Title of Invention

"STABLE FORMULATIONS OF PEPTIDES"

Abstract Stable pharmaceutical composition comprising insulinotropic peptide
Full Text STABLE FORMULATIONS OF PEPTIDES.
FIELD OF THE INVENTION
The present invention relates to the field of pharmaceutical formulations. More specifically the invention pertains to shelf-stable pharmaceutical formulations comprising an insulino-tropic peptide.
BACKGROUND OF THE INVENTION
Therapeutic peptides are widely used in medical practise. Pharmaceutical compositions of such therapeutic peptides are required to have a shelf life of several years in order to be suitable for common use. However, peptide compositions are inherently unstable due to sensitivity towards chemical and physical degradation. Chemical degradation involves change of covalent bonds, such as oxidation, hydrolysis, racemization or cross linking. Physical degradation involves conformational changes relative to the native structure of the peptide, which may lead to aggregation, precipitation or adsorption to surfaces.
Glucagon has been used for decades in medical practise within diabetes and several gluca-gon-like peptides are being developed for various therapeutic indications. The preprogluca-gon gene encodes glucagon as well as glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2). GLP-1 analogs and derivatives as well as the homologous lizard peptide, exendin-4, are being developed for the treatment of hyperglycemia within type 2 diabetes. GLP-2 are potentially useful in the treatment of gastrointestinal diseases. However, all these peptides encompassing 29-39 amino acids have a high degree of homology and they share a number of properties, notably their tendency to aggregate and formation of insoluble fibrils. This property seems to encompass a transition from a predominant alpha-helix conformation to beta-sheets (Blundell T.L. (1983) The conformation of glucagon. In: Lefebvre P.J. (Ed) Glucagon I. Springer Verlag, pp 37-55, Senderoff R.I. et al., J. Pharm. Sci. 87 (1998)183-189, WO 01/55213). Aggregation of the glucagon-like peptides are mainly seen when solutions of the peptides are stirred or shaken, at the interface between solution and gas phase (air), and at contact with hydrophobic surfaces such as Teflon®.
WO 01/77141 discloses heat treatment of Arg34-GLP-1 (7-37) at elevated temperatures for less than 30 seconds. WO 04/55213 discloses microfiltration of Arg34-GLP-1 (7-37) at pH 9.5.

WO 01/55213 discloses treatment of Val8-GLP-1 (7-37) atpH 12.3 for 10 minutes at room temperature. WO 03/35099 discloses the preparation of zinc crystals of GLP-1 at alkaline PH.
Thus, various treatments and addition of excipients must often be applied to pharmaceutical compositions of the glucagon-like peptides in order to improve their stability. Shelf life of liquid parenteral formulations of these peptides must be at least a year, preferably longer. The in-use period where the product may be transported and shaken daily at ambient temperature preferably should be several weeks. Thus, there is a need for pharmaceutical compositions of glucagon-like peptides which have improved stability.
BREIF DESCRIPTION OF THE DRAWINGS.
Figure 1. Both samples contain a formulation of 1.2 mM Liraglutide, 14 mg/ml propylene gly-
col, 40 mM phenol, 10 mM NaCI, pH 7.7. Poloxamer-188 is added to a final concentration of
200 ppm in one sample.
Figure 2. All samples contain 1.67 mM Liraglutide, 58 mM phenol, 14 mg/ml propylene gly-
col, 8 mM sodium phosphate pH 7.7. Poloxamer 188 is added to two samples.
Figure 3. Both samples contain 1.2 mM Liraglutide, 40 mM phenol, 14 mg/ml propylene gly-
col, 10 mM NaCI, pH 7.7. Polysorbate 20 added to one sample
Figure 4, Measurement of NTU versus time during a rotation test of liraglutide compositions
without surfactant (F1) and with surfactant (F2 and F3).
Figure 5. Measurement of ThT fluorescence versus time during a rotation test of liraglutide
compositions without surfactant (F1) and with surfactant (F2). The lower curve is the trace of
F2.
Figure 6. Time course for fibril formation.
Figure 7. Physical stability of liraglutide prepared by heat treatment at 60° C.
Figure 8. Purity of liraglutide after heat treatment at 60 °C.
Figure 9. Physical stability of liraglutide prepared by heat treatment at 80 ° C.
Figure 10. Purity of liraglutide after heat treatment at 80 °C.
Figure 11. Physical stability of liraglutide prepared by 15 min. of heat treatment at 22, 40, 60,
and 80° C.
Figure 12. Physical stability of liraglutide prepared by heat treatment at 50 and 80° C at pH
10.
Figure 13. Purity of liraglutide after heat treatment at 50 and 80 °C at pH 10.

Figure 14. Physical stability of liraglutide prepared by heat treatment at 60 and 80° C at pH 9 and 10.
Figure 15. This figure shows 5 different formulations. 4 different formulations containing various amounts of Solutol HS-15 in either phosphate ortricine buffer. One formulation (Ref. formulation) is liraglutide in phosphate buffer without surfactant.
Figure 16. This figure shows 5 different formulations. 4 different formulations containing various amounts of Pluronic F-127 in either phosphate ortricine buffer. One formulation (Ref. formulation) is liraglutide in phosphate buffer without surfactant.
Figure 17. Physical stability of liraglutide after heat treatment at 50-70 8C for 60-120 minutes. Figure 18. Penfill® heat treated at different times and temperatures which were subsequently subjected to rotation.
Figure 19: Stability of formulations containing different excipients. Figure 20:Penfill® rotation test of the formulations containing different excipients.
The following is a detailed definition of the terms used in the specification.
The term "effective amount" as used herein means a dosage which is sufficient in order for the treatment of the patient to be effective compared with no treatment.
The term "medicament" as used herein means a pharmaceutical composition suitable for administration of the pharmaceutically active compounds to a patient.
The term "pharmaceutical composition" as used herein .means a product comprising an active compound or a salt thereof together with pharmaceutical excipients such as buffer, preservative and tonicity modifier, said pharmaceutical composition being useful for treating, preventing or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a person. Thus a pharmaceutical composition is also known in the art as a pharmaceutical formulation. It is to be understood that pH of a pharmaceutical composition which is to be reconstituted is the pH value which is measured on the reconstituted composition produced by reconstitution in the prescribed reconstitution liquid at room temperature.
The term "shelf-stable pharmaceutical composition" as used herein means a pharmaceutical composition which is stable for at least the period which is required by regulatory agencies in connection with therapeutic proteins. Preferably, a shelf-stable pharmaceutical composition is stable for at least one year at 5 eC. Stability includes chemical stability as well as physical stability.
The term "stable solution" as used herein means a preparation of a compound which is used as intermediates in the preparation of shelf-stable pharmaceutical compositions as described above.

The term "pharmaceutically acceptable" as used herein means suited for normal pharmaceutical applications, i.e. giving rise to no adverse events in patients etc.
The term "buffer" as used herein refers to a chemical compound in a pharmaceutical composition that reduces the tendency of pH of the composition to change over time as would otherwise occur due to chemical reactions. Buffers include chemicals such as sodium phosphate, TRIS, glycine and sodium citrate.
The term "preservative" as used herein refers to a chemical compound which is added to a pharmaceutical composition to prevent or delay microbial activity (growth and metabolism). Examples of pharmaceutically acceptable preservatives are phenol, m-cresol and a mixture of phenol and m-cresol.
The term "isotonicity agent" as used refers to a chemical compound in a pharmaceutical composition that serves to modify the osmotic pressure of the pharmaceutical composition so that the osmotic pressure becomes closer to that of human plasma. Isotonicity agents include NaCI, glycerol, mannitol etc.
The term "stabilizer" as used herein refers to chemicals added to peptide containing pharmaceutical compositions in order to stabilize the peptide, i.e. to increase the shelf life and/or in-use time of such compositions. Examples of stabilizers used in pharmaceutical formulations are L-glycine, L-histidine, arginine, polyethylene glycol, and carboxymethylcellu-lose.
The term "Surfactant" as used herein refers to any molecules or ions that are comprised of a water-soluble (hydrophilic) part, the head, and a fat-soluble (iipophilic) segment. Surfactants accumulate preferably at interfaces, which the hydrophilic part is orientated towards the water (hydrophilic phase) and the Iipophilic part towards the oil- or hydrophobic phase (i.e. glass, air, oil etc.). The concentration at which surfactants begin to form micelles is known as the critical micelle concentration or CMC. Furthermore, surfactants lower the surface tension of a liquid. Surfactants are also known as amphipathic compounds. The term "Detergent" is a synonym used for surfactants in general.
Anionic surfactants may be selected from the group of: Chenodeoxycholic acid, Chenodeoxycholic acid sodium salt, Cholic acid, Dehydrocholic acid, Deoxycholic acid, Deoxycholic acid methyl ester, Digitonin, Digitoxigenin, N,N-Dimethyldodecylamine N-oxide, Docusate sodium, Glycochenodeoxycholic acid sodium, Glycocholic acid hydrate, Glycodeoxycholic acid monohydrate, Glycodeoxycholic acid sodium salt, Glycodeoxycholic acid sodium salt, Glycolithocholic acid 3-sulfate disodium salt, Glycolithocholic acid ethyl ester, N-Lauroylsarcosine sodium salt, N-Lauroylsarcosine sodium salt, N-Lauroylsarcosine, IM-Lauroylsarcosine, Lithium dodecyl sulfate, Lugol, 1 -Octanesulfonic acid sodium salt, 1 -Octanesulfonic acid sodium salt, Sodium 1-butanesulfonate, Sodium 1-decanesulfonate,

Sodium 1-dodecanesulfonate, Sodium 1-heptanesulfonate, Sodium 1-heptanesuifonate, Sodium 1-nonanesulfonate, Sodium 1-propanesulfonate monohydrate, Sodium 2-bromoethanesulfonate, Sodium cholate hydrate, ox or sheep bile, Sodium cholate hydrate, Sodium choleate, Sodium deoxycholate, Sodium dodecyl sulfate, Sodium dodecyl sulfate, Sodium hexanesulfonate, Sodium octyl sulfate, Sodium pentanesulfonate, Sodium taurocholate, Taurochenodeoxycholic acid sodium salt, Taurodeoxycholic acid sodium salt monohydrate, Taurolithocholic acid 3-sulfate disodium salt, Tauroursodeoxycholic acid sodium salt, Trizma® dodecyl sulfate, DSS (docusate sodium, CAS registry no [577-11-7]), docusate calcium, CAS registry no [128-49-4]); docusate potassium, CAS registry no [7491-09-0]), SDS (sodium dodecyl suifate or sodium lauryl sulfate), Dodecylphosphocholine (FOS-Choline-12), Decylphosphocholine (FOS-Choline-10), Nonylphosphocholine (FOS-Choline-9), dipalmitoyl phosphatidic acid, sodium caprylate, and/or Ursodeoxycholic acid.
Cationic surfactants may be selected from the group of: Alkyltrimethylammonium bromide
Benzalkonium chloride, Benzalkonium chloride,
Benzyldimethylhexadecylammonium chloride, Benzyldimethyltetradecylammonium chloride, Benzyltrimethylammonium tetrachloroiodate, Dimethyldioctadecylammonium bromide, Dodecylethyldimethylammonium bromide, Dodecyltrimethylammonium bromide, Dodecyltrimethylammonium bromide, Ethylhexadecyldimethylammonium bromide, Hexadecyltrimethylammonium bromide, Hexadecyltrimethylammonium bromide, Polyoxyethylene(10)-N-tallow-1,3-diaminopropane, Thonzonium bromide, and/or Trimethyl(tetradecyl)ammonium bromide.
Nonionic surfactants may be selected from the group of: BigCHAP, Bis(polyethylene glycol bis[imidazoyl carbonyl]), block copolymers as polyethyleneox-ide/polypropyleneoxide block copolymers such as poloxamers, poloxamer 188 and polox-amer 407, Brij®35, Brij® 56, Brij® 72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P, Cremophor® EL, Decaethylene glycol monododecyl ether, N-Decanoyl-N-methylglucamine, n-Dodecanoyl-N-methylglucamide, alkyl-polyglucosides, ethoxylated castor oil, Heptaethylene glycol monodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethylene glycol monotetradecyl ether, Hexaethylene giycol monododecyl ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol monooctadecyl ether, Hexaethylene glycol monotetradecyl ether, Igepal CA-630, Igepal CA-630, Methyl-6-O-(N-heptylcarbamoyl)-beta-D-glucopyranoside, Nonaethylene glycol monododecyl ether, N-Nonanoyl-N-methylglucamine, N-Nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether, Octaethylene glycol monododecyl ether, Octaethylene glycol monohexadecyl ether, Octaethylene glycol monooctadecyl ether, Octaethylene glycol monotetradecyl ether, Octyl-

(3-D-glucopyranoside, Pentaethylene glycol monodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethylene glycol monohexadecyl ether, Pentaethylene glycol monohexyl ether, Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctyl ether, Polyethylene glycol diglycidyl ether, Polyethylene glycol ether W-1, Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate, Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether, Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate, Polyoxyethylene 8 stearate, Polyoxyethyiene bis(imidazolyl carbonyl), Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillaja bark, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85, Tergitol, Type 15-S-12, Tergitol, Type 15-S-30, Tergitol, Type 15-S-5, Tergitol, Type 15-S-7, Tergitol, Type 15-S-9, Tergitol, Type NP-10, Tergitol, Type NP-4, Tergitol, Type NP-40, Tergitol, Type NP-7, Tergitol, Type NP-9, Tetradecyl-(3-D-maltoside, Tetraethylene glycol monodecyl ether, Tetraethylene glycol monododecyl ether, Tetraethylene glycol monotetradecyl ether, Triethylene glycol monodecyl ether, Triethylene glycol monododecyl ether, Triethylene glycol monohexadecyl ether, Triethylene glycol monooctyl ether, Triethylene glycol monotetradecyl ether, Triton CF-21, Triton CF-32, Triton DF-12, Triton DF-16, Triton GR-5M, Triton QS-15, Triton QS-44, Triton X-100, Triton X-102, Triton X-15, Triton X-151, Triton X-200, Triton X-207, Triton® X-100, Triton® X-114, Triton® X-165 solution, Triton® X-305 solution, Triton® X-405, Triton® X-45, Triton® X-705-70, TWEEN® 20, TWEEN® 40, TWEEN® 60, TWEEN® 6, TWEEN® 65, TWEEN® 80, TWEEN® 81, TWEEN® 85, Tyloxapol, sphingophospholipids (sphingomyelin), and sphingoglycolipids (ceramides, gangliosides), phospholipids, and/or n-Undecyl p-D-glucopyranoside. '
Zwitterionic surfactants may be selected from the group of: CHAPS, CHAPSO, 3-(Decyldimethylammonio)propanesulfonate inner salt, 3-(Dodecyldimethylammonio)-propanesulfonate inner salt, 3-(Dodecyldimethylammonio)propanesulfonate inner salt, 3-(N,N-Dimethylmyristylammonio)propanesulfonate, 3-(N,N-Dimethyloctadecylammonio)-propanesulfonate, 3-(N,N-Dimethyloctylammonio)propanesulfonate inner salt, 3-(N,N-Dimethylpalmitylammonio)propanesulfonate, N-alkyl-N,N-dimethylammonio-1-propanesulfonates, 3-cholamido-1 -propyldimethylammonio-1 -propanesulfonate, Dodecyl-phosphocholine, myristoyl lysophosphatidylcholine, Zwittergent3-12 (A/-dodecyl-/V,/V-dimethyl-3-ammonio-1-propanesulfonate), Zwittergent 3-10 (3-(Decyldimethylammonio)-propanesulfonate inner salt), Zwittergent 3-08 (3-(Octyldimethylammonio)pro-panesulfonate), glycerophospholipids (lecithins, kephalins, phosphatidyl serine), glyceroglycolipids (galac-topyranoside), alkyl, alkoxyl (alkyl ester), alkoxy (alkyl ether)- derivatives of lysophosphatidyl and phosphatidylcholines, e.g. lauroyl and myristoyl derivatives of lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and modifications of the polar head group, that is cholines,

ethanolamines, phosphatidic acid, serines, threonines, glycerol, inositol, lysophosphatidylser-ine and lysophosphatidylthreonine, acyicarnitines and derivatives, Nbeta-acylated derivatives of lysine, arginine or histidine, or side-chain acylated derivatives of lysine or arginine, Nbeta-acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, Nbeta-acylated derivative of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, or the surfactant may be selected from the group of imidazoline derivatives, long-chain fatty acids and salts thereof C6-Ci2 (eg. oleic acid and caprylic acid), N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate, anionic (alkyl-aryl-sulphonates) monovalent surfactants, palmitoyl lysophosphati-dyl-L-serine, lysophospholipids (e.g. 1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine orthreonine), or mixtures thereof.
The term "alkyl-polyglucosides" as used herein in relates to an straight or branched C5-2o-alkyl, -alkenyl or -alkynyl chain which is substituted by one or more glucoside moieties such as maltoside, saccharide etc. Embodiments of these alkyl-polyglucosides include C6-ia-alkyl-polyglucosides. Specific embodiments of these alkyl-polyglucosides includes the even numbered carbon-chains such as C6,C8, Cio, C12, C-|4, Ci6, Ci8and C20 alkyl chain. Specific embodiments of the glucoside moieties include pyranoside, glucopyranoside, maltoside, mal-totrioside and sucrose. In embodiments of the invention less than 6 glucosid moieties are attached to the alkyl group. In embodiments of the invention less than 5 glucosid moieties are attached to the alkyl group. In embodiments of the invention less than 4 glucosid moieties are attached to the alkyl group. In embodiments of the invention less than 3 glucosid moieties are attached to the alkyl group. In embodiments of the invention less than 2 glucosid moieties are attached to the alkyl group. Specific embodiments of alkyl-polyglucosides are alkyl glucosides such n-decyl p-D-glucopyranoside, decyl p-D-maltopyranoside, dodecyl B-D-glucopyranoside, n-dodecyl B-D-maltoside, n-dodecyl p-D-maltoside, n-dodecyl B-D-maltoside, tetradecyl p-D-glucopyranoside, decyl B-D-maltoside, hexadecyl B-D-maltoside, decyl B-D-maltotrioside, dodecyl B-D-maltotrioside, tetradecyl B-D-maltotrioside, hexadecyl p-D-maltotrioside, n-dodecyl-sucrose, n-decyl-sucrose, sucrose monocaprate, sucrose monolaurate, sucrose monomyristate, and sucrose monopalmitate.
The term 'treatment of a disease" as used herein 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, and prevention of the disease, condition or disorder.

The term "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.
The term "analogue" as used herein referring to a peptide means a modified peptide wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and/or wherein one or more amino acid residues have been deleted from the peptide and or wherein one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the peptide and/or at the C-terminal of the peptide. In one embodiment an analogue comprises less than 6 modifications (substitutions, deletions, additions) relative to the native peptide. In another embodiment an analogue comprises less than 5 modifications (substitutions, deletions, additions) relative to the native peptide. In another embodiment an analogue comprises less than 4 modifications (substitutions, deletions, additions) relative to the native peptide. In another embodiment an analogue comprises less than 3 modifications (substitutions, deletions, additions) relative to the native peptide. In another embodiment an analogue comprises less than 2 modifications (substitutions, deletions, additions) relative to the native peptide. In another embodiment an analogue comprises only a single modification (substitutions, deletions, additions) relative to the native peptide.
The term "derivative" as used herein in relation to a parent peptide means a chemically modified parent protein or an analogue thereof, wherein at least one substituent is not present in the parent protein or an analogue thereof, i.e. a parent protein which has been co-valently modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters, PEGylations and the like.
The term "GLP-1 compound" as used herein means GLP-1 (7-37) (SEQ ID NO. 1), insulinotropic analogue thereof and insulinotropic derivatives thereof. Non-limiting examples of GLP-1 analogues are GLP-1 (7-36) amide, Arg34-GLP-1 (7-37), Gly8-GLP-1(7-37), Val8-GLP-1(7-36)-amide and VaPAsp^-GLP-1(7-37). Non-limiting examples of GLP-1 derivatives are desamino-His7, Arg26, Lys34(Ne-(y-Glu(Na-hexadecanoyl)))-GLP-1(7-37), desamino-His7, Arg26, Lys34(N£-octanoyl)-GLP-1(7-37), Arg26'34, Lys38(Ne-(co-carboxypentadecanoyl))-GLP-1(7-38), Arg26'34, Lys36(Ne-(y-Glu(Na-hexadecanoyl)))-GLP-1(7-36) and Arg34, Lys26(NE-(y-Glu(Na-hexadecanoyl)))-GLP-1(7-37).

The term "dipeptidyl aminopeptidase IV protected" as used herein means a compound, e.g. a GLP-1 analogue, which is more resistant to dipeptidyl aminopeptidase IV (DPP-IV) than the native compound, e.g. GLP-1 (7-37). Resistance of a GLP-1 compound towards degradation by dipeptidyl aminopeptidase IV is determined by the following degradation assay:
Aliquots of the GLP-1 compound (5 nmol) are incubated at 37 QC with 1 u.L of purified dipeptidyl aminopeptidase IV corresponding to an enzymatic activity of 5 mil for 10-180 minutes in 100 ul of 0.1 M triethylamine-HCI buffer, pH 7.4. Enzymatic reactions are terminated by the addition of 5 u,L of 10% trifluoroacetic acid, and the peptide degradation products are separated and quantified using HPLC analysis. One method for performing this analysis is : The mixtures are applied onto a Vydac C18 widepore (30 nm pores, 5 u.m particles) 250 x 4.6 mm column and eluted at a flow rate of 1 ml/min with linear stepwise gradients of acetonitrile in 0.1% trifluoroacetic acid (0% acetonitrile for 3 min, 0-24% acetonitrile for 17 min, 24-48% acetonitrile for 1 min) according to Siegel et al., Regul. Pept. 1999;79:93-102 and Mentlein et al. Eur. J. Biochem. 1993;214:829-35. Peptides and their degradation products may be monitored by their absorbance at 220 nm (peptide bonds) or 280 nm (aromatic amino acids), and are quantified by integration of their peak areas related to those of standards. The rate of hydrolysis of a GLP-1 compound by dipeptidyl aminopeptidase IV is estimated at incubation times which result in less than 10% of the GLP-1 compound being hydrolysed.
The term "insulinotropic" as used herein referring to a peptide or a compound means the ability to stimulate secretion of insulin in response to an increased plasma glucose level. Insulinotropic peptides and compounds are agonists of the GLP-1 receptor. The insulinotropic property of a compound may be determined by in vitro or in vivo assays known in the art. The following in vitro assay may be used to determine the insulinotropic nature of a compound such as a peptide. Preferably insulinotropic compounds exhibit an EC50 value in below assay of less than 5 nM, even more preferably EC50 values less than 500 pM.
Baby hamster kidney (BHK) cells expressing the cloned human GLP-1 receptor (BHK 467-12A) are grown in DMEM media with the addition of 100 lU/mL penicillin, 100 nL/mL streptomycin, 10% foetal calf serum and 1 mg/mL Geneticin G-418 (Life Technologies). Plasma membranes are prepared by homogenization in buffer (10 mM Tris-HCI, 30 mM NaCI and 1 mM dithiothreitol, pH 7.4, containing, in addition, 5 mg/mL leupeptin (Sigma), 5 mg/L pepstatin (Sigma), 100 mg/L bacitracin (Sigma), and 16 mg/L aprotinin (Calbiochem-Novabiochem, La Jolla, CA)). The homogenate was centrifuged on top of a layer of 41% W7v sucrose. The white band between the two layers was diluted in buffer and centrifuged. Plasma membranes were stored at -80 -C until used.

The functional receptor assay is carried out by measuring cAMP as a response to stimulation by the insulinotropic peptide or insulinotropic compound. Incubations are carried out in 96-well microtiter plates in a total volume of 140 ml and with the following final concentrations: 50 mM Tris-HCI, 1 mM EGTA, 1.5 mM MgSO4, 1.7 mM ATP, 20 mM GTP, 2 mM 3-isobutyl-1 -methylxanthine (IBMX), 0.01% w/v Tween-20, pH 7.4. Compounds are dissolved and diluted in buffer. GTP is freshly prepared for each experiment: 2.5 u.g of membrane is added to each well and the mixture is incubated for 90 min at room temperature in the dark with shaking. The reaction is stopped by the addition of 25 mt_ 0.5 M HCI. Formed cAMP is measured by a scintillation proximity assay (RPA 542, Amersham, UK). A dose-response curves is plotted for the compound and the EC50 value is calculated using GraphPad Prism software.
The term "prodrug of an insulinotropic compound" as used herein means a chemically modified compound which following administration to the patient is converted to an insulinotropic compound. Such prodrugs are typically amino acid extended versions or esters of an insulinotropic compound.
The term "exendin-4 compound" as used herein is defined as exendin-4(1-39) (SEQ ID NO. 2), insulinotropic fragments thereof, insulinotropic analogs thereof and insulinotropic derivatives thereof. Insulinotropic fragments of exendin-4 are insulinotropic peptides for which the entire sequence can be found in the sequence of exendin-4 (SEQ ID NO. 2) and where at least one terminal amino acid has been deleted. Examples of insulinotropic fragments of exendin-4(1 -39) are exendin-4(1 -38) and exendin-4(1 -31). The insulinotropic property of a compound may be determined by in vivo or in vitro assays well known in the art. For instance, the compound may be administered to an animal and monitoring the insulin concentration over time. Insulinotropic analogs of exendin-4(1 -39) refer to the respective molecules wherein one or more of the amino acids residues have been exchanged with other amino acid residues and/or from which one or more amino acid residues have been deleted and/or from which one or more amino acid residues have been added with the proviso that said analogue either is insulinotropic or is a prodrug of an insulinotropic compound . An example of an insulinotropic analog of exendin-4(1-39) is Ser2Asp3-exendin-4(1-39) wherein the amino acid residues in position 2 and 3 have been replaced with serine and aspartic acid, respectively (this particular analog also being known in the art as exendin-3). Insulinotropic derivatives of exendin-4(1-39) and analogs thereof are what the person skilled in the art considers to be derivatives of these peptides, i.e. having at least one substituent which is not present in the parent peptide molecule with the proviso that said derivative either is insulinotropic or is a prodrug of an insulinotropic compound. Examples of substituents are amides, carbohydrates, alkyl groups, esters and lipophilic substituents. An example of an insulinotropic derivatives of exendin-4(1 -39) and analogs thereof is Tyr31-exendin-4(1 -31 )-amide.

The term "stable exendin-4 compound" as used herein means a chemically modified exendin-4(1-39), i.e. an analogue or a derivative which exhibits an in vivo plasma elimination half-life of at least 10 hours in man, as determined by the method described under the definition of "stable GLP-1 compound".
The term "dipeptidyl aminopeptidase IV protected exendin-4 compound" as used herein means an exendin-4 compound which is more resistant towards the plasma peptidase dipeptidyl aminopeptidase IV (DPP-IV) than exendin-4 (SEQ ID NO. 2), as determined by the assay described under the definition of dipeptidyl aminopeptidase IV protected GLP-1 compound.
The term "isoelectric point" as used herein means the pH value where the overall net charge of a macromolecule such as a peptide is zero. In peptides there may be several charged groups, and at the isoelectric point the sum of all these charges is zero. At a pH above the isoelectric point the overall net charge of the peptide will be negative, whereas at pH values below the isoelectric point the overall net charge of the peptide will be positive.
The term "reconstituted" as used herein referring to a pharmaceutical composition means an aqueous composition which has been formed by the addition of water to a solid material comprising the active pharmaceutical ingredient. Pharmaceutical compositions for reconstitution are applied where a liquid composition with acceptable shelf-life cannot be produced. An example of a reconstituted pharmaceutical composition is the solution which results when adding water to a freeze dried composition. The solution is often for parenteral administration and thus water for injection is typically used for reconstituting the solid material.
The term "about" as used herein means in reasonable vicinity of the stated numerical value, such as plus or minus 10%.
In a first aspect the present invention relates to a shelf-stable pharmaceutical composition
comprising an insulinotropic peptide, a pharmaceutically acceptable preservative, a polox-
amer or polysorbate 20 surfactant at a concentration of from about 10 mg/L to about 400
mg/L, and optionally a pharmaceutically acceptable tonicity modifier, where said composition
has a pH that is in the range from about 7.0 to about 8,5.
In one embodiment the concentration of surfactant is from about 20 mg/L to about 300 mg/L.
In another embodiment the concentration of surfactant is from about 50 mg/L to about 200
mg/L,
In another embodiment the concentration of surfactant is from about 10 mg/L to about 200
mg/L.

In another embodiment the concentration of surfactant is from about 50 mg/L to about 400 mg/L.
In another embodiment the concentration of surfactant is from about 50 mg/L to about 300 mg/L.
In another embodiment the surfactant is poloxamer 188.
In another embodiment the surfactant is selected from the group consisting of poloxamer 407, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 237, poloxamer 331 and poloxamer 338.
In another embodiment the surfactant is polysorbate 20.
In an embodiment the invention provides a composition comprising an insulinotropic peptide and an alkyl-polyglucosid, and optionally a pharmaceutically acceptable tonicity modifier. In an embodiment the invention provides a composition according to the embodiment above, wherein said composition has a pH that is in the range from about 7.0 to about 8.5 In an embodiment the invention provides a composition according to any of the embodiments above, wherein the alkyl-polyglucoside is present in a concentration from about 10mg/L. In an embodiment the invention provides a composition according to any of the embodiments above, wherein the alkyl-polyglucoside is present in a concentration from about 1000 mg/L. In an embodiment the invention provides a composition according to any of the embodiments above, wherein the alkyl-polyglucoside is present in a concentration from about 10 mg/L to about 15000 mg/L.
In an embodiment the invention provides a composition according to any of the embodiments above, wherein the alkyl-polyglucoside is present in a concentration from about 1000 mg/L to about 10000 mg/L.
In an embodiment the invention provides a composition according to any of the embodiments above, wherein the alkyl-polyglucoside is present in a concentration from about 2000 mg/L to about 5000 mg/L.
In an embodiment the invention provides a composition according to any one of the embodiments above, wherein the alkyl-polyglucoside is an C10-2o-alkyl-polyglucoside. In an embodiment the invention provides a composition according to any one of the embodiments above, wherein the alkyl-polyglucoside is selected from dodecyl p-D-glucopyranoside, dodecyl p-D-maltoside, tetradecy! p-D-glucopyranoside, decyl p-D-maltoside, dodecyl p-D-maltoside, tetradecyl p-D-maltoside, hexadecyl p-D-maltoside, decyl p-D-maltotrioside, dodecyl p-D-maltotrioside, tetradecyl P-D-maltotrioside, hexadecyl p-D-maltotrioside, n-dodecyl-sucrose, n-decyl-sucrose.

In another embodiment of the invention the pharmaceutical composition comprises two different surfactants.
In another embodiment of the invention the pharmaceutical composition comprises two different surfactants wherein at least one surfactant is a non-ionic surfactant. In another embodiment of the invention the pharmaceutical composition comprises two different surfactants wherein the two different surfactants are both non-ionic surfactants. In another embodiment of the invention the pharmaceutical composition comprises two different surfactants wherein all surfactants are non-ionic surfactants. In another embodiment of the invention the pharmaceutical composition comprises polox-amer 188 and polysorbate 20.
In another embodiment of the invention the pharmaceutical composition has a pH in the range from about 7.4 to about 8.0.
In another embodiment of the invention the pharmaceutical composition has a pH in the range from about 7.4 to about 8.5.
In another embodiment of the invention the pharmaceutical composition has a pH in the range from about 7.7 to about 8.2.
In another embodiment of the invention the pharmaceutical composition comprises a buffer which is a phosphate buffer.
In another embodiment of the invention the pharmaceutical composition comprises a buffer which is a zwitterionic buffer.
In another embodiment of the invention the pharmaceutical composition comprises a buffer which is selected from the group consisting of glycyl-glycine, TRIS, bicine, HEPES, MOBS, MOPS, TES and mixtures thereof.
In another embodiment of the invention the pharmaceutical composition comprises a tonicity modifier selected from the group consisting of glycerol, propylene glycol and mannitol. In another embodiment of the invention the pharmaceutical composition the preservative is selected from the group consisting of phenol, m-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, thiomerosal and mixtures thereof.
In another embodiment of the invention the pharmaceutical composition comprises an insuli-notropic peptide which is a DPP-IV protected peptide.
In another embodiment of the invention the pharmaceutical composition the insulinotropic peptide comprises a lipophilic substituent selected from the group consisting of CH3(CH2)nCO- wherein n is 4 to 38, and HOOC(CH2)mCO- wherein m is from 4 to 38. In another embodiment of the invention the pharmaceutical composition the insulinotropic peptide is acylated GLP-1 or an acylated GLP-1 analogue.

In another embodiment of the invention the pharmaceutical composition comprises an insuli-notropic peptide which is an acylated GLP-1 analogue wherein said GLP-1 analogue is selected from the group consisting of Arg34-GLP-1 (7-37), Gly8-GLP-1 (7-36)-amide, Gly8-GLP-1 (7-37), Va!8-GLP-1 (7-36)-amide, Val8-GLP-1 (7-37), Aib8-GLP-1 (7-36)-amide, Aib8-GLP-1 (7-37), Val8Asp22-GLP-1(7-36)-amide, Val8Asp22-GLP-1 (7-37), Val8Glu22-GLP-1 (7-36)-amide , Val8Glu22-GLP-1 (7-37), Val8Lys22-GLP-1 (7-36)-amide, Val8Lys22-GLP-1(7-37), Val8Arg22-GLP-1(7-36)-amide, Val8Arg22-GLP-1(7-37), Val8His22-GLP-1(7-36)-amide, Val8His22-GLP-1(7-37), Val8Trp19Glu22-GLP-1(7-37), Va^Glu^VaP-GLP-l (7-37), Val8Tyr16Glu22-GLP-1 (7-37), Val8Trp16Glu22-GLP-1(7-37), Val8Leu16Glu22-GLP-1(7-37), Val8Tyr18Glu22-GLP-1(7-37), VaPGIu^His^-GLP-l (7-37), Val8Glu22lle33-GLP-1 (7-37), Val8Trp16Glu22Val25lle33-GLP-1 (7-37), Val8Trp16Glu22lle33-GLP-1(7-37), Val8Glu22Val25lle33-GLP-1(7-37), Val8Trp16Glu22Val25-GLP-1 (7-37), and analogues thereof.
In another embodiment of the invention the pharmaceutical composition the insulinotropic peptide is Arg34, Lys26(N£-(y-Glu(Na-hexadecanoyl)))-GLP-1(7-37). In another embodiment of the invention the concentration of said insulinotropic peptide is in the range from about 0.1 mg/ml to about 25 mg/mi, in the range from about 1 mg/ml to about 25 mg/ml, in the range from about 2 mg/ml to about 15 mg/ml, in the range from about 3 mg/ml to about 10 mg/ml, or in the range from about 5 mg/ml to about 8 mg/ml. In another embodiment of the invention the insulinotropic peptide is exendin-4 or ZP-10, i.e. HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2. In another embodiment of the invention the pharmaceutical composition the insulinotropic peptide is acylated exendin-4 or an acylated exendin-4 analogue. In another embodiment of the invention the pharmaceutical composition the insulinotropic peptide is [N-epsilon(17-carboxyheptadecanoic acid)20 exendin-4(1-39)-amide



NH0~H GEGTFTSDLSKQMEEEA V-N.J L F i EWLKNGGPSSGAPP P-,„
* Y H O


or N-epsilon32-(17-carboxy-heptadecanoyl)[Lys32]exendin-4(1-39)amide

(Figure Removed)


In another embodiment of the invention the pharmaceutical composition the concentration of the insulinotropic peptide in the pharmaceutical composition is from about 5ug/mL to about 10mg/ml_, from about 5ug/mL to about 5mg/mL, from about 5ug/mL to about 5mg/mL, from about 0.1 mg/mL to about 3mg/ml_, or from about 0.2mg/ml_ to about 1 mg/mL.
In another aspect the present invention relates to a method for preparation of a pharmaceutical composition according to the invention, said method comprising dissolving said insulinotropic peptide and admixing the preservative and tonicity modifier.
The present invention also relates to a method for preparation of a stable solution of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound at alkaline pH to a temperature above 40 -C for at least 5 minutes. Concentrations of the GLP-1 compound during the heat treatment is generally preferred to be in the range from 10 g/L to 100 g/L The GLP-1 compound may be dissolved in an aqueous solution having the final temperature, or it may be dissolved in aqueous solution having room temperature followed by heating to the appropriate temperature for the specified time.
It has been shown that the physical stability of the GLP-1 compound, liraglutide, was significantly improved as the temperature of heat treatment was increased (22 to 80 °C). For temperatures of 60 and 80 °C, time of heat treatment was shown to have a strong influence on the physical stability of liraglutide, as 120 minutes of heat treatment showed to improve the physical stability significantly in comparison to 1 minute of heat treatment. It has also been shown that the physical stability of liraglutide was significantly improved by increasing the temperature from 22 to 50-80 °C at pH 9-10 (cn.f. examples). For all temperatures, time of heat treatment was shown to have an influence on the physical stability of liraglutide, as 15 to 20 minutes of heat treatment showed to improve the physical stability significantly compared to 1 minute of heat treatment.
Optimal conditions for heat treatment to dissolve fibril germs appear to be 3-20 minutes at pH 9-10.5 and 70-85°C. In production scale, this could be performed using common methods for fast heating and cooling of large volumes by heat exchangers.
In another aspect the present invention relates to a method for preparation of a stable solution of a GLP-1 compound, which method comprises heating a solution of said GLP-1 com-

pound having a pH between pH 8.0 to pH 10.5 to a temperature between 50e C and 80 eC for a period of time which is between 3 minutes and 180 minutes.
In one embodiment the present invention relates to a method for preparation of a stable solution of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound having a pH between pH 8.0 to pH 10.0 to a temperature between 50e C and 80 -C for a period of time which is between 3 minutes and 180 minutes.
In another embodiment the present invention relates to a method for preparation of a stable solution of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound having a pH between pH 8.0 to pH 10.0 to a temperature between 509 C and 80 6C for a period of time which is between 3 minutes and 120 minutes. In another embodiment the temperature is between 60 9C and 80 -C for a period of time which is between 5 minutes and 15 minutes.
In another embodiment the temperature is between 60 9C and 80 °C for a period of time which is between 1 minute and 15 minutes.
In another embodiment the temperature is between 60 eC and 80 BC for a period of time which is between 3 minutes and 30 minutes.
In another embodiment the temperature is between 60 9C and 80 eC for a period of time which is between 5 minutes and 30 minutes.
In another embodiment the present invention relates to a method for preparation of a stable solution of exendin-4, which method comprises heating a solution of exendin-4 having a pH between pH 8.0 to pH 10.0 to a temperature between 50- C and 80 SC for a period of time which is between 3 minutes and 120 minutes.
In another embodiment the present invention relates to a method for preparation of a stable solution of Aib8'35-GLP-1 (7-36)-amide, which method comprises heating a solution of Aib8'35-GLP-1(7-36)-amide having a pH between pH 8.0 to pH 10.0 to a temperature between 509 C and 80 eC for a period of time which is between 3 minutes and 120 minutes. In another embodiment the GLP-1 compound is Arg34, Lys26(NE-(y-Glu(Na-hexadecanoyl)))-GLP-1(7-37).
In an aspect the invention relates to a method for preparation of a stable solution of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound. In an aspect the invention relates to a method as above wherein the temperature is between 509 C and 95 QC.
In an aspect the invention relates to a method as above wherein the temperature is between 60Q C and 95 eC.
In an aspect the invention relates to a method as above wherein the temperature is between 50e C and 80 eC.

In an aspect the invention relates to a method as above wherein the temperature is between 70Q C and 80 eC
In an aspect the invention relates to a method as above wherein the temperature is between 609 C and 80 QC
In an aspect the invention relates to a method as above wherein the pH is between about 8.0 to 10.5.
In an aspect the invention relates to a method as above wherein the pH is between about 8.0 to 10.0.
In an aspect the invention relates to a method as above wherein the pH is between about 8.0 to about 9.7.
In an aspect the invention relates to a method as above wherein the pH is between about 7.5 to 8.5.
In an aspect the invention relates to a method as above wherein the pH is about 7.7 In an aspect the invention relates to a method as above wherein the pH is about 8.15; In an aspect the invention relates to a method as above wherein the heating is continued for a period of time which is between 3 minutes and 180 minutes.
In an aspect the invention relates to a method as above wherein the heating is continued for a period of time which is between 10 minutes and 90 minutes.
In an aspect the invention relates to a method as above wherein the heating is continued for a period of time which is between 3 minutes and 30 minutes.
In an aspect the invention relates to a method as above wherein the heating is continued for a period of time which is between 5 minutes and 15 minutes.
In an aspect the invention relates to a method as above, wherein the pH is between pH 8.0 to pH 10.5 and the method includes heating to a temperature between 50e C and 85 -C for a period of time which is between 3 minutes and 180 minutes.
In another aspect the present invention relates to a method for preparation of a shelf-stable pharmaceutical composition of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound having a pH between pH 8.0 to pH 10.0 to a temperature between 50e C and 80 QC for a period of time which is between 3 minutes and 180 minutes. In one embodiment the present invention relates to a method for preparation of a shelf-stable pharmaceutical composition of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound having a pH between pH 8.0 to pH 10.0 to a temperature between 509 C and 80 SC for a period of time which is between 3 minutes and 120 minutes. In an aspect the invention relates to a method for preparation of a stable solution of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound having a

pH between pH 8.0 to pH 10.0 to a temperature between 70s C and 80 QC for a period of time which is between 3 minutes and 30 minutes.
In an aspect the invention relates to a method for preparation of a stable solution of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound having a pH between pH 8.0 to pH 10.0 to a temperature between 609 C and 80 SC for a period of time which is between 5 minutes and 15 minutes.
In an aspect the invention relates to a method for preparation of a stable solution of a GLP-1 compound, which method comprises heating a solution of said GLP-1 compound to a temperature between 60e C and 95 9C for a period of time which is between 10 minutes and 90 minutes.
The above aspect includes pH values of the solutions of about 7.5 to about 8.5. In an aspect of the invention the pH is about 7.7. In an aspect of the invention the pH value is about 8.15 In an aspect the invention relates to a method for preparation of a shelf-stable pharmaceutical composition of a GLP-1 compound, which method comprises one or more of the methods according to any one of the above aspects followed by addition of pharmaceutically acceptable excipients.
In an aspect the invention relates to a method for preparation of a shelf-stable pharmaceutical composition of a GLP-1 compound, which method comprises the a bulk peptide product which has been produced by the procedure according to any of the aspects above followed by freeze drying of the solution or suspension of said glucagon-like peptide. In an aspect the invention relates to a method for preparation of a shelf-stable pharmaceutical composition of a GLP-1 compound, which method comprises that the pharmaceutical composition is prepared from a freeze dried product according to the aspect above followed by a treatment according to any of the aspects above.
In an aspect the invention relates to a method for preparation of a shelf-stable pharmaceutical composition of a GLP-1 compound, which method comprises that the pharmaceutical composition is prepared as described in the former aspect and followed by a treatment according to any of the aspects above, either before filling in a final delivery system or after filling the a final delivery system or both.
In an aspect the invention relates to a method according to any of the aspects above, wherein said GLP-1 compound is Arg34, Lys26(NE-(y-Glu(Na-hexadecanoyl)))-GLP-1 (7-37). In another aspect the present invention relates to a method for the treatment of hyperglyce-mia comprising parenteral administration of an effective amount of the pharmaceutical composition according to the invention to a mammal in need of such treatment. In another aspect the present invention relates to a method for the treatment of obesity, beta-cell deficiency, IGT or dyslipidemia comprising parenteral administration of an effective

amount of the pharmaceutical composition according to the invention to a mammal in need of such treatment.
EXAMPLES General procedure
Thioflavin T (ThT) fibrillation assay: Principle and examples
Low physical stability of a peptide may lead to amyloid fibril formation, which is observed as well-ordered, thread-like macromolecular structures in the sample eventually resulting in gel formation. This has traditionally been measured by visual inspection of the sample. However, that kind of measurement is very subjective and depending on the observer. Therefore, the application of a small molecule indicator probe is much more advantageous. Thioflavin T (ThT) is such a probe and has a distinct fluorescence signature when binding to fibrils [Naiki et al. (1989) Anal. Biochem. 177, 244-249; LeVine (1999) Methods. Enzymol. 309, 274-284].
The time course for fibril formation can be described by a sigmoidal curve with the following expression [Nielsen et al. (2001) Biochemistry 40, 6036-6046], cn.f figure 6 :
Here, F is the ThT fluorescence at the time t. The constant to is the time needed to reach 50% of maximum fluorescence. The two important parameters describing fibril formation are the lag-time calculated by t0 - 2t and the apparent rate constant /capp = 1/t.
Formation of a partially folded intermediate of the peptide is suggested as a general initiating mechanism for fibrillation. Few of those intermediates nucleate to form a template onto which further intermediates may assembly and the fibrillation proceeds. The lag-time corresponds to the interval in which the critical mass of nucleus is built up and the apparent rate constant is the rate with which the fibril itself is formed.
Sample preparation
Samples were prepared freshly before each assay. Each sample composition is described in the legends. The pH of the sample was adjusted to the desired value using appropriate amounts of concentrated NaOH and HCIO4. Thioflavin T was added to the samples from a stock solution in H2O to a final concentration of 1 jxM.
Sample aliquots of 200 |*l were placed in a 96 well microtiter plate (Packard OptiPlate™-96, white polystyrene). Usually, eight replica of each sample (corresponding to one test condition) were placed in one column of wells. The plate was sealed with Scotch Pad (Qiagen).
Incubation and fluorescence measurement
Incubation at given temperature, shaking and measurement of the ThT fluorescence emission were done in a Fluoroskan Ascent FL fluorescence platereader (Thermo Labsystems). The temperature was adjusted to 37 °C. The orbital shaking was adjusted to 960 rpm with an amplitude of 1 mm in all the presented data. Fluorescence measurement was done using excitation through a 444 nm filter and measurement of emission through a 485 nm filter. Each run was initiated by incubating the plate at the assay temperature for 10 min. The plate was measured every 20 minutes for typically 45 hours. Between each measurement, the plate was shaken and heated as described.
Data handling
The measurement points were saved in Microsoft Excel format for further processing and curve drawing and fitting was performed using GraphPad Prism. The background emission from ThT in the absence of fibrils was negligible. The data points are typically a mean of eight samples and shown with standard deviation error bars. Only data obtained in the same experiment (i.e. samples on the same plate) are presented in the same graph ensuring a relative measure of fibrillation between the individual samples of one assay rather than comparison between different assays.
The data set may be fitted to Eq. (1). However, since full sigmodial curves in this case are not usually achieved during the measurement time, the degree of fibrillation is expressed as ThT fluorescence at various time points calculated as the mean of the eight samples and shown with the standard deviation.
Example 1
The ThT fibrillation assay of a pharmaceutical composition of the acylated GLP-1 analogue liraglutide is shown in Figure 1 (experimental performed along procedures described in "General procedure"). After approximately 10 hours the ThT fluorescence emission increases indicating the on-set of fibrillation. This signal increases steadily and reaches a plateau before the assay is terminated. In the presence of 200 ppm Poloxamer 188, however, the ThT fluorescence signal remains at the background level. This indicates that no fibrillation occurs and, hence, the pharmaceutical composition is physical stable under these conditions. The pharmaceutical compositions used in example 1 (Figure 1) is not added a buffer.
Example 2
The effect of Poloxamer 188 in a pharmaceutical composition of liraglutide containing sodium phosphate as a buffer is shown in Figure 2 (experimental performed along procedures described in "General procedure"). Here, the presence of 50 ppm Poloxamer 188 prolongs the lag time before on-set of fibrillation, whereas 100 ppm Poloxamer 188 completely inhibits fibrillation during the assay time.
Example 3
Polysorbate 20 does also stabilise formulations of liraglutide. One such example is shown in Figure 3 (experimental performed along procedures described in "General procedure"). The presence of 200 ppm Polysorbate 20 attenuates the fibrillation, which is observed as a slower growth rate of the ThT fluorescence signal. Hence, a significantly smaller ThT
fluorescence signal is observed in the Polysorbate 20 sample than in the reference after 40 hours of incubation.
Example 4
Two pharmaceutical compositions are prepared :
F1.1.2 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 3 Zn/hexamer, aspart 0.6
mM, 8 mM bicine, 50 ppm poloxamer 188, pH 7,7.
F2. 1.2 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 3 Zn/hexamer, aspart 0.6
mM, 8 mM bicine, pH 7.7.
Physical stability of the pharmaceutical compositions are evaluated by means of an accelerated stressed test. The stressed test is performed as a rotation test. 50ul air is added to 5 cartridges (glass .vials) of each formulation. The cartridges are rotated with a frequency of 30 rotations per minute for 4 hours daily. The test is stopped after 22 days of rotation. The inspection of the cartridges is followed daily or as required. The turbidity of the pharmaceutical compositions is characterized by nephelometric measurement of the turbidity on a HACH Turbidimeter 2100AN. The turbidity measurement of a liquid is specified in "Nephelometric Turbidity Unit" (NTU). Physical instability of the protein is characterised by high turbidity measurements.
The experiment shows that composition F2 has a much more rapid increase in NTU as compared to that of the F1 composition.
Example 5
Three pharmaceutical compositions were prepared :
F1.1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate,
P.H 7.7.
F2.1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate,
100 ng/ml poloxamer 188, pH 7.7.
F3.1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate,
200 u.g/ml poloxamer 188, pH 7.7.
The pharmaceutical compositions F1-F3 were subjected to the rotation test as described in example 4. The resulting NTU measurements versus time are shown in figure 4.
Example 6
Two pharmaceutical compositions were prepared :
F1.1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate, 0 ng/ml poloxamer 407 (Pluronic F-127), pH 7.7.
F2.1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate, 200 jig/ml poloxamer 407 (Pluronic F-127), pH 7.7.
The formulations were tested with respect to physical stability using the Thioflavin T assay. The formulations are placed in 96-well plates (Black NUNC) and incubated at 37 °C for up to 72h at the BMG FLUOstar microtiterplate fluorimeter using the following program: [300 rpm 15 min, 5 min rest]n = 72. The resulting measurements are shown in figure 5 (lower curve being F2)
Example 7
Solution 1 was prepared by dissolving preservative, isotonic agent, and buffer in water, pH was adjusted to 7.3. In another container solution 2 was prepared: liraglutide was dissolved in 60 °C hot water and kept on a water bath at 60 °C for 1, 20, and 120 minutes. The heat treatment of liraglutide was carried out in solution having pHs of about 8 and 10. After heat treatment solution 2 was cooled to 22 °C where after the two solutions were mixed and pH adjusted to 7.7 using sodium hydroxide and/or hydrochloric acid. Finally, the formulation was filtered through a 0.22 u.m filter.
The physical stability of the liraglutide preparations was evaluated by the use of a florescence method; the Thioflavine T-test where the histological thiazole dye Thioflavine T (ThT) was used as an indicator of fibril formation. By the use of Thioflavine T-test it was possible to determine the presence of fibrils in the different formulations. The method was based on the fluorescent characteristics of ThT. In the presence of fibrils, the fluorescence of ThT exhibited an excitation maximum at 450 nm and enhanced emission at 482 nm. The ThT fluorescence intensity has been shown to be linear with an increase in fibril concentration. ThT was used in a stress test applying the different formulations in microtiter plates with ThT at 35 °C and shaken with 350 rpm until the formulations were fibrillated. Graphs of the fluorescence intensity (Fl) as a function of time (sec) were obtained. The response variable was; time (seconds) to achieve & fluorescence intensity of 400, e.g. the longer time to reach Fl = 400, the more stable a formulation.
The purity of the liraglutide preparations was measured by RP-HPLC. Results from the experiments are depicted in figures 7 and 8. the following experiments are without surfactant - heat treatment 3
Example 7a
Solution 1 is prepared by dissolving preservative, isotonic agent, and buffer in water, pH was adjusted to 7.9. In another container solution 2 is prepared: liraglutide is dissolved in 60°C hot water and kept on a water bath at 60°C for 1, 20, and 120 minutes. The heat treatment of liraglutide is carried out in a solution having a pH of about 8 to 10. The two solutions are mixed and pH adjusted to 8.15 using sodium hydroxide and/or hydrochloric acid. Finally, the formulation is filtered through a 0.22 u,m filter.
The physical stability of the liraglutide preparations is evaluated by the use of a florescence method; the Thioflavine T-test where the histological thiazole dye Thioflavine T (ThT) is used as an indicator of fibril formation. By the use of Thioflavine T-test it was possible to determine the presence of fibrils in the different formulations. The method is based on the fluorescent characteristics of ThT. In the presence of fibrils, the fluorescence of ThT exhibited an excitation maximum at 450 nm and enhanced emission at 482 nm. The ThT fluorescence intensity is shown to be linear with an increase in fibril concentration.
Example 8
Solution 1 was prepared by dissolving preservative, isotonic agent, and buffer in water, pH was adjusted to 7.3. In another container solution 2 was prepared: liraglutide was dissolved in 80 °C hot water and kept on a water bath at 80 °C for 1, 30, and 120 minutes. The heat treatment of liraglutide was carried out in solution having pHs of about 8 and 10. After heat treatment solution 2 was cooled to 22 °C where after the two solutions were mixed and pH adjusted to 7.7 using sodium hydroxide and/or hydrochloric acid. Finally, the formulation was filtered through a 0.22 u.m filter.
Physical stability and purity of the preparations were measured as described in example 7. Results from the experiments are depicted in figures 9 and 10.
Example 8a
Solution 1 is prepared by dissolving preservative, isotonic agent, and buffer in water, pH was adjusted to 7.9. In another container solution 2 is prepared: liraglutide is dissolved in 80°C hot water and kept on a water bath at 80 °C for 1, 20, and 120 minutes. The heat treatment of liraglutide is carried out in a solution having a pH of about 8 to 10. The two solutions are mixed and pH adjusted to 8.15 using sodium hydroxide and/or hydrochloric acid. Finally, the formulation is filtered through a 0.22 u.m filter.
The physical stability of the liraglutide preparations is evaluated by the use of a florescence method; the Thioflavine T-test where the histological thiazole dye Thioflavine T (ThT) is used as an indicator of fibril formation. By the use of Thioflavine T-test it was possible to determine the presence of fibrils in the different formulations. The method is based on the fluorescent characteristics of ThT. In the presence of fibrils, the fluorescence of ThT exhibited an excitation maximum at 450 nm and enhanced emission at 482 nm. The ThT fluorescence intensity is shown to be linear with an increase in fibril concentration.
Example 9
Solution 1 was prepared by dissolving preservative, isotonic agent, and buffer in water, pH was adjusted to 7.3. In another container solution 2 was prepared: liraglutide was dissolved in water of various temperatures: 22, 40, 60, and 80 °C and kept on a water bath for 15 minutes for all the investigated temperatures. The heat treatments of liraglutide were carried out in solution having a pH of about 10. After heat treatment solution 2 was cooled to 22 °C where after the two solutions were mixed and pH adjusted to 7.7 using sodium hydroxide and/or hydrochloric acid. Finally, the formulation was filtered through a 0.22 nm filter.
Physical stability of the preparations was measured as described in example 7. Results from the experiments are depicted in figure 1 1 .
Example 10
Prior to freeze-drying liraglutide drug substance is dissolved in 70-80 °C hot water at pH about 8.0-1 0.0 to a concentration of 1 0-1 00 g/L. The heat treatment is carried out for 3-30 minutes. Hereafter the DS is freeze-dried. Subsequently, the freeze-dried drug substance is dissolved in water. The concentration is about 10-100 g/L and the pH of the solution (solution 2) is about 8-10. Another solution (solution 1 ) is prepared by dissolving preservative, isotonic agent, and buffer in water. pH is adjusted to 7.9. The two solutions are mixed and pH is adjusted to 8.15 using sodium hydroxide and/or hydrochloric acid..
Example 10a.
The base treatment of example 1 0a may be performed with or without the described heat treatment of example 10 before freeze drying. In a special embodiment the treatment of drug substance in example 10a may be performed at 75°C for 8 min before freeze drying.
Prior to freeze-drying liraglutide drug substance is dissolved in 70-80°C hot water at pH about 8.0-10.0 to a concentration of 10-100 g/L. The heat treatment is carried out for 3-30 minutes. Hereafter the DS is freeze-dried. Subsequently, the freeze-dri^d drug substance is dissolved in water. The concentration is about 10-100 g/L and the pH of the solution (solution 2) is about 8-10. Another solution (solution 1) is prepared by dissolving preservative, isotonic agent, and buffer in water. pH is adjusted to 7.3. The two solutions are mixed and pH is adjusted to 7.7 using sodium hydroxide and/or hydrochloric acid..
Example 10c.
The base treatment of example 10c may be performed with or without the described heat treatment of example 10b before freeze drying. In a special embodiment the treatment of drug substance in example 10c may be performed at 75°C for 8 min before freeze drying.
Example 11
Liraglutide was dissolved in water at room temperature and pH was adjusted to pH 10. The solution was heated on a water bath at 50 and 80 °C for 1, 3, 5 and 20 minutes. After heat treatment, the solution was cooled to 22 °C on a water batch. The solution was then filtered through a 0.22 u,m filter and freeze dried. The powder was dissolved in a solution containing preservative, isotonic agent, and buffer components and pH was adjusted to pH 7.7 using sodium hydroxide and/or hydrochloric acid.
The physical stability of heat treated liraglutide preparations was evaluated by the use of the Thioflavin T method described in example 7. Chemical stability of the preparations were measured using reversed phase HPLC. The results are depicted in figures 12 and 13.
Example 12
Liraglutide was dissolved in water at room temperature and pH was adjusted to pH 9 and 10. The solution was heated on a water bath at 60 and 80°C for 1 and 15 minutes. After heat treatment, the solution was cooled to 22 °C on a water bath. The solution was then filtered through a 0.22 u,m filter and freeze dried. The powder was dissolved in a solution containing preservative, isotonic agent and buffer components and pH was adjusted to pH
The physical stability of heat treated liraglutide preparations was evaluated by the use of the Thioflavin T method described in example 7. Chemical stability of the preparations were measured using reversed phase HPLC.
The results are depicted in figure 14.
Example 13
The formulations were mixed according to tables 1 and 2.
(Table Removed) 8 x 250nl of each formulation (8 repeats) was pipetted into a 96-well plate (Black NUNC). Subsequently, the plates were sealed using "Sealing tape for plates, NUNC". The plate was inserted into a BMG FLUOstar microtiter plate fluorimeter. Excitation was measured at 440 ± 10 mm and emission at 480 ± 10 mm. Data were sampled for 72 h (approx. 260.000 sec). The BMG FLUOstar microtiter plate fluorimeter was programmed as indicated here: [600 rpm for 300 sec, rest 100 sec]n = 72 using double orbital rotation.
From what can be seen in fig. 1 and 2, the formulations containing Solutol HS-15 in phosphate buffer are only slightly more stable than the reference formulation. The formulations containing either 100 or 200 (ig/m! Pluronic F-127 in phosphate buffer are more stable. Interestingly, formulations containing either Solutol HS-15 or Pluronic F-127 in tricine buffer are exceptionally stable, especially the latter.
Example 14
Solution 1 was prepared by dissolving preservative, isotonic agent, and buffer in water, pH was adjusted to 7.9. In another container solution 2 was prepared: liraglutide was dissolved in 60 - 70°C hot water and kept on a water bath at 50, 60, and 70°C for 60, 90, and 120 minutes. The heat treatment of liraglutide was carried out in solution having pHs of about 8 and 10. After heat treatment solution 2 was coolecf to 22 °C where after the two solutions were mixed and pH adjusted to 8.15 using sodium hydroxide and/or hydrochloric acid. Finally, the formulation was filtered through a 0.22 urn filter.
The physical stability of the liraglutide preparations were evaluated by the use of a florescence method; the Thioflavine T-test where the histological thiazole dye Thioflavine T (ThT) was used as an indicator of fibril formation. By the use of Thioflavine T-test it was possible to determine the presence of fibrils in the different formulations. The method was based on the fluorescent characteristics of ThT. In the presence of fibrils, the fluorescence of ThT exhibited an excitation maximum at 450 nm and enhanced emission at 482 nm. The ThT fluorescence intensity has been shown to be linear with an increase in fibril concentration.
ThT was used in a stress test applying the different formulations in microtiter plates with ThT at 35°C and shaken with 350 rpm until the formulations were fibrillated. Graphs of the fluorescence intensity (Fl) as a function of time (sec) were obtained. The response variable was; time (sec) to achieve a fluorescence intensity of 400, e.g. the longer time to reach Fl = 400, the more stable a formulation. The results are depicted in figure 17.
Example 15
Solution 1 was prepared by dissolving preservative, isotonic agent, and buffer in water, pH was adjusted to 7.9. In another container solution 2 was prepared: liraglutide was dissolved in 60 - 70 ° C hot water and kept on a water bath at 50, 60, 65, and 70 ° C for 30,45,150, and 180 minutes. The heat treatment of liraglutide was carried out in solution having pHs of about 8 and 10. After heat treatment solution 2 was cooled to 22 °C where after the two solutions

were mixed and pH adjusted to 8.15 using sodium hydroxide and/or hydrochloric acid. Finally, the formulation was filtered through a 0.22 |am filter.
The physical stability of the liraglutide preparations were evaluated by the use of a florescence method as described in example 14.
The formulations as described above may all include surfactants as described previously in examples 8-15 and surfactants as described above. The surfactants are dissolved in solution 1 and subsequently admixed with solution 2 resulting in a final formulation. In an aspect of the invention the surfactants can be in concentrations of 0 - 50 mg/ml.
Example 16
Table 1. Penfill® containing fibrillated liraglutide were heat treated for 30 min at 85°C. Freshly produced liraglutide drug product has a turbidity of approx. 0,2-1,0 NTU. Thus, heat treatment of highly fibrillated liraglutide drug product can dissolve the otherwise very stable fibril structures.

Figure 18 shows Penfill® heat treated at different times and temperatures which were subsequently subjected to rotation.
The examples above can be performed individually or in combination.
(Table Removed) In an aspect of the invention the procedure is as follows:
Prior to freeze-drying liraglutide drug substance is dissolved in 70-80 °C hot water at pH about 8.0-10.0 to a concentration of 10-100 g/L. The heat treatment is carried out for 3-30 minutes. Hereafter the drug substance is freeze-dried. Subsequently, the freeze-dried drug substance is dissolved in 50-80 °C hot water for 30-180 min. The concentration is about 10-100 g/L and the pH of the solution (solution 2) is about 8-10. Another solution (solution 1) is prepared by dissolving preservative, isotonic agent, and buffer in water. pH is adjusted to 7.9. The two solutions are mixed and pH is adjusted to 8.15 using sodium hydroxide and/or hydrochloric acid. Finally, the formulation is filtered through a 0.22 u,m filter. Either before or after filling in container-closure systems, the resulting iiragiutide drug product can be exposed to heat treatment at 60-95°C for 10-90 min.
Example 17
Use of n-Oodecyl-(3-D-maltoside (DDM) and Zwittergent 3-10 in formulations comprising liraglutide: The formulations F1, F2 and F3 were tested.
Physical stability of the formulations is evaluated by means of an accelerated stressed test. The stressed test is performed as a rotation test at 37°C. 50u.L air is added to 5 cartridges (3 ml glass vials) of each formulation. The cartridges are rotated with a frequency of 30 rotations per minute for 4 hours daily. The test was stopped after 37 days of rotation. The inspection of the cartridges is followed daily or as required. The turbidity of the formulation is characterized by nephelometric measurement of the turbidity on a HACH Turbidimeter 2100AN. The turbidity measurement of a liquid is specified in "Nephelometric Turbidity Unit" (NTU). Physical instability of the protein is characterised by high turbidity measurements. The following experiments were performed:
Ret.: 6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate, pH 7.7.
F1.1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate, 10 mM Zwittergent 3-10, pH 7.7.
F2.1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate, lOmMDDM, pH 7.7.
F3.1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate, 25 mM DDM, pH 7.7. The results are depicted in Fig, 19.
After 37 days in rotation at 37°C one Penfill® from each formulation (F1, F2, and F3) was
analysed with respect to total amount of lirqglutide (Content, mg/ml), purity (%), and sum of
impurities (%) was measured. The following experiments were performed:
F1.1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate,
10 mM Zwittergent 3-10, pH 7.7.
F2.1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate,
lOmMDDM, pH 7.7.
F3. 1.6 mM liraglutide, 14 mg/ml propylene glycol, 40 mM phenol, 8 mM sodium phosphate,
25 mM DDM, pH 7.7.
The results are depicted in Fig. 20





We claim:
1. A method for preparation of a stable solution of a GLP-1 compound, which method
comprises heating a solution of said GLP-1 compound; wherein the temperature is
between 500C and 950C, the pH is between about 8.0 to 10.5 and the heating is
continued for a period of time which is between 3 minutes and 180 minutes.
2. A method according to claim 1, wherein the temperature is between 600C and
3. A method according to claim 1, wherein the temperature is between 500C and
80OC.
4. A method according to claim 1, wherein the temperature is between 700C and 800C
5. A method according to claim 1, wherein the temperature is between 600C and 80%
6. A method according to any one of the claims 1-5, wherein the pH is between about
8.0 to 10.0.
7. A method according to any one of the claims 1-5, wherein the pH is between about
7.5 to 8.5.
8. A method according to any one of the claims 1-5, wherein the pH is about 7.7
9. A method according to any one of the claims 1-5, wherein the pH is about 8.15;
10. A method according to any one of the claims 1-9, wherein the heating is continued
for a period of time which is between 15 minutes and 120 minutes.
11. A method according to any one of the claims 1-9, wherein the heating is continued
for a period of time which is between 10 minutes and 90 minutes.
12. A method according to any one of the claims 1-9, wherein the heating is continued
for a period of time which is between 3 minutes and 30 minutes.
13. A method according to any one of the claims 1-9, wherein the heating is continued
for a period of time which is between 5 minutes and 15 minutes.
Indian Patent App. No.: 2602/CHENP/2012 NN reference no.: 7001.204-IN Amended claims-Clean copy
1
07-December-2012
14. A method for preparation of a stable GLP-1 compound, which method comprises a
bulk peptide product which has been produced by the procedure according to any of
the claims 1-13 followed by freeze drying of the solution or suspension of said
glucagon-like peptide.
15. A method for preparation of a shelf-stable pharmaceutical composition of a GLP-1
compound, which method comprises that the pharmaceutical composition is
prepared from a freeze dried product according to claim 14 followed by one or more
of the methods according to any of the claims 1-13.
16. The method according to claim 15, which is performed either before filling in a final
delivery system or aRer filling in a final delivery system or both.
17. A method for preparation of a shelf-stable pharmaceutical composition of a GLP-1
compound, which method comprises the methods according to any one of claims 1-
16 followed by addition of the other pharmaceutically acceptable excipients.
18. A method according to any of claims 1-17, wherein said GLP-1 compound is ~ r g ~ ~ ,
~ys~~(~"-(~-~lu(~~-hexadecanoyl)))-~~~-l(7-37).
19. A stable solution of a GLP-1 compound obtainable by the methods according to
claims 1-18.
20. The use of a stable solution of a GLP-1 compound of claim 19 for the preparation of
a shelf-stable pharmaceutical composition.
21. A shelf-stable pharmaceutical composition of a GLP-1 compound obtainable by the
methods according to claims 1-18.

Documents:

2602-delnp-2007-Abstract-(10-12-2012).pdf

2602-delnp-2007-abstract.pdf

2602-delnp-2007-Claims-(10-12-2012).pdf

2602-delnp-2007-claims.pdf

2602-delnp-2007-Correspondence Others-(10-12-2012).pdf

2602-delnp-2007-correspondence-others.pdf

2602-delnp-2007-description (complete).pdf

2602-delnp-2007-drawings.pdf

2602-delnp-2007-form-1.pdf

2602-delnp-2007-Form-2-(10-12-2012).pdf

2602-delnp-2007-form-2.pdf

2602-delnp-2007-Form-3-(10-12-2012).pdf

2602-delnp-2007-form-3.pdf

2602-delnp-2007-Form-5-(10-12-2012).pdf

2602-delnp-2007-form-5.pdf

2602-delnp-2007-pct-101.pdf

2602-delnp-2007-pct-210.pdf

2602-delnp-2007-pct-220.pdf

2602-delnp-2007-pct-237.pdf

2602-delnp-2007-pct-304.pdf

2606-DELNP-2007-Correspondence Others-(16-09-2011).pdf

2606-DELNP-2007-Form-1-(16-09-2011).pdf

2606-DELNP-2007-Form-13-(16-09-2011).pdf


Patent Number 257528
Indian Patent Application Number 2602/DELNP/2007
PG Journal Number 42/2013
Publication Date 18-Oct-2013
Grant Date 10-Oct-2013
Date of Filing 05-Apr-2007
Name of Patentee NOVO NORDISK A/S
Applicant Address NOVO ALLE, DK-2880 BAGSVAERD DENMARK
Inventors:
# Inventor's Name Inventor's Address
1 LUDVIGSEN SVEND BAUNEDALEN 13, DK-3540 LYNGE DENMARK
2 SCHLEIN MORTEN UNDER ELMENE 16, 4.TV, DK-KOBENHAVN S 2300 DENMARK
3 BOVING TINE ELISABETH GOTTSCHALK BOVESKOVVEJ 4, DK-2800 LYNGBY DENMARK
4 BONDE CLAUDE BORGEVEJ 41 B, DK-2800 LYNGBY DENMARK
5 LILLEORE ANNE-METTE SCHIOLDANNSVEJ 1, 1.SAL, DK-2920 CHARLOTTENLUND DENMARK
6 ENGELUND DORTHE KOT GASSEHAVEN 39, DK-2840 HOLTE DENMARK
7 NIELSEN BJARNE RONFELDT LIGUSTERVAENGET 27, DK-2830 VIRUM DENMARK
PCT International Classification Number A61K 38/26
PCT International Application Number PCT/EP2005/055068
PCT International Filing date 2005-11-14
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 05104050.9 2005-05-13 Denmark
2 PA 2004 01906 2004-12-08 Denmark
3 05104172.1 2005-05-18 Denmark
4 PA 2004 01753 2004-11-12 Denmark
5 PCT/EP2005/055916 2005-11-11 Denmark