US 2016.0002311A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0002311 A1 Lau et al. (43) Pub. Date: Jan. 7, 2016
(54) NOVEL GLUCAGON ANALOGUES Publication Classification (71) Applicant: NOVO NORDISKA/S, Bagsvaerd (DK) (51) Int. Cl. C07K I4/605 (2006.01) (72) Inventors: Jesper F. Lau, Farum (DK); Thomas A647/48 (2006.01) Kruse, Herlev (DK); Lars Linderoth, A638/28 (2006.01) Alleroed (DK); Henning Thoegersen, A638/26 (2006.01) Farum (DK); Jacob Kofoed, Vaerloese (52) U.S. Cl. (DK); Kirsten Dahl, Smoerum (DK) CPC ...... C07K 14/605 (2013.01); A61K 38/26 (2013.01); A61K 47/48038 (2013.01); A61 K (21) Appl. No.: 14/827,539 38/28 (2013.01) (22) Filed: Aug. 17, 2015 (57) ABSTRACT Related U.S. Application Data The present invention relates to novel peptide compounds (63) Continuation of application No. 136375 filed on which have a protracted profile of action and improved solu Oct. 16, 2012, filed as application No. PCT/EP2011/ bility and stability, to the use of the compounds in therapy, to 054712 on Mar. 28, 2011. methods of treatment comprising administration of the com (60) Provisional application No. 61/319,994, filed on Apr. pounds to patients in need thereof, and to the use of the 1, 2010. compounds in the manufacture of medicaments. The com pounds of the invention are of particular interest in relation to (30) Foreign Application Priority Data the treatment of hyperglycemia, diabetes and obesity, as well as a variety of diseases or conditions associated with hyper Mar. 26, 2010 (EP) ...... 1 O157901.9 glycemia, diabetes and obesity. Patent Application Publication Jan. 7, 2016 Sheet 1 of 9 US 2016/0002311 A1
6 O
5 O
4.O
Vehicle 2 O f Ex. 3, 1000nmol/kg 1030 Ex. 3, 300nmol/kg Ex. 3, 100nmol/kg O O 10 20 30 40 Time (hours) Fig. 2 Patent Application Publication Jan. 7, 2016 Sheet 2 of 9 US 2016/0002311 A1
$2$3$º -- Ex. 4, 300nmol/kg
Time (hours)
©
$2$3$º - Ex. 5, 300nmol/kg
Time (hours) Patent Application Publication Jan. 7, 2016 Sheet 3 of 9 US 2016/0002311 A1
1000000
1 O O O 0. O
100 O O
1000
LoC O 20 40 60 80 100 time (hours) x Ex. 3, 25nmol/kg IV, n = 3 - Ex. 3, 50nmol/kg SC, n = 2 Fig. 5
Wehicle HF G3, inmol/kg Vehicle HF pairfed (to 30+1) Ex. 3, 3nmol/kg Ex. 3, 30nmol/kg Ex. 3, 3nmol/kg + G3, 1 nmol/kg Ex. 3, 30nmol/kg + G3, 1 nmol/kg & Wehicle F 600
-5 O 5 10 15 time (days) Fig. 6 Patent Application Publication Jan. 7, 2016 Sheet 4 of 9 US 2016/0002311 A1
o -100 3. an < 200
-300 & s s s s s sy s ess S S S & aV SS s S S * S S * S s\9 aS SS g & b' oos s o' c SSS. S.Sg NN SSSS g ? & ? V Patent Application Publication Jan. 7, 2016 Sheet 5 of 9 US 2016/0002311 A1
1 2 Vehicle HF G3, 1nmol/kg 1 O Vehicle HF pairfed (to 30+1) Ex. 3, 3nmol/kg Ex. 3, 30nmol/kg Ex. 3, 3nmol/kg + G3, 1 nmol/kg Ex. 3, 30nmol/kg+ G3, 1 nmol/kg : Vehicle F
Time (hours) Fig. 8
25
Vehicle HF G3, 1 nmol/kg Vehicle HF pairfed (to 30+1) Ex. 3, 3nmol/kg Ex. 3, 30nmol/kg Ex. 3, 3nmol/kg + G3, 1 nmol/kg Ex. 3, 30nmol/kg + G3, nmol/kg s: Vehicle LF Patent Application Publication Jan. 7, 2016 Sheet 6 of 9 US 2016/0002311 A1
300
2 O O
Patent Application Publication Jan. 7, 2016 Sheet 7 of 9 US 2016/0002311 A1
Solubility
Analogue Fig. 12
Solubility & Stability Area (214 nm, UPC)
OOOOOO SOCOCOO -- 4000000 3}}{C} - 2OOOOOO -- COOOO ----- deuexB Analogue Fig. 13 Patent Application Publication Jan. 7, 2016 Sheet 8 of 9 US 2016/0002311 A1
Ø4. ???????????????????????????ºZZZZZZZZZZZZZZZZZ. Ø4. ZZZZZZZZZZZZZZZZZ!) ØA. Recovery(%)
Fig. 14
100 200 300 Time (min)
Fig. 15 Patent Application Publication Jan. 7, 2016 Sheet 9 of 9 US 2016/0002311 A1
Lag-time - to - 2t to Time Fig. 16 US 2016/0002311 A1 Jan. 7, 2016
NOVEL, GLUCAGON ANALOGUES 0006 Glucagon is however of limited potential use in pharmaceuticals due to fast clearance in human plasma with CROSS-REFERENCE TO RELATED a half life of approximately 5 minutes A high clearance of a APPLICATIONS therapeutic agent is inconvenient in cases where it is desired to maintain a high blood level thereof over a prolonged period 0001. This application is a continuation of U.S. patent of time since repeated administrations will then be necessary. application Ser. No. 13/637,454, filed Oct. 16, 2012 which is In some cases it is possible to influence the release profile of a 35 U.S.C. S371 national stage application of International peptides by applying Suitable pharmaceutical compositions, Patent Application PCT/EP2011/054712 (published as WO but this approach has various shortcomings and is not gener 2011/117415A1), filed Mar. 28, 2011, which claims priority ally applicable. to European Patent Application 10157901.9, filed Mar. 26, 0007 Glucagon is currently available in recombinant 2010; this application further claims priority under 35 U.S.C. form as a freeze-dried formulation, with a short duration of S119 of U.S. Provisional Application 61/319,994, filed Apr. action, restricted to a few hours in spite of a glucagon level 1, 2010; the contents of which are incorporated herein by that peaks at levels far higher than endogenous glucagon reference. levels. There is therefore a need for chemically modified glucagon compounds in order to be delivered at continuous FIELD OF THE INVENTION levels, so that longer biological half-life is achieved, i.e. 0002 The present invention relates to novel glucagon pep modified glucagon peptides with a protracted profile of tide analogues with improved physical stability and solubil action. ity, and with a protracted profile of action, to the use of said 0008 Furthermore, glucagon is not stable for very long peptides in therapy to methods of treatment comprising when dissolved in aqueous solution since physical stability of administration of said peptides to patients, and to the use of glucagonis very poor and solutions of glucagonform gels and said peptides in the manufacture of medicaments. fibrils within hours or days, depending on the purity of the peptide, Salt concentration, pH and temperature. In addition INCORPORATION-BY-REFERENCE OF THE the solubility of human glucagon is very poor at pH 3.5-9.5. SEQUENCE LISTING 0009. Several patent applications disclosing different glu cagon-based analogues and GLP-1/glucagon receptor 0003. In accordance with 37 C.F.R. S.1.52(e)(5), Appli co-agonists are known in the art, such as e.g. patents cants enclose herewith the Sequence Listing for the above WO2008/086086, WO2008/101017, WO2007/056362, captioned application entitled “SeqList 8132 US03', cre WO2008/1524.03 and WO96/29342. Some of the GLP-1/ ated on Aug. 5, 2015. The Sequence Listing is made up of 494 glucagon receptor co-agonists disclosed in these patents refer bytes, and the information contained in the attached to specific mutations relative to native human glucagon. “SeqList 8132 US03” is identical to the information in the Other glucagon analogs disclosed are PEGylated (e.g. specification as originally filed. No new matter is added. WO2007/056362) or acylated in specific positions of native human glucagon (e.g. WO96/29342). Glucagon for preven BACKGROUND OF THE INVENTION tion of hypoglycaemia have been disclosed, as e.g. in patent 0004. The precise control of blood glucose levels is of vital application U.S. Pat. No. 7,314,859. importance to humans as well as other mammals. It is well 0010. The peptides of the present invention provide novel established that the two hormones insulin and glucagon are modified glucagon peptides with a protracted profile of action important for maintenance of correct blood glucose levels. in addition to providing Such modified glucagon peptides in While insulin acts in the liver and peripheral tissues by reduc stable pharmaceutical compositions at physiological pH. ing blood glucose levels via increased peripheral uptake of glucose and reduced glucose output from the liver, glucagon SUMMARY OF THE INVENTION acts mainly on the pancreas and liver, by increasing blood 0011. The present invention relates to novel glucagon pep glucose levels via up-regulation of gluconeogenesis and gly tides with improved physical stability and solubility, to the cogenolysis. Glucagon has also been reported to increase use of said peptides in therapy to methods of treatment com lipolysis, to induce ketosis and to reduce plasma triglyceride prising administration of said peptides to patients, and to the levels in plasma Schade and Eaton, Acta Diabetologica, use of said peptides in the manufacture of medicaments for 1977, 14, 62. use in the treatment of diabetes, eating-disorders, obesity and 0005 Glucagon is an important part of the defence mecha related diseases and conditions. nism against hypoglycaemia and administration of a low dose 0012. The present inventors have surprisingly found a of glucagon may prevent insulin-induced hypoglycaemia or number of positions in human glucagon where acylation with improve the ability to recover from hypoglycaemia. Studies a Substituent comprising a lipophilic moiety and two nega have also shown that glucagon does reduce food intake and tively charged moieties in combination with specific muta body weight in rats and in humans Schulman et al. J. Appl. tions in the glucagon peptide sequence, leads to glucagon Physiol. 1957, 11,419. Hence, glucagon is a plausible signal agonists with improved physical stability and solubility and that may contribute to the termination of food intake. Further preserved activity on the glucagon receptor. more, administration of a lower dose of glucagon may induce 0013. In a first embodiment (Embodiment 1), the present Satiety without affecting the blood glucose. A large number of invention relates to a glucagon peptide comprising SEQID 1. people Suffering from diabetes, in particular Type 2 diabetes, wherein X, represents Lys, Xs represents Lys and X rep are over-weight or obese. Obesity represents a high risk factor resents Glu, up to five amino acid Substitutions in amino acid in serious and even fatal common diseases and for most positions X2, Xio. X2, X16, X20, X24, X2s, X27, X2s. X29 diabetics it is highly desirable that their treatment does not and/or Xo of said glucagon peptide, and a substituent com cause weight gain. prising two or more negatively charged moieties, wherein one US 2016/0002311 A1 Jan. 7, 2016 of the said negatively charged moieties is distal of a lipohilic G3. Groups are compared using 1-way ANOVA and Dunnet's moiety and where the said moiety is attached at the epsilon post-test comparing groups to vehicle high fat fed group. position of a LyS, at the delta position of an Orn, or at the Data mean+/-Sem. Sulphur of a Cys, in one or more of the following amino acid 0026 FIG. 12 shows the solubility of glucagon analogues positions of said a glucagon peptide: Xio. X2, X20, X24, X2s, in 10 mM HEPES buffer (pH=7.5). Buffer was added to X29, and/or Xso glucagon analogues to a nominal concentration of 250 LM or a pharmaceutically acceptable salt, amide, acid or prodrug and the concentration was measured after one hour, upon thereof. centrifugation. The concentrations were assessed using a 0014. The present invention further relates to the use of the chemiluminescent nitrogen specific HPLC detector. compounds of the present invention in therapy, to pharma 0027 FIG. 13 shows the stability of glucagon analogues. ceutical compositions comprising compounds of the inven Glucagon analogues were added buffer to a nominal concen tion and the use of the compounds of the invention in the tration of 250 uM and a UPLC chromatogram was recorded manufacture of medicaments. after one hour. The solutions were kept for 6 days at 30° C. whereupon the samples were filtered and a new UPLC was DESCRIPTION OF THE DRAWINGS recorded. The areas under the curves of the peaks (214 nM) 0015 FIG. 1 shows pH dependant solubility of glucagon were used as a measure of concentration of peptide in solu and analogue. Peptides were dissolved in water to app. 1 tion. mg/ml and aliquots were adjusted to various pH. The samples 0028 FIG. 14 shows the lagtime (leftY-axis) and recovery were stored for 5 days at room temperature. After centrifuga (right Y-axis) obtained in a ThT (thioflavin T) fibrillation tion pH was measured and concentration determined by assay. Column 1: Lagtime and recovery for Formulation 1. reverse phase HPLC using an internal glucagon Standard. “1” Column 2A: Lagtime and recovery of glucagon analogue of is glucagon (black line and ()), '2' is glucagon analogue of Example 3 in Formulation 2. Column 2B: Recovery of insulin Example 3 (grey line and (D)). analogue G5 in Formulation 2. Column 3A: Lag time and recovery of glucagon analogue of Example 3 in Formulation 0016 FIG. 2 shows the accumulated food intake in rats 3. Column 3B: Recovery of GLP-1 analogue G1 in formula after sc adm. of 100 nmol/kg, 300 nmol/kg or 1000 nmol/kg tion 3. Column 4: Lag time and recovery of glucagon ana glucagon analogue of Example 3. Data mean+/-Sem, n=5-6. logue of Example 3 in Formulation 4 (GLP-1 analogue G3 0017 FIG. 3 shows the accumulated food intake in rats recovery not determined due to technical reasons). Column 5: after sc adm. of 300 nmol/kg of glucagon analogue of Lagtime and recovery for insulin analogue G5 in Formula Example 4. Data-mean+/-sem, n=5-6 tion 5. Column 6: Lagtime and recovery for GLP-1 analogue 0.018 FIG. 4 shows the accumulated food intake in rats G1 in Formulation 6. after Sc. adm. of 300 nmol/kg of glucagon analogue of 0029 FIG. 15 shows GLP-1, glucagon and glucagon ana Example 5. Data-mean+/-sem, n=5-6. logue of Example 3, incubated with DPP-IV (2 ug/ml) at 37° 0019 FIG. 5 shows the PK of glucagon analogue of C. in a HEPES buffer. The half-lives were determined to 11 Example 3, after iv. and sc. dosing in rats. Halflife (iv.) -8.6 min, 32 min and 260 min, respectively. hit-0.5, Half life (sc.) -9.4 hit-0.9, Data-mean+/-sem. 0020 FIG. 6 shows the reduction of body weight in diet 0030 FIG. 16 depicts ThT fluorescence as a function of induced obese (DIO) rats dosed with glucagon analogue of time t. Example 3 alone, or with GLP-1 analogue G3. Stippled lines indicate start of dosing and reduction of doses, respectively. DESCRIPTION OF THE INVENTION Data mean+/-Sem. 0031. Among further embodiments of the present inven 0021 FIG. 7 shows delta body weight at day 14 in diet tion are the following: induced obese rats dosed with glucagon analogue of Example 3 alone, or with GLP-1 analogue G3. Bars show significant 0032. 2. The glucagon peptide according to embodiment difference (1-way ANOVA, Bonferroni’s post-test). 1, wherein said glucagon peptide comprises Zero, one, two, Data mean+/-Sem. three, four or five amino acid residues Substitutions in said 0022 FIG. 8 shows blood glucose profiles 11" day of glucagon peptide. dosing in diet induced obese rats dosed with glucagon ana 0033 3. The glucagon peptide according to any one of the logue of Example 3 alone, or with GLP-1 analogue G3. previous embodiments, wherein said glucagon peptide com prises Zero amino acid residues Substitutions in said glucagon Stippled lines indicate dosing. Data mean+/-Sem. peptide. 0023 FIG. 9 shows food intake in diet induced obese rats in diet induced obese rats dosed with glucagon analogue of 0034 4. The glucagon peptide according to any one of the Example 3 alone, or with GLP-1 analogue G3. previous embodiments, wherein said glucagon peptide com Data mean+/-Sem. prises one amino acid residues Substitutions in said glucagon 0024 FIG. 10 shows insulin levels measured at the end of peptide. the study in diet induced obese rats dosed with glucagon 0035. 5. The glucagon peptide according to any one of the analogue of Example 3 alone, or with GLP-1 analogue G3. previous embodiments, wherein said glucagon peptide com Groups are compared using 1-way ANOVA and Dunnet's prises two amino acid residues Substitutions in said glucagon post-test comparing groups to vehicle high fat fed group. peptide. Data mean+/-Sem. 0036 6. The glucagon peptide according to any one of the 0025 FIG. 11 shows cholesterol levels measured at the previous embodiments, wherein said glucagon peptide com end of the study in diet induced obese rats dosed with gluca prises three amino acid residues substitutions in said gluca gon analogue of Example 3 alone, or with GLP-1 analogue gon peptide. US 2016/0002311 A1 Jan. 7, 2016
0037 7. The glucagon peptide according to any one of the 0054 24. A glucagon peptide according to any one of the previous embodiments, wherein said glucagon peptide com previous embodiments, wherein X represents Ser. prises four amino acid residues Substitutions in said glucagon 0055 25. A glucagon peptide according to any one of the peptide. previous embodiments, wherein X represents Lys. 0038 8. The glucagon peptide according to any one of the 0056 26. A glucagon peptide according to any one of the previous embodiments, wherein said glucagon peptide com previous embodiments, wherein X2 represents Gln, Cys, prises five amino acid residues Substitutions in said glucagon Ala, Lys or Orn. peptide. 0057 27. A glucagon peptide according to any one of the 0039. 9. A glucagon peptide according to embodiment 1, previous embodiments, wherein X represents Gln. wherein said amino acid substitutions may be in the following 0.058 28. A glucagon peptide according to any one of the positions of said glucagon peptide: previous embodiments, wherein Xo represents Cys. X represents Ser, Aib or D-Ser; 0059 29. A glucagon peptide according to any one of the Xo represents Tyr, Lys, CyS or Orn; previous embodiments, wherein Xo represents Lys. X represents Lys, Orn, Cys, Arg, Leu, Ile or His: 0060 30. A glucagon peptide according to any one of the X represents Ser, Glu, Thr, Val, Phe, Tyr, Ile, Leu, Lys or previous embodiments, wherein Xo represents Orn. Orn; 0061 31. A glucagon peptide according to any one of the Xo represents Gln, Cys, Ala, Lys or Orn; previous embodiments, wherein X represents Gln, Lys, X represents Gln, LyS, Cys, Ala, Arg, His, Glu, Asp, Gly, Ser Cys, Ala, Arg, His, Glu, Asp, Gly, Ser or Orn. or Orn; 0062 32. A glucagon peptide according to any one of the Xs represents Trp, Phe, Tyr, (p)Tyr, His, Gln, Lys or Orn; previous embodiments, wherein X2 represents Lys, Orn or X, represents Met, Met(O), Leu, Lys, Orn, Ile, Leu, Gln or CyS and X, represents Leu. Glu: 0063. 33. A glucagon peptide according to any one of the Xs represents ASn, Lys, Cys, Ser, Thr, Glu, Asp, Gln or Orn; previous embodiments, wherein X2 represents Lys, CyS or Xo represents Thr, Glu, Cys, Asp, Lys, Pro or Orn; and Orn. X is absent or represents Cys, Lys, Pro or Orn. 0064. 34. A glucagon peptide according to any one of the 0040 10. A glucagon peptide according to any one of the previous embodiments, wherein X2 represents Gln, Lys or previous embodiments, wherein said amino acid substitu Orn. tions may be in the following positions of said glucagon 0065 35. A glucagon peptide according to any one of the peptide: X represents Ser, Xo represents Tyr, X2 represents previous embodiments, wherein X represents Lys or Orn. Lys, X represents Ser or Lys, X represents Gln, X rep 0.066 36. A glucagon peptide according to any one of the resents Gln, Lys or Orn, Xs represents Trp, X, represents previous embodiments, wherein X represents Gln. Leu, Xs represents ASn, Ser or Asp, Xo represents Thr, Lys 0067 37. A glucagon peptide according to any one of the and Xso is absent or represents Lys. previous embodiments, wherein X represents Cys. 0041 11. A glucagon peptide according to any one of the 0068 38. A glucagon peptide according to any one of the previous embodiments, wherein X represents Ser, Aib or previous embodiments, wherein X represents Lys. D-Ser. 0069. 39. A glucagon peptide according to any one of the 0.042 12. A glucagon peptide according to any one of the previous embodiments, wherein X represents Orn. previous embodiments, wherein X represents Ser. 0070 40. A glucagon peptide according to any one of the 0.043 13. A glucagon peptide according to any one of the previous embodiments, wherein Xs represents Trp, Phe, Tyr, previous embodiments, wherein X represents Tyr, Lys, Cys (p)Tyr, His, Gln, Lys or Orn. or Orn. 0071. 41. A glucagon peptide according to any one of the 0044 14. A glucagon peptide according to any one of the previous embodiments, wherein Xis represents Phe, Tyr. previous embodiments, wherein Xo represents Tyr. (p)Tyr, His, Gln, Lys or Orn 0045 15. A glucagon peptide according to any one of the 0072 42. A glucagon peptide according to any one of the previous embodiments, wherein X represents Lys. previous embodiments, wherein Xis represents Lys. His or 004.6 16. A glucagon peptide according to any one of the (p)Tyr. previous embodiments, wherein Xo represents Cys. 0073. 43. A glucagon peptide according to any one of the 0047 17. A glucagon peptide according to any one of the previous embodiments, wherein Xs represents Trp. previous embodiments, wherein Xo represents Orn. 0074 44. A glucagon peptide according to any one of the 0.048. 18. A glucagon peptide according to any one of the previous embodiments, wherein X, represents Met, Met(O), previous embodiments, wherein X represents Lys, Orn, Leu, Lys, Orn, Ile, Leu, Gln or Glu. Cys, Arg, Leu, Ile or His. 0075 45. A glucagon peptide according to any one of the 0049 19. A glucagon peptide according to any one of the previous embodiments, wherein X, represents Leu. previous embodiments, wherein X2 represents Lys. 0076 46. A glucagon peptide according to any one of the 0050. 20. A glucagon peptide according to any one of the previous embodiments, whereinXs represents ASn, Lys, Ser, previous embodiments, wherein X represents Orn. Cys, Thr, Glu, Asp, Gln or Orn. 0051. 21. A glucagon peptide according to any one of the 0077 47. A glucagon peptide according to any one of the previous embodiments, wherein X2 represents Cys. previous embodiments, wherein Xs represents ASn, Ser or 0.052 22. A glucagon peptide according to any one of the Asp. previous embodiments, wherein X represents Ser, Glu, Thr, 0078 48. A glucagon peptide according to any one of the Val, Phe, Tyr, Ile, Leu, Lys or Orn. previous embodiments, wherein Xis represents Asn. 0053 23. A glucagon peptide according to any one of the 0079 49. A glucagon peptide according to any one of the previous embodiments, wherein X represents Ser or Lys. previous embodiments, wherein Xs represents Ser. US 2016/0002311 A1 Jan. 7, 2016
0080 50. A glucagon peptide according to any one of the -continued previous embodiments, wherein Xs represents Asp. 0081 51. A glucagon peptide according to any one of the previous embodiments, wherein Xis represents Lys, Cys or IIb Orn. Ns N O 0082 52. A glucagon peptide according to any one of the Nw previous embodiments, wherein Xis represents Lys. N : 0083 53. A glucagon peptide according to any one of the H previous embodiments, wherein Xis represents Cys. 0084. 54. A glucagon peptide according to any one of the IIc previous embodiments, wherein Xs represents Orn. OH O 0085 55. A glucagon peptide according to any one of the previous embodiments, wherein X represents Thr, Glu, Asp, Cys, Lys, Pro or Orn. I0086 56. A glucagon peptide according to any one of the previous embodiments, wherein X2 represents Cys, Lys or Orn. 0087 57. A glucagon peptide according to any one of the wherein n in formula IIa is 6-20, previous embodiments, wherein X represents Lys or Orn. 0088 58. A glucagon peptide according to any one of the m in formula IIc is 5-11 previous embodiments, wherein X represents Thr or Lys. the COOH group in formula IIc can be attached to position 2, 0089 59. A glucagon peptide according to any one of the 3 or 4 on the phenyl ring, previous embodiments, wherein X represents Thr. 0090 60. A glucagon peptide according to any one of the the symbol * in formula IIa, IIb and IIc represents the attach previous embodiments, wherein X represents Lys. ment point to the nitrogen in Z; 0091 61. A glucagon peptide according to any one of the if Z is absent, Z is attached to the nitrogen on Z at symbol previous embodiments, wherein X represents Cys. * and if Z and Z are absent Z is attached to the nitrogen on 0092 62. A glucagon peptide according to any one of the Zaat symbol * previous embodiments, wherein X represents Orn. 0093. 63. A glucagon peptide according to any one of the Z2 is absent or represents a structure according to one of the previous embodiments, wherein X is absent or represents formulas IId, IIe, IIf IIg, IIh, II or IIk; Cys, Lys, Pro or Orn. 0094) 64. A glucagon peptide according to any one of the previous embodiments, wherein X is absent or represents Cys, Lys or Orn. 0095 65. A glucagon peptide according to any one of the IId previous embodiments, wherein X is absent or represents Lys. 0096 66. A glucagon peptide according to any one of the previous embodiments, wherein X represents Cys. 0097 67. A glucagon peptide according to any one of the N previous embodiments, wherein X represents Orn. 0098 68. A glucagon peptide according to any one of the previous embodiments, wherein X is absent 0099 69. A glucagon peptide according to any one of the IIe previous embodiments, wherein Xso represents Lys. O OH 0100 Further embodiments of the present invention relate O tO: : n N : 0101 70. A glucagon peptide according to any of the pre H vious embodiments, wherein said substituent has the formula O II: O OH wherein, Z represents a structure according to one of the formulas IIa, IIf IIb or IIc; O OH O OH O
IIa N N O O H H O O ---. O OH US 2016/0002311 A1 Jan. 7, 2016
-continued -continued II O OH IIg O OH O : H H O O n N o NH N : O O O OH O OH H O
IIh O OH O OH IIk O OH O OH
O O
: 1 N N H H O O II O OH O OH O OH
O wherein each amino acid moiety independently has the Ste
: reochemistry L or D; wherein Z is connected via the carbonatom denoted * to the H nitrogen of Z denoted *; O if Z is absent, Z is connected via the carbonatom denoted * to the nitrogen of Z denoted * and if Z and Z are absent Z. is connected via the carbon denoted * to the epsilon nitrogen ofalysine or the delta nitrogenofan ornithine of the glucagon O OH peptide. Z is absent or represents a structure according to one of the formulas IIm, In, IIo or IIp;
Im. -N-----.O
IIn --~~~O N----.O
IIo
-N-----~~ ^------. US 2016/0002311 A1 Jan. 7, 2016
Z is connected Vi the carbon of Z with symbol * to the -continued nitrogen of Z with symbol, if Z is absent Z is connected IIf via the carbon with symbol * to the epsilon nitrogen of a O OH O OH lysine or the delta nitrogen of an ornithine of the glucagon O peptide N N Z is absent or represents a structure according to one of the H H formulas IId, IIe, IIf IIg, IIh, II or IIk; wherein each amino O O acid moiety is independently either L or D, wherein Z is O OH connected via the carbon with symbol * to the epsilon nitro II 9. O OH O OH gen of a lysine or the delta nitrogen of an ornithine of the O O glucagon peptide. : NN N : 0102 71. A glucagon peptide according to embodiments H H O O 28, wherein said substituent has the formula II: O1 OH O1 OH IIh Z-Z2-Z-Za- II) O wherein, : 1 : Z represents a structure according to one of the formulas IIa, IIb or IIc;
O OH IIa II. O OH
O IIb
:
H O
O OH II O OH HO
O
H wherein n in formula IIa is 6-20, O Z2 is absent or represents a structure according to one of the formulas IId, IIe, IIf IIg, IIh, II or IIk; O OH O OH IIk IId O OH O OH O OH
"n : O O N H : O 1 N N H H IIe O OH O O O
: n N : O OH O OH H wherein each amino acid moiety independently has the Ste O reochemistry L or D. Z is absent or represents a structure according to one of the formulas IIm, In, IIo or IIp; US 2016/0002311 A1 Jan. 7, 2016
Im. -N-----.O IIn O O --~~~~.H IIo --~~~~.O O O IIp -N------~~O O ~------.O O H H
Z is absent or represents a structure according to one of the 0105 74. A glucagon peptide according to any one of the formulas IId, IIe, IIf IIg, IIh, II or IIk; previous embodiments, wherein Z of said substituent of for wherein each amino acid moiety independently has the Ste mula II is absent when Z is present. reochemistry L or D. 0106 75. A glucagon peptide according to any one of the 0103 72. A glucagon peptide according to embodiment previous embodiments, wherein Z of said substituent of for 29, wherein the structures of formulas IIa-IIp have the stere mula II is absent when Z is present. ochemistry L. 0107 76. A glucagon peptide according to any of the pre 0104 73. A glucagon peptide according to embodiment vious embodiments, wherein said Substituent represents a 29, wherein the structures of formulas IIa-IIp have the stere structure according to one of the formulas IIIa, IIIb, IIIc, IIId, ochemistry D. IIIe, IIIfor IIIg: US 2016/0002311 A1 Jan. 7, 2016
B.III QIII 3.III ÞIII 3.III
US 2016/0002311 A1 Jan. 7, 2016 10
0108) 77. A glucagon peptide according to any of the pre vious embodiments, wherein said substituent a structure of formula IIIa:
IIIa. O 's--" s H HO H ~~~~N O O O OH O Y1 Nulls 1-O n-r N r : O O O OH
0109) 78. A glucagon peptide according to any of the pre vious embodiments, wherein Z of said Substituent is absent. 0110 79. A glucagon peptide according to any of the pre vious embodiments, wherein Z and Z of said substituent are absent. 0111 80. A glucagon peptide according to any of the pre vious embodiments, said Substituent represents a structure according to one of the formulas IVa., IVb. IVc or IVd: IV a O OH O Ys1 H O
for--~~~~ H N-o~------~~~H O O O IVb O OH o S1 O HO : ouls H r-r n-1N1\-1 8.s O O IVc O OH o Y O HO s ouls H r-n N-1 no-1N1 O O o OS1 OH NN1N- n-1No N s H H O O O OH IVd O o OS-1 OH HO E
NH N 1N1 N. O H
O OH o OS1 OH H s N *: Y N r-r O O O OH US 2016/0002311 A1 Jan. 7, 2016 11
-continued IWe MNN N O
O N N-11N1'N-1O O O O --~ O n-r *; or O IVf
N N N-1-1N1'N-1O O O us 1N1O N-1 no~ 8. O
0112 81. A glucagon peptide according to any of the pre 0.122 91. A glucagon peptide according to any of the pre vious embodiments, wherein Z and Z of said substituent are vious embodiments, wherein Z2 and Z of said Substituent are independently represented by negatively charged moieties independently represented by Asp, Asp-Asp, Asp-Asp-Asp Such as Glu, Glu and/or Asp. or Asp-Asp-Asp-Asp. 0113 82. A glucagon peptide according to any of the pre 0123 92. A glucagon peptide according to any of the pre vious embodiments, wherein Z and Z of said substituent are vious embodiments, wherein Z2 and Z of said Substituent are independently represented by up to ten of said moieties. independently represented by Glu moieties. 0114 83. A glucagon peptide according to any of the pre vious embodiments, wherein Z2 and Z of said Substituent are 0.124 93. A glucagon peptide according to any of the pre independently represented by three of said moieties. vious embodiments, wherein Z2 and Z of said Substituent are 0115 84. A glucagon peptide according to any of the pre independently represented by Glu, Glu-Glu, Glu-Glu-Glu, vious embodiments, wherein Z2 and Z of said Substituent are Glu-Glu-Glu-Glu, Glu-Glu-Glu-Glu-Glu. independently represented by four of said moieties. 0.125 94. A glucagon peptide according to any of the pre 0116 85. A glucagon peptide according to any of the pre vious embodiments, wherein Z2 and Z of said Substituent are vious embodiments, wherein Z2 and Z of said Substituent are independently represented by YGlu moieties. independently represented by five of said moieties. 0.126 95. A glucagon peptide according to any of the pre 0117 86. A glucagon peptide according to any of the pre vious embodiments, wherein Z2 and Z of said Substituent are vious embodiments, wherein Z and Z of said substituent are independently represented by YGlu, YGlu-YGlu, YGlu-YGlu independently represented by Glu and/or YGlu moieties. YGlu, YGlu-yGlu-YGlu-YGlu, YGlu-yGlu-YGlu-YGlu-yGlu. 0118 87. A glucagon peptide according to any of the pre I0127 96. A glucagon peptide according to any of the pre vious embodiments, wherein Z2 and Z of said Substituent are vious embodiments, wherein said Substituent comprises a independently represented by YGlu, YGlu-Glu, YGlu-Glu-Glu, lipophilic moiety. YGlu-Glu-Glu-Glu, YGlu-Glu-Glu-Glu-Glu. 0119 88. A glucagon peptide according to any of the pre I0128 97. A glucagon peptide according to any of the pre vious embodiments, wherein Z2 and Z of said Substituent are vious embodiments, wherein said Substituent comprises a independently represented by Glu and/or Asp moieties. straight chain alkyl group or a branched alkyl group. 0120 89. A glucagon peptide according to any of the pre I0129. 105. A glucagon peptide according to any of the vious embodiments, wherein Z2 and Z of said Substituent are previous embodiments, wherein said Substituent binds non independently represented by YGlu and/or Asp moieties. covalently to albumin. 0121 90. A glucagon peptide according to any of the pre 0.130 106. A glucagon peptide according to any of the vious embodiments, wherein Z2 and Z of said Substituent are previous embodiments, wherein said Substituent is negatively independently represented by Asp moieties charged at physiological pH. US 2016/0002311 A1 Jan. 7, 2016
0131 Further embodiments of the present invention relate biological activity is prolonged. Effect is defined as being tO: protracted when a compound significantly reduces food 0132) 107. A glucagon peptide according to any of the intake in the period from 24 hours to 48 hours in test animals previous embodiments, wherein said Substituent is attached at compared to the food intake in the same time period in the the epsilon position of a Lys or at the delta position of an Orn, vehicle-treated control group of animals in Assay IV”. The or at the sulphur of a Cys. protracted effect can be evaluated through different binding 0.133 108. A glucagon peptide according to any of the assays, for example the protracting effect may be evaluated in previous embodiments, wherein said Substituent is attached at an indirect albumin-binding assay, in which Ki determined the epsilon position of a Lys or at the delta position of an Orn. for binding in the presence of ovalbuminis compared with the 0134 109. A glucagon peptide according to any of the ECso value determined in the presence of human serum albu previous embodiments, wherein said Substituent is attached at min (HSA). the epsilon position of a Lys. 0151. The inventors surprisingly found that the com 0135) 110. A glucagon peptide according to any of the pounds of the present invention, show improved aqueous previous embodiments, wherein said Substituent is attached at solubility at neutral pH or slightly basic pH. Furthermore, the the delta position of an Orn. present inventors have also Surprisingly found that the gluca 0136 111. A glucagon peptide according to any of the gon analogues of the present invention have improved Stabil previous embodiments, wherein said Substituent is attached at the Sulphur position of a Cys. ity towards formation of gels and fibrils in aqueous Solutions. 0.137 112. A glucagon peptide according to any of the The stability of the compounds of the present invention may previous embodiments, wherein said Substituent is attached be measured by a method as described in example 63. in one or more of the following amino acid positions of the 0152. A better control of blood glucose levels in Type 1 glucagon glucagon peptide: Xio. X2: X20, X2: X2s. X29. and 2 diabetes may be achieved by co-administration of glu and/or Xso cagon with known antidiabetic agents such as insulin, GLP-1 0138 113. A glucagon peptide according to any of the agonists and GIP. The glucagon analogues of the invention previous embodiments, wherein said Substituent is in one or show anorectic effects in rats when administered a single more of following amino acid positions of said glucagon dose, and the effect at day two were observed to be as least as peptide: X2, X24, X29 and Xso. good as the effect at the day of dosing, clearly demonstrating 0139 114. A glucagon peptide according to any of the the protracted effect of these analogues. Furthermore, the previous embodiments, wherein said Substituent is at amino compounds of the present invention give a high reduction of acid position X of said glucagon peptide. body weight when administered to diet induced obese rats. An 0140 115. A glucagon peptide according to any of the even more pronounced reduction of body weight can be previous embodiments, wherein said Substituent is at amino obtained by co-administration with a protracted GLP-1 ana acid position X of said glucagon peptide. logue, which in addition leads to a better control of blood 0141 116. A glucagon peptide according to any of the glucose. 0153. In one embodiment, the glucagon analogues of this previous embodiments, wherein said Substituent is at amino invention can be co-formulated with GLP-1 analogues or acid position X of said glucagon peptide. insulin analogues, forming stable pharmaceutical composi 0142 117. A glucagon peptide according to any of the tions. previous embodiments, wherein said Substituent is at amino 0154 Combination of insulin and glucagon therapy may acid position X of said glucagon peptide. be advantageous compared to insulin-only therapy comes 0143) 118. The glucagon peptide according to any of the from the architecture of the human defence against hypogly previous embodiments, wherein said Substituent is in up to caemia. Normally, in a postprandial situation when blood five amino acid positions of said glucagon peptide. glucose levels become low the first hormonal response is 0144. 119. The glucagon peptide according to any of the reduction in the production of insulin. When blood glucose previous embodiments, wherein said Substituent is in up to drops further the second line response is production of glu four amino acid positions of said glucagon peptide. cagon—resulting in increased glucose output from the liver. 0145 120. The glucagon peptide according to any of the When diabetics receive an exogenous dose of insulin that is previous embodiments, wherein said Substituent is in up to too high the natural response of raised glucagon is prevented three amino acid positions of said glucagon peptide. by the presence of exogenous insulin, since insulin has an 0146 121. The glucagon peptide according to any of the inhibiting effect on glucagon production. Consequently, previous embodiments, wherein said Substituent is in two slight overdosing of insulin may cause hypoglycaemia. Pres amino acid position of said glucagon peptide. ently, many diabetic patients tend to prefer to use a little less 0147 122. The glucagon peptide according to any of the insulin than optimal in fear of hypoglycaemic episodes which previous embodiments, wherein said Substituent is in one may be life-threatening. amino acid position of said glucagon peptide. 0155 The fact that the compounds of the present invention 0148. The present invention relates to novel glucagonana are soluble at neutral pH, may allow a co-formulation with logues with improved solubility, improved physical stability insulin and allow for more stable blood glucose levels and a toward gel and fibril formation and with increased half life. reduced number of hypoglycaemic episodes, as well as a 014.9 The inventors have surprisingly found that the com reduced risk of diabetes related complications. pounds of the present invention have a prolonged halflife and 0156 Further embodiments of the present invention relate that they show improved pharmacokinetic properties, i.e., they have prolonged exposure in vivo due to prolonged to intramolecular bridges: plasma elimination half-life and a prolonged absorption 0157 123. A glucagon peptide according to any one of the phase. Furthermore, the compounds of the present invention previous embodiments, further comprising an intramolecular show a significant reduction in food intake when adminis bridge between the side chains of an amino acid at positionXi tered s.c. with a protracted effect up to 48 hours. This is to our and an amino acid at position Xi+4 or Xi+3. best knowledge, the first demonstration of reduced food 0158 124. A glucagon peptide according to any of intake of a protracted glucagon analogue. embodiment 50, wherein the amino acid at position Xi and an 0150 Protracted effect of the compounds of the present amino acid at position Xi+4 are linked through a lactam invention means that the period of time in which they exert a bridge or a salt bridge. US 2016/0002311 A1 Jan. 7, 2016
0159 125. A glucagon peptide according to any of 0.165. 131. A glucagon peptide according to any one of the embodiment 50, wherein the amino acid at position Xi and an previous embodiments, wherein said glucagon peptide com amino acid at position Xi+4 are linked through a lactam prises C-terminal extensions of up to two amino acid resi bridge. dues. 0160 126. A glucagon peptide according to embodiment 0166 132. A glucagon peptide according to any one of the 50, wherein the amino acid at position Xi and an amino acid previous embodiments, wherein said glucagon peptide com at position Xi+4 are linked through a salt bridge. prises C-terminal extensions of one amino acid residue. 0161 127. A glucagon peptide according to embodiments 0167. 133. A glucagon peptide according to any one the 50-53, wherein Xi is selected from positions X, XX or previous embodiments, wherein the glucagon peptide is a X24. C-terminal amide or a C-terminal carboxylic acid. 0162. 128. A glucagon peptide according to any one of 0168 134. A glucagon peptide according to any one of the embodiments 53-54, wherein one, two, three or more of posi previous embodiments, wherein said glucagon peptide is a tions X, Xo, or X of said glucagon peptide, are substi C-terminal amide. tuted with an O. amino acid and/or an O-disubstituted amino 0169. 135. A glucagon peptide according to any one of the acid. previous embodiments, wherein said glucagon peptide is a 0163) 129. A glucagon peptide according to any one of the C-terminal carboxylic acid. previous embodiments, wherein X represents Glu and Xo 0170 136. A glucagon peptide according to any one of the represents Lys. previous embodiments, selected from the group consisting of 0164 130. A glucagon peptide according to any one of the (0171 N'-(2-2-[2-(4S)-5-hydroxy-4-(18-hydroxy previous embodiments, wherein said glucagon peptide com 18-oxooctadecanoyl)amino5-oxopentanoylamino prises C-terminal extensions of up to three amino acid resi ethoxyethoxyacetylaminoethoxyethoxyacetyl) dues. Lys7.Lys'.Glu'.Lys''...Leu’Glucagon O H-H SQG TFTs D Y S KYLDS K KAQEF v-u-WLLNH T-OH
O HO H N O h O "N- --~~~~~. O O O
(0172 N'-(2-2-[2-(4S)-5-hydroxy-4-(18-hydroxy 18-oxooctadecanoyl)amino5-oxopentanoylamino ethoxyethoxyacetylaminoethoxyethoxyacetyl) Lys'.Lys'7Lys'Glu'...Leu’Glucagon H-H SQG TFTS DYSKYLD-NJ-KKAQEFH v QWLLN T-OH
o Os-OH O to-a-M N-a ~-o- 1-O-1 NH
(0173 N'-(2-2-2-2-22-(4S)-5-hydroxy-4-(18 hydroxy-18-oxooctadecanoyl)amino-5-oxopentanoyl aminoethoxyethoxyacetylaminoethoxyethoxy acetyl) Lys7.Lys'.Glu'.Lys. Leu’.Ser'Glucagon
O H-HSQG TFTs DYSKYLDS KKA QEF v-u-WLLST-OHH
o Os-OH O * ------~~~ s N-a ~-os- 1-On-1a NH
US 2016/0002311 A1 Jan. 7, 2016 16
0181 N'-(2-2-[2-(4S)-4-carboxy-4-(17-carboxyhep tadecanoylamino)butanoylaminoethoxyethoxyacetyl Lys'7.Lys' Glu'.Lys’.Leu'. Ser-Glucagon
O H-H so GTFTs DYSKYLDs KKAQEF v-u-WLLH st-OH
O O s-OH NH H HO N Na1a -N-N-( H O O ~ O
0182 N'-(2-2-[2-(2-2-[2-(2S)-4-carboxy-2-(17 carboxyheptadecanoylamino)butanoylaminoethoxy ethoxyacetylaminolethoxyethoxyacetyl-Lys'". Lys'.Glu'.Lys' Leu’.Ser-Glucagon
O H-H SQG TFTs DYSKYLD S K KAQEF v-Nuy-WLLST-OHH
O Hos---~~~~ N---~~~~~~);O O l H O
OD. OH
0183 N'-(2-2-2-2-2-[2-(4S)-4-carboxy-4-(17 carboxyheptadecanoylamino)butanoylaminoethoxy ethoxyacetylaminolethoxyethoxyacetyl-Lys'". Lys'.Glu',Orn.Leu7,Ser-Glucagon
H-H SQG TFT S D Y S KY LDS K KAQE F V-Nu-WH O L L S T-OH o, On-OH O H HO N O O NH
~~~~ n-1-o1n- ~~ n-norO
0184 Further embodiments of the present invention relate 0189 141. A glucagon peptide according to any one of the to administration of the compounds of the present invention previous embodiments, in combination with a glucagon-like with antidiabetic agents or anti-obesity agents: 0185. 137. A glucagon peptide according to any one of the peptide 1 (GLP-1) compound, for the preparation of a medi previous embodiments, in combination with a glucagon-like cament for the treatment of diabetes and/or obesity. peptide 1 (GLP-1) compound. 0.190 142. A glucagon peptide according to any one of the 0186 138. A glucagon peptide according to any one of the previous embodiments, in combination with an insulinic previous embodiments, in combination with an insulinic compound. compound, for the preparation of a medicament for the treat 0187. 139. A glucagon peptide according to any one of the ment of diabetes and/or obesity. previous embodiments, in combination with exendin-4. 0191) 143. A glucagon peptide according to any one of the 0188 140. A glucagon peptide according to any one of the previous embodiments, in combination with exendin-4, for previous embodiments, which is in a dual chamber, deposi the preparation of a medicament for the treatment of diabetes tory and/or micro-encapsulation formulation. and/or obesity.
US 2016/0002311 A1 Jan. 7, 2016 18
(0196) N-epsilon37-2-(2-2-[2-(2-2-(S)-4-carboxy-4- (15-carboxy-pentadecanoylamino)-butyrylamino ethoxy-ethoxy)-acetylamino-ethoxy-ethoxy)-acetyl Aib8,22,35,Lys37GLP-1-(7-37):
(compound G4) O OH O O * ----~~~ N -> N- O~-os- N 1n-On-1a orne O and O O NH HN\, s O O H H OH ...O vil-sociative O - O
(0197) NeB29-hexadecandiyol-y-Glu-(desB30) human insulin
(compound G5)
— HG IV E Q C C T S I C S LY QLE NY CN-OH
S I S H-F V N Q H LCGS H L V E A LY L V C G E R G F FY TP-N
0198 GLP-1 is an incretin hormone produced by the administration of exendin-4(7-45) (SEQ ID NO.1 in the US endocrine cells of the intestine following ingestion of food. patent). GLP-1 is a regulator of glucose metabolism, and the Secretion 0201 The term “GLP-1 compound” as used herein refers of insulin from the beta cells of the islets of Langerhans in the to human GLP-1 (7-37) (amino acids 1-31 of SEQID NO:3), pancreas. GLP-1 also causes insulin secretion in the diabetic exendin-4(7-45) (amino acids 1-39 of SEQID NO:4), as well state. The half-life in vivo of GLP-1 itself is, however, very as analogues, fusion peptides, and derivatives thereof, which short, thus, ways of prolonging the half-life of GLP-1 in vivo maintain GLP-1 activity. has attracted much attention. 0202 As regards position numbering in GLP-1 com (0199 WO 98/08871 discloses protracted GLP-1 ana pounds: for the present purposes any amino acid substitution, logues and derivatives based on human GLP-1 (7-37) (amino deletion, and/or addition is indicated relative to the sequences acids 1-31 of SEQ ID NO:3) which have an extended half of SEQ ID NO:3, and/or 4. However, the numbering of the life, including liraglutide, a GLP-1 derivative for once daily amino acid residues in the sequence listing always starts with administration developed by Novo Nordisk A/S marketed for no. 1, whereas for the present purpose we want, following the the treatment of type 2 diabetes. established practice in the art, to start with amino acid residue 0200 Exenatide is a commercial incretin mimetic for the no. 7 and assign number 7 to it. Therefore, generally, any treatment of diabetes mellitus type 2 which is manufactured reference herein to a position number of the GLP-1 (7-37) or and marketed by Amylin Pharmaceuticals and Eli Lilly & Co. exendin-4 sequence is to the sequence starting with His at Exenatide is based on exendin-4, a hormone found in the position 7 in both cases, and ending with Gly at position 37, saliva of the Gila monster. It displays biological properties or Ser at position 45, respectively. similar to human GLP-1. U.S. Pat. No. 5,424,286 relates i.a. 0203 GLP-1 compounds may be prepared as exemplified to a method of stimulating insulin release in a mammal by in example 65. US 2016/0002311 A1 Jan. 7, 2016
0204 GLP-1 activity may be determined using any 0208 iii) be a GLP-1 derivative comprising an albumin method known in the art, e.g. the assay (II) herein (stimulation binding moiety that comprises an acyl radical of a dicarboxy of cAMP formation in a cell line expressing the human GLP-1 lic acid, preferably comprising a total of from 12 to 24 carbon receptor). atoms, such as C12, C14, C16, C18, C20, C22, or C24, most 0205 Furthermore, the GLP-1 compound is a compound preferably C16, C18, or C20; wherein preferably a) the acyl which may: radical is attached to the epsilon amino group of a lysine 0206 i) comprise at least one of the following: Desami residue of the GLP-1 peptide via a linker; b) the linker com noHis7, Aib8, Aib22, Arg26, Arg34, Aib35, and/or Lys37; prises at least one OEG radical, and/or at least one Trx radical, 0207 ii) be a GLP-1 derivative comprising an albumin and, optionally, additionally at least one Glu, and/or binding moiety which comprises at least one, preferably at 0209 iv) be selected from the group consisting of com least two, more preferably two, free carboxylic acid groups; pounds N-epsilon26-(S)-4-Carboxy-4-hexadecanoylamino or a pharmaceutically acceptable salt thereof; butyryl)Arg34GLP-1-(7-37):
(compound G1) O H N n-n-n-n-n-n-n-r NH O O OH
OH: H-H AEG TFTs DV SS YLE GQAA-N EFlawl v GR-1) O O
0210 N-epsilon37-2-(2-2-[2-(2-2-(S)-4-Carboxy-4- (trans-4-(19-carboxynonadecanoylamino)methyl cyclohexanecarbonyl)amino)butyrylaminol ethoxyethoxy) acetylaminolethoxyethoxy)acetyl DesaminoHis7.Glu22. Arg26. Arg34.Lys37IGLP-1-(7- 37):
(compound G2) O H O O H N N1N1 O~orn YO n101-5H Ho-ha-n--~NawO OY OH H O O HN N s
O AE GT FTSD vs SYLE EQA A RE FIA W L V RGR-N O OH:
0211 N-epsilon26-2-(2-2-[2-(2-2-(S)-4-Carboxy-4- (17-carboxyheptadecanoylamino)butyrylamino ethoxyethoxy)acetylaminolethoxyethoxy)acetyl Aib8. Arg34GLP-1-(7-37):
(compound G3) I-ii-k-horrisovssyH O Ego-su-prawlH O vior-Su-oH O O O l O Hosa-N-o-o-ra-o-o-NHH O US 2016/0002311 A1 Jan. 7, 2016 20
0212 N-epsilon37-2-(2-2-[2-(2-2-(S)-4-carboxy-4- 0217. By “simultaneous' dosing of a preparation of a (15-carboxy-pentadecanoylamino)-butyrylamino- compound of the present invention and a preparation of anti ethoxy-ethoxy)-acetylamino-ethoxy-ethoxy)-acetyl obesity or anti-diabetic agents is meant administration of the Aib8,22,35,Lys37GLP-1-(7-37): compounds in single-dosage form, or administration of a first
(compound G4) O OH O is- N- ~-os- 1n-On-1a O O O HN1 N s O O H H OH
HN O --torneysvir---orrisviv- O R-N O and their pharmaceutically acceptable salts, amides, alkyls, or agent followed by administration of a second agent with a esterS. time separation of no more than 15 minutes, preferably 10, 0213. An “insulin' according to the invention is herein to more preferred 5, more preferred 2 minutes. Either factor may be understood as human insulin, an insulin analogue or an be administered first. insulin derivative. 0218. By “sequential dosing is meant administration of a 0214. The insulinic compound is a compound which may first agent followed by administration of a second agent with for example, be represented by: a time separation of more than 15 minutes. Either of the two 0215 NeB29-hexadecandiyol-y-Glu-(desB30) human unit dosage form may be administered first. Preferably, both insulin products are injected through the same intravenous access.
(compound G5)
O
— N O HG IV EQ CCTS I C S LY QLE NYC N-OH l H-F V N Q H LCGS H L V E A LY L V C G E R G F FY TP-N O O
0216 The compounds of the present invention and anti 0219. As already indicated, in all of the therapeutic meth obesity oranti-diabetic agents as defined in the present speci ods or indications disclosed above, a compound of the present fication, may be administered simultaneously or sequentially. invention may be administered alone. However, it may also be administered in combination with one or more additional The factors may be supplied in single-dosage form wherein therapeutically active agents, Substances or compounds, the single-dosage form contains both compounds, or in the either sequentially or concomitantly. form of a kit-of-parts comprising a preparation of a com 0220. A typical dosage of a compound of the invention pound of the present invention as a first unit dosage form and when employed in a method according to the present inven a preparation of a anti-obesity or anti-diabetic agents as a tion is in the range of from about 0.001 to about 100 mg/kg second unit dosage form. Whenever a first or second or third, body weight per day, preferably from about 0.01 to about 10 etc., unit dose is mentioned throughout this specification this mg/kg body weight, more preferably from about 0.01 to about does not indicate the preferred order of administration, but is 5 mg/kg body weight per day, e.g. from about 0.05 to about 10 merely done for convenience purposes. mg/kg body weight per day or from about 0.03 to about 5 US 2016/0002311 A1 Jan. 7, 2016 mg/kg body weight per day administered in one or more intake; as well as PPAR (peroxisome proliferator-activated doses, such as from 1 to 3 doses. The exact dosage will depend receptor)agonists and RXR (retinoidxreceptor)agonists Such upon the frequency and mode of administration, the sex, age, as ALRT-268, LG-1268 or LG-1069. weight and general condition of the Subject treated, the nature 0226. Other examples of suitable additional therapeuti and severity of the condition treated, any concomitant dis cally active Substances include insulin or insulin analogues; eases to be treated and other factors evident to those skilled in Sulfonylureas, e.g. tolbutamide, chlorpropamide, tolaZamide, the art. glibenclamide, glipizide, glimepiride, glicazide or glyburide; 0221 Compounds of the invention may conveniently be biguanides, e.g. metformin; and meglitinides, e.g. repaglinide formulated in unit dosage form using techniques well known or senaglinide/nateglinide. to those skilled in the art. A typical unit dosage form intended 0227 Further examples of suitable additional therapeuti for oral administration one or more times per day, Such as cally active Substances include thiazolidinedione insulin sen from one to three times per day, may suitably contain from sitizers, e.g. troglitaZone, ciglitaZone, pioglitaZone, rosiglita about 0.05 to about 1000 mg, preferably from about 0.1 to Zone, isaglitaZone, dargilitaZone, englitaZone, CS-011/CI about 500 mg, such as from about 0.5 to about 200 mg of a 1037 or T 174, or the compounds disclosed in WO 97/41097 compound of the invention. (DRF-2344), WO 97/41119, WO 97/41120, WO 00/41121 0222 Compounds of the invention comprise compounds and WO 98/.45292 (Dr. Reddy's Research Foundation), the that are believed to be well-suited to administration with contents of all of which are incorporated herein by reference. longer intervals than, for example, once daily, thus, appropri 0228. Additional examples of suitable additional thera ately formulated compounds of the invention may be suitable peutically active Substances include insulin sensitizers, e.g. for, e.g., twice-weekly or once-weekly administration by a GI 262570, YM-440, MCC-555, JTT-501, AR-HO39242, Suitable route of administration, such as one of the routes KRP-297, GW-409544, CRE-16336, AR-HO49020, disclosed herein. LY510929, MBX-102, CLX-0940, GW-501516 and the com 0223) As described above, compounds of the present pounds disclosed in WO99/19313 (NN622/DRF-2725), WO invention may be administered or applied in combination 00/50414, WO 00/63191, WO 00/631.92 and WO 00/63193 with one or more additional therapeutically active com (Dr. Reddy's Research Foundation), and in WO 00/23425, pounds or Substances, and suitable additional compounds or WO 00/23415, WO 00/23451, WO 00/23445, WO 00/23417, Substances may be selected, for example, from antidiabetic WO 00/23416, WO 00/63153, WO 00/63196, WO 00/63209, agents, antihyperlipidemic agents, antiobesity agents, antihy WO 00/63190 and WO 00/63189 (Novo Nordisk A/S), the pertensive agents and agents for the treatment of complica contents of all of which are incorporated herein by reference. tions resulting from, or associated with, diabetes. 0229. Still further examples of suitable additional thera 0224 Suitable antidiabetic agents include insulin, insulin peutically active Substances include: C-glucosidase inhibi derivatives or analogues, GLP-1 (glucagon like peptide-1) tors, e.g. Voglibose, emiglitate, miglitol or acarbose; glyco derivatives or analogues such as those disclosed in WO gen phosphorylase inhibitors, e.g. the compounds described 98/08871 (Novo Nordisk A/S), which is incorporated herein in WO97/09040 (Novo NordiskNS); glucokinase activators: by reference, or other GLP-1 analogues such as exenatide agents acting on the ATP-dependent potassium channel of the (Byetta, Eli Lilly/Amylin; AVE0010, Sanofi-Aventis), taspo pancreatic B-cells, e.g. tolbutamide, glibenclamide, glipizide, glutide (Roche), albiglutide (Syncria, GlaxoSmithKline), glicazide, BTS-67582 or repaglinide; amylin, amylin analogues (e.g. SymlinTM/Pramlintide) as 0230. Other suitable additional therapeutically active sub well as orally active hypoglycemic agents. stances include antihyperlipidemic agents and antilipidemic 0225 Suitable orally active hypoglycemic agents include: agents, e.g. cholestyramine, colestipol, clofibrate, gemfi metformin, imidazolines; Sulfonylureas; biguanides; megli brozil, lovastatin, pravastatin, simvastatin, probucol or dex tinides; oxadiazolidinediones; thiazolidinediones; insulin trothyroxine. sensitizers; C-glucosidase inhibitors; agents acting on the 0231. Further agents which are suitable as additional ATP-dependent potassium channel of the pancreatic B-cells, therapeutically active substances include antiobesity agents e.g. potassium channel openerS Such as those disclosed in WO and appetite-regulating agents. Such Substances may be 97/26265, WO 99/03861 and WO 00/37474 (Novo Nordisk selected from the group consisting of CART (cocaine A/S) which are incorporated herein by reference; potassium amphetamine regulated transcript) agonists, NPY (neuropep channel openers such as ormitiglinide; potassium channel tide Y receptor 1 and/or 5) antagonists, MC3 (melanocortin blockers such as nateglinide or BTS-67582: glucagon recep receptor 3) agonists, MC3 antagonists, MC4 (melanocortin tor antagonists such as those disclosed in WO99/01423 and receptor 4) agonists, orexin receptor antagonists, TNF (tumor WO 00/39088 (Novo Nordisk A/S and Agouron Pharmaceu necrosis factor) agonists, CRF (corticotropin releasing fac ticals, Inc.), all of which are incorporated herein by reference; tor) agonists, CRFBP (corticotropin releasing factor binding GLP-1 receptor agonists such as those disclosed in WO protein) antagonists, urocortin agonists, neuromedin U ana 00/42026 (Novo Nordisk A/S and Agouron Pharmaceuticals, logues (agonists on the neuromedin U receptor Subtypes 1 Inc.), which are incorporated herein by reference; amylin and 2), B3 adrenergic agonists such as CL-316243, AJ-9677, analogues (agonists on the amylin receptor); DPP-IV (dipep GW-0604, LY362884, LY377267 or AZ-40140, MC1 (mel tidyl peptidase-IV) inhibitors; PTPase (protein tyrosine phos anocortin receptor 1) agonists, MCH (melanocyte-concen phatase) inhibitors; glucokinase activators, such as those trating hormone) antagonists, CCK (cholecystokinin) ago described in WO 02/08209 to Hoffmann LaRoche: inhibitors nists, serotonin reuptake inhibitors (e.g. fluoxetine, seroXator of hepatic enzymes involved in stimulation of gluconeogen citalopram), serotonin and norepinephrine reuptake inhibi esis and/or glycogenolysis; glucose uptake modulators; tors, 5HT (serotonin) agonists, 5HT6 agonists, 5HT2c ago GSK-3 (glycogen synthase kinase-3) inhibitors; compounds nists such as APD356 (U.S. Pat. No. 6,953,787), bombesin modifying lipid metabolism, such as antihyperlipidemic agonists, galanin antagonists, growth hormone, growth fac agents and antilipidemic agents; compounds lowering food tors such as prolactin or placental lactogen, growth hormone US 2016/0002311 A1 Jan. 7, 2016 22 releasing compounds, TRH (thyrotropin releasing hormone) 0239. The term “polypeptide' and “peptide' as used agonists, UCP2 or 3 (uncoupling protein 2 or 3) modulators, herein means a compound composed of at least five constitu chemical uncouplers, leptin agonists, DA (dopamine) ago ent amino acids connected by peptide bonds. The constituent nists (bromocriptin, doprexin), lipase/amylase inhibitors, amino acids may be from the group of the amino acids PPAR modulators, RXR modulators, TR Bagonists, adrener encoded by the genetic code and they may be natural amino gic CNS Stimulating agents, AGRP (agouti-related protein) acids which are not encoded by the genetic code, as well as inhibitors, histamine H3 receptor antagonists such as those synthetic amino acids. Natural amino acids which are not disclosed in WOOO/42023, WOOO/63208 and WOOO/64884, encoded by the genetic code are e.g. hydroxyproline, Y-car the contents of all of which are incorporated herein by refer boxyglutamate, ornithine, phosphoserine, D-alanine and ence, exendin-4 analogues, GLP-1 analogues, ciliary neu D-glutamine. Synthetic amino acids comprise amino acids rotrophic factor, amylin analogues, peptide YY. (PYY3 manufactured by chemical synthesis, i.e. D-isomers of the 36) (Batterham etal, Nature 418, 650-654 (2002)), PYY3-36 amino acids encoded by the genetic code such as D-alanine analogues, NPYY2 receptoragonists, NPYY4 receptorago and D-leucine, Aib (CL-aminoisobutyric acid), Abu (CL-ami nists and substances acting as combined NPYY2 and NPYY4 nobutyric acid), Tle (tert-butylglycine), B-alanine, 3-ami agonists, FGF21 and analogues thereof, u-opioid receptor nomethylbenzoic acid, anthranilic acid. antagonists, oxyntomodulin or analogues thereof. 0240. The term “analogue' as used herein referring to a 0232 Further suitable antiobesity agents are bupropion polypeptide means a modified peptide wherein one or more (antidepressant), topiramate (anticonvulsant), ecopipam amino acid residues of the peptide have been substituted by (dopamine D1/D5 antagonist) and naltrexone (opioidantago other amino acid residues and/or wherein one or more amino nist), and combinations thereof. Combinations of these anti acid residues have been deleted from the peptide and/or obesity agents would be e.g.: phentermine+topiramate, wherein one or more amino acid residues have been deleted bupropion sustained release (SR)+naltrexone SR, Zonisa from the peptide and or wherein one or more amino acid mide SR and bupropion SR. Among embodiments of suitable residues have been added to the peptide. Such addition or antiobesity agents for use in a method of the invention as deletion of amino acid residues can take place at the N-ter additional therapeutically active Substances in combination minal of the peptide and/or at the C-terminal of the peptide. A with a compound of the invention are leptin and analogues or simple system is used to describe analogues. Formulae of derivatives of leptin. peptide analogs and derivatives thereofare drawn using stan 0233. Additional embodiments of suitable antiobesity dard single letter or three letter abbreviations for amino acids agents are serotonin and norepinephrine reuptake inhibitors, used according to IUPAC-IUB nomenclature. e.g. Sibutramine. 0241 The term "derivative' as used herein in relation to a 0234. Other embodiments of suitable antiobesity agents peptide means a chemically modified peptide oran analogue are lipase inhibitors, e.g. orlistat. thereof, wherein at least one substituent is not present in the 0235 Still further embodiments of suitable antiobesity unmodified peptide or an analogue thereof, i.e. a peptide agents are adrenergic CNS stimulating agents, e.g. dexam which has been covalently modified. Typical modifications phetamine, amphetamine, phentermine, mazindol, phen are amides, carbohydrates, alkyl groups, acyl groups, esters dimetrazine, diethylpropion, fenfluramine or dexfenflu and the like. ramine. 0242 All amino acids for which the optical isomer is not stated is to be understood to mean the L-isomer. 0236. Other examples of suitable additional therapeuti cally active compounds include antihypertensive agents. 0243 The term “distal as used herein, means most remote Examples of antihypertensive agents are n-blockers such as (terminal) from the point of attachment. alprenolol, atenolol, timolol, pindolol, propranolol and meto 0244. The term “negative charged moiety” as used herein, prolol, ACE (angiotensin converting enzyme) inhibitors such means a negatively chargeable chemical moiety such as, but as benazepril, captopril, enalapril, fosinopril, lisinopril, not limited to a carboxylic acid, Sulphonic acid or a tetrazole quinapril and ramipril, calcium channel blockers such as nife moiety. dipine, felodipine, nicardipine, isradipine, nimodipine, dilt 0245. The term “lipophilic moiety” as used herein, means iaZemand Verapamil, and C.-blockers such as doxazosin, ura an alkyl chain —(CH)n- where n=5-20. pidil, praZosin and teraZosin. 0246 The term “substituent as used herein, means a 0237. The compounds of the present invention have higher chemical moiety or group replacing a hydrogen. glucagon receptor selectivity in relation to previously dis 0247 The term “glucagon peptide' as used herein means closed peptides in the art. The peptides of the present inven glucagon peptide, glucagon compound, compound according tion also have prolonged in vivo half-life. The compounds of to the present invention, compound of the present invention, the present invention can be a soluble glucagon receptor compound of formula I, a glucagon analogue, a glucagon agonist, for example with solubility of at least 0.2 mmol/l, at derivative or a derivative of a glucagon analogue human glu least 0.5 mmol/l, at least 2 mmol/l, at least 4 mmol/l, at least cagon, human glucagon(1-29), glucagon(1-30), glucagon(1- 8 mmol/l, at least 10 mmol/l, or at least 15 mmol/l. 31), glucagon(1-32) as well as analogues, fusion peptides, 0238. In the present context, if not stated otherwise, the and derivatives thereof, which maintain glucagon activity. terms “soluble”, “solubility”, “soluble in aquous solution', 0248 AS regards position numbering in glucagon com “aqueous solubility”, “water soluble”, “water-soluble'. pounds: for the present purposes any amino acid substitution, “water solubility” and “water-solubility”, refer to the solubil deletion, and/or addition is indicated relative to the sequences ity of a compound in water or in an aqueous salt or aqueous of native human glucagon (1-29) (SEQID1). Human gluca buffer solution, for example a 10 mM phosphate solution, or gon amino acids positions 1-29 are herein to be the same as in an aqueous Solution containing other compounds, but no amino acid positions X to X. The human glucagon (1-29) organic solvents. sequence is His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser US 2016/0002311 A1 Jan. 7, 2016
Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln chemically modified in order to render said compound resis Trp-Leu-Met-Asn-Thr (SEQID 1). tant to the plasma peptidase dipeptidyl aminopeptidase-4 0249 Glucagon(1-30) means human glucagon with an (DPP-IV). The DPP-IV enzyme in plasma is known to be extension of one amino acid in the C-terminal, glucagon(1- involved in the degradation of several peptide hormones, e.g. 31) means human glucagon with an extension of two amino glucagon, GLP-1, GLP-2, oxyntomodulin etc. Thus, a con acid in the C-terminal and glucagon(1-32) means human siderable effort is being made to develop analogues and glucagon with an extension of three amino acid in the C-ter derivatives of the polypeptides susceptible to DPP-IV medi minal. ated hydrolysis in order to reduce the rate of degradation by 0250. In embodiments of the invention a maximum of 17 DPP-IV. amino acids in the glucagon analogue have been modified 0263. Furthermore, the compounds of the present inven (substituted, deleted, added or any combination thereof) rela tion are stabilized against DPP-IV cleavage in an albumin free tive to human glucagon (1-29). In embodiments of the inven assay as described in Assay VI. tion a maximum of 15 amino acids in the glucagon analogue 0264. The term “glucagonagonist’ as used herein refers to have been modified. In embodiments of the invention a maxi any glucagon peptide which fully or partially activates the mum of 10 amino acids in the glucagon analogue have been human glucagon receptor. In another embodiment, the “glu modified. In embodiments of the invention a maximum of 8 cagon agonist' is any glucagon peptide that binds to a gluca amino acids in the glucagon analogue have been modified. In gon receptor, preferably with an affinity constant (KD) or a embodiments of the invention a maximum of 7 amino acids in potency (ECs) of below 1 uM, e.g., below 100 nM or below the glucagon analogue have been modified. In embodiments 1 nM, as measured by methods known in the art and exhibits of the invention a maximum of 6 amino acids in the glucagon insulinotropic activity, where insulinotropic activity may be analogue have been modified. In embodiments of the inven measured in vivo or in vitro assays known to those of ordinary tion a maximum of 5 amino acids in the glucagon analogue skill in the art. For example, the glucagon agonist may be have been modified. In embodiments of the invention a maxi administered to an animal and the insulin concentration mea mum of 4 amino acids in the glucagon analogue have been sured over time. modified. In embodiments of the invention a maximum of 3 0265. In the present context, the term “agonist' is intended amino acids in the glucagon analogue have been modified. In to indicate a Substance (ligand) that activates the receptor type embodiments of the invention a maximum of 2 amino acids in in question. the glucagon analogue have been modified. In embodiments 0266. In the present context, the term “antagonist' is of the invention 1 amino acid in the glucagon analogue has intended to indicate a substance (ligand) that blocks, neutral been modified. izes or counteracts the effect of an agonist. 0251 Further embodiments of the present invention relate 0267 More specifically, receptor ligands may be classi tO: fied as follows: 0252) 145. A glucagon peptide according to any of the 0268 Receptor agonists, which activate the receptor; par previous embodiments, wherein said glucagon peptide is a tial agonists also activate the receptor, but with lower efficacy DPPIV protected compound. than full agonists. A partial agonist will behave as a receptor 0253) 146. A glucagon peptide according to any of the partial antagonist, partially inhibiting the effect of a full ago previous embodiments, wherein said glucagon peptide is nist. DPPIV Stabilised. 0269. Receptor neutral antagonists, which block the 0254. 147. A glucagon peptide according to any of the action of an agonist, but do not affect the receptor-constitutive previous embodiments, wherein said glucagon peptide is an activity. agonist of the glucagon receptor. 0270. Receptor inverseagonists, which block the action of 0255 148. A glucagon peptide according to any of the an agonist and at the same time attenuate the receptor-consti previous embodiments, wherein said glucagon peptide is an tutive activity. A full inverse agonist will attenuate the recep agonist of the glucagon receptor, with an ECso<1 nM. tor-constitutive activity completely; a partial inverse agonist 0256 149. A glucagon peptide according to any of the will attenuate the receptor-constitutive activity to a lesser previous embodiments, wherein said glucagon peptide has eXtent. more than 70% recovery in the ThT fibrillation assay. 0271 As used herein the term “antagonist' includes neu 0257 150. A glucagon peptide according to any of the tral antagonists and partial antagonists, as well as inverse previous embodiments, wherein said glucagon peptide has agonists. The term "agonist includes full agonists as well as more than 90% recovery in the ThT fibrillation assay. partial agonists. 0258 151. A glucagon peptide according to any of the 0272. In the present context, the term “pharmaceutically previous embodiments, wherein said glucagon peptide has acceptable salt' is intended to indicate a salt which is not about 100% recovery in the ThT fibrillation assay. harmful to the patient. Such salts include pharmaceutically 0259 152. A glucagon peptide according to any of the acceptable acid addition salts, pharmaceutically acceptable previous embodiments, wherein said glucagon peptide has metal salts, ammonium and alkylated ammonium salts. Acid more than 7 hours lag time in the ThT fibrillation assay. addition salts include salts of inorganic acids as well as 0260 153. A glucagon peptide according to any of the organic acids. Representative examples of Suitable inorganic previous embodiments, wherein said glucagon peptide has acids include hydrochloric, hydrobromic, hydroiodic, phos more than 20 hours lag time in the ThT fibrillation assay. phoric, Sulfuric and nitric acids, and the like. Representative 0261) 154. A glucagon peptide according to any of the examples of Suitable organic acids include formic, acetic, previous embodiments, wherein said glucagon peptide has 45 trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, hours lag time or more in the ThT fibrillation assay. citric, fumaric, glycolic, lactic, maleic, malic, malonic, man 0262 The term “DPP-IV protected” as used herein refer delic, oxalic, picric, pyruvic, Salicylic, Succinic, methane ring to a polypeptide means a polypeptide which has been Sulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismeth US 2016/0002311 A1 Jan. 7, 2016 24 ylene-Salicylic, ethanedisulfonic, gluconic, citraconic, wherein aspartic, Stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids and the Z1 may be a lipophillic hydrocarbon chain with a negatively like. Further examples of pharmaceutically acceptable inor charged group Such as a carboxylic acid or a 5-yl tetrazole in ganic or organic acid addition salts include the pharmaceuti the terminus, cally acceptable salts listed in J. Pharm. Sci. (1977) 66, 2. Z and Z may comprise one or more moieties of gamma which is incorporated herein by reference. Examples of rel glutamic acid or glutamic acid and, evant metal salts include lithium, Sodium, potassium and magnesium salts, and the like. Examples of alkylated ammo Z may comprise one or more units of Ado. An example of an nium salts include methylammonium, dimethylammonium, Substituent of the present invention, in which moiety Z is trimethylammonium, ethylammonium, hydroxyethylammo absent, may be:
Z O HO H N O O "N-N-----~~~~ O O O nium, diethylammonium, butylammonium and tetramethy Where the symbol * indicates attachment point to the peptide. lammonium salts, and the like. 0277. The substituent may be attached via the epsilon 0273. As use herein, the term “therapeutically effective position of alysine or via the delta position of an ornithine and amount of a compound refers to an amount Sufficient to cure, can reside on one or more of the following positions of glu alleviate or partially arrest the clinical manifestations of a cagon peptide: Xio. X2, X-20X24, X2s, X27,X2s. X29, and/or given disease and/or its complications. An amount adequate Xso to accomplish this is defined as a “therapeutically effective (0278. Further embodiment of the present invention relate amount. Effective amounts for each purpose will depend on to a substituent: the severity of the disease or injury, as well as on the weight 0279 155. A substituent with the formula II: and general state of the subject. It will be understood that determination of an appropriate dosage may be achieved Z-Z2-Z-Z II) using routine experimentation, by constructing a matrix of wherein, values and testing different points in the matrix, all of which Z represents a structure according to one of the formulas IIa, is within the level of ordinary skill of a trained physician or IIb or IIc; Veterinarian. 0274 The terms “treatment”, “treating and other variants thereofas used herein refer to the management and care of a IIa patient for the purpose of combating a condition, Such as a disease or a disorder. The terms are intended to include the full spectrum of treatments for a given condition from which --- : the patient is suffering, such as administration of the active IIb compound(s) in question to alleviate symptoms or complica -N O tions thereof, to delay the progression of the disease, disorder y or condition, to cure or eliminate the disease, disorder or ( N ) : condition, and/or to prevent the condition, in that prevention H is to be understood as the management and care of a patient IIc for the purpose of combating the disease, condition, or disor OH O der, and includes the administration of the active compound (s) in question to prevent the onset of symptoms or compli cations. The patient to be treated is preferably a mammal, in particular a human being, but treatment of other animals, such as dogs, cats, cows, horses, sheep, goats or pigs, is within the Scope of the invention. wherein n in formula IIa is 6-20, 0275. As used herein, the term “solvate” refers to a com m in formula IIc is 5-11, plex of defined stoichiometry formed between a solute (in the COOH group in formula IIc can reside on position 2, 3 or casu, a compound according to the present invention) and a 4 on the phenyl ring, Solvent. Solvents may include, by way of example, water, the symbol * in formula IIa, IIb and IIc represents the attach ethanol, or acetic acid. ment point to the nitrogen in Z; 0276. The present invention also relates to substituents, if Z is absent, Z is attached to the nitrogen on Z at symbol which may have the general formula II: * and if Z and Z are absent Z is attached to the nitrogen on Zaat symbol *, US 2016/0002311 A1 Jan. 7, 2016 25
Z2 is absent or represents a structure according to one of the Z is connected Vi the carbon of Z with symbol * to the formulas IId, IIe, IIf IIg, IIh, II or IIk; nitrogen of Z with symbol, if Z is absent Z is connected via the carbon with symbol * to the epsilon nitrogen of a lysine or the delta nitrogen of an ornithine of the glucagon IId peptide; OH Z is absent or represents a structure according to one of the formulas IId, IIe, IIf IIg, IIh, II or IIk; wherein each amino acid moiety is independently either L or D, wherein Z is N connected via the carbon with symbol * to the epsilon nitro O gen of a lysine or the delta nitrogen of an ornithine of the IIe glucagon peptide. O OH 0280 156. A substituent according to embodiment 155, O wherein : n N : Z represents a structure according to one of the formulas IIa, H IIb or IIc; O O OH IIf IIa O O O OH O OH O ---. IIb N N H H O O O 7 O OH N. N > --~~~~ : H wherein each amino acid has the stereochemistry L or D; wherein Z is connected via the carbonatom denoted * to the nitrogen of Z denoted *; if Z is absent, Z is connected via the carbon atom denoted * to the nitrogen of Z denoted * and if Z and Z are absent Z. HO is connected via the carbon denoted * to the epsilon nitrogen ofalysine or the delta nitrogenofan ornithine of the glucagon peptide; Z is absent or represents a structure according to one of the formulas IIm, In, IIo or IIp;
Im. -N-----.O
IIn -N------.O O
IIo -N-~~~~.O O O IIp O O O -N-no-n- N------> N~---.O US 2016/0002311 A1 Jan. 7, 2016 26 wherein n in formula IIa is 6-20, -continued II. Z2 is absent or represents a structure according to one of the O OH formulas IId, IIe, IIf IIg, IIh, II or IIk;
O IId O OH : H N O IIe O OH O OH II O O OH
: NN : H O O H H O OH -N N IIf : N : H O OH O OH O O
: : NN N H H O OH O OH O O IIk O OH O OH O O H IIg O OH O OH H O H O *N N N O O N N : H H H H N N : O O 1 N N O OH O OH H H IIh O O O
1 : O OH O OH
wherein each amino acid moiety is independently either L or D. O OH Z is absent or represents a structure according to one of the formulas IIm, In, IIo or IIp;
Im. -N-N-N-N-N.O IIn -N-N-N-N------.O O IIo -N-~~~~.O O O IIp O O O -N-no-n- N-~~~~ US 2016/0002311 A1 Jan. 7, 2016 27
-continued O
Z is absent or represents a structure according to one of the formulas IId, IIe, IIf IIg, IIh, II or IIk; wherein each amino acid moiety is independently either L or D. 0281 157. A substituent according to any one of embodi ments 155-156, wherein Z is absent when Z is present. 0282 158. A substituent according to any one of embodi ments 155-157, wherein Z is absent when Z2 is present. 0283 159. A substituent according to any one of embodi ments 155-158, which is selected from the structures accord ing to one of the formulas IIIa, IIIb, IIIc, IIId, IIIe, III for IIIg: US 2016/0002311 A1 Jan. 7, 2016 28
B.III QIII 3.III ÞIII 3.III
O s X"( O Z, - O s Z, O 3.
Z O- S - & &
-,O & s O Z s X"K. O
O S. S.
US 2016/0002311 A1 Jan. 7, 2016 30
0284. 160. A substituent according to any one of embodi ments 78-80, which represents a structure of formula IIIa:
IIIa O 's--" H HO N N N-1 no-1a H O ~ O O O st-'" N-N-----~~ r-r : O O O OH
0285 161. A substituent according to any one of embodi ments 155-160, wherein Z is absent. 0286 162. A substituent according to any one of embodi ments 155-161, wherein Z and Z are absent. 0287. 163. A substituent according to any one of embodi ments 155-162, selected from a structure according to one of the formulas Iva, IVb, IVc or IVd:
IV a O OH O s1 H O
for--~~~~ H N-o~---~~~H O O O IVb O OH O O HO ouls NH -n. N-1 no-1N1 8.s O O IVc O OH o S-1 O HO r-r n-1N1\-1 ous O O o O Y OH N1a- n-1No N s H H O O O OH IVd O O OH O HO N N N O H
O OH O OH O N1 H N 8. NH ~ O O O OH US 2016/0002311 A1 Jan. 7, 2016
0288 The term “albumin binding residue' as used herein 0291 165. A pharmaceutical composition according to means a residue which binds non-covalently to human serum embodiment 164, further comprising one or more additional albumin. The albumin binding residue attached to the thera therapeutically active compounds or Substances. peutic polypeptide typically has an affinity below 10 uM to human serum albuminand preferably below 1 uM. A range of 0292 166. A pharmaceutical composition according to albumin binding residues are known among linear and any one of embodiments 164-165, further comprising a branched lipohophillic moieties containing 4-40 carbon GLP-1 compound. atOmS. 0293 167. A pharmaceutical composition according to 0289. Other embodiments of the present relates to phar maceutical compositions: any one of embodiments 164-166, wherein the GLP-1 com 0290) 164. A pharmaceutical composition comprising a pound is selected from the group consisting of: glucagon peptide according to any one of embodiments 0294 N-epsilon26-(S)-4-Carboxy-4-hexadecanoy 1-154. lamino-butyryl)Arg34GLP-1-(7-37): (compound G1) O H N n-n-n-n-n-n-n- NH O O OH
OH: H-H AE GT FTSD vs SYLE GQAA-N EFAwl v GR-1) O O
0295 N-epsilon37-2-(2-2-[2-(2-2-(S)-4-Carboxy-4- (trans-4-(19-carboxynonadecanoylamino)methyl cyclohexanecarbonyl)amino)butyrylaminol ethoxyethoxy) acetylaminolethoxyethoxy)acetyl DesaminoHis7.Glu22. Arg26. Arg34.Lys37IGLP-1-(7- 37): (compound G2) O H O O H O H c ^-o-o-n Y n1orn. Hon-n-n-n-n-n-n-n-n-naO O1 OH H O O 1. HNr.s
O A E G TFTSD V SS YLE EQA A RE FIA WLV RGR-N O OH:
0296 N-epsilon26-2-(2-2-[2-(2-2-(S)-4-Carboxy-4- (17-carboxyheptadecanoylamino)butyrylamino ethoxyethoxy)acetylaminolethoxyethoxy)acetyl Aib8. Arg34GLP-1-(7-37):
(compound G3) O O O H-H-Nu-EH GT FTSD V SS YLE G QAA-NJ-EH FIA W L V RGR-N-OH:H O Ho----~~~~ NH H O l O Hon-NS-or-o-ra-o-o-NH O O H O US 2016/0002311 A1 Jan. 7, 2016 32
0297 N-epsilon37-2-(2-2-[2-(2-2-(S)-4-carboxy-4- (15-carboxy-pentadecanoylamino)-butyrylamino ethoxy-ethoxy)-acetylamino-ethoxy-ethoxy)-acetyl Aib8,22,35,Lys37GLP-1-(7-37):
(compound G4) O OH O is- N -n. N- O ~s N 1nu-On-1a or? O; O NH HN1\ N S. O O OH HN NH E GT FTSDWSSYLE-NH QA A K E FIA W L V K-N R-N O O O and their pharmaceutically acceptable salts, amides, alkyls, or esterS. 0298 168. A pharmaceutical composition according to embodiments 164-167, further comprising an insulinic com pound. 0299) 169. A pharmaceutical composition according to embodiment 168, wherein the insulin compound is: 0300 NeB29-hexadecandiyol-y-Glu-(desB30) human insulin
(compound G5)
O
N O — HG IV EQ C CT S I C S LY QLE NYC NOH S / S H-F V N Q H LCGS H L V E A LY L V C G E R G F FY TP-N O O
0301 170. The pharmaceutical composition according to 0304 Further embodiments of the present invention relate any one of embodiments 164-169, in unit dosage form com to the following: prising from about 0.05 mg to about 1000 mg, such as from 0305 173. A glucagon peptide according to any of about 0.1 mg to about 500 mg, from about 2 mg to about 5 mg. embodiments 1-154154, optionally in combination with one e.g. from about 0.5 mg to about 200 mg. ofaglucagon peptide or more additional therapeutically active compounds, for use according to any of embodiments 1-154 in treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes and obesity 0302) 171. The pharmaceutical composition according to 0306 174. A glucagon peptide according to any of any one of embodiments 164-170, which is suited for embodiments 1-154154, optionally in combination with one parenteral administration. or more additional therapeutically active compounds, for use 0303 172. A glucagon peptide according to any of any one in delaying or preventing disease progression in type 2 dia of embodiments 1-154, for use in therapy. betes. US 2016/0002311 A1 Jan. 7, 2016
0307 175. A glucagon peptide according to any of 0322, 190. A glucagon peptide according to any of embodiments 1-154154, optionally in combination with one embodiments 1-154, optionally in combination with one or or more additional therapeutically active compounds, for use more additional therapeutically active glucagon peptides, for treating obesity or preventing overweight. use in treating impaired glucose tolerance. 0308 176. A glucagon peptide according to any of 0323, 191. A glucagon peptide according to any of embodiments 1-154, optionally in combination with one or embodiments 1-154, optionally in combination with one or more additional therapeutically active compounds, for use in more additional therapeutically active glucagon peptides, for for decreasing food intake. use in treating dyslipidemia. 0309 177. A glucagon peptide according to any of 0324 192. A glucagon peptide according to any of embodiments 1-154, optionally in combination with one or embodiments 1-154, optionally in combination with one or more additional therapeutically active glucagon peptides, for more additional therapeutically active glucagon peptides, for use in increasing energy expenditure. use in treating coronary heart disease. 0310) 178. A glucagon peptide according to any of 0325 193. A glucagon peptide according to any of embodiments 1-154, optionally in combination with one or embodiments 1-154, optionally in combination with one or more additional therapeutically active glucagon peptides, for more additional therapeutically active glucagon peptides, for use in reducing body weight. use in treating hepatic steatosis. 0311 179. A glucagon peptide according to any of 0326 194. A glucagon peptide according to any of embodiments 1-154, optionally in combination with one or embodiments 1-154, optionally in combination with one or more additional therapeutically active glucagon peptides, for more additional therapeutically active glucagon peptides, for use in delaying the progression from impaired glucose toler use in treating hepatic steatosis. ance (IGT) to type 2 diabetes. 0327 195. A glucagon peptide according to any of 0312 180. A glucagon peptide according to any of embodiments 1-154, optionally in combination with one or embodiments 1-154, optionally in combination with one or more additional therapeutically active glucagon peptides, for more additional therapeutically active glucagon peptides, for use in treating beta-blocker poisoning. use in delaying the progression from type 2 diabetes to insu 0328 196. A glucagon peptide according to any of lin-requiring diabetes. embodiments 1-154, optionally in combination with one or 0313 181. A glucagon peptide according to any of more additional therapeutically active glucagon peptides, for embodiments 1-154, optionally in combination with one or use in inhibition of the motility of the gastrointestinal tract, more additional therapeutically active glucagon peptides, for useful in connection with investigations of the gastrointesti nal tract using techniques such as X-ray, CT- and NMR use regulating appetite. Scanning. 0314 182. A glucagon peptide according to any of 0329 197. A glucagon peptide according to any of embodiments 1-154, optionally in combination with one or embodiments 1-154, optionally in combination with one or more additional therapeutically active glucagon peptides, for more additional therapeutically active glucagon peptides, for use inducing Satiety. use in treatment or prevention of hypoglycaemia. 0315, 183. A glucagon peptide according to any of 0330 198. A glucagon peptide according to any of embodiments 1-154, optionally in combination with one or embodiments 1-154, optionally in combination with one or more additional therapeutically active glucagon peptides, for more additional therapeutically active glucagon peptides, for use in preventing weight regain after Successful weight loss. use in treatment or prevention of insulin induced hypoglycae 0316 184. A glucagon peptide according to any of 18. embodiments 1-154, optionally in combination with one or 0331) 199. A glucagon peptide according to any of more additional therapeutically active glucagon peptides, for embodiments 1-154, optionally in combination with one or use in treating a disease or state related to overweight or more additional therapeutically active glucagon peptides, for obesity. use in treatment or prevention of reactive hypoglycaemia. 0317 185. A glucagon peptide according to any of 0332 200. A glucagon peptide according to any of embodiments 1-154, optionally in combination with one or embodiments 1-154, optionally in combination with one or more additional therapeutically active glucagon peptides, for more additional therapeutically active glucagon peptides, for use in treating bulimia. use in treatment or prevention of diabetic hypoglycaemia. 0318, 186. A glucagon peptide according to any of 0333 201. A glucagon peptide according to any of embodiments 1-154, optionally in combination with one or embodiments 1-154, optionally in combination with one or more additional therapeutically active glucagon peptides, for more additional therapeutically active glucagon peptides, for use in treating binge-eating. use in treatment or prevention of non-diabetic hypoglycae 0319 187. A glucagon peptide according to any of 18. embodiments 1-154, optionally in combination with one or 0334 202. A glucagon peptide according to any of more additional therapeutically active glucagon peptides, for embodiments 1-154, optionally in combination with one or use in treating atherosclerosis. more additional therapeutically active glucagon peptides, for 0320 188. A glucagon peptide according to any of use in treatment or prevention of fasting hypoglycaemia. embodiments 1-154, optionally in combination with one or 0335 203. A glucagon peptide according to any of more additional therapeutically active glucagon peptides, for embodiments 1-154, optionally in combination with one or use in treating hypertension. more additional therapeutically active glucagon peptides, for 0321 189. A glucagon peptide according to any of use in treatment or prevention of drug-induced hypoglycae embodiments 1-154, optionally in combination with one or 18. more additional therapeutically active glucagon peptides, for 0336 204. A glucagon peptide according to any of use in treating type 2 diabetes. embodiments 1-154, optionally in combination with one or US 2016/0002311 A1 Jan. 7, 2016 34 more additional therapeutically active glucagon peptides, for amount of a glucagon peptide according to any of embodi use in treatment or prevention of gastric by-pass induced ments 1-154, optionally in combination with one or more hypoglycaemia. additional therapeutically active compounds. 0337 205. A glucagon peptide according to any of 0350 217. A method for use in regulating appetite, com embodiments 1-154, optionally in combination with one or prising administering to a patient in need thereof, an effective more additional therapeutically active glucagon peptides, for amount of a glucagon peptide according to any of embodi use in treatment or prevention of hypoglycemia in pregnancy. ments 1-154, optionally in combination with one or more 0338 206. A glucagon peptide according to any of additional therapeutically active compounds. embodiments 1-154, optionally in combination with one or 0351. 218. A method for use in inducing satiety, compris more additional therapeutically active glucagon peptides, for ing administering to a patient in need thereof, an effective use in treatment or prevention of alcohol-induced hypogly amount of a glucagon peptide according to any of embodi caemia. ments 1-154, optionally in combination with one or more 0339 207. A glucagon peptide according to any of additional therapeutically active compounds. embodiments 1-154, optionally in combination with one or 0352. 219. A method for use in preventing weight regain more additional therapeutically active glucagon peptides, for after Successful weight loss, comprising administering to a use in treatment or prevention of insulinoma. patient in need thereof, an effective amount of a glucagon 0340 208. A glucagon peptide according to any of peptide according to any of embodiments 1-154, optionally in embodiments 1-154, optionally in combination with one or combination with one or more additional therapeutically more additional therapeutically active glucagon peptides, for active compounds. use in treatment or prevention of Von Girkes disease. 0353 220. A method for use in treating a disease or state 0341 Further embodiments of the present invention relate related to overweight or obesity, comprising administering to to the following methods: a patient in need thereof, an effective amount of a glucagon 0342. 209. A method for treating or preventing hypergly peptide according to any of embodiments 1-154, optionally in cemia, type 2 diabetes, impaired glucose tolerance, type 1 combination with one or more additional therapeutically diabetes and obesity, comprising administering to a patient in active compounds. need thereof, an effective amount of a glucagon peptide 0354) 220a). A method for use in treating bulimia, com according to any of embodiments 1-154, optionally in com prising administering to a patient in need thereof, an effective bination with one or more additional therapeutically active amount of a glucagon peptide according to any of embodi compounds. ments 1-154, optionally in combination with one or more 0343 210. A method for delaying or preventing disease additional therapeutically active compounds. progression in type 2 diabetes, comprising administering to a 0355 221. A method for use intreating binge-eating, com patient in need thereof, an effective amount of a glucagon prising administering to a patient in need thereof, an effective peptide according to any of embodiments 1-154, optionally in amount of a glucagon peptide according to any of embodi combination with one or more additional therapeutically ments 1-154, optionally in combination with one or more active compounds. additional therapeutically active compounds. 0344) 211. A method for treating obesity or preventing overweight, comprising administering to a patient in need 0356. 222. A method for use in treating atherosclerosis, thereof, an effective amount of a glucagon peptide according comprising administering to a patient in need thereof, an to any of embodiments 1-154, optionally in combination with effective amount of a glucagon peptide according to any of one or more additional therapeutically active compounds. embodiments 1-154, optionally in combination with one or 0345 212. A method for decreasing food intake, compris more additional therapeutically active compounds. ing administering to a patient in need thereof, an effective 0357 223. A method for use in treating hypertension, amount of a glucagon peptide according to any of embodi comprising administering to a patient in need thereof, an ments 1-154, optionally in combination with one or more effective amount of a glucagon peptide according to any of additional therapeutically active compounds. embodiments 1-154, optionally in combination with one or 0346 213. A method for use in increasing energy expen more additional therapeutically active compounds. diture, comprising administering to a patient in need thereof, 0358 224. A method for use in treating type 2 diabetes, an effective amount of a glucagon peptide according to any of comprising administering to a patient in need thereof, an embodiments 1-154, optionally in combination with one or effective amount of a glucagon peptide according to any of more additional therapeutically active compounds. embodiments 1-154, optionally in combination with one or 0347 214. A method for use in reducing body weight, more additional therapeutically active compounds. comprising administering to a patient in need thereof, an 0359 225. A method for use in treating impaired glucose effective amount of a glucagon peptide according to any of tolerance, comprising administering to a patient in need embodiments 1-154, optionally in combination with one or thereof, an effective amount of a glucagon peptide according more additional therapeutically active compounds. to any of embodiments 1-154, optionally in combination with 0348 215. A method for use in delaying the progression one or more additional therapeutically active compounds. from impaired glucose tolerance (IGT) to type 2 diabetes, 0360 226. A method for use in treating dyslipidemia, comprising administering to a patient in need thereof, an comprising administering to a patient in need thereof, an effective amount of a compound according to any of embodi effective amount of a glucagon peptide according to any of ments 1-154, optionally in combination with one or more embodiments 1-154, optionally in combination with one or additional therapeutically active compounds. more additional therapeutically active compounds. 0349. 216. A method for use in delaying the progression 0361) 227. A method for use in treating coronary heart from type 2 diabetes to insulin-requiring diabetes, compris disease, comprising administering to a patient in need ing administering to a patient in need thereof, an effective thereof, an effective amount of a glucagon peptide according US 2016/0002311 A1 Jan. 7, 2016
to any of embodiments 1-154, optionally in combination with istering to a patient in need thereof, an effective amount of a one or more additional therapeutically active compounds. glucagon peptide according to any of embodiments 1-154, 0362 228. A method for use in treating hepatic steatosis, optionally in combination with one or more additional thera comprising administering to a patient in need thereof, an peutically active compounds. effective amount of a glucagon peptide according to any of 0373) 239. A method for use in treatment or prevention of embodiments 1-154, optionally in combination with one or hypoglycemia in pregnancy, comprising administering to a more additional therapeutically active compounds. patient in need thereof, an effective amount of a glucagon 0363. 229. A method for use in treating beta-blocker poi peptide according to any of embodiments 1-154, optionally in soning, comprising administering to a patient in need thereof, combination with one or more additional therapeutically an effective amount of a glucagon peptide according to any of active compounds. embodiments 1-154, optionally in combination with one or 0374 240. A method for use in treatment or prevention of more additional therapeutically active compounds. alcohol-induced hypoglycaemia, comprising administering 0364. 230. A method for use in inhibition of the motility of to a patient in need thereof, an effective amount of a glucagon the gastrointestinal tract, useful in connection with investiga peptide according to any of embodiments 1-154, optionally in tions of the gastrointestinal tract using techniques such as combination with one or more additional therapeutically X-ray, CT- and NMR-Scanning, comprising administering to a active compounds. patient in need thereof, an effective amount of a glucagon 0375 241. A method for use in treatment or prevention of peptide according to any of embodiments 1-154, optionally in insulinoma, comprising administering to a patient in need combination with one or more additional therapeutically thereof, an effective amount of a compound according to any active compounds. of embodiments 1-154, optionally in combination with one or 0365. 231. A method for use in treatment or prevention of more additional therapeutically active compounds. hypoglycaemia, comprising administering to a patient in 0376 242. A method for use in treatment or prevention of need thereof, an effective amount of a glucagon peptide Von Girkes disease, comprising administering to a patient in according to any of embodiments 1-154, optionally in com need thereof, an effective amount of a glucagon peptide bination with one or more additional therapeutically active according to any of embodiments 1-154, optionally in com compounds. bination with one or more additional therapeutically active 0366 232. A method for use in treatment or prevention of compounds. insulin induced hypoglycaemia, comprising administering to 0377. Further embodiments of the present invention relate a patient in need thereof, an effective amount of a glucagon to the following uses: peptide according to any of embodiments 1-154, optionally in 0378 243. Use of a glucagon peptide according to any one combination with one or more additional therapeutically of the embodiments 1-154, for the preparation of a medica active compounds. ment. 0367 233. A method for use in treatment or prevention of 0379 244. Use of a glucagon peptide according to any one reactive hypoglycaemia, comprising administering to a of embodiments 1-154, for the preparation of a medicament patient in need thereof, an effective amount of a glucagon for the treatment or prevention of hyperglycemia, type 2 peptide according to any of embodiments 1-154, optionally in diabetes, impaired glucose tolerance, type 1 diabetes and combination with one or more additional therapeutically obesity. active compounds. 0380 245. Use of a glucagon peptide according to any one 0368 234. A method for use in treatment or prevention of of the embodiments 1-154, for the preparation of a medica diabetic hypoglycaemia, comprising administering to a ment for delaying or preventing disease progression in type 2 patient in need thereof, an effective amount of a glucagon diabetes, treating obesity or preventing overweight, for peptide according to any of embodiments 1-154, optionally in decreasing food intake, increase energy expenditure, reduc combination with one or more additional therapeutically ing body weight, delaying the progression from impaired active compounds. glucose tolerance (IGT) to type 2 diabetes; delaying the pro 0369) 235. A method for use in treatment or prevention of gression from type 2 diabetes to insulin-requiring diabetes; non-diabetic hypoglycaemia, comprising administering to a regulating appetite; inducing Satiety; preventing weight patient in need thereof, an effective amount of a glucagon regain after Successful weight loss; treating a disease or state peptide according to any of embodiments 1-154, optionally in related to overweight or obesity; treating bulimia; treating combination with one or more additional therapeutically binge-eating; treating atherosclerosis, hypertension, type 2 active compounds. diabetes, IGT, dyslipidemia, coronary heart disease, hepatic 0370 236. A method for use in treatment or prevention of Steatosis, treatment of beta-blocker poisoning, use for inhibi fasting hypoglycaemia, comprising administering to a patient tion of the motility of the gastrointestinal tract, useful in in need thereof, an effective amount of a glucagon peptide connection with investigations of the gastrointestinal tract according to any of embodiments 1-154, optionally in com using techniques such as X-ray, CT- and NMR-Scanning. bination with one or more additional therapeutically active 0381 246. Use of a glucagon peptide according to any one compounds. of the embodiments 1-154, for the preparation of a medica 0371) 237. A method for use in treatment or prevention of ment for treatment or prevention of hypoglycemia, insulin drug-induced hypoglycaemia, comprising administering to a induced hypoglycemia, reactive hypoglycemia, diabetic patient in need thereof, an effective amount of a glucagon hypoglycemia, non-diabetic hypoglycemia, fasting hypogly peptide according to any of embodiments 1-154, optionally in cemia, drug-induced hypoglycemia, gastric by-pass induced combination with one or more additional therapeutically hypoglycemia, hypoglycemia in pregnancy, alcohol induced active compounds. hypoglycemia, insulinoma and Von Girkes disease. 0372 238. A method for use in treatment or prevention of 0382. In certain embodiments of the uses and methods of gastric by-pass induced hypoglycaemia, comprising admin the present invention, the compound of the present invention US 2016/0002311 A1 Jan. 7, 2016 36 may be administered or applied in combination with more X, represents Thr, Lys or Orn; than one of the above-mentioned, suitable additional thera Xo represents Tyr, Lys, Ornor (p)Tyr; peutically active compounds or Substances, e.g. in combina X represents Lys, Orn or Arg; tion with: metformin and a Sulfonylurea Such as glyburide; a X represents Ser, Glu, Thr, Lys or Orn; Sulfonylurea and acarbose; nateglinide and metformin; acar X, represents Arg, Gln, Lys or Orn; bose and metformin; a Sulfonylurea, metformin and troglita Xs represents Arg, Gln, Ala, Lys or Orn; Zone; insulinanda Sulfonylurea; insulin and metformin; insu Xo represents Arg, Gln, Lys or Orn; lin, metformin and a Sulfonylurea; insulin and troglitaZone; X represents Asp, Glu or Lys; insulin and lovastatin; etc. X represents Gln, Lys, Arg, His, Glu, Asp, Gly, Pro, Ser or 0383. In the case, in particular, of administration of a com Orn; pound of the invention, optionally in combination with one or Xs represents Trp, Arg, Lys. His, Glu, Asp, Gly, Pro, Phe, more additional therapeutically active compounds or Sub Ser, Tyr (p)Tyr or Orn; stances as disclosed above, for a purpose related to treatment X, represents Met, Met(O), Val, Pro, Leu, Arg, Lys or Orn; or prevention of obesity or overweight, i.e. related to reduc Xs represents ASn, Lys, Arg, Ser, Thr, Glu, Asp, Ala, Gln, Pro tion or prevention of excess adiposity, it may be of relevance or Orn; to employ Such administration in combination with Surgical X represents Thr, Glu, Asp, Lys, Arg, Pro or Ornand intervention for the purpose of achieving weight loss or pre X is absent or represents Lys, Gly, Pro or Orn, venting weight gain, e.g. in combination with bariatric Surgi and an albumin binding residue comprising two or more cal intervention. Examples of frequently used bariatric Surgi negatively charged groups, wherein one of the said negatively cal techniques include, but are not limited to, the following: charged groups is terminal of the said albumin binding resi Vertical banded gastroplasty (also known as 'stomach sta due and where the albumin binding residue is attached at the pling'), wherein a part of the stomach is stapled to create a epsilon position of a Lys or at the delta position of an Orn, in Smaller pre-stomach pouch which serves as a new stomach; one or more of the following amino acid positions of the gastric banding, e.g. using an adjustable gastric band system compound of formula I: X7, Xio X2, Xs, X17, Xs, X20, (such as the Swedish Adjustable Gastric Band (SAGB), the X21, X24, X2s, X27, X2s. X29, and/or Xso. LAP-BANDTM or the MIDbandTM), wherein a small pre or a pharmaceutically acceptable salt, amide, acid or prodrug stomach pouch which is to serve as a new stomach is created thereof. using an elastomeric (e.g. silicone) band which can be 0388 248. A compound according to embodiment 247, adjusted in size by the patient; and gastric bypass Surgery, e.g. Selected from the group consisting of the glucagon peptides “Roux-en-Y” bypass wherein a small stomach pouch is cre of the examples. ated using a stapler device and is connected to the distal Small 0389 249. A compound according to embodiment 247, intestine, the upperpart of the Small intestine being reattached in a Y-shaped configuration. wherein said albumin binding residue has the formula II: 0384 The administration of a compound of the invention (optionally in combination with one or more additional thera wherein, peutically active compounds or Substances as disclosed Z represents a structure according to one of the formulas IIa, above) may take place for a period prior to carrying out the IIb or IIc; bariatric Surgical intervention in question and/or for a period of time Subsequent thereto. In many cases it may be preferable to begin administration of a compound of the invention after IIa bariatric Surgical intervention has taken place. O O 0385. The term “obesity' implies an excess of adipose tissue. When energy intake exceeds energy expenditure, the --- : excess calories are stored in adipose tissue, and if this net IIb positive balance is prolonged, obesity results, i.e. there are two components to weight balance, and an abnormality on -N O either side (intake or expenditure) can lead to obesity. In this f N. N > --~~~~ : context, obesity is best viewed as any degree of excess adi H pose tissue that imparts a health risk. The distinction between IIc normal and obese individuals can only be approximated, but OH O the health risk imparted by obesity is probably a continuum with increasing adipose tissue. However, in the context of the present invention, individuals with a body mass index (BMI=body weight in kilograms divided by the square of the height in meters) above 25 are to be regarded as obese. 0386 Further embodiments of the present invention relate to the following: wherein n in formula IIa is 6-20, 0387 247. A compound according to formula I: m in formula IIc is 5-9 His-X-Gln-Gly-Thr-X-X7-Ser-Asp-Xo-Ser-X- the COOH group in formula IIc can reside on position 2, 3 or 4 on the phenyl ring, the symbol * in formula IIa, IIb and IIc represents the attach wherein ment point to the nitrogen in Z, Z or Z; X represents Ser, Aib or D-Ser; Z2 is absent or represents a structure according to one of the X represents Phe or Gln; formulas IId, IIe, IIf IIg, IIh, II or IIk; US 2016/0002311 A1 Jan. 7, 2016 37
-continued II. O OH IId O OH
O "n : N : H : 1 N O H O IIe O OH O O OH
: II n N : O OH H O O OH O
IIf H O OH O OH O O
N N H H O OH O OH O O IIk O OH O OH O O H IIg O OH O OH O O O O : : NN N : H H 1 N N O O H H O OH O OH O O
IIh O O OH O OH
: 1 : wherein each amino acid moiety is independently either L or D; wherein Z is connected via the carbonatom with symbol * to the nitrogen of Z, Z or to the epsilon nitrogen of a lysine or O OH the delta nitrogen of an ornithine of the glucagon peptide; Z is absent or represents a structure according to one of the formulas IIm, In, IIo or IIp;
Im. O
1 N-1N1\-1 : IIn -N------.O O IIo -N-~~~~.O O O US 2016/0002311 A1 Jan. 7, 2016 38
-continued IIp O O O -N-~~~~-NH N~~---.O
Z is connected vi the carbon of Z with symbol * to the connected via the carbon with symbol * to the epsilon nitro nitrogen of Z with symbol * or to the epsilon nitrogen of a gen of a lysine or the delta nitrogen of an ornithine of the lysine or the delta nitrogen of an ornithine of the glucagon glucagon peptide. peptide; 0390 250. An albumin binding residue according to Z is absent or represents a structure according to one of the embodiment 249, which is selected from the structures formulas IId, IIe, IIf IIg, IIh, II or IIk; wherein each amino according to one of the formulas IIIa, IIIb, IIIc, IIId, IIIe, IIIf acid moiety is independently either L or D, wherein Z is or IIIg: US 2016/0002311 A1 Jan. 7, 2016 39
B.III QIII 3.III ÞIII 3.III
O s X"( O Z, - O s Z, O 3. -
Z O- S - & &
-,O
& O s O Z s X"K. O
O S.
US 2016/0002311 A1 Jan. 7, 2016
0391 251. An albumin binding residue according to embodiment 249, selected from a structure according to one of the formulas Iva, IVb, IVc or IVd:
IV a O O n1 OH O H HO N H N-o~...~~~~~H O O O IVb O s-ori O
"~...~N~~.H O O IVc O OH O N1 O "~~N~~ ouls O O o O Y OH N1-1 O n-1No Rul N : *...s or H H O O O OH IVd O O 's--" HO s N 1N1N H
O OH o OS-1 OH H N 8. NH ~ O O O OH IWe O N N-N--~~~~N N
NY-N-~O O
0392) 252. A pharmaceutical composition comprising a 0397 257. A compound according to any of any one of compound according to any one of embodiments 247-249. embodiments 247-249, for use in therapy. 0393 253. A pharmaceutical composition according to 0398 258. Use of a compound according to any one of embodiments 252, further comprising one or more additional therapeutically active compounds or Substances. embodiments 247-249, for the preparation of a medicament. 0394 254. A pharmaceutical composition according to 0399 259. Use of a compound according to any one of any one of embodiment 253, further comprising a GLP-1 embodiments 247-249, for the preparation of a medicament compound. for the treatment or prevention of hyperglycemia, type 2 0395. 255. A pharmaceutical composition according to diabetes, impaired glucose tolerance, type 1 diabetes and any one of embodiments 252-254, further comprising an obesity. insulinic compound. 0400 260. Use of a compound according to any one of the 0396 256. The pharmaceutical composition according to embodiments 247-249, for the preparation of a medicament any one of embodiments 252-255, which is suited for for delaying or preventing disease progression in type 2 dia parenteral administration. betes, treating obesity or preventing overweight, for decreas US 2016/0002311 A1 Jan. 7, 2016 42 ing food intake, increase energy expenditure, reducing body -continued weight, delaying the progression from impaired glucose tol erance (IGT) to type 2 diabetes; delaying the progression Ser Serine from type 2 diabetes to insulin-requiring diabetes; regulating Thr Threonine appetite; inducing satiety; preventing weight regain after Suc Tyr Tyrosine cessful weight loss; treating a disease or state related to over Tyr OH weight or obesity; treating bulimia; treating binge-eating: p(Tyr) OS/ treating atherosclerosis, hypertension, type 2 diabetes, IGT. -oil dyslipidemia, coronary heart disease, hepatic steatosis, treat O ment of beta-blocker poisoning, use for inhibition of the motility of the gastrointestinal tract, useful in connection with investigations of the gastrointestinal tract using techniques OH Such as X-ray, CT- and NMR-Scanning. HN 04.01 261. Use of a compound according to any one of O embodiments 247-249, for the preparation of a medicament Trp Tryptophan for treatment or prevention of hypoglycemia, insulin induced Wall Valine hypoglycemia, reactive hypoglycemia, diabetic hypoglyce mia, non-diabetic hypoglycemia, fasting hypoglycemia, drug-induced hypoglycemia, gastric by-pass induced 0403 Amino acid abbreviations beginning with D- fol hypoglycemia, hypoglycemia in pregnancy, alcohol induced lowed by a three letter code, such as D-Ser, D-His and so on, hypoglycemia, insulinoma and Von Girkes disease. refer to the D-enantiomer of the corresponding amino acid, 0402. The amino acid abbreviations used in the present for example D-serine, D-histidine and so on. context have the following meanings: Pharmaceutical Compositions 0404 Pharmaceutical compositions containing a com Ado O pound according to the present invention may be prepared by conventional techniques, e.g. as described in Remington's HN n-1\o1N1 NullsOH Pharmaceutical Sciences, 1985 or in Remington: The Sci Aib 2-Aminoisobutyric acid ence and Practice of Pharmacy, 19" edition, 1995. Ala Alanine 04.05 As already mentioned, one aspect of the present ASn Asparagine invention is to provide a pharmaceutical formulation com Asp Aspartic acid prising a compound according to the present invention which Arg Arginine Cit Citrulline is present in a concentration from about 0.01 mg/mL to about Cys Cysteine 25 mg/mL, Such as from about 0.1 mg/mL to about 5 mg/mL Gln Glutamine and from about 2 mg/mL to about 5 mg/mL, and wherein said Glu Glutamic acid formulation has a pH from 2.0 to 10.0. The pharmaceutical formulation may comprise a compound according to the Y-Glu O OH present invention which is present in a concentration from about 0.1 mg/ml to about 50 mg/ml, and wherein said formu '' OH lation has a pH from 2.0 to 10.0. The formulation may further comprise a buffer system, preservative(s), isotonicity agent HN '1. (S), chelating agent(s), stabilizers and Surfactants. In one embodiment of the invention the pharmaceutical formulation f / 3 is an aqueous formulation, i.e. formulation comprising water. C-nitrogen and Y-carboxy group form the Such formulation is typically a solution or a Suspension. In a amide bonds to the two neighboring residues Gly Glycine further embodiment of the invention the pharmaceutical for His Histidine mulation is an aqueous solution. The term “aqueous formu Hyp 4-hydroxyproline lation is defined as a formulation comprising at least 50% Ile Isoleucine w/w water. Likewise, the term “aqueous solution' is defined Leu Leucine Lys Lysine as a solution comprising at least 50% w/w water, and the term Met Methionine “aqueous Suspension' is defined as a Suspension comprising at least 50% w/w water. Met(O) s 1. 0406. In another embodiment the pharmaceutical formu lation is a freeze-dried formulation, whereto the physician or the patient adds solvents and/or diluents prior to use. 0407. In another embodiment the pharmaceutical formu OH lation is a dried formulation (e.g. freeze-dried or spray-dried) HN ready for use without any prior dissolution. 0408. In a further aspect the invention relates to a pharma O ceutical formulation comprising an aqueous solution of a Orn Ornithine compound according to the present invention, and a buffer, Phe Phenylalanine wherein said compound is present in a concentration from 0.1 Pro Proline mg/ml or above, and wherein said formulation has a pH from about 2.0 to about 10.0. US 2016/0002311 A1 Jan. 7, 2016
04.09. In a further aspect the invention relates to a pharma soluble glucans, including for example fructose, glucose, ceutical formulation comprising an aqueous solution of a mannose, Sorbose, Xylose, maltose, lactose, Sucrose, treha compound according to the present invention, and a buffer, lose, dextran, pullulan, dextrin, cyclodextrin, Soluble starch, wherein said compound is present in a concentration from 0.1 hydroxyethyl starch and carboxymethylcellulose-Na may be mg/ml or above, and wherein said formulation has a pH from used. In one embodiment the Sugar additive is Sucrose. Sugar about 7.0 to about 8.5. alcohol is defined as a C4-C8 hydrocarbon having at least one 0410. In a another embodiment of the invention the pH of —OH group and includes, for example, mannitol, Sorbitol, the formulation is selected from the list consisting of 2.0.2.1. inositol, galacititol, dulcitol. Xylitol, and arabitol. In one 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, embodiment the Sugar alcohol additive is mannitol. The Sug 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, ars or Sugar alcohols mentioned above may be used individu 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, ally or in combination. There is no fixed limit to the amount 6.4., 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, used, as long as the Sugar or Sugar alcohol is soluble in the 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, liquid preparation and does not adversely effect the stabiliz 9.2, 9.3, 9.4, 9.5, 9.6, 9.7. 9.8, 9.9, and 10.0. Preferably, the ing effects achieved using the methods of the invention. In pH of the formulation is at least 1 pH unit from the isoelectric one embodiment, the Sugar or Sugar alcohol concentration is point of the compound according to the present invention, between about 1 mg/ml and about 150 mg/ml. In a further even more preferable the pH of the formulation is at least 2 pH embodiment of the invention the isotonic agent is present in a unit from the isoelectric point of the compound according to concentration from 1 mg/ml to 50 mg/ml. In a further embodi the present invention. ment of the invention the isotonic agent is present in a con 0411. In a further embodiment of the invention the buffer centration from 1 mg/ml to 7 mg/ml. In a further embodiment is selected from the group consisting of sodium acetate, of the invention the isotonic agent is present in a concentra Sodium carbonate, citrate, glycylglycine, histidine, glycine, tion from 8 mg/ml to 24 mg/ml. In a further embodiment of lysine, arginine, Sodium dihydrogen phosphate, disodium the invention the isotonic agent is present in a concentration hydrogen phosphate, sodium phosphate, and tris(hydroxym from 25 mg/ml to 50 mg/ml. Each one of these specific ethyl)-aminomethane, hepes, bicine, tricine, malic acid, suc isotonic agents constitutes an alternative embodiment of the cinate, maleic acid, fumaric acid, tartaric acid, aspartic acidor invention. The use of an isotonic agent in pharmaceutical mixtures thereof. Each one of these specific buffers consti compositions is well-known to the skilled person. For conve tutes an alternative embodiment of the invention. nience reference is made to Remington: The Science and 0412. In a further embodiment of the invention the formu Practice of Pharmacy, 19" edition, 1995. lation further comprises a pharmaceutically acceptable pre 0414. In a further embodiment of the invention the formu servative. In a further embodiment of the invention the pre lation further comprises a chelating agent. In a further servative is selected from the group consisting of phenol, embodiment of the invention the chelating agent is selected o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, pro from salts of ethylenediaminetetraacetic acid (EDTA), citric pyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxy acid, and aspartic acid, and mixtures thereof. In a further benzoate, 2-phenylethanol, benzyl alcohol, ethanol, chlo embodiment of the invention the chelating agent is present in robutanol, and thiomerosal, bronopol, benzoic acid, a concentration from 0.1 mg/ml to 5 mg/ml. In a further imidurea, chlorohexidine, sodium dehydroacetate, chlorocre embodiment of the invention the chelating agent is present in sol, ethyl p-hydroxybenzoate, benzethonium chloride, chlo a concentration from 0.1 mg/ml to 2 mg/ml. In a further rphenesine (3p-chlorphenoxypropane-1,2-diol) or mixtures embodiment of the invention the chelating agent is present in thereof. In a further embodiment of the invention the preser a concentration from 2 mg/ml to 5 mg/ml. Each one of these vative is present in a concentration from 0.1 mg/ml to 30 specific chelating agents constitutes an alternative embodi mg/ml. In a further embodiment of the invention the preser ment of the invention. The use of a chelating agent in phar Vative is present in a concentration from 0.1 mg/ml to 20 maceutical compositions is well-known to the skilled person. mg/ml. In a further embodiment of the invention the preser For convenience reference is made to Remington: The Sci Vative is present in a concentration from 0.1 mg/ml to 5 ence and Practice of Pharmacy, 19' edition, 1995. mg/ml. In a further embodiment of the invention the preser 0415. In a further embodiment of the invention the formu Vative is present in a concentration from 5 mg/ml to 10 mg/ml. lation further comprises a stabiliser. The use of a stabilizer in In a further embodiment of the invention the preservative is pharmaceutical compositions is well-known to the skilled present in a concentration from 10 mg/ml to 20 mg/ml. Each person. For convenience reference is made to Remington: The one of these specific preservatives constitutes an alternative Science and Practice of Pharmacy, 19' edition, 1995. embodiment of the invention. The use of a preservative in 0416) More particularly, compositions of the invention are pharmaceutical compositions is well-known to the skilled stabilized liquid pharmaceutical compositions whose thera person. For convenience reference is made to Remington: peutically active components include a polypeptide that pos The Science and Practice of Pharmacy, 19" edition, 1995. sibly exhibits aggregate formation during storage in liquid 0413. In a further embodiment of the invention the formu pharmaceutical formulations. By "aggregate formation' is lation further comprises an isotonic agent. In a further intended a physical interaction between the polypeptide mol embodiment of the invention the isotonic agent is selected ecules that results in formation of oligomers, which may from the group consisting of a salt (e.g. sodium chloride), a remain soluble, or large visible aggregates that precipitate Sugar or Sugar alcohol, an amino acid (e.g. L-glycine, L-his from the solution. By “during storage' is intended a liquid tidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, pharmaceutical composition or formulation once prepared, is threonine), an alditol (e.g. glycerol (glycerine), 1,2-pro not immediately administered to a subject. Rather, following panediol (propyleneglycol), 1,3-propanediol. 1,3-butane preparation, it is packaged for storage, either in a liquid form, diol) polyethyleneglycol (e.g. PEG400), or mixtures thereof. in a frozen state, or in a dried form for later reconstitution into Any Sugar Such as mono-, di-, or polysaccharides, or water a liquid form or other form suitable for administration to a US 2016/0002311 A1 Jan. 7, 2016 44 subject. By “dried form' is intended the liquid pharmaceuti results in greater retention of the polypeptide in its proper cal composition or formulation is dried either by freeze dry molecular form. Any stereoisomer of methionine (L, D or a ing (i.e., lyophilization; see, for example, Williams and Polli mixture thereof) can be used. The amount to be added should (1984) J. Parenteral Sci. Technol. 38:48-59), spray drying bean amount sufficient to inhibit oxidation of the methionine (see Masters (1991) in Spray-Drying Handbook (5th ed; residues such that the amount of methionine sulfoxide is Longman Scientific and Technical, Essez, U.K.), pp. 491 acceptable to regulatory agencies. Typically, this means that 676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. the composition contains no more than about 10% to about 18:1169-1206; and Mumenthaler et al. (1994) Pharm. Res. 30% methionine sulfoxide. Generally, this can beachieved by 11:12-20), or air drying (Carpenter and Crowe (1988) Cryo adding methionine Such that the ratio of methionine added to biology 25:459-470; and Roser (1991) Biopharm. 4:47-53). methionine residues ranges from about 1:1 to about 1000:1, Aggregate formation by a polypeptide during storage of a such as 10:1 to about 100:1. liquid pharmaceutical composition can adversely affect bio 0419. In a further embodiment of the invention the formu logical activity of that polypeptide, resulting in loss of thera lation further comprises a stabiliser selected from the group peutic efficacy of the pharmaceutical composition. Further of high molecular weight polymers or low molecular com more, aggregate formation may cause other problems such as pounds. In a further embodiment of the invention the stabi blockage of tubing, membranes, or pumps when the polypep lizer is selected from polyethylene glycol (e.g. PEG 3350), tide-containing pharmaceutical composition is administered polyvinylalcohol (PVA), polyvinylpyrrolidone, carboxy-/hy using an infusion system. droxycellulose or derivates thereof (e.g. HPC, HPC-SL, 0417. The pharmaceutical compositions of the invention HPC-L and HPMC), cyclodextrins, sulphur-containing sub may further comprise an amount of an amino acid base Suf stances as monothioglycerol, thioglycolic acid and 2-meth ficient to decrease aggregate formation by the polypeptide ylthioethanol, and different salts (e.g. sodium chloride). Each during storage of the composition. By “amino acid base' is one of these specific stabilizers constitutes an alternative intended an amino acid or a combination of amino acids, embodiment of the invention. where any given amino acid is present either in its free base 0420. The pharmaceutical compositions may also com form or in its salt form. Where a combination of amino acids prise additional stabilizing agents, which further enhance is used, all of the amino acids may be present in their free base stability of a therapeutically active polypeptide therein. Sta forms, all may be present in their salt forms, or some may be bilizing agents of particular interest to the present invention present in their free base forms while others are present in include, but are not limited to, methionine and EDTA, which their salt forms. In one embodiment, amino acids used for protect the polypeptide against methionine oxidation, and a preparing the compositions of the invention are those carry nonionic Surfactant, which protects the polypeptide against ing a charged side chain, such as arginine, lysine, aspartic aggregation associated with freeze-thawing or mechanical acid, and glutamic acid. In one embodiment, the amino acid shearing. used for preparing the compositions of the invention is gly 0421. In a further embodiment of the invention the formu cine. Any stereoisomer (i.e. L or D) of a particular amino acid lation further comprises a surfactant. In a further embodiment (e.g. methionine, histidine, imidazole, arginine, lysine, iso of the invention the Surfactant is selected from a detergent, leucine, aspartic acid, tryptophan, threonine and mixtures ethoxylated castor oil, polyglycolyzed glycerides, acetylated thereof) or combinations of these stereoisomers, may be monoglycerides, Sorbitan fatty acid esters, polyoxypropy present in the pharmaceutical compositions of the invention lene-polyoxyethylene block polymers (eg. poloxamers such So long as the particular amino acid is present either in its free as Pluronic R. F68, poloxamer 188 and 407, Triton X-100), base form or its salt form. In one embodiment the L-stereoi polyoxyethylene sorbitan fatty acid esters, starshaped PEO, Somer is used. Compositions of the invention may also be polyoxyethylene and polyethylene derivatives such as alky formulated with analogues of these amino acids. By “amino lated and alkoxylated derivatives (tweens, e.g. Tween-20, acid analogue' is intended a derivative of the naturally occur Tween-40, Tween-80 and Brij-35), polyoxyethylene hydrox ring amino acid that brings about the desired effect of yStearate, monoglycerides or ethoxylated derivatives thereof, decreasing aggregate formation by the polypeptide during diglycerides or polyoxyethylene derivatives thereof, alco storage of the liquid pharmaceutical compositions of the hols, glycerol, lecitins and phospholipids (eg. phosphatidyl invention. Suitable arginine analogues include, for example, serine, phosphatidyl choline, phosphatidyl ethanolamine, aminoguanidine, ornithine and N-monoethyl L-arginine, phosphatidyl inositol, diphosphatidylglycerol and sphingo Suitable methionine analogues include ethionine and buthion myelin), derivates of phospholipids (eg. dipalmitoyl phos ine and Suitable cystein analogues include S-methyl-L cys phatidic acid) and lysophospholipids (eg. palmitoyl lyso tein. As with the otheramino acids, the amino acid analogues phosphatidyl-L-serine and 1-acyl-sn-glycero-3-phosphate are incorporated into the compositions in either their free base esters of ethanolamine, choline, serine or threonine) and form or their salt form. In a further embodiment of the inven alkyl, alkoxyl (alkyl ester), alkoxy (alkyl ether)-derivatives of tion the amino acids or amino acid analogues are used in a lysophosphatidyl and phosphatidylcholines, e.g. lauroyl and concentration, which is sufficient to prevent or delay aggre myristoyl derivatives of lysophosphatidylcholine, dipalmi gation of the protein. toylphosphatidylcholine, and modifications of the polar head 0418 Inafurther embodiment of the invention methionine group, that is cholines, ethanolamines, phosphatidic acid, (or other Sulphuric amino acids oramino acid analogous) may serines, threonines, glycerol, inositol, and the positively be added to inhibit oxidation of methionine residues to charged DODAC, DOTMA, DCP BISHOP, lysophosphati methionine Sulfoxide when the polypeptide acting as the dylserine and lysophosphatidylthreonine, and glycerophos therapeutic agent is a polypeptide comprising at least one pholipids (eg. cephalins), glyceroglycolipids (eg. galactopy methionine residue susceptible to such oxidation. By ransoide), Sphingoglycolipids (eg. ceramides, gangliosides), “inhibit is intended minimal accumulation of methionine dodecylphosphocholine, hen egg lysolecithin, fusidic acid oxidized species over time. Inhibiting methionine oxidation derivatives—(e.g. sodium tauro-dihydrofusidate etc.), long US 2016/0002311 A1 Jan. 7, 2016
chain fatty acids and salts thereof C6-C12 (eg. oleic acid and capsules and Soft gelatine capsules, Suppositories, rectal cap caprylic acid), acylcarnitines and derivatives, Na-acylated Sules, drops, gels, sprays, powder, aerosols, inhalants, eye derivatives of lysine, arginine or histidine, or side-chain acy drops, ophthalmic ointments, ophthalmic rinses, vaginal pes lated derivatives of lysine or arginine, Na-acylated derivatives saries, vaginal rings, vaginal ointments, injection solution, in of dipeptides comprising any combination of lysine, arginine situ transforming solutions, for example in situ gelling, in situ or histidine and a neutral or acidic amino acid, Na-acylated setting, in situ precipitating, in situ crystallization, infusion derivative of a tripeptide comprising any combination of a Solution, and implants. neutral amino acid and two charged amino acids, DSS (docu 0427 Compositions of the invention may further be com sate sodium, CAS registry no 577-11-7), docusate calcium, pounded in, or attached to, for example through covalent, CAS registry no 128-49-4), docusate potassium, CAS reg hydrophobic and electrostatic interactions, a drug carrier, istry no 7491-09-0), SDS (sodium dodecyl sulfate or drug delivery system and advanced drug delivery system in Sodium lauryl Sulfate), Sodium caprylate, cholic acid or order to further enhance stability of the compound, increase derivatives thereof, bile acids and salts thereof and glycine or bioavailability, increase solubility, decrease adverse effects, taurine conjugates, urSodeoxycholic acid, Sodium cholate, achieve chronotherapy well known to those skilled in the art, Sodium deoxycholate, Sodium taurocholate, Sodium glyco and increase patient compliance or any combination thereof. cholate, N-Hexadecyl-N,N-dimethyl-3-ammonio-1-pro Examples of carriers, drug delivery systems and advanced panesulfonate, anionic (alkyl-aryl-Sulphonates) monovalent drug delivery systems include, but are not limited to, poly surfactants, Zwitterionic surfactants (e.g. N-alkyl-N,N-dim mers, for example cellulose and derivatives, polysaccharides, ethylammonio-1-propanesulfonates, 3-cholamido-1-propy for example dextran and derivatives, starch and derivatives, ldimethylammonio-1-propanesulfonate, cationic Surfactants poly(vinyl alcohol), acrylate and methacrylate polymers, (quarternary ammonium bases) (e.g. cetyl-trimethylammo polylactic and polyglycolic acid and block co-polymers nium bromide, cetylpyridinium chloride), non-ionic Surfac thereof, polyethylene glycols, carrier proteins, for example tants (eg. Dodecyl B-D-glucopyranoside), poloxamines (eg. albumin, gels, for example, thermogelling systems, for Tetronics), which are tetrafunctional block copolymers example block co-polymeric systems well known to those derived from sequential addition of propylene oxide and eth skilled in the art, micelles, liposomes, microspheres, nano ylene oxide to ethylenediamine, or the Surfactant may be particulates, liquid crystals and dispersions thereof. L2 phase selected from the group of imidazoline derivatives, or mix and dispersions there of well known to those skilled in the art tures thereof. Each one of these specific Surfactants consti of phase behaviour in lipid-water systems, polymeric tutes an alternative embodiment of the invention. micelles, multiple emulsions, self-emulsifying, self-micro 0422 The use of a surfactant in pharmaceutical composi emulsifying, cyclodextrins and derivatives thereof, and den tions is well-known to the skilled person. For convenience drimers. reference is made to Remington: The Science and Practice of Pharmacy, 19' edition, 1995. 0428 Compositions of the current invention are useful in 0423. Additional ingredients may also be present in the the formulation of Solids, semisolids, powder and solutions pharmaceutical formulation of the present invention. Such for pulmonary administration of the compound, using, for additional ingredients may include wetting agents, emulsifi example a metered dose inhaler, dry powder inhaler and a ers, antioxidants, bulking agents, tonicity modifiers, chelat nebulizer, all being devices well known to those skilled in the ing agents, metal ions, oleaginous Vehicles, proteins (e.g., art. human serum albumin, gelatin or proteins) and a Zwitterion 0429 Compositions of the current invention are specifi (e.g., an amino acid Such as betaine, taurine, arginine, gly cally useful in the formulation of controlled, Sustained, pro cine, lysine and histidine). Such additional ingredients, of tracting, retarded, and slow release drug delivery systems. course, should not adversely affect the overall stability of the More specifically, but not limited to, compositions are useful pharmaceutical formulation of the present invention. in formulation of parenteral controlled release and Sustained 0424 Pharmaceutical compositions containing a com release systems (both systems leading to a many-fold reduc pound according to the present invention may be adminis tion in number of administrations), well known to those tered to a patient in need of such treatment at several sites, for skilled in the art. Even more preferably, are controlled release example, at topical sites, for example, skin and mucosal sites, and Sustained release systems administered subcutaneous. at sites which bypass absorption, for example, administration Without limiting the scope of the invention, examples of in an artery, in a vein, in the heart, and at sites which involve useful controlled release system and compositions are hydro absorption, for example, administration in the skin, under the gels, oleaginous gels, liquid crystals, polymeric micelles, skin, in a muscle or in the abdomen. microspheres, nanoparticles, 0425 Administration of pharmaceutical compositions 0430 Methods to produce controlled release systems use according to the invention may be through several routes of ful for compositions of the current invention include, but are administration, for example, lingual, Sublingual, buccal, in not limited to, crystallization, condensation, co-cystalliza the mouth, oral, in the stomach and intestine, nasal, pulmo tion, precipitation, co-precipitation, emulsification, disper nary, for example, through the bronchioles and alveoli or a Sion, high pressure homogenization, encapsulation, spray combination thereof, epidermal, dermal, transdermal, vagi drying, microencapsulation, coacervation, phase separation, nal, rectal, ocular, for examples through the conjunctiva, ure Solvent evaporation to produce microspheres, extrusion and tal, and parenteral to patients in need of Such a treatment. Supercritical fluid processes. General reference is made to 0426 Compositions of the current invention may be Handbook of Pharmaceutical Controlled Release (Wise, D. administered in several dosage forms, for example, as Solu L., ed. Marcel Dekker, New York, 2000) and Drug and the tions, Suspensions, emulsions, microemulsions, multiple Pharmaceutical Sciences Vol. 99: Protein Formulation and emulsion, foams, salves, pastes, plasters, ointments, tablets, Delivery (MacNally, E. J., ed. Marcel Dekker, New York, coated tablets, rinses, capsules, for example, hard gelatine 2000). US 2016/0002311 A1 Jan. 7, 2016 46
0431 Parenteral administration may be performed by sub amino acids, such as phenylalanine, leucine, isoleucine, cutaneous, intramuscular, intraperitoneal or intravenous methionine, and valine, or the like. injection by means of a syringe, optionally a pen-like syringe. 0435 The term "chemical stability” of the protein formu Alternatively, parenteral administration can be performed by lation as used herein refers to chemical covalent changes in means of an infusion pump. A further option is a composition the protein structure leading to formation of chemical degra which may be a solution or suspension for the administration dation products with potential less biological potency and/or of the compound according to the present invention in the potential increased immunogenic properties compared to the form of a nasal or pulmonal spray. Alternatively, the peptide native protein structure. Various chemical degradation prod can be administrated via a rectal suppository. As a still further ucts can be formed depending on the type and nature of the option, the pharmaceutical compositions containing the com native protein and the environment to which the protein is pound of the invention can also be adapted to transdermal exposed. Elimination of chemical degradation can most prob administration, e.g. by needle-free injection or from a patch, ably not be completely avoided and increasing amounts of optionally an iontophoretic patch, or transmucosal, e.g. buc chemical degradation products is often seen during storage cal, administration. and use of the protein formulation as well-known by the person skilled in the art. Most proteins are prone to deamida 0432. The term "stabilized formulation” refers to a formu tion, a process in which the side chain amide group in lation with increased physical stability, increased chemical glutaminyl or asparaginyl residues is hydrolysed to form a stability or increased physical and chemical stability. free carboxylic acid. Other degradations pathways involves 0433. The term “physical stability” of the protein formu formation of high molecular weight transformation products lation as used herein refers to the tendency of the protein to where two or more protein molecules are covalently bound to form biologically inactive and/or insoluble aggregates of the each other through transamidation and/or disulfide interac protein as a result of exposure of the protein to thermo tions leading to formation of covalently bound dimer, oligo mechanical stresses and/or interaction with interfaces and mer and polymer degradation products (Stability of Protein Surfaces that are destabilizing, such as hydrophobic surfaces Pharmaceuticals, Ahern. T. J. & Manning M. C., Plenum and interfaces. Physical stability of the aqueous protein for Press, New York 1992). Oxidation (of for instance methionine mulations is evaluated by means of visual inspection and/or residues) can be mentioned as another variant of chemical turbidity measurements after exposing the formulation filled degradation. The chemical stability of the proteinformulation in Suitable containers (e.g. cartridges or vials) to mechanical/ can be evaluated by measuring the amount of the chemical physical stress (e.g. agitation) at different temperatures for degradation products at various time-points after exposure to various time periods. Visual inspection of the formulations is different environmental conditions (the formation of degra performed in a sharp focused light with a dark background. dation products can often be accelerated by for instance The turbidity of the formulation is characterized by a visual increasing temperature). The amount of each individual deg score ranking the degree of turbidity for instance on a scale radation product is often determined by separation of the from 0 to 3 (a formulation showing no turbidity corresponds degradation products depending on molecule size and/or to a visual score 0, and a formulation showing visual turbidity charge using various chromatography techniques (e.g. SEC in daylight corresponds to visual score 3). A formulation is HPLC and/or RP-HPLC). classified physical unstable with respect to protein aggrega 0436 Hence, as outlined above, a “stabilized formulation tion, when it shows visual turbidity in daylight. Alternatively, refers to a formulation with increased physical stability, the turbidity of the formulation can be evaluated by simple increased chemical stability or increased physical and chemi turbidity measurements well-known to the skilled person. cal stability. In general, a formulation must be stable during Physical stability of the aqueous protein formulations can use and storage (in compliance with recommended use and also be evaluated by using a spectroscopic agent or probe of storage conditions) until the expiration date is reached. the conformational status of the protein. The probe is prefer 0437. In one embodiment of the invention the pharmaceu ably a small molecule that preferentially binds to a non-native tical formulation comprising the compound according to the conformer of the protein. One example of a small molecular present invention is stable for more than 6 weeks of usage and spectroscopic probe of protein structure is Thioflavin T. for more than 3 years of storage. Thioflavin T is a fluorescent dye that has been widely used for 0438. In another embodiment of the invention the pharma the detection of amyloid fibrils. In the presence of fibrils, and ceutical formulation comprising the compound according to perhaps other protein configurations as well. Thioflavin T the present invention is stable for more than 4 weeks of usage gives rise to a new excitation maximum at about 450 nm and and for more than 3 years of storage. enhanced emission at about 482 nm when bound to a fibril 0439. In a further embodiment of the invention the phar protein form. Unbound Thioflavin T is essentially non-fluo maceutical formulation comprising the compound according rescent at the wavelengths. to the present invention is stable for more than 4 weeks of 0434) Other small molecules can be used as probes of the usage and for more than two years of storage. changes in protein structure from native to non-native states. 0440. In an even further embodiment of the invention the For instance the “hydrophobic patch” probes that bind pref pharmaceutical formulation comprising the compound is erentially to exposed hydrophobic patches of a protein. The stable for more than 2 weeks of usage and for more than two hydrophobic patches are generally buried within the tertiary years of storage. Structure of a protein in its native state, but become exposed as 0441 Pharmaceutical compositions containing a gluca a protein begins to unfold or denature. Examples of these gon peptide according to the present invention may be admin Small molecular, spectroscopic probes are aromatic, hydro istered parenterally to patients in need of such a treatment. phobic dyes, such as antrhacene, acridine, phenanthroline or Parenteral administration may be performed by subcutane the like. Other spectroscopic probes are metal-amino acid ous, intramuscular or intravenous injection by means of a complexes, such as cobalt metal complexes of hydrophobic Syringe, optionally a pen-like syringe. Alternatively, US 2016/0002311 A1 Jan. 7, 2016 47 parenteral administration can be performed by means of an treatment and/or prevention of complications resulting from infusion pump. A further option is a composition which may or associated with diabetes and agents for the treatment and/ be a powder or a liquid for the administration of the glucagon or prevention of complications and disorders resulting from peptide in the form of a nasal or pulmonal spray. As a still or associated with obesity. In the present context the expres further option, the glucagon peptides of the invention can also sion “antidiabetic agent' includes compounds for the treat be administered transdermally, e.g. from a patch, optionally a ment and/or prophylaxis of insulin resistance and diseases iontophoretic patch, or transmucosally, e.g. bucally. wherein insulin resistance is the pathophysiological mecha 0442. Thus, the injectable compositions of the glucagon 1S. peptide of the present invention can be prepared using the conventional techniques of the pharmaceutical industry 0451 Examples of these pharmacologically active sub which involves dissolving and mixing the ingredients as stances are: Insulin, GLP-1 agonists, Sulphonylureas (e.g. appropriate to give the desired end product. tolbutamide, glibenclamide, glipizide and gliclazide), bigu 0443 According to one embodiment of the present inven anides e.g. metformin, meglitinides, glucosidase inhibitors tion, the glucagon peptide is provided in the form of a com (e.g. acorbose), glucagon antagonists, DPP-IV (dipeptidyl position Suitable for administration by injection. Such a com peptidase-IV) inhibitors, inhibitors of hepatic enzymes position can either be an injectable solution ready for use or it involved in stimulation of gluconeogenesis and/or glyco can be an amount of a Solid composition, e.g. a lyophilised genolysis, glucose uptake modulators, thiazolidinediones product, which has to be dissolved in a solvent before it can be Such as troglitaZone and ciglitaZone, compounds modifying injected. The injectable solution preferably contains not less the lipid metabolism Such as antihyperlipidemic agents as than about 2 mg/ml, preferably not less than about 5 mg/ml. more preferred not less than about 10 mg/ml of the glucagon HMG CoA inhibitors (statins), compounds lowering food peptide and, preferably, not more than about 100 mg/ml of the intake, RXR agonists and agents acting on the ATP-depen glucagon peptide. dent potassium channel of the B-cells, e.g. glibenclamide, 0444 The glucagon peptides of this invention can be used glipizide, gliclazide and repaglinide; Cholestyramine, in the treatment of various diseases. The particular glucagon colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, peptide to be used and the optimal dose level for any patient simvastatin, probucol, dextrothyroxine, neteglinide, repa will depend on the disease to be treated and on a variety of glinide; B-blockers such as alprenolol, atenolol, timolol, pin factors including the efficacy of the specific peptide derivative dolol, propranolol and metoprolol, ACE (angiotensin con employed, the age, body weight, physical activity, and diet of verting enzyme) inhibitors such as benazepril, captopril, the patient, on a possible combination with other drugs, and enalapril, fosinopril, lisinopril, alatriopril, quinapril and on the severity of the case. It is recommended that the dosage of the glucagon peptide of this invention be determined for ramipril, calcium channel blockers such as nifedipine, felo each individual patient by those skilled in the art. dipine, nicardipine, isradipine, nimodipine, diltiazem and 0445. In particular, it is envisaged that the glucagon pep Verapamil, and C.-blockers such as doxazosin, urapidil, pra tide will be useful for the preparation of a medicament with a Zosin and teraZosin; CART (cocaine amphetamine regulated protracted profile of action for the treatment of non-insulin transcript) agonists, NPY (neuropeptideY) antagonists, MC4 dependent diabetes mellitus and/or for the treatment of obe (melanocortin 4) agonists, orexin antagonists, TNF (tumor S1ty. necrosis factor) agonists, CRF (corticotropin releasing fac 0446. In another aspect the present invention relates to the tor) agonists, CRFBP (corticotropin releasing factor binding use of a compound according to the invention for the prepa protein) antagonists, urocortin agonists, 33 agonists, MSH ration of a medicament. (melanocyte-stimulating hormone) agonists, MCH (melano 0447. In one embodiment the present invention relates to cyte-concentrating hormone) antagonists, CCK (cholecysto the use of a compound according to the invention for the preparation of a medicament for the treatment of hypergly kinin) agonists, serotonin re-uptake inhibitors, serotonin and cemia, type 2 diabetes, impaired glucose tolerance, type 1 noradrenaline re-uptake inhibitors, mixed serotonin and diabetes, obesity, hypertension, syndrome X, dyslipidemia, noradrenergic compounds, 5HT (serotonin) agonists, bomb B-cell apoptosis, B-cell deficiency, myocardial infarction, esin agonists, galanin antagonists, growth hormone, growth inflammatory bowel syndrome, dyspepsia, cognitive disor hormone releasing compounds, TRH (thyreotropin releasing ders, e.g. cognitive enhancing, neuroprotection, atheroschle hormone) agonists, UCP2 or 3 (uncoupling protein 2 or 3) rosis, coronary heart disease and other cardiovascular disor modulators, leptin agonists, DA agonists (bromocriptin, ders. doprexin), lipase/amylase inhibitors, RXR (retinoidxrecep 0448. In another embodiment the present invention relates tor) modulators, TR Bagonists; histamine H3 antagonists. to the use of a compound according to the invention for the preparation of a medicament for the treatment of small bowel 0452. It should be understood that any suitable combina syndrome, inflammatory bowel syndrome or Crohns disease. tion of the glucagon peptides according to the invention with 0449 In another embodiment the present invention relates one or more of the above-mentioned compounds and option to the use of a compound according to the invention for the ally one or more further pharmacologically active substances preparation of a medicament for the treatment of hypergly are considered to be within the scope of the present invention. cemia, type 1 diabetes, type 2 diabetes or n-cell deficiency. 0453 The present invention is further illustrated by the 0450. The treatment with a compound according to the following examples which, however, are not to be construed present invention may also be combined with combined with as limiting the scope of protection. The features disclosed in a second or more pharmacologically active Substances, e.g. the foregoing description and in the following examples may, selected from antidiabetic agents, antiobesity agents, appetite both separately and in any combination thereof, be material regulating agents, antihypertensive agents, agents for the for realizing the invention in diverse forms thereof. US 2016/0002311 A1 Jan. 7, 2016 48
EXAMPLES was protected with Mitt (e.g. Fmoc-Lys(Mtt)-OH) and the List of Abbreviations Used N-terminal alpha amino group was protected with Boc. Like 0454 DCM; dichloromethane wise when the Substituent was present on an ornithine 0455 Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene) sidechain the delta aminogroup of the ornithine to be acylated ethyl was protected with Mtt (e.g. Fmoc-Orn(Mtt)-OH. 0456 DIC: diisopropylcarbodiimide 0487. A suitable resin for the synthesis of a glucagon 0457 DIPEA: diisopropylethylamine analogues with a C-terminal carboxylic acid is a pre-loaded, 0458 Fmoc: 9-fluorenylmethyloxycarbonyl low-load Wang resin available from Novabiochem (e.g. low 0459. HATU: (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tet load fmoc-Thr(tBu)-Wang resin, LL, 0.27 mmol/g). A suit ramethyluronium hexafluorophosphate) able resin for the synthesis of glucagon analogues with a 0460 HBTU: (2-(1H-benzotriazol-1-yl-)-1,1,3,3 tetram C-terminal amide is PAL-ChemMatrix resin available from ethyluronium hexafluorophosphate) Matrix-Innovation. Fmoc-deprotection was achieved with 0461. HFIP 1,1,1,3,3,3-hexafluoro-2-propanol or hexaflu 20% piperidine in NMP for 2x3 min. The coupling chemistry oroisopropanol was DIC/HOAt/collidine in NMP. Amino acid/HOAt solu 0462 HOAt: 1-hydroxy-7-azabenzotriazole tions (0.3 M/0.3 M in NMP at a molar excess of 3-10 fold) 0463 HOBt: 1-hydroxybenzotriazole were added to the resin followed by the same molar equiva 0464 HPLC: High Performance Liquid Chromatography lent of DIC (3 M in NMP) followed by collidine (3 M in 0465 ivDde: 1-(4.4-dimethyl-2,6-dioxocyclohexy NMP). For example, the following amounts of 0.3 Mamino lidene)-3-methylbutyl acid/HOAt solution were used per coupling for the following 0466 LCMS: Liquid Chromatography Mass Spectros scale reactions: Scale/ml, 0.05 mmol/1.5 mL, 0.10 mmol/3.0 copy mL, 0.25 mmol/7.5 mL. Coupling time were in general 30 0467 MeOH: methanol min. All couplings were repeated to ensure complete cou 0468 Mmt: 4-methoxytrityl plings. 0469 Mtt: 4-methyltrityl 0488. Deprotection of the Mtt protected lysine was per 0470 NMP: N-methyl pyrrollidone formed on a Prelude Solid Phase Peptide Synthesizer or by 0471 OEG: 8-amino-3,6-dioxaoctanic acid manual synthesis. 0472. OthBu: tert butyl ester 0473 PBS: Phosphate Buffered Saline 0489. Manual synthesis; the Mtt group was removed by 0474 RP: Reverse Phase washing the resin with DCM and suspending the resin in 0475 RP-HPLC: Reverse Phase High Performance Liq HFIP/DCM/TIPS (70:28:2) (2x20 min) and subsequently uid Chromatography washed in sequence with DCM (3x), 5% DIPEA in DCM 0476 RT: Room Temperature (1x), DCM 4x) and NMP-DCM (4:1). Prelude Synthesizer; 0477 Rt. Retention time the Mtt group was removed by washing the resin with HFIP/ 0478 SPPS: Solid Phase Peptide Synthesis DCM (75:25) (2x2 min), washed with DCM and suspending 0479 TFA. trifluoroacetic acid the resin in HFIP/DCM (75:25)(2x20 min) and subsequently 0480 TIPS: triisopropylsilane washed in sequence with Piperidine/NMP (20:80), DCM 0481 Trt: triphenylmethyl or trityl (1x), NMP (1x), DCM (1x), NMP (1x) 0482 UPLC: Ultra High Performance Liquid Chromatog SPPS Method B. Attachment of the Preformed Albumin raphy Binding Moiety General Methods 0490 A solution the carboxylic acid of the preformed 0483 This section relates to methods for synthesising albumin binding moiety such as 2-2-2-2-2-[2-(4S)-5- resin bound peptide (SPPS methods, including methods for tert-butoxy-4-(18-tert-butoxy-18-oxo-octadecanoyl) de-protection of amino acids, methods for cleaving the pep amino-5-oxo-pentanoyl)aminoethoxyethoxyacetyl tide from the resin, and for its purification), as well as meth aminoethoxyethoxyacetic acid. (4 eq.), HOAt (4 eq.) and ods for detecting and characterising the resulting peptide DIC (4 eq.) in NMP-DCM (4:1) was stirred for 30 min before (LCMS and UPLC methods). it was added to the resin. The resin was agitated for 30 min in Synthesis of Resin Bound Peptide the mixture before collidine (4 eq.) was added. The resin was agitated for 16 h. before it was washed with NMP (5x) and SPPS Method A DCM (5x). 0484 SPPS method A refers to peptide synthesis by Fmoc chemistry on a Prelude Solid Phase Peptide Synthesizer from SPPS Method C. Attachment of the Albumin Binding Protein Technologies (Tucson, Ariz. 85714 U.S.A.). Moiety—Stepwise Procedure 0485 The Fmoc-protected amino acid derivatives used were the standard recommended: Fmoc-Ala-OH, Fmoc-Arg 0491. The albumin binding moiety can be introduced in a (Pbf)-OH, Fmoc-ASn(Trt)-OH, Fmoc-Asp(OtBu)-OH, stepwise procedure by the Prelude peptide synthesizer as Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)- described above (SPPC method A) using suitably protected OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, building blocks, with the modification that the amino acids Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc and fatty acid derivatives including Fmoc-Ado-OH, Fmoc Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(thBu)-OH, Fmoc-Thr Glu-OtBu, and octadecanedioic acid mono-tert-butyl ester (thBu)-OH, Fmoc-Trp.(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc (or the analogous C12-, C16-, C20-diacid mono tert-butyl Lys(Mtt)-OH and Fmoc-Val-OH, supplied from e.g. esters) were coupled for 6 hrs in each step. Anaspec, Bachem, Iris Biotech, or Novabiochem. Cleavage from the Resin 0486. When the substituent was present on a lysine 0492. After synthesis the resin was washed with DCM, sidechain the epsilon amino group of lysine to be acylated and the peptide was cleaved from the resin by a 2-3 hour US 2016/0002311 A1 Jan. 7, 2016 49 treatment with TFA/TIS/water (95/2.5/2.5) followed by pre UPLC Methods cipitation with diethylether. The precipitate was washed with diethylether. Method 04 A3 1 (0500 UPLC (method 04 A3 1): The RP-analysis was Purification and Quantification performed using a Waters UPLC system fitted with a dual 0493. The crude peptide is dissolved in a suitable mixture band detector. UV detections at 214 nm and 254 nm were of water and MeCN such as water/MeCN (4:1) and purified collected using an ACQUITY UPLC BEH 130, C18, 130 A, by reversed-phase preparative HPLC (Waters Deltaprep 4000 1.7 um, 2.1 mmx150 mm column, 40°C. or Gilson) on a column containing C18-silica gel. Elution is (0501) The UPLC system was connected to two eluent performed with an increasing gradient of MeCN in water reservoirs containing: containing 0.1%TFA. Relevant fractions are checked by ana A: 90% HO, 10% CHCN, 0.25 Mammonium bicarbonate lytical HPLC or UPLC. Fractions containing the pure target peptide are mixed and concentrated under reduced pressure. B: 70% CHCN, 30% HO The resulting solution is analyzed (HPLC, LCMS) and the 0502. The following linear gradient was used: 75% A, product is quantified using a chemiluminescent nitrogen spe 25% B to 45% A, 55% Bover 16 minutes at a flow-rate of 0.35 cific HPLC detector (Antek 8060 HPLC-CLND) or by mea ml/min. suring UV-absorption at 280 nm. The product is dispensed into glass vials. The vials are capped with Millipore glassfibre Method 04 A4 1 prefilters. Freeze-drying affords the peptide trifluoroacetate as a white solid. (0503 UPLC (method 04 A4 1): The RP-analysis was performed using a Waters UPLC system fitted with a dual Methods for Detection and Characterization band detector. UV detections at 214 nm and 254 nm were collected using an ACQUITY UPLC BEH 130, C18, 130 A, LC-MS Methods 1.7 um, 2.1 mmx150 mm column, 40°C. 0504 The UPLC system was connected to two eluent Method: LCMS 2 reservoirs containing: A: 90% HO, 10% CHCN, 0.25 Mammonium bicarbonate 0494. A Perkin Elmer Sciex API3000 mass spectrometer was used to identify the mass of the sample after elution from B: 70% CHCN, 30% HO a Perkin Elmer Series 200 HPLC system. Eluents: A: 0.05% Trifluoro acetic acid in water, B: 0.05% Trifluoroacetic acid 0505. The following linear gradient was used: 65% A, in acetonitrile. Column: Waters Xterra MS C-18x3 mm id 5 35% B to 25% A, 65% Bover 16 minutes at a flow-rate of 0.35 um. Gradient: 5%-90% B over 7.5 min at 1.5 ml/min. ml/min. Method: LCMS 4 Method: 04 A2 1 0506. The RP-analysis was performed using a Waters 0495 LCMS 4 was performed on a setup consisting of UPLC system fitted with a dual band detector. UV detections Waters Acquity UPLC system and LCT Premier XE mass at 214 nm and 254 nm were collected using an ACQUITY spectrometer from Micromass.” UPLC BEH130, C18, 130 A, 1.7 um, 2.1 mmx150 mm col 0496 Eluents: A: 0.1% Formic acid in water umn, 40°C. The UPLC system was connected to two eluent 0497 B: 0.1% Formic acid in acetonitrile The analysis reservoirs containing: A: 90% HO, 10% CHCN, 0.25 M was performed at RT by injecting an appropriate Volume ammonium bicarbonate; B: 70% CHCN, 30% HO. The of the sample (preferably 2-10 ul) onto the column following linear gradient was used: 90% A, 10% B to 60% A. which was eluted with a gradient of A and B. The UPLC 40% B over 16 minutes at a flow-rate of 0.40 ml/min. conditions, detector settings and mass spectrometer set tings were: Column: Waters Acquity UPLCBEH, C-18, Method: 04 A6 1 1.7 m, 2.1 mmx50 mm. Gradient: Linear 5%-95% 0507. The RP-analysis was performed using a Waters acetonitrile during 4.0 min (alternatively 8.0 min) at 0.4 UPLC system fitted with a dual band detector. UV detections ml/min. Detection: 214 nm (analogue output from TUV at 214 nm and 254 nm were collected using an ACQUITY (Tunable UV detector)) MS ionisation mode: API-ES UPLC BEH130, C18, 130 A, 1.7 um, 2.1 mmx150 mm col 0498 Scan: 100-2000 amu (alternatively 500-2000 umn, 40°C. The UPLC system was connected to two eluent amu), step 0.1 amu. reservoirs containing: A: 10 mM TRIS, 15 mM ammonium sulphate,80%HO, 20%, pH 7.3; B: 80% CHCN, 20% H.O. Method: LCMS AP The following linear gradient was used: 95% A, 5% B to 10% 0499. A Micromass Quatro micro API mass spectrometer A, 90% B over 16 minutes at a flow-rate of 0.35 ml/min. was used to identify the mass of the sample after elution from a HPLC system composed of Waters 2525 binary gradient Method: 04 A7 1 modul, Waters 2767 sample manager, Waters 2996 Photo 0508. The RP-analysis was performed using a Waters diode Array Detector and Waters 2420 ELS. Detector. Eluents: UPLC system fitted with a dual band detector. UV detections A: 0.1% Trifluoro acetic acid in water, B: 0.1% Trifluoro at 214 nm and 254 nm were collected using an ACQUITY acetic acid in acetonitrile. Column: Phenomenex Synergi UPLC BEH130, C18, 130 A, 1.7 um, 2.1 mmx150 mm col MAXRP 4 um, 75x4.6 mm. Gradient: 5%-95% B over 7 min umn, 40°C. The UPLC system was connected to two eluent at 1.0 ml/min. reservoirs containing: A: 10 mM TRIS, 15 mM ammonium US 2016/0002311 A1 Jan. 7, 2016 50 sulphate, 80% HO, 20%, pH 7.3; B: 80% CHCN, 20% H.O. linear gradient was used: 40% A, 60% B to 20% A, 80% B The following linear gradient was used: 95% A, 5% B to 40% over 8 minutes at a flow-rate of 0.40 ml/min. A, 60% B over 16 minutes at a flow-rate of 0.40 ml/min. Method: 07 B4 1 Method 05 B5 1 0509 UPLC (method 05 B5 1): The RP-analysis was 0516. The RP-analysis was performed using a Waters performed using a Waters UPLC system fitted with a dual UPLC system fitted with a dual band detector. UV detections band detector. UV detections at 214 nm and 254 nm were at 214 nm and 254 nm were collected using an ACQUITY collected using an ACQUITY UPLC BEH 130, C18, 130 A, UPLC BEH130, C18, 130 A, 1.7 um, 2.1 mmx150 mm col 1.7 um, 2.1 mmx150 mm column, 40°C. umn, 40°C. 0510. The UPLC system was connected to two eluent 0517. The UPLC system was connected to two eluent reservoirs containing: reservoirs containing: A: 99.95% HO, 0.05% TFA: B: 99.95% CHCN, 0.05% TFA. The following linear gradient A: 0.2 MNaSO, 0.04 MHPO, 10% CHCN (pH 3.5) was used: 95% A, 5% B to 5% A, 95% B over 16 minutes at a flow-rate of 0.40 ml/min. B: 70% CHCN, 30% HO 0511. The following linear gradient was used: 60% A, Method: 09 B2 1 40% B to 30% A, 70% B over 8 minutes at a flow-rate of 0.35 0518. The RP-analysis was performed using a Waters ml/min. UPLC system fitted with a dual band detector. UV detections at 214 nm and 254 nm were collected using an ACQUITY Method: 05 B7 1 UPLC BEH130, C18, 130 A, 1.7 um, 2.1 mmx150 mm col 0512. The RP-analysis was performed using a Waters umn, 40°C. The UPLC system was connected to two eluent UPLC system fitted with a dual band detector. UV detections reservoirs containing: A: 99.95% HO, 0.05% TFA: B: at 214 nm and 254 nm were collected using an ACQUITY 99.95% CHCN, 0.05% TFA. The following linear gradient UPLC BEH130, C18, 130 A, 1.7 um, 2.1 mmx150 mm col was used: 95% A, 5% B to 40% A, 60% B over 16 minutes at umn, 40°C. The UPLC system was connected to two eluent a flow-rate of 0.40 ml/min. reservoirs containing: A: 0.2 MNaSO, 0.04 MHPO, 10% CHCN (pH 3.5); B: 70% CHCN, 30% H.O.The following Method: 09 B4 1 linear gradient was used: 80% A, 20% B to 40% A, 60% B over 8 minutes at a flow-rate of 0.40 ml/min. 0519. The RP-analysis was performed using a Waters UPLC system fitted with a dual band detector. UV detections Method: 05 B8 1 at 214 nm and 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 A, 1.7 um, 2.1 mmx150 mm col 0513. The RP-analysis was performed using a Waters umn, 40°C. The UPLC system was connected to two eluent UPLC system fitted with a dual band detector. UV detections reservoirs containing: A: 99.95% HO, 0.05% TFA: B: at 214 nm and 254 nm were collected using an ACQUITY 99.95% CHCN, 0.05% TFA. The following linear gradient UPLC BEH130, C18, 130 A, 1.7 um, 2.1 mmx150 mm col was used: 95% A, 5% B to 5% A, 95% B over 16 minutes at umn, 40°C. The UPLC system was connected to two eluent a flow-rate of 0.40 ml/min. reservoirs containing: A: 0.2 MNaSO, 0.04 MHPO, 10% CHCN (pH 3.5); B: 70% CHCN, 30% H.O.The following linear gradient was used: 50% A, 50% B to 20% A, 80% B Method 08 B2 1 over 8 minutes at a flow-rate of 0.40 ml/min. 0520 UPLC (method 08 B2 1): The RP-analysis was performed using a Waters UPLC system fitted with a dual Method: 05 B9 1 band detector. UV detections at 214 nm and 254 nm were 0514. The RP-analysis was performed using a Waters collected using an ACQUITY UPLC BEH 130, C18, 130 A, UPLC system fitted with a dual band detector. UV detections 1.7 um, 2.1 mmx150 mm column, 40°C. at 214 nm and 254 nm were collected using an ACQUITY 0521. The UPLC system was connected to two eluent UPLC BEH130, C18, 130 A, 1.7 um, 2.1 mmx150 mm col reservoirs containing: umn, 40°C. The UPLC system was connected to two eluent reservoirs containing: A: 0.2 MNaSO, 0.04 MHPO, 10% CHCN (pH 3.5); B: 70% CHCN, 30% H.O.The following A: 99.95% HO, 0.05%TFA linear gradient was used: 70% A, 30% B to 20% A, 80% B over 8 minutes at a flow-rate of 0.40 ml/min. B: 99.95% CHCN, 0.05%TFA 0522 The following linear gradient was used: 95% A, 5% Method: 05 B10 1 B to 40% A, 60% B over 16 minutes at a flow-rate of 0.40 0515. The RP-analyses was performed using a Waters ml/min. UPLC system fitted with a dual band detector. UV detections at 214 nm and 254 nm were collected using an ACQUITY Method 08 B4 1 UPLC BEH130, C18, 130 A, 1.7 um, 2.1 mmx150 mm col umn, 40°C. The UPLC system was connected to two eluent 0523 UPLC (method 08 B4 1): The RP-analysis was reservoirs containing: A: 0.2 MNaSO, 0.04 MHPO, 10% performed using a Waters UPLC system fitted with a dual CHCN (pH 3.5); B: 70% CHCN, 30% HO. The following band detector. UV detections at 214 nm and 254 nm were US 2016/0002311 A1 Jan. 7, 2016 51 collected using an ACQUITY UPLC BEH 130, C18, 130 A, voirs containing: A: 99.95% HO, 0.05% TFA; B: 99.95% 1.7 um, 2.1 mmx150 mm column, 40°C. CHCN, 0.05%TFA. The following linear gradient was used: 0524. The UPLC system was connected to two eluent 70% A, 30% B to 40% A, 60% B over 12 minutes at a reservoirs containing: flow-rate of 0.40 ml/min. Method: AP B4 1 A: 99.95% HO, 0.05%TFA 0533. The RP-analysis was performed using a Waters UPLC system fitted with a dual band detector. UV detections at 214 nm and 254 nm were collected using an ACQUITY B: 99.95% CHCN, 0.05%TFA UPLC BEH130, C18, 130 A, 1.7 um, 2.1 mmx150 mm col umn, 30°C. The UPLC system was connected to two eluent 0525. The following linear gradient was used: 95% A, 5% reservoirs containing: A: 99.95% HO, 0.05% TFA: B: B to 5% A, 95% B over 16 minutes at a flow-rate of 0.40 99.95% CHCN, 0.05% TFA. The following linear gradient ml/min. was used: 95% A, 5% B to 5% A, 95% B over 16 minutes at a flow-rate of 0.30 ml/min. Method 10 B4 2 Example 1 0526 UPLC (method 08 B4 1): The RP-analysis was N'-(2-2-[2-(4S)-5-hydroxy-4-(18-hydroxy performed using a Waters UPLC system fitted with a dual 18-oxooctadecanoyl)amino5-oxopentanoylamino band detector. UV detections at 214 nm and 254 nm were ethoxyethoxyacetylaminoethoxyethoxyacetyl) collected using an ACQUITY UPLC BEH 130, C18, 130 A, D-Ser-Lys, Leu7|Glucagon 1.7 um, 2.1 mmx150 mm column, 50° C. 0534
OH 1 O H WLLN T-OH H-H-N1)-QGO TFTs D Y S KYLDS R R A QDF V-N O for---~.N O l to-N-or-H u ^-N-nor NH O O H O
0527. The UPLC system was connected to two eluent 0535 The peptide was prepared essentially as described in reservoirs containing: SPPS method A and Busing 2-2-2-2-2-[2-(4S)-5-tert butoxy-4-(18-tert-butoxy-18-oxo-octadecanoyl)amino-5- A: 99.95% HO, 0.05%TFA oXo-pentanoylaminoethoxyethoxyacetylaminoethoxy B: 99.95% CHCN, 0.05%TFA ethoxyacetic acid. 0536 UPLC 08 B4 1:8.3 min 0528. The following linear gradient was used: 95% A, 5% 0537 UPLC 04 A4 1: 6.3 min B to 5% A, 95% B over 12 minutes at a flow-rate of 0.40 0538 UPLC 05 B5 1:5.8 min ml/min. 0539 LCMS 4: m/z 1494.8 (M+3H)3+, 1046.6 (M+4H) Method 10 B5 2 4+, 837.5 (M+5)5+ 0529 UPLC (method 08 B4 1): The RP-analysis was Preparation of building block 2-2-2-2-2-2- performed using a Waters UPLC system fitted with a dual (4S)-5-tert-butoxy-4-(18-tert-butoxy-18-oxo-octa band detector. UV detections at 214 nm and 254 nm were decanoyl)amino-5-oxo-pentanoyl)aminoethoxy collected using an ACQUITY UPLC BEH 130, C18, 130 A, ethoxyacetylaminoethoxyethoxyacetic acid 1.7 um, 2.1 mmx150 mm column, 50° C. 0530. The UPLC system was connected to two eluent reservoirs containing: A: 70% MeCN, 30% Water B: 0.2M NaSO, 0.04 MHPO, 10% MeCN, pH 2.25 0531. The following linear gradient was used: 40% A in 1 min, 40->70% A in 7 min at a flow-rate of 0.40 ml/min. Curon, 1. Method: 10 B14 1 0532. The RP-analyses was performed using a Waters UPLC system fitted with a dual band detector. UV detections at 214 nm and 254 nm were collected using an ACQUITY UPLC BEH ShieldRP18, 1.7 um, 2.1 mmx 150 mm column, -ururuO 50° C. The UPLC system was connected to two eluent reser US 2016/0002311 A1 Jan. 7, 2016 52
(0541 2-Chlorotrityl resin 100-200 mesh (42.6 g., 42.6 ethylamine (16.7 mL. 95.7 mmol) in N.N- mmol) was left to swell in dry dichloromethane (205 mL) for dimethylformamide/dichloromethane mixture (1:4, 200 mL) 20 min. A solution of {2-[2-(9H-fluoren-9-ylmethoxycarbo was added to resin. Resin was shaken for 2 hrs, filtered and nylamino)-ethoxy-ethoxy-acetic acid (13.7g, 35.5 mmol) washed with N.N-dimethylformamide (3x150 mL), dichlo and N,N-diisopropylethylamine (23.5 mL, 135 mmol) in dry romethane (2x150 mL), methanol (2x150 mL) and dichlo dichloromethane (30 mL) was added to resin and the mixture romethane (300 mL, 6x150 mL). The product was cleaved was shaken for 3 hrs. Resin was filtered and treated with a from resin by treatment with 2.2.2-trifluoethanol (200 mL) solution of N,N-diisopropylethylamine (12.4 mL, 70.9 for 19 hrs. Resin was filtered off and washed with dichlo mmol) in methanol/dichloromethane mixture (4:1, 250 mL. romethane (2x150 mL), 2-propanol/dichloromethane mix 2x5 min). Then resin was washed with N,N-dimethylforma ture (1:1, 2x150 mL), 2-propanol (150 mL) and dichlo romethane (2x150 mL). Solutions were combined; solvent mide (2x150 mL), dichloromethane (3x150 mL) and N,N- evaporated and crude product was purified by flash column dimethylformamide (3x150 mL). Fmoc group was removed chromatography (Silicagel 60, 0.040-0.060 mm; eluent: by treatment with 20% piperidine in dimethylformamide dichloromethane/methanol 1:0-9:1). Pure product was dried (1x5 min, 1x30 min, 2x150 mL). Resin was washed with N,N-dimethylformamide (3x150 mL), 2-propanol (2x150 in vacuo and obtained as yellow oil. (0542. Yield: 25.85 g (86%). mL) and dichloromethane (200 mL, 2x150 mL). Solution of (0543. R. (SiO, chloroform/methanol 85:15): 0.25. {2-2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethoxy (0544 'H NMR spectrum (300 MHz, CDC1,): 7.38 (bs, ethoxy-acetic acid (20.5g, 53.2 mmol), O-(6-chloro-benzo 1H); 7.08 (bs, 1H); 6.61 (d. J–7.5 Hz, 1H); 4.43 (m. 1H); 4.15 triazol-1-yl)-N.N.N',N'-tetramethyluronium tetrafluorobo (s. 2H); 4.01 (s. 2H); 3.78-3.39 (m, 16H); 2.31 (t, J=6.9 Hz, rate (TCTU, 18.9 g, 53.2 mmol) and N,N- 2H); 2.27-2.09 (m, 5H); 2.01-1.84 (m, 1H); 1.69-1.50 (m, diisopropylethylamine (16.7 mL. 95.7 mmol) in N.N- 4H); 1.46 (s.9H); 1.43 (s.9H); 1.24 (bs, 24H). dimethylformamide (100 mL) and dichloromethane (50 mL) (0545 LC-MS purity: 100%. was added to resin and mixture was shaken for 1 hr. Resin was (0546 LC-MS Rt (Sunfire 4.6 mmx100 mm, acetonitrile/ filtered and washed with N,N-dimethylformamide (2x150 water 60:40 to 0:100+0.1%FA): 7.89 min. LC-MS m/z: 846.6 mL), dichloromethane (3x150 mL) and N,N-dimethylforma (M+H)". mide (155 mL). Fmoc group was removed by treatment with 20% piperidine in dimethylformamide (1x5 min, 1x30 min, Example 2 2x150 mL). Resin was washed with N,N-dimethylformamide N'-(2-2-[2-(4S)-5-hydroxy-4-(18-hydroxy-18 (3x150 mL), 2-propanol (2x150 mL) and dichloromethane oxooctadecanoyl)amino5-oxopentanoylamino (200 mL, 2x150 mL). Solution of Fmoc-Glu-OtBu (22.6 g. ethoxyethoxyacetylaminoethoxyethoxyacetylD 53.2 mmol), O-(6-chloro-benzotriazol-1-yl)-N.N.N',N'-tet D-Ser'.Glu'.Lys’.Leu’Glucagon ramethyluronium tetrafluoroborate (TCTU, 18.9 g, 53.2 mmol) and N,N-diisopropylethylamine (16.7 mL. 95.7 0547
OH 1 O H WLLN T-OH H-H-N1)-QGO TFTs D Y S K YLE R R A QDF v-N O HO H S^-n-n-n-n-n-n-n-l O l O "...Y N-o~~~~~.H O O O mmol) in N,N-dimethylformamide (155 mL) was added to 0548. The peptide was prepared essentially as described in resin and mixture was shaken for 1 hr. Resin was filtered and SPPS method A and Busing 2-2-2-2-2-[2-(4S)-5-tert washed with N,N-dimethylformamide (2x150 mL), dichlo butoxy-4-(18-tert-butoxy-18-oxo-octadecanoyl)amino-5- romethane (2x150 mL) and N,N-dimethylformamide (150 oXo-pentanoylaminoethoxyethoxyacetylaminoethoxy mL). Fmoc group was removed by treatment with 20% pip ethoxyacetic acid. eridine in dimethylformamide (1x5 min, 1x30 min, 2x150 (0549 UPLC 08 B4 1: 8.4 min mL). Resin was washed with N,N-dimethylformamide 0550 UPLC 08 B2 1: 12.6 min (3x150 mL), 2-propanol (2x150 mL) and dichloromethane (200 mL, 2x150 mL). Solution of octadecanedioic acid 0551 UPLC 05 B5 1: 6.2 min mono-tert-butyl ester (19.7g, 53.2 mmol), O-(6-chloro-ben 05:52 UPLC 04 A3 1: 9.3 min Zotriazol-1-yl)-N.N.N',N'-tetramethyluronium tetrafluo 1056.08 roborate (TCTU, 18.9 g, 53.2 mmol) and N,N-diisopropyl
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0847 The peptide was prepared essentially as described in SPPS method A and Busing 2-2-2-2-2-[2-(4S)-5-tert butoxy-4-(18-tert-butoxy-18-oxo-octadecanoyl)amino-5- oXo-pentanoylaminoethoxyethoxyacetylaminoethoxy ethoxyacetic acid. 0848 UPLC 08 B4 1: Rt=8.3 0849 UPLC 05 B5 1: Rt=5.0 0850 LCMS m/z.: 1382.18 ((M/3)+3); 1036.89 ((M/4)+ 4); 829.72 ((M/5)+5). Example 58 N'-(2-2-2-2-2-[2-(4S)-5-hydroxy-4-(18 hydroxy-18-oxooctadecanoyl)amino-5-oxopen tanoylaminoethoxyethoxyacetylaminoethoxy ethoxyacetyl)(Glu'.Lys.Arg, Leu'Glucagon 0851
non-n-n-n-n-n-n-n-nO --~H n-nor O n-1No.1n 1 NH O OH
H-HSQGTFTSDYS KYLDSRRAQEFV-N RLLNT-OH
0852. The peptide was prepared essentially as described in SPPS method A and Busing 2-2-2-2-2-[2-(4S)-5-tert butoxy-4-(18-tert-butoxy-18-oxo-octadecanoyl)amino-5- oXo-pentanoylaminoethoxyethoxyacetylaminoethoxy ethoxyacetic acid. 0853 UPLC 08 B4 1: Rt=8.55 0854 LCMS 4: 4164.8 Example 59 N'-(Lys'7.Lys' Glu'...Leu7|Glucagonyl)N-(2-2- 2-2-2-2-(4S)-5-hydroxy-4-(18-hydroxy-18 oxooctadecanoyl)amino-5-oxopentanoylamino ethoxyethoxyacetylaminoethoxyethoxy-acetyl) Lysin 0855
no-n-n-n-n-n-n-n-nO --~H n-nor O n-1-o-1-1 NH O OH
OH H-HSQGTFTS DYSKYLDSKKAQEFVQWLLNT-N
0856. The peptide was prepared essentially as described in Example 60 SPPS method A and Busing 2-2-2-2-2-[2-(4S)-5-tert butoxy-4-(18-tert-butoxy-18-oxo-octadecanoyl)amino-5- ThT Fibrillation Assays for the Assessment of oXo-pentanoylaminoethoxyethoxyacetylaminoethoxy Physical Stability of Protein Formulations ethoxyacetic acid. 0859 Low physical stability of a peptide may lead to 0857 UPLC 08 B4 1: Rt=8.45 amyloid fibril formation, which is observed as well-ordered, 0858 LCMS 4:4266.5 thread-like macromolecular structures in the sample eventu US 2016/0002311 A1 Jan. 7, 2016 79 ally resulting in gel formation. This has traditionally been 0868. The concentration of peptide in the initial sample measured by visual inspection of the sample. However, that and in the filtered supernatant was determined by reverse kind of measurement is very Subjective and depending on the phase HPLC using an appropriate standard as reference. The observer. Therefore, the application of a small molecule indi percentage fraction the concentration of the filtered sample cator probe is much more advantageous. Thioflavin T (ThT) constituted of the initial sample concentration was reported as is such a probe and has a distinct fluorescence signature when the recovery. binding to fibrils Naiki et al. (1989) Anal. Biochem. 177, 0869. The measurement points were saved in Microsoft 244-249; LeVine (1999) Methods. Enzymol. 309, 274-284). Excel format for further processing and curve drawing and 0860. The time course for fibril formation can be fitting was performed using GraphPad Prism. The back described by a sigmoidal curve with the following expression ground emission from ThT in the absence of fibrils was neg Nielsen et al. (2001) Biochemistry 40, 6036-6046: ligible. The data points are typically a mean of four or eight samples and shown with standard deviation error bars. Only ff + m fit Eq. (1) data obtained in the same experiment (i.e. samples on the same plate) are presented in the same graph ensuring a rela tive measure of fibrillation between experiments. 0861. Here, F is the ThT fluorescence at the time t, as 0870. The data set may be fitted to Eq. (1). However, the depicted in FIG. 16. The constant t0 is the time needed to lag time before fibrillation may be assessed by visual inspec reach 50% of maximum fluorescence. The two important tion of the curve identifying the time point at which ThT parameters describing fibril formation are the lag-time calcu fluorescence increases significantly above the background lated by t0-2t and the apparent rate constant kapp 1?t. level. 0862 Formation of a partially folded intermediate of the peptide is suggested as a general initiating mechanism for Example 61 fibrillation. Few of those intermediates nucleate to form a template onto which further intermediates may assembly and Peptide Solubility the fibrillation proceeds. The lag-time corresponds to the interval in which the critical mass of nucleus is built up and 0871. The solubility of peptides and proteins depends on the apparent rate constant is the rate with which the fibril itself the pH of the solution. Often a protein or peptide precipitates is formed. at or close to its isoelectric point (pl), at which its net charge 0863 Samples were prepared freshly before each assay. is zero. At low pH (i.e. lower than the pl) proteins and peptides Each sample composition is described in the legends. The pH are typically positively charged, at pH higher than the p they of the sample was adjusted to the desired value using appro are negatively charged. priate amounts of concentrated NaOH and HC1. Thioflavin T 0872. It is advantageous for a therapeutic peptide if it is was added to the samples from a stock solution in HO to a soluble in a Sufficient concentration at a given pH, which is final concentration of 1 uM. suitable for both formulating a stable drug product and for 0864 Sample aliquots of 200 ul were placed in a 96 well administrating the drug product to the patient e.g. by Subcu microtiterplate (Packard OptiPlateTM-96, white polystyrene). taneous injection. Usually, four or eight replica of each sample (corresponding to one test condition) were placed in one column of wells. The 0873 Solubility versus pH curves were measured as plate was sealed with Scotch Pad (Qiagen). described: A formulation or a peptide solution in water was 0865 Incubation at given temperature, shaking and mea prepared and aliquots were adjusted to pH values in the surement of the ThT fluorescence emission were done in a desired range by adding HCl and NaOH. These samples were Fluoroskan Ascent FL fluorescence platereader (Thermo left equilibrating at room temperature for 2-3 days. Then the Labsystems). The temperature was adjusted to the desired samples were centrifuged. A Small aliquot of each sample was value, typically 30° C. or 37°C. The plate was either incu withdrawn for reverse HPLC analysis for determination of bated without shaking (no external physical stress) or with orbital shaking adjusted to 960 rpm with an amplitude of 1 the concentration of the proteins in solution. The pH of each mm. Fluorescence measurement was done using excitation sample was measured after the centrifugation, and the con through a 444 nm filter and measurement of emission through centration of each protein was depicted versus the measured a 485 nm filter. pH. 0866. Each run was initiated by incubating the plate at the assay temperature for 10 min. The plate was measured every Example 62 20 minutes for a desired period of time. Between each mea Surement, the plate was shaken and heated as described. Peptide Solubility at pH 7.5 0867. After completion of the ThT assay the four or eight replica of each sample was pooled and centrifuged at 20000 0874. A solubility test at pH 7.5 of native glucagon and rpm for 30 minutes at 18°C. The supernatant was filtered glucagon analogues was performed in order to establish if the through a 0.22 Lum filter and an aliquot was transferred to a solubility of the glucagon analogues near physiological pH HPLC via1. was improved compared to native glucagon. US 2016/0002311 A1 Jan. 7, 2016
0875. A sample of native glucagon or glucagon analogue 0879 1. 250 uM glucagon analogue (Example 3), 10 mM (typical250 nmol) was added HEPES buffer (typical 1 mL) to Hepes pH 7.5 a nominal concentration of 250 uM. The mixture was kept for 0880 2.250 Mglucagon analogue (Example 3), 0.6 mM 1 hat room temperature and was occasionally shaken where insulin analogue G5, 0.5 mM Zn(Ac)2, 16 mM m-cresol, 16 upon a sample of 200 uL was taken from the solution. The mM phenol, 213 mM glycerol, pH 7.6 sample was centrifuged (6000 rpm, 5 min) whereupon the 0881 3.250 Mglucagon analogue (Example 3), 1.6 mM Supernatant was quantified using a chemiluminescent nitro GLP-1 analogue G1, 58 mM phenol, 10 mM phosphate pH gen specific HPLC detector (Antek 8060 HPLC-CLND). 8.15 0882 4.250 Mglucagon analogue (Example 3), 1.2 mM GLP-1 analogue G3, 58 mM phenol, 10 mM phosphate pH Example 63 7.4 0883) 5. 0.6 mM insulin analogue G5, 0.5 mM Zn(Ac)2, Peptide Solubility/Stability 16 mM m-cresol, 16 mM phenol, 213 mM glycerol, pH 7.6 0884 6.1.6 mM GLP-1 analogue G1, 58 mM phenol, 10 0876. A stability test of glucagon analogues was per mM phosphate pH 8.15 formed in order to establish if the stability of the solutions 0885 Formulation 2 was prepared by diluting an appro were improved compared to solutions of native glucagon. priate insulin analogue G5 stock solution in water, adding 0877. A sample of glucagon analogue (typical 250 nmol) m-cresol and phenol, and then adding Zinc acetate. The glu cagon analogue was added as the last component. Formula was added HEPES buffer (typical 1 mL) to a nominal con tion 5 was prepared in a similar fashion. centration of 250 uM. The mixture was kept for 1 h at room 0886. These 6 formulations were subjected to the ThT temperature and was occasionally shaken whereupon a fibrillation assay. Samples were incubated at 37° C. for 45 sample of 200 uL was taken from the solution. The sample hours and with vigorously shaking (960 rpm). Under these was centrifuged (6000 rpm, 5 min) and the Supernatant was conditions no samples exhibited any ThT fluorescence signal analyzed on a UPLC and the area under the peak (UV absorp and full recovery of both the glucagon analogue and the tion at 214 nm) was measured as t—0. Due to the poor solu combined peptides (GLP-1 analogue G3 was not analysed bility of glucagon at pH 7.5 a sample of glucagon (Glu due to technical reasons) were found in formulations. Thus caGen(R) hypokit, Novo Nordisk in water, 250 uM, pH 2-3)) co-formulating glucagon analogue (Example 3) with other was included for comparison. The solutions were kept at 30° peptides did not result in less stable formulations compared to C. for 6 days whereupon the solution was filtered (Millex(R)- the individual peptides (Formulations 1, 5, and 6). GV, 0.22um filter unit, Durapore(R) Membrane) and analyzed on a UPLC. The area under the peak (UV absorption at 214 Example 65 nm) was measured as t-6 days. Preparation of GLP-1 Derivatives Example 64 0887. The following GLP-1 compounds were prepared (all being derivatives of analogues of GLP-1 (7-37)): Co-Formulation of a Glucagon Analogue (Example Compound G1 3) with GLP-1 Analogue G1, GLP-1 Analogue G3 and Insulin Analogue G5 N-epsilon26-((S)-4-Carboxy-4-hexadecanoylamino butyryl)Arg34GLP-1-(7-37), which may also be 0878 Co-formulation of the glucagon analogue (Example designated Arg'Lys'(Ne-(y-glutamyl(Na-hexade 3) was investigated with a number of peptides with potential canoyl)))-GLP-1 (7-37)-OH for treatment of obesity and diabetes. The following formu lations were prepared: 0888
O H N n-n-n-n-n-n- NH O O OH
OH H- HAEGTFTSDVSSYLEGQAA-N Elswivic-1) O O
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0928 The peptide was prepared essentially as described in SPPS method A and C. 0929 UPLC Method: AP B4 1: Rt=9.11 min 0930 LCMS Method: LCMS AP: Rt. 9.04 min m/z: m/3=1366.62 Pharmacological Methods Assay (I) Glucagon Activity 0931. The glucagon receptor was cloned into HEK-293 cells having a membrane bound cAMP biosensor (AC TOneTM). The cells (14000 per well) were incubated (37° C. 5% CO2) overnight in 384-well plates. Next day the cells were loaded with a calcium responsive dye that only distrib uted into the cytoplasm. Probenecid, an inhibitor of the organic anion transporter, was added to prevent the dye from leaving the cell. A PDE inhibitor was added to prevent for matted cAMP from being degraded. The plates were placed into a FLIPRTETRA and the glucagon analogues were added. End point data were collected after 6 minutes. An increase in intracellular cAMP was proportional to an increased in cal cium concentrations in the cytoplasm. When calcium was bound the dry a fluorescence signal was generated. EC50 values were calculated in Prism5.
US 2016/0002311 A1 Jan. 7, 2016 88
OO (~~~~9<_^<,pºs:(~~~~~~~~"LH OHOOO US 2016/0002311 A1 Jan. 7, 2016 89
US 2016/0002311 A1 Jan. 7, 2016 90
O O
no-1s-1)-r^x-x)dovassa1xxsxas??oosh-n#I O „.^^^^^^^~~~~pºs:(~~~~~~~~~). OO