U.S. patent application number 15/194025 was filed with the patent office on 2016-10-20 for glucagon analogues.
The applicant listed for this patent is Zealand Pharma A/S. Invention is credited to Jens Rosengren DAUGAARD, Eddi MEIER, Ditte RIBER, Marie SKOVGAARD.
Application Number | 20160304576 15/194025 |
Document ID | / |
Family ID | 44630045 |
Filed Date | 2016-10-20 |
United States Patent
Application |
20160304576 |
Kind Code |
A1 |
MEIER; Eddi ; et
al. |
October 20, 2016 |
GLUCAGON ANALOGUES
Abstract
The invention provides materials and methods for promoting
weight loss or preventing weight gain without affecting glycemic
control. In particular, the invention provides novel glucagon
analogue peptides effective in such methods. The peptides may
mediate their effect by having increased selectivity for the GLP-1
receptor as compared to human glucagon.
Inventors: |
MEIER; Eddi; (Vaerlose,
DK) ; RIBER; Ditte; (Bronshoj, DK) ; DAUGAARD;
Jens Rosengren; (Virum, DK) ; SKOVGAARD; Marie;
(Copenhagen O, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zealand Pharma A/S |
Glostrup |
|
DK |
|
|
Family ID: |
44630045 |
Appl. No.: |
15/194025 |
Filed: |
June 27, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13702841 |
Feb 25, 2013 |
9403894 |
|
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PCT/DK2011/000067 |
Jun 23, 2011 |
|
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15194025 |
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61358623 |
Jun 25, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/12 20180101; A61P
3/06 20180101; A61P 43/00 20180101; A61P 5/48 20180101; A61P 9/00
20180101; A61P 3/04 20180101; A61K 38/26 20130101; C07K 14/605
20130101; A61P 9/10 20180101; A61K 45/06 20130101 |
International
Class: |
C07K 14/605 20060101
C07K014/605; A61K 45/06 20060101 A61K045/06; A61K 38/26 20060101
A61K038/26 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 23, 2010 |
DK |
PA 2010 00550 |
Claims
1. A compound having the formula R.sup.1--X--Z--R.sup.2 wherein
R.sup.1 is H, C.sub.1-4 alkyl, acetyl, formyl, benzoyl or
trifluoroacetyl; R.sup.2 is OH or NH.sub.2; X is a peptide which
has the formula I: TABLE-US-00005 (I) (SEQ ID NO: 11)
His-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-
Tyr-Leu-Asp-X16-X17-Arg-Ala-X20-Asp-Phe-Ile-Glu-
Trp-Leu-X27-X28-X29
wherein X2 is selected from Ser and Aib; X16 is selected from Glu
and Y; X17 is selected from Arg and Y; X20 is selected from Lys and
Y; X27 is selected from Leu and Y; X28 is selected from Ser and Y
or absent; X29 is Ala or absent; wherein at least one of X16, X17,
X20, X27 and X28 is Y; wherein each residue Y is independently
selected from Lys, Cys and Orn; wherein the side chain of at least
one amino acid residue Y of X is conjugated to a lipophilic
substituent having the formula: (i) Z.sup.1, wherein Z.sup.1 is a
lipophilic moiety conjugated directly to the side chain of Y; or
(ii) Z.sup.1Z.sup.2, wherein Z.sup.1 is a lipophilic moiety,
Z.sup.2 is a spacer, and Z.sup.1 is conjugated to the side chain of
Y via Z.sup.2; and Z is absent or is a sequence of 1-20 amino acid
units independently selected from the group consisting of Ala, Leu,
Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn; or a
pharmaceutically acceptable salt thereof.
2. A compound according to claim 1 having the sequence
TABLE-US-00006 (SEQ ID NO: 12) HSQGTFTSDYSKYLDERRAKDFIEWLKSA (SEQ
ID NO: 13) HSQGTFTSDYSKYLDERRAKDFIEWLLSA (SEQ ID NO: 14)
HSQGTFTSDYSKYLDERRAKDFIEWLLKA (SEQ ID NO: 15)
HSQGTFTSDYSKYLDKRRAKDFIEWLLSA (SEQ ID NO: 16)
HSQGTFTSDYSKYLDEKRAKDFIEWLLSA or (SEQ ID NO: 17)
H-Aib-QGTFTSDYSKYLDEKRAKDFIEWLLSA.
3. A compound according to claim 1, wherein said lipophilic
substituent is attached to the amino acid residue at position 16,
17, 20, 27 or 28.
4. A compound according to claim 1 wherein R.sup.1 is H.
5. A compound according to claim 1 wherein R.sup.2 is NH.sub.2.
6. A compound according to claim 1, having the sequence
TABLE-US-00007 (SEQ ID NO: 5)
H-HSQGTFTSDYSKYLDERRAKDFIEWL-K(Hexadecanoyl- isoGlu)-SA-NH.sub.2
(SEQ ID NO: 6) H-HSQGTFTSDYSKYLDERRA-K(Hexadecanoyl-isoGlu)-
DFIEWLLSA-NH.sub.2 (SEQ ID NO: 7)
H-HSQGTFTSDYSKYLDERRAKDFIEWLL-K(Hexadecanoyl- isoGlu)-A-NH.sub.2
(SEQ ID NO: 8) H-HSQGTFTSDYSKYLD-K(Hexadecanoyl-isoGlu)-
RRAKDFIEWLLSA-NH.sub.2 (SEQ ID NO: 9)
H-HSQGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- RAKDFIEWLLSA-NH.sub.2 or
(SEQ ID NO: 10) H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-
RAKDFIEWLLSA-NH.sub.2.
7. A compound according to claim 1 wherein one or more of the amino
acid side chains in the compound is conjugated to a polymeric
moiety.
8. A compound according to claim 7 wherein one or more of the amino
acid side chains in peptide X is conjugated to a polymeric
moiety.
9. A composition comprising a compound according to claim 1, or a
salt or derivative thereof, in admixture with a carrier.
10. A composition according to claim 9 wherein the composition is a
pharmaceutically acceptable composition, and the carrier is a
pharmaceutically acceptable carrier.
11. (canceled)
12. A method of preventing weight gain or promoting weight loss in
an individual in need thereof, said method comprising administering
to said individual a therapeutically effective amount of a compound
according to claim 1.
13. A method of lowering circulating LDL levels, and/or increasing
HDL/LDL ratio in an individual in need thereof, said method
comprising administering to said individual a therapeutically
effective amount of a compound according to claim 1.
14. The method of claim 12, wherein the subject has a condition
caused or characterised by excess body weight.
15. (canceled)
16. The method according to claim 13 wherein the compound is
administered as part of a combination therapy together with an
agent for treatment of obesity, dyslipidemia or hypertension.
17. The method according to claim 16, wherein the agent for
treatment of obesity is a glucagon-like peptide receptor 1 agonist,
peptide YY receptor agonist or analogue thereof, cannabinoid
receptor 1 antagonist, lipase inhibitor, melanocortin receptor 4
agonist, or melanin concentrating hormone receptor 1
antagonist.
18. The method according to claim 16 wherein the agent for
treatment of hypertension is an angiotensin-converting enzyme
inhibitor, angiotensin II receptor blocker, diuretic, beta-blocker,
or calcium channel blocker.
19. The method according to claim 16 wherein the agent for
treatment of dyslipidaemia is a statin, a fibrate, a niacin or a
cholesterol absorbtion inhibitor.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to glucagon analogues and
their medical use, for example in the treatment of excess food
intake, obesity and excess weight, elevated cholesterol, and with
no or only little effect on glycemic control.
BACKGROUND OF THE INVENTION
[0002] Preproglucagon is a 158 amino acid precursor polypeptide
that is differentially processed in the tissues to form a number of
structurally related proglucagon-derived peptides, including
glucagon (Glu), glucagon-like peptide-1 (GLP-1), glucagon-like
peptide-2 (GLP-2), and oxyntomodulin (OXM). These molecules are
involved in a wide variety of physiological functions, including
glucose homeostasis, insulin secretion, gastric emptying and
intestinal growth, as well as regulation of food intake.
[0003] Glucagon is a 29-amino acid peptide that corresponds to
amino acids 53 to 81 of pre-proglucagon and has the sequence
His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-A-
la-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 1).
Oxyntomodulin (OXM) is a 37 amino acid peptide which includes the
complete 29 amino acid sequence of glucagon with an octapeptide
carboxyterminal extension (amino acids 82 to 89 of pre-proglucagon,
having the sequence Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala (SEQ ID NO: 2)
and termed "intervening peptide 1" or IP-1; the full sequence of
human oxyntomodulin is thus
His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-A-
la-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala-
) (SEQ ID NO: 3). The major biologically active fragment of GLP-1
is produced as a 30-amino acid, C-terminally amidated peptide that
corresponds to amino acids 98 to 127 of pre-proglucagon.
[0004] Glucagon helps maintain the level of glucose in the blood by
binding to glucagon receptors on hepatocytes, causing the liver to
release glucose--stored in the form of glycogen--through
glycogenolysis. As these stores become depleted, glucagon
stimulates the liver to synthesize additional glucose by
gluconeogenesis. This glucose is released into the bloodstream,
preventing the development of hypoglycemia.
[0005] OXM is released into the blood in response to food ingestion
and in proportion to meal calorie content. OXM has been shown to
suppress appetite and inhibit food intake in humans (Cohen et al,
Journal of Endocrinology and Metabolism, 88, 4696-4701, 2003; WO
2003/022304). In addition to those anorectic effects, which are
similar to those of GLP-1, OXM must also affect body weight by
another mechanism, since rats treated with oxyntomodulin show less
body weight gain than pair-fed rats (Bloom, Endocrinology 2004,
145, 2687). Treatment of obese rodents with OXM also improves their
glucose tolerance (Parlevliet et al, Am J Physiol Endocrinol Metab,
294, E142-7, 2008) and suppresses body weight gain (WO
2003/022304).
[0006] OXM activates both the glucagon and the GLP-1 receptors with
a two-fold higher potency for the glucagon receptor over the GLP-1
receptor, but is less potent than native glucagon and GLP-1 on
their respective receptors. Human glucagon is also capable of
activating both receptors, though with a strong preference for the
glucagon receptor over the GLP-1 receptor. GLP-1 on the other hand
is not capable of activating glucagon receptors. The mechanism of
action of oxyntomodulin is not well understood. In particular, it
is not known whether some of the extrahepatic effects of the
hormone are mediated through the GLP-1 and glucagon receptors, or
through one or more unidentified receptors.
[0007] Other peptides have been shown to bind and activate both the
glucagon and the GLP-1 receptor (Hjort et al, Journal of Biological
Chemistry, 269, 30121-30124, 1994) and to suppress body weight gain
and reduce food intake (WO 2006/134340, WO 2007/100535, WO
2008/10101, WO 2008/152403, WO 2009/155257 and WO 2009/155258).
[0008] Obesity is a globally increasing health problem is
associated with various diseases, particularly cardiovascular
disease (CVD), type 2 diabetes, obstructive sleep apnea, certain
types of cancer, and osteoarthritis. As a result, obesity has been
found to reduce life expectancy. According to 2005 projections by
the World Health Organization there are 400 million adults (age
>15) classified as obese worldwide. In the US, obesity is now
believed to be the second-leading cause of preventable death after
smoking.
[0009] The rise in obesity drives an increase in diabetes, and
approximately 90% of people with type 2 diabetes may be classified
as obese. There are 246 million people worldwide with diabetes, and
by 2025 it is estimated that 380 million will have diabetes. Many
have additional cardiovascular risk factors, including
high/aberrant LDL and triglycerides and low HDL.
[0010] Accordingly, there is a strong medical need for treating
obesity.
SUMMARY OF THE INVENTION
[0011] The invention provides a compound having the formula
R.sup.1--X--Z--R.sup.2
[0012] wherein
[0013] R.sup.1 is H, C.sub.1-4 alkyl, acetyl, formyl, benzoyl or
trifluoroacetyl;
[0014] R.sup.2 is OH or NH.sub.2;
[0015] X is a peptide which has the formula I:
TABLE-US-00001 (I) His-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-
Tyr-Leu-Asp-X16-X17-Arg-Ala-X20-Asp-Phe-Ile-
X24-Trp-Leu-X27-X28-X29
[0016] wherein
[0017] X2 is selected from Ser and Aib;
[0018] X16 is selected from Glu and Y;
[0019] X17 is selected from Arg and Y;
[0020] X20 is selected from Lys and Y;
[0021] X24 is selected from Glu and Y;
[0022] X27 is selected from Leu and Y;
[0023] X28 is selected from Ser and Y or absent;
[0024] X29 is Ala or absent;
[0025] wherein at least one of X16, X17, X20, X24, X27 and X28 is
Y;
[0026] wherein each residue Y is independently selected from Lys,
Cys and Orn;
[0027] wherein the side chain of at least one amino acid residue Y
of X is conjugated to a lipophilic substituent having the
formula:
[0028] (i) Z.sup.1, wherein Z.sup.1 is a lipophilic moiety
conjugated directly to the side chain of X; or
[0029] (ii) Z.sup.1Z.sup.2, wherein Z.sup.1 is a lipophilic moiety,
Z.sup.2 is a spacer, and Z.sup.1 is conjugated to the side chain of
X via Z.sup.2;
[0030] and Z is absent or is a sequence of 1-20 amino acid units
independently selected from the group consisting of Ala, Leu, Ser,
Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn;
[0031] or a pharmaceutically acceptable salt thereof.
[0032] In one example, X may have the sequence:
TABLE-US-00002 HSQGTFTSDYSKYLDERRAKDFIEWLKSA
HSQGTFTSDYSKYLDERRAKDFIEWLLSA HSQGTFTSDYSKYLDERRAKDFIEWLLKA
HSQGTFTSDYSKYLDKRRAKDFIEWLLSA HSQGTFTSDYSKYLDEKRAKDFIEWLLSA or
H-Aib-QGTFTSDYSKYLDEKRAKDFIEWLLSA
[0033] In some embodiments, the lipophilic substituent is attached
to the amino acid residue at position 16, 17, 20, 27 or 28.
[0034] For example, the compound of the invention may have the
sequence:
TABLE-US-00003 H-HSQGTFTSDYSKYLDERRAKDFIEWL-K(Hexadecanoyl-
isoGlu)-SA-NH.sub.2 H-HSQGTFTSDYSKYLDERRA-K(Hexadecanoyl-isoGlu)-
DFIEWLLSA-NH.sub.2 H-HSQGTFTSDYSKYLDERRAKDFIEWLL-K(Hexadecanoyl-
isoGlu)-A-NH.sub.2 H-HSQGTFTSDYSKYLD-K(Hexadecanoyl-isoGlu)-
RRAKDFIEWLLSA-NH.sub.2 H-HSQGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-
RAKDFIEWLLSA-NH or H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-
RAKDFIEWLLSA-NH.sub.2
[0035] The invention further provides a nucleic acid (which may be
DNA or RNA) encoding a compound of the invention, an expression
vector comprising such a nucleic acid, and a host cell containing
such a nucleic acid or expression vector.
[0036] In a further aspect, the present invention provides a
composition comprising a glucagon analogue peptide as defined
herein, or a salt or derivative thereof, a nucleic acid encoding
such a glucagon analogue peptide, an expression vector comprising
such a nucleic acid, or a host cell containing such a nucleic acid
or expression vector, in admixture with a carrier. In preferred
embodiments, the composition is a pharmaceutically acceptable
composition and the carrier is a pharmaceutically acceptable
carrier. The glucagon peptide analogue may be in the form of a
pharmaceutically acceptable salt of the glucagon analogue.
[0037] In still a further aspect, the present invention provides a
composition for use in a method of medical treatment.
[0038] The compounds described find use, inter alia, in preventing
weight gain or promoting weight loss. By "preventing" is meant
inhibiting or reducing when compared to the absence of treatment,
and is not necessarily meant to imply complete cessation of weight
gain. The peptides may cause a decrease in food intake and/or
increased energy expenditure, resulting in the observed effect on
body weight. Independently of their effect on body weight, the
compounds of the invention may have a beneficial effect on
circulating cholesterol levels, being capable of lowering
circulating LDL levels and increasing HDL/LDL ratio. Thus the
compounds of the invention can be used for direct or indirect
therapy of any condition caused or characterised by excess body
weight, such as the treatment and/or prevention of obesity, morbid
obesity, obesity linked inflammation, obesity linked gallbladder
disease, obesity induced sleep apnea. They may also be used for the
prevention of metabolic syndrome, hypertension, atherogenic
dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart
disease, or stroke. Their effects in these conditions may be as a
result of or associated with their effect on body weight, or may be
independent thereof.
[0039] In certain embodiments, the compounds described may find use
in preventing weight gain or promoting weight loss with no or only
little effect on glycemic control. It has been found in the present
invention that certain of the compounds described have marked
effect on weight loss with no or only little effect on the HbA1c
level in a well-established animal model.
[0040] As already described, the invention extends to expression
vectors comprising the above-described nucleic acid sequence,
optionally in combination with sequences to direct its expression,
and host cells containing the expression vectors. Preferably the
host cells are capable of expressing and secreting the compound of
the invention. In a still further aspect, the present invention
provides a method of producing the compound, the method comprising
culturing the host cells under conditions suitable for expressing
the compound and purifying the compound thus produced.
[0041] The invention further provides a nucleic acid of the
invention, an expression vector of the invention, or a host cell
capable of expressing and secreting a compound of the invention,
for use in a method of medical treatment. It will be understood
that the nucleic acid, expression vector and host cells may be used
for treatment of any of the disorders described herein which may be
treated with the compounds of the invention themselves. References
to a therapeutic composition comprising a compound of the
invention, administration of a compound of the invention, or any
therapeutic use thereof, should therefore be construed to encompass
the equivalent use of a nucleic acid, expression vector or host
cell of the invention, except where the context demands
otherwise.
DESCRIPTION OF THE FIGURES
[0042] FIG. 1: Effect of s.c. administration of the Glu-GLP-1 dual
agonist compound 6 on the change in HbA1c over the 6 weeks
treatment period in db/db mice. Data are shown as mean+SEM with
n=11/group. *p<0.05, **p<0.01, ***p<0.001 compared to
vehicle at the same time point.
[0043] FIG. 2: Effect of s.c. administration of the Glu-GLP-1
agonist compound 6 on body weight gain over the 6 weeks treatment
period in db/db mice. Data are given as mean+SEM with n=11/group.
**p<0.01, ***p<0.001 compared to vehicle.
[0044] FIG. 3: Effect of s.c. administration the Glu-GLP-1 agonist
compound 6 on body weight in high fat fed C57BL/6J mice. Data are
mean.+-.SEM.
[0045] FIG. 4: Effect of s.c. administration of the Glu-GLP-1
agonist compound 6 on area under the glucose curve (AUC) during
OGTT in high fat fed C57BL/6J mice. Data are mean+SEM.
*p<0.05.
[0046] FIG. 5: Effect of 3 weeks treatment of mice that have been
on 30 weeks High Fat Diet for 30 weeks prior treatment (s.c.) with
vehicle (PBS), 10 nmol/kg exendin-4 or 10 nmol/kg compound 6 twice
daily for 3 weeks on lipids. CHO: Total Cholesterol; HDL: High
Density Cholesterol; LDL: Low Density Cholesterol; TRIG:
Triglycerides; HDL/LDL: Ratio between HDL and LDL.
[0047] FIG. 6: The effect of compound 6 and compound 7 on body
weight gain in high fat fed C57BL/6J mice.
DETAILED DESCRIPTION OF THE INVENTION
[0048] Throughout this specification, the conventional one letter
and three letter codes for naturally occurring amino acids are
used, as well as generally accepted three letter codes for other
amino acids, such as Aib (.alpha.-aminoisobutyric acid), Orn
(ornithine), Dbu (2,4 diaminobutyric acid) and Dpr
(2,3-diaminopropanoic acid).
[0049] The term "native glucagon" refers to native human glucagon
having the sequence
H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-
-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH (SEQ ID NO: 1).
[0050] The invention provides compounds as defined above. For the
avoidance of doubt, in the definitions provided herein, it is
generally intended that the sequence of X only differs from Formula
I at those positions which are stated to allow variation. Amino
acids within the sequence X can be considered to be numbered
consecutively from 1 to 29 in the conventional N-terminal to
C-terminal direction. Reference to a "position" within X should be
construed accordingly, as should reference to positions within
native human glucagon and other molecules.
[0051] The compounds of the invention may carry one or more
intramolecular bridge within the peptide sequence X. Each such
bridge is formed between the side chains of two amino acid residues
of X which are typically separated by three amino acids in the
linear sequence of X (i.e. between amino acid A and amino acid
A+4).
[0052] More particularly, the bridge may be formed between the side
chains of residue pairs 12 and 16, 16 and 20, 17 and 21, 20 and 24,
or 24 and 28. The two side chains can be linked to one another
through ionic interactions, or by covalent bonds. Thus these pairs
of residues may comprise oppositely charged side chains in order to
form a salt bridge by ionic interactions. For example, one of the
residues may be Glu or Asp, while the other may be Lys or Arg. The
pairs Lys and Glu and Lys and Asp, respectively, may also be
capable of reacting to form a lactam ring. Likewise, a Tyr and a
Glu or a Tyr and an Asp are capable of forming a lactone ring.
[0053] Without wishing to be bound by any particular theory, it is
believed that such intramolecular bridges stabilise the alpha
helical structure of the molecule and thereby increase potency
and/or selectivity at the GLP-1 receptor and possibly also the
glucagon receptor.
[0054] Without wishing to be bound by any particular theory, the
arginine residues at positions 17 and 18 of native glucagon appear
to provide significant selectivity for the glucagon receptor
[0055] Without wishing to be bound by any particular theory, the
residues at positions 27, 28 and 29 of native glucagon appear to
provide significant selectivity of the peptide for the glucagon
receptor. Substitutions at one, two or all three of these positions
with respect to the native glucagon sequence may increase potency
at and/or selectivity for the GLP-1 receptor, potentially without
significant reduction of potency at the glucagon receptor.
Particular examples include Leu at position 27, Ser at position 28
and Ala at position 29.
[0056] Substitution of the naturally-occurring Met residue at
position 27 (e.g. with Leu or Lys, especially with Leu) also
reduces the potential for oxidation, thereby increasing the
chemical stability of the compounds.
[0057] Substitution of the naturally-occurring Asn residue at
position 28 (e.g. by Ser, Arg or Ala) also reduces the potential
for deamidation in acidic solution, so increasing the chemical
stability of the compounds.
[0058] Potency and/or selectivity at the GLP-1 receptor may also be
increased by introducing residues that are likely to form an
amphipathic helical structure, potentially without significant loss
of potency at the glucagon receptor. This may be achieved by
introduction of charged residues at one or more of positions 16,
20, 24, and 28. Thus the residues at positions 16 and 20 may both
be charged, the residues at positions 16 and 24 may both be
charged, the residues at positions 20 and 24 may both be charged,
the residues at positions 16, 20 and 24 may all be charged, or the
residues at positions 16, 20, 24 and 28 may all be charged. For
example, the residue at position 16 may be Glu or Lys. The residue
at position 20 may be Lys. The residue at position 24 may be Glu.
The residue at position 28 may be Lys.
[0059] Substitution of one or both of the naturally-occurring Gln
residues at positions 20 and 24 also reduces the potential for
deamidation in acidic solution, thereby increasing the chemical
stability of the compounds.
[0060] Substitution of one or more of the naturally occurring amino
acids at positions 16, 17, 20, 27 and 28 with a charged amino acid
enables conjugation to a lipophilic substituent. For example, the
residues at positions 16, 17, 20, 27 or 28 may be Cys, Orn or Lys.
More particularly, one or more of the residues at positions 16, 17,
20, 27 and 28 may be Cys. Further, one or more of the the residues
at positions 16, 17, 20, 27 and 28 may be Lys.
[0061] As already disclosed, a compound of the invention may
comprise a C-terminal peptide sequence Z of 1-20 amino acids, for
example to stabilise the conformation and/or secondary structure of
the glucagon analogue peptide, and/or to render the glucagon
analogue peptide more resistant to enzymatic hydrolysis, e.g. as
described in WO99/46283.
[0062] When present, Z represents a peptide sequence of 1-20 amino
acid residues, e.g. in the range of 1-15, more preferably in the
range of 1-10, in particular in the range of 1-7 amino acid
residues, e.g., 1, 2, 3, 4, 5, 6 or 7 amino acid residues, such as
6 amino acid residues. Each of the amino acid residues in the
peptide sequence Z may independently be selected from Ala, Leu,
Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu (2,4 diaminobutyric acid),
Dpr (2,3-diaminopropanoic acid) and Orn (ornithine). Preferably,
the amino acid residues are selected from Ser, Thr, Tyr, Glu, Lys,
Arg, Dbu, Dpr and Orn, more preferably selected exclusively from
Glu, Lys, and Cys. The above-mentioned amino acids may have either
D- or L-configuration, but preferably have an L-configuration.
Particularly preferred sequences Z are sequences of four, five, six
or seven consecutive lysine residues (i.e. Lys.sub.3, Lys.sub.4,
Lys.sub.5, Lys.sub.6or Lys.sub.7), and particularly five or six
consecutive lysine residues. Other exemplary sequences of Z are
shown in WO 01/04156. Alternatively the C-terminal residue of the
sequence Z may be a Cys residue. This may assist in modification
(e.g. PEGylation, or conjugation to albumin) of the compound. In
such embodiments, the sequence Z may, for example, be only one
amino acid in length (i.e. Z=Cys) or may be two, three, four, five,
six or even more amino acids in length. The other amino acids
therefore serve as a spacer between the peptide X and the terminal
Cys residue.
[0063] The peptide sequence Z has no more than 25% sequence
identity with the corresponding sequence of the IP-1 portion of
human OXM (which has the sequence
Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala).
[0064] "Percent (%) amino acid sequence identity" of a given
peptide or polypeptide sequence with respect to another polypeptide
sequence (e.g. IP-1) is calculated as the percentage of amino acid
residues in the given peptide sequence that are identical with
correspondingly positioned amino acid residues in the corresponding
sequence of that other polypeptide when the two are aligned with
one another, introducing gaps for optimal alignment if necessary. %
identity values may be determined using WU-BLAST-2 (Altschul et
al., Methods in Enzymology, 266:460-480 (1996)). WU-BLAST-2 uses
several search parameters, most of which are set to the default
values. The adjustable parameters are set with the following
values: overlap span=1, overlap fraction=0.125, word threshold
(T)=11. A % amino acid sequence identity value is determined by the
number of matching identical residues as determined by WU-BLAST-2,
divided by the total number of residues of the reference sequence
(gaps introduced by WU-BLAST-2 into the reference sequence to
maximize the alignment score being ignored), multiplied by 100.
[0065] Thus, when Z is aligned optimally with the 8 amino acids of
IP-1, it has no more than two amino acids which are identical with
the corresponding amino acids of IP-1.
[0066] In a specific embodiment, the present invention provides a
compound wherein Z is absent.
[0067] One or more of the amino acid side chains in the compound of
the invention may be conjugated to a lipophilic substituent. The
lipophilic substituent may be covalently bonded to an atom in the
amino acid side chain, or alternatively may be conjugated to the
amino acid side chain by a spacer. A lipophilic substituent may be
conjugated to a side chain of an amino acid which is part of the
peptide X, and/or part of the peptide Z.
[0068] Without wishing to be bound by any particular theory, it is
thought that the lipophilic substituent binds albumin in the blood
stream, thus shielding the compounds of the invention from
enzymatic degradation and thereby enhancing the half-life of the
compounds. The spacer, when present, is used to provide spacing
between the compound and the lipophilic substituent.
[0069] The lipophilic substituent may be attached to the amino acid
side chain or to the spacer via an ester, a sulphonyl ester, a
thioester, an amide or a sulphonamide. Accordingly it will be
understood that preferably the lipophilic substituent includes an
acyl group, a sulphonyl group, an N atom, an O atom or an S atom
which forms part of the ester, sulphonyl ester, thioester, amide or
sulphonamide. Preferably, an acyl group in the lipophilic
substituent forms part of an amide or ester with the amino acid
side chain or the spacer.
[0070] The lipophilic substituent may include a hydrocarbon chain
having 4 to 30 C atoms. Preferably it has at least 8 or 12 C atoms,
and preferably it has 24 C atoms or fewer, or 20 C atoms or fewer.
The hydrocarbon chain may be linear or branched and may be
saturated or unsaturated. It will be understood that the
hydrocarbon chain is preferably substituted with a moiety which
forms part of the attachment to the amino acid side chain or the
spacer, for example an acyl group, a sulphonyl group, an N atom, an
0 atom or an S atom. Most preferably the hydrocarbon chain is
substituted with acyl, and accordingly the hydrocarbon chain may be
part of an alkanoyl group, for example palmitoyl, caproyl, lauroyl,
myristoyl or stearoyl.
[0071] Accordingly, the lipophilic substituent may have the formula
shown below:
##STR00001##
[0072] A may be, for example, an acyl group, a sulphonyl group, NH,
N-alkyl, an O atom or an S atom, preferably acyl. n is an integer
from 3 to 29, preferably at least 7 or at least 11, and preferably
23 or less, more preferably 19 or less.
[0073] The hydrocarbon chain may be further substituted. For
example, it may be further substituted with up to three
substituents selected from NH.sub.2, OH and COOH. If the
hydrocarbon chain is further substituted, preferably it is further
substituted with only one substituent. Alternatively or
additionally, the hydrocarbon chain may include a cycloalkane or
heterocycloalkane, for example as shown below:
##STR00002##
[0074] Preferably the cycloalkane or heterocycloalkane is a
six-membered ring. Most preferably, it is piperidine.
[0075] Alternatively, the lipophilic substituent may be based on a
cyclopentanophenanthrene skeleton, which may be partially or fully
unsaturated, or saturated. The carbon atoms in the skeleton each
may be substituted with Me or OH. For example, the lipophilic
substituent may be cholyl, deoxycholyl or lithocholyl.
[0076] As mentioned above, the lipophilic substituent may be
conjugated to the amino acid side chain by a spacer. When present,
the spacer is attached to the lipophilic substituent and to the
amino acid side chain. The spacer may be attached to the lipophilic
substituent and to the amino acid side chain independently by an
ester, a sulphonyl ester, a thioester, an amide or a sulphonamide.
Accordingly, it may include two moieties independently selected
from acyl, sulphonyl, an N atom, an O atom or an S atom. The spacer
may have the formula:
##STR00003##
[0077] wherein B and D are each independently selected from acyl,
sulphonyl, NH, N-alkyl, an O atom and an S atom, preferably from
acyl and NH. Preferably, n is an integer from 1 to 10, preferably
from 1 to 5. The spacer may be further substituted with one or more
substituents selected from C.sub.0-6 alkyl, C.sub.0-6 alkyl amine,
C.sub.0-6 alkyl hydroxy and C.sub.0-6 alkyl carboxy.
[0078] Alternatively, the spacer may have two or more repeat units
of the formula above. B, D and n are each selected independently
for each repeat unit. Adjacent repeat units may be covalently
attached to each other via their respective B and D moieties. For
example, the B and D moieties of the adjacent repeat units may
together form an ester, a sulphonyl ester, a thioester, an amide or
a sulphonamide. The free B and D units at each end of the spacer
are attached to the amino acid side chain and the lipophilic
substituent as described above.
[0079] Preferably the spacer has five or fewer, four or fewer or
three or fewer repeat units. Most preferably the spacer has two
repeat units, or is a single unit.
[0080] The spacer (or one or more of the repeat units of the
spacer, if it has repeat units) may be, for example, a natural or
unnatural amino acid. It will be understood that for amino acids
having functionalised side chains, B and/or D may be a moiety
within the side chain of the amino acid. The spacer may be any
naturally occurring or unnatural amino acid. For example, the
spacer (or one or more of the repeat units of the spacer, if it has
repeat units) may be Gly, Pro, Ala, Val, Leu, Ile, Met, Cys, Phe,
Tyr, Trp, His, Lys, Arg, Gln, Asn, .alpha.-Glu, .gamma.-Glu, Asp,
Ser Thr, Gaba, Aib, .beta.-Ala, 5-aminopentanoyl, 6-aminohexanoyl,
7-aminoheptanoyl, 8-aminooctanoyl, 9-aminononanoyl or
10-aminodecanoyl.
[0081] For example, the spacer may be a single amino acid selected
from .gamma.-Glu, Gaba, .beta.-Ala and .alpha.-Glu.
[0082] A lipophilic substituent may be conjugated to any amino acid
side chain in a compound of the invention. Preferably, the amino
acid side chain includes a carboxy, hydroxyl, thiol, amide or amine
group, for forming an ester, a sulphonyl ester, a thioester, an
amide or a sulphonamide with the spacer or lipophilic substituent.
For example, the lipophilic substituent may be conjugated to Asn,
Asp, Glu, Gln, His, Lys, Arg, Ser, Thr, Tyr, Trp, Cys or Dbu, Dpr
or Orn. Preferably, the lipophilic substituent is conjugated to
Lys. An amino acid shown as Lys in the formulae provided herein may
be replaced by, e.g., Dbu, Dpr or Orn where a lipophilic
substituent is added.
[0083] An example of a lipophilic substituent and spacer is shown
in the formula below:
##STR00004##
[0084] Here, a Lys residue in the compound of the present invention
is covalently attached to .gamma.-Glu (the spacer) via an amide
moiety. Palmitoyl is covalently attached to the .gamma.-Glu spacer
via an amide moiety.
[0085] Alternatively or additionally, one or more amino acid side
chains in the compound of the invention may be conjugated to a
polymeric moiety, for example, in order to increase solubility
and/or half-life in vivo (e.g. in plasma) and/or bioavailability.
Such modification is also known to reduce clearance (e.g. renal
clearance) of therapeutic proteins and peptides.
[0086] The polymeric moiety is preferably water-soluble
(amphiphilic or hydrophilic), non-toxic, and pharmaceutically
inert. Suitable polymeric moieties include polyethylene glycol
(PEG), homo- or co-polymers of PEG, a monomethyl-substituted
polymer of PEG (mPEG), and polyoxyethylene glycerol (POG). See, for
example, Int. J. Hematology 68:1 (1998); Bioconjugate Chem. 6:150
(1995); and Crit. Rev. Therap. Drug Carrier Sys. 9:249 (1992).
[0087] Other suitable polymeric moieties include poly-amino acids
such as poly-lysine, poly-aspartic acid and poly-glutamic acid (see
for example Gombotz, et al. (1995), Bioconjugate Chem., vol. 6:
332-351; Hudecz, et al. (1992), Bioconjugate Chem., vol. 3, 49-57;
Tsukada, et al. (1984), J. Natl. Cancer Inst., vol 73, : 721-729;
and Pratesi, et al. (1985), Br. J. Cancer, vol. 52: 841-848).
[0088] The polymeric moiety may be straight-chain or branched. It
may have a molecular weight of 500-40,000 Da, for example
500-10,000 Da, 1000-5000 Da, 10,000-20,000 Da, or 20,000-40,000
Da.
[0089] A compound of the invention may comprise two or more such
moieties, in which case the total molecular weight of all such
moieties will generally fall within the ranges provided above.
[0090] The polymeric moiety may be coupled (by covalent linkage) to
an amino, carboxyl or thiol group of an amino acid side chain.
Preferred examples are the thiol group of Cys residues and the
epsilon amino group of Lys residues. The carboxyl groups of Asp and
Glu residues may also be used.
[0091] The skilled reader will be well aware of suitable techniques
that can be used to perform the coupling reaction. For example, a
PEG moiety carrying a methoxy group can be coupled to a Cys thiol
group by a maleimido linkage using reagents commercially available
from Nektar Therapeutics AL. See also WO 2008/101017, and the
references cited above, for details of suitable chemistry.
[0092] In another aspect, one or more of the amino acid side chains
in a compound in the present invention, for example in peptide X,
is/are conjugated to a polymeric moiety.
[0093] In a further aspect, the present invention provides a
composition comprising a compound of the invention as described
herein, or a salt or derivative thereof, in admixture with a
carrier.
[0094] The term "derivative thereof" refers to a derivative of any
one of the compounds. Derivatives include all chemical
modifications, all conservative variants, all prodrugs and all
metabolites of the compounds.
[0095] The invention also provides the use of a compound of the
present invention in the preparation of a medicament for the
treatment of a condition as described below.
[0096] The invention also provides a composition wherein the
composition is a pharmaceutically acceptable composition, and the
carrier is a pharmaceutically acceptable carrier.
[0097] Peptide Synthesis
[0098] The compounds of the present invention may be manufactured
either by standard synthetic methods, recombinant expression
systems, or any other state of the art method. Thus the glucagon
analogues may be synthesized in a number of ways, including, for
example, a method which comprises:
[0099] (a) synthesizing the peptide by means of solid-phase or
liquid-phase methodology, either stepwise or by fragment assembly,
and isolation and purifying of the final peptide product; or
[0100] (b) expressing a nucleic acid construct that encodes the
peptide in a host cell, and recovering the expression product from
the host cell culture; or
[0101] (c) effecting cell-free in vitro expression of a nucleic
acid construct that encodes the peptide, and recovering the
expression product;
[0102] or any combination of methods of (a), (b), and (c) to obtain
fragments of the peptide, subsequently ligating the fragments to
obtain the peptide, and recovering the peptide.
[0103] It is preferred to synthesize the analogues of the invention
by means of solid-phase or liquid-phase peptide synthesis. In this
context, reference is made to WO 98/11125 and, among many others,
Fields, G B et al., 2002, "Principles and practice of solid-phase
peptide synthesis". In: Synthetic Peptides (2nd Edition), and the
Examples herein.
[0104] For recombinant expression, the nucleic acid fragments of
the invention will normally be inserted in suitable vectors to form
cloning or expression vectors carrying the nucleic acid fragments
of the invention; such novel vectors are also part of the
invention. The vectors can, depending on purpose and type of
application, be in the form of plasmids, phages, cosmids,
mini-chromosomes, or virus, but also naked DNA which is only
expressed transiently in certain cells is an important vector.
Preferred cloning and expression vectors (plasmid vectors) of the
invention are capable of autonomous replication, thereby enabling
high copy-numbers for the purposes of high-level expression or
high-level replication for subsequent cloning.
[0105] In general outline, an expression vector comprises the
following features in the 5'.fwdarw.3' direction and in operable
linkage: a promoter for driving expression of the nucleic acid
fragment of the invention, optionally a nucleic acid sequence
encoding a leader peptide enabling secretion (to the extracellular
phase or, where applicable, into the periplasma), the nucleic acid
fragment encoding the peptide of the invention, and optionally a
nucleic acid sequence encoding a terminator. They may comprise
additional features such as selectable markers and origins of
replication. When operating with expression vectors in producer
strains or cell lines it may be preferred that the vector is
capable of integrating into the host cell genome. The skilled
person is very familiar with suitable vectors and is able to design
one according to their specific requirements.
[0106] The vectors of the invention are used to transform host
cells to produce the compound of the invention. Such transformed
cells, which are also part of the invention, can be cultured cells
or cell lines used for propagation of the nucleic acid fragments
and vectors of the invention, or used for recombinant production of
the peptides of the invention.
[0107] Preferred transformed cells of the invention are
micro-organisms such as bacteria [such as the species Escherichia
(e.g. E. coli), Bacillus (e.g. Bacillus subtilis), Salmonella, or
Mycobacterium (preferably non-pathogenic, e.g. M. bovis BCG),
yeasts (e.g., Saccharomyces cerevisiae and Pichia pastoris), and
protozoans. Alternatively, the transformed cells may be derived
from a multicellular organism, i.e. it may be fungal cell, an
insect cell, an algal cell, a plant cell, or an animal cell such as
a mammalian cell. For the purposes of cloning and/or optimised
expression it is preferred that the transformed cell is capable of
replicating the nucleic acid fragment of the invention. Cells
expressing the nucleic fragment are useful embodiments of the
invention; they can be used for small-scale or large-scale
preparation of the peptides of the invention.
[0108] When producing the peptide of the invention by means of
transformed cells, it is convenient, although far from essential,
that the expression product is secreted into the culture
medium.
[0109] Efficacy
[0110] Binding of the relevant compounds to GLP-1 or glucagon (Glu)
receptors may be used as an indication of agonist activity, but in
general it is preferred to use a biological assay which measures
intracellular signalling caused by binding of the compound to the
relevant receptor. For example, activation of the glucagon receptor
by a glucagon agonist will stimulate cellular cyclic AMP (cAMP)
formation. Similarly, activation of the GLP-1 receptor by a GLP-1
agonist will stimulate cellular cAMP formation. Thus, production of
cAMP in suitable cells expressing one of these two receptors can be
used to monitor the relevant receptor activity. Use of a suitable
pair of cell types, each expressing one receptor but not the other,
can hence be used to determine agonist activity towards both types
of receptor.
[0111] The skilled person will be aware of suitable assay formats,
and examples are provided below. The GLP-1 receptor and/or the
glucagon receptor may have the sequence of the receptors as
described in the examples. For example, the assays may employ the
human glucagon receptor (Glucagon-R) having primary accession
number GI:4503947 and/or the human glucagon-like peptide 1 receptor
(GLP-1R) having primary accession number GI:166795283. (in that
where sequences of precursor proteins are referred to, it should of
course be understood that assays may make use of the mature
protein, lacking the signal sequence).
[0112] EC.sub.50 values may be used as a numerical measure of
agonist potency at a given receptor. An EC.sub.50 value is a
measure of the concentration of a compound required to achieve half
of that compound's maximal activity in a particular assay. Thus,
for example, a compound having EC.sub.50[GLP-1] lower than the
EC.sub.50[GLP-1] of glucagon in a particular assay may be
considered to have higher GLP-1 receptor agonist potency than
glucagon.
[0113] The compounds described in this specification are typically
Glu-GLP-1 dual agonists, as determined by the observation that they
are capable of stimulating cAMP formation at both the glucagon
receptor and the GLP-1 receptor. The stimulation of each receptor
can be measured in independent assays and afterwards compared to
each other.
[0114] By comparing the EC.sub.50 value for the glucagon receptor
(EC.sub.50[Glucagon-R]) with the EC.sub.50 value for the GLP-1
receptor, (EC.sub.50 [GLP-1R]) for a given compound, the relative
glucagon selectivity (%) of that compound can be found as
follows:
Relative Glucagon-R selectivity [compound]=(1/EC.sub.50
[Glucagon-R]).times.100/(1/EC.sub.50 [Glucagon-R]+1/EC.sub.50
[GLP-1R])
[0115] The relative GLP-1R selectivity can likewise be found:
Relative GLP-1R selectivity
[compound]=(1/EC.sub.50[GLP-1R]).times.100/(1/EC.sub.50
[Glucagon-R]+1/EC.sub.50 [GLP-1R])
[0116] A compound's relative selectivity allows its effect on the
GLP-1 or glucagon receptor to be compared directly to its effect on
the other receptor. For example, the higher a compound's relative
GLP-1 selectivity is, the more effective that compound is on the
GLP-1 receptor as compared to the glucagon receptor.
[0117] Using the assays described below, we have found the relative
GLP-1 selectivity for human glucagon to be approximately 5%.
[0118] The compounds of the invention have a higher relative GLP-1R
selectivity than human glucagon in that for a particular level of
glucagon-R agonist activity, the compound will display a higher
level of GLP-1R agonist activity (i.e. greater potency at the GLP-1
receptor) than glucagon. It will be understood that the absolute
potency of a particular compound at the glucagon and GLP-1
receptors may be higher, lower or approximately equal to that of
native human glucagon, as long as the appropriate relative GLP-1R
selectivity is achieved.
[0119] Nevertheless, the compounds of this invention may have a
lower EC.sub.50[GLP-1R] than human glucagon. The compounds may have
a lower EC.sub.50[GLP-1-R] than glucagon while maintaining an
EC.sub.50[Glucagon-R] that is less than 10-fold higher than that of
human glucagon, less than 5-fold higher than that of human
glucagon, or less than 2-fold higher than that of human
glucagon.
[0120] The compounds of the invention may have an
EC.sub.50[Glucagon-R] that is less than two-fold that of human
glucagon. The compounds may have an EC.sub.50 [Glucagon-R] that is
less than two-fold that of human glucagon and have an
EC.sub.50[GLP-1R] that is less than half that of human glucagon,
less than a fifth of that of human glucagon, or less than a tenth
of that of human glucagon.
[0121] The relative GLP-1R selectivity of the compounds may be
between 5% and 95%. For example, the compounds may have a relative
selectivity of 5-20%, 10-30%, 20-50%, 30-70%, or 50-80%; or of
30-50%, 40-60,%, 50-70% or 75-95%. The compounds of the invention
may also have effect on other Class B GPCR receptors, such as, but
not limited to, Calcitonin gene-related peptide 1 (CGRP1),
corticotropin-releasing factor 1 & 2 (CRF1 & CRF2), gastric
inhibitory polypeptide (GIP), glucagon-like peptide 1 & 2
(GLP-1 & GLP-2, glucagon (GCGR), secretin, gonadotropin
releasing hormone (GnRH), parathyroid-hormone 1 & 2 (PTH1 &
PTH2), vasoactive intestinal peptide (VPAC1 & VPAC2).
[0122] Therapeutic Uses
[0123] The compounds of the invention may provide an attractive
treatment option for, inter alia, obesity and metabolic
diseases.
[0124] Metabolic syndrome is characterized by a group of metabolic
risk factors in one person. They include abdominal obesity
(excessive fat tissue around the abdominal internal organs),
atherogenic dyslipidemia (blood fat disorders including high
triglycerides, low HDL cholesterol and/or high LDL cholesterol,
which foster plaque buildup in artery walls), elevated blood
pressure (hypertension), insulin resistance and glucose
intolerance, prothrombotic state (e.g. high fibrinogen or
plasminogen activator inhibitor-1 in the blood), and
proinflammatory state (e.g., elevated C-reactive protein in the
blood).
[0125] Individuals with the metabolic syndrome are at increased
risk of coronary heart disease and other diseases related to other
manifestations of arteriosclerosis (e.g., stroke and peripheral
vascular disease). The dominant underlying risk factors for this
syndrome appear to be abdominal obesity.
[0126] Without wishing to be bound by any particular theory, it is
believed that the compounds of the invention act as GluGLP-1 dual
agonists. The dual agonist combines the effect of glucagon on fat
metabolism with the effects of GLP-1 on food intake. They might
therefore act in a synergistic fashion to accelerate elimination of
excessive fat deposition and induce sustainable weight loss.
[0127] The synergistic effect of dual GluGLP-1 agonists may also
result in reduction of cardiovascular risk factors such as high
cholesterol and LDL, which may be entirely independent of their
effect on body weight.
[0128] The compounds of the present invention may therefore be used
as pharmaceutical agents for preventing weight gain, promoting
weight loss, reducing excess body weight or treating obesity (e.g.
by control of appetite, feeding, food intake, calorie intake,
and/or energy expenditure), including morbid obesity, as well as
associated diseases and health conditions including but not limited
to obesity linked inflammation, obesity linked gallbladder disease
and obesity induced sleep apnea. The compounds of the invention may
also be used for treatment of metabolic syndrome, hypertension,
atherogenic dyslipidimia, atherosclerois, arteriosclerosis,
coronary heart disease and stroke. These are all conditions which
can be associated with obesity. However, the effects of the
compounds of the invention on these conditions may be mediated in
whole or in part via an effect on body weight, or may be
independent thereof.
[0129] In particular, the compounds described in the present
invention may find use in preventing weight gain or promoting
weight loss with no or little effect on glucose tolerance. It has
been found that the compounds described has marked effect on weight
loss with no or little effect on the HbA1c level in an suitable
glycemic control animal model.
[0130] Thus the invention provides use of a compound of the
invention in the treatment of a condition as described above, in an
individual in need thereof.
[0131] The invention also provides a compound of the invention for
use in a method of medical treatment, particularly for use in a
method of treatment of a condition as described above.
[0132] In a preferred aspect, the compounds described may be used
in preventing weight gain or promoting weight loss.
[0133] In a specific embodiment, the present invention comprises
use of a compound for preventing weight gain or promoting weight
loss in an individual in need thereof.
[0134] In a specific embodiment, the present invention comprises
use of a compound in a method of treatment of a condition caused or
characterised by excess body weight, e.g. the treatment and/or
prevention of obesity, morbid obesity, morbid obesity prior to
surgery, obesity linked inflammation, obesity linked gallbladder
disease, obesity induced sleep apnea, hypertension, atherogenic
dyslipidimia, atherosclerois, arteriosclerosis, coronary heart
disease, peripheral artery disease, stroke or microvascular disease
in an individual in need thereof.
[0135] In another aspect, the compounds described may be used in a
method of lowering circulating LDL levels, and/or increasing
HDL/LDL ratio.
[0136] In a specific embodiment, the present invention comprises
use of a compound in a method of lowering circulating LDL levels,
and/or increasing HDL/LDL ratio in an individual in need
thereof.
[0137] Pharmaceutical Compositions
[0138] The compounds of the present invention, or salts thereof,
may be formulated as pharmaceutical compositions prepared for
storage or administration, which typically comprise a
therapeutically effective amount of a compound of the invention, or
a salt thereof, in a pharmaceutically acceptable carrier.
[0139] The therapeutically effective amount of a compound of the
present invention will depend on the route of administration, the
type of mammal being treated, and the physical characteristics of
the specific mammal under consideration. These factors and their
relationship to determining this amount are well known to skilled
practitioners in the medical arts. This amount and the method of
administration can be tailored to achieve optimal efficacy, and may
depend on such factors as weight, diet, concurrent medication and
other factors, well known to those skilled in the medical arts. The
dosage sizes and dosing regimen most appropriate for human use may
be guided by the results obtained by the present invention, and may
be confirmed in properly designed clinical trials.
[0140] An effective dosage and treatment protocol may be determined
by conventional means, starting with a low dose in laboratory
animals and then increasing the dosage while monitoring the
effects, and systematically varying the dosage regimen as well.
Numerous factors may be taken into consideration by a clinician
when determining an optimal dosage for a given subject. Such
considerations are known to the skilled person.
[0141] The term "pharmaceutically acceptable carrier" includes any
of the standard pharmaceutical carriers. Pharmaceutically
acceptable carriers for therapeutic use are well known in the
pharmaceutical art, and are described, for example, in Remington's
Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit.
1985). For example, sterile saline and phosphate-buffered saline at
slightly acidic or physiological pH may be used. pH buffering
agents may be phosphate, citrate, acetate,
tris/hydroxymethyl)aminomethane (TRIS),
N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS),
ammonium bicarbonate, diethanolamine, histidine, which is a
preferred buffer, arginine, lysine, or acetate or mixtures thereof.
The term further encompases any agents listed in the US
Pharmacopeia for use in animals, including humans.
[0142] The term "pharmaceutically acceptable salt" refers to a salt
of any one of the compounds. Salts include pharmaceutically
acceptable salts such as acid addition salts and basic salts.
Examples of acid addition salts include hydrochloride salts,
citrate salts and acetate salts. Examples of basic salts include
salts where the cation is selected from alkali metals, such as
sodium and potassium, alkaline earth metals, such as calcium, and
ammonium ions .sup.+N(R.sup.3).sub.3(R.sup.4), where R.sup.3 and
R.sup.4 independently designates optionally substituted
C.sub.1-6-alkyl, optionally substituted C.sub.2-6-alkenyl,
optionally substituted aryl, or optionally substituted heteroaryl.
Other examples of pharmaceutically acceptable salts are described
in "Remington's Pharmaceutical Sciences", 17th edition. Ed. Alfonso
R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., U.S.A.,
1985 and more recent editions, and in the Encyclopaedia of
Pharmaceutical Technology.
[0143] "Treatment" is an approach for obtaining beneficial or
desired clinical results. For the purposes of this invention,
beneficial or desired clinical results include, but are not limited
to, alleviation of symptoms, diminishment of extent of disease,
stabilized (i.e., not worsening) state of disease, delay or slowing
of disease progression, amelioration or palliation of the disease
state, and remission (whether partial or total), whether detectable
or undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment.
"Treatment" is an intervention performed with the intention of
preventing the development or altering the pathology of a disorder.
Accordingly, "treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented
[0144] The pharmaceutical compositions can be in unit dosage form.
In such form, the composition is divided into unit doses containing
appropriate quantities of the active component. The unit dosage
form can be a packaged preparation, the package containing discrete
quantities of the preparations, for example, packeted tablets,
capsules, and powders in vials or ampoules. The unit dosage form
can also be a capsule, cachet, or tablet itself, or it can be the
appropriate number of any of these packaged forms. It may be
provided in single dose injectable form, for example in the form of
a pen. In certain embodiments, packaged forms include a label or
insert with instructions for use. Compositions may be formulated
for any suitable route and means of administration.
Pharmaceutically acceptable carriers or diluents include those used
in formulations suitable for oral, rectal, nasal, topical
(including buccal and sublingual), vaginal or parenteral (including
subcutaneous, intramuscular, intravenous, intradermal, and
transdermal) administration. The formulations may conveniently be
presented in unit dosage form and may be prepared by any of the
methods well known in the art of pharmacy.
[0145] Subcutaneous or transdermal modes of administration may be
particularly suitable for the compounds described herein.
[0146] Compositions of the invention may further be compounded in,
or attached to, for example through covalent, hydrophobic and
electrostatic interactions, a drug carrier, drug delivery system
and advanced drug delivery system in order to further enhance
stability of the compound, increase bioavailability, increase
solubility, decrease adverse effects, achieve chronotherapy well
known to those skilled in the art, and increase patient compliance
or any combination thereof. Examples of carriers, drug delivery
systems and advanced drug delivery systems include, but are not
limited to, polymers, for example cellulose and derivatives,
polysaccharides, for example dextran and derivatives, starch and
derivatives, poly(vinyl alcohol), acrylate and methacrylate
polymers, polylactic and polyglycolic acid and block co-polymers
thereof, polyethylene glycols, carrier proteins, for example
albumin, gels, for example, thermogelling systems, for example
block co-polymeric systems well known to those skilled in the art,
micelles, liposomes, microspheres, nanoparticulates, liquid
crystals and dispersions thereof, L2 phase and dispersions there
of, well known to those skilled in the art of phase behaviour in
lipid-water systems, polymeric micelles, multiple emulsions,
self-emulsifying, self-microemulsifying, cyclodextrins and
derivatives thereof, and dendrimers.
[0147] Combination Therapy
[0148] As noted above, it will be understood that reference in the
following to a compound of the invention also extends to a
pharmaceutically acceptable salt or solvate thereof as well as to a
composition comprising more than one different compounds of the
invention.
[0149] A compound of the invention may be administered as part of a
combination therapy with an agent for treatment of obesity,
hypertension dyslipidemia or diabetes.
[0150] In such cases, the two active agents may be given together
or separately, and as part of the same pharmaceutical formulation
or as separate formulations.
[0151] Thus a compound or salt thereof can further be used in
combination with an anti-obesity agent, including but not limited
to a glucagon-like peptide receptor 1 agonist, peptide YY or
analogue thereof, cannabinoid receptor 1 antagonist, lipase
inhibitor, melanocortin receptor 4 agonist, or melanin
concentrating hormone receptor 1 antagonist.
[0152] A compound of the invention or salt thereof can be used in
combination with an anti-hypertension agent, including but not
limited to an angiotensin-converting enzyme inhibitor, angiotensin
II receptor blocker, diuretics, beta-blocker, or calcium channel
blocker.
[0153] A compound of the invention or salt thereof can be used in
combination with a dyslipidaemia agent, including but not limited
to a statin, a fibrate, a niacin and/or a cholesterol absorbtion
inhibitor.
[0154] Further, a compound of the invention or salt thereof can be
used in combination with an anti-diabetic agent, including but not
limited to metformin, a sulfonylurea, a glinide, a DPP-IV
inhibitor, a glitazone, a different GLP-1 agonist or an insulin. In
a preferred embodiment, the compound or salt thereof is used in
combination with insulin, DPP-IV inhibitor, sulfonylurea or
metformin, particularly sulfonylurea or metformin, for achieving
adequate glycemic control. In an even more preferred embodiment the
compound or salt thereof is used in combination with an insulin or
an insulin analogue for achieving adequate glycemic control.
Examples of insulin analogues include but are not limited to
Lantus, Novorapid, Humalog, Novomix, and Actraphane HM.
[0155] Methods
[0156] General Synthesis of Glucagon Analogues
[0157] Solid phase peptide synthesis (SPPS) was performed on a
microwave assisted synthesizer using standard Fmoc strategy in NMP
on a polystyrene resin (TentaGel S Ram). HATU was used as coupling
reagent together with DIPEA as base. Piperidine (20% in NMP) was
used for deprotection. Pseudoprolines: Fmoc-Phe-Thr(.Psi. Me, Me
pro)-OH and Fmoc-Asp-Ser(.Psi., Me, Me pro)-OH (purchased from
NovaBiochem) were used where applicable.
[0158] Abbreviations Employed are as Follows: [0159] ivDde:
1-(4,4-dimethyl-2,6-dioxocyclohexylidene)3-methyl-butyl [0160] Dde:
1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl [0161] DCM:
dichloromethane [0162] DMF: N,N-dimethylformamide [0163] DIPEA:
diisopropylethylamine [0164] EtOH: ethanol [0165] Et.sub.2O:
diethyl ether [0166] HATU:
N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridine-1-ylmethylene]-N-methy-
lmethanaminium hexafluorophosphate N-oxide [0167] MeCN:
acetonitrile [0168] NMP: N-methylpyrrolidone [0169] TFA:
trifluoroacetic acid [0170] TIS: triisopropylsilane
[0171] Cleavage:
[0172] The crude peptide was cleaved from the resin by treatment
with 95/2.5/2.5% (v/v) TFA/TIS/water at r.t. for 2 h. For peptides
with a methionine in the sequence a mixture of 95/5% (v/v) TFA/EDT
was used. Most of the TFA was removed at reduced pressure and the
crude peptide was precipitated and washed with diethylether and
allowed to dry to constant weight at ambient temperature.
[0173] General Synthesis of Acylated Glucagon Analogues
[0174] The peptide backbone was synthesized as described above for
the general synthesis of glucagon analogues, with the exception
that it was acylated on the side chain of a lysine residue with the
peptide still attached to the resin and fully protected on the side
chain groups, except the epsilon-amine on the lysine to be
acylated. The lysine to be acylated was incorporated with the use
of Fmoc-Lys(ivDde)-OH or Fmoc-Lys(Dde)-OH. The N-terminal of the
peptide was protected with a Boc group using Boc.sub.2O in NMP.
While the peptide was still attached to the resin, the ivDde
protecting group was selectively cleaved using 5% hydrazine hydrate
in NMP. The unprotected lysine side chain was then first coupled
with a spacer amino acid like Fmoc-Glu-OtBu, which was deprotected
with piperidine and acylated with a fatty acid using standard
peptide coupling methodology as described above. Alternatively, the
histidine at the N-terminal may be incorporated from the beginning
as Boc-His(Boc)-OH. Cleavage from the resin and purification were
performed as described above.
[0175] Generation of Cell Lines Expressing Human Glucagon- and
GLP-1 Receptors
[0176] The cDNA encoding either the human glucagon receptor
(Glucagon-R) (primary accession number P47871) or the human
glucagon-like peptide 1 receptor (GLP-1R) (primary accession number
P43220) were cloned from the cDNA clones BC104854
(MGC:132514/IMAGE:8143857) or BC112126 (MGC:138331/IMAGE:8327594),
respectively. The DNA encoding the Glucagon-R or the GLP-1-R was
amplified by PCR using primers encoding terminal restriction sites
for subcloning. The 5'-end primers additionally encoded a near
Kozak consensus sequence to ensure efficient translation. The
fidelity of the DNA encoding the Glucagon-R and the GLP-1-R was
confirmed by DNA sequencing. The PCR products encoding the
Glucagon-R or the GLP-1-R were subcloned into a mammalian
expression vector containing a neomycin (G418) resistance
marker.
[0177] The mammalian expression vectors encoding the Glucagon-R or
the GLP-1-R were transfected into HEK293 cells by a standard
calcium phosphate transfection method. 48 hr after transfection
cells were seeded for limited dilution cloning and selected with 1
mg/ml G418 in the culture medium. Three weeks later 12 surviving
colonies of Glucagon-R and GLP-1-R expressing cells were picked,
propagated and tested in the Glucagon-R and GLP-1-R efficacy assays
as described below. One Glucagon-R expressing clone and one GLP-1-R
expressing clone were chosen for compound profiling.
[0178] Glucagon Receptor and GLP-1-Receptor Efficacy Assays
[0179] HEK293 cells expressing the human Glucagon-R, or human
GLP-1-R were seeded at 40,000 cells per well in 96-well microtiter
plates coated with 0.01% poly-L-lysine and grown for 1 day in
culture in 100 .mu.l growth medium. On the day of analysis, growth
medium was removed and the cells washed once with 200 .mu.l Tyrode
buffer. Cells were incubated in 100 .mu.l Tyrode buffer containing
increasing concentrations of test peptides, 100 .mu.M IBMX, and 6
mM glucose for up 15 min at 37.degree. C. The reaction was stopped
by addition of 25 .mu.l 0.5 M HCl and incubated on ice for 60 min.
The cAMP content was estimated using the Flash Plate.RTM. cAMP kit
from Perkin-Elmer according to manufacturer instructions. EC.sub.50
and relative efficacies compared to reference compounds (glucagon
and GLP-1) were estimated by computer aided curve fitting.
[0180] HbA1c Determination
[0181] It is possible to assess the long term effect of a compound
on a subject's glucose level by determining the level of
haemoglobin A1C (HbA1c). HbA1c is a glycosylated form of
haemoglobin whose level in a cell reflects the average level of
glucose to which the cell has been exposed during its lifetime. In
mice, HbA1c is a relevant biomarker for the average blood glucose
level during the preceding 4 weeks, because conversion to HbA1c is
limited by the erythrocyte's life span of approximately 47 days
(Abbrecht & Littell, 1972; J. Appl. Physiol. 32, 443-445).
[0182] The HbA1c determination is based on Turbidimetric INhibition
ImmunoAssay (TINIA) in which HbA1c in the sample reacts with
anti-HbA1c to form soluble antigen-antibody complexes. Additions of
polyhaptens react with excess anti-HbA1c antibodies to form an
insoluble antibody-polyhapten complex, which can be measured
turbidimetrically. Liberated hemoglobin in the hemolyzed sample is
converted to a derivative having a characteristic absorption
spectrum, which is measured bichromatically during the
preincubation phases. The final result is expressed as percent
HbA1c of total hemoglobin (Cobas.RTM.Application note A1C-2).
[0183] Cholesterol Level Determination
[0184] The assay is an enzymatic colorimetric method. In the
presence of magnesium ions, dextran sulfate selectively forms
water-soluble complexes with LDL, VLDLA and chylomicrons, which are
resistant to PEG-modified enzymes. The HDL cholesterol is
determined enzymatically by cholesterol esterase and cholesterol
oxidase coupled with PEG to the amino groups. Cholesterol esters
are broken down quantitatively to free cholesterol and fatty acids.
HDL cholesterol is enzymatically oxidized to choles-4-en-3-one and
H.sub.2O.sub.2, and the formed H.sub.2O.sub.2 is measured
colorimetrically (Cobas.RTM.; Application note HDLC3).
[0185] The direct determination of LDL takes advantage of the
selective micellary solubilization of LDL by a nonionic detergent
and the interaction of a sugar compound and lipoproteins (VLDL and
chylomicrons). The combination of a sugar compound with detergent
enables the selective determination of LDL in plasma. The test
principle is the same as that of cholesterol and HDL, but due to
the sugar and detergent only LDL-cholesterol esters are broken down
to free cholesterol and fatty acids. Free cholesterol is then
oxidized and the formed H.sub.2O.sub.2 is measured colorimetrically
(Application note LDL_C, Cobas.RTM.).
[0186] Tested GPCR-B Targets
[0187] Receptors for: Calcitonin gene-related peptide 1 (CGRP1),
corticotropin-releasing factor 1 & 2 (CRF1 & CRF2), gastric
inhibitory polypeptide (GIP), glucagon-like peptide 1 & 2
(GLP-1 & GLP-2, glucagon (GCGR), secretin, gonadotropin
releasing hormone (GnRH), parathyroid-hormone 1 & 2 (PTH1 &
PTH2), vasoactive intestinal peptide (VPAC1 & VPAC2).
[0188] Assay Design for Other Class B Receptors
[0189] Agonist percentage activation determinations were obtained
by assaying sample compounds (glucagon analogues) and referencing
the Emax control for each GPCR profiled. Antagonist percentage
inhibition determinations were obtained by assaying sample
compounds and referencing the control EC80 wells for each GPCR
profiled. The samples were run using a "Double Addition" assay
protocol for the agonist and antagonist assay run. The protocol
design is as follows:
[0190] Compound Preparation Master Stock Solution
[0191] Unless specified otherwise, the sample compounds were
diluted in 100% anhydrous DMSO including all dilutions. If the
sample compound is provided in a different solvent all master stock
dilutions are performed in the specified solvent. All control wells
contained identical solvent final concentrations as the sample
compound wells.
[0192] Compound Plate for Assay
[0193] The glucagon analogues were transferred from a master stock
solution into a daughter plate that was used in the assay. Each
sample compound was diluted into assay buffer (1.times. HBSS with
20mM HEPES and 2.5 mM Probenecid) at an appropriate concentration
to obtain final specified concentrations.
[0194] Calcium Flux Assay Agonist Assay Format
[0195] The glucagon analogues were plated in duplicate at 100 nM
and 1 nM. The concentrations described here reflect the final
concentrations of the compounds during the antagonist assay.
Reference agonists were handled as mentioned above serving as assay
control. The reference agonists were handled as described above for
Emax. Assay was read for 90 seconds using the FLIPR.sup.TETRA
[0196] Antagonist Assay Format
[0197] Using the EC80 values determined previously, stimulated all
pre-incubated sample compound and reference antagonist (if
applicable) wells with EC.sub.80 of reference agonist.
[0198] Read for 180 seconds using the FLIPR.sup.TETRA (this
addition added reference agonist to respective wells)--then
fluorescence measurements were collected to calculate
IC.sub.50-values.
[0199] PK in Cynomolgus Monkeys
[0200] Cynomolgus monkeys (male, N=2) were dosed with 20 nmol
compound 6/kg i.v. and 20 nmol compound 6/kg s.c. as single dose at
day 1 and 8, respectively. Plasma samples were withdrawn at the
following time points; predose, 5, 10, 30 min, 1, 2, 4, 8, 12, 24
and 36 h post i.v. dosing and predose, 10, 30 min, 1, 2, 4, 8, 12,
24, 36 and 48 h post s.c. dosing. The vehicle consisted of 25 mM
phosphate buffered saline at pH 7.4 (25 mM sodium phosphate, 25 mM
NaCl). Plasma samples were analyzed using protein precipitation
followed by solid phase extraction and LC-MS/MS analysis.
Pharmacokinetic parameters were calculated using non-compartmental
analysis in WinNonLin version 4.1.
[0201] Effect of 6 Weeks Subcutaneous Administration of Glu-GLP-1
Agonist Compound 6 on HbA1c in db/db Mice
[0202] db/db (BKS.Cg-m+/+Lepr.sup.db/J) male mice, 5-6 weeks old,
were obtained from Charles River Laboratories, Germany, and
acclimatized in their new environment with free access to normal
chow and water. The animals were stratified into groups with
similar average HbA1c and treated twice daily s.c. with compound 6
(1, 2.5, and 5 nmol/kg) or vehicle for six weeks. Throughout the
study, body weights were recorded daily and used to administer the
body weight-corrected doses of peptide. HbA1c levels were measured
on blood samples before treatment, and then once weekly during the
study. Immediately following final blood sampling all animals were
sacrificed.
[0203] Effect of three weeks treatment with the dual Glu-GLP-1
agonist compound 6 on oral glucose tolerance and body weight in
diet induced obese mice (30 weeks high-fat diet).
[0204] C57Bl/6J male mice, 6 weeks old, were acclimatized in their
new environment with free access to a high fat diet and water. 36
weeks old, the animals were randomized into groups with similar
average fasting (6 hours) blood glucose (assessed from blood
samples taken from the tip of the tail). The mice were treated
twice daily s.c. for three weeks with compound 6 or vehicle. Body
weight was recorded daily. After peptide treatment an oral glucose
tolerance test (OGTT) were performed after subjecting the animals
to a 6 hour fast. Animals were dosed with peptide or vehicle in the
morning. Approximately four hours later an initial blood sample
(fasting blood glucose level) was taken. Thereafter an oral dose of
glucose was given and the animals were returned to their home cages
(t=0). BG was measured at t=15 min, t=30 min, t=60 min, t=90 min
and t=120 min. All animals were sacrificed immediately following
blood sampling by CO.sub.2 anesthesia followed by cervical
dislocation.
[0205] The invention is further illustrated by the following
examples.
EXAMPLE 1
Efficacy on Glucagon and GLP-1 Receptors
TABLE-US-00004 [0206] TABLE 1 EC.sub.50 values were measured as
described above Com- GLP-1R GluR pound SEQ ID EC.sub.50 EC.sub.50
No No Sequence (nM) (nM) 1 4 H-HSQGTFTSDYSKYLDERRAKDFIEWLLSA- 0.06
0.06 NH2 2 5 H-HSQGTFTSDYSKYLDERRAKDFIEWL- 3.09 0.81
K(Hexadecanoyl-isoGlu)-SA-NH2 3 6
H-HSQGTFTSDYSKYLDERRA-K(Hexadecanoyl- 0.64 0.60
isoGlu)-DFIEWLLSA-NH2 4 7 H-HSQGTFTSDYSKYLDERRAKDFIEWLL- 0.31 0.25
K(Hexadecanoyl-isoGlu)-A-NH2 5 8 H-HSQGTFTSDYSKYLD-K(Hexadecanoyl-
0.35 0.19 isoGlu)-RRAKDFIEVVLLSA-NH2 6 9
H-HSQGTFTSDYSKYLDE-K(Hexadecanoyl- 0.13 0,16
isoGlu)-RAKDFIEVVLLSA-NH2 7 10
H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl- 0.10 0.16
isoGlu)-RAKDFIEWLLSA-NH2
EXAMPLE 2
Pharmacokinetic Study following Subcutaneous and Intravenous
Administration in the Monkey
[0207] Compound 6 was dosed to monkeys in order to test the
pharmacokinetics and bioavailability after subcutaneous
administration, which is the intended route in humans. Compound 6
showed a bioavailability of 43%.+-.8.1, t.sub.1/2 of 8.2 h.+-.2.0
and t.sub.max between 4 and 8 h. The pharmacokinetic profile, of
compound 6 is likely to predict constant exposure above EC.sub.50
at both the glucagon and GLP-1 receptors in humans after once daily
administration of a feasible dose by the subcutaneous route.
EXAMPLE 3
Effect of 6 Weeks Subcutaneous Administration of Glu-GLP-1 Agonist
Compound 6 on HbA1c in db/db Mice
[0208] The animals were injected s.c. with 100 .mu.l vehicle (once
a day) for a period of three days to acclimatize the animals to
handling and injections. The animals were randomized into groups
with similar average HbA1c. Then mice were treated twice daily s.c.
with compound 6 (1, 2.5, or 5 nmol/kg) or vehicle. Before treatment
and once weekly for six weeks of treatment, blood samples were
taken from the retroorbital venous plexus.
[0209] Blood samples were analyzed for HbA1c using the Cobas c111
analyzer (Roche Diagnostics, Mannheim, Germany). Samples for HbA1c
analysis were analyzed within 24 hours of sampling.
[0210] Throughout the study, body weights were recorded daily and
used to administer the body weight-corrected doses of peptide.
Solutions were prepared immediately before dosing.
[0211] As expected from the db/db mouse model an increase in HbA1c
over time was seen (FIG. 1). During the six weeks treatment we
observed a 27% increase in HbA1c in the vehicle group.
[0212] Compound 6, at all doses, did not at any time-point reduce
the increase in HbA1c seen over time in vehicle-treated animals
(FIG. 1).
[0213] During the six weeks treatment we observed a 22% increase in
body weight in the vehicle group. Compound 6 (5 nmol/kg)
significantly decreased body weight gain compared to vehicle
(p<0.0001, FIG. 2).
[0214] The effect of six weeks treatment with the dual Glu-GLP-1
agonist compound 6 on glycemic control as assessed by HbA1c in
db/db mice was investigated.
[0215] The db/db mouse model, as expected, showed an increase in
HbA1c over time. Treatment with compound 6 for six weeks did not
significantly reduce the increase in HbA1c seen over time in
vehicle-treated db/db mice.
[0216] Treatment with compound 6 (5 nmol/kg) for six weeks
significantly reduced the increase in body weight seen over time in
vehicle-treated db/db mice.
EXAMPLE 4
Effect of 3 Weeks Subcutaneous Administration of Glu-GLP-1 Agonist
Compound 6 on Oral Glucose Tolerance and Body Weight in Diet
Induced Obese C57BL/6J Mice (6 Months High Fat Diet)
[0217] Body Weight
[0218] Compound 6 decreased the body weight 38.7% (p<0.05) (FIG.
3). Interestingly, the body weight obtained by high fat fed animals
treated with compound 6 for three weeks stabilized to the same body
weight as obtained by non-treated control animals on a regular chow
diet (FIG. 3).
[0219] OGTT (Oral Glucose Tolerence Test)
[0220] Treatment with compound 6 for three weeks had no significant
effect on glucose tolerance (measured as decrease in AUC) (FIG.
4).
[0221] Compound 6 significantly decreased body weight in diet
induced obese mice (30 weeks high fat diet) to a level as that seen
in non-treated control animals on a regular chow diet (FIG. 3). The
effect of Compound 6 significantly decreased fasting blood glucose.
Compound 6 did not significantly increase oral glucose
tolerance.
[0222] These differences in weight loss and glucose handling could
reflect the potency (Table 1) and/or the exposure on the GLP-1
receptor of the Glu-GLP-1 agonist compound 6. The difference could
also be related to differences between GLP-1 and GLP-1 analogs and
Glu-GLP-1 agonists in mechanism of action. Exendin-4 and other
GLP-1 analogs are known to regulate blood glucose via stimulation
of glucose-dependent insulin secretion, inhibition of gastric
emptying, and inhibition of glucagon secretion. In addition to
effects such as these on the GLP-1 receptor, compound 6 also binds
and activates the GluR (see Table 1).
[0223] In diet induced obese mice, compound 6 significantly
decreased body weight. Compound 6 did not improve glucose tolerance
measured during and oral glucose tolerance test.
EXAMPLE 5
Effect of 3 Weeks Subcutaneous Administration of Glu-GLP-1 Agonist
Compound 6 on Lipids in 30 Weeks High Fat Diet Feeded Mice
[0224] Effect of 3 weeks treatment of mice that have been on a High
Fat Diet for 30 weeks prior to treatment (s.c.) with vehicle (PBS),
10 nmol/kg exendin-4 or 10 nmol/kg compound 6 twice daily for 3
weeks on lipids (FIG. 5). The effect was measured on LDL, HDL and
triglycerides (CHO: Total Cholesterol; HDL: High Density
Cholesterol; LDL: Low Density Cholesterol; TRIG: Triglycerides;
HDL/LDL: Ratio between HDL and LDL).
[0225] Compound 6 demonstrated significantly decreased total
cholesterol, HDL, LDL (P<0.001) and triglycerides (P<0.05),
while the ratio HDL/LDL was increased significantly (p<0.001)
(FIG. 5). The HDL/LDL ratio is considered to be a risk indicator
for heart disease. The higher the ratio, the lower the risk of
heart attack or other cardiovascular problems.
EXAMPLE 6
Counterscreen of Compound 6 Against CGRP and Other Receptors
[0226] Compound 6 was tested for agonist and antagonist activity
against Calcitonin gene-related peptide 1 (CGRP1),
corticotropin-releasing factor 1 & 2 (CRF1 & CRF2), gastric
inhibitory polypeptide (GIP), glucagon-like peptide 1 & 2
(GLP-1 & GLP-2, glucagon (GCGR), secretin, gonadotropin
releasing hormone (GnRH), parathyroid-hormone 1 & 2 (PTH1 &
PTH2), vasoactive intestinal peptide (VPAC1 & VPAC2) at 1
(1.25), 100 (125) and 10,000 (12,500) nM.
[0227] Agonist activity against the following receptors was
observed:
[0228] GLP-1 receptor: compound 6 exhibited agonist activities at
12.5 .mu.M and 125 nM
[0229] Glucagon receptor compound 6 exhibited agonist activities at
12.5 .mu.M and 125 nM
[0230] Antagonist activity against the following receptors was
observed:
[0231] CRF2 receptor. Compound 6 exhibited antagonist activity at
10 .mu.M
[0232] GIP receptor: Compound 6 exhibited antagonist activity at 10
.mu.M and 100 nM
[0233] Secretin receptor: Compound 6 exhibited antagonist activity
at 10 .mu.M.
EXAMPLE 7
The Effect of Compounds 6 and 7 on Body Weight Gain in High Fat Fed
C57BL/6J Mice
[0234] C57BL/6J mice were acclimatized in their new environment for
4 weeks with free access to a high fat diet and water. Mice were
then treated twice daily s.c. with compound 6 and compound 7 (at
the two doses: 0.5 and 5 nmol/kg) or vehicle for 10 days.
Throughout the study, body weights were recorded daily and used to
administer the body weight-corrected doses of peptide (FIG. 6).
Mice were sacrificed by cervical dislocation.
[0235] Compound 6 and compound 7 significantly decreased body
weight gain at both doses (0.5 and 5 nmol/kg) in high fat fed
C57BL/6J mice.
Sequence CWU 1
1
17129PRTArtificialSynthetic peptide sequence 1His Ser Gln Gly Thr
Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala
Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25
28PRTArtificialSynthetic peptide sequence 2Lys Arg Asn Arg Asn Asn
Ile Ala 1 5 337PRTArtificialSynthetic peptide sequence 3His Ser Gln
Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg
Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn 20 25
30 Arg Asn Asn Ile Ala 35 429PRTArtificialSynthetic peptide
sequence 4His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu 1 5 10 15 Arg Arg Ala Lys Asp Phe Ile Glu Trp Leu Leu Ser
Ala 20 25 529PRTArtificialSynthetic peptide sequence 5His Ser Gln
Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg
Arg Ala Lys Asp Phe Ile Glu Trp Leu Xaa Ser Ala 20 25
629PRTArtificialSynthetic peptide sequence 6His Ser Gln Gly Thr Phe
Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Arg Ala Xaa
Asp Phe Ile Glu Trp Leu Leu Ser Ala 20 25 729PRTArtificialSynthetic
peptide sequence 7His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys
Tyr Leu Asp Glu 1 5 10 15 Arg Arg Ala Lys Asp Phe Ile Glu Trp Leu
Leu Xaa Ala 20 25 829PRTArtificialSynthetic peptide sequence 8His
Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa 1 5 10
15 Arg Arg Ala Lys Asp Phe Ile Glu Trp Leu Leu Ser Ala 20 25
929PRTArtificialSynthetic peptide sequence 9His Ser Gln Gly Thr Phe
Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Xaa Arg Ala Lys
Asp Phe Ile Glu Trp Leu Leu Ser Ala 20 25
1029PRTArtificialSynthetic peptide sequence 10His Xaa Gln Gly Thr
Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Xaa Arg Ala
Lys Asp Phe Ile Glu Trp Leu Leu Ser Ala 20 25
1129PRTArtificialSynthetic peptide sequence 11His Xaa Gln Gly Thr
Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Xaa Arg Ala
Xaa Asp Phe Ile Xaa Trp Leu Xaa Xaa Xaa 20 25
1229PRTArtificialSynthetic peptide sequence 12His Ser Gln Gly Thr
Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Arg Ala
Lys Asp Phe Ile Glu Trp Leu Lys Ser Ala 20 25
1329PRTArtificialSynthetic peptide sequence 13His Ser Gln Gly Thr
Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Arg Ala
Lys Asp Phe Ile Glu Trp Leu Leu Ser Ala 20 25
1429PRTArtificialSynthetic peptide sequence 14His Ser Gln Gly Thr
Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Arg Ala
Lys Asp Phe Ile Glu Trp Leu Leu Lys Ala 20 25
1529PRTArtificialSynthetic peptide sequence 15His Ser Gln Gly Thr
Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys 1 5 10 15 Arg Arg Ala
Lys Asp Phe Ile Glu Trp Leu Leu Ser Ala 20 25
1629PRTArtificialSynthetic peptide sequence 16His Ser Gln Gly Thr
Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala
Lys Asp Phe Ile Glu Trp Leu Leu Ser Ala 20 25
1729PRTArtificialSynthetic peptide sequence 17His Xaa Gln Gly Thr
Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala
Lys Asp Phe Ile Glu Trp Leu Leu Ser Ala 20 25
* * * * *