Description

Pharmaceutical Composition for Treating a Metabolic Syndrome
The present invention is directed to a pharmaceutical composition containing at least
one FGF-21 (fibroblast growth factor 2 1) compound, at least one GLP-1 R (glucagon-like
peptide-1 receptor) agonist and optionally at least one anti-diabetic drug and/or at least
one DPP-4 (dipeptidyl peptidase-4) inhibitor for the treatment of at least one metabolic
syndrome and/or atherosclerosis, in particular diabetes, dyslipidemia, obesity and/or
adipositas.
Diabetes mellitus is characterized by its clinical manifestations, namely the non-insulin-dependent
or maturity onset form, also known as Type 2 diabetes and the insulin-dependent
or juvenile onset form, also known as Type 1 diabetes. The manifestations of
clinical symptoms of Type 2 diabetes and the underlying obesity usually appear at an
age over 40. In contrast, Type 1 diabetes usually shows a rapid onset of the disease
often before 30. The disease is a metabolic disorder in humans with a prevalence of
approximately one percent in the general population, with one-fourth of these being
Type 1 and three-fourth of these being Type 2 diabetes. Type 2 diabetes is a disease
characterized by high-circulating blood glucose, insulin and corticosteroid levels.
Currently, there are various pharmacological approaches for the treatment of Type 2
diabetes, which may be utilized individually or in combination, and which act via
different modes of action:
1) sulfonylurea stimulate insulin secretion;
2) biguanides (metformin) act by promoting glucose utilization, reducing hepatic glucose
production and diminishing intestinal glucose output;
3) oc-glucosidase inhibitors (acarbose, miglitol) slow down carbohydrate digestion and
consequently absorption from the gut and reduce postprandial hyperglycemia;
4) thiazolidinediones (troglitazone) enhance insulin action, thus promoting glucose
utilization in peripheral tissues; and
5) insulin stimulates tissue glucose utilization and inhibits hepatic glucose output.
However, most of the drugs have limited efficacy and do not address the most important
problems, the declining -cell function and the associated obesity.
Obesity is a chronic disease that is highly prevalent in modern society and is associated
with numerous medical problems including diabetes mellitus, insulin resistance,
hypertension, hypercholesterolemia, and coronary heart disease. It is further highly
correlated with diabetes and insulin resistance, the latter of which is generally
accompanied by hypehnsulinemia or hyperglycemia, or both. In addition, Type 2
diabetes is associated with a two to fourfold risk of coronary artery disease.
Type 1 diabetics characteristically show very low or immeasurable plasma insulin with
elevated glucagon. An immune response specifically directed against -cells leads to
Type 1 diabetes because -cells secrete insulin.
Current therapeutic regimens for Type 1 diabetes try to minimize hyperglycemia
resulting from the lack of natural insulin.
Fibroblast growth factor 2 1 (FGF21 ) is a novel metabolic regulator produced primarily
by the liver that exerts potent antidiabetic and lipid-lowering effects in animal models of
obesity and type 2 diabetes mellitus. This hormone contributes to body weight
regulation and is involved in the response to nutritional deprivation and ketogenic state
in mice. The principal sites of metabolic actions of FGF21 are adipose tissue, liver and
pancreas. Experimental studies have shown improvements in diabetes compensation
and dyslipidemia after FGF21 administration in diabetic mice and primates (Dostalova
et al. 2009). FGF21 has been shown to stimulate glucose uptake in mouse 3T3-L1
adipocytes in the presence and absence of insulin, and to decrease fed and fasting
blood glucose, triglycerides, and glucagon levels in ob/ob and db/db mice and 8 week
olf ZDF rats in a dose dependant manner, thus, providing the basis for the use of FGF-
2 1 as a therapy for treating diabetes and obesity (see e.g. WO03/01 12 13).
Fibroblast growth factors (FGFs) are polypeptides widely expressed in developing and
adult tissues. The FGF family currently consists of twenty-two members, FGF-1 to FGF-
23. The members of the FGF family are highly conserved in both gene structure and
amino acid sequence between vertebrate species. There are 18 mammalian fibroblast
growth factors (FGF1-FGF1 0 and FGF1 6-FGF23) which are grouped into 6 subfamilies
based on differences in sequence homology and phylogeny. The numbered 'FGFs' that
are unassigned to subfamilies - the FGF homologous factors (previously known as
FGF1 1-FGF14) - have high sequence identity with the FGF family but do not activate
FGF receptors (FGFRs) and are therefore not generally considered members of the
FGF family.
While most of FGFs act as local regulators of cell growth and differentiation, recent
studies indicated that FGF1 9 subfamily members including FGF1 5/1 9, FGF21 and
FGF23 exert important metabolic effects by an endocrine fashion. The members of
FGF1 9 subfamily regulate diverse physiological processes that are not affected by
classical FGFs. The wide variety of metabolic activities of these endocrine factors
include the regulation of the bile acid, carbohydrate and lipid metabolism as well as
phosphate, calcium and vitamin D homeostasis (Tomlinson et al. 2002, Holt et al. 2003,
Shimada et al. 2004, Kharitonenkov et al. 2005, Inagaki et al. 2005, Lundasen et al.
2006).
FGF21 was originally isolated from mouse embryos. FGF21 mRNA was most
abundantly expressed in the liver, and to lesser extent in the thymus (Nishimura et al.
2000). Human FGF21 is highly identical (approximately 75 % amino acid identity) to
mouse FGF21 . Among human FGF family members, FGF21 is the most similar
(approximately 35 % amino acid identity) to FGF19 (Nishimura et al. 2000). FGF21 is
free of the proliferative and tumorigenic effects (Kharitonenkov et al. 2005, Huang et al.
2006, Wente et al. 2006) that are typical for majority of the members of FGF family
(Ornitz and Itoh 2001 , Nicholes et al. 2002, Eswarakumar et al. 2005).
The administration of FGF21 to obese leptin-deficient ob/ob and leptin receptor-deficient
db/db mice and obese ZDF rats significantly lowered blood glucose and triglycerides,
decreased fasting insulin levels and improved glucose clearance during an oral glucose
tolerance test. FGF21 did not affect food intake or body weight/composition of diabetic
or lean mice and rats over the course of 2 weeks of administration. Importantly, FGF21
did not induce mitogenicity, hypoglycemia, or weight gain at any dose tested in diabetic
or healthy animals or when overexpressed in transgenic mice (Kharitonenkov et al.
2005). FGF21 -overexpressing transgenic mice were resistant to diet-induced obesity.
The administration of FGF21 to diabetic rhesus monkeys for 6 weeks reduced fasting
plasma glucose, fructosamine, triglyceride, insulin and glucagone levels. Importantly,
hypoglycemia was not observed during the study despite of significant glucose-lowering
effects. FGF21 administration also significantly lowered LDL-cholesterol and increased
HDL-cholesterol and, in contrast to mice (Kharitonenkov et al. 2005), slightly but
significantly decreased body weight (Kharitonenkov et al. 2007).
Further information can be taken from the following references:
1. DOSTALOVA I. et al.: Fibroblast Growth Factor 21: A Novel Metabolic Regulator
With Potential Therapeutic Properties in Obesity/Type 2 Diabetes Mellitus. Physiol
Res 58: 1-7, 2009.
2 . ESWARAKUMAR V.P. et al.: Cellular signaling by fibroblast growth factor receptors.
Cytokine Growth Factor Rev 16: 139-149, 2005.
3 . HOLT J.A. et al.: Definition of a novel growth factor-dependent signal cascade for
the suppression of bile acid biosynthesis. Genes Dev 17: 1581 - 1591, 2003.
4 . HUANG X. et al.: Forced expression of hepatocytespecific fibroblast growth factor
2 1 delays initiation of chemically induced hepatocarcinogenesis. Mol Carcinog 45:
934-942, 2006.
5 . INAGAKI T. et al.: Endocrine regulation of the fasting response by PPARa-mediated
induction of fibroblast growth factor 21. Cell Metab 5: 4 15-425, 2007.
6 . KHARITONENKOV A. et al.: FGF-21 as a novel metabolic regulator. J Clin Invest
5: 1627-1 635, 2005.
7 . KHARITONENKOV A. et al.: The metabolic state of diabetic monkeys is regulated
by fibroblast growth factor-21 . Endocrinology 148: 774-781 , 2007.
8 . LUNDASEN T. et al.: Circulating intestinal fibroblast growth factor 19 has a
pronounced diurnal variation and modulates hepatic bile acid synthesis in man. J
Intern Med 260: 530-536, 2006.
9 . NICHOLES K. et al.: A mouse model of hepatocellular carcinoma: ectopic
expression of fibroblast growth factor 19 in skeletal muscle of transgenic mice. Am J
Pathol 160: 2295-2307, 2002.
10. NISHIMURA T. et al.: Identification of a novel FGF, FGF-21, preferentially
expressed in the liver. Biochim Biophys Acta 1492: 203-206, 2000.
11. ORNITZ D.M. et al.: Fibroblast growth factors. Genome Biol 2: REVIEWS3005,
2001.
12. SHIMADA T. et al.: FGF-23 is a potent regulator of vitamin D metabolism and
phosphate homeostasis. J Bone Miner Res 19: 429-435, 2004.
13 . TOMLINSON E. et al.: Transgenic mice expressing human fibroblast growth factor-
19 display increased metabolic rate and decreased adiposity. Endocrinology 143:
1741 - 1747, 2002.
14. WENTE W. et al.: Fibroblast growth factor-21 improves pancreatic beta-cell function
and survival by activation of extracellular signal-regulated kinase 1/2 and Akt
signaling pathways. Diabetes 55: 2470-2478, 2006.
The gut peptide glucagon-like peptide-1 (GLP-1 ) is an incretin hormone and secreted in
a nutrient-dependent manner. It stimulates glucose-dependent insulin secretion. GLP-1
also promotes -cell proliferation and controls glycemia via additional actions on
glucose sensors, inhibition of gastric emptying, food intake and glucagons secretion.
Furthermore, GLP-1 stimulates insulin secretion and reduces blood glucose in human
subjects with Type 2 diabetes. Exogenous administration of bioactive GLP-1, GLP-1 (7-
27) or GLP-1 (7-36 amide), in doses elevating plasma concentrations to approximately
3-4 fold physiological postprandial levels fully normalizes fasting hyperglycaemia in
Type 2 diabetic patients (Nauck, M. A. et al. (1997) Exp Clin Endocrinol Diabetes, 105,
187-1 97). The human GLP-1 receptor (GLP-1 R) is a 463 amino acid heptahelical G
protein-coupled receptor widely expressed in pancreatic islets, kidney, lung, heart and
multiple regions of the peripheral and central nervous system. Within islets, the GLP-1 R
is predominantly localized to islet -cells. Activation of GLP-1 R signalling initiates a
program of differentiation toward a more endocrine-like phenotype, in particular the
differentiation of progenitors derived from human islets into functioning -cells (Drucker,
D. J. (2006) Cell Metabolism, 3, 153-1 65).
Unfortunately, both, FGF-21 and bioactive GLP-1, as well as other known drugs have
limited efficacy by themselves to the complex and multifactorial metabolic dysfunctions
which can be observed in Type 2 diabetes or othe metabolic disorders. This applies
also for the efficacy in lowering the blood glucose levels by said compounds themselves.
According to the present invention it has surprisingly been found that the combination of
FGF-21 and a GLP-1 R agonist significantly lowered blood glucose levels in a
synergistic manner up to normo-glycaemic levels.
One embodiment of the present invention is, therefore, directed to a pharmaceutical
composition containing at least one FGF-21 (fibroblast growth factor 2 1) compound and
at least one GLP-1 R (glucagon-like peptide-1 receptor) agonist.

A "FGF-21 compound" is defined as a compound showing FGF-21 activity, in particular
comprising (i) native FGF-21 , especially human FGF-21 , in particular human FGF-21 as
shown in SEQ ID NO: 1, or (ii) a FGF-21 mimetic with FGF-21 activity.
"FGF-21 activity" is usually measured in a FGF-21 activity assay generally known to a
person skilled in the art. An FGF-21 activity assay is e.g. a "glucose uptake assay" as
described in Kharitonenkov, A. et al. (2005), 115; 1627, No. 6 . As an example for the
glucose uptake assay, adipocytes are starved for 3 hours in DMEM/0.1 % BSA,
stimulated with FGF-21 for 24 hours, and washed twice with KRP buffer ( 15 mM
HEPES, pH 7.4, 118 mM NaCI, 4.8 mM KCI, 1.2 mM MgSO4, 1.3 mM CaCI2, 1.2 mM
KH2PO4, 0.1 % BSA), and 100 of KRP buffer containing 2-deoxy-D-[ 14C]glucose (2-
DOG) (0.1 \, 100 ) is added to each well. Control wells contains 100 of KRP
buffer with 2-DOG (0.1 \, 10 mM) to monitor for nonspecificity. The uptake reaction is
carried out for 1 hour at 37°C, terminated by addition of cytochalasin B (20 ) , and
measured using Wallac 1450 MicroBeta counter (PerkinElmer, USA).
Examples of FGF-21 mimetics are (a) proteins having at least about 96%, in particular
99% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 1
and having FGF-21 activity, (b) a FGF-21 fusion protein or a (c) FGF-21 conjugate, e.g.
a FGF-21 mutein, a FGF-21 -Fc fusion protein, a FGF-21 -HSA fusion protein or a
PEGylated FGF-21 .
Examples of FGF-21 muteins are described in e.g. WO2005/061 712, WO2006/028595,
WO2006/028714, WO2006/065582 or WO2008/121 563. Exemplary muteins are
muteins which have a reduced capacity for O-glycosylation when e.g. expressed in
yeast compared to wild-type human FGF-21 , e.g. human FGF-21 with a substitution at
position 167 (serine), e.g. human FGF-21 with one of the following substitutions:
Ser1 67Ala, Ser1 67Glu, Ser167Asp, Ser1 67Asn, Ser1 67Gln, Ser1 67Gly, Ser1 67Val,
Ser1 67His, Ser167Lys or Ser167Tyr. Another example is a mutein which shows
reduced deamidation compared to wild-type human FGF-21 , e.g. a mutein with a
substitution at position 12 1 (asparagine) of human FGF-21 , e.g. Asn121Ala, Asn1 21Val,
Asn1 21Ser, Asn1 21Asp or Asn1 2 1Glu. An alternative mutein is human FGF-21 having
one or more non-naturally encoded amino acids, e.g. as described by the general
formula in claim 29 of WO2008/1 21563. Other muteins comprise a substitution of a
charged (e.g. aspartate, glutamate) or polar but uncharged amino acids (e.g. serine,
threonine, asparagine, glutamine) for e.g. a polar but uncharged or charged amino acid,
respectively. Examples are Leu1 39Glu, Ala145Glu, Leu146Glu, lle152Glu, Gln156Glu,
Ser163Glu, lle1 52Glu, Ser1 63Glu or Gln54Glu. Another mutein is a mutein showing a
reduced susceptibility for proteolytic degradation when expressed in e.g. yeast
compared to human FGF-21 , in particular human FGF-21 with a substitution of Leu153
with an amino acid selected from Gly, Ala, Val, Pro, Phe, Tyr, Trp, Ser, Thr, Asn, Asp,
Gin, Glu, Cys or Met. A preferred FGF-21 mutein is the mutated FGF-21 according to
SEQ ID NO: 2 which carries a deletion of amino acids 1-28 of human FGF-21 (SEQ ID
NO: 1) and contains an additional glycine at the N-terminus.
Examples of FGF-21 fusion proteins are described in e.g. WO2004/1 10472 or
WO2005/1 13606, for example a FGF-21 -Fc fusion protein or a FGF-21 -HAS fusion
protein. "Fc" means the Fc portion of an immunoglobulin, e.g. the Fc portion of lgG4.
"HSA" means human serum albumin.
Examples of FGF-21 conjugates are described in e.g. WO2005/091 944,
WO2006/050247 or WO2009/089396, for example glycol-linked FGF-21 compounds.
Such glycol-linked FGF21 compounds usually carry a polyethylene glycol (PEG), e.g. at
a cysteine or lysine amino acid residue or at an introduced N-linked or O-linked
glycosylation site, (herein referred to as "PEGylated FGF-21 "). Such PEGylated FGF-21
compounds generally show an extended time action compared to human FGF-21 .
Suitable PEGs have a molecular weight of about 20,000 to 40,000 daltons.
A "GLP-1 R agonist" is defined as a compound which binds to and activates the GLP-1
receptor, like GLP-1 (glucagon-like peptide 1) . Physiological actions of GLP-1 and/or of
the GLP-1 R agonist are described e.g. in Nauck, M. A. et al. ( 1997) Exp. Clin.
Endocrinol. Diabetes, 105, 187-1 95. These physiological actions in normal subjects, in
particular humans, include e.g. glucose-dependent stimulation of insulin secretion,
suppression of glucagon secretion, stimulation of (pro)insulin biosynthesis, reduction of
food intake, deceleration of gastric emptying and/or equivocal insulin sensitivity.
Suitable assays to discover GLP-1 R agonists are described in e.g. Thorkildsen, Chr. et
al. (2003), Journal of Pharmacology and Experimental Therapeutics, 307, 490-496;
Knudsen, L. B. et al. (2007), PNAS, 04, 937-942, No. 3; Chen, D. et al. (2007), PNAS,
104, 943-948, No. 3; or US2006/0003417 A 1 (see e.g. Example 8). In short, in a
"receptor binding assay", a purified membrane fraction of eukaryotic cells harbouring e.g.
the human recombinant GLP-1 receptor, e.g. CHO, BHK or HEK293 cells, is incubated
with the test compound or compounds in the presence of e.g. human GLP-1 , e.g. GLP-1
(7-36) amide which is marked with e.g. 125 l (e.g. 80 kBq/pmol). Usually different
concentrations of the test compound or compounds are used and the IC5o values are
determined as the concentrations diminishing the specific binding of human GLP-1 . In a
"receptor functional assay", isolated plasma membranes from eukaryotic cells, as e.g.
described above, expressing e.g. the human GLP-1 receptor were prepared and
incubated with a test compound. The functional assay is carried out by measuring
cAMP as a response to stimulation by the test compound. In a "reporter gene assay",
eukaryotic cells, as e.g. described above, expressing e.g. the human GLP-1 receptor
and containing e.g. a multiple response element/cAMP response element-driven
luciferase reporter plasmid are cultured in the presence of a test compound. cAMP
response element-driven luciferase activities are measured as a response to stimulation
by the test compound.
Suitable GLP-1 R agonists are selected from a bioactive GLP-1, a GLP-1 analog or a
GLP-1 substitute, as e.g. described in Drucker, D. J. (2006) Cell Metabolism, 3, 153-
165; Thorkildsen, Chr. (2003; supra); Chen, D. et al. (2007; supra); Knudsen, L. B. et al.
(2007; supra); Liu, J. et al. (2007) Neurochem Int., 5 1, 361-369, No. 6-7; Christensen, M.
et al. (2009), Drugs, 12, 503-51 3; Maida, A. et al. (2008) Endocrinology, 149, 5670-
5678, No. 11 and US2006/0003417. Exemplary compounds are GLP-1 (7-37), GLP-1 (7-
36)amide, extendin-4, liraglutide, CJC-1 13 1, albugon, albiglutide, exenatide, exenatide-
LAR, oxyntomodulin, lixisenatide, geniproside, AVE-0010, a short peptide with GLP-1 R
agonistic activity and/or a small organic compound with GLP-1 R agonistic activity.
In detail, Human GLP-1 (7-37) possesses the amino acid sequence of SEQ ID NO: 3.
Human GLP-1 (7-36)amide possesses the amino acid sequence of SEQ ID NO: 4 .
Extendin-4 possesses the amino acid sequence of SEQ ID NO: 5 . Exenatide possesses
the amino acid sequence of SEQ ID NO: 6 and oxyntomodulin the amino acid sequence
of SEQ ID NO: 7 . The amino acid sequence of lixisenatide is shown in SEQ ID NO: 8.
The structure of lixisenatide is based on exendin-4(1 -39) modified C-terminally with six
additional lysine residues in order to resist immediate physiological degradation by
DPP-4 (dipeptidyl peptidase-4). The amino acid sequence of AVE0010 is shown in SEQ
ID NO: 9
The chemical structure of liraglutide is shown in Fig. 1. Liraglutide was obtained by
substitution of Lys 34 of GLP-1 (7-37) to Arg, and by addition of a C 16 fatty acid at
position 26 using a -glutamic acid spacer. The chemical name is [N-epsilon(gamma-Lglutamoyl(
N-alpha-hexadecanoyl)-Lys 2 ,Arg34-GLP-1 (7-37)].
The chemical structure of CJC-1 13 1 is shown in Fig. 2. Albumin is attached at the Cterminal
of GLP-1 with a d-alanine substitution at position 8 . CJC-1 13 1 shows a very
good combination of stability and bioactivity.
Other peptides with GLP-1 R agonistic activity are exemplary disclosed in US
2006/000341 7 and small organic compound with GLP-1 R agonistic activity are
exemplary disclosed in Chen et al. 2007, PNAS, 104, 943-948, No. 3 or Knudsen et al.,
2007, PNAS, 104, 937-942.
In a further embodiment of the present invention the pharmaceutical composition
additionally contains at least one anti-diabetic drug and/or at least one DPP-4 inhibitor.
Exemplary anti-diabetic drugs are
a) insulin,
b) thiazolidinedione, e.g. rosiglitazone or pioglitazone (see e.g. WO2005/072769),
metformin ( /,/V-dimethylimidodicarbonimidic-diamide), or
c) sulphonylurea, such as chlorpropamide (4-chloro-/V-(propylcarbamoyl)-
benzenesulfonamide), tolazamide (A/-[(azepan-1 -ylamino)carbonyl]-4-methylbenzenesulfonamide),
gliclazide (A/-(hexahydrocyclopenta[c]pyrrol-2(1 H)-ylcarbamoyl)-
4-methylbenzenesulfonamide), or glimepiride (3-ethyl-4-methyl-/V-(4-[/V-
((1 r,4r)-4-methylcyclohexylcarbamoyl)-sulfamoyl]phenethyl)-2-oxo-2,5-dihydro-1 Hpyrrole-
1 -carboxamide).
According to the present invention "insulin" means naturally occurring insulin, modified
insulin or an insulin analogue, including salts thereof, and combinations thereof, e.g.
combinations of a modified insulin and an insulin analogue, for example insulins which
have amino acid exchanges/deletions/additions as well as further modifications such as
acylation or other chemical modification. One example of this type of compound is
insulin detemir, i.e. LysB29-tetradecanoyl/des(B30) human insulin. Another example
may be insulins in which unnatural amino acids or amino acids which are normally noncoding
in eukaryotes, such as D-amino acids, have been incorporated (Geiger, R. et al.,
Hoppe Seylers Z. Physiol. Chem. (1976) 357, 1267-1 270; Geiger, R. et al., Hoppe
Seylers Z. Physiol. Chem. ( 1975) 356, 1635-1 649, No. 10; Krail, G. et al., Hoppe
Seylers Z. Physiol. Chem. (1971 ) 352, 1595-1 598, No. 11) . Yet other examples are
insulin analogues in which the C-terminal carboxylic acid of either the A-chain or the Bchain,
or both, are replaced by an amide.
"Modified insulin" is preferably selected from acylated insulin with insulin activity, in
particular wherein one or more amino acid(s) in the A and/or B chain of insulin is/are
acylated, preferably human insulin acylated at position B29 (Tsai, Y. J. et al. ( 1997)
Journal of Pharmaceutical Sciences, 86, 1264-1 268, No. 11). Other acetylated insulins
are desB30 human insulin or B01 bovine insulin (Tsai, Y. J. et al., supra). Other
Examples of acylated insulin are e.g. disclosed in US 5,750,497 and US 6,01 1,007. An
overview of the structure-activity relationships for modified insulins, is provided in Mayer,
J. P. et al. (2007) Biopolymers, 88, 687-71 3, No. 5. Modified insulins are typically
prepared by chemical and/or enzymatic manipulation of insulin, or a suitable insulin
precursor such as preproinsulin, proinsulin or truncated analogues thereof.
An "insulin analogue" is preferably selected from insulin with insulin activity having one
or more mutation(s), substitution(s), deletion(s) and/or addition(s), in particular an
insulin with a C- and/or N-terminal truncation or extension in the A and/or B chain,
preferably des(B30) insulin, PheB1 insulin, B 1-4 insulin, AspB28 human insulin (insulin
aspart), LysB28/ProB29 human insulin (insulin lispro), LysB03/GluB29 human insulin
(insulin glulisine) or GlyA21/ArgB31/ArgB32 human insulin (insulin glargine). The only
proviso of an insulin analogue is that it has a sufficient insulin activity. An overview of
the structure-activity relationships for insulin analogues, with discussion of which amino
acid exchanges, deletions and/or additions are tolerated is provided in Mayer, J. P. et al.
(2007; supra). The insulin analogues are preferably such wherein one or more of the
naturally occurring amino acid residues, preferably one, two or three of them, have been
substituted by another amino acid residue. Further examples of insulin analogues are
C-terminal truncated derivatives such as des(B30) human insulin; B-chain N-terminal
truncated insulin analogues such as des PheB1 insulin or des B 1-4 insulin; insulin
analogues wherein the A-chain and/or B-chain have an N-terminal extension, including
so-called "pre-insulins" where the B-chain has an N-terminal extension; and insulin
analogues wherein the A-chain and/or the B-chain have C-terminal extension. For
example one or two Arg may be added to position B 1 . Examples of insulin analogues
are described in the following patents and equivalents thereto: US 5,61 8,91 3, EP 0 254
5 16 A2 and EP 0 280 534 A2. An overview of insulin analogues in clinical use is
provided in Mayer J. P. et al. (2007, supra). Insulin analogues or their precursors are
typically prepared using gene technology techniques well known to those skilled in the
art, typically in bacteria or yeast, with subsequent enzymatic or synthetic manipulation if
required. Alternatively, insulin analogues can be prepared chemically (Cao, Q. P. et al.
( 1986) Biol. Chem. Hoppe Seyler, 367, 135-140, No. 2). Examples of specific insulin
analogues are insulin aspart (i.e. AspB28 human insulin); insulin lispro (i.e. LysB28,
ProB29 human insulin); insulin glulisine (ie. LysB03, GluB29 human insulin); and insulin
glargine (i.e. GlyA21 , ArgB31 , ArgB32 human insulin).
Exemplary DPP-4 Inhibitors are
sitagliptin: (R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1 ,2,4]triazolo[4,3-a]-pyrazin-
7(8/-/)-yl]-1 -(2,4,5-trifluorophenyl)butan-2-amine,
vildagliptin: (S)-1 -[/V-(3-hydroxy-1-adamantyl)glycyl]pyrrolidine-2-carbonitrile,
saxagliptin: ( 1S,3S,5S)-2-[(2S)-2-amino-2-(3-hydroxy-1 -adamantyl)-acetyl]-2-
azabicyclo[3.1 .0]hexane-3-carbonitrile,
linagliptin 8-[(3R)-3-aminopiperidin-1 -yl]-7-(but-2-yn-1 -yl)-3- methyl-1 -[(4-methylquinazolin-
2-yl)methyl]-3,7-dihydro-1 H-purine-2,6-dione) adogliptin (2-({6-[(3R)-3-
aminopiperidin-1-yl]-3-methyl-2,4-dioxo-3,4-dihydropyrimidin-1 (2/-/)-yl}methyl)-
benzonitrile, and
berberine which is a quaternary ammonium salt from the group of isoquinoline alkaloids
found in in the roots, rhizomes, stems, and bark of plants such as Berberis, goldenseal
(Hydrastis canadensis), and Coptis chinensis.
The individual compounds of the pharmaceutical composition of the present invention
can be combined in one formulation or contained in several formulations for e.g.
simultaneous or subsequent, i.e. sequential administration(s), or combinations thereof.
According to the present invention the combination of at least one FGF-21 compound
and at least one GLP-1 R agonist surprisingly resulted in a synergistic effect in lowering
plasma glucose levels as shown with the animal models in the Examples. The animal
models are an ob/ob or obese mouse and a db/db mouse. The ob/ob mouse is a mutant
mouse which cannot produce the hormone leptin which regulates the appetite.
Consequently, the ob/ob mouse eats excessively and becomes profoundly obese. It is a
standard animal model for hyperglycemia, insulin resistance and obesity. Another
standard animal model for diabetes is the db/db mouse carrying a deficient leptin
receptor activity. Also this mouse is characterized by obesity, hyperglycemia and insulin
resistance.
The pharmaceutical composition of the present invention contains therapeutically
effective amounts of the individual compounds and generally an acceptable
pharmaceutical carrier, diluent or excipient, e.g. sterile water, physiological saline,
bacteriostatic saline, i.e. saline containing about 0.9% mg/ml benzyl alcohol, phosphatebuffered
saline, Hank's solution, Ringer's-lactate, lactose, dextrose, sucrose, trehalose,
sorbitol, Mannitol, and the like. The composition is generally a solution or suspension. It
can be administered orally, subcutaneously, intramuscularly, pulmonary, by inhalation
and/or through sustained release administrations. Preferably, the composition is
administered subcutaneously.
The term "therapeutically effective amount" generally means the quantity of a compound
that results in the desired therapeutic and/or prophylactic effect without causing
unacceptable side-effects. A typical dosage range is from about 0.01 mg per day to
about 1000 mg per day. A preferred dosage range for each therapeutically effective
compound is from about 0.1 mg per day to about 100 mg per day and a most preferred
dosage range is from about 1.0 mg/day to about 10 mg/day, in particular about 1-5
mg/day.
In case of subsequent administration(s), the individual compounds of the
pharmaceutical composition are administered during a time period where the synergistic
effect of the FGF-21 compound and the GLP-1 R agonist are still measurable e.g. in a
"glucose tolerance test", as e.g. shown in the Examples. The glucose tolerance test is a
test to determine how quickly glucose is cleared from the blood after administration of
glucose. The glucose is most often given orally ("oral glucose tolerance test" or "OGTT").
The time period for the subsequent administration of the individual compounds, in
particular of the FGF-21 compound and the GLP-1 R agonist, is usually within one hour,
preferably, within half an hour, most preferably within 15 minutes, in particular within 5
minutes.
Generally, the application of the pharmaceutical composition to a patient is one or
several times per day, or one or several times a week, or even during longer time
periods as the case may be. The most preferred application of the pharmaceutical
composition of the present invention is a subcutaneous application one to three times
per day in a combined dose.
The pharmaceutical composition of the present invention lowers blood glucose levels up
to normo-glycaemic levels and increase energy expenditure by faster and more efficient
glucose utilization, and thus is useful for treating at least one metabolic syndrome
and/or atherosclerosis, in particular Type 1 or Type 2 diabetes, dyslipidemia, obesity
and/or adipositas, in particular Type 2-diabetes.
Consequently, the present invention is also directed to the use of the described
pharmaceutical composition(s) for the preparation of a medicament for treating at least
one of the above-mentioned diseases or disorders, and to a method for treating at least
one of the above-mentioned diseases in a patient. The patient is especially selected
from a Type 1-diabetic patient, a Type 2-diabetic patient, in particular a diet-treated
Type 2-diabetic patient, a sulfonyl urea-treated Type 2-diabetic patient, a far-advanced
stage Type 2-diabetic patient and/or a long-term insulin-treated Type 2-diabetic patient.
The medicament can be prepared by methods known to a person skilled in the art, e.g.
by mixing the pharmaceutically effective amounts of the compound or compounds with
an acceptable pharmaceutical carrier, diluent or excipient, as described above.
The following figures and examples are for the purpose of illustration only and are not
intended to be limiting of the present invention.
Figures
shows the chemical structure of liraglutide.
Fig. 2 shows the chemical structure of CJC-1 13 1.
Fig. 3 shows the results of an oral glucose tolerance test (OGTT) after ten days
subcutaneous injection of FGF-21 together with AVE0010 in ob/ob mice.
Fig. 4 shows the plasma glucose levels over time after subcutaneous injection of FGF-
2 1 together with AVE0010 in ob/ob mice.
Fig. 5 shows the results of an OGTT after after twenty-one days subcutaneous
injection of FGF-21 together with AVE0010 in db/db mice.
Fig. 6 shows the plasma glucose levels over time after subcutaneous injection of FGF-
2 1 together with AVE0010 in db/db mice.
Examples
1. Treatment of ob/ob mice
Female ob/ob mice (B6.V-LEP OB/J, age of 6 weeks) were obtained from Charles
Rivers Laboratories (Sulzfeld, Germany). Mice were randomly assigned to treatment or
vehicle groups, and the randomization was stratified by body weight and fed blood
glucose levels. The animals were housed in groups of 6 at 23°C and on a 12 h lightdark
cycle. All experimental procedures were conducted according to German Animal
Protection Law.
Ob/ob mice were treated with vehicle (PBS), 0.05 mg • kg 1 • day 1 AVE0010 (SEQ ID
NO:9), 0.75 mg • kg 1 • day 1 recombinant human FGF-21 (SEQ ID NO: 2) or a combined
dose of FGF-21 (SEQ ID NO: 2) and AVE0010 (SEQ ID NO:9), (0.75 + 0.05 mg • kg 1 •
day 1) subcutaneously once daily. Mice were fed ad libitum with standard rodent chow
during the drug treatment periods. Body weight was recorded every other day, and food
intake was measured once a week throughout the study. One day before the first
treatment and at study day 10 blood glucose was measured by tail tip bleeding under
fed conditions. As shown in Figure 4 the blood glucose levels of the treated mice
became normo-glycaemic. On study day 8 a glucose tolerance test (OGTT) was
performed. Fasted mice were orally challenged with 2 g • kg 1 glucose. Blood glucose
was measured at indicated time points by tail tip bleeding without anaesthesia. The
results of the OGTT are shown in Figure 3 . Compared to the administration of only
FGF-21 or only AVE0010 glucose tolerance was markedly stronger improved by
combination treatment. The combination treated obese animals were even more
glucose tolerant than lean control animals.
2 . Treatment of db/db mice
Female db/db mice (BKS.Cg-m +/+ Leprdb /J, age of 6 weeks) were treated with vehicle
(PBS), 0.05 mg • kg 1 • day 1 AVE0010, 0.75 mg • kg 1 • day 1 recombinant human FGF-
2 1 (SEQ ID NO: 2) or a combined dose of FGF-21 (SEQ ID NO: 2) and AVE0010 (SEQ
ID NO:9), (0.75 + 0.05 mg • kg 1 • day 1) subcutaneously once daily. Mice were fed ad
libitum. Before the first treatment, after one week and 4 weeks blood glucose and
HbA1 c were measured under fed conditions. After 2 1 days of treatment an oral glucose
tolerance test (OGTT) was initiated. Fasted mice were orally challenged with 2 g • kg 1
glucose solution and blood glucose was measured at indicated time points. The results
are shown in Figure 5 and 6 . The administration of the FGF21 plus AVE0010
combination results in normalisation of blood glucose and improved dramatically the
glucose tolerance compared to the vehicle treated obese control. On the other hand
leads the single treatment of FGF21 or AVE001 0 compared to the combination only to
inhibition of blood glucose increase and a small improvement in glucose tolerance.
Sequences
Human FGF-21 (SEQ ID NO: 1) :
MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQPGGQVRQRYLYTDDAQQTEAHLEIREDGT
VGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQ
SEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRS
PSYAS
Mutated FGF-21 (G + FGF21 H29-S209; SEQ ID NO: 2):
GHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILG
VKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPAR
FLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS
Human GLP-1 (7-37) (SEQ ID NO: 3):
HAEGTFTSDVSSYLEGQAAKEFiAWLVKGRG-NH 2
Human GLP-1 (7-36)NH 2 (SEQ ID NO: 4):
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH 2
Exendin-4 (SEQ ID NO: 5):
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH 2
Exenatide (SEQ ID NO: 6):
HGEGTFTSDLSKQMEEEAVRLFIETLKNGGPSSGAPPPS-NH 2
Oxyntomodulin (SEQ ID NO: 7):
HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-NH 2
Lixisenatide (SEQ ID NO: 8)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2
AVE0010 (SEQ ID NO: 9):
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2

Claims
A pharmaceutical composition containing at least one FGF-21 (fibroblast growth
factor 2 1) compound and at least one GLP-1 R (glucagon-like peptide-1 receptor)
agonist.
The pharmaceutical composition of claim 1, wherein the composition further
contains at least one anti-diabetic drug and/or at least one DPP-4 (dipeptidyl
peptidase-4) inhibitor.
The pharmaceutical composition of claim 1 or 2, wherein the FGF-21 compound(s),
the GLP-1 R agonist(s), optionally the anti-diabetic drug(s) and optionally the DPP-4
inhibitor are combined in one formulation or contained in several formulations.
The pharmaceutical composition of claim 3, wherein the formulations of the FGF-21
compound(s), the GLP-1 R agonist(s), optionally the anti-diabetic drug(s) and
optionally the DPP-4 inhibitor are suitable for simultaneous or subsequent
administration(s).
The pharmaceutical composition of at least one of the claims 1-4, wherein the FGF-
2 1 compound is selected from FGF-21 or a FGF-21 mimetic.
The pharmaceutical composition of claim 5, wherein the FGF-21 mimetic is selected
from a protein having at least about 96% amino acid sequence identity to the amino
acid sequence shown in SEQ ID NO: 1 and having FGF-21 activity, a FGF-21
fusion protein and/or a FGF-21 conjugate.
The pharmaceutical composition of claim 6, wherein the FGF-21 mimetic is selected
from a FGF-21 mutein, a FGF-21 -Fc fusion protein, a FGF-21 -HSA fusion protein
and/or a PEGylated FGF-21 .
The pharmaceutical composition of at least one of the claims 1-7, wherein the GLP-
1R agonist is selected from a bioactive GLP-1, a GLP-1 analog or a GLP-1
substitute.
The pharmaceutical composition of claim 8, wherein the GLP-1 R agonist is selected
from GLP-1 (7-37), GLP-1 (7-36)amide, extendin-4, liraglutide, CJC-1 13 1, albugon,
albiglutide, exenatide, exenatide-LAR, oxyntomodulin, lixisenatide, geniproside,
AVE-0010 (SEQ ID NO: 9), a short peptide with GLP-1 R agonistic activity and/or a
small organic compound with GLP-1 R agonistic activity.
10 . The pharmaceutical composition of at least one of the claims 1-9, wherein the antidiabetic
drug is selected from metformin, a thiazolidinedione, a sulphonylurea,
and/or insulin.
11. The pharmaceutical composition of at least one of the claims 1- 10, wherein the
DPP-4 inhibitor is selected from sitagliptin, vildagliptin, saxagliptin, linagliptin,
adogliptin and/or berberine.
12 . The pharmaceutical composition of at least one of the claims 1- 1 1S for use in treating
at least one metabolic syndrome and/or atherosclerosis.
13 . The pharmaceutical composition of claim 12, wherein the metabolic syndrome is
selected from diabetes, dyslipidemia, obesity and/or adipositas, in particular Type 2-
diabetes.
14. Use of a pharmaceutical composition as defined in at least one of the claims 1- 1 1
for the preparation of a medicament for treating at least one metabolic syndrome
and/or atherosclerosis in a patient.
15 . The use of claim 14, wherein the metabolic syndrome is selected from diabetes,
dyslipidemia, obesity and/or adipositas, in particular Type 2-diabetes.
16 . The use of claim 14 or 15, wherein the patient is selected from a Type 1-diabetic
patient, a Type 2-diabetic patient, in particular a diet-treated Type 2-diabetic patient,
a sulfonyl urea-treated Type 2-diabetic patient, a far-advanced stage Type 2-diabetic
patient and/or a long-term insulin-treated Type 2-diabetic patient.