EP1888104A2 - Insuline acylee tres pure - Google Patents

Insuline acylee tres pure

Info

Publication number
EP1888104A2
EP1888104A2 EP06763218A EP06763218A EP1888104A2 EP 1888104 A2 EP1888104 A2 EP 1888104A2 EP 06763218 A EP06763218 A EP 06763218A EP 06763218 A EP06763218 A EP 06763218A EP 1888104 A2 EP1888104 A2 EP 1888104A2
Authority
EP
European Patent Office
Prior art keywords
insulin
des
acid
human insulin
lys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06763218A
Other languages
German (de)
English (en)
Inventor
Jan Markussen
Svend Havelund
Jes Kristian Jacobsen
Aage Hvass
Ib Jonassen
Georg Wilhelm Jensen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP1888104A2 publication Critical patent/EP1888104A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins

Definitions

  • the present invention relates to highly purified acylated insulins which are soluble at physiological pH values and have a prolonged profile of action.
  • the invention also relates to methods of providing such acylated insulins, to pharmaceutical compositions containing them and to the use of such acylated insulins in the treatment of diabetes and hyperglycaemia.
  • WO 99/21888 discloses insulin derivatives prone to aggregate forming high molecu- lar weight assemblies and a gel permeation method (SEC) to access this property.
  • the present invention is related to a pharmaceutical composition com- prising an acylated insulin and an isomer thereof in a weight ratio of greater than 97:3 and pharmaceutically acceptable adjuvants, wherein said acylated insulin has the formula I
  • X at position A 18 is Asn or GIn
  • X1 in position B1 is Phe or deleted
  • X2 at position B2 is VaI or deleted
  • X3 at position B3 is Asn or modified to Thr
  • X4 at posi- tion A21 is Asn or modified to Ala
  • X5 at position B30 may be any codable amino acid residue except Lys, Arg and Cys or is deleted
  • Acyl is an acyl group derived from the group consisting of mono- or dicarboxylic, unsaturated or saturated fatty acids with a chain length of from about 6 to about 40, lithocholic acids, cholic acid, hyocholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, hyodeoxycholic acid and chola
  • the weight ratio of the acylated insulin to the isomer thereof is greater than about 98:2.
  • the weight ratio of the acylated insulin to the isomer thereof is greater than about 99:1 .
  • the weight ratio of the acylated insulin to the isomer thereof is greater than 99.5:0.5.
  • the weight ratio of the acylated insulin to the isomer thereof is greater than 99.8:0.2. In a still further embodiment the weight ratio of the acylated insulin to the isomer thereof is greater than 99.9:01 .
  • the weight ratio is in the range from 97:3 to 99.9:0.1.
  • the weight ratio is in the range from 98:2 to 99.9:0.1 .
  • the weight ratio is in the range from 99:1 to 99.9:0.1 . In another embodiment the weight ratio is in the range from 97:3 to 99.5:0.5.
  • the weight ratio is in the range from 98:2 to 99.5:0.5.
  • the weight ratio is in the range from 99:1 to 99.5:0.5.
  • X is Asn.
  • X1 is Phe. In another embodiment X2 is VaI.
  • X3 is Asn.
  • X4 is Asn.
  • X5 is deleted or is Thr.
  • X is Asn
  • X1 is Phe
  • X2 is VaI
  • X3 is Asn
  • X4 is Asn
  • X5 is deleted.
  • the acyl group is a mono- or dicarboxylic, saturated or unsaturated fatty acid with a chain length of from about 6 to about 24 carbon atoms.
  • the acyl group is a mono- or dicarboxylic, saturated or unsaturated fatty acid with a chain length from 14-16 carbon atoms.
  • the acyl group is lithocholic acid.
  • the pharmaceutical formulation may contain the insulin derivative Lys B29 (N ⁇ — litrocholyl- ⁇ - glutamyl)des(B30) human insulin and the isomer thereof is Lys B29 (N ⁇ -lithocholyl- ⁇ - glutamyl)des(B30) human insulin.
  • the present invention is related to a method for producing a solu- tion containing an acylated insulin with formula I and an isomer thereof with formula Il in a weight ratio of greater than about 97:3, said method comprising a) subjecting a solution with a weight ratio of the acylated insulin with formula I and the isomer thereof with formula Il of less than 97:3 to ion exchange or RP-HPLC chromatography under conditions effective to separate the isomer from the acylated insulin with formula I, and b) collecting the fractions from said chromatography containing said acylated insulin and the isomer thereof in a weight ratio greater than 97:3.
  • the pH may vary under the separation process depending on the insulin compound in question.
  • the pH of the RP-HPLC chromatographic step is be- tween about 3 and about 7. More typically the pH of this step will be between about 4 and about 7, between about 5 and about 6.5, between about 4 and about 6, between about 4 and about 5, between about 3.5 and about 6, between about 4.5 and about 6.5 or between about 4.75 and about 6.5
  • the RP-HPLC step is conducted at pH of about 2.5 to about 5 in water-acetonitrile or water-ethanol mixtures.
  • the temperature of the chromatographic step may also vary but will typically be between about 15 and about 5O 0 C.
  • the temperature of the chromatographic step is between about 20 and about 45 0 C. In another embodiment the temperature of the chromatographic step is between about 25 and about 45 0 C, between about 20 to about 45 0 C, between about 25 and about 45 0 C, between about 30 to about 45 0 C or between about 35 and about 4O 0 C.
  • the RP-HPLC step is typically conducted in water-acetonitrile or water-ethanol mixtures.
  • the solvent in the RP-HPLC step will comprise a salt such as Na 2 SO 4 , (NhU) 2 SO 4 , NaCI, KCI, and buffer systems such as phosphate, and citrate and maleic acid.
  • the required concentration of salt in the solvent may be from about 0.1 M to about 1 M, preferably between 0.2 M to 0.5 M, most preferable between 0.3 to 0.4 M. In- crease of the concentration of salt requires an increase in the concentration of organic solvent in order to achieve elution from the column within a suitable time.
  • the principles of RP-HPLC and ion exchange chromatography can be combined, e.g. by using a silica matrix only partially substituted with the organic ligand leaving free silanol sites capable of binding cat-ions. Eluation from such columns us- ing the combination of binding principles typically requires higher concentrations of salts and organic solvent as compared to using the principle separately on separate columns.
  • the temperature of the chromatographic step will typically be between about 15 and about 5O 0 C.
  • the temperature will be in the range of about 20 to about 45 0 C. In another embodiment the temperature will be in the range of about 25 to about
  • the present invention is related to a method for treating hy- perglycemia in a patient, said method comprising administering to a patient in need of such treatment an effective amount of a pharmaceutical composition according to the invention.
  • the invention is related to a method of treating type 1 diabetes, type 2 diabetes and other states that are associated with hyperglycaemia in a patient, comprising administering to the patient in need of such a treatment a therapeutically effective amount of the pharmaceutical preparation according to the invention.
  • the acyl group is a lithocholic acid selected from 5- ⁇ litho- cholic acid or 5- ⁇ lithocholic acid.
  • the invention is related to a solution of Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) in which the sum of all impurities is less than 1 % based on total protein.
  • the acyl group attached to the parent insulin molecule may be a lipophilic group containing from 6 to 40 carbon atoms.
  • examples of such groups are fatty dicarboxylic or monocarboxylic groups having from 6 to 40, from 6 to 36, from 6 to 24, from 6 to 18, from 8 to 36, from 8 to 24, from 8 to 20, from 8 to 18, from 12 to 18 or from 14 to 18 carbon atoms.
  • the acyl group is selected from the following group: CH 3 -(CH 2 ) n - CO- , (COOH)-(CH 2 ) n -CO-, (NH 2 -CO)-(CH 2 ) n -CO-, HO-(CH 2 ) n -CO- , where 4 ⁇ n ⁇ 38.
  • the acyl group is 5- ⁇ lithocholic acid or 5- ⁇ lithocholic acid.
  • the dicarboxylic fatty acid will typically comprise from about 4 to about 26, from 4 to about 18, from about 6 to about 18, from about 8 to about 16, from about 8 to about 22, from about 8 to about 17, from about 8 to about 15, from about 10 to about 18, from about 10 to about 16 and from about 6 to about 17 carbon atoms in the carbon chain.
  • dicarboxylic fatty acids are diacids with the formula HOOC-(CH 2 ) r1 - COOH, where r1 is 4 to 22
  • Lys B29 N ⁇ -lithocholyl- ⁇ -glutamyl
  • B30 human insulin a contamination of the corresponding ⁇ -glutamyl isomer, in the range of 3.0-3.6 %, has been identified.
  • the Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin derivative may be syn- thesized by acylation of des(B30) human insulin using a litrocholyl-Glu derivative, in which the ⁇ -carboxyl group may be protected in the form of an ester and the ⁇ -carboxyl group may be activated in the form of an active ester or as an active amide.
  • the conditions for the selective acylation of the ⁇ -amino group of the lysine B29 residue of insulin with fatty acids are disclosed in US patent No. 5,646,242 and US patent No. 5,905,140.
  • Table 1 shows that the fraction of high molecular assemblies in mixtures of the ⁇ - glutamyl isomer and Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin is highly dependent of the content of the ⁇ -glutamyl isomer when using the SEC method described in PCT WO 99/21888, in this case adapted to a smaller column.
  • Lys B29 N ⁇ -lithocholyl- ⁇ -glutamyl
  • B30 human insulin derivative that contains 3.5 % of the ⁇ -glutamyl isomer as compared to the gamma-glutamyl isomer on a weight/weight basis only 66.7 % of the acylated insulin was found in the high molecular part of the chromatogram, defined as the fraction eluting before aldolase in SEC
  • the small content of the ⁇ -glutamyl isomer as compared to the gamma- glutamyl isomer has a marked ability to decrease the percentage of the high molecular frac- tion estimated by SEC, much higher than the percentage of ⁇ -glutamyl isomer would suggest.
  • the percentage of self-assembled hexamers increases by 10%, from 67 to 77%, when the content of the ⁇ -glutamyl isomer in the mixture is lowered only 3.5%, from 3.5 to 0 %.
  • the increased tendency to form high molecular assemblies in purified Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin results in increased prolongation of the absorption time after subcutaneous injection. Consequently, as the content of the ⁇ - glutamyl isomer is lowered towards zero the possible variation in the day-to-day pharmacokinetics of the absorption, due to variations in the ⁇ -glutamyl isomer, will decrease accordingly.
  • the pharmaceutical preparations according to the present invention will comprise less that 3 percent by weight of the unwanted isomer as compared to the desired compound. Typically it will contain less than 2 percent by weight, more typically less 1 percent by weight and the content of the isomer may be as low as 0.5 to 0.05 percent by weight as compared to the desired acylated insulin.
  • the content of the alpha isomer may even be zero.
  • the insulin moiety - in the present text also referred to as the parent insulin - of an insulin according to the invention can be a naturally occurring insulin such as human insulin or porcine insulin.
  • the parent insulin can be an insulin analogue.
  • the amino acid residue at position A21 is
  • the amino acid residue at position B1 has been deleted.
  • a specific example from this group of parent insulin analogues is desB1 human insulin.
  • the amino acid residue at position B30 has been deleted.
  • a specific example from this group of parent insulin analogues is desB30 human insulin.
  • the present invention is related to a method for producing a solution containing an acylated insulin with formula I and an isomer thereof with formula Il in a weight ratio of greater than about 97:3, said method comprising a) subjecting a solution with a weight ratio of the acylated insulin with formula I and the isomer thereof with formula Il of less than 97:3 to ion exchange or RP-HPLC chromatography under conditions effective to separate the isomer from the acylated insulin with formula I, and b) collecting the fractions from said chromatography containing said acylated insulin and the isomer thereof in a weight ratio greater than 97:3.
  • the method according to the present invention may comprise a step wherein the weight ratio between the acylated insulin derivative and the isomer thereof in fractions obtained from step a) is determined and compared to the corresponding weight ratio in the solution before step a) where an increase in the weight ratio in said fractions indicates that the fractions contain a solution of an acylated insulin that exhibits an increased percentage of self-assembled hexamers as compared to the solution of said acylated insulin before step a).
  • the method according to the present invention may fur- thermore comprise a step wherein the weight ratio between the acylated insulin derivative and the isomer thereof in fractions obtained from step a) is determined and compared to the corresponding weight ratio in the solution before step a) where an increase in the weight ratio in said fractions indicates that the fractions contain a solution of an acylated insulin that exhibits less day to day variation in pharmacokinetics upon administration to humans as compared to that exhibited by the solution of acylated insulin before step a).
  • the medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared accord- ing to published recipes (e.g. in catalogues of the American Type Culture Collection).
  • the peptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like, dependent on the type of peptide in question.
  • a salt e.g. ammonium sulphate
  • the DNA sequence encoding the parent insulin may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridisation using synthetic oligonucleotide probes in accordance with standard techniques (see, for example, Sambrook, J, Fritsch, EF and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989).
  • the DNA sequence encoding the polypeptide may also be prepared synthetically by established standard methods, e.g.
  • the DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., Science 239 (1988), 487 - 491.
  • the DNA sequence may be inserted into any vector which may conveniently be sub- jected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and repli- cated together with the chromosome(s) into which it has been integrated.
  • the vector is preferably an expression vector in which the DNA sequence encoding the peptide is operably linked to additional segments required for transcription of the DNA, such as a promoter.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the parent insulin in a variety of host cells are well known in the art, cf. for instance Sambrook et al., supra.
  • the DNA sequence encoding the parent insulin may also, if necessary, be operably connected to a suitable terminator, polyadenylation signals, transcriptional enhancer se- quences, and translational enhancer sequences.
  • the recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell or one which confers resistance to a drug, e.g. ampicil- Nn, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
  • a secretory signal sequence also known as a leader sequence, prepro sequence or pre sequence
  • the secretory signal sequence is joined to the DNA sequence encoding the peptide in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the peptide.
  • the secretory signal sequence may be that normally associated with the peptide or may be from a gene encoding another secreted protein.
  • the host cell into which the DNA sequence or the recombinant vector is introduced may be any cell which is capable of producing the parent insulin and includes bacteria, yeast, fungi and higher eukaryotic cells.
  • suitable host cells well known and used in the art are, without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.
  • compositions according to the invention are typically solutions and may be administered parenterally to patients in need of such a treatment.
  • Parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe.
  • parenteral administration can be performed by means of an infusion pump.
  • Further options are to administer the insulin preparation nasally or pulmonally, preferably in compositions, powders or liquids, specifically designed for the purpose.
  • Injectable preparations of the invention can be prepared using the conventional techniques of the pharmaceutical industry which involve dissolving and mixing the ingredients as appropriate to give the desired end product.
  • an insulin derivative according to the invention is dissolved in an amount of water which is somewhat less than the final volume of the composition to be prepared.
  • An isotonic agent, a preservative and a buffer is added as required and the pH value of the solution is adjusted - if necessary - using an acid, e.g. hydrochloric acid, or a base, e.g. aqueous sodium hydroxide as needed.
  • the volume of the solution is adjusted with water to give the desired concentration of the ingredients.
  • the buffer may be selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phos- phate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)- aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof.
  • Each one of these specific buffers constitutes an alternative embodiment of the invention.
  • the preservative may be selected from the group consisting of phenol, o-cresol, m- cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride, chlorphenesine (3p-chlorphenoxypropane-1 ,2- diol) or mixtures thereof.
  • the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In a further embodiment of the invention the preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In another embodiment of the invention the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. In a further embodiment of the invention the preservative is present in a concentration from 10 mg/ml to 20 mg/ml.
  • the use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.
  • the isotonic agent may be selected from the group consisting of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid (e.g. glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g. glycerol (glycerine), 1 ,2- propanediol (propyleneglycol), 1 ,3-propanediol, 1 ,3-butanediol) polyethyleneglycol (e.g. PEG400), or mixtures thereof.
  • a salt e.g. sodium chloride
  • a sugar or sugar alcohol e.g. sodium chloride
  • an amino acid e.g. glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine
  • Any sugar such as mono-, di-, or polysaccharides, or water- soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hy- droxyethyl starch and carboxymethylcellulose-Na may be used.
  • the sugar additive is sucrose.
  • Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one -OH group and includes, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xyli- tol, and arabitol.
  • the sugar alcohol additive is mannitol.
  • the sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely effect the stabilizing effects achieved using the methods of the invention.
  • the sugar or sugar alcohol concentration is between about 1 mg/ml and about 150 mg/ml.
  • the isotonic agent may be present in a concentration from 1 mg/ml to 50 mg/ml, from 1 mg/ml to 7 mg/ml, from 8 mg/ml to 24 mg/ml, or from 25 mg/ml to 50 mg/ml.
  • the use of an isotonic agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.
  • Typical isotonic agents are sodium chloride, mannitol, dimethyl sulfone and glycerol and typical preservatives are phenol, m-cresol, methyl p-hydroxybenzoate and benzyl alco- hoi.
  • buffers examples include sodium acetate, glycylglycine, HEPES (4-(2- hydroxyethyl)-1 -piperazineethanesulfonic acid) and sodium phosphate.
  • the insulin preparation of this invention can be used in the treatment of states which are sensitive to insulin. Thus, they can be used in the treatment of type 1 diabetes, type 2 diabetes and hyperglycaemia for example as sometimes seen in seriously injured persons and persons who have undergone major surgery.
  • the optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific insulin derivative employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the state to be treated. It is recommended that the daily dosage of the insulin derivative of this invention be determined for each individual patient by those skilled in the art in a similar way as for known insulin compositions.
  • the insulin derivatives of this invention may be used in mixture with other types of insulin, e.g. insulin analogues with a more rapid onset of action.
  • insulin analogues e.g. in the European patent applications having the publication Nos. EP 214826 (Novo Nordisk A/S), EP 375437 (Novo Nordisk A/S) and EP 383472 (EIi Lilly & Co.).
  • Examples of pharmaceutical preparations are neutral solutions from pH 6.5 to 8.3 containing from 300 to 4800 nmol/ml of the drug substance, isotonic agents, NaCI, buffers preservatives, zinc and stabilizers.
  • desB30 or “B(1-29)” is meant a natural insulin B chain or an analogue thereof lacking the B30 amino acid residue and "A(1-21)” means the natural insulin A chain or an analogue thereof.
  • the C-peptide and its amino acid sequence are indicated in the three letter amino acid code.
  • DesB30,desB29 human insulin is a human insulin lacking B29 and B30. With “B1 ", "A1 " etc.
  • amino acid residue in position 1 in the B chain of insulin counted from the N-terminal end
  • amino acid residue in position 1 in the A chain of insulin counted from the N-terminal end
  • the amino acid residue in a specific position may also be denoted as e.g. Phe B1 which means that the amino acid residue in position B1 is a phenylalanine residue.
  • A-chain is understood the sequence of amino acids in the A-chain of human insulin.
  • B-chain is understood the sequence of amino acids in the B-chain of human insulin.
  • Insulin as used herein is meant human insulin with disulfide bridges between Cys A7 and Cys B7 and between Cys A20 and Cys B19 and an internal disulfide bridge between Cys A6 and Cys A11 , porcine insulin and bovine insulin.
  • insulin analogue as used herein is meant a polypeptide which has a molecular structure which formally can be derived from the structure of a naturally occurring insulin, for example that of human insulin, by deleting and/or substituting at least one amino acid residue occurring in the natural insulin and/or by adding at least one amino acid residue.
  • the added and/or substituted amino acid residues can either be codable amino acid residues or other naturally occurring amino acid residues or purely synthetic amino acid residues.
  • the insulin analogues may in one embodiment comprise up to 5 changes compared to the human insulin molecule, more typically up to 4, or up 3 and even more typically 1 or 2 changes compared to human insulin.
  • the insulin analogues may be such wherein position Asn at position A21 may be modified to Ala, GIn, GIu, GIy, His, He, Leu, Met, Ser, Thr, Trp, Tyr or VaI, in particular to GIy, Ala, Ser, or Thr and in particular to GIy. Furthermore, Asn at position B3 may be modified to Lys or Asp. Further examples of insulin analogues are des(B30) human insulin, insulin analogues wherein one or both of B1 and B2 have been deleted; insulin analogues wherein the A-chain and/or the B-chain have an N-terminal extension and insulin analogues wherein the A-chain and/or the B-chain have a C-terminal extension.
  • mutation is understood a substitution with a codable amino acid.
  • insulin derivative as used herein is meant a naturally occurring insulin or an insulin analogue which has been chemically modified by introducing a side chain in one or more positions of the insulin backbone or by oxidizing or reducing groups of the amino acid residues in the insulin or by acylating a free amino group or a hydroxy group
  • a codable amino acid or "a codable amino acid residue” is used to indicate an amino acid or amino acid residue which can be coded for by a triplet ("codon") of nucleotides.
  • insulin hexamer is meant all the possible conformations of 6 non-covalently associated molecules of insulin, some of which are known as the R6, T6, and R3T3 forms.
  • acylation is understood the chemical reaction whereby a hydrogen of an amino group or hydroxy group is exchanged with an acyl group.
  • protracted insulin an insulin peptide which has a time-action of more than 8 hours in standard models of diabetes.
  • the insulin pep- tide has a time-action of at least 9 hours or at least 10 hours. More preferably, the protracted insulin has a time-action in the range from at least 9 to at least 15 hours.
  • rapid acting insulin is meant an insulin compound which has a faster onset of action than human insulin.
  • rapid acting insulins is analogues wherein the amino acid residue at position B28 is Asp.
  • a specific example from this group of insulin ana- logues is Asp B28 human insulin.
  • Another example of rapid acting insulin analogues are analogues wherein the amino acid residue at position B28 is Lys and the amino acid residue at position B29 is Pro.
  • a specific example from this group of parent insulin analogues is
  • fatty acid is meant any saturated aliphatic monocarboxylic acid of the general formula C n H 2n+ iCOOH.
  • saturated fatty acid is meant a fatty acid featuring one or several double bonds.
  • branched fatty acid is meant a fatty acid featuring at least one carbon atom bound to 3 or 4 other carbon atoms. Branched fatty acids may be saturated or unsaturated.
  • dicarboxylic acid is meant an acid containing two carboxylic moieties.
  • ⁇ , ⁇ -dicarboxylic acid is meant a dicarboxylic acid where the carboxyl groups are located in the opposite terminal positions of the main carbon chain.
  • linker is meant a bridge having the function of binding a substituent to the insulin moiety or forming a bridge between two sites of the insulin derivative.
  • the linker be- comes part of the substituent and may by itself contribute to the properties of the insulin derivative.
  • Examples of linkers are amino acids, dicarboxylic acids, and functionalized PEG (polyethylene glycol) such as amino and/or carboxy PEG.
  • an amino acid residue having a carboxylic acid group in the side chain designates amino acid residues like Asp, GIu and hGlu.
  • the amino acids can be in either the L- or D-configuration. If nothing is specified it is understood that the amino acid residue is in the L configuration.
  • amino acids mentioned herein are L-amino acids.
  • left and right ends of an amino acid sequence of a peptide are, respectively, the N- and C-termini unless otherwise specified.
  • ALP is the Achromobacter lyticus protease which is a lysine specific protease from Acromobacter lyticus.
  • RP-HPLC reverse phase high pressure liquid chromatography
  • SEC size exclusion chromatography
  • t 50% is meant the time at which 50% of the injected insulin has disappeared from the subcutaneous injection site.
  • Tris Tris(hydroxymethyl)aminomethane.
  • GIu is acylated in its amino group, the ⁇ -carboxyl group is protected as an ester function and the ⁇ -carboxyl group activated as the N- hydroxysuccinimide ester.
  • ID is meant internal diameter.
  • a 125 X 4 mm I. D. column was packed with a dimethylbutyldimethylsilyl substituted silica having pore size of about 100 A and particle diameter of about 5 ⁇ m and equilibrated at
  • Lys B29 N ⁇ -lithocholyl- ⁇ -glutamyl des(B30) hu- man insulin emerged from the column after about 20 min and Lys B29 (N ⁇ -lithocholyl- ⁇ - glutamyl) des(B30) human insulin after about 23.5 min.
  • Example 2 Preparative purification of Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin from Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin using RP-HPLC in (NhU) 2 SO 4 ZH 2 SO 4 buffer of pH 2.5 in water-acetonitril.
  • a 300 X 50 mm I. D. column is packed with a dimethylbutyldimethylsilyl substituted silica having pore size of about 100 A and particle diameter of about 15 ⁇ m is equilibrated at 5O 0 C and at a flow rate of 50 ml/min with a buffer of 0.075 M NH 4 SO 4 adjusted to pH 2.50 with H 2 SO 4 and comprising 30 % (v/v) of acetonitrile.
  • a sample of 1 g of impure Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin containing 3.5% of Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin is applied to the column followed by isocratic elution using the conditions of equilibration.
  • the chromatography is monitored by absorption at 280 nm. After about 2.5 h the product emerges from the column.
  • the product in the first half of the peak is isolated by dilution with 2 volumes of water followed by adjustment of the pH to 5.2 whereby it precipitates.
  • Analysis of the purity using the method of example 1 shows a content of 1 % of Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin.
  • Lys B29 N ⁇ -lithocholyl- ⁇ -glutamyl des(B30) human insulin and Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin using cat-ion exchange in wa- ter-ethanol at pH 4.8.
  • Lys B29 N ⁇ -lithocholyl- ⁇ -glutamyl des(B30) human insulin containing 3.5% of Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin dissolved in 60 % ethanol at pH 3.0 was applied to a 150 x 4 I. D. mm column packed with the cat-ion exchanger Source 15 S. The column was equilibrated and eluted with 0.02 M citrate buffer in 60% ethanol, adjusted to pH 4.8 with NaOH at a rate of 0.3 ml/min. The product eluted from about 75 column volumes to about 170 column volumes. The impurity of Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin was absent in the pool eluting between 75 and 142 column volumes.
  • Lichro- sorb RP C18 having particle diameter of about 5 ⁇ m was equilibrated at 35 0 C and at a flow rate of 1 ml/min with a buffer comprising 0.04 M Na 2 SO 4 , 0.008 M H 3 PO 4 , 34% (v/v) of ace- tonitrile and adjusted to pH 4.0 with NaOH after addition of acetonitrile.
  • a 40 ⁇ g sample was applied to the column dissolved in 10 ⁇ l of the above mentioned buffer. The separation was achieved by isocratic elution using the above mentioned conditions for equilibration.
  • Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin emerged from the column after about 21 min and Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin after about 34 min.
  • the concentration of acetonitrile may be adjusted if needed, as the elution times are sensitive to this parameter.
  • the sample contained 3.5 % of Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin.
  • the separation was achieved by isocratic elution using a 36/64 (v/v) mixture of the above mentioned solvents A and B.
  • the Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin product emerging from the column after 24-37 min (fraction 1 ) was collected and precipitated at pH 5.2 after 2 fold dilution with water.
  • the impurity of Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin emerged after about 40-43 min fraction 2).
  • the concentration of ethanol may be adjusted if needed, as the elution times are sensitive to this parameter.
  • Lys B29 N ⁇ -lithocholyl- ⁇ -glutamyl des(B30) human insulin and Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin using cat-ion exchange in water- ethanol in a pH gradient from pH 4 to 6.
  • Lys B29 N ⁇ -lithocholyl- ⁇ -glutamyl des(B30) human insulin containing 3.5% of Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl) des(B30) human insulin dissolved in 60 % ethanol at pH 3.0 was applied to a 150 x 4 I. D. mm column packed with the cat-ion ex- changer Source 15 S. The column was equilibrated with 0.02 M citrate buffer in 60% ethanol, adjusted to pH 4.0 with triethanolamine at a rate of 0.3 ml/min. Elution was accomplished by a linear gradient from this buffer to the same adjusted to pH 6.0 with triethanolamine over a total of 40 column volumn.
  • Lys B29 N ⁇ -lithocholyl- ⁇ -glutamyl des(B30) human insulin was absent in the first fractions from pH 4.76 to 4.91.
  • a 150 X 4.6 mm I. D. column was packed with a octyldimethylsilyl substituted silica having pore size of about 100 A and particle diameter of about 3.5 ⁇ m and equilibrated at 4O 0 C at a flow rate of 1 ml/min with a mixture consisting of 1 : a buffer of 2OmM NaH 2 PO 4 -H 2 O and I OOmmol Na 2 SO 4 adjusted to pH 5.9 with NaOH in the aqueous buffer containing 7.8% (w/w) acetonitrile,and 2: acetonitrile solvent containing 42,8% w/w acetonitrile, to make 25 % (w/w).
  • Lys B29 N ⁇ -hexadecandioyl- ⁇ - glutamyl
  • Lys B29 N ⁇ -hexadecandioyl - ⁇ -glutamyl
  • Lys B29 N ⁇ -hexadecandioyl- ⁇ - glutamyl des(B30) human insulin and Lys B29 ( N ⁇ -hexadecandioyl- ⁇ -glutamyl) des(B30) human insulin using RP-HPLC in Na 2 SO 4 / NaH 2 PO 4 buffer of pH 5.9 in water-acetonitril.
  • RP-HPLC Na 2 SO 4 / NaH 2 PO 4 buffer of pH 5.9 in water-acetonitril.
  • Example 9 Preparative purification of Lys B29 ( N ⁇ -hexadecandioyl- ⁇ - glutamyl) des(B30) human insulin and Lys B29 ( N ⁇ -hexadecandioyl- ⁇ -glutamyl) des(B30) human insulin using RP-HPLC in Maleic acid/KCI buffer of pH 5.5 in water-ethanol.
  • a 250 X 10 mm I. D. column packed with an octadecyldimethylsilyl substituted silica having particle diameter of about 15 ⁇ m and pore size about 200 A was equilibrated at 5O 0 C and with a flow rate of 1 .96 ml/min with solvent A comprising 20 mmol/kg maleic acid 250 mmol/kg KCI, 15% (w/w) of ethanol and adjusted to pH 5.5 with NaOH.
  • a sample of 50 ml containing 49 mg Lys B29 ( N ⁇ -hexadecandioyl- ⁇ - glutamyl) des(B30) human insulin was applied to the column.
  • the sample contained also 0.5mg of Lys B29 ( N ⁇ -hexadecandioyl- ⁇ - glutamyl) des(B30) human insulin.
  • the separation was achieved by gradient elution going from 57 to 67%B in 15 column volumes.
  • Solvent B comprising 20 mmol/kg maleic acid 250 mmol/kg KCI, 40% (w/w) of ethanol and adjusted to pH 5.5 with NaOH. After about 2.5 h the product emerged from the column. The product was collected between OD 0.25 and OD 0.5 on the leading and trailing edge was collected. The concentration of ethanol may be adjusted if needed, as the elution times are sensitive to this parameter.
  • Analysis of the purity using the method of example 7 shows a content of Lys B29 ( N ⁇ - hexadecandioyl - ⁇ -glutamyl) des(B30) human insulin below limit of detection (LOD).
  • LOD limit of detection
  • Lys B29 (N ⁇ -lithocholyl- ⁇ -glutamyl)des(B30) human insulin and Lys B29 (N ⁇ -lithocholyl- ⁇ - glutamyl)des(B30) human insulin.
  • Zinc ions were added in a ratio of 2, 3 or 4 per hexamer of insulin derivative.

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Abstract

L'invention concerne une préparation pharmaceutique très purifiée comprenant une insuline acylée, le groupe acyle étant fixé au moyen d'une molécule de liant au groupe e- amino du résidu B29Lys de la fraction d'insuline mère. La formulation pharmaceutique d'insuline comprend moins d'environ 3 % en poids/poids d'un isomère de l'insuline acylée.
EP06763218A 2005-05-26 2006-05-22 Insuline acylee tres pure Withdrawn EP1888104A2 (fr)

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DKPA200500767 2005-05-26
PCT/EP2006/062495 WO2006125765A2 (fr) 2005-05-26 2006-05-22 Insuline acylee tres pure

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EP3341401A1 (fr) 2015-08-25 2018-07-04 Novo Nordisk A/S Nouveaux dérivés de l'insuline et leurs utilisations médicales
WO2017032797A1 (fr) 2015-08-25 2017-03-02 Novo Nordisk A/S Nouveaux dérivés de l'insuline et leurs utilisations médicales
EP3856141A1 (fr) * 2018-09-25 2021-08-04 Amphastar Pharmaceuticals, Inc. Principe actif pharmaceutique d'insuline humaine recombinée (rhi) hautement purifiée et ses procédés de production
JP2022501546A (ja) 2018-10-01 2022-01-06 ベーリンガー インゲルハイム フェトメディカ ゲーエムベーハーBoehringer Ingelheim Vetmedica GmbH 蠕動ポンプ及びサンプルを検査するための分析器

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RO112873B1 (ro) * 1993-09-17 1998-01-30 Novo Nordisk As Derivati de insulina
WO1999021888A1 (fr) * 1997-10-24 1999-05-06 Novo Nordisk A/S Agregats de derives de l'insuline humaine
US20040138099A1 (en) * 2002-11-29 2004-07-15 Draeger Eberhard Kurt Insulin administration regimens for the treatment of subjects with diabetes

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See references of WO2006125765A2 *

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