Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/50—Fibroblast growth factor [FGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • 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
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to novel analogues of Fibroblast Growth Factor 21 (FGF21 ) and to de- rivatives thereof having a modifying moiety covalently attached.
• FGF21 Fibroblast Growth Factor 21
• the invention also relates to pharmaceutical use of these analogues and derivatives, in particular for the treatment of diabetes, dyslipide- mia, obesity, cardiovascular diseases, metabolic syndrome, and/or Non Alcoholic Fatty Liver Disease (NAFLD).
• FGF21 Fibroblast Growth Factor 21
• NAFLD Non Alcoholic Fatty Liver Disease
• the derivatives of the invention are protracted, e.g. capable of maintaining a low blood glu- cose level for a longer period of time, capable of increasing the in vivo half-life of FGF21 , and/or result in a lower clearance of FGF21.
• Fibroblast growth factors are polypeptides expressed in developing and adult tissues. They are in- volved in several physiological mechanisms including for example metabolic regulation and cellular differentiation. A whole family of more than twenty fibroblast growth factors exists (the FGF family). Three members of the FGF family including FGF19, FGF21 , and FGF23 form a subfamily functioning as endocrine factors involved in metabolic regulation.
• Fibroblast Growth Factor 21 or FGF-21 is expressed preferentially in the liver and has been shown to exert hormone-like metabolic effects.
• FGF21 has been demonstrated to activate glucose uptake in mouse adipocytes, to protect mice from diet induced obesity when over-expressed in transgenic mice, and to lower blood glucose and triglyceride levels when administered to diabetic rodents (Kharitonenkov ef al., J. Clin. Invest. (2005), 115:1627-1635).
• the lowering effect of FGF21 on blood glucose and triglycerides has also been shown in diabetic monkeys.
• FGF21 was also able to decrease LDL and to increase HDL significantly in diabetic monkeys (Kharitonenkov ef al., Endocrinology (2007), 148(2):774-81 ).
• FGF21 was furthermore shown to lower body weight, predominantly by an increase in energy expenditure and a reduction in adiposity (Coskun ef al., Endocrinology (2008), 149(12): 6018-6027).
• FGF21 has been suggested as a pharmacological agent with the potential to treat diabetes, dyslipidemia, obesity, cardiovascular diseases, and metabolic syndrome.
• Metabolic syndrome includes aspects like insulin resistance, dyslipidemia, visceral obesity and hypertension, see e.g. the definition of metabolic syndrome in Grundy ef al., Circulation (2004), (109): 433- 438.
• FGF21 may furthermore be used as a pharmacological agent with a potential to treat Non
• NAFLD Alcoholic Fatty Liver Disease
• WO 2003/01 1213 A2 discloses a method for treating diabetes of type 1 and 2, or obesity, by use of FGF21 compounds with at least 95% identity to the FGF21 precursor amino acid sequence.
• WO 2003/061712 A1 discloses muteins of FGF21 with improved pharmaceutical properties, e.g. A145E.
• WO 2005/091944 A2 discloses PEGylated derivatives of FGF21 , FGF21-K59C, and FGF21-
• WO 2005/1 13606 A2 discloses various FGF21 fusion proteins with the Fc portion of an lgG4 immunoglobulin, or human serum albumin.
• WO 2006/028595 A2 discloses further muteins of FGF21 with reduced capacity of O- glycosylation when expressed in yeast, e.g. L1 18C-A134C-S167A.
• WO 2006/028714 A1 discloses additional muteins of FGF21 with reduced susceptibility for proteolytic degradation when expressed in yeast, e.g. L153I.
• WO 2006/065582 A2 discloses still further muteins of FGF21 with reduced deamidation, e.g. des-H P I P-L 1 18C-A134C-N 121 D .
• WO 2006/078463 A2 discloses a method for treating cardiovascular disease by use of native mature FGF21 or specified variants thereof.
• WO 2008/121563 discloses FGF21 polypeptides modified to include non-naturally encoded amino acids, as well as derivatives thereof.
• the object of this invention is to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
• Another aspect of this invention relates to the furnishing of analogues or derivatives of FGF21 which have improved properties for the treatment of diabetes, for example compared with human FGF21.
• Another aspect of this invention relates to the furnishing of analogues or derivatives of FGF21 which have improved properties for the treatment of obesity, for example compared with human FGF21.
• Another aspect of this invention relates to the furnishing of analogues or derivatives of FGF21 which have improved properties for the treatment of non-alcoholic fatty liver disease (NAFLD), for example compared with human FGF21.
• NAFLD non-alcoholic fatty liver disease
• Another aspect of this invention relates to the furnishing of analogues or derivatives of FGF21 which can relatively easy be prepared recombinant in E. coli.
• Another aspect of this invention relates to the furnishing of analogues or derivatives of FGF21 being protected against N-terminal degradation.
• Another aspect of this invention relates to the furnishing of analogues or derivatives of FGF21 which have increased potency with respect to glucose uptake in 3T3-L1 cells, for example compared with human FGF21.
• Another aspect of this invention relates to the furnishing of analogues and derivatives of FGF- 21 having increased mean half-life time compared with the mean half life time of Met-FGF-21 , vide the test in example 9, below.
• Further objects of this invention are to furnish compounds which can effectively be used to treat hypertension, critical illness, the metabolic syndrome, epilepsy, cancer, acromegaly, dyslipidemia (high TG, high LDL and low HDC) and cardiovascular diseases, e.g., atherosclerosis and hypercholesterolemia.
• the sequence of the native human FGF21 protein is available from the UNIPROT database with accession no. Q9NSA1.
• the 209 amino acid precursor protein includes a signal peptide (amino acids 1- 28) and a mature protein (amino acids 29-209).
• the mature protein is included herein as SEQ ID NO: 1 (amino acids 1-181 ), and the signal peptide as SEQ ID NO:2 (amino acids 1-28).
• native human FGF21 are: SEQ ID NO:1 , SEQ ID NO: 1 having the substitution L146P, as well as any of these sequences preceded by the 27 or 28 amino acids signal peptide referred to above.
• Preferred examples of native human FGF21 are the mature parts, viz. SEQ ID NO:1 and the L146P isoform thereof.
• an FGF21 analogue refers to polypeptides that are or can be, deduced or derived from native FGF21 , from SEQ ID NO:1 in particular, by modification of the amino acid sequence thereof. Such modification, amendment or change may include substitution, deletion, and/or addition of one or more amino acids. For example, an amino acid may be added at the N-terminus end.
• amino acid or "amino acid residue” as referred to herein in the context of FGF21 modifications includes the twenty standard alpha-amino acids being used by cells in protein biosynthesis and specified by the genetic code, viz. alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine (the amino acid residues being the corre- sponding residues from which hydrogen has been removed from an amino group and/or a hydroxy group has been removed from a carboxy group and hydrogen may have been removed from any mer- capto group).
• an amino acid is preferably one which can be prepared by genetic engineering.
• derivative refers to an analogue of FGF21 which has been cova- lently modified.
• the term is not limiting as such, rather descriptive, as it is intended to mark a distinction between changes made to the constituent FGF21 polypeptide as such ("analogues"), and the co- valent binding of a side chain to the FGF21 compound, whereby the compound is "derivatised”. If desired, this term can be substituted with other general chemical terms, for example compound.
• a substitution in an analogue may be indicated as: "Original amino acid- position-substituted amino acid” (or as “position-substituted amino acid”).
• the three or one letter code may be used. Accordingly, the notation “S71 C” or “Ser71 Cys” means, that the analogue comprises a substitution of serine with cysteine in the amino acid position corresponding to the amino acid at position 71 in human FGF21 (SEQ ID NO:1 ).
• the analogue designated [-1A, L166F, M168L, G174V, Y179F] FGF21 is human FGF21 having Ala (A) in position -1 , Phe (F) in position 166, Leu (L) in position 168, Val (V) in position 174, and Phe (F) in position 179.
• An extension can be described by reference to SEQ ID NO: 1 by addition of position numbers (continued positive numbers in the C-terminal end and negative numbers in the N-terminal end) or, more simply, by adding the amino acids of the extension in question, using the correct sequence thereof, to the compound in question, which is then often given a trivial name, such as FGF21 , again in order to ease the understanding of the relevant technical point.
• [-1A, L166F, M168L, G174V, Y179F] FGF21 can also be designated [L166F, M168L, G174V, Y179F] Ala-FGF21.
• the present invention relates to novel analogues and derivatives of FGF21.
• a modifying group is covalently attached to the FGF21 analogue.
• the invention also relates to the use of said analogues and derivatives in pharmaceutical compositions, in particular for the treatment of diabetes, dyslipidemia, obesity, cardiovascular diseases, metabolic syndrome, and/or Non Alcoholic Fatty Liver Disease (NAFLD).
• NAFLD Non Alcoholic Fatty Liver Disease
• the derivatives of the invention are protracted, e.g. capable of maintaining a low blood glu- cose level for a longer period of time, capable of increasing the in vivo half-life of FGF21 , and/or result in a lower clearance of FGF21.
• the derivatives of FGF21 retain satisfactory biological activity and may be administered less frequently than the parent FGF21 analogues. Furthermore, said derivatives have a reduced risk of deamidation.
• this invention relates to analogues of FGF21.
• the analogues and derivatives of this invention are [-1A, L166F, M168L, G174V, Y179F] FGF21 , analogues of [-1A, L166F, M168L, G174V, Y179F] FGF21 optionally containing one or more of the following amino acid substitutions (exchanges): 71 C, 121 Q, 173A and/or desl 81 , optionally, having up to four further mutations and/or, optionally, the 179 and/or 180 amino acid is not present and derivatives of such analogues containing Cys in position 71 which derivatives have a group of the general formula HOOC-(CH 2 )n-CONH-CH(COOH)-CH 2 -CH 2 -CONH-(CH 2 CH 2 0) m -CH 2 -CONH- (CH 2 CH 2 0) p -CH 2 -CONH-(CH 2 ) q -NHCO-CH 2 - (modifying
• the above aspect covers, e.g., 1 ) analogues of [-1A, L166F, M168L, G174V, Y179F] FGF21 containing one or more of the following amino acid substitutions (exchanges): 71 C, 121 Q, 173A and/or desl 81 , 2) analogues of [-1A, L166F, M168L, G174V, Y179F] FGF21 having up to four further mutations (apart from any mutation(s) selected from the group consisting of 71 C, 121 Q, 173A and/or des181 ), 3) analogues of [-1A, L166F, M168L, G174V, Y179F] FGF21 wherein the 179 and/or 180 amino acid is not present and 4) any combination of 1 ), 2) and/or 3).
• FGF-21 analogues wherein the 179 and/or 180 amino acid is not present can also be designated des179 and/or des 180 analogues.
• analogues and derivatives of this invention are [-1A, 71 C, L166F,
• FGF21 analogues of [-1A, 71 C, L166F, M168L, G174V, Y179F] FGF21 optionally containing one or more of the following amino acid substitutions (exchanges): 121 Q, 173A and/or des181 , optionally, having up to four further mutations and/or, optionally, the 179 and/or 180 amino acid is not present and derivatives of such analogues which derivatives have a group of the general formula HOOC-(CH 2 )n-CONH-CH(COOH)-CH 2 -CH 2 -CONH-(CH 2 CH 2 0) m -CH 2 -CONH-(CH 2 - CH 2 0) p -CH 2 -CONH-(CH 2 ) q -NHCO-CH 2 - (modifying moiety), wherein n is an integer in the range 10-20, m is an integer in the range 1-3, p is an
• the analogues of this invention are [-1A, L166F, S167G, M168L, G174V, Y179F, A180E] FGF21 and analogues of [-1A, L166F, S167G, M168L, G174V, Y179F, A180E] FGF21 additionally containing one or more of the following amino acid substitutions (exchanges): 71 C, 121 Q, 171 L, 172E, 173A and/or desl 81.
• analogues of[-1A, L166F, S167G, M168L, G174V, Y179F, A180E] FGF21 additionally containing one or more of the following amino acid substitutions (exchanges): 71 C, 121Q, 171 L, 172E, 173A and/or desl 81" is that, compared with human FGF21 (SEQ ID NO:1 ), said analogues contain Ala (A) in position -1 , Phe (F) in position 166, Gly (G) in position 167, Leu (L) in position 168, Val (V) in position 174, Phe (F) in position 179, and Glu (E) in position 180 and, furthermore, either Cys (C) in position 71 , Gin (Q) in position 121 , Leu (L) in position 171 , Glu (E) in position 172, Ala (A) in position 173, and/or no amino acid residue in position 181.
• the analogues of this invention are [-1 A, S71 C, L166F, S167G, M168L, G174V, Y179F, A180E] FGF21 and [-1A, S71 C, L166F, S167G, M168L, G174V, Y179F, A180E] FGF21 analogues thereof additionally, contains one or more of the following amino acid substitutions (exchanges): 121 Q, 171 L, 172E, 173A and/or desl 81.
• these FGF21 analogues are [-1A, S71 C, L166F, S167G, M168L, G174V, Y179F, A180E] FGF21 and FGF21 analogues thereof which in addition to Ala (A) in position -1 , Cys (C) in position 71 , Phe (F) in position 166, Gly (G) in position 167, Leu (L) in position 168, Val (V) in position 174, Phe (F) in position 179, and Glu (E) in position 180, additionally, contains either Gin (Q) in position 121 , Leu (L) in position 171 , Glu (E) in position 172, Ala (A) in position 173, and/or no amino acid residue in position 181.
• Gin (Q) in position 121 is herein also expressed as "121Q, 171L, 172E, 173A and/or desl 81".
• one analogue according to this invention is [-1A, S71 C, L166F, S167G, M168L, G174V, Y179F, A180E] FGF21 which can, alternatively, be designated [S71 C, L166F, S167G, M168L, G174V, Y179F, A180E] Ala-FGF21 or [71 C, 166F, 167G, 168L, 174V, 179F, 180E] Ala-FGF21.
• this invention relates to derivatives of FGF21 analogues.
• the derivatives of this invention are [-1A, S71 C, L166F, S167G, M168L, G174V, Y179F, A180E] FGF21 and [-1A, S71 C, L166F, S167G, M168L, G174V, Y179F, A180E] FGF21 analogues which FGF21 analogues additionally contain one or more of the following amino acid substitutions (exchanges): 121 Q, 171 L, 172E, 173A and/or desl 81 , carrying a group of the general formula HOOC-(CH 2 ) n -CONH- CH(COOH)-CH 2 -CH 2 -CONH-(CH 2 CH 2 0) m -CH 2 -CONH-(CH 2 CH 2 0)p-CH 2 -CONH-(CH 2 ) q -NHCO-CH 2 - (modifying moiety
• a pharmaceutical composition comprising an analogue or a derivative of FGF21 may further comprise a pharmaceutically acceptable carrier.
• the carrier may be water, if desired supplemented with other materials, e.g. saline, such as physiological saline.
• Other pharmaceutically acceptable agents such as diluents and appropriate buffers may also be used.
• additional pharmaceutically acceptable agents such as emulsifiers, suspending agents, solvents, fillers, bulking agents, adjuvants, preservatives, antioxidants, colouring agents, and/or flavouring agents may also be used.
• the analogue or derivative of FGF21 may be used in the form of a purified polypeptide or a derivative thereof, or formulated using appropriate pharmaceutically acceptable excipients, as is known in the art.
• the pharmaceutical composition may be administered in any way as is known in the art, e.g. injected, for example intravenously (i.v.) or subcutaneously (s.c).
• the analogue or derivative of FGF21 may be included in the pharmaceutical composition in a therapeutically or prophylactically effective amount.
• the amount to be administered to the patient depends upon the therapeutic or prophylactic objective, such as the indication in question, the condition of the patient in need of treatment, the desired route of administration, etc.
• the skilled medical practitioner may have to adjust dosage and modify the administration depending on these factors, as is routine in the art.
• the compounds of this invention can be administered once daily or one or more times per week.
• n is an integer in the range 10-20
• m is an integer in the range 1-3
• p is an integer in the range 1-3
• q is an integer in the range 2-4, covalently attached to the sulphur atom in the mercapto group present in the cysteine residue in position 71.
• [-1A, L166F, M168L, G174V, Y179F] FGF21 analogues of [-1A, L166F, M168L, G174V, Y179F] FGF21 optionally containing one or more of the following amino acid substitutions (exchanges): 71 C, 121 Q, 173A and/or des181 , optionally, having up to four further mutations and/or, optionally, the 179 and/or 180 amino acid is not present, according to the precvious clause.
• FGF21 [-1A, L166F, S167G, M168L, G174V, Y179F, A180E] FGF21 , analogues of [-1A, L166F, S167G, M168L, G174V, Y179F, A180E] FGF21 additionally containing one or more of the following amino acid substitutions (exchanges): 71 C, 121 Q, 171 L, 172E, 173A and/or desl 81 and derivatives of such analogues containing Cys in position 71 which derivatives have a group of the general formula HOOC- (CH 2 )n-CONH-CH(COOH)-CH 2 -CH 2 -CONH-(CH 2 CH 2 0) m -CH 2 -CONH-(CH 2 CH 2 0)p-CH 2 -CONH-(CH 2 ) q - NHCO-CH 2 - (modifying moiety), wherein n is an integer in the range 10-20, m is an integer
• FGF21 additionally containing one or more of the following amino acid substitutions (exchanges): 71 C, 121 Q, 171 L, 172E, 173A and/or desl 81.
• FGF21 or analogues of [-1A, S71 C, L166F, S167G, M168L, G174V, Y179F, A180E] FGF21 additionally containing one or more of the following amino acid substitutions (exchanges) 121 Q, 171 L, 172E, 173A and/or desl 81 and derivatives thereof having a group of the general formula HOOC-(CH 2 )n-CONH-CH(COOH)-CH 2 -CH 2 -CONI-l- (CH 2 CH 2 0) m -CH 2 -CONH-(CH 2 CH 2 0) p -CH 2 -CONH-(CH 2 ) q -NHCO-CH 2 - (modifying moiety), wherein n is an integer in the range 10-20, m is an integer in the range 1-3, p is an integer in the range
• the analogue according to any one of the preceding clauses to the extent possible which is [-1A, S71 C, L166F, S167G, M168L, G174V, Y179F, A180E] FGF21.
• the analogue according to any one of the preceding clauses to the extent possible which is [-1 A, S71 C, L166F, S167G, M168L, P171 L, S172E, Q173A, G174V, Y179F, A180E] FGF-21.
• the analogue according to any one of the preceding clauses to the extent possible which is [-1 A, S71 C, N121 Q, L166F, S167G, M168L, G174V, Y179F, A180E, des181] FGF-21.
• the analogue or derivative according to any one of the preceding clauses to the extent possible which has a potency of at least 1 %, preferably at least 5%, more preferably at least 10%, or most preferably at least 20% relative to the potency of Met-FGF21 , wherein the potency is determined by measuring glucose uptake in 3T3-L1 adipocytes.
• NAFLD Non Alcoholic Fatty Liver Disease
• NAFLD Non Alcoholic Fatty Liver Disease
• NAFLD Non Alcoholic Fatty Liver Disease
• BG blood glucose
• BW body weight
• DCM dichloromethane
• DIC diisopropylcarbodiimide
• DIPEA diisopropylethyl- amine
• DPBS Dulbecco's Phosphate-Buffered Saline
• DVB divinyl benzene
• EDAC is (3-dimethyl- aminopropyl) ethyl carbodiimide hydrochloride
• Fmoc 9H-fluoren-9-ylmethoxycarbonyl
• HEPES 4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid
• HOAt is 1-hydroxy-7-azabenzotriazole
• HOBt 1- hydroxybenzotriazole
• HP CD hydroxypropyl beta cyclodextrin
• HPLC High Performance Liquid Chromatography
• IBMX is 3-isobutyl-1-methylxanthine
• OEG 8-amino-3,6-dioxaoctanic acid
• OtBu tert. butyl ester
• PBS phosphate buffered saline
• RT room temperature
• TFA trifluoroacetic acid
• TG triglyceride
• THF tetrahydrofuran
• TIPS triiso- propylsilane.
• Tris is tris(hydroxymethyl)aminomethane or 2-amino-2-hydroxymethylpropane-1 ,3-diol
• Trx is tranexamic acid
• TSTU is 0-(N-succimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate
• UPLC Ultra Performance Liquid Chromatography.
• a Perkin Elmer Sciex API 3000 mass spectrometer was used to identify the mass of the sample after elution from a Perkin Elmer Series 200 HPLC system.
• a Waters Micromass ZQ mass spectrometer was used to identify the mass of the sample after elution from a Waters Alliance HT HPLC system.
• the DNA and amino acid sequences for human FGF21 have been disclosed by, e.g., Nishi- mura ef al. in Biochim. Biophys. Acta 1492(1 ):203-206 (2000).
• the sequences are also available from public databases with accession nos. EMBL:AB021975 and UNIPROT:Q9NSA1 , respectively.
• the native polypeptide is synthesised with a signal peptide of 28 amino acids for secretion:
• the signal peptide shown in italics above, is included in the appended sequence listing as SEQ ID NO:2.
• the mature FGF21 polypeptide consisting of the remaining 181 amino acids is included in the sequence listing as SEQ ID NO: 1.
• the mature FGF21 polypeptide was cloned and expressed as an intracellular protein in E.coli, without the signal peptide, but with an added N-terminal methionine or an N-terminal Met-Ala which is processed in E. coli resulting in N-terminal Ala (-1Ala). More in particular, a 550 bp coding region including at the 3'-end the ATG codon for Met, as well as Nde1 and BamH1 restriction sites at the 3'- and 5'-ends, respectively, was inserted into the expression vector pET 1 1 c in Nde1-BamH1 under control of the phage T7 promoter, and transformed into E.coli B BL21 (DE3).
• the cells were grown in LB amp 100 ug/mL to OD 450 0.5, and expression was induced with 0.3 mM IPTG for 4 hours at 37°C. Crude extracts of cells were made by sonication for analysis of FGF21 expression.
• FGF21 analogues 1 1 1 -1A, 71 C, 121 Q, 166F, 168L, 173A, 174V, 179F
• the same FGF21 analogues can be expressed and prepared in S. cerevisiae in ways suitable and per se known for this organism.
• Example 3 Purification of FGF21 analogues
• FGF21 analogues described in Examples 1-2 may be further purified as follows or using similar technics:
• the polypeptide was pelleted by centrifugation (10,000 x g, for 30 minutes), re-solubilised by sonication in 50 mM Tris pH 8.0, and debris removed by centrifugation (10,000 x g, for 30 minutes).
• the polypeptide in the resulting supernatant was purified by anion exchange chromatography (50 mM Tris pH 8.0, 50-250 mM NaCI) using Q Sepharose Fast Flow resin (GE Healthcare), as generally described in Protein Purification.
• Example 4 Preparation of reagents which can be used to modify the free Cys of FGF21 analogues in position 71 Preparation of 17-
• Step 1 17-[(S)-3-(2- ⁇ 2-[(2- ⁇ 2-[(2-Aminoethylcarbamoyl)methoxy]ethoxy ⁇ ethylcarbamoyl)methoxy]- ethoxy ⁇ ethylcarbamoyl)-1-carboxypropylcarbamoyl]heptadecanoic acid.
• Example 5 Derivatisation of FGF21 compounds at 71Cys with modifying group Preparation of a 71Cys derivative of an FGF21 analogue
• FGF21 analogue 1 1 prepared as generally described in Examples 2 and 3, was modified at the thiol
• the mixture was purified using anion exchange on a MonoQ 10/100 column using A-buffer: 20 mM TRIS, pH 7.5; B-buffer: 20 mM TRIS, 500 mM NaCI, pH 7.5, flow 0.5 mL/min, gradient: 0-100%B over 60CV.
• the collected fractions were buffer exchanged to a phosphate buffer using a HiPrep 26/10 desalting column.
• the eluate was collected and filtered through a Millex GV sterile 0.22 urn. Yield: 4.65 mg.
• Derivative 102 could be prepared by reacting analogue 1 1 which is (-1A, S71 C, 121 Q, L166F, M168L, Q173A, G174V, Y179F)-FGF21 together with 17- ⁇ (S)-1-carboxy-3-[2-(2- ⁇ [2-(2- ⁇ [4-(2- iodoacetylamino)butylcarbamoyl]methoxy ⁇ ethoxy)ethylcarbamoyl]methoxy ⁇ ethoxy)ethylcarbamoyl]- propylcarbamoyl ⁇ heptadecanoic acid, such that the free thiol of the cysteine at postion 71 becomes modified with the modifying moiety III ( ⁇ 4-[2-(2- ⁇ 2-[2-(2- ⁇ 2-[(S)-4-carboxy-4-(17-carboxyheptadecanoyl- amino)butyrylamino]ethoxy ⁇ ethoxy
• the modifying moiety co- valently attached to the sulphur atom from the mercapto group in 71 Cys is as stated in the following table using the same "modifying moiety numbers" as used for the corresponding modifying reagents.
• the following assay was used for determining the biological activity, or potency, of FGF21 compounds of the invention.
• Mouse 3T3-L1 fibroblasts (e.g. available from ATCC, catalogue no. CL-173) are maintained in basal medium (DMEM (4500 mg/l Glucose) with 10 % Fetal Bovine Serum (FBS) and Penicillin/Streptomycin). The cells are not allowed to reach confluence and should be passed (transferred to new vials) before reaching approx. 60 % of confluency (by visual inspection).
• DMEM 500 mg/l Glucose
• FBS Fetal Bovine Serum
• Penicillin/Streptomycin Penicillin/Streptomycin
• cells are plated 80,000 cells/well in a 24 well plate, or 20,000 cells/well in a 96 well plate, and when they reach confluency (high density, with a view to have differentiated adipose cells made), the medium is changed from basal medium to basal medium containing Troglitazone, IBMX, Dexamethasone (commercially available from, e.g., Sigma) and human insulin (commercially available from, e.g., Novo Nordisk A/S).
• the cells are used 7-14, preferably 7-10, days after initiation of differentiation.
• the cells are stimulated with increasing concentrations (0-300 nM) of the FGF21 polypeptides or derivatives of the invention for 20 hours in basal medium.
• 3H-deoxyglucose in what follows: the tracer
• the cells are washed in warm (approximately 37°C) assay buffer (PBS with 1 mM MgCI 2 and 2 mM CaCI 2 ), HEPES and 0.1 % Human serum albumin) and the cells are incubated with the tracer for 1 hour. This incubation is terminated by washing twice in ice cold assay buffer.
• the cells are lysed with Triton X-100 and lysates transferred to a 96 wells plate, microscint-40 (commercially available from, e.g., Perkin Elmer) is added and amount of tracer counted in a TOP-counter (e.g. a Packard top- counter from Perkin Elmer).
• a TOP-counter e.g. a Packard top- counter from Perkin Elmer.
• Erk phosphorylation assay was performed in HEK293 cells that were stably transfected with human beta-Klotho.
• the HEK293T/b-klotho stable cells were seeded at 30000 cells/well on 96-well plates. After two days, fresh media was added, and after 2 hours more the FGF21 proteins were added. The plates were incubated for 12 minutes. And total ERK phosphorylation was assessed using an AlphaScreen SureFire Phospho-ERK1/2 Assay Kit (Perkin Elmer, Waltham, MA) according to the manufacturer's instructions and an EnVision Multilabel Microplate Reader Model 2103 (Perkin Elmer) with the AlphaScreen HTS Turbo option was used for signal detection.
• This analogue can, for example, be derivatised by covalently attaching the above modifying moiety number III being ⁇ 2-[2-(2- ⁇ 2-[2-(2- ⁇ 2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy ⁇ - ethoxy)acetylamino]ethoxy ⁇ ethoxy)acetylamino]ethylcarbamoyl ⁇ methyl to the sulphur atom from the mercapto group in 71 Cys.
• the db/db mouse is a mouse model for Type 2 diabetes.
• the mice lack the leptin receptor and they are characterized by hyperglycemia, insulin resistance, hyperphagia and obesity.
• mice Male db/db mice (9-10 weeks old) were used to measure the effect on blood glucose and body weight of the following FGF21 analogue and derivatives.
• Body weight was measured before dosing and again after 7 days treatment. Non-fasting blood glucose was measured before dosing and again 2 hours after dosing day 7. Blood glucose was measured using a glucose analyzer (Biosen 5040) based on the glucose oxidase method. The results are shown in Table 1 below.
• Table 3 Difference from vehicle in delta blood glucose and delta body weight (day 1-7)
• the mean half-life (T 1 ⁇ 2 ) of the comparative compound Met-FGF21 has been determined to be 10.8 hours with a standard deviation of 2.7 hours.
• the mean half-life (T 1 ⁇ 2 ) of the compound to be tested is determined.
• the plasma levels of the FGF21 compounds can be determined using Fibroblast Growth Factor-21 Human ELISA (available from BioVendor, catalogue no. RD191 108200R).
• the PC based software, WinNonLin version 5.2 from Pharsight Corportion, Cary N.C., can be used for the pharmacokinetic calculation.

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