Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/26—Glucagons
• • 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
  • A61K38/2278—Vasoactive intestinal peptide [VIP]; Related peptides (e.g. Exendin)
• • 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
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • 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
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • 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
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • 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
  • A61P3/04—Anorexiants; Antiobesity agents
• • 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
  • A61P3/06—Antihyperlipidemics
• • 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

Definitions

• the present invention relates to the field of pharmaceutical formulations. More specifically the invention pertains to soluble and stable pharmaceutical formulations.
• 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 crosslinking. Physical degra- dation 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.
• 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.
• FIG. 1 ThT fluorescence assay of fibril formation in acylated GLP-1 samples.
• the samples contain 6 mg/ml acylated GLP-1 , 5 ⁇ M ThT dissolved in water and adjusted to the stated pH. Experimental conditions are described in "Examples”. Briefly, the samples were incubated at 40 °C and shaken with 960 rpm in an Ascent Fluoroskan fluorescence plate reader. All data points are from the same experiment (i.e. all samples are from the same microtiterplate) and are means of eight replica and shown with standard deviations as error bars. Furthermore, these values at 20 and 40 hours are tabulated for each sample.
• FIG. 10 Proton NMR spectrum of acylated GLP-1 with different additives at pH 7.9. N- terminal histidine signals of the imidazol sidechain are observed at approx 7.80 ppm and 7.00 ppm. The linewidth and position reflects the reduced flexibility of the N-terminal. NMR samples were prepared with 6mg/ml acylated GLP-1 at pH 7.9 dissolved in 90%/10% H2O/D2O. NMR spectra were recorded at 600 MHz using a Varian Inova 600 MHz NMR in- strument using 5mm samples tubes. Sample volumes were 800 ul and spectra were measured at 27 degrees Celcius
• FIG. 12 Schematic representation of the time dependence of ThT fluorescence on fibril formation.
• the curve is Eq.(1) fitted to theoretically data points.
• the graphical meaning of lag time and k app are shown.
• soluble as used herein referring to a formulation means a liquid formulation wherein substantially all of the active ingredient is on a soluble form. Thus soluble formulations typically are optically clear.
• shelf-stable as used herein referring to a formulation means that the formulation remains suitable for its intended medical use until its expiration date.
• Parenteral liquid formulations typically must have a long shelf-life due to the distribution and stockpile before the product reaches doctors and patients.
• shelf-life of liquid parenteral formulations of peptides are more than 1 year, more than 3 years, such as 5 years at the prescribed condition for keeping the product.
• buffer as used herein means a chemical compound added to a formulation in order to prevent pH from changing over time.
• salts as used herein means compounds formed, together with water, by reaction of an acid with a metallic base.
• effective amount as used herein means a dosage which is sufficient to be effec- tive for the treatment of the patient compared with no treatment.
• terapéuticaally effective concentration means a concentration which renders treatment effective applying volumes of the pharmaceutical formulation which are typical in the art, e.g. 5 mL, 1 mL or lower than 500 ⁇ L.
• treatment of a disease means the management and care of a pa- tient 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.
• GLP glucagon-like peptide
• GLP-1 glucagon-like peptide 1
• GLP-2 glucagon-like peptide 2
• exendins and analogues and derivatives thereof.
• exendins which are found in the Gila monster are homologous to GLP-1 and also exert an insulinotropic effect. Examples of exendins are exendin-4 and exendin-3.
• the glucagon-like peptides have the following sequences : 1 5 10 15 20 25 30 35 GLP-1 HAEGT FTSDV SSYLE GQAAK EFIA LVKGR G GLP-2 HADGS FSDE NTILD NLAAR DFI LIQTK ITD Exendin-4 HGEGT FTSDL SKQME EEAVR LFIE LKGG PSSGA PPPS-NH2 Exendin-3 HSDGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS-NH2
• 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.
• Arg ⁇ -GLP-I (7-37) or K34R-GLP-1 (7-37) designates a GLP-1 analogue wherein amino acid residues at position 1-6 have been deleted, and the naturally oc- curing lysine at position 34 has been substituted with arginine (standard single letter abbreviation for amino acids used according to IUPAC-IUB nomenclature).
• derivative as used herein in relation to a parent peptide means a chemically modi- fied 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 covalently modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters, pegylations and the like.
• An examples of a derivative of GLP-1 (7-37) is Arg 34 , Lys 26 (N ⁇ -( ⁇ - Glu(N ⁇ -hexadecanoyl)))-GLP-1 (7-37).
• GLP-1 peptide as used herein means GLP-1 (7-37), a GLP-1 analogue, a GLP-1 derivative or a derivative of a GLP-1 analogue.
• GLP-2 peptide as used herein means GLP-2(1-33), a GLP-2 analogue, a GLP-2 derivative or a derivative of a GLP-2 analogue.
• exendin-4 peptide as used herein means exendin-4(1-39), an exendin-4 ana- logue, an exendin-4 derivative or a derivative of an exendin-4 analogue.
• stable exendin-4 compound as used herein means a chemically modified ex- endin-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 following method.
• the method for determination of plasma elimination half-life of an exendin-4 compound in man is :
• the com- pound is dissolved in an isotonic buffer, pH 7.4, PBS or any other suitable buffer.
• the dose is injected peripherally, preferably in the abdominal or upper thigh. Blood samples for determination of active compound are taken at frequent intervals, and for a sufficient duration to cover the terminal elimination part (e.g.
• Pre-dose 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24 (day 2), 36 (day 2), 48 (day 3), 60 (day 3), 72 (day 4) and 84 (day 4) hours post dose).
• Determination of the concentration of active compound is performed as described in Wilken et al., Diabetolo- gia 43(51 ):A143, 2000.
• Derived pharmacokinetic parameteres are calculated from the concentration-time data for each individual subject by use of non-compartmental methods, using the commercially available software WinNonlin Version 2.1 (Pharsight, Cary, NC, USA).
• the terminal elimination rate constant is estimated by log-linear regression on the terminal log- linear part of the concentration-time curve, and used for calculating the elimination half-life.
• DPP-IV protected exendin-4 compound as used herein means an exendin-4 compound which has been chemically modified to render said compound resistant to the plasma peptidase dipeptidyl aminopeptidase-4 (DPP-IV).
• immunomodulated exendin-4 compound as used herein means an exendin-4 compound which is an analogue or a derivative of exendin-4(1-39) having a reduced immune response in humans as compared to exendin-4(1-39).
• the method for assessing the immune response is to measure the concentration of antibodies reactive to the exendin-4 compound after 4 weeks of treatment of the patient.
• isoelectric point as used herein means the pH value where the overall net charge of a macromolecule such as a peptide is zero.
• peptides there may be many charged groups, and at the isoelectric point the sum of all these charges is zero, i.e. the number of negative charges balances the number of positive charges.
• the isoelectric point of a peptide may be determined by isoelectric focusing or it may be estimated from the sequence of the peptide by computational algorithms known in the art.
• the present invention relates to a soluble and shelf-stable pharmaceutical formulation
• a soluble and shelf-stable pharmaceutical formulation comprising a therapeutically effective concentration of a glucagon-like peptide, a pharmaceutically acceptable preservative, a pharmaceutically acceptable tonicity modifier, optionally a pharmaceutically acceptable buffer, and a pH that is in the range from about 7.0 to about 8.0, characterized in that the content of salts is lower than about 5 mM, preferably lower than about 2 mM, even more preferable lower than about 1 mM.
• the present invention relates to a soluble and shelf-stable pharmaceutical formulation
• a soluble and shelf-stable pharmaceutical formulation comprising a therapeutically effective concentration of a glucagon-like peptide, a pharmaceutically acceptable preservative, a pharmaceutically acceptable tonicity modifier, and a pH that is in the range from about 7.0 to about 8.0, characterized in that no buffer is present or low concentration of a buffer is present.
• no buffer is present in the formulation. In another embodiment of the invention substantially no buffer is present in the formulation.
• a low concentration of buffer is present in the formulation.
• the concentration of buffer in the formulation is less than about 8 mM, less than about 6 mM, or less than about 4 mM. In another embodiment of the invention the buffer comprises no phosphorous.
• the buffer is a zwitterion.
• the buffer is glycyl-glycine.
• the buffer is histidine or bicine.
• the tonicity modifier is not a salt.
• the tonicity modifier is selected from the group consisting of glycerol, mannitol and dimethylsulphone.
• the formulation has a pH in the range from about 7.4 to about 8.0
• the formulation has a pH in the range from about 7.6 to about 7.9.
• the isoelectric point of said glucagon-like peptide is from 3.0 to 7.0, preferably from 4.0 to 6.0.
• the glucagon-like peptide is GLP-1 , a GLP-1 analogue, a derivative of GLP-1 or a derivative of a GLP-1 analogue.
• the GLP-1 analogue is selected from the group consisting of Gly 8 -GLP-1(7-36)-amide, Gly 8 -GLP-1 (7-37), Val 8 -GLP-1 (7-36)-amide, Val 8 -GLP- 1 (7-37), Val 8 Asp 22 -GLP-1 (7-36)-amide, Val 8 Asp 22 -GLP-1 (7-37) , Val 8 Glu 22 -GLP-1 (7-36)- amide , Val 8 Glu 22 -GLP-1 (7-37), Val 8 Lys 22 -GLP-1 (7-36)-amide, Val 8 Lys 22 -GLP-1 (7-37), Val 8 Arg 22 -GLP-1 (7-36)-amide, Val 8 Arg 22 -GLP-1 (7-37), VaI 8 His 22 -GLP-1 (7-36)-amide, Val 8 His 22 -GLP-1 (7-37), Va ⁇ Trp ⁇ Glu ⁇ -GLP-l (7-37), Val 8 Glu 22 -GLP-1
• the derivative of a GLP-1 analogue is Arg 34 , Lys 26 (N ⁇ - ( ⁇ -Glu(N ⁇ -hexadecanoyl)))-GLP-1(7-37).
• the glucagon-like peptide is GLP-1 , a GLP-1 analogue, a derivative of GLP-1 or a derivative of a GLP-1 analogue and the concentration in the pharmaceutical composition is higher than 1 mg/ml, preferably higher than 2 mg/ml, more preferred higher than 3 mg/ml, even more preferred higher than 5 mg/ml.
• the concentration of the glucagon-like peptide in the pharmaceutical composition is in the range from about 1 mg/ml to about 25 mg/ml, preferably in the range from about 2 mg/ml to about 15 mg/ml, more preferred in the range from about 3 mg/ml to about 10 mg/ml, even more preferred in the range from about 5 mg/ml to about 8 mg/ml.
• the glucagon-like peptide is exendin-4, an exendin-4 analogue, a derivative of exendin-4, or a derivative of an exendin-4 analogue.
• the peptide is exendin-4.
• the peptide is a stable exendin-4 compound. In another embodiment of the invention the peptide is a DPP-IV protected exendin-4 compound. In another embodiment of the invention the peptide is an immunomodulated exendin-4 compound.
• the peptide is ZP10, i.e. HGEGT- FTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2.
• concentration of the glucagon-like peptide is ex- endin-4, an exendin-4 analogue, a derivative of exendin-4, or a derivative of an exendin-4 analogue in the pharmaceutical composition is from about 5//g/mL to about 10mg/mL, from about 5//g/mL to about 5mg/mL, from about 5 ⁇ g/mL to about 5mg/mL, from about 0.1mg/mL to about 3mg/mL, or from about 0.2mg/mL to about 1 mg/mL.
• the glucagon-like peptide is GLP-2, a GLP-2 analogue, a derivative of GLP-2 or a derivative of a GLP-2 analogue.
• the glucagon-like peptide is Gly 2 -GLP-2(1-33).
• the derivative of GLP-2 or a derivative of a GLP-2 analogue has a lysine residue, such as one lysine, wherein a lipophilic substituent optionally via a spacer is attached to the epsilon amino group of said lysine.
• the derivative of GLP-2 or said derivative of a GLP-2 analogue is an acylated GLP-2 compound.
• the derivative of a GLP-2 analogue is Arg 30 ,Lys 17 (N ⁇ - (1-propyl-3-amino-hexadecanoyl)) GLP-2 (1-33).
• the glucagon-like peptide is GLP-2, a GLP-2 analogue, a derivative of GLP-2 or a derivative of a GLP-2 analogue and the concentration of said glucagon-like peptide in the pharmaceutical composition is from 0.1mg/mL to 100mg/mL, from 0.1mg/mL to 25mg/mL, or from 1mg/mL to 25mg/mL.
• the preservative is selected from phenol, m-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p- hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, or mixtures thereof.
• the invention in another aspect relates to a method for preparation of a pharmaceutical com- position, comprising dissolving the GLP compound and admixing the preservative and tonicity modifier.
• the invention in another aspect relates to a pharmaceutical formulation having a pH between about 7.4 to about 8.0, said composition comprising a glucagon-like peptide and at least one pharmaceutically acceptable excipient, wherein said composition is shelf stable as measured in a Thioflavin T assay as described herein which shows less than three fold increase of the Thioflavin T fluorescence from 20 hours to 40 hours during incubation of the sample at 40 °C (based on the mean Thioflavin T fluorescence at each time point).
• the invention in another aspect relates to a pharmaceutical formulation having a pH between about 7.4 to about 8.0, said composition comprising a glucagon-like peptide and at least one pharmaceutically acceptable excipient, wherein said composition is shelf stable as measured in a Thioflavin T assay as described herein which shows less Thioflavin T fluorescence after storage of the composition for 40 hours at 40 °C than a similar formulation buffered by 8 mM phosphate at the same pH.
• the present invention relates to a method for the treatment of hyperglyce- mia comprising parenteral administration of an effective amount of the pharmaceutical composition comprising a GLP-1 peptide to a mammal in need of such treatment.
• the invention relates to a method for the treatment of obesity, beta-cell deficiency, IGT or dyslipideamia comprising parenteral administration of an effective amount of the pharmaceutical composition comprising a GLP-1 peptide to a mammal in need of such treatment.
• the present invention relates to a method for the treatment of short bowels syndrome comprising the administration of a formulation comprising a GLP-2 compound to a mammal in need of such treatment.
• excipients such as preservatives, isotonic agents and surfactants in pharmaceutical compositions are well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.
• the parent glucagon-like peptide can be produced by peptide synthesis, e.g. solid phase peptide synthesis using t-Boc or F-Moc chemistry or other well established techniques.
• the parent glucagon-like peptide can also be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the polypeptide and capable of expressing the polypeptide in a suitable nutrient medium under conditions permitting the expression of the peptide, after which the resulting peptide is recovered from the culture.
• the medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g.
• the peptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the pro- teinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like, dependent on the type of peptide in question.
• a salt e.g. ammonium sulphate
• the DNA sequence encoding the parent peptide may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the peptide by hybridisation using synthetic oligonucleotide probes in accordance with standard techniques (see, for example, Sambrook, J, Fritsch, EF and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989).
• the DNA sequence encoding the peptide may also be prepared synthetically by estab- lished standard methods, e.g.
• the DNA sequence may also be prepared by poly- merase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., Science 239 (1988), 487 - 491.
• the DNA sequence may be inserted into any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
• the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
• the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
• the vector is preferably an expression vector in which the DNA sequence encoding the peptide is operably linked to additional segments required for transcription of the DNA, such as a pro- moter.
• the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the peptide of the invention in a variety of host cells are well known in the art, cf. for instance Sambrook et al., supra.
• the DNA sequence encoding the peptide may also, if necessary, be operably connected to a suitable terminator, polyadenylation signals, transcriptional enhancer sequences, and transla- tional enhancer sequences.
• the recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
• the vector may also comprise a selectable marker, e.g. a gene the product of which comple- ments a defect in the host cell or one which confers resistance to a drug, e.g. ampicillin, kana- mycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
• a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector.
• the secretory signal sequence is joined to the DNA sequence encoding the peptide in the correct reading frame.
• Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the peptide.
• the secretory signal sequence may be that normally associated with the peptide or may be from a gene encoding another secreted protein.
• the host cell into which the DNA sequence or the recombinant vector is introduced may be any cell which is capable of producing the present peptide and includes bacteria, yeast, fungi and higher eukaryotic cells.
• suitable host cells well known and used in the art are, without limitation, £. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.
• Thioflavin T 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].
• F is the ThT fluorescence at the time t (see Fig. 12).
• the constant t 0 is the time needed to reach 50% of maximum fluorescence.
• 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.
• acylated GLP-1 The physico-chemical properties of the solvent/solution may affect the degree of self- assembly as well as the flexibility and partial unfolding of acylated GLP-1 molecules, i.e. factors responsible for initiation of amyloid fibril formation.
• Sample preparation Samples were prepared freshly before each assay. Usually, acylated GLP-1 was dissolved to 6 mg/ml in desired buffer or solvent. The pH of the sample was adjusted to the desired value using appropriate amounts of concentrated NaOH and HCIO 4 . Thioflavin T was added to the samples from a 1 mM stock solution in H 2 O to a final concentration of 5 ⁇ M.
• Sample aliquots of 100 ⁇ l were placed in a 96 well microtiter plate (Packard Opti- PlateTM-96, white polystyrene). Usually, eight replica of each sample (corresponding to one test condition) was placed in one column of wells. The plate was sealed with Scotch Pad (Qiagen).
• the measurement points were saved in Microsoft Excel format for further process- ing 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 experiments.
• 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 20 and 40 hours, calculated as the mean of the eight samples and shown with the standard deviation.
• the physical stability is always better in water at a given pH compared to phosphate buffer.
• Using water adjusted to pH7.7 results in similar physical stability as achieved with 8 mM phosphate pH8.15, and in water at pH7.53 the physical stability is comparable with 8 mM phosphate pH7.88. Less tendency to fibril formation is observed when lowering the phosphate concentration at a given pH.
• the phosphate concentration is gradually lowered from 8 mM phosphate to 1 mM phosphate at pH7.9.
• 1 mM phosphate buffer, pH7.90 the physical stability is similar to that of an acylated GLP-1 solution in water, pH7.90.
• zwitterionic buffer substances may be added without compromising the increased physical stability achieved in water in the absence of phosphate ions.
• Solutions of acylated GLP-1 with 10 mM MOPS or TES are both more physical stable than a solution in 8 mM phosphate at pH8.14, see figure 4, and 10 mM MOBS pH7,9 has similar physical stability as water pH7.9, see figure 5.
• HEPES or BICINE may be used as buffer at 10 mM without significantly increasing the fibrillation compared to water at pH7.89, see figure 6.
• acylated GLP-1 are in all cases significantly more physically stable than in aqueous solution with 8 mM phosphate, pH7.92.
• Buffered aqueous solutions using 10 mM HEPES become slightly more physically unstable when pH is gradually lowered from pH7.9 to pH7.51 , see figure 7.
• these are still more stable than solutions buffered with phosphate, see figure 6.
• acylated GLP-1 in 10 mM HEPES pH7.73 is as physically stable as in 8 mM phosphate pH8.15, compare figure 7 with figure 2.
• Some amino acids may also be used as zwitterionic buffers.
• an added tonicity modifier must not be an electrolyte/salt. This is illustrated in figure 9, where the addition of 10 mM NaCI to an acylated GLP- 1 solution in water promotes fibrillation. This effect is even more pronounced when adding 100 mM NaCI. However, both concentrations are much lower than the physiological NaCI concentration of 154 mM, usually applied when using NaCI as a tonicity modifier.
• a skilled artisan in the field of protein NMR spectroscopy will be able to assign most of the resonance peaks of a proton NMR spectrum of a protein to specific protons (or proton groups) in the protein given that the actual behavior of the protein in solution give rise to a well resolved proton NMR spectrum (well known to the skilled artisan in the field).
• the line width of the resonance peaks in the protein NMR spectrum reflects to some extend the size and dynamic properties of the protein in solution.
• Aqueous solution of acylated GLP-1 at concentrations between 1 and 30 mg/ml, with or without buffers in the pH range 7.0 to 8.2 give rise to NMR spectra that to a high extend allow the afore mentioned resonance assignment.
• Narrow proton resonances of the imidazol side chain of the N-terminal histidine is reflecting a higher degree of structural flexibility of the N-terminal part of acylated GLP-1 in so- lution as compared to more broad proton resonances of the imidazol side chain of the N- terminal histidine which reflects that this part of the structure is more rigid and ordered.
• amide protons of glutamic acid residue 9 and glycine residue 10 of acylated GLP-1 can be separately monitored under the influence of different buffer substances or variation in additives and their concentration under otherwise constant conditions.
• the exchange rate of amide protons in the pH range 7.0 to 8.2 reflects clearly the degree to which the specific amide protons are protected from direct access to the solvent water molecules.
• Figure 11 shows that the two mentioned amide proton resonances vary in line width and intensity as buffers or additives are changed. Surprisingly, but very pronounced, is the almost disappearance of amides protons resonances in the NMR spectrum of acylated GLP- 1 in aqueous solution buffered with 8 mM phosphate at pH 7.9 compared to the non-buffered situation.

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