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US20090306337A1 - Pegylated, Extended Insulins - Google Patents

Pegylated, Extended Insulins Download PDF

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Publication number
US20090306337A1
US20090306337A1 US12/375,678 US37567807A US2009306337A1 US 20090306337 A1 US20090306337 A1 US 20090306337A1 US 37567807 A US37567807 A US 37567807A US 2009306337 A1 US2009306337 A1 US 2009306337A1
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Prior art keywords
insulin
human insulin
propionyl
insulin analogue
pegylated
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Peter Madsen
Thomas Børglum Kjeldsen
Tina Møller Tagmose
Palle Jakobsen
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Novo Nordisk AS
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Novo Nordisk AS
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Assigned to NOVO NORDISK A/S reassignment NOVO NORDISK A/S ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MADSEN, PETER, JAKOBSEN, PALLE, TAGMOSE, TINA MOLLER, KJELDSEN, THOMAS BORGLUM
Publication of US20090306337A1 publication Critical patent/US20090306337A1/en
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones

Definitions

  • the present invention is related to PEGylated, extended insulins which have insulin activity and can be used for the treatment of diabetes.
  • the PEGylated, extended insulins have higher bioavailability and a longer time-action profile than regular insulin and are in particular suited for pulmonary administration. They will also have a high physical stability and a low tendency to fibrillation and will be soluble at neutral pH.
  • This invention is also related to pharmaceutical compositions containing the PEGylated, extended insulins.
  • the inherited physical and chemical stability of the insulin molecule is a basic condition for insulin therapy of diabetes mellitus. These basic properties are fundamental for insulin formulation and for applicable insulin administration methods, as well as for shelf-life and storage conditions of pharmaceutical preparations.
  • Use of solutions in administration of insulin exposes the molecule to a combination of factors, e.g., elevated temperature, variable air-liquid-solid interphases as well as shear forces, which may result in irreversible conformation changes, e.g., fibrillation.
  • Efficient pulmonary delivery of a protein is dependent on the ability to deliver the protein to the deep lung alveolar epithelium. Proteins that are deposited in the upper airway epithelium are not absorbed to a significant extent. This is due to the overlying mucus which is approximately 30-40 ⁇ m thick and acts as a barrier to absorption. In addition, proteins deposited on this epithelium are cleared by mucociliary transport up the airways and then eliminated via the gastrointestinal tract. This mechanism also contributes substantially to the low absorption of some protein particles. The extent to which proteins are not absorbed and instead eliminated by these routes depends on their solubility, their size, as well as other less understood characteristics.
  • the organic chain-like molecules often used to enhance properties are polyethylene glycolbased chains, i.e., chains that are based on the repeating unit —CH 2 CH 2 O—.
  • PEG polyethylene glycolbased chains
  • PEG polyethyleneglycol
  • Insulin compositions for pulmonary administration comprising a conjugate of two-chain insulin covalently coupled to one or more molecules of non-naturally hydrophilic polymers including polyalkylene glycols and methods for their preparation are disclosed in WO 02/094200 and WO 03/022996.
  • An aspect of this invention deals with furnishing of a medicament which can conveniently be administered pulmonary to treat diabetic patients.
  • Another aspect of this invention deals with the furnishing of a medicament which can conveniently be administered pulmonary to treat diabetic patients and to reduce the risk of some of or all of the late complications often associated with diabetes.
  • Another aspect of this invention deals with the furnishing of a medicament which can conveniently be administered pulmonary to treat diabetic patients and which is more convenient to use for many patients that the use of injections.
  • Another aspect of this invention deals with the furnishing of a medicament which can conveniently be administered pulmonary to treat diabetic patients and which has a sufficient chemical stability.
  • Another aspect of this invention deals with the furnishing of a medicament which can conveniently be administered pulmonary to treat diabetic patients and which has a sufficient physical stability.
  • Another aspect of this invention deals with the furnishing of a medicament having a sufficiently high insulin receptor affinity.
  • the object of this invention is to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
  • Insulin is a polypeptide hormone secreted by ⁇ -cells of the pancreas and consists of two polypeptide chains designated the A and B chains which are linked together by two inter-chain disulphide bridges.
  • the hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acid followed by proinsulin containing 86 amino acids in the configuration: prepeptide-B-Arg-Arg-C-Lys-Arg-A, in which C is a connecting peptide of 31 amino acids, and A and B are the A and B chains, respectively, of insulin.
  • Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting peptide between the A and B chains to form the two-chain insulin molecule. Insulin is essential in maintaining normal metabolic regulation.
  • insulin covers natural occurring insulins, e.g., human insulin, as well as insulin analogues thereof.
  • amino acid residue covers an amino acid from which a hydrogen atom has been removed from an amino group and/or a hydroxy group has been removed from a carboxy group and/or a hydrogen atom has been removed from a mercapto group. Imprecise, an amino acid residue may be designated an amino acid.
  • insulin analogue covers a polypeptide which has a molecular structure which formally can be derived from the structure of a naturally occurring insulin, e.g., human insulin, by deleting and/or substituting (replacing) one or more amino acid residue occurring in the natural insulin and/or by adding one or more 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.
  • extended insulin covers an insulin analogue wherein there (compared with human insulin) is added one or more amino acid residue either C- or N-terminally to the A- or B-chain of insulin.
  • the A chain may be extended at its C-terminal end, e.g., by 1, 2, 3 or 4 amino acid residues (compared with human insulin) which extensions are denoted A22, A23, A24 and A25, respectively.
  • the amino acid residue in position A23 is PEGylated
  • the amino acid in position A22 may be any amino acid residue except Cys and Lys, and so forth.
  • the A chain may be extended at its N-terminal end, e.g., by 1, 2, 3 or 4 amino acid residues (compared with human insulin) which extensions are denoted A-1, A-2, A-3 and A-4, respectively.
  • A-1 amino acid residues
  • A-2 amino acid residues
  • A-3 amino acid residues
  • A-4 amino acid residues
  • the amino acid in position A-1 may be any amino acid residue except Cys and Lys, and so forth.
  • the extended insulin has an extension at one of the four termini, there may be deletions at other positions in said extended insulin.
  • the extended insulin consists of two chains, i.e., the A chain and B chain.
  • cysteine residues there are six cysteine residues, two of which are present in the A chain forming an intra-chain disulphide bridge (corresponding to A6 and A11 in human insulin) and four of which form two inter-chain disulphide bridges (corresponding to positions A7, A20, B7 and B19 in human insulin).
  • cysteine residues are designated inter-chain cysteine residues.
  • each chain A and B chain
  • one of the inter-chain cysteine residues is closest to the N terminal end of each chain and the other inter-chain cysteine residues is closest to the C terminal end of each chain and, herein, such inter-chain cysteine residues are designated an N terminal inter-chain cysteine residue and a C terminal inter-chain cysteine residue, respectively.
  • an insulin analogue is an extended insulin
  • parent insulin means the extended insulin without appended PEG moieties.
  • mutation covers any change in amino acid sequence (substitutions and insertions with codable amino acids as well as deletions).
  • analogues of the A chain and analogues of the B chains of human insulin covers A and B chains of human insulin, respectively, having one or more substitutions, deletions and or extensions (additions) of the A and B amino acid chains, respectively, relative to the A and B chains, respectively, of human insulin.
  • A-1 indicates the positions of the first amino acids N-terminally to the A1 and B1 positions, respectively, and so forth.
  • desB29 and desB30 indicate an insulin analogue lacking the B29 or B30 amino acid residue, respectively.
  • single chain insulin covers a polypeptide sequence of the general structure BC-A, wherein A is the A chain of human insulin or an analogue thereof, B is the B chain of human insulin or an analogue thereof, and C is a bond or the so-called connecting peptide, e.g., a peptide chain of about 1-35 amino acid residues connecting the C-terminal amino acid residue in the B-chain, e.g., B30, with the N-terminal amino acid residue in the A-chain, e.g., A1. If the B chain is a desB30 chain, the connecting peptide (C) will connect B29 with A1.
  • A is the A chain of human insulin or an analogue thereof
  • B is the B chain of human insulin or an analogue thereof
  • C is a bond or the so-called connecting peptide, e.g., a peptide chain of about 1-35 amino acid residues connecting the C-terminal amino acid residue in the B-chain, e.g., B30, with
  • the single-chain insulin will contain the three, correctly positioned disulphide bridges as in human insulin, i.e., between Cys A7 and Cys B7 , between Cys A20 and Cys B19 and between Cys A6 and Cys A11 .
  • connecting peptide covers a peptide chain which can connect the C-terminal amino acid residue of the B-chain with the N-terminal amino acid residue of the A-chain in insuin.
  • B′A means a single chain insulin wherein the connecting peptide does not consist an any amino acids but simply is a bond, i.e. there is a bond between the B-chain C-terminal and the A-chain N-terminal.
  • fast acting insulin an insulin having a faster onset of action than normal or regular human insulin.
  • long acting insulin is meant an insulin having a longer duration of action than normal or regular human insulin.
  • the numbering of the positions in insulin analogues, extended insulins and A and B chains is done so that the parent compound is human insulin with the numbering used for it.
  • basal insulin as used herein means an insulin peptide which has a time-action of more than 8 hours, in particularly of at least 9 hours. Preferably, the basal insulin has a time-action of at least 10 hours.
  • the basal insulin may thus have a time-action in the range from about 8 to 24 hours, preferably in the range from about 9 to about 15 hours.
  • linker covers a chemical moiety which connects an —HN— group of the extended insulin with the —O— group of the PEG moiety.
  • the linker does not have any influence on the desired action of the final PEGylated extended insulin, especially it does not have any adverse influence.
  • PEG polyethylene glycol
  • polyethylene glycol any water soluble poly(ethylene glycole) or poly(ethylene oxide).
  • the expression PEG will comprise the structure —(CH 2 CH 2 O) n —, where n is an integer from 2 to about 1000.
  • a commonly used PEG is end-capped PEG, wherein one end of the PEG termini is end-capped with a relatively inactive group such as alkoxy, while the other end is a hydroxyl group that may be further modified by linker moieties.
  • An often used capping group is methoxy and the corresponding end-capped PEG is often denoted mPEG.
  • mPEG is CH 3 O(CH 2 CH 2 O) n —, where n is an integer from 2 to about 1000 sufficient to give the average molecular weight indicated for the whole PEG moiety, e.g., for mPEG Mw 2,000, n is approximately 44 (a number that is subject for batch-to-batch variation).
  • PEG is often used instead of mPEG.
  • “PEG” followed by a number (not being a subscript) indicates a PEG moiety with the approximate molecular weight equal the number.
  • PEG2000 is a PEG moiety having an approximate molecular weight of 2000.
  • PEG forms of this invention are branched, linear, forked, dumbbell PEGs, and the like and the PEG groups are typically polydisperse, possessing a low polydispersity index of less than about 1.05.
  • the PEG moieties present in an extended insulin will for a given molecular weight typically consist of a range of ethyleneglycol (or ethyleneoxide) monomers.
  • a PEG moiety of molecular weight 2000 will typically consist of 44 ⁇ 10 monomers, the average being around 44 monomers.
  • the molecular weight (and number of monomers) will typically be subject to some batch-to-batch variation.
  • PEG forms are monodisperse that can be branched, linear, forked, or dumbbell shaped as well. Being monodisperse means that the length (or molecular weight) of the PEG polymer is specifically defined and is not a mixture of various lengths (or molecular weights).
  • mdPEG is used to indicate that the mPEG moiety is monodisperse, using “d” for “discrete”.
  • the number in subscript after mdPEG, for example “12” in mdPEG 12 indicates the number of ethyleneglycol monomers within the monodisperse polymer (oligomer).
  • PEGylation covers modification of insulin by attachment of one or more PEG moieties via a linker.
  • the PEG moiety can either be attached by nucleophilic substitution (acylation) on N-terminal alpha-amino groups or on lysine residue(s) on the gamma-positions, e.g., with OSu-activated esters, or PEG moieties can be attached by reductive alkylation—also on amino groups present in the extended insulin molecule—using PEG-aldehyde reagents and a reducing agent, such as sodium cyanoborohydride, or, alternatively, PEG moieties can be attached to the sidechain of an unpaired cysteine residue in a Michael addition reaction using eg. PEG maleimide reagents.
  • PEGylated extended insulin having insulin activity is meant a PEGylated, extended insulin with either the ability to lower the blood glucose in mammalians as measured in a suitable animal model, which may be a rat, rabbit, or pig model, after suitable administration e.g., by intravenous, subcutaneous, or pulmonary administration, or an insulin receptor binding affinity.
  • alkyl covers a saturated, branched or straight hydrocarbon group.
  • alkoxy covers the radical “alkyl-O—”.
  • Representative examples are methoxy, ethoxy, propoxy (e.g., 1-propoxy and 2-propoxy), butoxy (e.g., 1-butoxy, 2-butoxy and 2-methyl-2-propoxy), pentoxy (1-pentoxy and 2-pentoxy), hexoxy (1-hexoxy and 3-hexoxy), and the like.
  • alkylene covers a saturated, branched or straight bivalent hydrocarbon group having from 1 to 12 carbon atoms.
  • Representative examples include, but are not limited to, methylene, 1,2-ethylene, 1,3-propylene, 1,2-propylene, 1,3-butylene, 1,4-butylene, 1,4-pentylene, 1,5-pentylene, 1,5-hexylene, 1,6-hexylene, and the like.
  • high physical stability is meant a tendency to fibrillation being less than 50% of that of human insulin. Fibrillation may be described by the lag time before fibril formation is initiated at a given conditions.
  • a polypeptide with insulin receptor and IGF-1 receptor affinity is a polypeptide which is capable of interacting with an insulin receptor and a human IGF-1 receptor in a suitable binding assay.
  • Such receptor assays are well-know within the field and are further described in the examples.
  • the present PEGylated, extended insulin will not bind to the IGF-1 receptor or will have a rather low affinity to said receptor. More precisely, the PEGylated, extended insulins of this invention will have an affinity towards the IGF-1 receptor of substantially the same magnitude or less as that of human insulin
  • treatment and treating means the management and care of a patient for the purpose of combating a disease, disorder or condition.
  • the term is intended to include the delaying of the progression of the disease, disorder or condition, the alleviation or relief of symptoms and complications, and/or the cure or elimination of the disease, disorder or condition.
  • the patient to be treated is preferably a mammal, in particular a human being.
  • treatment of a disease means the management and care of a patient having developed the disease, condition or disorder.
  • the purpose of treatment is to combat the disease, condition or disorder.
  • Treatment includes the administration of the active compounds to eliminate or control the disease, condition or disorder as well as to alleviate the symptoms or complications associated with the disease, condition or disorder.
  • prevention of a disease as used herein is defined as the management and care of an individual at risk of developing the disease prior to the clinical onset of the disease.
  • the purpose of prevention is to combat the development of the disease, condition or disorder, and includes the administration of the active compounds to prevent or delay the onset of the symptoms or complications and to prevent or delay the development of related diseases, conditions or disorders.
  • effective amount means a dosage which is sufficient in order for the treatment of the patient to be effective compared with no treatment.
  • POT is the Schizosaccharomyces pombe triose phosphate isomerase gene
  • TPI1 is the S. cerevisiae triose phosphate isomerase gene.
  • a leader an amino acid sequence consisting of a pre-peptide (the signal peptide) and a pro-peptide.
  • signal peptide is understood to mean a pre-peptide which is present as an N-terminal sequence on the precursor form of a protein.
  • the function of the signal peptide is to allow the heterologous protein to facilitate translocation into the endoplasmic reticulum.
  • the signal peptide is normally cleaved off in the course of this process.
  • the signal peptide may be heterologous or homologous to the yeast organism producing the protein.
  • a number of signal peptides which may be used with the DNA construct of this invention including yeast aspartic protease 3 (YAP3) signal peptide or any functional analog (Egel-Mitani et al. (1990) YEAST 6:127-137 and U.S. Pat. No.
  • pro-peptide means a polypeptide sequence whose function is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e. exportation of the polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast cell).
  • the pro-peptide may be the yeast ⁇ -factor pro-peptide, vide U.S. Pat. Nos. 4,546,082 and 4,870,008.
  • the pro-peptide may be a synthetic pro-peptide, which is to say a pro-peptide not found in nature. Suitable synthetic pro-peptides are those disclosed in U.S. Pat.
  • the pro-peptide will preferably contain an endopeptidase processing site at the C-terminal end, such as a Lys-Arg sequence or any functional analogue thereof.
  • 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.
  • amino acids present in the PEGylated insulins of this invention are, preferably, amino acids which can be coded fro by a nucleic acid.
  • Da is Dalton (molecular weight)
  • mPEG-SBA is mPEG-CH 2 CH 2 CH 2 —CO—OSu (N-hydroxysuccinimidyl ester of mPEG-butanoic acid)
  • mPEG-SMB is mPEG-CH 2 CH 2 CH(CH 3 )—CO—OSu (N-hydroxysuccinimidyl ester of mPEG- ⁇ -methylbutanoic acid)
  • mPEG-SPA is mPEG-CH 2 CH 2 —CO—OSu (N-hydroxysuccinimidyl ester of mPEG-propionic acid)
  • Mw is molecular weight
  • R room temperature
  • SA is sinapinic acid
  • Su is 1-succinimidyl
  • this invention is related to a PEGylated insulin analogue which, compared with human insulin, has one or more extensions extended from the A1, B1, A21 and/or B30 position(s), said extension(s) consist(s) of amino acid residue(s) and wherein the PEG moiety, via a linker, is attached to one or more of the amino acid residues in the extension(s).
  • a PEG group can be attached to side chain(s) of lysine or cysteine residue(s) when present or attached to the N-terminal amino group(s) or at both places in the parent insulin.
  • the linker is typically a derivative of a carboxylic acid, where the carboxylic acid functionality is used for attachment to the parent insulin via an amide bond.
  • the linker may be an acetic acid moiety with the linking motif: —CH 2 CO—, a propionic acid moiety with the linking motif: —CH 2 CH 2 CO— or —CHCH 3 CO—, or a butyric acid moiety with the linking motif: —CH 2 CH 2 CH 2 CO— or —CH 2 CHCH 3 CO—.
  • the linker may be a —CO— group.
  • the parent insulin molecule may have a limited number of the naturally occurring amino acid residues substituted with other amino acid residues as explained in the detailed part of the specification.
  • this invention relates to a PEGylated, extended insulin, wherein the parent insulin analogue deviates from human insulin in one or more of the following deletions or substitutions: E or D in position A14, Q in position A18, A, G or Q in position A21, G or Q in position B1 or no amino acid residue in position B1, Q, S or T in position B3 or no amino acid residue in position B3, Q in position B13, H in position B25 or no amino acid residue in position B25, no amino acid residue in position B27, D, E or R in position B28, P, Q or R in position B29 or no amino acid residue in position B29, no amino acid residue in position B30.
  • the PEG group may vary in size within a large range as is well known within the art. However, too large PEG groups may interfere in a negative way with the biological activity of the PEGylated, extended insulin molecule.
  • this invention is related to pharmaceutical preparations comprising the PEGylated, extended insulin of this invention and suitable adjuvants and additives such as one or more agents suitable for stabilization, preservation or isotoni, e.g., zinc ions, phenol, cresol, a parabene, sodium chloride, glycerol or mannitol.
  • suitable adjuvants and additives such as one or more agents suitable for stabilization, preservation or isotoni, e.g., zinc ions, phenol, cresol, a parabene, sodium chloride, glycerol or mannitol.
  • the zinc content of the present formulations may be between 0 and about 6 zinc atoms per insulin hexamer.
  • the pH value of the pharmaceutical preparation may be between about 4 and about 8.5, between about 4 and about 5 or between about 6.5 and about 7.5.
  • this invention is related to the use of the PEGylated, extended insulin as a pharmaceutical for the reducing of blood glucose levels in mammalians, in particularly for the treatment of diabetes.
  • this invention is related to the use of the PEGylated, extended insulin for the preparation of a pharmaceutical preparation for the reducing of blood glucose level in mammalians, in particularly for the treatment of diabetes.
  • this invention is related to a method of reducing the blood glucose level in mammalians by administrating a therapeutically active dose of a PEGylated, extended insulin of this invention to a patient in need of such treatment.
  • the PEGylated, extended insulins are administered in combination with one or more further active substances in any suitable ratios.
  • Such further active agents may be selected from human insulin, fast acting insulin analogues, antidiabetic agents, antihyperlipidemic agents, antiobesity agents, antihypertensive agents and agents for the treatment of complications resulting from or associated with diabetes.
  • the two active components are administered as a mixed pharmaceutical preparation. In another embodiment, the two components are administered separately either simultaneously or sequentially.
  • the PEGylated, extended insulins of this invention may be administered together with fast acting human insulin or human insulin analogues.
  • Such fast acting insulin analogue may be such wherein the amino acid residue in position B28 is Asp, Lys, Leu, Val, or Ala and the amino acid residue in position B29 is Lys or Pro, des(B28-B30), des(B27) or des(B30) human insulin, and an analogue wherein the amino acid residue in position B3 is Lys and the amino acid residue in position B29 is Glu or Asp.
  • the PEGylated, extended insulin of this invention and the rapid acting human insulin or human insulin analogue can be mixed in a ratio from about 90/10%; about 70/30% or about 50/50%.
  • the PEGylated, extended insulins of this invention may also be used on combination treatment together with an antidiabetic agent.
  • Antidiabetic agents will include insulin, GLP-1 (1-37) (glucagon like peptide-1) described in WO 98/08871, WO 99/43706, U.S. Pat. No. 5,424,286 and WO 00/09666, GLP-2, exendin-4(1-39), insulinotropic fragments thereof, insulinotropic analogues thereof and insulinotropic derivatives thereof.
  • Insulinotropic fragments of GLP-1 (1-37) are insulinotropic peptides for which the entire sequence can be found in the sequence of GLP-1 (1-37) and where at least one terminal amino acid has been deleted.
  • the PEGylated, extended insulins of this invention may also be used on combination treatment together with an oral antidiabetic such as a thiazolidindione, metformin and other type 2 diabetic pharmaceutical preparation for oral treatment.
  • an oral antidiabetic such as a thiazolidindione, metformin and other type 2 diabetic pharmaceutical preparation for oral treatment.
  • the PEGylated, extended insulin of this invention may be administered in combination with one or more antiobesity agents or appetite regulating agents.
  • this invention is related to a pulmonal pharmaceutical preparation
  • a pulmonal pharmaceutical preparation comprising the PEGgylated extended insulin of this invention and suitable adjuvants and additives such as one or more agents suitable for stabilization, preservation or isotoni, e.g., zinc ions, phenol, cresol, a parabene, sodium chloride, glycerol, propyleneglycol or mannitol.
  • suitable adjuvants and additives such as one or more agents suitable for stabilization, preservation or isotoni, e.g., zinc ions, phenol, cresol, a parabene, sodium chloride, glycerol, propyleneglycol or mannitol.
  • the stability and solubility properties of insulin are important underlying aspects for current insulin therapy.
  • This invention is addressed to these issues by providing stable, PEGylated, extended insulin analogues wherein the PEGylation in the extension decreases molecular flexibility and concomitantly reduce the fibrillation propensity and limit or modify the pH precipitation zone.
  • the PEGylated, extended insulins of this invention are in particularly intended for pulmonal administration due to their relatively high bioavailability compared to, e.g., human insulin. Furthermore, the PEGylated, extended insulins will have a protracted insulin activity.
  • N i is the mole-fraction (or the number-fraction) of molecules with molecular weight M i in the polymer mixture.
  • M i is the mole-fraction (or the number-fraction) of molecules with molecular weight M i in the polymer mixture.
  • the ratio of M w to M n is known as the polydispersity index (PDI), and provides a rough indication of the breadth of the distribution.
  • the PDI approaches 1.0 (the lower limit) for special polymers with very narrow MW distributions.
  • high molecular weight PEG chains e.g., having an average molecular weight of 4000-6000 daltons or greater, although generally found to decrease the bioactivity of the insulin molecule, may be preferred for increasing half-life, e.g., in the case of formulations for pulmonary administration.
  • the PEG groups of this invention will typically comprise a number of (—OCH 2 CH 2 —) subunits.
  • the PEG groups of the invention will for a given molecular weight typically consist of a range of ethyleneglycol (or ethyleneoxide) monomers.
  • a PEG group of molecular weight 2000 dalton will typically consist of 43 ⁇ 10 monomers, the average being around 43-44 monomers.
  • the parent insulin molecule which is PEGylated in this invention is an extended insulin molecule, i.e., an insulin molecule having one or more amino acid residues attached to the N-terminal end of the parent A and/or B chain, e.g., to A1 and/or B1, and/or attached to the C-terminal end of the parent A and/or B chain, e.g., A21 and/or B30, referring to human insulin.
  • the extended insulin molecule i.e., the parent insulin, contains at least 52 amino acid residues.
  • the PEGgylated extended insulins of this invention may be mono-substituted having only one PEG group attached to a lysine amino acid residue in the parent insulin molecule or to a N-terminal amino acid residue.
  • the PEGylated, extended insulins of this invention may comprise two, three- or four PEG groups. If the extended insulin comprises more than one PEG group, it will typically have the same PEG moiety attached to each lysine group or to the N-terminal amino acid residue.
  • the individual PEG groups may also vary from each other in size and length.
  • an extended insulin having the following deviations as compared to human insulin: A22K, B29R, desB30 and being PEGylated in the lysine residue in position A22 with mPEG-propionic acid, 2 kDa, e.g., using mPEG-SPA is named A22K(N ⁇ mPEG2000-propionyl) B29R desB30 human insulin. It is obvious that if any of the corresponding other PEGylation reagents (Mw 2000 Da), containing other linkers, e.g. the butyric acid linkers, were used for preparation of that particular compound, the “exact” name of that particular compound would be different, but the small molecular differences will not result in any differences in biological properties.
  • the PEGylated extended insulins are, to a great extent, named as if the linking moiety is a propionic acid linker, irrespective of the actual linker.
  • the linking moiety is a propionic acid linker, irrespective of the actual linker.
  • the important variables are, with respect to biological properties: Size (in Daltons) and shape of the PEG moiety and position of the PEG attachment within the protein.
  • the parent insulins are produced by expressing a DNA sequence encoding the extended insulin in question in a suitable host cell by well known technique as disclosed in, e.g., U.S. Pat. No. 6,500,645.
  • the parent insulin is either expressed directly or as a precursor molecule which has an N-terminal extension on the B-chain.
  • This N-terminal extension may have the function of increasing the yield of the directly expressed product and may be of up to 15 amino acid residues long.
  • the N-terminal extension is to be cleaved of in vitro after isolation from the culture broth and will therefore have a cleavage site next to B1.
  • N-terminal extensions of the type suitable in this invention are disclosed in U.S. Pat. No. 5,395,922, and European Patent No. 765,395A.
  • the polynucleotide sequence coding for the parent insulin may be prepared synthetically by established standard methods, e.g., the phosphoamidite method described by Beaucage et al. (1981) Tetrahedron Letters 22:1859-1869, or the method described by Matthes et al. (1984) EMBO Journal 3: 801-805.
  • oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, duplexed and ligated to form the synthetic DNA construct.
  • a currently preferred way of preparing the DNA construct is by polymerase chain reaction (PCR).
  • the polynucleotide sequences may also be of mixed genomic, cDNA, and synthetic origin.
  • a genomic or cDNA sequence encoding a leader peptide may be joined to a genomic or cDNA sequence encoding the A and B chains, after which the DNA sequence may be modified at a site by inserting synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination in accordance with well-known procedures or preferably generating the desired sequence by PCR using suitable oligonucleotides.
  • the recombinant method will typically make use of a vector which is capable of replicating in the selected microorganism or host cell and which carries a polynucleotide sequence encoding the parent insulin.
  • the recombinant vector may be an autonomously replicating vector, i.e., a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
  • the vector may be linear or closed circular plasmids and will preferably contain an element(s) that permits stable integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
  • the recombinant expression vector is capable of replicating in yeast.
  • sequences which enable the vector to replicate in yeast are the yeast plasmid 2 ⁇ m replication genes REP 1-3 and origin of replication.
  • the vector may contain one or more selectable markers which permit easy selection of trans-formed cells.
  • a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus lichenifonnis , or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
  • Selectable markers for use in a filamentous fungal host cell include amdS (acetamidase), argB (or nithine carbamoyltransferase), pyrG (orotidine-5′-phosphate decarboxylase) and trpC (anthranilate synthase.
  • Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
  • a well suited selectable marker for yeast is the Schizosaccharomyces pompe TPI gene (Russell (1985) Gene 40:125-130).
  • the polynucleotide sequence is operably connected to a suitable promoter sequence.
  • the promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extra-cellular or intra-cellular polypeptides either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription in a bacterial host cell are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus lichenifonnis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and Bacillus lichenifonnis penicillinase gene (penP).
  • dagA Streptomyces coelicolor agarase gene
  • sacB Bacillus subtilis levansucrase gene
  • amyL Bacillus stearothermophilus maltogenic amylase gene
  • amyQ Bacillus amyloliquefaciens alpha-amylase gene
  • penP Bacillus
  • promoters for directing the transcription in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, and Aspergillus niger acid stable alpha-amylase.
  • useful promoters are the Saccharomyces cerevisiae Mal, TPI, ADH or PGK promoters.
  • the polynucleotide sequence encoding the parent insulin will also typically be operably connected to a suitable terminator.
  • a suitable terminator is the TPI terminator (Alber et al. (1982) J. Mol. Appl. Genet. 1:419-434).
  • the procedures used to ligate the polynucleotide sequence encoding the parent insulin, the promoter and the terminator, respectively, and to insert them into a suitable vector containing the information necessary for replication in the selected host are well known to persons skilled in the art. It will be understood that the vector may be constructed either by first preparing a DNA construct containing the entire DNA sequence encoding the extended insulins of this invention, and subsequently inserting this fragment into a suitable expression vector, or by sequentially inserting DNA fragments containing genetic information for the individual elements (such as the signal, pro-peptide, connecting peptide, A and B chains) followed by ligation.
  • the vector comprising the polynucleotide sequence encoding the parent insulin is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector.
  • the term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • the host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote.
  • Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, Streptomyces cell, or gram negative bacteria such as E. coli and Pseudomonas sp.
  • Eukaryote cells may be mammalian, insect, plant, or fungal cells.
  • the host cell is a yeast cell.
  • the yeast organism may be any suitable yeast organism which, on cultivation, produces large amounts of the single chain insulin of the invention.
  • yeast organisms are strains selected from the yeast species Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Sacchoromyces uvarum, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp., and Geotrichum fermentans.
  • the transformation of the yeast cells may for instance be effected by protoplast formation followed by transformation in a manner known per se.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing yeast organisms.
  • the secreted extended insulin a significant proportion of which will be present in the medium in correctly processed form, may be recovered from the medium by conventional procedures including separating the yeast cells from the medium by centrifugation, filtration or catching the insulin precursor by an ion exchange matrix or by a reverse phase absorption matrix, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g., ammonium sulphate, followed by purification by a variety of chromatographic procedures, e.g., ion exchange chromatography, affinity chromatography, or the like.
  • a salt e.g., ammonium sulphate
  • the PEGylated, extended insulins of this invention may be administered subcutaneously, orally, or pulmonary.
  • the PEGylated, extended insulins of this invention are formulated analogously with the formulation of known insulins. Furthermore, for subcutaneous administration, the PEGylated, extended insulins of this invention are administered analogously with the administration of known insulins and, generally, the physicians are familiar with this procedure.
  • PEGylated, extended insulins of this invention may be administered by inhalation in a dose effective to increase circulating insulin levels and/or to lower circulating glucose levels. Such administration can be effective for treating disorders such as diabetes or hyperglycemia. Achieving effective doses of insulin requires administration of an inhaled dose of more than about 0.5 ⁇ g/kg to about 50 ⁇ g/kg of PEGylated, extended insulins of this invention.
  • a therapeutically effective amount can be determined by a knowledgeable practitioner, who will take into account factors including insulin level, blood glucose levels, the physical condition of the patient, the patient's pulmonary status, or the like.
  • the PEGylated, extended insulins of this invention may be delivered by inhalation to achieve slow absorption and/or reduced systemical clearance thereof.
  • Different inhalation devices typically provide similar pharmacokinetics when similar particle sizes and similar levels of lung deposition are compared.
  • the PEGylated, extended insulins of this invention may be delivered by any of a variety of inhalation devices known in the art for administration of a therapeutic agent by inhalation. These devices include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Preferably, the PEGylated, extended insulins of this are delivered by a dry powder inhaler or a sprayer.
  • an inhalation device for administering PEGylated, extended insulins of this invention is advantageously reliable, reproducible, and accurate.
  • the inhalation device should deliver small particles or aerosols, e.g., less than about 10 ⁇ m, for example about 1-5 ⁇ m, for good respirability.
  • Some specific examples of commercially available inhalation devices suitable for the practice of this invention are Cyclohaler, TurbohalerTM (Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), SpirosTM inhaler (Dura), devices marketed by Inhale Therapeutics, AERxTM (Aradigm), the Ultravent® nebulizer (Mallinckrodt), the Acorn II® nebulizer (Marquest Medical Products), the Ventolin® metered dose inhaler (Glaxo), the Spinhaler® powder inhaler (Fisons), or the like.
  • the formulation of PEGylated, extended insulins of this invention depends on the type of inhalation device employed.
  • the frequency of administration and length of time for which the system is activated will depend mainly on the concentration of PEGylated, extended insulins in the aerosol.
  • shorter periods of administration can be used at higher concentrations of PEGylated, extended insulins in the nebulizer solution.
  • Devices such as metered dose inhalers can produce higher aerosol concentrations, and can be operated for shorter periods to deliver the desired amount of the PEGylated, extended insulins.
  • Devices such as powder inhalers deliver active agent until a given charge of agent is expelled from the device.
  • the amount of insulin PEGylated, extended insulins of this invention in a given quantity of the powder determines the dose delivered in a single administration.
  • the particle size of PEGylated, extended insulins of this invention in the formulation delivered by the inhalation device is critical with respect to the ability of insulin to make it into the lungs, and preferably into the lower airways or alveoli.
  • the PEGylated, extended insulins of this invention ion is formulated so that at least about 10% of the PEGylated, extended insulins delivered is deposited in the lung, preferably about 10 to about 20%, or more. It is known that the maximum efficiency of pulmonary deposition for mouth breathing humans is obtained with particle sizes of about 2 ⁇ m to about 3 ⁇ m. When particle sizes are above about 5 ⁇ m, pulmonary deposition decreases substantially.
  • particles of the PEGylated, extended insulins delivered by inhalation have a particle size preferably less than about 10 ⁇ m, more preferably in the range of about 1 ⁇ m to about 5 ⁇ m.
  • the formulation of the PEGylated, extended insulins is selected to yield the desired particle size in the chosen inhalation device.
  • a PEGylated, extended insulin of this invention is prepared in a particulate form with a particle size of less than about 10 ⁇ m, preferably about 1 to about 5 ⁇ m.
  • the preferred particle size is effective for delivery to the alveoli of the patient's lung.
  • the dry powder is largely composed of particles produced so that a majority of the particles have a size in the desired range.
  • at least about 50% of the dry powder is made of particles having a diameter less than about 10 ⁇ m.
  • Such formulations can be achieved by spray drying, milling, micronisation, or critical point condensation of a solution containing the PEGylated, extended insulin of this invention and other desired ingredients. Other methods also suitable for generating particles useful in the current invention are known in the art.
  • the particles are usually separated from a dry powder formulation in a container and then transported into the lung of a patient via a carrier air stream.
  • a carrier air stream typically, in current dry powder inhalers, the force for breaking up the solid is provided solely by the patient's inhalation.
  • air flow generated by the patient's inhalation activates an impeller motor which deagglomerates the particles.
  • Formulations of PEGylated, extended insulins of this invention for administration from a dry powder inhaler typically include a finely divided dry powder containing the derivative, but the powder can also include a bulking agent, carrier, excipient, another additive, or the like.
  • Additives can be included in a dry powder formulation of PEGylated, extended insulin, e.g., to dilute the powder as required for delivery from the particular powder inhaler, to facilitate processing of the formulation, to provide advantageous powder properties to the formulation, to facilitate dispersion of the powder from the inhalation device, to stabilize the formulation (for example, antioxidants or buffers), to provide taste to the formulation, or the like.
  • the additive does not adversely affect the patient's airways.
  • the PEGylated, extended insulin can be mixed with an additive at a molecular level or the solid formulation can include particles of the PEGylated, extended insulin mixed with or coated on particles of the additive.
  • Typical additives include mono-, di-, and polysaccharides; sugar alcohols and other polyols, such as, e.g., lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, starch, or combinations thereof; surfactants, such as sorbitols, diphosphatidyl choline, or lecithin; or the like.
  • an additive such as a bulking agent
  • an additive is present in an amount effective for a purpose described above, often at about 50% to about 90% by weight of the formulation.
  • Additional agents known in the art for formulation of a protein such as insulin analogue protein can also be included in the formulation.
  • a spray including the PEGylated, extended insulins of this invention can be produced by forcing a suspension or solution of the PEGylated, extended insulin through a nozzle under pressure.
  • the nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size.
  • An electrospray can be produced, e.g., by an electric field in connection with a capillary or nozzle feed.
  • particles of insulin conjugate delivered by a sprayer have a particle size less than about 10 ⁇ m, preferably in the range of about 1 ⁇ m to about 5 ⁇ m.
  • Formulations of PEGylated, extended insulins of this invention suitable for use with a sprayer will typically include the PEGylated, extended insulins in an aqueous solution at a concentration of from about 1 mg to about 500 mg of the PEGylated, extended insulin per ml of solution.
  • the upper limit may be lower, e.g., 450, 400, 350, 300, 250, 200, 150, 120, 100 or 50 mg of the PEGylated insulin per ml of solution.
  • the formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc.
  • the formulation can also include an excipient or agent for stabilization of the PEGylated, extended insulin, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.
  • Bulk proteins useful in formulating insulin conjugates include albumin, protamine, or the like.
  • Typical carbohydrates useful in formulating the PEGylated, extended insulin include sucrose, mannitol, lactose, trehalose, glucose, or the like.
  • the PEGylated, extended insulins formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the insulin conjugate caused by atomization of the solution in forming an aerosol.
  • Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between about 0.001 and about 4% by weight of the formulation.
  • compositions containing a PEGylated, extended insulin of this invention may also 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.
  • compositions of the PEGylated, extended insulins of this 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.
  • a PEGylated, extended insulin is dissolved in an amount of water which is somewhat less than the final volume of the composition to be prepared.
  • Zink, an isotonic agent, a preservative and/or a buffer is/are 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 is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, 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 this invention.
  • the formulation further comprises a pharmaceutically acceptable preservative which 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 (3-(4-chlorophenoxy)-1,2-propanediol) or mixtures thereof.
  • a pharmaceutically acceptable preservative which may be selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,
  • the preservative is present in a concentration from about 0.1 mg/ml to 20 mg/ml. In a further embodiment of this invention the preservative is present in a concentration from about 0.1 mg/ml to 5 mg/ml. In a further embodiment of this invention the preservative is present in a concentration from about 5 mg/ml to 10 mg/ml. In a further embodiment of this invention the preservative is present in a concentration from about 10 mg/ml to 20 mg/ml. Each one of these specific preservatives constitutes an alternative embodiment of this invention.
  • 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, 1 gth edition, 1995.
  • the formulation further comprises an isotonic agent which may be selected from the group consisting of a salt (e.g., sodium chloride), a sugar or sugar alcohol, an amino acid (for example, L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine), an alditol (e.g. glycerol (glycerine), 1,2-propanediol (propyleneglycol), 1,3-propanediol or 1,3-butanediol), polyethyleneglycol (e.g., PEG400) or mixtures thereof.
  • a salt e.g., sodium chloride
  • an amino acid for example, L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine
  • an alditol e.g. glyce
  • 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, hydroxyethyl 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, e.g., mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, 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 this invention.
  • the sugar or sugar alcohol concentration is between about 1 mg/ml and about 150 mg/ml.
  • the isotonic agent is present in a concentration from about 1 mg/ml to 50 mg/ml. In a further embodiment of this invention the isotonic agent is present in a concentration from about 1 mg/ml to 7 mg/ml. In a further embodiment of this invention the isotonic agent is present in a concentration from about 8 mg/ml to 24 mg/ml. In a further embodiment of this invention the isotonic agent is present in a concentration from about 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agents constitutes an alternative embodiment of this invention.
  • 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 alcohol.
  • buffers examples include sodium acetate, glycylglycine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and sodium phosphate.
  • a composition for nasal administration of a PEGylated, extended insulins of this invention may, e.g., be prepared as described in European Patent No. 272097.
  • compositions containing PEGylated, extended insulins 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 PEGylated, extended insulin of this invention be determined for each individual patient by those skilled in the art in a similar way as for known insulin compositions.
  • this invention does not relate to PEGylated insulin analogues wherein the parent insulin analogue is so-called single-chain insulin (Danish appl. No.: 2005/00400 and WO appl. No.: EP2006/060816; our ref.: 7148).
  • the parent insulin analogue is so-called single-chain insulin (Danish appl. No.: 2005/00400 and WO appl. No.: EP2006/060816; our ref.: 7148).
  • PEGylation reagents are listed as activated N-hydroxysuccinimide esters (OSu).
  • active esters such as 4-nitrophenoxy and many other active esters known to those skilled in the art.
  • the PEG (or mPEG) moiety, CH 3 O—(CH 2 CH 2 O) n —, can be of any size up to Mw 40.000 Da, e.g., 750 Da, 2000 Da, 5000 Da, 20.000 Da and 40.000 Da.
  • the mPEG moiety can be polydisperse but also monodisperse consisting of mPEG's with well defined chain lengths (and, thus, molecular weights) of, e.g., 12 or 24 repeating ethylene glycol units—denoted mdPEGx for m: methyl/methoxy end-capped, d: discrete and x for the number of repeating ethylene glucol residues, e.g., 12 or 24.
  • the PEG moiety can be either straight chain or branched.
  • the structure/sequence of the PEG-residue on the extended insulin can formally be obtained by replacing the leaving group (e.g., “—OSu”) from the various PEGylation reagents with “NH-insulin”, where the insulin is PEGylated either in an epsilon position in a lysine residue or in the alpha-amino position in the A- or B-chain (or both):
  • larger PEGylation reagents can be prepared by assembling two or more smaller PEG reagents.
  • end-capped PEG reagents as N-hydroxysuccinimide esters like any of the ones above can be coupled to—optionally protected—PEG moieties that are functionalised by amino-groups in one end and carboxylic acid (esters) in the other end.
  • carboxylic acid esters
  • the carboxylic acid is activated eg. as the N-hydroxysuccinimide ester to furnish a longer PEGylation reagent.
  • the obtained PEGylation reagent can be further extended by repeating the cycle one or more times. This principle and methodology is illustrated in the examples.
  • This methodology enables construction of larger monodisperse (and polydisperse) PEGylation reagents of tailored sizes.
  • PEG residues of the invention includes:
  • mPEG750 (where “750” indicates the average molecular weight in Da), mPEG2000, mPEG5000, mPEG10000, mPEG20000, mPEG30000, mPEG40000, mdPEG 12 , (wherein “12” in subscript indicates the number of PEG monomers—as defined herein and eg. by Quanta BioDesign Ltd.) mdPEG 24 , mdPEG 3x12 (wherein “3 ⁇ 12” in subscript indicates that PEG is branched and composed of 3 arms each composed of 12 PEG monomers—as defined herein and eg.
  • PEGylation reagents are listed as maleimide derivatives.
  • other Michael acceptors may be employed, such as vinylsulfones and many other Michael acceptors known to those skilled in the art.
  • the PEG (or mPEG) moiety, CH 3 O—(CH 2 CH 2 O) n —, can be of any size up to Mw about 40.000 Da.
  • the structure/sequence of the PEG-residue on the extended insulin can formally be obtained by replacing the maleimide “MAL” from the various PEGylation reagents with “3-thio-succinimidyl-Ala-insulin”, where the insulin is PEGylated at a free cysteine residue according to the scheme below:
  • the PEGylated, extended insulins of this invention have in the following all been named as if the linker connecting the PEG moiety to the insulin in all cases is a (3-)propionyl linker (—CH 2 —CH 2 —CO—). It is evident from the foregoing that many types of linkers are commercially available and since it is not the exact structure/composition of the linker that governs the beneficial effects of placing the PEG moiety at residues outside the sequence of regular insulin, it is to be understood that all types of linkers (cf. above) are within the scope of this invention.
  • Parent extended insulins of the invention comprise the following:
  • A22K, B29R, desB30 human insulin A21Q, A22G, A23K, B29R, desB30 human insulin; A21G, A22G, A23K, B29R, desB30 human insulin; A22G, A23K, B29R, desB30 human insulin; A21Q, A22G, A23G, A24K, B29R, desB30 human insulin; A21G, A22G, A23G, A24K, B29R, desB30 human insulin; A21Q, A22G, A23G, A24G, A25K, B29R, desB30 human insulin; A21G, A22G, A23G, A24G, A25K, B29R, desB30 human insulin; A21G, A22K, B29R, desB30 human insulin; A21G, A22G, A23K, B29R, desB30 human insulin; A21G, A22G, A23K, B29R, desB30 human insulin; A21G, A
  • plasmids are of the C-POT type, similar to those described in EP 171142, which are characterized by containing the Schizosaccharomyces pombe triose phosphate isomerase gene (POT) for the purpose of plasmid selection and stabilization in S. cerevisiae .
  • POT Schizosaccharomyces pombe triose phosphate isomerase gene
  • the plasmids also contain the S. cerevisiae triose phosphate isomerase promoter and terminator. These sequences are similar to the corresponding sequences in plasmid pKFN1003 (described in WO 90/10075) as are all sequences except the sequence of the EcoRI-XbaI fragment encoding the fusion protein of the leader and the insulin product.
  • EcoRI-XbaI fragment of pKFN1003 is simply replaced by an EcoRI-XbaI fragment encoding the leader-insulin fusion of interest.
  • EcoRI-XbaI fragments may be synthesized using synthetic oligonucleotides and PCR according to standard techniques.
  • Yeast transformants were prepared by transformation of the host strain S. cerevisiae strain MT663 (MATa/MAT ⁇ pep4-3/pep4-3 HIS4/his4 tpi::LEU2/tpi::LEU2 Cir + ).
  • the yeast strain MT663 was deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen in connection with filing WO 92/11378 and was given the deposit number DSM 6278.
  • plasmid DNA 0.1 mg
  • the mixture was centrifuged and the pellet resuspended in 0.1 ml of SOS (1.2 M sorbitol, 33% v/v YPD, 6.7 mM CaCl 2 ) and incubated at 30° C. for 2 hours.
  • the suspension was then centrifuged and the pellet resuspended in 0.5 ml of 1.2 M sorbitol.
  • insulin precursors were produced as described above and isolated from the culture medium and purified.
  • the insulin precursors were PEGylated and processed as described in the examples below to produce the final insulin derivatives (General Procedure (A)).
  • the precursors can be processed by trypsin prior to PEGylation (General Procedure (B)).
  • These insulin derivatives were tested for biological insulin activity as measured by binding affinity to the human insulin receptor relative to that of human insulin as described below.
  • the insulin derivatives of this invention can be purified by employing one or more of the following procedures which are typical within the art. These procedures can—if needed—be modified with regard to gradients, pH, salts, concentrations, flow, columns and so forth. Depending on factors such as impurity profile, solubility of the insulins in question etcetera, these modifications can readily be recognised and made by a person skilled in the art.
  • Step 1 Preparation and Purification of the Insulin Precursor LysA22 ArqB29 B29R desB30 B′A
  • the insulin precursor A22K, B29R, desB30, B′A single chain insulin can be purified as described in the purification steps A to C below.
  • a 300 ml SP Big Beads Sepharose column (100-300 ⁇ m, Amersham Biosciences) was equilibrated with 1 litre of 0.1 M citric acid pH 3.5 (flow app. 20 ml/min), before loading the 15.25 litres of prepared culture media over night (flow app. 10 ml/min). After loading the column was again washed with 1 litre of 0.1 M citric acid pH 3.5 followed by 1 liter of 40 vol % ethanol (flow app.
  • the bound insulin precursor A22K, B29R, desB30, B′A single chain insulin was then eluted with 1.5 litres of 0.2 M sodium acetate, 35 vol % ethanol, pH 5.75 (flow: 1.5 ml/min, volume of eluted precursor: 400 ml, amount of precursor: 220 mg).
  • step B the eluate was evaporated to dryness and the pellet re-dissolved in 0.25 M acetic acid.
  • the pH was lowered further to 1.5 immediately before purification by reverse-phase HPLC on a C18 column (ODDMS C18, 20 ⁇ 250 mm, 200 ⁇ , 10 ⁇ m, FeF Chemicals A/S).
  • the precursor solution was sterile filtrated (22 ⁇ m, Low Protein Binding Durapore® (PVDF), Millipore).
  • a gradient from 15% B to 50% B was run over the column, where Buffer A: 0.2 M (NH 4 ) 2 SO 4 , 0.04 M ortho-phosphoric acid, 10 vol % ethanol, pH 2.5 and Buffer B: 70 vol % ethanol.
  • the gradient was run over 120 min with a flow of 5 ml/min, column temperature at 40° C.
  • the insulin precursor A22K, B29R, desB30, B′A single chain insulin was eluted and pooled (total volume 75 ml).
  • step C the ethanol content in the eluate from reverse-phase HPLC was lowered to less than 5 vol % using a rotary evaporator (new volume: ⁇ 50 ml).
  • a 1000 ml G25 Sephadex column (5 ⁇ 55 cm, Amersham Biosciences) was washed in 0.5 M acetic acid and the insulin precursor A22K, B29R, desB30, B′A single chain insulin was then applied to the column and thereby de-salted by gelfiltration in 0.5 M acetic acid.
  • the insulin precursor was followed by UV detection at 280 nm, while the salt was followed by conductivity measurement. Immediately after de-salting, the insulin precursor was lyophilized.
  • Step 2 Synthesis of A22K(N ⁇ -mPEG2000-propionyl), B29R, desB30 B′A human insulin precursor 0.15 mmol of lyophilized insulin precursor LysA22 ArgB29 desB30 B′A is dissolved in aqueous sodium carbonate (3 ml, 100 mM). A solution of the PEGylation reagent mPEG2000-SPA-OSu in acetonitrile (0.15 mmol in 3 ml) is added to the solution of the precursor, and the mixture is gently stirred for 1 hour. The mixture is lyophilised, purified by HPLC and lyophilised to afford the PEGylated precursor.
  • Step 3 Conversion to A22K(N ⁇ -mPEG2000-Propionyl), B29R, desB30 Human Insulin
  • the PEGylated insulin precursor A22K(N ⁇ -mPEG2000-propionyl), desB30 B′A single chain human insulin precursor (3.9 ⁇ mol) is dissolved in 4.2 ml 50 mM glycine, 20 vol % ethanol pH 10.0. 3.6 mg of lyophilized porcine trypsin (Novo Nordisk A/S) is also dissolved in 3.5 ml 50 mM glycine, 20 vol % ethanol pH 10.0. Of this trypsin solution 0.5 ml is then added to the insulin precursor solution (hereby the insulin precursor is in 200 times excess).
  • the PEGylated insulin analogue A22K(N ⁇ -mPEG2000-propionyl), B29R, desB30 human insulin is then purified (removing trypsin and any un-acylated, doubly-acylated etc. or un-cleaved insulin molecules) by reverse-phase HPLC an lyophilised to afford the title insulin.
  • A22K, B29R, desB30 human insulin (125 mg) was dissolved in 0.1 M Na 2 CO 3 (2.8 ml).
  • mPEG-SPA 2000 50 mg dissolved in acetonitrile (1.25 ml) was added. pH was adjusted from 10.2 to 10.4 with 0.1 N NaOH.
  • mPEG-SPA 2000 25 mg dissolved in acetonitrile (1.25 ml) was added.
  • water 4.5 ml was added and pH was adjusted to 5 with 1 N HCl. The mixture was lyophilized.
  • the title compound was obtained by preparative HPLC purification. Column: C4, 2 cm.
  • A-Buffer 0.1% TFA in MiliQ Water
  • B-buffer 0.1% TFA in acetonitrile. Gradient 30-65% B over 30 min. Yield 43 mg.
  • MALDI-MS matrix: sinapinic acid
  • m/z 8114.
  • MALDI-MS matrix: sinapinic acid
  • m/z 5862.
  • MALDI matrix: sinapinic acid
  • m/z 6432.
  • MALDI matrix: sinapinic acid
  • m/z 6962.
  • MALDI-MS matrix: sinapinic acid
  • m/z 8167.
  • MALDI-MS matrix: sinapinic acid
  • m/z 6807
  • MALDI-MS matrix: sinapinic acid
  • m/z 8170
  • MALDI-MS matrix: sinapinic acid
  • m/z 6258.
  • MALDI-MS matrix: sinapinic acid
  • m/z 6082.
  • MALDI-MS matrix: sinapinic acid
  • m/z around 11600.
  • MALDI-MS matrix: sinapinic acid
  • m/z around 21500.
  • MALDI-MS matrix: sinapinic acid
  • m/z 7520.
  • MALDI-MS matrix: sinapinic acid
  • m/z 7520.
  • the PEGylation reagent was prepared as described in the following:
  • Omega-(methoxy-PEG 11 -propanoylamino)PEG 24 -propanoic acid (249 mg, 0.145 mmol) was dissolved in acetonitrile (10 mL) and pH was adjusted to 8 by addition of DIPEA (measurement of pH was done using wet indicator strips).
  • TSTU 48 mg, 0.16 mmol
  • acetonitrile (10 mL) was added and the mixture was stirred at room temperature for 1.5 h, and evaporated to dryness.
  • the residue was dissolved in DCM and washed with hydrochloric acid (0.01 M), the organic phase was dried (MgSO 4 ), filtered and the filtrate was evaporated to dryness.
  • the resulting omega-(methoxy-PEG 11 -propanoylamino)PEG 24 propanoic acid N-hydroxysuccinimide ester was used for coupling to insulin without further purification.
  • MALDI-MS matrix: sinapinic acid
  • m/z 7519.
  • N-hydroxysuccinimide activated PEG reagent was prepared similarly as described above from mdPEG 24 NHS ester (Quanta BiodDesign Ltd. Product No 10304) and amino-dPEG 12 tert-butyl ester (Quanta BioDesign Ltd. Product No 10281) via omega-(methoxy-PEG 23 -propanoylamino)PEG 12 propanoic acid tert-butyl ester, omega-(methoxy-PEG 23 -propanoylamino)PEG 12 propanoic acid, and omega-(methoxy-PEG 23 -propanoylamino)PEG 12 propanoic acid NHS ester
  • MALDI-MS matrix: sinapinic acid
  • m/z around 8200.
  • MALDI-MS matrix: sinapinic acid
  • m/z 8123.
  • This insulin was prepared using the PEG reagent NHS-dPEG 4 -(m-dPEG 12 ) 3 ester (Quanta BioDesign Ltd. Product No 10401).
  • MALDI-MS matrix: sinapinic acid
  • m/z 8724.
  • This insulin was prepared using the PEG reagent NHS-dPEG 4 -(m-dPEG 12 ) 3 ester (Quanta BioDesign Ltd. Product No 10401) and amino-dPEG 12 tert-butyl ester (Quanta BioDesign Product No 10281) similarly as described above.
  • MALDI-MS matrix: sinapinic acid
  • m/z 8768.
  • This insulin was prepared using the PEG reagent NHS-dPEG 4 -(m-dPEG 12 ) 3 ester (Quanta BioDesign Ltd. Product No 10401) and amino-dPEG 12 tert-butyl ester (Quanta BioDesign Product No 10281) similarly as described above.
  • MALDI-MS matrix: sinapinic acid
  • m/z 6918.
  • MALDI-MS matrix: sinapinic acid
  • m/z around 11400.
  • MALDI-MS matrix: sinapinic acid
  • m/z around 8268.
  • the affinity of the insulin derivatives of this invention for the human insulin receptor is determined by a SPA assay (Scintillation Proximity Assay) microtiterplate antibody capture assay.
  • SPA-PVT antibody-binding beads, anti-mouse reagent (Amersham Biosciences, Cat No. PRNQ0017) are mixed with 25 ml of binding buffer (100 mM HEPES pH 7.8; 100 mM sodium chloride, 10 mM MgSO 4 , 0.025% Tween-20).
  • Reagent mix for a single Packard Optiplate Packard No.
  • 6005190 is composed of 2.4 ⁇ l of a 1:5000 diluted purified recombinant human insulin receptor (either with or without exon 11), an amount of a stock solution of A14Tyr[ 125 ]-human insulin corresponding to 5000 cpm per 100 ⁇ l of reagent mix, 12 ⁇ l of a 1:1000 dilution of F12 antibody, 3 ml of SPA-beads and binding buffer to a total of 12 ml. A total of 100 ⁇ l reagent mix is then added to each well in the Packard Optiplate and a dilution series of the insulin derivative is made in the Optiplate from appropriate samples. The samples are then incubated for 16 hours while gently shaken. The phases are the then separated by centrifugation for 1 min and the plates counted in a Topcounter. The binding data were fitted using the nonlinear regression algorithm in the GraphPad Prism 2.01 (GraphPad Software, San Diego, Calif.).
  • Insulin receptor binding, A-isoform (without exon 11) Ex. No: Relative to human insulin: 1, 2 90% 3 123% 4 188% 6 120% 5 118% 7 44% 8 58% 9 128% 10 123% 15 20% 13 24% 14 16% 17 19% 18 16% 19 106% 20 24% 22 14% 16 15%
  • Hypnorm-Dormicum s.c. (1.25 mg/ml Dormicum, 2.5 mg/ml fluanisone, 0.079 mg/ml fentanyl citrate) 2 ml/kg as a priming dose (to timepoint ⁇ 30 min prior to test substance dosing) and additional 1 ml/kg every 20 minutes.
  • the animals are dosed with an intravenous injection (tail vein), 1 ml/kg, of control and test compounds (usual dose range 0.125-20 nmol/kg).
  • Blood samples for the determination of whole blood glucose concentration are collected in heparinized 10 ⁇ l glass tubes by puncture of the capillary vessels in the tail tip to time ⁇ 20 min and 0 min (before dosing), and to time 10, 20, 30, 40, 60, 80, 120, and 180 min after dosing.
  • Blood glucose concentrations are measured after dilution in analysis buffer by the immobilized glucose oxidase method using an EBIO Plus autoanalyzer (Eppendorf, Germany).
  • Mean plasma glucose concentrations courses (mean ⁇ SEM) are made for each dose and each compound.
  • Sprague Dawley male rats weighing 238-383 g on the experimental day are used for the clamp experiment.
  • the rats have free access to feed under controlled ambient conditions and are fasted overnight (from 3 ⁇ m) prior to the clamp experiment.
  • the rats are acclimatized in the animal facilities for at least 1 week prior to the surgical procedure. Approximately 1 week prior to the clamp experiment, Tygon catheters are inserted under halothane anaesthesia into the jugular vein (for infusion) and the carotid artery (for blood sampling) and exteriorised and fixed on the back of the neck. The rats are given Streptocilin vet. (Boehringer Ingelheim; 0.15 ml/rat, i.m.) post-surgically and placed in an animal care unit (25° C.) during the recovery period.
  • Anorphin (0.06 mg/rat, s.c.) is administered during anaesthesia and Rimadyl (1.5 mg/kg, s.c.) is administered after full recovery from the anaesthesia (2-3 h) and again once daily for 2 days.
  • rats are weighed and connected to the sampling syringes and infusion system (Harvard 22 Basic pumps, Harvard, and Perfectum Hypodermic glass syringe, Aldrich) and then placed into individual clamp cages where they rest for ca. 45 min before start of experiment.
  • the rats are able to move freely on their usual bedding during the entire experiment and have free access to drinking water.
  • the insulin derivative to be tested and human insulin are infused (i.v.) at a constant rate for 300 min.
  • Plasma glucose levels are measured at 10 min intervals throughout and infusion of 20% aqueous glucose is adjusted accordingly in order to maintain euglyceamia.
  • Samples of re-suspended erythrocytes are pooled from each rat and returned in about 1 ⁇ 2 ml volumes via the ⁇ -rotid catheter.
  • test substance will be dosed pulmonary by the drop instillation method.
  • male Wistar rats (app.250 g) are anaesthesized in app. 60 ml fentanyl/dehydrodenzperidol/-dormicum given as a 6.6 ml/kg sc primingdose and followed by 3 maintenance doses of 3.3 ml/kg sc with an interval of 30 min.
  • a special cannula with rounded ending is mounted on a syringe containing the 200 ul air and test substance (1 ml/kg). Via the orifice, the cannula is introduced into the trachea and is forwarded into one of the main bronchi—just passing the bifurcature. During the insertion, the neck is palpated from the exterior to assure intratracheal positioning. The content of the syringe is injected followed by 2 sec pause. Thereafter, the cannula is slowly drawn back. The rats are kept anaesthesized during the test (blood samples for up to 4 or 8 hrs) and are euthanized after the experiment.
  • PEGylated extended insulins in the following examples may be prepared similarly as described above:
  • FIG. 1 and FIG. 2 is the rat intratracheal drop instillation of the insulin of example 1 and 2.
  • FIG. 3 is the rat intratracheal drop instillation of the insulin of example 6.
  • FIG. 4 is the rat intratracheal drop instillation of the insulin of example 5.
  • FIG. 5 is the rat intratracheal drop instillation of the insulin of example 16.
  • FIG. 6 is the rat intratracheal drop instillation of the insulin of example 18.
  • FIG. 7 is the rat intratracheal drop instillation of the insulin of example 17.
  • FIG. 8 is the rat intratracheal drop instillation of the insulin of example 19.
  • FIG. 9 is the rat intratracheal drop instillation of the insulin of example 22.
  • FIG. 10 is the blood glucose profile by pulmonary administration of a spray dried powder of the insulin of examples 1 and 2 to mini-pigs where the mean dose delivered was 0.037 ⁇ 0.009 mg/kg.
  • FIG. 11 is the pharmacokinetic profile by pulmonary administration of a spray dried powder of the insulin of examples 1 and 2 to mini-pigs where the mean dose delivered was 0.037 ⁇ 0.009 mg/kg.

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CN101573133A (zh) 2009-11-04
US20110319591A1 (en) 2011-12-29
US8710001B2 (en) 2014-04-29
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CN101573133B (zh) 2014-08-27
JP2009545550A (ja) 2009-12-24

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