1538809A (en) * 2001-06-04 2004-10-20 诺贝克斯公司 Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, used thereof and methods of making same
CN103223160A (en) * 2004-07-19 2013-07-31 比奥孔有限公司 Cation complexes of insulin compund conjugates, formulation and uses thereof

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Publication number Priority date Publication date Assignee Title
CN1538809A (en) * 2001-06-04 2004-10-20 诺贝克斯公司 Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, used thereof and methods of making same
CN103223160A (en) * 2004-07-19 2013-07-31 比奥孔有限公司 Cation complexes of insulin compund conjugates, formulation and uses thereof

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