N-terminal modified FGF21 compounds

Abstract

Analogs of FGF21 carrying a modifying, substituted ethylene or benzyl group can be used for treatment or prevention of diabetes, dyslipidemia, obesity, cardiovascular diseases, metabolic syndrome, and/or Non Alcoholic Fatty Liver Disease (NAFLD).

Claims

1. A compound comprising an FGF21 polypeptide or an analogue thereof, derivatized with A-B-C-D- covalently attached to the alpha amino-group of the N-terminal amino acid of the FGF21 polypeptide (SEQ ID NO:1) or an analogue thereof, wherein the FGF21 analogue has at least 80% identity to SEQ ID NO:1; and wherein: A- is an element of formula I: ##STR00112## wherein n is 12, 14, or 16, and * is the point of attachment to -B-, -B- is an element of formula VIII ##STR00113## wherein b is 2, c is 0, * is the point of attachment to -C-, and # is the point of attachment to A-; -C- is an element of formula XI: ##STR00114## wherein p is 1, q is 2, * is the point of attachment to -D-, and # is the point of attachment to -B-; -D- is an element of formula XIV: ##STR00115## wherein s is 1, * is the point of attachment to the FGF21 polypeptide or an analogue thereof and # is the point of attachment to -C-; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein A-B-C-D- is selected from the group consisting of: ##STR00116##

3. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier.

4. The compound of claim 1, wherein the FGF21 analogue is a Gly-FGF21 analogue consisting of a Lysine (K) to Arginine (R) substitutions at residues 56, 59, 69 and 122 of the native FGF21 polypeptide (SEQ ID NO:1).

Description

DETAILED DESCRIPTION OF THIS INVENTION

(1) The present invention relates to derivatives of an FGF21 compound having one or two modifying groups of the formula A-B-C-D- covalently attached to the N-terminal alpha amino group in said FGF21 compound, wherein

(2) A- is an element of formula I, II or III:

(3) ##STR00002##
wherein n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, and * is the point of attachment to -B-;
-B- is absent, represents a bond or is selected from -B1-, -B2- or combinations thereof, wherein -B1- is an element of formula IV, V, VI or VII:

(4) ##STR00003##
wherein * is the point of attachment to -B2- or -C-, and # is the point of attachment to A- or -B2-; and
-B2- is an element of formula VIII, IX or X or a combination of up to four elements of formula VIII and/or formula IX and/or formula X:

(5) ##STR00004##
wherein b is 0, 1 or 2, c is 0, 1 or 2, with the proviso that b is 1 or 2 when c is 0, and b is 0 when c is 1 or 2, * is the point of attachment to -C-, -B1- or another -B2-, and # is the point of attachment to A-, -B1- or another -B2-;

(6) ##STR00005##
wherein d is 1 or 2, e is 0, 1 or 2, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-; or
#-NHSO.sub.2(CH.sub.2).sub.uCO.(formula X)
wherein u is 2, 3 or 4, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-;
-C- is absent, represents a bond or is an element of formula XI or XII:

(7) ##STR00006##
wherein p is 0, 1, 2, 3, 4, 5 or 6, q is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-; or

(8) ##STR00007##
wherein k is 0, 1, 2, 3, 4, 5, 11 or 23, m is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound; and # is the point of attachment to -B-;
-D- is an element of formula XIII, XIIIa, XIV or XV or two of these elements together:
#-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII),
#-NHCH(CH.sub.3).sub.2CH.sub.2*(formula XIIIa),

(9) ##STR00008##
wherein r is an integer in the range 1-5, or s is an integer in the range 0-5 or y is an integer in the range 0-5, * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-;
or a pharmaceutically acceptable salt thereof.

(10) In those cases where -D- is two of the elements of formula XIII, XIIIa, XIV or XV together, those two elements can be the same or different. In those cases where -D- is two of the elements of formula XIII, XIIIa, XIV or XV together, the point of attachment where those two elements are joined together is one attachment point identified by the symbol # from one of the elements of formula XIII, XIIIa, XIV or XV and one attachment point from the other element identified by the symbol * and in this joined element, * is the point of attachment from one of the elements of formula XIII, XIIIa, XIV or XV to the FGF21 compound, and # is the point of attachment from the other element to -C-.

(11) In those cases where it is mentioned that a first moiety is attached to a second moiety identified by a letter (such as A-, -B- or -C-) which is absent, said first moiety is attached to the following moiety which is present. To take one example: If it is mentioned that, for the moiety designated -D-, # is the point of attachment to -C- and -C- is absent, then the point of attachment identified by # of the moiety designated -D- is attached to the moiety designated -B-.

(12) In one aspect, the present invention relates to derivatives of an FGF21 compound having one or two modifying groups of the formula A-B-C-D- covalently attached to the N-terminal alpha amino group in said FGF21 compound, wherein

(13) A- is an element of formula I, II or III:

(14) ##STR00009##
wherein n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, and * is the point of attachment to -B-;
-B- is absent, represents a bond or is selected from -B1-, -B2- or combinations thereof, wherein -B1- is an element of formula IV, V, VI or VII:

(15) ##STR00010##
wherein * is the point of attachment to -B2- or -C-, and # is the point of attachment to A- or -B2-; and
-B2- is an element of formula VIII, IX or X or a combination of up to four elements of formula VIII and/or formula IX and/or formula X:

(16) ##STR00011##
wherein b is 0, 1 or 2, c is 0, 1 or 2, with the proviso that b is 1 or 2 when c is 0, and b is 0 when c is 1 or 2, * is the point of attachment to -C-, -B1- or another -B2-, and # is the point of attachment to A-, -B1- or another -B2-;

(17) ##STR00012##
wherein d is 1 or 2, e is 0, 1 or 2, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-; or
#-NHSO.sub.2(CH.sub.2).sub.uCO.(formula X)
wherein u is 2, 3 or 4, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-;
-C- is absent, represents a bond or is an element of formula XI or XII:

(18) ##STR00013##
wherein p is 0, 1, 2, 3, 4, 5 or 6, q is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-; or

(19) ##STR00014##
wherein k is 0, 1, 2, 3, 4, 5, 11 or 23, m is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound; and # is the point of attachment to -B-;
-D- is an element of formula XIII, XIV or XV:
#-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII) or

(20) ##STR00015##
wherein r is an integer in the range 1-5, or s is an integer in the range 0-5 or y is an integer in the range 0-5, * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-;
or a pharmaceutically acceptable salt thereof.

(21) In one embodiment of this invention, -D- is not an element of formula XIII as defined herein. In other words, -D- is an element of formula XIV or XV as defined herein.

(22) Examples of FGF21 compounds are FGF21 analogues comprising at least one of the following modifications as compared to SEQ ID NO:1: -1G, -1A, -1S, -1F, -1M, R17A, R17H, Q27E, Q28R, Q28E, A31E, R36A, R36H, K56R, K59R, K69R, S71C, D102E, D102N, D102T, N121Q, N121D, K122R, D159E, L166F, S167G, M168L, D169E, V169aT, des170, G170T, G170P, P171L, P171G, P171S, S172E, S172L, S172Q, Q173E, 173aA, Q173A, G174A, G174V, Y179F, A180E, des181, S181K, S181R and/or 182G.

(23) It has been found that the modifying moiety defined herein has a certain capability of binding in vivo to human serum albumin.

(24) Furthermore, the invention relates to pharmaceutical compositions comprising a therapeutically effective amount of the analogues or derivatives of the invention, and a pharmaceutically acceptable carrier; as well as to methods for treating a patient exhibiting diabetes, dyslipidemia, obesity, cardiovascular diseases, metabolic syndrome, and/or Non Alcoholic Fatty Liver Disease (NAFLD) comprising administering to the patient a therapeutically effective amount of an analogue, derivative or composition of the invention.

(25) The derivatives of the invention has one or two modifying groups of the formula A-B-C-D- covalently attached to the N-terminal alpha amino group of the FGF21 compound.

(26) The derivatives of this invention and the analogues of this invention shall have biological effect similar to or better than that of human FGF21, for example, in relation to glucose lowering effect, improvement in dyslipidemia, obesity, cardiovascular diseases, metabolic syndrome, and/or Non Alcoholic Fatty Liver Disease (NAFLD).

PREFERRED FEATURES OF THIS INVENTION

(27) The following are particular embodiments of the derivative of the invention, in particular of the first aspect of the invention, in which only one modifying group of the formula A-B-C-D- is covalently attached to the alpha amino group of the N-terminal amino acid of the FGF21 compound:

(28) In one embodiment, A- is an element of formula I, II or III:

(29) ##STR00016##
wherein n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, and * is the point of attachment to -B-;
-B- is absent, represents a bond or is selected from -B1-, -B2- or combinations thereof, wherein -B1- is an element of formula IV, V, VI or VII:

(30) ##STR00017##
wherein * is the point of attachment to -B2- or -C-, and # is the point of attachment to A- or -B2-; and
-B2- is an element of formula VIII, IX or X or a combination of up to four elements of formula VIII and/or formula IX and/or formula X:

(31) ##STR00018##
wherein b is 0, 1 or 2, c is 0, 1 or 2, with the proviso that b is 1 or 2 when c is 0, and b is 0 when c is 1 or 2, * is the point of attachment to -C-, -B1- or another -B2-, and # is the point of attachment to A-, -B1- or another -B2-;

(32) ##STR00019##
wherein d is 1 or 2, e is 0, 1 or 2, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-; or
#-NHSO.sub.2(CH.sub.2).sub.uCO.(formula X)
wherein u is 2, 3 or 4, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-;
-C- is absent, represents a bond or is an element of formula XI or XII:

(33) ##STR00020##
wherein p is 0, 1, 2, 3, 4, 5 or 6, q is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-; or

(34) ##STR00021##
wherein k is 0, 1, 2, 3, 4, 5, 11 or 23, m is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound; and # is the point of attachment to -B-;
-D- is an element of formula XIII, XIIIa, XIV or XV or two of these formulae together:
#-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII),
#-NHCH(CH.sub.3).sub.rCH.sub.2*(formula XIIIa),

(35) ##STR00022##
wherein r is an integer in the range 1-5, or s is an integer in the range 0-5 or y is an integer in the range 0-5, * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-;
or a pharmaceutically acceptable salt thereof.

(36) In another embodiment, A- is an element of formula I, II or III:

(37) ##STR00023##
wherein n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, and * is the point of attachment to -B-;
-B- is absent, represents a bond or is selected from -B1-, -B2- or combinations thereof, wherein -B1- is an element of formula IV, V, VI or VII:

(38) ##STR00024##
wherein * is the point of attachment to -B2- or -C-, and # is the point of attachment to A- or -B2-; and
-B2- is an element of formula VIII, IX or X or a combination of up to four elements of formula VIII and/or formula IX and/or formula X:

(39) ##STR00025##
wherein b is 0, 1 or 2, c is 0, 1 or 2, with the proviso that b is 1 or 2 when c is 0, and b is 0 when c is 1 or 2, * is the point of attachment to -C-, -B1- or another -B2-, and # is the point of attachment to A-, -B1- or another -B2-;

(40) ##STR00026##
wherein d is 1 or 2, e is 0, 1 or 2, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-; or
#-NHSO.sub.2(CH.sub.2).sub.uCO.(formula X)
wherein u is 2, 3 or 4, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-;
-C- is absent, represents a bond or is an element of formula XI or XII:

(41) ##STR00027##
wherein p is 0, 1, 2, 3, 4, 5 or 6, q is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-; or

(42) ##STR00028##
wherein k is 0, 1, 2, 3, 4, 5, 11 or 23, m is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound; and # is the point of attachment to -B-;
-D- is an element of formula XIII, XIV or XV:
#-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII) or

(43) ##STR00029##
wherein r is an integer in the range 1-5, or s is an integer in the range 0-5 or y is an integer in the range 0-5, * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-;
or a pharmaceutically acceptable salt thereof.

(44) In one embodiment, A- is an element of formula I:

(45) ##STR00030##
in which n is preferably 12, 14, 16 or 18, more preferred 12, 14 or 16, and wherein * is the point of attachment to -B-.

(46) In another embodiment, A- is an element of formula I:

(47) ##STR00031##
in which n is preferably 14, 16 or 18, and wherein * is the point of attachment to -B-.

(48) In one embodiment, -B- comprises -B1-, preferably an element of formula IV or V:

(49) ##STR00032##
and wherein * is the point of attachment to -B2- or -C-, and # is the point of attachment to A- or -B2-.

(50) In one embodiment, -B- comprises -B2-, preferably one or two elements of formula VIII:

(51) ##STR00033##
in which more preferably c is 0 and b is 2 (gamma-Glu), or c is 0 and b is 1 (beta-Asp), and wherein * is the point of attachment to -C-, -B1- or another -B2-, and # is the point of attachment to A-, -B1- or another -B2-.

(52) In one embodiment, -B- comprises -B2-, preferably four elements of formula VIII:

(53) ##STR00034##
in which more preferably c is 0 and b is 2, and wherein * is the point of attachment to -C-, -B1- or another -B2-, and # is the point of attachment to A-, -B1- or another -B2-.

(54) In one embodiment, -B- comprises -B2-, preferably one or two elements of formula IX:

(55) ##STR00035##
in which more preferably d is 1 and e is 2, or d is 2 and e is 1, and wherein * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-.

(56) In one embodiment, -B- comprises -B2-, preferably one or two elements of formula X: #-NHSO.sub.2(CH.sub.2).sub.uCO. (formula X), in which more preferably u is 3, and wherein * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-.

(57) In one embodiment, -C- is an element of formula XI:

(58) ##STR00036##
in which preferably q is 0, 1, 2, 3 or 4, more preferably q is 1 or 2, and wherein * is the point of attachment to -D-, and # is the point of attachment to -B-.

(59) In one embodiment, -C- is an element of formula XI:

(60) ##STR00037##
in which preferably p is 1 or 2, more preferably p is 1, and wherein * is the point of attachment to -D-, and # is the point of attachment to -B-.

(61) In one embodiment, -C- is an element of formula XI:

(62) ##STR00038##
in which p is 1 and q is 1 or 2, preferably p is 1 and q is 2, and wherein * is the point of attachment to -D-, and # is the point of attachment to -B-.

(63) In one embodiment, -C- is an element of formula XII:

(64) ##STR00039##
in which preferably m is 0, 1 or 2, more preferably m is 1 or 2, and wherein * is the point of attachment to -D-, and # is the point of attachment to -B-.

(65) In one embodiment, -C- is an element of formula XII:

(66) ##STR00040##
in which preferably k is 1, 2, 3, 4, 5 or 11, more preferably k is 5, even more preferably m is 1 and k is 4, 5 or 11, or most preferably m is 1 and k is 5, and wherein * is the point of attachment to -D-, and # is the point of attachment to -B-.

(67) In one embodiment, -D- is an element of formula XIII, XIIIa, XIV or XV or two of these elements together:
#-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII)
#-NHCH(CH.sub.3).sub.2CH.sub.2*(formula XIIIa) or

(68) ##STR00041##
wherein r is an integer in the range 1-5, or s is an integer in the range 0-5 or y is an integer in the range 0-5, * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-.

(69) In those cases where -D- is two of the elements of formula XIII, XIIIa, XIV or XV together, those two elements can be the same or different. In those cases where -D- is two of the elements of formula XIII, XIIIa, XIV or XV together, the point of attachment where those two elements are joined together is one attachment point identified by the symbol # from one of the elements of formula XIII, XIIIa, XIV or XV and one attachment point from the other element identified by the symbol * and in this joined element, * is the point of attachment from one of the elements of formula XIII, XIIIa, XIV or XV to the FGF21 compound, and # is the point of attachment from the other element to -C-.

(70) In one embodiment, -D- is an element of formula XIII, XIV or XV:
#-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII) or

(71) ##STR00042##
wherein r is an integer in the range 1-5, or s is an integer in the range 0-5 or y is an integer in the range 0-5, * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-.

(72) In one embodiment of this invention, -D- is not an element of formula XIII as defined herein. In other words, -D- is an element of formula XIV or XV as defined herein.

(73) In one embodiment, -D- is an element of formula XIII
#-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII)
in which preferably r is 1, 2 or 3, more preferably r is 1, and wherein # is the point of attachment to -C- and * is the point of attachment to the FGF21 compound.

(74) In one embodiment, -D- is an element of formula XIV

(75) ##STR00043##
in which preferably s is 0 or 1, more preferably s is 1, and wherein * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-.

(76) In one embodiment, -D- is an element of formula XV

(77) ##STR00044##
in which preferably y is 0 or 1, more preferably y is 1, and wherein * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-.

(78) In one embodiment, -D- is the reaction product of the FGF21 compound with an aldehyde e.g. but not limited to an element of formula XVI or XVII:
#-NH(CH.sub.2).sub.tCHO(formula XVI)

(79) ##STR00045##
wherein # is the point of attachment to -C- or -B-, t is 1, 2, 3, 4 or 5, more preferably t is 1, 2 or 3 or most preferably t is 1 or preferably u is 0 or 1, more preferably u is 1.

(80) In one embodiment, -D- is the reaction product of the FGF21 compound with an aldehyde precursor, which is activated in situ, e.g. but not limited to an element of formula XVIII:

(81) ##STR00046##
# is the point of attachment to -C- or -B-, v is 1, 2, 3, 4 or 5, and preferably x is 1 or 2, more preferably v is 1, 2 or 3 and preferably x is 1 or most preferably v is 1 and x is 1, and wherein * are the point of attachments to the FGF21 compound.

(82) In one embodiment, the moiety A-B-C-D- is selected from the following formulas (1-2)

(83) ##STR00047##
where * is the point of attachment to the FGF21 compound, or a pharmaceutically acceptable salts of the corresponding derivatives.

(84) The following are particular embodiments of the derivative of the invention, in particular of the second aspect of the invention, in which two modifying groups of the formula A-B-C-D- are covalently attached to the alpha amino-group of the N-terminal amino acid of the FGF21 compound:

(85) In an embodiment, A- is an element of formula I, II or III:

(86) ##STR00048##
wherein n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, and * is the point of attachment to -B-;
-B- is absent, represents a bond or is selected from -B1-, -B2- or combinations thereof, wherein -B1- is an element of formula IV, V, VI or VII:

(87) ##STR00049##
wherein * is the point of attachment to -B2- or -C-, and # is the point of attachment to A- or -B2-; and
-B2- is an element of formula VIII, IX or X or a combination of up to four elements of formula VIII and/or formula IX and/or formula X:

(88) ##STR00050##
wherein b is 0, 1 or 2, c is 0, 1 or 2, with the proviso that b is 1 or 2 when c is 0, and b is 0 when c is 1 or 2, * is the point of attachment to -C-, -B1- or another -B2-, and # is the point of attachment to A-, -B1- or another -B2-;

(89) ##STR00051##
wherein d is 1 or 2, e is 0, 1 or 2, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-; or
#-NHSO.sub.2(CH.sub.2).sub.uCO.(formula X)
wherein u is 2, 3 or 4, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-;
-C- is absent, represents a bond or is an element of formula XI or XII:

(90) ##STR00052##
wherein p is 0, 1, 2, 3, 4, 5 or 6, q is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-; or

(91) ##STR00053##
wherein k is 0, 1, 2, 3, 4, 5, 11 or 23, m is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound; and # is the point of attachment to -B-;
-D- is an element of formula XIII, XIIIa, XIV or XV or two of these elements together:
#-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII),
#-NHCH(CH.sub.3).sub.2CH.sub.2*(formula XIIIa),

(92) ##STR00054##
wherein r is an integer in the range 1-5, s is an integer in the range 0-5, and y is an integer in the range 0-5, * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-;
or a pharmaceutically acceptable salt thereof.

(93) In another embodiment, A- is an element of formula I, II or III:

(94) ##STR00055##
wherein n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, and * is the point of attachment to -B-;
-B- is absent, represents a bond or is selected from -B1-, -B2- or combinations thereof, wherein -B1- is an element of formula IV, V, VI or VII:

(95) ##STR00056##
wherein * is the point of attachment to -B2- or -C-, and # is the point of attachment to A- or -B2-; and
-B2- is an element of formula VIII, IX or X or a combination of up to four elements of formula VIII and/or formula IX and/or formula X:

(96) ##STR00057##
wherein b is 0, 1 or 2, c is 0, 1 or 2, with the proviso that b is 1 or 2 when c is 0, and b is 0 when c is 1 or 2, * is the point of attachment to -C-, -B1- or another -B2-, and # is the point of attachment to A-, -B1- or another -B2-;

(97) ##STR00058##
wherein d is 1 or 2, e is 0, 1 or 2, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-; or
#-NHSO.sub.2(CH.sub.2).sub.uCO.(formula X)
wherein u is 2, 3 or 4, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-;
-C- is absent, represents a bond or is an element of formula XI or XII:

(98) ##STR00059##
wherein p is 0, 1, 2, 3, 4, 5 or 6, q is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-; or

(99) ##STR00060##
wherein k is 0, 1, 2, 3, 4, 5, 11 or 23, m is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound; and # is the point of attachment to -B-;
-D- is an element of formula XIII, XIV or XV:
#-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII) or

(100) ##STR00061##
wherein r is an integer in the range 1-5, s is an integer in the range 0-5, and y is an integer in the range 0-5, * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-;
or a pharmaceutically acceptable salt thereof.

(101) In one embodiment, A- is an element of formula I:

(102) ##STR00062##
in which n is preferably 12, 14, 16 or 18, more preferred 12, 14 or 16, wherein * is the point of attachment to -B-.

(103) In another embodiment, A- is an element of formula I:

(104) ##STR00063##
in which n is preferably 14, 16 or 18, wherein * is the point of attachment to -B-.

(105) In one embodiment, -B- comprises -B1-, preferably an element of formula IV or V:

(106) ##STR00064##
and wherein * is the point of attachment to -B2- or -C-, and # is the point of attachment to A- or -B2-.

(107) In one embodiment, -B- comprises -B2-, preferably one or two elements of formula VIII:

(108) ##STR00065##
in which more preferably c is 0 and b is 2 (gamma-Glu), or c is 0 and b is 1 (beta-Asp), and wherein * is the point of attachment to -C-, -B1- or another -B2-, and # is the point of attachment to A-, -B1- or another -B2-.

(109) In one embodiment, -B- comprises -B2-, preferably one or two elements of formula IX:

(110) ##STR00066##
in which more preferably d is 1 and e is 2, or d is 2 and e is 1, and wherein * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-.

(111) In one embodiment, -B- comprises -B2-, preferably one or two elements of formula X: #-NHSO.sub.2(CH.sub.2).sub.uCO. (formula X), in which more preferably u is 3, and wherein * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-.

(112) In one embodiment, -C- is an element of formula XI:

(113) ##STR00067##
in which preferably q is 0, 1, 2, 3 or 4, more preferably q is 1 or 2, and wherein * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-

(114) In one embodiment, -C- is an element of formula XI:

(115) ##STR00068##
in which preferably p is 1 or 2, more preferably p is 1, and wherein * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-.

(116) In one embodiment, -C- is an element of formula XI:

(117) ##STR00069##
in which p is 1 and q is 1 or 2, preferably p is 1 and q is 2, and wherein * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-.

(118) In one embodiment, -C- is an element of formula XII:

(119) ##STR00070##
in which preferably m is 0, 1 or 2, more preferably m is 1 or 2, and wherein * is the point of attachment to -D-, and # is the point of attachment to -B-.

(120) In one embodiment, -C- is an element of formula XII:

(121) ##STR00071##
in which preferably k is 1, 2, 3, 4, 5 or 11, more preferably k is 5, even more preferably m is 1 and k is 4, 5 or 11, or most preferably m is 1 and k is 5, and wherein * is the point of attachment to -D-, and # is the point of attachment to -B-.

(122) In one embodiment of this invention, -D- is not an element of formula XIII as defined herein. In other words, -D- is an element of formula XIV or XV as defined herein.

(123) In one embodiment, -D- is an element of formula XIII
#-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII)
in which preferably r is 1, 2 or 3, more preferably r is 1, and wherein * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-.

(124) In one embodiment, -D- is an element of formula XIV

(125) ##STR00072##
in which preferably s is 0 or 1, more preferably s is 1, and wherein * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-.

(126) In one embodiment, -D- is an element of formula XV

(127) ##STR00073##
in which preferably y is 1, 2 or 3, more preferably y is 1, and wherein * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-.

(128) In one embodiment, -D- is the reaction product of the FGF21 compound with an aldehyde e.g. but not limited to an element of formula XV or XVI:
#-NH(CH.sub.2).sub.tCHO(formula XV)

(129) ##STR00074##
wherein # is the point of attachment to -C- or -B-, t is 1, 2, 3, 4 or 5, more preferably t is 1, 2 or 3 or most preferably t is 1 or preferably u is 0 or 1, more preferably u is 1.

(130) In one embodiment, -D- is the reaction product of the FGF21 compound with an aldehyde precursor, which is activated in situ, e.g. but not limited to an element of formula XVII:

(131) ##STR00075##
wherein # is the point of attachment to -C- or -B-, v is 1, 2, 3, 4 or 5, and preferably x is 1 or 2, more preferably v is 1, 2 or 3 and preferably x is 1 or most preferably v is 1 and x is 1, and wherein * are the point of attachments to the FGF21 compound.

(132) In one embodiment, the moiety A-B-C-D- is selected from the following formulas (1 or 3):

(133) ##STR00076##
wherein * is the point of attachment to the FGF21 compound, or a pharmaceutically acceptable salts of the corresponding derivatives.

(134) In one embodiment, the modifying groups are attached to the alpha-amino group of the N-terminal amino acid to form two alkylamine bonds.

(135) In one embodiment, the compound of this invention is a compound of example 3, 4, 5 and 6, preferably the following N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [Arg56, Arg59, Arg69, Arg122] Gly-FGF21 (Compound I); N-alpha-bis-(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [Arg56, Arg59, Arg69, Arg122] Gly-FGF21 (Compound II); N-alpha-(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]-ethoxy}ethoxy)acetylamino]methyl}benzyl) [K59R, K69R, D102T, D121Q, K12NR, L166F, S167G, M168L, G170T] Ala-FGF21 (Compound III); N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]-methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Ala-FGF21 (Compound IV); or a pharmaceutically acceptable salts of the corresponding derivatives.

(136) The following are additional particular embodiments of the derivative of the invention, as characterized by the structure of the constituent FGF21 compound:

(137) Preferably, the FGF21 compound has an identity of at least 80%, to SEQ ID NO:1, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%.

(138) Preferably, the FGF21 compound has a maximum of 36 amino acid changes (modifications) as compared to SEQ ID NO:1, preferably a maximum of 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid change(s); more preferably a maximum of 15, 14, 13, 12, 11 or 10 amino acid modifications; even more preferably a maximum of 9, 8, 7, 6 or 5 amino acid modifications; or most preferably a maximum of 4, 3, 2 or 1 amino acid modification(s).

(139) In one embodiment, the FGF21 compound comprises (a) at least one of the following modifications as compared to SEQ ID NO:1: -1G, -1A, -1S, -1F, -1M, R17A, R17H, Q27E, Q28R, Q28E, A31E, R36A, R36H, K56R, K59R, K69R, S71C, D102E, D102N, D102T, N121Q, N121D, K122R, D159E, L166F, S167G, M168L, D169E, V169aT, des170, G170T, G170P, P171L, P171G, P171S, S172E, S172L, S172Q, Q173E, Q173aA, Q173A, G174A, G174V, Y179F, A180E, des181, S181K, S181R and/or 182G.

(140) In one embodiment, the FGF21 compound comprises Y179F, A180E and des 181 (i); preferably it additionally comprises P171L, S172E, Q173A and G174V (ii); more preferably it additionally comprises L166F, S167G and M168L (iii); most preferably it comprises (i), (ii) and (iii). Each of these embodiments (i)-(iii) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(141) In one embodiment, the FGF21 compound comprises S71C (i), preferably it additionally comprises Y179F, A180E and des 181 (ii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii), (iii) and (iv). Each of these embodiments (i)-(iv) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(142) In one embodiment, the FGF21 compound comprises N121Q (i), Y179F, A180E and des 181 (Ii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii) (iii) and (iv). Each of these embodiments (i)-(iv) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(143) In one embodiment, the FGF21 compound comprises S71C (i), N121Q (ii), preferably it additionally comprises Y179F, A180E and des 181 (iii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iv); more preferably it additionally comprises L166F, S167G and M168L (v); most preferably it comprises (i), (ii), (iii), (iv) and (v). Each of these embodiments (i)-(iv) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(144) In one embodiment, the FGF21 compound comprises N121Q (i), Y179F, A180E and des 181 (ii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii), (iii) and (iv). Each of these embodiments (i)-(iii) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(145) In one embodiment, the FGF21 compound comprises S71C(i), N121Q (ii), Y179F, A180E and des 181 (iii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iv); more preferably it additionally comprises L166F, S167G and M168L (v); most preferably it comprises (i), (ii), (iii), (iv) and (v). Each of these embodiments (i)-(v) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(146) In one embodiment, the FGF21 compound comprises Y179F, A180E and des 181 (i); preferably it additionally comprises des170, 173aA and G174V (ii); more preferably it additionally comprises L166F, S167G and M168L (iii); most preferably it comprises (i), (ii) and (iii). Each of these embodiments (i)-(iii) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(147) In one embodiment, the FGF21 compound comprises S71C (i), preferably it additionally comprises Y179F, A180E and des 181 (ii); preferably it additionally comprises des170, 173aA and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii), (iii) and (iv). Each of these embodiments (i)-(iv) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(148) In one embodiment, the FGF21 compound comprises N121Q (i), Y179F, A180E and des 181 (ii); preferably it additionally comprises des170, 173aA and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii), (iii) and (iv). Each of these embodiments (i)-(iii) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(149) In one embodiment, the FGF21 compound comprises S71C(i), N121Q (ii), Y179F, A180E and des 181 (iii); preferably it additionally comprises des170, 173aA and G174V (iv); more preferably it additionally comprises L166F, S167G and M168L (v); most preferably it comprises (i), (ii), (iii), (iv) and (v). Each of these embodiments (i)-(v) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(150) In one embodiment, the FGF21 compound comprises Y179F and A180E (i); preferably it additionally comprises P171L, S172E, Q173A and G174V (ii); more preferably it additionally comprises L166F, S167G and M168L (iii); most preferably it comprises (i), (ii) and (iii). Each of these embodiments (i)-(iii) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(151) In one embodiment, the FGF21 compound comprises S71C (i), preferably it additionally comprises Y179F and A180E (ii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii), (iii) and (iv). Each of these embodiments (i)-(iv) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(152) In one embodiment, the FGF21 compound comprises N121Q (i), Y179F and A180E (ii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii) (iii) and (iv). Each of these embodiments (i)-(iv) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(153) In one embodiment, the FGF21 compound comprises S71C (i), N121Q (ii), preferably it additionally comprises Y179F and A180E (iii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iv); more preferably it additionally comprises L166F, S167G and M168L (v); most preferably it comprises (i), (ii), (iii), (iv) and (v). Each of these embodiments (i)-(iv) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(154) In one embodiment, the FGF21 compound comprises N121Q (i), Y179F and A180E (ii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii), (iii) and (iv). Each of these embodiments (i)-(iii) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(155) In one embodiment, the FGF21 compound comprises S71C(i), N121Q (ii), Y179F and A180E (iii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iv); more preferably it additionally comprises L166F, S167G and M168L (v); most preferably it comprises (i), (ii), (iii), (iv) and (v). Each of these embodiments (i)-(v) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(156) In one embodiment, the FGF21 compound comprises Y179F and A180E (i); preferably it additionally comprises des170, 173aA and G174V (ii); more preferably it additionally comprises L166F, S167G and M168L (iii); most preferably it comprises (i), (ii) and (iii). Each of these embodiments (i)-(iii) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(157) In one embodiment, the FGF21 compound comprises S71C (i), preferably it additionally comprises Y179F and A180E (ii); preferably it additionally comprises des170, 173aA and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii), (iii) and (iv). Each of these embodiments (i)-(iv) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(158) In one embodiment, the FGF21 compound comprises N121Q (i), Y179F and A180E (ii); preferably it additionally comprises des170, 173aA and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii), (iii) and (iv). Each of these embodiments (i)-(iii) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(159) In one embodiment, the FGF21 compound comprises S71C(i), N121Q (ii), Y179F and A180E (iii); preferably it additionally comprises des170, 173aA and G174V (iv); more preferably it additionally comprises L166F, S167G and M168L (v); most preferably it comprises (i), (ii), (iii), (iv) and (v). Each of these embodiments (i)-(v) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).

(160) In one embodiment, the FGF21 compound has a proline at position 146, wherein the position number refers to SEQ ID NO:1, which is an allelic wild type form of FGF21 known from the prior art.

(161) The following are additional particular embodiments of the derivative of the invention, as characterized by biological and chemical properties:

(162) In one embodiment, the derivative of this invention is protracted.

(163) The derivative of this invention preferably has an acceptable potency, cf. example 38 of the Experimental section herein.

(164) In one embodiment, the potency of the derivative of this invention is at least 1%, preferably at least 5%, more preferably at least 10%, even more preferably at least 20%, or most preferably at least 30% of the potency of Met-FGF21, wherein the potency is determined by measuring glucose uptake in 3T3-L1 adipocytes, vide example 38.

(165) In one embodiment, the potency of the derivative of this invention is at least 40%, preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, relative to the potency of Met-FGF21.

(166) In one embodiment, the potency of the derivative of this invention may even be at least 80%, preferably at least 90%, more preferably at least 100%, even more preferably at least 110%, or most preferably at least 120%, relative to the potency of Met-FGF21, vide example 38 below.

(167) For each of these embodiments the potency of the derivative of this invention is preferably determined by measuring glucose uptake in 3T3-L1 adipocytes.

(168) The potency of the derivative of this invention is calculated as the EC.sub.50 of the derivative relative to the EC.sub.50 of Met-FGF21.

(169) The 3T3-L1 adipocytes derive from mouse 3T3-L1 fibroblasts, preferably ATCC CL-173.

(170) The glucose uptake in 3T3-L1 adipocytes of the derivative of this invention may be measured as outlined in example 38.

(171) In one embodiment, the FGF21 analogue of this invention has a potency of at least 1% relative to the potency of Met-FGF21, wherein the potency is determined by measuring glucose uptake in 3T3-L1 adipocytes, vide example 38.

(172) In one embodiment, the FGF21 analogue of this invention has a potency of at least 5%, preferably at least 10%, more preferably at least 20%, even more preferably at least 30%, or most preferably at least 40%, relative to the potency of Met-FGF21.

(173) In one embodiment, the FGF21 analogue of this invention has a potency of at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, or most preferably at least 90%, relative to the potency of Met-FGF21.

(174) In one embodiment, the FGF21 analogue of this invention has a potency of at least 100%, preferably at least 120%, more preferably at least 140%, even more preferably at least 160%, or most preferably at least 180%, relative to the potency of Met-FGF21.

(175) In one embodiment, the FGF21 analogue of this invention has a potency of at least 200%, preferably at least 250%, more preferably at least 300%, even more preferably at least 350%, or most preferably at least 400%, and even more preferred at least 500% or even at least 600%, especially at least 1000%, relative to the potency of Met-FGF21.

(176) Potency is determined as described for FGF21 derivatives herein.

(177) The FGF21 analogues of this invention are capable of lowering blood glucose in db/db mice relative to a vehicle control.

(178) In one embodiment, the blood glucose of the derivative of this invention is lowered by at least 1%, preferably by at least 2%, more preferably by at least 3%, even more preferably by at least 4%, or most preferably by at least 5%, based on the mean blood glucose measurements in mM and relative to the corresponding vehicle control.

(179) In one embodiment, the FGF21 derivative of this invention has a pERK-HEK293-beta-klotho without HSA [EC50 (nM)] value of below 10 nM, preferably below 5 nM, and especially preferred below 2 nM, and even more preferred below about 1 nM, vide the test described below (example 39).

(180) In one embodiment, the derivative of this invention has a T when dosed s.c. in mice of at least 1.5 hours, preferably at least 2 hours, more preferably at least 4 hours, even more preferably at least 5 hours, or most preferably at least 6 hours, vide example 40, below.

(181) In one embodiment, the derivative of this invention has a T when dosed s.c. in mice of at least 10 hours, preferably at least 15 hours, more preferably at least 24 hours, or most preferably at least 48 hours. The mice are db/db mice, preferably mice that lack the leptin receptor.

(182) In one embodiment, the dosage of the derivative of this invention is 0.5 mg/kg, although a dosage in the range of 0.1 to 1.0 mg/kg may also be used.

(183) In another embodiment, the derivative of this invention has a T when dosed i.v. in mini pig of at least 15 hours, preferably at least 20 hours, more preferably at least 30 hours, even more preferably at least 40 hours, or most preferably at least 50 hours.

(184) The derivative of this invention may even have a T when dosed i.v. in mini pig of at least 60 hours, preferably of at least 70 hours, more preferably of at least 75 hours. The mini pigs are preferably normal male Gttingen mini pigs. The number of pigs in each treatment group is preferably n=3-4. The pigs are preferably 12-15 months old, and more preferably of a weight of approximately 25 kg. The pigs are preferably dosed a single intravenous dose of preferably 0.1 mg/kg (approximately 5 nmol/kg).

(185) The plasma concentration of the derivatives and analogues of the invention as well as comparative FGF21 compounds may be determined by any suitable method known in the art. A preferred assay is Fibroblast Growth Factor-21 Human ELISA, available from BioVendor with catalogue no. RD191108200R (e.g. BioVendor GmbH, Im Neuenheimer Feld 583, D-69120 Heidelberg, Germany).

(186) The derivative of this invention has effect in vivo on the blood glucose level of db/db mice, cf. example 41 below.

(187) The derivative of this invention is capable of lowering blood glucose in vivo in db/db mice relative to a vehicle control.

(188) In one embodiment, the blood glucose value of the derivative of this invention is lowered 24 hours, preferably 48 hours, after a last dose of the derivative has been administered.

(189) In one embodiment, the blood glucose value of the derivative of this invention is lowered by at least 10%, preferably by at least 15%, more preferably by at least 20%, even more preferably by at least 25%, or most preferably by at least 30%, based on the mean blood glucose measurements in mM and relative to the corresponding vehicle control.

(190) In one embodiment, the blood glucose value of the derivative of this invention is lowered by at least 35%, preferably by at least 40%, more preferably by at least 45%, or most preferably by at least 50%.

(191) The derivative of this invention preferably has an acceptable potency and an extended half-life.

(192) In one embodiment, the derivative of this invention has a T when dosed s.c. in mice of at least 1.5 hours, preferably at least 2 hours, more preferably at least 4 hours, even more preferably at least 5 hours, or most preferably at least 6 hoursand at the same time a potency as described in any one of the embodiments herein, vide example 40.

(193) In one embodiment, the derivative of this invention has a T when dosed s.c. in mice of at least 10 hours, preferably at least 15 hours, more preferably at least 24 hours, or most preferably at least 48 hoursand at the same time a potency as described in any one of the embodiments herein.

(194) In one embodiment, the derivative of this invention has a T when dosed i.v. in mini pig of at least 15 hours, preferably at least 20 hours, more preferably at least 30 hours, even more preferably at least 40 hours, or most preferably at least 50 hoursand at the same time a potency as described in any one of the embodiments herein.

(195) In one embodiment, the derivative of this invention has a T when dosed i.v. in mini pig of at least 60 hours, preferably of at least 70 hours, more preferably of at least 75 hoursand at the same time a potency as described in any one of the embodiments herein.

(196) The term stable or stability refers to potency, determined as described herein.

(197) In one embodiment, the potency of the analogue of this invention after incubation with H.sub.2O.sub.2 is at least 15%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, or most preferably at least 50%, wherein the potency is relative to Met-FGF21 treated in the same way, however without H.sub.2O.sub.2.

(198) The FGF21 compounds, including FGF21 analogues, and derivatives of FGF21 compounds of this invention can be prepared analogously as described for similar compounds. More specifically, reference is made to the specific working examples below and other publications describing the preparation of such compounds.

(199) Example 1 and 2 below describes the cloning and expression of FGF21 and FGF21 analogues in E. coli. Alternatively, FGF21 and FGF21 analogues, optionally with N-terminal extensions, can be expressed in yeast as follows:

(200) FGF21 with N-terminal amino acid extensions can be expressed in S. cerevisiae. In one embodiment, this requires strain design in which a strain disrupted in PMT2, PEP4 and YPS1 is created. This strain can be designed using classical techniques relying on homologous recombination allowing specific integration at the respective loci. FGF21 with N-terminal extension is coded for on an S. cerevisiae expression vector which can be maintained in S. cerevisiae. To direct the FGF21 analogue to the secretory pathway a pre-pro sequence including a signal peptide (for example the MFalpha prepro leader sequence) may be provided in the recombinant expression vector. This sequence is joined to the DNA encoding the FGF21 analogue in correct reading frame. This signal peptide ensures secretion to the media. Upstream and adjacent to the FGF21 analogue sequence a dibasic amino acid sequence is placed ensuring cleavage of the prepro sequence from the FGF21 analogue before secretion to the media. The cleavage is likely to be caused by KEX2 activity. The FGF21 analogue can be harvested from the media.

(201) The derivatives of an FGF compound and the FGF21 analogues can, if desired, be formulated together with other medicaments such as insulin.

(202) To sum up and supplement the above statements, certain embodiments of this invention are as follows:

(203) 1. A derivative of an FGF21 compound, wherein a modifying group of the formula A-B-C-D- is covalently attached to the alpha amino-group of the N-terminal amino acid of the FGF21 compound wherein A- is an element of formula I, II or III:

(204) ##STR00077##
wherein n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, and * is the point of attachment to -B-;
-B- is absent, represents a bond or is selected from -B1-, -B2- or combinations thereof, wherein -B1- is an element of formula IV, V, VI or VII:

(205) ##STR00078##
wherein * is the point of attachment to -B2- or -C-, and # is the point of attachment to A- or -B2-; and
-B2- is an element of formula VIII, IX or X or a combination of up to four elements of formula VIII and/or formula IX and/or formula X:

(206) ##STR00079##
wherein b is 0, 1 or 2, c is 0, 1 or 2, with the proviso that b is 1 or 2 when c is 0, and b is 0 when c is 1 or 2, * is the point of attachment to -C-, -B1- or another -B2-, and # is the point of attachment to A-, -B1- or another -B2-;

(207) ##STR00080##
wherein d is 1 or 2, e is 0, 1 or 2, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-; or
#-NHSO.sub.2(CH.sub.2).sub.uCO.(formula X)
wherein u is 2, 3 or 4, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-;
-C- is absent, represents a bond or is an element of formula XI or XII:

(208) ##STR00081##
wherein p is 0, 1, 2, 3, 4, 5 or 6, q is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-; or

(209) ##STR00082##
wherein k is 0, 1, 2, 3, 4, 5, 11 or 23, m is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound; and # is the point of attachment to -B-;
-D- is an element of formula XIII, XIIIa, XIV or XV or two of these elements together:
#-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII),
#-NHCH(CH.sub.3).sub.2CH.sub.2*(formula XIIIa),

(210) ##STR00083##
wherein r is an integer in the range 1-5, y is an integer in the range 0-5, and s is an integer in the range 0-5, * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-;
or a pharmaceutically acceptable salt thereof.
2. A derivative of an FGF21 compound according to the preceding embodiment, wherein a modifying group of the formula A-B-C-D- is covalently attached to the alpha amino-group of the N-terminal amino acid of the FGF21 compound wherein A- is an element of formula I, II or III:

(211) ##STR00084##
wherein n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, and * is the point of attachment to -B-;
-B- is absent, represents a bond or is selected from -B1-, -B2- or combinations thereof, wherein -B1- is an element of formula IV, V, VI or VII:

(212) ##STR00085##
wherein * is the point of attachment to -B2- or -C-, and # is the point of attachment to A- or -B2-; and
-B2- is an element of formula VIII, IX or X or a combination of up to four elements of formula VIII and/or formula IX and/or formula X:

(213) ##STR00086##
wherein b is 0, 1 or 2, c is 0, 1 or 2, with the proviso that b is 1 or 2 when c is 0, and b is 0 when c is 1 or 2, * is the point of attachment to -C-, -B1- or another -B2-, and # is the point of attachment to A-, -B1- or another -B2-;

(214) ##STR00087##
wherein d is 1 or 2, e is 0, 1 or 2, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-; or
#-NHSO.sub.2(CH.sub.2).sub.uCO.(formula X)
wherein u is 2, 3 or 4, * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-;
-C- is absent, represents a bond or is an element of formula XI or XII:

(215) ##STR00088##
wherein p is 0, 1, 2, 3, 4, 5 or 6, q is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-; or

(216) ##STR00089##
wherein k is 0, 1, 2, 3, 4, 5, 11 or 23, m is 0, 1, 2, 3, 4, 5 or 6, * is the point of attachment to -D- or the FGF21 compound; and # is the point of attachment to -B-;
-D- is an element of formula XIII, XIV or XV:
#-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII) or

(217) ##STR00090##
wherein r is an integer in the range 1-5, y is an integer in the range 0-5, and s is an integer in the range 0-5, * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-;
or a pharmaceutically acceptable salt thereof.
3. The derivative of embodiment 1, wherein if A is an element of formula (III), then at least one of -B- and -C- is present and/or does not represent a bond.
4. The derivative of any of the preceding embodiments to the extent possible, wherein -B- and -C- are present and do not represent a bond.
5. The derivative according to any of the preceding embodiments to the extent possible, wherein A- is an element of formula I, II or III:

(218) ##STR00091##
wherein n is 12, 14 or 16, and * is the point of attachment to -B-.
6. The derivative according to any of the preceding embodiments to the extent possible, wherein -A- is an element of formula I:

(219) ##STR00092##
wherein n is as defined herein, and * is the point of attachment to -B-.
7. The derivative according to any one of the preceding embodiments to the extent possible, in which n is 12, 14, 16 or 18, preferably 12, 14 or 16.
8. The derivative according to any one of the preceding embodiments to the extent possible, in which n is 14, 16 or 18, preferably 14 or 16.
9. The derivative according to any one of the preceding embodiments to the extent possible, in which A- is an element of formula I, preferably one wherein n is 13.
10. The derivative according to any one of the preceding embodiments to the extent possible, in which -B- comprises -B1-, preferably an element of formula IV or V:

(220) ##STR00093##
wherein * is the point of attachment to -B2- or -C-, and # is the point of attachment to A- or -B2-.
11. The derivative according to any one of the preceding embodiments to the extent possible, in which -B- is -B2-, preferably one or two elements of formula VIII:

(221) ##STR00094##
in which more preferably c is 0 and b is 2, or c is 0 and b is 1, and wherein * is the point of attachment to -C-, -B1- or another -B2-, and # is the point of attachment to A-, -B1- or another -B2-.
12. The derivative according to any one of the preceding embodiments to the extent possible, in which -B- is -B2-, preferably four elements of formula VIII:

(222) ##STR00095##
in which more preferably c is 0 and b is 2, and wherein * is the point of attachment to -C-, -B1- or another -B2-, and # is the point of attachment to A-, -B1- or another -B2-.
13. The derivative according to any one of the preceding embodiments to the extent possible, in which -B- is -B2-, more precisely an element of formula IX:

(223) ##STR00096##
preferably one wherein d is 1 and e is 2, and wherein * is the point of attachment to -C- or -B1-, and # is the point of attachment to A- or -B1-.
14. The derivative according to any one of the preceding embodiments to the extent possible, in which -B- is a bond.
15. The derivative according to any one of the preceding embodiments to the extent possible, in which -C- is an element of formula XI:

(224) ##STR00097##
in which preferably q is 0, 1, 2, 3 or 4, more preferably q is 1 or 2, and even more preferred q is 2, and p is as defined herein, and wherein * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-.
16. The derivative according to any one of the preceding embodiments to the extent possible, in which -C- is an element of formula XI:

(225) ##STR00098##
in which preferably p is 1 or 2, more preferably p is 1, and q is as defined herein, and wherein * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-.
17. The derivative according to any one of the preceding embodiments to the extent possible, in which p is 1 and q is 1 or 2, preferably p is 1 and q is 2.
18. The derivative according to any one of the preceding embodiments to the extent possible, in which -C- is an element of formula XII:

(226) ##STR00099##
in which preferably m is 0, 1 or 2, more preferably m is 1 or 2, and k is as defined herein, and wherein * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-.
19. The derivative according to any one of the preceding embodiments to the extent possible, in which in which -C- is an element of formula XII:

(227) ##STR00100##
in which preferably k is 1, 2, 3, 4, 5 or 11, more preferably k is 5, and m is as defined herein, and wherein * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-.
20. The derivative according to any one of the preceding embodiments to the extent possible, in which in which -C- is an element of formula XII:

(228) ##STR00101##
in which m is 1 and k is 4, 5 or 11, preferably m is 1 and k is 5, and m is as defined herein, and wherein * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -B-.
21. The derivative according to any one of the preceding embodiments to the extent possible, in which -D- is an element of formula XIII, XIIIa, XIV or XV or two of these elements together:
#-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII),
#-NHCH(CH.sub.3).sub.2CH.sub.2*(formula XIIIa),

(229) ##STR00102##
wherein r is an integer in the range 1-5, y is an integer in the range 0-5, and s is an integer in the range 0-5, * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-.
22. The derivative according to any one of the preceding embodiments to the extent possible, in which -D- is an element of formula XIII, XIV or XV:
#-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII),

(230) ##STR00103##
wherein r is an integer in the range 1-5, y is an integer in the range 0-5, and s is an integer in the range 0-5, * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-.
23. The derivative according to any one of the preceding embodiments to the extent possible, in which -D- is an element of formula XIII: #-NH(CH.sub.2).sub.rCH.sub.2*(formula XIII), in which preferably r is 1, 2 or 3, more preferably r is 1, and wherein * is the point of attachment to the FGF21 compound, and # is the point of attachment to -C-.
24. The derivative according to any one of the preceding embodiments to the extent possible, in which -D- is an element of formula XIV:

(231) ##STR00104##
in which preferably s is 0 or 1, more preferably s is 1, and wherein * is the point of attachment to * is the point of attachment to -D- or the FGF21 compound, and # is the point of attachment to -C-.
25. The derivative according to any one of the preceding embodiments to the extent possible, wherein -D- is not an element of formula XIII as defined herein (in other words, -D- is an element of formula XIV or XV as defined herein).
26. The derivative according to any one of the preceding embodiments to the extent possible, wherein the modifying group of the formula A-B-C-D- is selected from the following formulas (1)-(3):

(232) ##STR00105##
wherein * is the point of attachment to the FGF21 compound, or a pharmaceutically acceptable salt of any one of the corresponding derivatives.
27. The derivative according of any one of the preceding embodiments to the extent possible, which has only one modifying group (A-B-C-D-).
28. The derivative according of any one of the preceding embodiments to the extent possible, which has two modifying groups (A-B-C-D-) which are the same or different.
29. The derivative according of any one of the preceding embodiments to the extent possible, which has one or two modifying group(s) (A-B-C-D-).
30. The derivative according to any one of the preceding embodiments to the extent possible, which is a compound selected from the derivatives in examples 3, 4, 5 and 6 herein, preferably the following: N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [Arg56, Arg59, Arg69, Arg122] Gly-FGF21 (Compound I); N-alpha-bis-(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [Arg56, Arg59, Arg69, Arg122] Gly-FGF21 (Compound II); N-alpha-(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]-methyl}benzyl) [K56R, K59R, K69R, D102T, D121Q, K122R, L166F, S167G, M168L, G170T] Ala-FGF21 (Compound III); N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Ala-FGF21 (Compound IV); or a pharmaceutically acceptable salts of the corresponding derivatives.
31. The derivative according to any one of the preceding embodiments to the extent possible, which is a compound selected from the derivatives in examples 3, 4, 5 and 6 herein, preferably the following: N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [Arg56, Arg59, Arg69, Arg122] Gly-FGF21 (Compound I); N-alpha-bis-(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [Arg56, Arg59, Arg69, Arg122] Gly-FGF21 (Compound II); N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]-methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Ala-FGF21 (Compound IV); or a pharmaceutically acceptable salts of the corresponding derivatives.
32. The derivative according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound has an identity of at least 80%, to SEQ ID NO:1, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%.
33. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound has a maximum of 36 amino acid modifications as compared to SEQ ID NO:1, preferably a maximum of 30, 25, 20, 15, 10 or 5 amino acid changes, or a maximum of 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid change(s); more preferably a maximum of 15, 14, 13, 12, 11 or 10 amino acid modifications; even more preferably a maximum of 9, 8, 7, 6 or 5 amino acid modifications; or most preferably a maximum of 4, 3, 2 or 1 amino acid modification(s).
34. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises the amino acid sequence of SEQ ID NO:1.
35. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises at least one of the following modifications as compared to SEQ ID NO:1 -1G, -1A, -1S, -1F, -1M, R17A, 17H, Q27E, A31E, R36A, R36H, S71C, D102E, D102N, D102T, N121Q, des121N, N121D, D159E, L166F, S167G, M168L, D169E, V169aT, des170, G170T, P171L, P171G, S172E, S172L, Q173E, 173aA, Q173A, G174A, G174V, Y179F, A180E, des181, S181K and/or S181R, and preferably said FGF21 compound comprises at least three of said modifications, more preferred at least five of said modifications.
36. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises at least one of the following modifications as compared to SEQ ID NO:1 -1G, -1A, -1S, -1F, -1M, R17A, 17H, Q27E, A31E, R36A, R36H, S71C, D102E, D102N, D102T, N121Q, des121N, N121D, D159E, L166F, S167G, M168L, D169E, V169aT, des170, G170T, P171L, P171G, S172E, S172L, Q173E, 173aA, Q173A, G174A, G174V, Y179F, A180E, S181K and/or S181R, and preferably said FGF21 compound comprises at least three of said modifications, more preferred at least five of said modifications, and even more preferred at least seven modifications.
37. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises at least one of the following modifications as compared to SEQ ID NO:1 -1G, -1A, -1S, -1F, -1M, R17A, 17H, Q27E, A31E, R36A, R36H, K56R, K59R, K69R, S71C, D102E, D102N, D102T, N121Q, des121N, N121D, K122R, D159E, L166F, S167G, M168L, D169E, V169aT, des170, G170T, P171L, P171G, S172E, S172L, Q173E, 173aA, Q173A, G174A, G174V, Y179F, A180E, des181, S181K and/or S181R, and preferably said FGF21 compound comprises at least three of said modifications, more preferred at least five of said modifications, and even more preferred at least seven modifications.
38. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises at least one of the following modifications as compared to SEQ ID NO:1 -1G, -1A, -1S, -1F, -1M, R17A, 17H, Q27E, A31E, R36A, R36H, K56R, K59R, K69R, S71C, D102E, D102N, D102T, N121Q, des121N, N121D, K122R, D159E, L166F, S167G, M168L, D169E, V169aT, des170, G170T, P171L, P171G, S172E, S172L, Q173E, 173aA, Q173A, G174A, G174V, Y179F, A180E, des181, S181K, S181R and/or 182G, and preferably said FGF21 compound comprises at least three of said modifications, more preferred at least five of said modifications, and even more preferred at least seven modifications.
39. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises Y179F, A180E and des 181 (i); preferably it additionally comprises P171L, S172E, Q173A and G174V (ii); more preferably it additionally comprises L166F, S167G and M168L (iii); most preferably it comprises (i), (ii) and (iii). Each of these embodiments (i)-(iii) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).
40. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises S71C (i), preferably it additionally comprises Y179F, A180E and des 181 (ii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii), (iii) and (iv). Each of these embodiments (i)-(iv) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).
41. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises N121Q (i), Y179F, A180E and des 181 (ii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii), (iii) and (iv). Each of these embodiments (i)-(iii) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).
42. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises S71C(i), N121Q (ii), Y179F, A180E and des 181 (iii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iv); more preferably it additionally comprises L166F, S167G and M168L (v); most preferably it comprises (i), (ii), (iii), (iv) and (v). Each of these embodiments (i)-(v) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).
43. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises Y179F and A180E (i); preferably it additionally comprises P171L, S172E, Q173A and G174V (ii); more preferably it additionally comprises L166F, S167G and M168L (iii); most preferably it comprises (i), (ii) and (iii). Each of these embodiments (i)-(iii) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).
44. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises S71C (i), preferably it additionally comprises Y179F and A180E (ii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii), (iii) and (iv). Each of these embodiments (i)-(iv) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).
45. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises N121Q (i), Y179F and A180E (ii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii), (iii) and (iv). Each of these embodiments (i)-(iii) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).
46. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises S71C(i), N121Q (ii), Y179F and A180E (iii); preferably it additionally comprises P171L, S172E, Q173A and G174V (iv); more preferably it additionally comprises L166F, S167G and M168L (v); most preferably it comprises (i), (ii), (iii), (iv) and (v). Each of these embodiments (i)-(v) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).
47. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises Y179F, A180E and des 181 (i); preferably it additionally comprises des170, 173aA and G174V (ii); more preferably it additionally comprises L166F, S167G and M168L (iii); most preferably it comprises (i), (ii) and (iii). Each of these embodiments (i)-(iii) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).
48. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises S71C (i), preferably it additionally comprises Y179F, A180E and des 181 (ii); preferably it additionally comprises des170, 173aA and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii), (iii) and (iv). Each of these embodiments (i)-(iv) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).
49. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises N121Q (i), Y179F, A180E and des 181 (ii); preferably it additionally comprises des170, 173aA and G174V (iii); more preferably it additionally comprises L166F, S167G and M168L (iv); most preferably it comprises (i), (ii), (iii) and (iv). Each of these embodiments (i)-(iv) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).
50. The derivative, according to any one of the preceding embodiments to the extent possible, wherein, the FGF21 compound comprises S71C(i), N121Q (ii), Y179F, A180E and des 181 (iii); preferably it additionally comprises des170, 173aA and G174V (iv); more preferably it additionally comprises L166F, S167G and M168L (v); most preferably it comprises (i), (ii), (iii), (iv) and (v). Each of these embodiments (i)-(v) optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).
51. The derivative, according to any one of the preceding embodiments to the extent possible, wherein, the FGF21 compound comprises at least one of the following modifications as compared to SEQ ID NO:1 -1G, -1A, -1S, -1F, -1M, R17A, 17H, Q27E, A31E, R36A, R36H, S71C, D102E, D102N, D102T, N121Q, des121N, N121D, D159E, L166F, S167G, M168L, D169E, V169aT, des170, G170T, P171L, P171G, S172E, S172L, Q173E, 173aA, Q173A, G174A, G174V, Y179F, A180E, des181, S181K and/or S181R, and preferably said FGF21 compound comprises at least three of said modifications, more preferred at least five of said modifications, and even more preferred at least seven modifications.
52. The derivative, according to any one of the preceding embodiments to the extent possible, wherein, the FGF21 compound comprises at least one of the following modifications as compared to SEQ ID NO:1 -1G, -1A, -1S, -1F, -1M, R17A, 17H, Q27E, A31E, R36A, R36H, S71C, D102E, D102N, D102T, N121Q, des121N, N121D, D159E, L166F, S167G, M168L, D169E, V169aT, des170, G170T, P171L, P171G, S172E, S172L, Q173E, 173aA, Q173A, G174A, G174V, Y179F, A180E, S181K and/or S181R, and preferably said FGF21 compound comprises at least three of said modifications, more preferred at least five of said modifications, and even more preferred at least seven modifications.
53. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises N121Q and optionally includes an N-terminal amino acid M, G, S, F or A (e.g., -1A).
54. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound comprises the amino acid sequence of SEQ ID NO:1.
55. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF21 compound has a proline at position 146, wherein the position number refers to SEQ ID NO:1.
56. The derivative, according to any one of the preceding embodiments to the extent possible, which has a T when dosed s.c. in mice of at least 1.5 hours, preferably at least 2 hours, more preferably at least 4 hours, even more preferably at least 5 hours, or most preferably at least 6 hours.
57. The derivative, according to any one of the preceding embodiments to the extent possible, which has a T when dosed s.c. in mice of at least 10 hours, preferably at least 15 hours, more preferably at least 24 hours, or most preferably at least 48 hours.
58. The derivative, according to any one of the preceding embodiments to the extent possible, which has a potency of at least 1%, preferably of at least 5%, more preferably of at least 10%, even more preferably of at least 20%, or most preferably of at least 30%, relative to the potency of Met-FGF21 and the potency can be determined by measuring glucose uptake in 3T3-L1 adipocytes.
59. The derivative, according to any one of the preceding embodiments to the extent possible, which has a potency of at least 40%, preferably of at least 50%, more preferably of at least 60%, even more preferably of at least 70%, relative to the potency of Met-FGF21.
60. The derivative according to any one of the preceding embodiments to the extent possible, which has a potency of at least 80%, preferably at least 90%, more preferably at least 100%, even more preferably at least 110%, or most preferably at least 120%, relative to the potency of Met-FGF21.
61. The derivative according to any one of the preceding embodiments to the extent possible, wherein the potency is at least 80%, preferably at least 90%, more preferably at least 100%, even more preferably at least 110%, or most preferably at least 120%, relative to the potency of Met-FGF21;
62. The derivative according to any one of the preceding embodiments to the extent possible, wherein the potency is at least 100%, preferably at least 120%, more preferably at least 140%, even more preferably at least 160%, or most preferably at least 180%, relative to the potency of Met-FGF21.
63. The derivative according to any one of the preceding embodiments to the extent possible, wherein the potency is at least 200%, preferably at least 250%, more preferably at least 300%, even more preferably at least 350%, or most preferably at least 400%, and even more preferred at least 500% or even at least 600%, especially at least 1000%, relative to the potency of Met-FGF21.
64. The derivative, according to any one of the preceding embodiments to the extent possible, in which the potency is calculated as the EC.sub.50 of the derivative relative to the EC.sub.50 of Met-FGF21.
65. The derivative, according to any one of the preceding embodiments to the extent possible, in which the 3T3-L1 adipocytes derive from mouse 3T3-L1 fibroblasts, preferably ATCC CL-173.
66. The derivative, according to any one of the preceding embodiments to the extent possible, in which the glucose uptake in 3T3-L1 adipocytes is measured as outlined in example 38 herein.
67. The derivative, according to any one of the preceding embodiments to the extent possible, in which the FGF21 derivative has a pERK-HEK293-beta-klotho without HSA [EC50 (nM)] value of below about 10 nM, preferably below about 5 nM, and especially preferred below about 2 nM, and even more preferred below about 1 nM, vide the test described in example 39 herein.
68. The derivative, according to any one of the preceding embodiments to the extent possible, which is capable of lowering blood glucose in vivo in db/db mice relative to a vehicle control.
69. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the blood glucose is lowered 24 hours, preferably 48 hours, after a last dose of the derivative has been administered.
70. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the blood glucose is lowered by at least about 10%, preferably by at least about 15%, more preferably by at least about 20%, even more preferably by at least about 25%, or most preferably by at least about 30%, based on the mean blood glucose measurements in mM and relative to the corresponding vehicle control.
71. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the blood glucose is lowered by at least 35%, preferably by at least 40%, more preferably by at least 45%, or most preferably by at least 50%.
72. The derivative, according to any one of the preceding embodiments to the extent possible, wherein (i) the db/db mice are male and 9-11 weeks old, (ii) the derivative is administered s.c., (iii) the derivative dosage is in the range of 0.2-1.0 mg/kg, preferably 0.2, 0.4, 0.6, 0.8 or 1.0 mg/kg, (iv) the derivative is dissolved in PBS, (v) the derivative is dosed once daily, preferably on day 1, day 2 and day 3, (vi) the vehicle control is treated with PBS, preferably 250 ul/50 g mouse, and/or (vii) blood glucose is measured using the glucose oxidase method, preferably using a glucose analyzer such as Biosen 5040.
73. The derivative, according to any one of the preceding embodiments to the extent possible, wherein the vehicle control is replaced by Met-FGF21.
74. The derivative, according to any one of the preceding embodiments to the extent possible, in which (i) the derivative and Met-FGF21 are both dissolved in PBS, pH 7.2, and/or (ii) the concentration of the derivative and Met-FGF21 is 1 mg/mL.
75. The derivative, according to any one of the preceding embodiments to the extent possible, which is any one of the specific derivatives mentioned in examples 3, 4 and 5.
76. The FGF21 derivative, according to any one of the preceding embodiments to the extent possible, which is capable of lowering blood glucose in db/db mice relative to a vehicle control.
77. The FGF21 derivative, according to any one of the preceding embodiments to the extent possible, which, when administered to humans, results in a lowering of the blood glucose by at least 1%, preferably by at least 2%, more preferably by at least 3%, even more preferably by at least 4%, or most preferably by at least 5%, based on the mean blood glucose measurements in mM and relative to the corresponding vehicle control.
78. The FGF21 derivative, according to any one of the preceding embodiments to the extent possible, for which the following features can be used when determining the blood glucose lowering effect: (i) the db/db mice are male and 9-11 weeks old, (ii) the analogue is administered s.c., (iii) the analogue dosage is in the range of 0.1-1.0 mg/kg, preferably 0.1, 0.2, 0.4, 0.6, 0.8 or 1.0 mg/kg, (iv) the analogue is dosed once daily, preferably on day 1, day 2 and day 3, and/or (v) blood glucose is measured using the glucose oxidase method, preferably using a glucose analyzer such as Biosen 5040.
79. The FGF derivative, according to any one of the preceding embodiments to the extent possible, wherein the FGF compound is any one of the specific analogues mentioned in the above examples 2 (i.e., 2a-2d).
80. A composition comprising a derivative according to any one of the above embodiments and a pharmaceutically acceptable carrier.
81. A derivative according to any one of the preceding embodiments to the extent possible for use as a medicament.
82. A derivative according to any one of the above embodiments for use as a medicament in the treatment or prevention of diabetes, dyslipidemia, obesity, cardiovascular diseases, metabolic syndrome, and/or Non Alcoholic Fatty Liver Disease (NAFLD).
83. A derivative according to any one of the preceding embodiments for use in the preparation of a medicament for the treatment or prevention of diabetes, dyslipidemia, obesity, cardiovascular diseases, metabolic syndrome, and/or Non Alcoholic Fatty Liver Disease (NAFLD).
84. A method for treating a patient exhibiting diabetes, dyslipidemia, obesity, cardiovascular diseases, metabolic syndrome, and/or Non Alcoholic Fatty Liver Disease (NAFLD) comprising administering to the patient a therapeutically effective amount of a derivative according to any one of the preceding embodiments.
85. A method for treating a patient needing intensive care comprising administering to the patient a therapeutically effective amount of a derivative according to any one of the preceding embodiments.
86. Any novel combination of embodiments, features and claims described herein.
Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

(233) Combining one or more of the clauses and embodiments described herein, optionally also with one or more of the claims below, results in further embodiments and the present invention relates to all possible combinations of said clauses, embodiments and claims.

(234) The following examples are offered by way of illustration, not by limitation.

EXAMPLES

(235) The following examples serve to illustrate the invention.

(236) Abbreviations

(237) The following abbreviations are used in the following, in alphabetical order: AcOH is acetic acid, CV is column volume, DCM is dichloromethane, DIPEA is diisopropylethylamine, DPBS is Dulbecco's Phosphate-Buffered Saline, DVB is divinyl benzene, EtOH is ethanol, EtOAc is ethyl acetate, HEPES is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HPCD is Hydroxypropyl Beta Cyclodextrin, HPLC is High Performance Liquid Chromatography, IBMX is 3-isobutyl-1-methylxanthine, Inp is isonipecotic acid, IPTG is isopropyl -D-1-thiogalactopyranoside check, LCMS is Liquid Chromatography Mass Spectroscopy, MALDI-TOF MS is Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectroscopy, NaAc is sodium acetate, OtBu is tert.butyl ester, PBS is phosphate buffered saline, RT is room temperature, TFA is trifluoroacetic acid, THF is tetrahydrofuran, Tris is tris(hydroxymethyl)aminomethane or 2-amino-2-hydroxymethylpropane-1,3-diol, Trx is tranexamic acid, TSTU is O(N-succimidyl)-N,N,N,N-tetramethyluronium tetrafluoroborate and UPLC is Ultra Performance Liquid Chromatography.

(238) General Methods

(239) LCMS Method 1 (LCMS1)

(240) An Agilent Technologies LC/MSD TOF (G1969A) mass spectrometer was used to identify the mass of the sample after elution from an Agilent 1200 series HPLC system. The deconvolution of the protein spectra was calculated with Agilent's protein confirmation software.

(241) Eluents:

(242) A: 0.1% Trifluoroacetic acid in water

(243) B: 0.1% Trifluoroacetic acid in acetonitrile

(244) Column: Zorbax 5u, 300SB-C3, 4.850 mm

(245) Gradient: 25%-95% acetonitrile over 15 min

(246) LCMS Method 2 (LCMS2)

(247) A Waters Micromass ZQ mass spectrometer was used to identify the mass of the sample after elution from a Waters Alliance HT HPLC system.

(248) Eluents:

(249) A: 0.1% Trifluoroacetic acid in water

(250) B: 0.1% Trifluoroacetic acid in acetonitrile

(251) Column: Phenomenex, Kinetex C18 504.60 mm id 2.6 m, 100AA

(252) Gradient: 10%-90% B over 7.5 min at 1.0 ml/min

Example 1

Cloning and Expression of FGF21

(253) The DNA and amino acid sequences for human FGF21 have been disclosed by, e.g., Nishimura et al. in Biochim. Biophys. Acta 1492(1):203-206 (2000). The sequences are also available from public databases with accession nos. EMBL:AB021975 and UNIPROT:Q9NSA1, respectively.

(254) The native polypeptide is synthesised with a signal peptide of 28 amino acids for secretion:

(255) 1 MDSDETGFEH SGLWVSVLAG LLLGACQAHP IPDSSPLLQF GGQVRQRYLY

(256) 51 TDDAQQTEAH LEIREDGTVG GAADQSPESL LQLKALKPGV IQILGVKTSR

(257) 101 FLCQRPDGAL YGSLHFDPEA CSFRELLLED GYNVYQSEAH GLPLHLPGNK

(258) 151 SPHRDPAPRG PARFLPLPGL PPALPEPPGI LAPQPPDVGS SDPLSMVGPS

(259) 201 QGRSPSYAS

(260) The signal peptide, shown in italics above, is included in the appended sequence listing as SEQ ID NO:2. The mature FGF21 polypeptide consisting of the remaining 181 amino acids is included in the sequence listing as SEQ ID NO:1.

(261) The mature FGF21 polypeptide was cloned and expressed as an intracellular protein in E. coli, without the signal peptide, but with an added N-terminal methionine or an N-terminal Met-Ala which is processed in E. coli resulting in N-terminal Ala (-1Ala). More in particular, a 550 bp coding region including at the 3-end the ATG codon for Met, as well as Nde1 and BamH1 restriction sites at the 3- and 5-ends, respectively, was inserted into the expression vector pET 11c in Nde1-BamH1 under control of the phage T7 promoter, and transformed into E. coli B BL21(DE3). The cells were grown in LB amp 100 ug/mL to OD.sub.450 0.5, and expression was induced with 0.3 mM IPTG for 4 hours at 37 C. Crude extracts of cells were made by sonication for analysis of FGF21 expression.

(262) A Coomassie stained SDS-PAGE showed successful expression of FGF21 which was identified mainly in the soluble supernatant fraction, with very little in the insoluble pellet. Although the calculated MW of the thus expressed FGF21 (Met-FGF21) (Compound A) is 19.5 kD, it migrated on the gel as a 25 kD protein, which is likely due to the high content of prolines, delaying the movement of the protein.

Example 2

Cloning, Expression and Purification of FGF21 Analogues

(263) The following analogues of Met-FGF21 were designed as is known in the art and expressed in E. coli as generally described in example 1:

(264) 2a) [K56R, K59R, K69R, K122R] Gly-FGF21.

(265) 2b) [Q28R, K56R, K59R, K69R, D102T, D121Q, K122R, L166F, S167G, M168L, G170T] Ala-FGF21.

(266) 2c) [S71C, N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Ala-FGF21.

(267) 2d) [S71C, N121Q, L166F, S167G, M168L, des170, 173aA, G174V, Y179F, A180E, des181] Ala-FGF21.

(268) 2e) [S71C, N121Q, L166F, S167G, M168L, 173aA, G174V, Y179F, A180E, des181] Ala-FGF21.

(269) 2f) [N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Ala-FGF21.

(270) 2g) [N121Q] Met-FGF21.

(271) 2h) [N121Q] Ala-FGF21.

(272) 2i) [N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Phe-FGF21.

(273) The same analogues can be expressed and prepared in S. cerevisiae by methods known per se and suitable for this organism.

(274) The FGF21 polypeptide and its analogues prepared as described in examples 1-2 were further purified as follows or using similar technics:

(275) A slurry (20% w/v) of E. coli in 10 mM potassium phosphate buffer pH 7.5 was sonicated (3 seconds on/off intervals on ice for 5 minutes). The polypeptide was pelleted by centrifugation (10,000g, for 30 minutes), re-solubilised by sonication in 50 mM Tris pH 8.0, and debris removed by centrifugation (10,000g, for 30 minutes). The polypeptide in the resulting supernatant was purified by anion exchange chromatography (50 mM Tris pH 8.0, 50-250 mM NaCl) using Q Sepharose Fast Flow resin (GE Healthcare), as generally described in Protein Purification. Principles and Practice Series: Springer Advanced Texts in Chemistry Scopes, Robert K. 3rd ed., 1994. In some instances, further purification was done by size exclusion chromatography using a HiLoad 26/60 Superdex pg 75 column (GE Healthcare) operated with 50 mM Tris pH 8.0 and 200 mM NaCl. For storage the polypeptide was transferred to DPBSS, and stored frozen.

Example 3

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(276) Modifying groups containing an aldehyde may be synthesised as described in the following and FGF21 and analogues thereof may be derivatised with such modifying groups as also described in the following.

Preparation of 17-[(S)-1-carboxy-3-(2-{2-[(2-{2-[(4-formylbenzylcarbamoyl)methoxy]ethoxy}ethylcarbamoyl)methoxy]ethoxy}ethylcarbamoyl)propylcarbamoyl]heptadecanoic acid

(277) t-Bu-N-(4-Formylbenzyl)carbamate (100 mg) was treated with TFA/DCM (1:1) for 1 h. The mixture was concentrated in vacuo and co-concentrated with toluene (twice). The residue was dissolved in THF (2.5 ml) and a solution of 17-((S)-1-carboxy-3-{2-[2-({2-[2-(2,5-dioxopyrrolidin-1-yloxycarbonyl-methoxy)ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)heptadecanoic acid (320 mg, prepared as described previously in WO2009/083549) in THF (5 ml) was added. DIPEA (0.5 ml) was added slowly. After 130 min, the mixture was concentrated in vacuo. The residue was dissolved in EtOAc and 1N HCl. The organic layer was extracted with 1N HCl and brine. The organic layer was dried (Na.sub.2SO.sub.4) and concentrated in vacuo to give the title compound as a white solid, which was used without further purification. Yield 234 mg (72%)

(278) LCMS2: Theoretical mass: 851.0 Found: 851.5 (M+1).

Preparation of the (K56R, K59R, K69R, K122R) Gly-FGF21 derivative N-alpha-(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}-ethoxy)acetylamino]methyl}benzyl) [Arg56, Arg59, Arg69, Arg122] Gly-FGF21 (Compound I)

(279) The N-terminal Gly residue in the K56R, K59R, K69R, K122R Gly-FGF21 analogue, prepared as generally described in examples 1 and 2 (SEQ ID NO:1 with K56R, K59R, K69R, and K122R and an N-terminal G), was modified at the alpha amino group with the following reagent:

(280) ##STR00106##

(281) To a 6.3 mg/ml solution of K56R, K59R, K69R, K122R Gly-FGF21 in PBS-buffer (2 mg, 0.32 ml, 102 nmol) was added a 20 mg/ml solution of Preparation of 17-[(S)-1-carboxy-3-(2-{2-[(2-{2-[(4-formylbenzylcarbamoyl)methoxy]ethoxy}ethylcarbamoyl)methoxy]ethoxy}ethylcarbamoyl)propylcarbamoyl]-heptadecanoic acid in 1M NaAc (6.1 ul, 1.5 eq). pH was adjusted to 6.0 with 10% AcOH. After 15 min 1M NaCNBH.sub.3 (7 ul) was added. The mixture was left at RT. for 0.5 h and then stored at 5 C. over night. The mixture was diluted with 20 mM Tris, 5% EtOH, pH 7.5 and purified by anion exchange chromatography on a MonoQ 5/50 GL, flow 2 ml with A-Buffer: 20 mM Tris, 5% EtOH, pH 7.5 and B-Buffer: 20 mM Tris, 0.5M NaCl, 5% EtOH, pH 7.5 using a gradient 0-20% B over 5CV, 20-80% B over 60 CV. The compound was buffer exchanged to DPBS-buffer on a HiPrep 26/10 desalting column. The collected fractions were concentrated in viva spin ultrafiltration device MWCO 10.000 Da, at 3000 g

(282) LCMS1: Theoretical mass=20413.1. found 20413.4.

Example 4

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

Preparation of the (K56R, K59R, K69R, K122R) Gly-FGF21 derivative N-alpha-bis-(4-{[(2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}-ethoxy)acetylamino]methyl}benzyl) [Arg56, Arg59, Arg69, Arg122] Gly-FGF21 (Compound II)

(283) The N-terminal Gly residue in the K56R, K59R, K69R, K122R Gly-FGF21 analogue, prepared as generally described in examples 1 and 2 (SEQ ID NO:1 with K56R, K59R, K69R, and K122R and an N-terminal G), was modified at the alpha amino group with the following reagent:

(284) ##STR00107##

(285) The compound was prepared as described in example 3 using 5 equivalents of 17-[(S)-1-carboxy-3-(2-{2-[(2-{2-[(4-formylbenzylcarbamoyl)methoxy]ethoxy}ethylcarbamoyl)methoxy]ethoxy}ethylcarbamoyl)propylcarbamoyl]heptadecanoic.

(286) LCMS1: Theoretical mass=21248.2. found 21248.9.

Example 5

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

Preparation of the (K56R, K59R, K69R, D102T, D121Q, K122R, L166F, S167G, M168L, G170T) Ala-FGF21 derivative N-alpha-(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [K56R, K59R, K69R, D102T, D121Q, K122R, L166F, S167G, M168L, G170T] Ala-FGF21 (Compound III)

(287) The N-terminal Ala residue in the K56R, K59R, K69R, D102T, D121Q, K122R, L166F, S167G, M168L, G170T Ala-FGF21 analogue, prepared as generally described in examples 1 and 2 (SEQ ID NO:1 with K56R, K59R, K69R, D102T, D121Q, K122R, L166F, S167G, M168L, G170T and an N-terminal A), was modified at the alpha amino group with the following reagent:

(288) ##STR00108##

(289) The compound was prepared as described in example 3 using 5 equivalents of 17-[(S)-1-carboxy-3-(2-{2-[(2-{2-[(4-formylbenzylcarbamoyl)methoxy]ethoxy}ethylcarbamoyl)methoxy]ethoxy}ethylcarbamoyl)propylcarbamoyl]heptadecanoic.

(290) LCMS1: Theoretical mass=20485.2. found 20485.5.

Example 6

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(291) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]-ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Ala-FGF21 (Compound IV)

(292) The N-terminal Ala residue in the S71C, N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181Ala-FGF21 analogue, prepared as generally described in examples 1 and 2 (SEQ ID NO:1 with S71C, N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181 and an N-terminal A), was modified at the alpha amino group with the following reagent:

(293) ##STR00109##

(294) The compound was prepared as described in example 3 using 5 equivalents of 17-[(S)-1-carboxy-3-(2-{2-[(2-{2-[(4-formylbenzylcarbamoyl)methoxy]ethoxy}ethylcarbamoyl)methoxy]ethoxy}ethylcarbamoyl)propylcarbamoyl]heptadecanoic at pH 6.0.

(295) LCMS1: Theoretical mass=20329.14. found 20329.14.

(296) The FGF21 derivatives of the invention in the following examples may be prepared similarly:

Example 7

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(297) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Gly-FGF21 (Compound V)

Example 8

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(298) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Gly-FGF21 (Compound VI)

Example 9

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(299) N-alpha-Bis(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]-ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Gly-FGF21 (Compound VII)

Example 10

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(300) N-alpha-Bis(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]-ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Gly-FGF21 (Compound VIII)

Example 11

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(301) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q, L166F, S167G, M168L, des170, 173aA, G174V, Y179F, A180E, des181] Gly-FGF21 (Compound IX)

Example 12

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(302) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L des170, 173aA, G174V, Y179F, A180E, des181]Gly-FGF21 (Compound X)

Example 13

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(303) N-alpha-Bis(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]-ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q, L166F, S167G, M168L, des170, 173aA, G174V, Y179F, A180E, des181]Gly-FGF21 (Compound XI)

Example 14

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(304) N-alpha-Bis(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]-ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L des170, 173aA, G174V, Y179F, A180E, des181]Gly-FGF21 (Compound XII)

Example 15

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(305) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181]Ala-FGF21 (Compound XIII)

Example 16

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(306) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]-ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q, L166F, S167G, M168L, des170, 173aA, G174V, Y179F, A180E, des181] Ala-FGF21 (Compound XIV)

Example 17

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(307) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L des170, 173aA, G174V, Y179F, A180E, des181] Ala-FGF21 (Compound XV)

Example 18

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(308) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Ser-FGF21 (Compound XVI)

Example 19

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(309) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Ser-FGF21 (Compound XVII)

Example 20

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(310) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q, L166F, S167G, M168L, des170, 173aA, G174V, Y179F, A180E, des181] Ser-FGF21 (Compound XVIII)

Example 21

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(311) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L des170, 173aA, G174V, Y179F, A180E, des181] Ser-FGF21 (Compound XIX)

Example 22

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(312) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Phe-FGF21 (Compound XX)

Example 23

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(313) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Phe-FGF21 (Compound XXI)

Example 24

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(314) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q, L166F, S167G, M168L, des170, 173aA, G174V, Y179F, A180E, des181] Phe-FGF21 (Compound XXII)

Example 25

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(315) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L des170, 173aA, G174V, Y179F, A180E, des181] Phe-FGF21 (Compound XXIII)

Example 26

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(316) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Met-FGF21 (Compound XXIV)

Example 27

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(317) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L, P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Met-FGF21 (Compound XXV)

Example 28

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(318) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q, L166F, S167G, M168L, des170, 173aA, G174V, Y179F, A180E, des181] Met-FGF21 (Compound XXVI)

Example 29

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(319) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [S71C, N121Q, L166F, S167G, M168L des170, 173aA, G174V, Y179F, A180E, des181] Met-FGF21 (Compound XXVII)

Example 30

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(320) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q] Gly-FGF21 (Compound XXVII)

Example 31

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(321) N-alpha-Bis(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]-ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q] Gly-FGF21 (Compound XXIX)

Example 32

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(322) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q] Ala-FGF21 (Compound XXX)

Example 33

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(323) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q] Ser-FGF21 (Compound XXXI)

Example 34

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(324) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q] Phe-FGF21 (Compound XXXII)

Example 35

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(325) N-alpha(4-{[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]methyl}benzyl) [N121Q] Met-FGF21 (Compound XXXIII)

Example 36

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(326) Preparation of the Met-FGF21 derivative N-alpha-bis-(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetylamino]-methyl}benzyl) Met-FGF21 (Compound XXXIV)

(327) The N-terminal Met residue in the Met-FGF21 analogue, prepared as generally described in Examples 1 and 2, was modified at the alpha amino group with the following reagent:

(328) ##STR00110##

(329) The compound was prepared as described in example 3 using 1.2 equivalents of 17-[(S)-1-carboxy-3-(2-{2-[(2-{2-[(4-formylbenzylcarbamoyl)methoxy]ethoxy}ethylcarbamoyl)methoxy]ethoxy}ethylcarbamoyl)propylcarbamoyl]heptadecanoic.

Example 37

Derivatisation of FGF21 Compounds at the N-Terminus with Modifying Groups

(330) Preparation of the Met-FGF21 derivative N-alpha-(4-{[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}-ethoxy)acetylamino]methyl}benzyl) Met-FGF21 (Compound XXXV)

(331) The N-terminal Gly residue in the Met-FGF21 analogue, prepared as generally described in examples 1 and 2 (SEQ ID NO:1 and an N-terminal M), was modified at the alpha amino group with the following reagent:

(332) ##STR00111##

(333) The compound was prepared as described in example 3 using 1.5 equivalents of 17-[(S)-1-carboxy-3-(2-{2-[(2-{2-[(4-formylbenzylcarbamoyl)methoxy]ethoxy}ethylcarbamoyl)methoxy]ethoxy}ethyl-carbamoyl)propylcarbamoyl]heptadecanoic acid in 1M NaAc (6.1 ul, 1.5 eq). pH was adjusted to 6.0 with 10% AcOH followed by the addition of 8 mg of hydroxypropyl-beta-cyclodextrin. After 15 min 1M NaCNBH.sub.3 (7 ul) was added. The mixture was left at RT for 2 h and analyzed.

(334) LCMS1: Theoretical mass=20374.2. found 20375.25.

Example 38

Potency Assay-Glucose Uptake in 3T3-L1 Adipocytes

(335) The following assay was used for determining the biological activity, or potency, of FGF21 compounds of the invention.

(336) Mouse 3T3-L1 fibroblasts (e.g. available from ATCC, catalogue no. CL-173) are maintained in basal medium (DMEM (4500 mg/l Glucose) with 10% Fetal Bovine Serum (FBS) and Penicillin/Streptomycin). The cells are not allowed to reach confluence and should be passed (transferred to new vials) before reaching approx. 60% of confluency (by visual inspection).

(337) For the glucose uptake assay, cells are plated 80,000 cells/well in a 24 well plate, or 20,000 cells/well in a 96 well plate, and when they reach confluency (high density, with a view to have differentiated adipose cells made), the medium is changed from basal medium to basal medium containing Troglitazone, IBMX, Dexamethasone (commercially available from, e.g., Sigma) and human insulin (commercially available from, e.g., Novo Nordisk A/S).

(338) The cells are used 7-14, preferably 7-10, days after initiation of differentiation. The cells are stimulated with increasing concentrations (0-300 nM) of the FGF21 polypeptides or derivatives of the invention for 20 hours in basal medium. Before addition of 3H-deoxyglucose (in what follows: the tracer) the cells are washed in warm (approximately 37 C.) assay buffer (PBS with 1 mM MgCl.sub.2 and 2 mM CaCl.sub.2), HEPES and 0.1% Human serum albumin) and the cells are incubated with the tracer for 1 hour. This incubation is terminated by washing twice in ice cold assay buffer. The cells are lysed with Triton X-100 and lysates transferred to a 96 wells plate, microscint-40 (commercially available from, e.g., Perkin Elmer) is added and amount of tracer counted in a TOP-counter (e.g. a Packard top-counter from Perkin Elmer). The EC.sub.50 of the polypeptide in question is calculated. The results which are shown in Table 1 below indicate the EC.sub.50 (potency) of the FGF21 compounds of the invention relative to that of Met-FGF21.

(339) TABLE-US-00002 TABLE 1 Potency of FGF21 compounds Compound Glucose uptake from 3T3-L1 Example Potency (%) rel. number Compound to Met-FGF21 1 100 2a 66 2b 416 2c 872 2d 663 3 I n.a. 4 II 8.5 5 III 100 6 IV 352 [N121Q] Met-FGF21 119 [S71C, N121Q, L166F, S167G, 394 M168L, 173aA, G174V, Y179F, A180E, des181] Ala-FGF21 [N121Q, L166F, S167G, M168L, 1593 P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Ala-FGF21 [N121Q] Ala-FGF21 116

Example 39

HEK293/Beta-Klotho Erk Phosphorylation Assay

(340) Erk phosphorylation assay was performed in HEK293 cells that were stably transfected with human beta-Klotho. The HEK293T/b-klotho stable cells were seeded at 30000 cells/well on 96-well plates. After two days, fresh media was added, and after 2 hours more the FGF21 proteins were added. The plates were incubated for 12 minutes. And total ERK phosphorylation was assessed using an AlphaScreen SureFire Phospho-ERK1/2 Assay Kit (Perkin Elmer, Waltham, Mass.) according to the manufacturer's instructions and an EnVision Multilabel Microplate Reader Model 2103 (Perkin Elmer) with the AlphaScreen HTS Turbo option was used for signal detection. Data are represented as means+/S.E.M. EC50 values were determined from a 4-parameter logistic nonlinear regression analysis using GraphPad Prism version 5.02. References: Yie, J. et al.: FGF21 N- and C-termini play different roles in receptor interaction and activation, FEBS Letters 583 (2009) 19-24, and Micanovic R. et al.: Different roles of N- and C-termini in the functional activity of FGF21. J. Cell. Physiol. 2009 May; 219(2):227-34.

(341) TABLE-US-00003 TABLE 2 ERK phosphorylation Compound pERK-HEK293- from Beta-klotho without example HSA [EC50 (nM)] number Compound Mean Value 1 1.6 2a 8.3 2b 0.8 2c 0.6 2d 1.8 3 I n.a. 4 II 4.6 5 III 1.0 6 IV 0.4 [N121Q] Met-FGF21 1.0 [S71C, N121Q, L166F, S167G, 0.4 M168L, 173aA, G174V, Y179F, A180E, des181] Ala-FGF21 [N121Q, L166F, S167G, M168L, 0.7 P171L, S172E, Q173A, G174V, Y179F, A180E, des181] Ala-FGF21 [N121Q] Ala-FGF21 2.3

Example 40

In Vivo Test for FGF21 Compounds

Pharmacokinetics

(342) Mini Pig

(343) The pharmacokinetic profile of Met-FGF21 can be tested in normal male Gttingen mini pigs, n=4 (8-15 months old, weighing approximately 18-30 kg). The plasma concentration of the compound to be tested is monitored for 14 days. The Met-FGF21 is dosed as a single intravenous dose of 0.1 mg/kg (approximately 5 nmol/kg).

(344) The mean half-life (T.sub.1/2) of the comparative compound Met-FGF21 has been determined to be 10.8 hours with a standard deviation of 2.7 hours.

(345) The pharmokinetic profile of the FGF21 compound of the invention is tested in normal male Gttingen mini pigs, n=4 (8-15 months old, weighing approximately 18-30 kg). The plasma concentration is monitored for 19 days. The compound is dosed as a single intravenous dose of 0.01-0.5 mg/kg.

(346) The mean half-life (T.sub.1/2) of the compound to be tested is determined.

(347) The plasma levels of the FGF21 compounds can be determined using Fibroblast Growth Factor-21 Human ELISA (available from BioVendor, catalogue no. RD191108200R). The PC based software, WinNonLin version 5.2 from Pharsight Corporation, Cary N.C., can be used for the pharmacokinetic calculation.

(348) This test will confirm the protracted effect of the FGF21 derivatives of this invention.

Example 41

In Vivo Test of FGF21 Compounds

Pharmacodynamics

(349) The db/db mouse is a mouse model for Type 2 diabetes. The mice lack the leptin receptor and they are characterized by hyperglycemia, insulin resistance, hyperphagia and obesity.

(350) Male db/db mice (9-10 weeks old) can be used to measure the effect on blood glucose of the FGF21 analogue and derivatives according to this invention.

(351) The compounds can be administered s.c. 0.1 mg/kg in 50 mM phosphate, 145 mM NaCl, 0.05% Tween-80, pH=7.4 (2 ml/kg) once daily for 7 days (n=7-9). The respective vehicle treated groups (control) are treated with 50 mM phosphate, 145 mM NaCl, 0.05% Tween-80, pH=7.4, (2 ml/kg) s.c. once daily for 7 days. Non-fasting blood glucose can be measured before dosing and again 2 hours after dosing day 7. Blood glucose can be measured using a glucose analyzer (Biosen 5040) based on the glucose oxidase method.

(352) All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law).

(353) The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents. The mentioning herein of references is no admission that they constitute prior art.

(354) All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

(355) The use of any and all examples, or exemplary language (e.g., such as) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

(356) Herein, the word comprise is to be interpreted broadly meaning include, contain or comprehend (vide, EPO guidelines C, III, 4.13).

(357) This invention includes all modifications and equivalents of the subject matter recited in the claims and clauses appended hereto as permitted by applicable law.