U.S. patent application number 12/593708 was filed with the patent office on 2010-08-19 for treatment of obesity.
Invention is credited to Mark Anthony Febbraio, Stefan Rose-John.
Application Number | 20100209385 12/593708 |
Document ID | / |
Family ID | 39807709 |
Filed Date | 2010-08-19 |
United States Patent
Application |
20100209385 |
Kind Code |
A1 |
Febbraio; Mark Anthony ; et
al. |
August 19, 2010 |
TREATMENT OF OBESITY
Abstract
The present invention relates generally to a method of
increasing lipid oxidation in a mammal and to agents useful for
same. More particularly, the present invention relates to a method
of increasing lipid oxidation in a mammal by administering a ligand
which interacts with the IL-6 receptor and signals via interaction
with a gp130/LIF receptor heterodimer. In a related aspect, the
present invention provides a method of increasing insulin
sensitivity in a mammal. The method of present invention is useful,
inter alia, in the treatment and/or prophylaxis of conditions
characterised by unwanted lipid accumulation (such as obesity,
obesity induced-metabolic disorders, type II diabetes,
dyslipidemia, glucose intolerance, insulin resistance, obstructive
sleep apnea, cardiovascular disease or non-alcoholic fatty liver
disease) or inadequate insulin sensitivity.
Inventors: |
Febbraio; Mark Anthony;
(Lower Plenty, AU) ; Rose-John; Stefan;
(Schellhorn, DE) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
39807709 |
Appl. No.: |
12/593708 |
Filed: |
March 28, 2008 |
PCT Filed: |
March 28, 2008 |
PCT NO: |
PCT/AU08/00438 |
371 Date: |
March 30, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60920822 |
Mar 30, 2007 |
|
|
|
Current U.S.
Class: |
424/85.2 ;
530/351 |
Current CPC
Class: |
C07K 14/48 20130101;
A61P 5/50 20180101; C07K 14/475 20130101; A61K 38/00 20130101; C07K
14/5412 20130101; A61P 3/00 20180101; A61P 3/08 20180101; A61P 3/10
20180101; C07K 2319/00 20130101; A61P 3/04 20180101 |
Class at
Publication: |
424/85.2 ;
530/351 |
International
Class: |
A61K 38/20 20060101
A61K038/20; C07K 14/54 20060101 C07K014/54; A61P 3/10 20060101
A61P003/10 |
Claims
1. A method of inducing lipid oxidation or increasing insulin
sensitivity in a mammal, said method comprising administering to
said mammal a ligand which binds to the IL-6 receptor and signals
via a gp130/LIF receptor heterodimer.
2. A method of treating a condition in a mammal, which condition is
characterized by either unwanted lipid accumulation or inadequate
insulin sensitivity, said method comprising administering to said
mammal a ligand which binds to the IL-6 receptor and signals via a
gp130/LIF receptor heterodimer.
3. (canceled)
4. The method according to claim 1, wherein said ligand has greater
affinity for the IL-6 receptor than the CNTF receptor.
5. The method of claim 1 wherein said ligand comprises an IL-6
receptor binding site, a gp130 binding site and a LIF receptor
binding site.
6. The method according to claim 5 wherein the IL-6 receptor
binding site of said ligand is substantially similar to the IL-6
receptor binding site of IL-6.
7. The method according to claim 6 wherein said ligand comprises a
binding site substantially similar to the IL-6R binding site of
IL-6, a binding site substantially similar to the gp130 binding
site of IL-6 and a binding site substantially similar to the LIF
receptor binding site of CNTF.
8. The method according to claim 7 wherein said ligand is an
IL-6/CNTF chimera.
9. The method according to claim 8 wherein said ligand is one in
which the site III loop of CNTF is inserted in IL-6 in place of the
site III loop of IL-6.
10. The method according to claim 9 wherein amino acid residues
Glu36-Met56 (SEQ ID NO: 4) are substituted in place of the IL-6
residues between Arg40-Asn60 of SEQ ID NO: 2, the amino acid
residue Gly147-Leu162 (SEQ ID NO: 6) are substituted in place of
IL-6 residues between Leu151-Arg168 or SEQ ID NO: 2 and amino acid
residues Leu91-Ile109 (SEQ ID NO: 5) are substituted in place of
the IL-6 residues between Leu101-Arg113 of SEQ ID NO: 2.
11. The method according to claim 10 wherein said ligand is IC7 or
a ligand comprising the SEQ ID NO: 7 amino acid sequence or a
substantially similar ligand or a functional fragment thereof.
12. The method according to claim 2 wherein said condition is
selected from the group consisting of obesity, insulin resistance,
glucose intolerance, dyslipidemia, non-alcoholic fatty liver
disease, sleep apnea, obesity associated metabolic disorders such
as osteoarthritis, type II diabetes mellitis, hypertension, stroke
or cardiovascular disease, unwanted weight gain or body mass index
and excessive appetite resulting in unwanted weight gain.
13. The method according to claim 1 wherein said ligand comprises a
region or is complexed to a molecule which prevents or retards said
ligand from crossing the blood-brain barrier.
14. The method according to claim 13 wherein said region or
molecule is all or part of an antibody.
15. The method according to claim 14 wherein said region or
molecule is the Fc portion of IgG.
16. An isolated ligand which binds to the IL-6 receptor and signals
via a gp130/LIF receptor heterodimer, which ligand is not IC7.
17. The ligand according to claim 16 wherein said ligand has
greater affinity for the IL-6 receptor than the CNTF receptor.
18. The ligand according to claim 17 wherein the IL-6 receptor
binding site of said ligand is substantially similar to the IL-6
receptor binding site of IL-6.
19. The ligand according claim 16 wherein said ligand comprises an
IL-6 receptor binding site, a gp130 binding site and a LIF receptor
binding site.
20. The ligand according to claim 19 wherein said ligand comprises
a binding site substantially similar to the IL-6R binding site of
IL-6, a binding site substantially similar to the gp130 binding
site of IL-6 and a binding site substantially similar to the LIF
receptor binding site of CNTF.
21. The ligand according to claim 20 wherein said ligand is an
IL-6/CNTF chimera.
22. The ligand according to claim 21 wherein said ligand is one in
which the site III loop of CNTF is inserted in IL-6 in place of the
site III loop of IL-6.
23. The ligand according to claim 16 wherein said ligand comprises
a region or is complexed to a proteinaceous or non-proteinaceous
molecule such that the ligand is prevented or retarded from
crossing the blood brain barrier.
24. A pharmaceutical composition comprising a ligand according to
claim 16 together with a pharmaceutically acceptable carrier.
25. A ligand according to claim 16 for use in therapy.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to a method of
increasing lipid oxidation in a mammal and to agents useful for
same. More particularly, the present invention relates to a method
of increasing lipid oxidation in a mammal by administering a ligand
which interacts with the IL-6 receptor and signals via interaction
with a gp130/LIF receptor heterodimer. In a related aspect, the
present invention provides a method of increasing insulin
sensitivity in a mammal. The method of present invention is useful,
inter alia, in the treatment and/or prophylaxis of conditions
characterised by unwanted lipid accumulation (such as obesity,
obesity induced-metabolic disorders, type II diabetes,
dyslipidemia, glucose intolerance, insulin resistance, obstructive
sleep apnea, cardiovascular disease or non-alcoholic fatty liver
disease) or inadequate insulin sensitivity.
BACKGROUND OF THE INVENTION
[0002] Bibliographic details of the publications referred to by
author in this specification are collected alphabetically at the
end of the description.
[0003] The reference to any prior art in this specification is not,
and should not be taken as, an acknowledgment or any form of
suggestion that that prior art forms part of the common general
knowledge in Australia.
[0004] Obesity is a condition in which the natural energy reserve,
stored in the fatty tissue of humans and other mammals, exceeds
healthy limits. It is commonly defined as a body mass index (weight
divided by height squared) of 30 kg/m.sup.2 or higher.
[0005] Although obesity is an individual clinical condition, some
authorities view it as a serious and growing public health problem,
particularly since excessive body weight has been linked to the
onset of diseases such as cardiovascular diseases, insulin
resistance, dyslipidemia, hypertension, diabetes mellitus type 2
and sleep apnea.
[0006] The prevalence of adult obesity has increased approximately
75% in the last quarter century (Flegal, K M et. al., (1999-2000),
Prevalence and trends in obesity among US adults, JAMA
288:1723-1727). The prevalence of overweight and obese children is
also increasing in both developed and developing countries
(Mascie-Taylor, C G, and Karim, E, (2003), The burden of chronic
disease, Science 302:1921-1922). Current therapies to treat obesity
centre on lifestyle modifications, but for those individuals who do
not respond to such treatment, or cannot adhere to lifestyle
intervention programs, bariatric surgery is often used. As this is
neither a feasible nor a desirable treatment for a pandemic, drug
therapy is a viable intervention for those in whom lifestyle
modification has failed. Currently, there are three obesity drugs
commonly prescribed. Xenical (orlistat) is a gastrointestinal
lipase inhibitor, Sibutramine, a monoamine reuptake inhibitor, and
Rimonabant, the first of the endocannabinoid receptor agonists.
Disappointingly, none has resulted in consistent and effective
weight loss, and to date, all anti-obesity drug trials have been
limited by their high attrition rates and lack of long-term
morbidity and mortality data (Padwal, R S, and Majumdar, S R (2007)
Drug treatment for obesity: orlistat, sibutramine, and rimonbant,
Lancet 369:71-77). Importantly, these drugs do not act by
increasing energy metabolism and, currently, this is the focus of
many pharmaceutical approaches.
[0007] The discovery of leptin (Zhang, Y et. al. (1994) Positional
cloning of the mouse obese gene and its human homologue, Nature
372:425-432, Halaas, J L et. al. (1995) Weight-reducing effects of
the plasma protein encoded by the obese gene, Science 269:543-546)
and the leptin receptor (Tartaglia, L A et. al., (1995)
Identification and expression cloning of a leptin receptor, OB-R,
Cell 83:1263-1271), over a decade ago, led to the hope that
researchers had at last identified a highly effective molecule
and/or pathway that could be targeted in the treatment of obesity.
However, it soon became apparent that obesity, in which high
circulating concentrations of leptin develop, resulted in leptin
resistance whereby endogenous leptin was no longer effective (Van
Heek, M et. al. (1997) Diet-induced obese mice develop peripheral,
but not central, resistance to leptin, J. Clin. Invest.
99:385-390).
[0008] Over the past decade a metabolic role for gp130 receptor
cytokines has been elucidated. Often termed the "interleukin (IL)-6
family" of cytokines these include IL-6, leukemia inhibitory factor
(LIF), IL-11, oncostatin-M, cardiotrophin-1 and ciliary neurotropic
factor (CNTF). In particular, CNTF and IL-6 enhance fat oxidation
in skeletal muscle and increase insulin sensitivity in vivo,
principally via the activation of AMP activated protein kinase
(AMPK) in both animals and humans. These results have generated a
great deal of excitement as gp130 receptor ligands are now becoming
recognised as a potential therapeutic target for obesity-induced
insulin resistance. However, despite these major advanced in the
understanding of the molecular processes as to how gp130 receptor
ligands may enhance insulin sensitivity and act as
"anti-obesogenic" agents, clinical trials have not been successful.
This has been due principally to two major complications. The first
is that IL-6 is pro-inflammatory and while it has positive effects
on energy balance and insulin sensitivity when administered
acutely, it has negative effects on the progression of many
diseases. Secondly, CNTF failed in clinical trials because patients
developed antibodies to Axokine.RTM., the human recombinant variant
of CNTF (Ettinger, M P, et. al. (2003) Recombinant variant of
ciliary neurotrophic factor for weight loss in obese adults: a
randomized, dose-ranging study, JAMA 289: 1826-1832). This was not
entirely surprising since CNTF lacks a secretory signal sequence
peptide and, therefore, does not circulate. Still further, due to
the low level of CNTF receptors present in the periphery and the
lower level of affinity of CNTF for the more light expressed IL-6
receptor, quite high concentration of CNTF were required to be
used.
[0009] Accordingly, there exists an ongoing need to develop new
methods for treating obesity. In work leading up to the present
invention it has been determined that in terms of the functioning
of IL-6 and CNTF, the unwanted side effects known to be associated
with the administration of these molecules can be minimised by
activating the IL-6 receptor and facilitating induction of the
subsequent signalling via a gp130/LIF receptor heterodimer, rather
than the gp130 homodimer which is used by IL-6. The findings of the
present invention have now facilitated the development of
methodology for increasing lipid oxidation in mammals without the
concomitant problems of the induction of an inflammatory state, or
the use of high concentrations of cytokine of the generation of
autoantibodies. Accordingly, there is now provided a means for
therapeutically or prophylactically treating conditions associated
with unwanted lipid accumulation.
SUMMARY OF THE INVENTION
[0010] Throughout this specification and the claims that follow,
unless the context requires otherwise, the word "comprise", or
variations such as "comprises" or "comprising" will be understood
to imply the inclusion of a stated element or integer or group of
elements or integers, but not the exclusion of any other element or
integer or group of elements or integers.
[0011] As used herein, the term "derived from" shall be taken to
indicate that a particular integer or group of integers has
originated from the species specified, but has not necessarily been
obtained directly from the specified source. Further, as used
herein the singular forms of "a", "and" and "the" include plural
referents unless the context clearly dictates otherwise.
[0012] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0013] The subject specification contains amino acid sequence
information prepared using the programme Patent In Version 3.1,
presented herein after the bibliography. Each amino acid sequence
is identified in the sequence listing by the numeric indicator
<210> followed by the sequence identifier (e.g. <210>1,
<210>2, etc). The length, type of sequence (amino acid, etc.)
and source organism for each sequence is indicated by information
provided in the numeric indicator fields <211>m <212>
and <213>, respectively. Amino acid sequences referred to in
the specification are identified by the indicator SEQ ID NO:
followed by the sequence identifier (e.g. SEQ ID NO:1, SEQ ID NO:
2, etc). The sequence identifier referred to in the specification
correlates to the information provided in numeric indicator field
<400> in the sequence listing, which is followed by the
sequence identifier (e.g. <400>1, <400>2, etc). That is
SEQ ID NO: 1 as detailed in the specification correlates to the
sequence indicated as <400>1 in the sequence listing.
[0014] One aspect of the present invention is directed to a method
of inducing lipid oxidation in a mammal, said method comprising
administering to said mammal a ligand which binds to the IL-6
receptor and signals via a gp130/LIF receptor heterodimer.
[0015] In another aspect the present invention is directed to a
method of inducing lipid oxidation in a mammal, said method
comprising administering to said mammal a ligand which binds to the
IL-6 receptor and signals via a gp130/LIF receptor heterodimer
wherein the IL-6 receptor binding site of said ligand has a greater
affinity for the IL-6 receptor than the CNTF receptor.
[0016] In yet another aspect the present invention is directed to a
method of inducing lipid oxidation in a mammal, said method
comprising administering to said mammal a ligand comprising an IL-6
receptor binding site, a gp130 binding site and a LIF receptor
binding site.
[0017] In still another aspect the present invention provides a
method of inducing lipid oxidation in a mammal, said method
comprising administering to said mammal a ligand comprising a
binding site substantially similar to the IL-6R binding site of
IL-6, a gp130 binding site and a LIF receptor binding site.
[0018] In a further aspect there is provided a method of inducing
lipid oxidation in a mammal, said method comprising administering
to said mammal a ligand comprising a binding site substantially
similar to the IL-6R binding site of IL-6, a binding site
substantially similar to the gp130 binding site of IL-6 and a
binding site substantially similar to the LIF receptor binding site
of CNTF.
[0019] In yet another aspect, said ligand comprises sites I and II
of IL-6, or substantially similar sites as hereinbefore defined and
site III of CNTF or substantially similar site.
[0020] In yet still another aspect, the subject ligand is an
IL-6/CNTF chimeric protein. More specifically, an exemplary chimera
is one in which the site III loop of CNTF is inserted in IL-6 in
place of the site III loop of IL-6. In such chimeras, amino acid
residues located in the C-terminal A-helix, the N-terminal AB loop
(Glu36-Met56; SEQ ID NO:4) of CNTF may be substituted in place of
the IL-6 residues between Arg40-Asn60 (i.e., Arg40 and Asn60 are
retained), the C-terminal CD loop with the adjoining N-terminal D
helix (Gly147-Leu162; SEQ ID NO:6) of CNTF may be substituted in
place of IL-6 residues between Leu151-Arg168 and the BC loop with
adjacent parts of B- and C-helix (Leu91-Ile109; SEQ ID NO:5) of
CNTF may be substituted in place of the IL-6 residues between
Leu101-Arg113.
[0021] Preferably, said ligand is IC7, as described in Kallen, K J
et. al, (1999) Receptor recognition sites of cytokines are
organized as exchangeable modules: transfer of the LIFR binding
site from CNTF to IL-6, J. Biol. Chem. 274:11859-11867 or SEQ ID
NO: 7 or a substantially similar ligand.
[0022] In a further aspect there is provided a method of inducing
lipid oxidation in a mammal, said method comprising administering
to said mammal IC7, a ligand comprising the SEQ ID NO:7 amino acid
sequence or a substantially similar ligand or functional fragment
thereof.
[0023] In another aspect of the present invention is directed to a
method of therapeutically or prophylactically treating a condition
in a mammal, which condition is characterised by unwanted lipid
accumulation, said method comprising administering to said mammal a
ligand which binds to the IL-6 receptor and signals via a gp130/LIF
receptor heterodimer.
[0024] In still another aspect the present invention is directed to
a method of therapeutically or prophylactically treating a
condition in a mammal, which condition is characterised by unwanted
lipid accumulation, said method comprising administering to said
mammal a ligand which binds to the IL-6 receptor and signals via a
gp130/LIF receptor heterodimer, wherein the IL-6 receptor binding
site of said ligand has a greater affinity for the IL-6 receptor
than the CNTF receptor.
[0025] In yet still another aspect there is provided a method of
therapeutically or prophylactically treating a condition in a
mammal, which condition is characterised by unwanted lipid
accumulation, said method comprising administering to said mammal a
ligand comprising a binding site substantially similar to the IL-6R
binding site of IL-6, a binding site substantially similar to the
gp130 binding site of IL-6, and a binding site substantially
similar to the LIF receptor binding site of CNTF.
[0026] In still yet another aspect there is provided a method of
therapeutically or prophylactically treating a condition in a
mammal, which condition is characterised by unwanted lipid
accumulation, said method comprising administering to said mammal
IC7, a ligand comprising the SEQ ID NO:7 amino acid sequence or a
substantially similar ligand or functional fragment thereof.
[0027] Yet another aspect of the present invention is directed to
the use of a ligand, which ligand binds to the IL-6 receptor and
signals via a gp130/LIF receptor heterodimer, in the manufacture of
a medicament for the treatment of a condition characterised by
unwanted lipid accumulation.
[0028] Yet still another aspect of the present invention is
directed to the use of a ligand, which ligand binds to the IL-6
receptor and signals via a gp130/LIF receptor heterodimer, wherein
the IL-6 receptor binding site of said ligand has a greater
affinity for the IL-6 receptor than the CNTF receptor, in the
manufacture of a medicament for the treatment of a condition
characterised by unwanted lipid accumulation.
[0029] A further aspect of the invention provides the use of a
ligand, which ligand comprises a binding site substantially similar
to the IL-6R binding site of IL-6, a binding site substantially
similar to the gp130 binding site of IL-6, and a binding site
substantially similar to the LIF receptor binding site of CNTF, in
the manufacture of a medicament for the treatment of a condition
characterised by unwanted lipid accumulation.
[0030] Another further aspect provides the use of IC7 or a ligand
comprising the SEQ ID NO: 7 amino acid sequence or a substantially
similar ligand or a functional fragment thereof in the manufacture
of a medicament for the treatment of a condition characterised by
unwanted lipid accumulation.
[0031] In another aspect, said condition is obesity, insulin
resistance, glucose intolerance, dyslipidemia, non-alcoholic fatty
liver disease, sleep apnea, obesity associated metabolic disorders
such as osteoarthritis, type II diabetes, mellitus, hypertension,
stroke or cardiovascular disease, unwanted weight gain (even where
that weight gain is below the level of obesity) or body mass index
and excessive appetite resulting in unwanted weight gain.
[0032] Yet another aspect of the present invention is directed to a
ligand, as hereinbefore defined, which ligand is not IC7.
[0033] Still another aspect provides a pharmaceutical composition
comprising a ligand as hereinbefore defined together with a
pharmaceutically acceptable carrier.
[0034] In yet another aspect there is provided a ligand as
hereinbefore defined for use in therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 is a schematic structure of IL-6.
[0036] FIG. 2 depicts the site III gp130 and LIFR binding epitope
of IL-6 and CNTF, respectively. (a) schematic drawing of the common
four-helix bundle cytokine fold. (b) ribbon models of the IL-6 NMR
and CNTF x-ray structures. the different parts of site III are
colour coded: yellow (site IIIA), green (site IIIB), and blue (site
IIIC). (c) bar representation of IL-6, CNTF, and chimeras IC1 to
IC7. Sequence stretches that are part of the exchanged epitopes of
IL-6 and CNTF are hatched. On CNTF the N- and C-terminal amino acid
residues of the transferred stretches are designated in single
letter code, on IL-6 the residues adjacent to the transferred CNTF
stretches are denoted.
[0037] FIG. 3 is (a) an epitope shuffle of receptor-binding sites
of IL-6-like cytokines. The figure shows the typical four-helix
bundle fold of IL-6, CNTF and IC7, with the characteristic
up-up-down-down orientation of the .alpha.-helices. Consequently,
two long loops (AB and CD) and one short loop (BC) connect the
helices. (b) A ribbon model of IL-6, CNTF IC-7. (c) Receptor
requirements of IL-6, CNTF and IC-7
[0038] FIG. 4 is a graphical representation depicting that IC-7
stimulates glucose uptake into skeletal muscle. All values are
relative to basal glucose uptake, adjusted to 100% (dotted line).
Insulin and IC-7 values were derived from three animals, while
co-treatment values were derived from two animals.
[0039] FIG. 5 is a graphical representation depicting that IC-7
increases fatty acid oximation in soleus and EDL muscle (p
value=0.005 and 0.034 respectively, n-12).
[0040] FIG. 6 is a graphical representation depicting that CNTF
increases fatty acid oxidation in EDL by not Soleus.
[0041] FIG. 7 is an image depicting that IC-7 activates gp130
receptor signalling.
[0042] FIG. 8 depicts a bar representation of IL-6, CNTF and the
chimeras IC1-IC7. Sequence stretches that are part of the exchanged
epitopes of IL-6 and CNTF are hatched. On CNTF the N and C terminal
amino acid residues of the transferred stretches are designated in
single letter code, on IL-6 the residues adjacent to the
transferred CNTF stretches are denoted.
[0043] FIG. 9 shows the complex ligand receptor signalling for the
gp130R.beta. cytokine CNTF. In contrast to the mechanism of
IL-6R.alpha./gp130R.beta. homodimer binding of IL-6 ligand, CNTF
can signal via a heterodimer containing CNTFR.alpha., gp130R.beta.
and LIFR.beta. (A) or via a heterodimer containing IL-6R.alpha.,
gp130R.beta. and LIFR.beta. (B). While CNTF can signal via
IL-6R.alpha., it cannot do so via a IL-6R.alpha./130R.beta.
homodimer as LIF is an absolute requirement.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention is predicated, in part, on the
determination that lipid oxidation can be induced in a mammal by
administering a ligand which interacts with the IL-6 receptor and
signals via a gp130/LIF receptor heterodimer. However, whereas IL-6
induced interaction with the IL-6 receptor and subsequent gp130
homodimer induced signalling can lead to inflammation, while CNTF
induced interaction with the CNTF receptor, or even the IL-6
receptor, and subsequent gp130/LIF receptor heterodimer induced
signalling requiring the use of high concentrations of CNTF and can
lead to anti-CNTF autoantibody generation, the use of a ligand
which is designed to be directed to the IL-6 receptor but signals
through a gp130/LIF receptor heterodimer minimises these problems
while nevertheless achieving lipid oxidation. Accordingly, this
finding has now facilitated the rational design of a means for
inducing lipid oxidation and, in particular, for therapeutically or
prophylactically treating conditions which are characterised by
unwanted lipid accumulation such as obesity, hypertension, obesity
induced type II diabetes, glucose intolerance or insulin
resistance.
[0045] Accordingly, one aspect of the present invention is directed
to a method of inducing lipid oxidation in a mammal, said method
comprising administering to said mammal a ligand which binds to the
IL-6 receptor and signals via a gp130/LIF receptor heterodimer.
[0046] Without limiting the present invention to any one theory or
mode of action, both IL-6 and CNTF have been found to enhance lipid
oxidation and increase insulin sensitivity in mammals. To this end,
IL-6 interacts with the IL-6 receptor and then signals via its
further interaction with a gp130 homodimer. CNTF functions by
interacting with the CNTF receptor (or with the IL-6 receptor at a
significantly lower affinity) and signalling via its further
interaction with a gp130/LIF receptor heterodimer. However, in
addition to lipid oxidation induction, both IL-6 and CNTF are
associated with less desirable functional outcomes. Specifically,
the interaction of a soluble IL-6 receptor/IL-6 complex with a
gp130 homodimer is linked to the induction of inflammation while
CNTF, due to its lack of a secretory signal and the high
concentrations at which it is required to be used in order to
effect lipid oxidation in the periphery, results in CNTF
autoantibody production. The inventors have determined, however,
that the induction of lipid oxidation can still be effected if IL-6
receptor binding is followed by signalling via a gp130/LIF receptor
heterodimer, rather than a gp130 homodimer, as is characteristic of
IL-6 stimulation, but that this mechanism reduces the incidence of
IL-6 related induction of inflammation. Similarly, by using a
ligand which is designed to interact with the IL-6 receptor with a
degree of affinity greater than that of the interaction of CNTF
with the IL-6 receptor (which interaction naturally occurs due to
the low level of expression of the CNTF receptor in the periphery),
lower concentrations of this molecule are required to be used than
if CNTF is used to induce lipid oxidation. Still further, by
designing this ligand to more closely resemble IL-6 rather than
CNTF, which is not secreted, the possibility of autoantibody
generation is still further minimised.
[0047] Accordingly, in one embodiment the present invention is
directed to a method of inducing lipid oxidation in a mammal, said
method comprising administering to said mammal a ligand which binds
to the IL-6 receptor and signals via a gp130/LIF receptor
heterodimer wherein the IL-6 receptor binding site of said ligand
has a greater affinity for the IL-6 receptor than the CNTF
receptor.
[0048] In another embodiment, the IL-6 receptor binding site of
said ligand is substantially similar to the IL-6 receptor binding
site of IL-6.
[0049] Reference to "IL-6 receptor", "gp130" and "LIF receptor"
should be understood as a reference to all forms of these molecules
and to functional derivatives and homologues thereof. This
includes, for example, any isoforms which arise from alternative
splicing of the mRNA, allelic variants or mutants of these
receptors.
[0050] Without limiting the present invention to any one theory or
mode of action, the IL-6 receptor system consists of two membrane
proteins, a ligand binding receptor (IL-6R) and a non-binding
signal transducer (gp130). The human IL-6 receptor consists of 468
amino acids, including a signal peptide of 19 amino acids, an
extracellular region of 339 amino acids, a membrane-spanning region
of 28 amino acids, and a cytoplasmic region of 82 amino acids
(Yamasaki, K et. al. (1988) Cloning and expression of the human
interleukin-6 (BSF-2/IFN beta 2) receptor, Science 241:825-828)
(GenBank accession number M20566, X12830). The predicted molecular
weight of IL-6R is 50,000 Da, although the observed molecular
weight is 80,000 Da due to N-glycosylation.
[0051] Upon binding of IL-6, IL-6R is triggered to become
associated with a signal transducing receptor component, gp130
(Taga, T et. al. (1989) Interleukin-6 triggers the association of
its receptor with a possible signal transducer, gp130, Cell
58:573-581). gp130 has no intrinsic IL-6 binding capability, but is
involved in the formation of high-affinity IL-6 binding sites.
gp130 consists of 918 amino acids, including a leader sequence of
22 amino acids, an extracellular region of 597 amino acids, a
membrane-spanning region of 22 amino acids, and a cytoplasmic
region of 277 amino acids (Hibi, M et. al. (1990) Molecular cloning
and expression of an IL-6 signal transducer, gp130, Cell
63:1149-1157) (GenBank accession number M57230). The gp130 protein
serves as a signal transducer not only for IL-6 but also for
leukaemia inhibitory factor (LIF), oncostatin M (OSM), ciliary
neurotrophic factor (CNTF), and IL-11 (Taga, T and Kishimoto, T
1992, Cytokine receptors and signal transduction, FASEB J.
6:3387-3396; Yin, T et. al. (1993) Involvement of IL-6 signal
transducer gp130 in IL-11 mediated signal transduction, J. Immunol.
151: 2555-2561). Stimulation by these cytokines induces
oligomerization of the receptor components (Davis, S et. al. (1993)
LIFR beta and gp130 as heterodimerizing signal transducers of the
tripartite CNTF receptor, Science 260:1805-1808; Murakami, M et.
al. (1993) IL-6-induced homodimerization of gp130 and associated
activation of a tyrosine kinase, Science 260:1808-1810).
[0052] The CNTF receptor complex contains three proteins, a ligand
binding receptor (CNTFR) that directly binds to CNTF as well as two
signal transducing components--LIF receptor and gp130. cDNAs
encoding CNTFR.alpha. have been cloned from both human (GenBank
accession number M73238) (Davis, S et. al. (1991), The receptor for
ciliary neurotrophic factor, Science 253:59-63) and rat (GenBank
accession number S54212) (Ip, N Y et. al. (1993a) The alpha
component of the CNTF receptor is required for signalling and
defines potential CNTF targets in the adult and during development,
Neuron 10:89-102; Ip N Y et. al. (1993b) Injury-induced regulation
of ciliary neurotrophic factor mRNA in the adult rat brain, Eur. J.
Neurosci. 5:25-33). The cDNA for human CNTFR.alpha. predicts a
protein precursor of 372 amino acids with putative leader sequences
of approximately 20 amino acids and four conserved glycosylation
sites. Glycosylation at these sites partially accounts for the
difference between the observed molecular weight of CNTFR on
SDS-PAGE gels (.about.70 kDa) and the molecular weight predicted
from the amino acid sequence (.about.40 kDa) (Davis et. al. (1991)
supra). Unlike most other growth factor receptor components, CNTFR
lacks trans-membrane and intracytoplasmic domains; instead, it is
anchored to the cell membrane via a GPI linkage (Davis et. al.
(1991) supra). The closest known relative to CNTFR is IL-6R (30
percent amino acid identity) (Davis et. al. (1991) supra), which is
a transmembrane protein.
[0053] In the absence of CNTF, the receptor components comprising
the CNTF receptor complex are un-associated on the cell surface
(Davis et. al. (1993) supra; Stahl, N et. al. (1993) Cross-linking
identifies leukaemia inhibitory factor-binding protein as a ciliary
neurotrophic factor receptor component, J. Biol. Chem.
268:7628-7631). It is this last step in receptor assembly--which
involves heterodimerization between LIFR and gp130 that is
responsible for transducing a signal across a membrane (Davis et.
al. (1993) supra; Stahl et. al. (1993) supra).
[0054] As would be understood by the person of skill in the art,
the ligand binding subunit of a receptor is referred to as the
.alpha. chain while other signal transducing subunits are referred
to as .beta. chains and even .gamma. chains. Accordingly, within
the context of the present invention, IL-6 receptor (IL-6R), CNTF
receptor (CNTFR), gp130 and LIF receptor (LIFR) are interchangeably
referred to as IL-6R.alpha., CNTRF.alpha., gp130.beta. and
LIFR.beta..
[0055] Reference to a "ligand which binds to the IL-6 receptor and
signals via a gp130/LIF receptor heterodimer" should therefore be
understood as a reference to a molecule which, although initially
binding the IL-6 receptor thereafter engages a CNTF-like signalling
mechanism, being the association of the IL-6 receptor/ligand
complex with gp130 and its heterodimerization with LIF receptor in
order to effect intracellular signalling.
[0056] Reference to a "ligand" should be understood as a reference
to a molecule which binds to and activates a receptor complex. In
terms of designing a ligand molecule capable of binding to the IL-6
receptor and signalling via a gp130/LIF receptor heterodimer, and
as detailed above, the amino acid sequence and 3-dimensional
structure of these receptors are well known, as are the actual
ligand binding regions. Similarly, the sequences and 3-dimensional
structures of IL-6 and CNTF are also well known.
[0057] Specifically, human IL-6 consists of 212 amino acids
including a 28 amino acid signal peptide (SEQ ID NO: 1) (Hirano, T
et. al. (1986) Complementary DNA for a novel human interleukin
(BSF-2) that induces B lymphocytes to produce immunoglobulin,
Nature 324:73-6) (GenBank accession number X04602). Human IL-6 is a
secreted glycoprotein containing 184 amino acids in the mature
protein (SEQ ID NO: 2). The molecular weight of the core protein is
about 20,000 Da. The two disulphide bridges have been located
between Cys44-Cys50 and Cys73-Cys83 in human IL-6 (Simpson, R J et.
al. (1988) Characterization of a recombinant murine interleukin-6:
assignment of disulphide bonds, Biochem. Biophys. Res. Commun.
157:364-372). The molecular weight of natural IL-6 is 21-26,000 Da
depending on the cellular source. Its heterogeneity results from
post-translational modifications such as N- and O-linked
glycosylation and phosphorylation (position 45 and 144 in human
IL-6 are N-glycosylated). IL-6 has a tertiary fold which is similar
to the four-.alpha.-helix bundle structure found in growth hormone,
despite little similarity in amino acid sequence to growth hormone
(Bazan, J F (1990a) Haemopoietic receptors and helical cytokines,
Immunol. Today 11:350-354; Bazan, J F (1990b) Structural design and
molecular evolution of a cytokine receptor superfamily, Proc. Natl.
Acad. Sci. (USA) 87:6934-6938). The four .alpha.-helices (labelled
A to D in FIG. 1) and loops (two long A-B and C-D loops, and a
short B-C loop) predicted in the IL-6 protein are adopted in other
cytokines, such as CNTF.
[0058] Genes and cDNAs encoding CNTF have been cloned from human
(GenBank accession numbers 60477-8, 60542, 55889-90) (McDonald, J R
et. al. (1991), Expression and characterization of recombinant
human ciliary neurotrophic factor from Escherichia coli, Biochim.
Biophys. Acta 1090:70-80; Masiakowski, P et. al. (1991) Recombinant
human and rat ciliary neurotrophic factors, J. Neurochem.
57:1003-1012). The human CNTF protein is 200 amino acids in length
(SEQ ID NO:3). CNTF is not secreted but rather found in the
cytoplasm of cells, such as astrocytes (Rudge, J S et. al. (1992)
Expression of ciliary neurotrophic factor and the
neurotrophins--nerve growth factor brain-derived neurotrophic
factor and neurotrophin-3--in cultured rat hippocampal astrocytes,
Eur. J. Neurosci. 4:459-471; Rende, M et. al. (1992)
Immunolocalization of ciliary neuroneotrophic factor in adult rat
sciatic nerve, Glia 5:25-32; Friedman, B et. al. (1992) Regulation
of ciliary neurotrophic factor expression in myelin-related Schwann
cells in vivo, Neuron 9:295-305), which express CNTF. CNTF is a
member of a cytokine subfamily that includes LIF, IL-6, and OSM
(Bazan, J F (1991) Neuropoietic cytokines in the hematopoietic
fold, Neuron 7:197-208). Though these four factors exhibit minimal
primary sequence homology, they all share secondary structural
features which link them and allow them to conform generally to the
four-.alpha. helix bundle structure first described for growth
hormone and depicted in FIG. 1 (Bazan (1991) supra).
[0059] In terms of the binding of IL-6 and CNTF to their respective
.alpha. receptors, for IL-6 and CNTF the contact site with the
receptor .alpha.-unit can be mapped to a site that includes
residues of the C-terminal AB loop and the C-terminal D-helix (site
I) (McDonald et. al. (1995), EMBO J. 14:2689-2699; Panayotatos, N
et. al. (1995), J. Biol. Chem. 270:14007-14014; Grotzinger, J et.
al. (1997) Proteins Struct. Funct. Genet. 27:96-109). Residues of
the A- and C-helices of CNTF and IL-6 constitute a gp130-binding
site (site II). In IL-6, a second gp130-binding site consists of
amino acids residues of the N-terminal AB loop, the C-terminal CD
loop, and the N-terminal D-helix. This site is termed site III.
Crystallographic and mutagenesis studies of CNTF and LIF indicate
that residues of the C-terminal B-helix, possibly the BC loop, CD
loop, and the N-terminal D-helix constitute site III in these
cytokines (McDonald et. al. (1995), supra; Panayotatos, N et. al.
(1995), supra) (see FIG. 2).
[0060] Considering the conserved four-helical bundle structure of
IL-6 and CNTF these cytokines have evolved as discontinuous modules
which are exchangeable (FIG. 2).
[0061] Accordingly, in one embodiment the present invention is
directed to a method of inducing lipid oxidation in a mammal, said
method comprising administering to said mammal a ligand comprising
an IL-6 receptor binding site, a gp130 binding site and a LIF
receptor binding site.
[0062] In another embodiment, said IL-6 receptor binding site
exhibits greater affinity for IL-6R.alpha. than CNTFR.alpha..
[0063] In still another embodiment, the IL-6 receptor binding site
of said ligand is substantially similar to the IL-6 receptor
binding site of IL-6.
[0064] This aspect of the present invention therefore provides a
method of inducing lipid oxidation in a mammal, said method
comprising administering to said mammal a ligand comprising a
binding site substantially similar to the IL-6R binding site of
IL-6, a gp130 binding site and a LIF receptor binding site.
[0065] Still more particularly there is provided a method of
inducing lipid oxidation in a mammal, said method comprising
administering to said mammal a ligand comprising a binding site
substantially similar to the IL-6R binding site of IL-6, a binding
site substantially similar to the gp130 binding site of IL-6 and a
binding site substantially similar to the LIF receptor binding site
of CNTF.
[0066] The phrase "substantially similar" in the context of two
polypeptides, can refer to two or more sequences that have, e.g.,
at least about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more amino acid residue (sequence) identity, when
compared and aligned for maximum correspondence, as measured using
one any known sequence comparison algorithm, as discussed below, or
by visual inspection.
[0067] Because two polypeptides may each comprise (1) a sequence
(i.e. only a portion of the complete polynucleotide sequence) that
is similar between the two polypeptides, and (2) a sequence that is
divergent between the two polypeptides, sequence comparisons
between two (or more) polypeptides are typically performed by
comparing sequences of the two polypeptides over a "comparison
window" to identify and compare local regions of sequence
similarity. A "comparison window", as used herein, includes
reference to a segment of any one of the numbers of contiguous
residues. For example, in alternative aspects of the invention,
contiguous residues ranging anywhere from 5 to the full length of
an exemplary polypeptide sequence of the invention are compared to
a reference sequence of the same number of contiguous positions
after the two sequences are optimally aligned. If the reference
sequence has the requisite sequence identity to an exemplary
polypeptide of the invention, that sequence is within the scope of
the invention.
[0068] The comparison window may comprise additions or deletions
(i.e. gaps) of about 20% or less as compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. Optimal alignment of
sequences for aligning a comparison window may be conducted by
computerized implementations of algorithms (GAP, BESTFIT, FASTA,
and TFASTA in the Wisconsin Genetics Software Package Release 7.0,
Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or
by inspection and the best alignment (i.e. resulting in the highest
percentage homology over the comparison window) generated by any of
the various methods selected. Reference also may be made to the
BLAST family of programs as for example disclosed by Altschul et
al. (1997) Nucl. Acids Res. 25:3389. A detailed discussion of
sequence analysis can be found in Unit 19.3 of Ausubel et al.
("Current Protocols in Molecular Biology" John Wiley & Sons
Inc, Chapter 15, 1994-1998). A range of other algorithms may be
used to compare the nucleotide and amino acid sequences such as but
not limited to PILEUP, CLUSTALW, SEQUENCHER or Vector NTI.
[0069] The terms "sequence similarity" and "sequence identity" as
used herein refers to the extent that sequences are identical or
functionally or structurally similar on a residue-by-residue basis
over a window of comparison. Thus, a "percentage of sequence
identity", for example, is calculated by comparing two optimally
aligned sequences over the window of comparison, determining the
number of positions at which the identical amino acid occurs in
both sequences to yield the number of matched positions, dividing
the number of matched positions by the total number of positions in
the window of comparison (i.e., the window size), and multiplying
the result by 100 to yield the percentage of sequence identity.
[0070] Protein sequence identities (homologies) may be evaluated
using any of the variety of sequence comparison algorithms and
programs known in the art. The extent of sequence identity
(homology) may be determined using any computer program and
associated parameters, including those described herein, such as
BLAST 2.2.2. or FASTA version 3.0t78, with the default
parameters.
[0071] The terms "homology" and "identity" in the context of two or
more polypeptide sequences, refer to two or more sequences that are
the same or have a specified percentage of amino acid residues or
nucleotides that are the same when compared and aligned for maximum
correspondence over a comparison window or designated region as
measured using any number of sequence comparison algorithms or by
manual alignment and visual inspection. For sequence comparison,
one sequence can act as a reference sequence to which test
sequences are compared. When using a sequence comparison algorithm,
test and reference sequences are entered into a computer,
subsequence coordinates are designated, if necessary, and sequence
algorithm program parameters are designated. Default program
parameters can be used, or alternative parameters can be
designated. The sequence comparison algorithm then calculates the
percent sequence identities for the test sequences relative to the
reference sequence, based on the program parameters.
[0072] In yet another embodiment, said ligand comprises sites I and
II of IL-6, or substantially similar sites as hereinbefore defined
and site III of CNTF or substantially similar site.
[0073] Accordingly to these embodiments, the subject ligand is an
IL-6/CNTF chimeric protein. More specifically, an exemplary chimera
is one in which the site III loop of CNTF is inserted in IL-6 in
place of the site III loop of IL-6. In such chimeras, amino acid
residues located in the C-terminal A-helix, the N-terminal AB loop
(Glu36-Met56; SEQ ID NO:4) of CNTF may be substituted in place of
the IL-6 residues between Arg40-Asn60 (i.e., Arg40 and Asn60 are
retained), the C-terminal CD loop with the adjoining N-terminal D
helix (Gly147-Leu162; SEQ ID NO:6) of CNTF may be substituted in
place of IL-6 residues between Leu151-Arg168 and the BC loop with
adjacent parts of B- and C-helix (Leu91-Ile109; SEQ ID NO:5) of
CNTF may be substituted in place of the IL-6 residues between
Leu101-Arg113.
[0074] Preferably, said ligand is IC7, as described in Kallen et.
al. (1999) supra or SEQ ID NO: 7 or a substantially similar
ligand.
[0075] The method of the present invention should also be
understood to extend to the use of functional fragments of IC7 or
SEQ ID NO:7. Reference to "functional" should be understood as a
reference to a fragment which is capable of binding to the IL-6
receptor and signalling via a gp130/LIF receptor heterodimer.
[0076] According to this embodiment there is provided a method of
inducing lipid oxidation in a mammal, said method comprising
administering to said mammal IC7, a ligand comprising the SEQ ID
NO:7 amino acid sequence or a substantially similar ligand or
functional fragment thereof.
[0077] The invention also extends to functional variants of the
subject ligand which have one or more amino acid substitutions,
additions and deletions. There may be 1 to 5, 5 to 10, 10 to 15, 15
to 20, 20 to 30 or more residues substituted, added or deleted,
whilst maintaining functionality.
[0078] Substitutions encompass amino acid alterations in which an
amino acid is replaced with a different naturally-occurring or a
non-conventional amino acid residue. Such substitutions may be
classified as "conservative", in which case an amino acid residue
present in a peptide is replaced with another naturally-occurring
amino acid of similar character, for example Gly to Ala, Asp to
Glu, Asn to Gln or Trp to Tyr. Possible alternative amino acids
include Serine or Threonine, Aspartic acid or Glutamic acid or
.gamma.-Carboxyglutamate, Proline or Hydroxyproline, Arginine or
Lysine, Asparagine or Histidine, Histidine or Asparagine, Tyrosine
or Phenylalanine or Tryptophan, Aspartate or Glutamate, Isoleucine
or Leucine or Valine.
[0079] Such conservative substitutions are shown in Table 1 under
the heading of preferred substitutions. If such substitutions do
not result in a change in functional activity, then more
substantial changes, denoted exemplary substitutions in Table 1, or
as further described below in reference to amino acid classes, may
be introduced, and the resulting variant analyzed for functional
activity.
TABLE-US-00001 TABLE 1 Amino acid substitutions Original Exemplary
Preferred Residue Substitutions Substitutions Ala (A) val; leu; ile
val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp
(D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G)
pro pro His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala;
phe; leu norleucine Leu (L) norleucine; ile; val; ile met; ala; phe
Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu;
val; ile; ala leu Pro (P) gly gly Ser (S) thr thr Thr (T) ser ser
Trp (W) tyr tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu;
met; phe; leu ala; norleucine
[0080] Substitutions encompassed by the present invention may also
be "non-conservative", in which an amino acid residue which is
present in a polypeptide is substituted with an amino acid having
different properties, such as a naturally-occurring amino acid from
a different group (e.g. substituting a charged or hydrophilic or
hydrophobic amino acid with Alanine), or alternatively, in which a
naturally-occurring amino acid is substituted with a
non-conventional amino acid. Additions encompass the addition of
one or more naturally occurring or non-conventional amino acid
residues. Deletions encompass the deletion of one or more amino
acid residues.
[0081] Methods for combinatorial synthesis of analogues of the
ligand and for screening of the analogues to determine that they
retain activity are well known in the art (see for example Gallop
et al. (1994) J. Med. Chem. 37:1233-1251; Hogan (1997) Nature
Biotechnology 15:328-330).
[0082] Non-conventional amino acids or chemical amino acid
analogues can be used in place of naturally occurring amino acid
molecules. Thus for example Leucine may be replaced by Norleucine,
Valine may be replaced by Norvaline, Cysteine may be replaced by
Homocysteine, Serine may be replaced by Homoserine, Lysine may be
replaced by 5-Hydroxylysine, Proline by 4-Hydroxyproline, Arginine
may be replaced by Homoarginine, Ornithine or Citrulline, Alanine
may be replaced by .alpha.-Methylalanine or .beta.-Alanine, a
D-amino acid may be used instead of the corresponding L-amino acid,
any amino acid may be N-methylated, or the N-terminus may be
acetylated. A non-conventional amino acid further includes one
selected from the group consisting of D-amino acids, homo-amino
acids, N-alkyl amino acids, dehydroamino acids, aromatic amino
acids (other than phenylalanine, tyrosine and tryptophan), ortho-,
meta- or para-aminobenzoic acid, ornithine, citrulline, norleucine,
.gamma.-glutamic acid, aminobutyric acid (Abu), and
.alpha..alpha.disubstituted amino acids.
[0083] Non-conventional amino acids also include compounds which
have an amine and carboxyl functional group separated in a 1,3 or
larger substitution pattern, such as .beta.-alanine, .gamma.-amino
butyric acid, Freidinger lactam, the bicyclic dipeptide (BTD),
amino-methyl benzoic acid and others well known in the art.
Statine-like isosteres, hydroxyethylene isosteres, reduced amide
bond isosteres, thioamide isosteres, urea isosteres, carbamate
isosteres, thioether isosteres, vinyl isosteres and other amide
bond isosteres known to the art may also be used.
[0084] The use of analogues or non-conventional amino acids may
improve the stability and biological half-life of the subject
ligand. The person skilled in the art will be aware of similar
types of substitution which may be made.
[0085] A non limiting list of non-conventional amino acids which
may be used as suitable replacements for the naturally occurring
amino acids and their standard abbreviations is set out in Table
2.
TABLE-US-00002 TABLE 2 Non-conventional amino acids
Non-conventional Non-conventional amino acid Abbrev. amino acid
Abbrev. .alpha.-aminobutyric acid Abu L-N-methylalanine Nmala
.alpha.-amino-.alpha.-methylbutyrate Mgabu L-N-methylarginine Nmarg
aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate
L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib
L-N-methylcysteine Nmcys aminonorbomyl- Norb L-N-methylglutamine
Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine
Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen
L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp
L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine
Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid
Dglu L-N-methylornithine Nmorn D-histidine Dhis
L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline
Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys
L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan
Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine
Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine
Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine
Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine
Dtyr .alpha.-methyl-aminoisobutyrate Maib D-valine Dval
.alpha.-methyl-.gamma.-aminobutyrate Mgabu D-.alpha.-methylalanine
Dmala .alpha.-methylcyclohexylalanine Mchexa
D-.alpha.-methylarginine Dmarg .alpha.-methylcylcopentylalanine
Mcpen D-.alpha.-methylasparagine Dmasn
.alpha.-methyl-.alpha.-napthylalanine Manap
D-.alpha.-methylaspartate Dmasp .alpha.-methylpenicillamine Mpen
D-.alpha.-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
D-.alpha.-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
D-.alpha.-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
D-.alpha.-methylisoleucine Dmile N-amino-.alpha.-methylbutyrate
Nmaabu D-.alpha.-methylleucine Dmleu .alpha.-napthylalanine Anap
D-.alpha.-methyllysine Dmlys N-benzylglycine Nphe
D-.alpha.-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln
D-.alpha.-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-.alpha.-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu
D-.alpha.-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-.alpha.-methylserine Dmser N-cyclobutylglycine Ncbut
D-.alpha.-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-.alpha.-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-.alpha.-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-.alpha.-methylvaline Dmval N-cylcododecylglycine Ncdod
D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct
D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe
D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg
D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine Nthr
D-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine Nser
D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-.gamma.-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t-butylglycine
Mtbug L-.alpha.-methylcysteine Mcys L-methylethylglycine Metg
L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomophenylalanine
Mhphe L-.alpha.-methylisoleucine Mile N-(2-methylthioethyl)glycine
Nmet L-.alpha.-methylleucine Mleu L-.alpha.-methyllysine Mlys
L-.alpha.-methylmethionine Mmet L-.alpha.-methylnorleucine Mnle
L-.alpha.-methylnorvaline Mnva L-.alpha.-methylornithine Morn
L-.alpha.-methylphenylalanine Mphe L-.alpha.-methylproline Mpro
L-.alpha.-methylserine Mser L-.alpha.-methylthreonine Mthr
L-.alpha.-methyltryptophan Mtrp L-.alpha.-methyltyrosine Mtyr
L-.alpha.-methylvaline Mval L-N-methylhomophenylalanine Nmhphe
N-(N-(2,2-diphenylethyl)carbamylmethyl)glycine Nnbhm
N-(N-(3,3-diphenylpropyl)carbamylmethyl)glycine Nnbhe
1-carboxy-1-(2,2-diphenyl- Nmbc L-O-methyl serine Omser
ethylamino)cyclopropane L-O-methyl homoserine Omhser
[0086] It is to be understood that the invention also encompasses
analogues of the subject ligand which include but are not limited
to the following: [0087] (i) ligands in which one or more amino
acids is replaced by its corresponding D-amino acid. The skilled
person will be aware that such sequences, including retro-inverso
amino acid sequences where substantially all of the amino acids are
D-amino acids and the order is reversed can be synthesised by
standard methods; see for example Chorev and Goodman (1993) Acc.
Chem. Res. 26:266-273; [0088] (ii) peptidomimetic compounds, in
which a peptide bond of the ligand is replaced by a structure more
resistant to metabolic degradation. See for example Olson et al.
(1993) J. Med. Chem. 36:3039-3049; and [0089] (iii) ligands in
which individual amino acids are replaced by analogous structures,
for example, gem-diaminoalkyl groups or alkylmalonyl groups, with
or without modified termini or alkyl, acyl or amine substitutions
to modify their charge.
[0090] It should be understood that the subject ligand may be
glycosylated or unglycosylated and/or may contain a range of other
proteinaceous or non-proteinaceous molecules fused, linked, bound
or otherwise associated to the ligand such as amino acids, lipids,
carbohydrates or other peptides, polypeptides or proteins.
Reference hereinafter to a "ligand" includes a ligand comprising a
sequence of amino acids as well as a ligand associated with other
molecules such as amino acids, lipids, carbohydrates or other
peptides, polypeptides or proteins. For example, the ligand may be
linked to a proteinaceous or non-proteinaceous molecule which can
facilitate steric hindrance of the subject ligand in terms of it
crossing the blood-brain barrier is suitable for use. In one
embodiment, the invention provides a ligand complexed to the Fc
portion of IgG.
[0091] Ligands of the invention can be isolated from natural
sources, be synthetic, or be recombinantly generated polypeptides.
Peptides and proteins can be recombinantly expressed in vitro or in
vivo. The ligands of the invention can be made and isolated using
any method known in the art. The ligands can also be synthesized,
whole or in part, using chemical methods well known in the art. See
e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn
(1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K. (1995)
Therapeutic Peptides and Proteins, Formulation, Processing and
Delivery Systems, Technomic Publishing Co., Lancaster, Pa. For
example, peptide synthesis can be performed using various
solid-phase techniques (see e.g., Roberge (1995) Science 269:202;
Merrifield (1997) Methods Enzymol. 289:3-13) and automated
synthesis may be achieved, e.g., using the ABI 431A Peptide
Synthesizer (Perkin Elmer) in accordance with the instructions
provided by the manufacturer. For example, the CNTF/IL-6 chimeric
ligand of the present invention may be generated by directly
linking or joining via a linker the subject receptor binding
regions.
[0092] The ligand of the invention can also be synthesised and
expressed as a fusion protein with one or more additional domains
linked thereto for, e.g., to more readily isolate a recombinantly
synthesized ligand or to prevent transport of the ligand across the
blood-brain barrier. Detection and purification facilitating
domains include, e.g., metal chelating peptides such as
polyhistidine tracts and histidine-tryptophan modules that allow
purification on immobilized metals, protein A domains that allow
purification on immobilized immunoglobulin, and the domain utilized
in the FLAGS extension/affinity purification system (Immunex Corp,
Seattle Wash.) may also be used. The inclusion of a cleavable
linker sequences such as Factor Xa or enterokinase (Invitrogen, San
Diego Calif.) between a purification domain and the
motif-comprising protein to facilitate purification. For example,
an expression vector can include an epitope-encoding nucleic acid
sequence linked to six histidine residues followed by a thioredoxin
and an enterokinase cleavage site (see e.g., Williams (1995)
Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr. Purif.
12:404-414). The histidine residues facilitate detection and
purification while the enterokinase cleavage site provides a means
for purifying a region from the remainder of the fusion protein.
Technology pertaining to vectors encoding fusion proteins and
application of fusion proteins are well described in the scientific
and patent literature, see e.g., Kroll (1993) DNA Cell. Biol.
12:441-453.
[0093] The method of the present invention is directed to inducing
lipid oxidation in a mammal and thereby providing a means of
preventing, retarding or reversing lipid accumulation. Since lipid
accumulation leads to fat deposition and subsequently to a broad
range of direct or indirect complications including, but not
limited to, obesity, insulin resistance, glucose intolerance,
dyslipidemia, hypertension, osteoarthritis, type II diabetes,
stroke, cardiovascular diseases, the method of the present
invention provides a wide range of potential applications.
[0094] Reference to "lipid" should be understood in its broadest
sense to encompass any member of the group of oils, fats and
fat-like substances which are found in tissue including, for
example, (1) fatty acids; (2) neutral fats (i.e. triacylglycerols),
other fatty-acid esters; (3) long-chain (or fatty) alcohols and
waxes; (4) sphingoids and other long-chain bases; (5) glycolipids,
phospholipids, and sphingolipids; and (6) carotenes, polyprenols,
sterols (and related compounds), terpenes, and other
isoprenoids.
[0095] The term "mammal" as used herein includes humans, primates,
livestock animals (e.g. horses, cattle, sheep, pigs, donkeys),
laboratory test animals (e.g. mice, rats, guinea pigs), companion
animals (e.g. dogs, cats) and captive wild animal (e.g. kangaroos,
deer, foxes). Preferably, the mammal is a human or a laboratory
test animal. Even more preferably, the mammal is a human.
[0096] In a related aspect, it has also been determined that
although the ligand hereinbefore defined increases lipid oxidation,
this ligand is also useful for increasing the insulin sensitivity
of a mammal independently of the induction of changes to lipid
oxidation. Without limiting the present invention to any one theory
or mode of action it is thought that insulin sensitivity is
increased by virtue of the activation of AMP activated protein
kinase.
[0097] Accordingly, a related aspect of the present invention is
directed to a method of increasing insulin sensitivity in a mammal,
said method comprising administering to said mammal a ligand as
hereinbefore defined.
[0098] Reference to "insulin sensitivity" should be understood as a
reference to the functional responsiveness of a mammal or mammalian
tissue to stimulation by insulin. Without limiting the present
invention in any way, insulin stimulates glucose uptake by muscle
and adipose tissue and promotes glycogenesis, lipogenesis,
synthesis of protein and nucleic acid. Accordingly, reference to
"increased" insulin sensitivity should be understood as an
increased level of any one or more of the functional outcomes of
insulin stimulation relative to the levels evident in the mammal
prior to application of the method of the invention. For example,
one may observe increased glucose uptake.
[0099] As detailed hereinbefore, a further aspect of the present
invention relates to the use of the invention in relation to the
treatment or prophylaxis of disease conditions or other unwanted
conditions.
[0100] Accordingly, another aspect of the present invention is
directed to a method of therapeutically or prophylactically
treating a condition in a mammal, which condition is characterised
by unwanted lipid accumulation, said method comprising
administering to said mammal a ligand which binds to the IL-6
receptor and signals via a gp130/LIF receptor heterodimer.
[0101] More particularly, the present invention is directed to a
method of therapeutically or prophylactically treating a condition
in a mammal, which condition is characterised by unwanted lipid
accumulation, said method comprising administering to said mammal a
ligand which binds to the IL-6 receptor and signals via a gp130/LIF
receptor heterodimer, wherein the IL-6 receptor binding site of
said ligand has a greater affinity for the IL-6 receptor than the
CNTF receptor.
[0102] In one embodiment, the IL-6 receptor binding site of said
ligand is substantially similar to the IL-6 receptor binding site
of IL-6.
[0103] In another embodiment, said ligand comprises an IL-6
receptor binding site, a gp130 binding site and a LIF receptor
binding site.
[0104] This aspect of the invention therefore provides a method of
therapeutically or prophylactically treating a condition in a
mammal, which condition is characterised by unwanted lipid
accumulation, said method comprising administering to said mammal a
ligand comprising a binding site substantially similar to the IL-6R
binding site of IL-6, a binding site substantially similar to the
gp130 binding site of IL-6, and a binding site substantially
similar to the LIF receptor binding site of CNTF.
[0105] In yet another embodiment, said ligand comprises sites I and
II of IL-6 or substantially similar sites and site III of CNTF or
substantially similar site.
[0106] Preferably, said ligand is IC7 or a ligand comprising the
SEQ ID NO: 7 amino acid sequence or a substantially similar ligand
or a functional fragment thereof.
[0107] This embodiment therefore provides a method of
therapeutically or prophylactically treating a condition in a
mammal, which condition is characterised by unwanted lipid
accumulation, said method comprising administering to said mammal
IC7, a ligand comprising the SEQ ID NO:7 amino acid sequence or a
substantially similar ligand or functional fragment thereof.
[0108] Reference to a condition "characterised by unwanted lipid
accumulation" should be understood as a reference to any condition
in respect of which lipid accumulation is either a cause or
symptom. To this end, in the context of some conditions, increasing
lipid oxidation may either treat the cause of the disease condition
or at least relieve a symptom associated with the condition.
Examples of conditions characterised by unwanted lipid accumulation
include, but are not limited to obesity, insulin resistance,
glucose intolerance, dyslipidemia, non-alcoholic fatty liver
disease, sleep apnea, obesity associated metabolic disorders such
as osteoarthritis, type II diabetes mellitus, hypertension, stroke
or cardiovascular disease, unwanted weight gain (even where that
weight gain is below the level of obesity) or body mass index and
excessive appetite resulting in unwanted weight gain.
[0109] The terms "obesity" and "obese" generally refer to
individuals whose body weight is at least 20% above the average
body weight for the individual's age, gender and height. An
individual is also defined as "obese" if the individual is a male
whose body mass index is greater than 27.8 kg/m.sup.2 or a female
whose body mass index is greater than 27.3 kg/m.sup.2. Those of
skill in the art will recognize that individuals can be
significantly above the average weight for their age, gender, and
height and still technically not be "obese." Such individuals are
referred to as "overweight" herein, in accordance with normal
usage. This invention will be beneficial for such overweight
individuals, and may also be beneficial to individuals who are
prone to obesity or to being overweight and who wish to avoid a
recurrence of earlier episodes of obesity or being overweight.
[0110] The term "obesity-associated metabolic disorder" means a
disorder which results from, is a consequence of, is exacerbated by
or is secondary to obesity. Non-limiting examples of such a
disorder are osteoarthritis, Type II diabetes mellitus, increased
blood pressure, stroke, and heart disease.
[0111] To this end, it should be understood that although the
method of the present invention has particular application in the
context of obese individuals, this is not a limitation to the
application of the invention. Rather, the method of the invention
can be applied in any situation in which lipid accumulation is
unwanted, such as in the context of an athlete, where the
alteration of overall body mass composition may be sought, or
improving appearance or body image.
[0112] In a related aspect there is provided a method of
therapeutically or prophylactically treating a condition in a
mammal, which condition is characterised by inadequate insulin
sensitivity, said method comprising administering to said mammal a
ligand as hereinbefore defined.
[0113] Reference to a "condition characterised by inadequate
insulin sensitivity" should be understood as a reference to any
condition in which the level of insulin responsiveness of the
mammal is inadequate or otherwise insufficient for its
physiological needs, irrespective of whether said inadequate
insulin sensitivity is a cause or a symptom of said condition.
Examples of such conditions include, but are not limited to
diabetes mellitus, insulin resistance, glucose intolerance,
obesity, dyslipidemia, liver disease, metabolic disorders,
hypertension, cardiovascular disease or stroke.
[0114] These therapeutic and prophylactic aspects of the present
invention are preferably achieved by administering an effective
amount of the ligand, as hereinbefore defined, for a time and under
conditions sufficient to appropriately modulate lipid
oxidation.
[0115] An "effective amount" means an amount necessary to at least
partly to attain the desired response, or to delay the onset or
inhibit progression or halt altogether, the onset or progression of
the particular condition being treated. The amount varies depending
upon the health and physical condition of the individual to be
treated, the taxonomic group of the individual to be treated, the
degree of protection desired, the formulation of the composition,
the assessment of the medical situation, and other relevant
factors. It is expected that the amount will fall in a relatively
broad range that can be determined through routine trials.
[0116] Reference herein to "treatment" and "prophylaxis" is to be
considered in its broadest context. The term "treatment" does not
necessarily imply that a subject is treated until total recovery.
Similarly, "prophylaxis" does not necessarily mean that the subject
will not eventually contract a disease condition. Accordingly,
treatment and prophylaxis include amelioration of the symptoms of a
particular condition or preventing or otherwise reducing the risk
of developing a particular condition. The term "prophylaxis" may be
considered as reducing the severity or onset of a particular
condition. "Treatment" may also reduce or retard the severity or
progression of an existing condition.
[0117] The present invention further contemplates a combination of
therapies, such as the administration of the ligand together with
other proteinaceous or non-proteinaceous molecules which may
facilitate the desired therapeutic or prophylactic outcome. For
example, one may combine the method of the present invention with
appetite suppression therapy, cholesterol medication, insulin
administration or the like.
[0118] Administration of the ligand of the present invention in the
form of a pharmaceutical composition, may be performed by any
convenient means. The ligand is contemplated to exhibit therapeutic
activity when administered in an amount which depends on the
particular case. The variation depends, for example, on the human
or animal. A broad range of doses may be applicable. Considering a
patient, for example, from about 0.1 .mu.g to about 1 mg of ligand
may be administered per kilogram of body weight per day. Dosage
regimes may be adjusted to provide the optimum therapeutic
response. For example, several divided doses may be administered
daily, weekly, monthly or other suitable time intervals or the dose
may be proportionally reduced as indicated by the exigencies of the
situation.
[0119] The ligand may be administered in a convenient manner such
as by the oral, intravenous (where water soluble), respiratory,
transdermal, intraperitoneal, intramuscular, subcutaneous,
intradermal or suppository routes or implanting (e.g. using slow
release molecules). The ligand may be administered in the form of
pharmaceutically acceptable nontoxic salts, such as acid addition
salts or metal complexes, e.g. with zinc, iron or the like (which
are considered as salts for purposes of this application).
Illustrative of such acid addition salts are hydrochloride,
hydrobromide, sulphate, phosphate, maleate, acetate, citrate,
benzoate, succinate, malate, ascorbate, tartrate and the like. If
the ligand is to be administered in tablet form, the tablet may
contain a binder such as tragacanth, corn starch or gelatin; a
disintegrating agent, such as alginic acid; and a lubricant, such
as magnesium stearate.
[0120] Routes of administration include, but are not limited to,
respiratorally, transdermally, intratracheally, nasopharyngeally,
intravenously, intraperitoneally, subcutaneously, intracranially,
intradermally, intramuscularly, intraocularly, intrathecally,
intracerebrally, intranasally, infusion, orally, rectally, via IV
drip, patch and implant.
[0121] In accordance with these methods, the ligand may be
coadministered with one or more other compounds or molecules. By
"coadministered" is meant simultaneous administration in the same
formulation or in two different formulations via the same or
different routes or sequential administration by the same or
different routes. For example, the subject ligand may be
administered together with an agonistic agent in order to enhance
its effects. By "sequential" administration is meant a time
difference of from seconds, minutes, hours or days between the
administration of the two types of molecules. These molecules may
be administered in any order.
[0122] Yet another aspect of the present invention is directed to
the use of a ligand, which ligand binds to the IL-6 receptor and
signals via a gp130/LIF receptor heterodimer, in the manufacture of
a medicament for the treatment of a condition characterised by
unwanted lipid accumulation.
[0123] More particularly, the present invention is directed to the
use of a ligand, which ligand binds to the IL-6 receptor and
signals via a gp130/LIF receptor heterodimer, wherein the IL-6
receptor binding site of said ligand has a greater affinity for the
IL-6 receptor than the CNTF receptor, in the manufacture of a
medicament for the treatment of a condition characterised by
unwanted lipid accumulation.
[0124] In one embodiment, the IL-6 receptor binding site of said
ligand is substantially similar to the IL-6 receptor binding site
of IL-6.
[0125] In another embodiment, said ligand comprises an IL-6
receptor binding site, a gp130 binding site and a LIF receptor
binding site.
[0126] This aspect of the invention therefore provides the use of a
ligand, which ligand comprises a binding site substantially similar
to the IL-6R binding site of IL-6, a binding site substantially
similar to the gp130 binding site of IL-6, and a binding site
substantially similar to the LIF receptor binding site of CNTF, in
the manufacture of a medicament for the treatment of a condition
characterised by unwanted lipid accumulation.
[0127] In yet another embodiment, said ligand comprises sites I and
II of IL-6 or substantially similar sites and site III of CNTF or
substantially similar site.
[0128] Preferably, said ligand is IC7 or a ligand comprising the
SEQ ID NO: 7 amino acid sequence or a substantially similar ligand
or a functional fragment thereof.
[0129] This embodiment therefore provides the use of IC7 or a
ligand comprising the SEQ ID NO: 7 amino acid sequence or a
substantially similar ligand or a functional fragment thereof in
the manufacture of a medicament for the treatment of a condition
characterised by unwanted lipid accumulation.
[0130] Preferably, said condition is obesity, insulin resistance,
glucose intolerance, dyslipidemia, non-alcoholic fatty liver
disease, sleep apnea, obesity associated metabolic disorders such
as osteoarthritis, type II diabetes, mellitus, hypertension, stroke
or cardiovascular disease, unwanted weight gain (even where that
weight gain is below the level of obesity) or body mass index and
excessive appetite resulting in unwanted weight gain.
[0131] Yet another aspect of the present invention is directed to
the use of the ligand as hereinbefore defined in the manufacture of
a medicament for the treatment of a condition characterised by
inadequate insulin sensitivity.
[0132] Preferably, said condition is diabetes mellitus, insulin
resistance, glucose intolerance, obesity, dyslipidemia, liver
disease, metabolic disorders, hypertension, cardiovascular disease
or stroke.
[0133] The ligand of the invention can be combined with a
pharmaceutically acceptable carrier (excipient) to form a
pharmacological composition. Pharmaceutically acceptable carriers
can contain a physiologically acceptable compound that acts to,
e.g., stabilize, or increase or decrease the absorption or
clearance rates of the pharmaceutical compositions of the
invention. Physiologically acceptable compounds can include, e.g.,
carbohydrates, such as glucose, sucrose, or dextrans, antioxidants,
such as ascorbic acid or glutathione, chelating agents, low
molecular weight proteins, compositions that reduce the clearance
or hydrolysis of the peptides or polypeptides, or excipients or
other stabilizers and/or buffers. Detergents can also used to
stabilize or to increase or decrease the absorption of the
pharmaceutical composition, including liposomal carriers.
Pharmaceutically acceptable carriers and formulations for peptides
and polypeptides are known to the skilled artisan and are described
in detail in the scientific and patent literature, see e.g., the
latest edition of Remington's Pharmaceutical Science, Mack
Publishing Company, Easton, Pa. ("Remington's").
[0134] Other physiologically acceptable compounds include wetting
agents, emulsifying agents, dispersing agents or preservatives
which are particularly useful for preventing the growth or action
of microorganisms. Various preservatives are well known and
include, e.g., phenol and ascorbic acid. One skilled in the art
would appreciate that the choice of a pharmaceutically acceptable
carrier including a physiologically acceptable compound depends,
for example, on the route of administration of the peptide or
polypeptide of the invention and on its particular physio-chemical
characteristics.
[0135] In one aspect, a solution of ligand is dissolved in a
pharmaceutically acceptable carrier, e.g., an aqueous carrier if
the composition is water-soluble. Examples of aqueous solutions
that can be used in formulations for enteral, parenteral or
transmucosal drug delivery include, e.g., water, saline, phosphate
buffered saline, Hank's solution, Ringer's solution,
dextrose/saline, glucose solutions and the like. The formulations
can contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions, such as buffering
agents, tonicity adjusting agents, wetting agents, detergents and
the like. Additives can also include additional active ingredients
such as bactericidal agents, or stabilizers. For example, the
solution can contain sodium acetate, sodium lactate, sodium
chloride, potassium chloride, calcium chloride, sorbitan
monolaurate or triethanolamine oleate. These compositions can be
sterilized by conventional, well-known sterilization techniques, or
can be sterile filtered. The resulting aqueous solutions can be
packaged for use as is, or lyophilized, the lyophilized preparation
being combined with a sterile aqueous solution prior to
administration. The concentration of protein in these formulations
can vary widely, and will be selected primarily based on fluid
volumes, viscosities, body weight and the like in accordance with
the particular mode of administration selected and the patient's
needs.
[0136] Solid formulations can be used for enteral (oral)
administration. They can be formulated as, e.g., pills, tablets,
powders or capsules. For solid compositions, conventional nontoxic
solid carriers can be used which include, e.g., pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharin, talcum, cellulose, glucose, sucrose, magnesium
carbonate, and the like. For oral administration, a
pharmaceutically acceptable nontoxic composition is formed by
incorporating any of the normally employed excipients, such as
those carriers previously listed, and generally 10% to 95% of
active ingredient (e.g., peptide). A non-solid formulation can also
be used for enteral administration. The carrier can be selected
from various oils including those of petroleum, animal, vegetable
or synthetic origin, e.g., peanut oil, soybean oil, mineral oil,
sesame oil, and the like. Suitable pharmaceutical excipients
include e.g., starch, cellulose, talc, glucose, lactose, sucrose,
gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate,
sodium stearate, glycerol monostearate, sodium chloride, dried skim
milk, glycerol, propylene glycol, water, ethanol.
[0137] The ligands of the invention, when administered orally, can
be protected from digestion. This can be accomplished either by
complexing the peptide or polypeptide with a composition to render
it resistant to acidic and enzymatic hydrolysis or by packaging the
peptide or polypeptide in an appropriately resistant carrier such
as a liposome. Means of protecting compounds from digestion are
well known in the art, see, e.g., Fix (1996) Pharm Res.
13:1760-1764; Samanen (1996) J. Pharm. Pharmacol. 48:119-135; U.S.
Pat. No. 5,391,377, describing lipid compositions for oral delivery
of therapeutic agents (liposomal delivery is discussed in further
detail, infra).
[0138] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated can be used
in the formulation. Such penetrants are generally known in the art,
and include, e.g., for transmucosal administration, bile salts and
fusidic acid derivatives. In addition, detergents can be used to
facilitate permeation. Transmucosal administration can be through
nasal sprays or using suppositories. See, e.g., Sayani (1996)
Systemic delivery of peptides and proteins across absorptive
mucosae, Crit. Rev. Ther. Drug Carrier Syst. 13:85-184. For
topical, transdermal administration, the agents are formulated into
ointments, creams, salves, powders and gels. Transdermal delivery
systems can also include, e.g., patches.
[0139] The ligands of the invention can also be administered in
sustained delivery or sustained release mechanisms, which can
deliver the formulation internally. For example, biodegradeable
microspheres or capsules or other biodegradeable polymer
configurations capable of sustained delivery of a peptide can be
included in the formulations of the invention (see, e.g., Putney
(1998) Nat. Biotechnol. 16:153-157).
[0140] For inhalation, the ligands of the invention can be
delivered using any system known in the art, including dry powder
aerosols, liquids delivery systems, air jet nebulizers, propellant
systems, and the like. See, e.g., Patton (1998) Biotechniques
16:141-143; product and inhalation delivery systems for polypeptide
macromolecules by, e.g., Dura Pharmaceuticals (San Diego, Calif.),
Aradigm (Hayward, Calif.), Aerogen (Santa Clara, Calif.), Inhale
Therapeutic Systems (San Carlos, Calif.), and the like. For
example, the pharmaceutical formulation can be administered in the
form of an aerosol or mist. For aerosol administration, the
formulation can be supplied in finely divided form along with a
surfactant and propellant. In another aspect, the device for
delivering the formulation to respiratory tissue is an inhaler in
which the formulation vaporizes. Other liquid delivery systems
include, e.g., air jet nebulizers.
[0141] Further, in another embodiment, the ligand may be
administered by intravenous, intraarterial, or intramuscular
injection of a liquid preparation. Suitable liquid formulations
include solutions, suspensions, dispersions, emulsions, oils and
the like. In one embodiment, the pharmaceutical formulations are
administered intravenously, and are thus formulated in a form
suitable for intravenous administration. In another embodiment, the
pharmaceutical formulations are administered intraarterially, and
are thus formulated in a form suitable for intraarterial
administration. In another embodiment, the pharmaceutical
formulations are administered intramuscularly, and are thus
formulated in a form suitable for intramuscular administration.
[0142] Parenteral vehicles (for subcutaneous, intravenous,
intraarterial, or intramuscular injection) include sodium chloride
solution, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's and fixed oils. Intravenous vehicles include fluid and
nutrient replenishers, electrolyte replenishers such as those based
on Ringer's dextrose, and the like. Examples are sterile liquids
such as water and oils, with or without the addition of a
surfactant and other-pharmaceutically acceptable adjuvants. In
general, water, saline, aqueous dextrose and related sugar
solutions, and glycols such as propylene glycols or polyethylene
glycol are preferred liquid carriers, particularly for injectable
solutions. Examples of oils are those of petroleum, animal,
vegetable, or synthetic origin, for example, peanut oil, soybean
oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.
[0143] In addition, the formulations may further comprise binders
(e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar
gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,
povidone), disintegrating agents (e.g. cornstarch, potato starch,
alginic acid, silicon dioxide, croscarmellose sodium, crospovidone,
guar gum, sodium starch glycolate), buffers (e.g., Tris-HCI,
acetate, phosphate) of various pH and ionic strength, additives
such as albumin or gelatin to prevent absorption to surfaces,
detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid
salts), protease inhibitors, surfactants (e.g. sodium lauryl
sulfate), permeation enhancers, solubilizing agents (e.g.,
glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic
acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers
(e.g. hydroxypropyl cellulose, hydroxypropylmethyl cellulose),
viscosity increasing agents (e.g. carbomer, colloidal silicon
dioxide, ethyl cellulose, guar gum), sweetners (e.g. aspartame,
citric acid), preservatives (e.g., Thimerosal, benzyl alcohol,
parabens), lubricants (e.g. stearic acid, magnesium stearate,
polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g.
colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate,
triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl
cellulose, sodium lauryl sulfate), polymer coatings (e.g.,
poloxamers or poloxamines), coating and film forming agents (e.g.
ethyl cellulose, acrylates, polymethacrylates) and/or
adjuvants.
[0144] In preparing pharmaceuticals of the present invention, a
variety of formulation modifications can be used and manipulated to
alter pharmacokinetics and biodistribution. A number of methods for
altering pharmacokinetics and biodistribution are known to one of
ordinary skill in the art. Examples of such methods include
protection of the compositions of the invention in vesicles
composed of substances such as proteins, lipids (for example,
liposomes, see below), carbohydrates, or synthetic polymers
(discussed above). For a general discussion of pharmacokinetics,
see, e.g., Remington's, Chapters 37-39.
[0145] The ligands of the invention can be delivered alone or as
pharmaceutical compositions by any means known in the art, e.g.,
systemically, regionally, or locally (e.g., directly into, or
directed to, a tumor); by intraarterial, intrathecal (IT),
intravenous (IV), parenteral, intra-pleural cavity, topical, oral,
or local administration, as subcutaneous, intra-tracheal (e.g., by
aerosol) or transmucosal (e.g., buccal, bladder, vaginal, uterine,
rectal, nasal mucosa). Actual methods for preparing administrable
compositions will be known or apparent to those skilled in the art
and are described in detail in the scientific and patent
literature, see e.g., Remington's. Parenteral administration is a
preferred route of delivery if a high systemic dosage is needed.
Actual methods for preparing parenterally administrable
compositions will be known or apparent to those skilled in the art
and are described in detail, in e.g., Remington's. See also, Bai
(1997) J. Neuroimmunol. 80:65-75; Warren (1997) J. Neurol. Sci.
152:31-38; Tonegawa (1997) J. Exp. Med. 186:507-515.
[0146] The preparation of pharmaceutical formulations which contain
an active component is well understood in the art, for example by
mixing, granulating, or tablet-forming processes. The active
therapeutic ingredient is often mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient. For oral administration, the ligands or their
physiologically tolerated derivatives such as salts, esters,
N-oxides, and the like are mixed with additives customary for this
purpose, such as vehicles, stabilizers, or inert diluents, and
converted by customary methods into suitable forms for
administration, such as tablets, coated tablets, hard or soft
gelatin capsules, aqueous, alcoholic or oily solutions. For
parenteral administration, the ligands or their physiologically
tolerated derivatives such as salts, esters, N-oxides, and the like
are converted into a solution, suspension, or emulsion, if desired
with the substances customary and suitable for this purpose, for
example, solubilizers or other.
[0147] An active component can be formulated into the formulation
as neutralized pharmaceutically acceptable salt forms.
Pharmaceutically acceptable salts include the acid addition salts
(formed with the free amino groups of the polypeptide or antibody
molecule), which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed from
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the
like.
[0148] For use in medicine, salts of the ligands will be
pharmaceutically acceptable salts. Other salts may, however, be
useful in the preparation of the compounds according to the
invention or of their pharmaceutically acceptable salts. Suitable
pharmaceutically acceptable salts of the compounds of this
invention include acid addition salts which may, for example, be
formed by mixing a solution of the compound according to the
invention with a solution of a pharmaceutically acceptable acid
such as hydrochloric acid, sulphuric acid, methanesulphonic acid,
fumaric acid, maleic acid, succinic acid, acetic acid, benzoic:
acid, oxalic acid, citric acid, tartaric acid, carbonic acid or
phosphoric acid.
[0149] The pharmaceutical compositions of the invention can be
administered in a variety of unit dosage forms depending upon the
method of administration. Dosages for typical modulatory
pharmaceutical compositions are well known to those of skill in the
art. Such dosages are typically advisorial in nature and are
adjusted depending on the particular therapeutic context, patient
tolerance, etc. The amount of ligand adequate to accomplish this is
defined as a "therapeutically effective dose." The dosage schedule
and amounts effective for this use, i.e., the "dosing regimen,"
will depend upon a variety of factors, including the stage of the
disease or condition, the severity of the disease or condition, the
general state of the patient's health, the patient's physical
status, age, pharmaceutical formulation and concentration of active
agent, and the like. In calculating the dosage regimen for a
patient, the mode of administration also is taken into
consideration. The dosage regimen must also take into consideration
the pharmacokinetics, i.e., the pharmaceutical composition's rate
of absorption, bioavailability, metabolism, clearance, and the
like. See, e.g., the latest Remington's; Egleton (1997)
"Bioavailability and transport of peptides and peptide drugs into
the brain" Peptides 18:1431-1439; Langer (1990) Science
249:1527-1533.
[0150] Yet another aspect of the present invention is directed to a
ligand, as hereinbefore defined, which ligand is not IC7.
[0151] In one embodiment, said ligand is complexed to a
proteinaceous or non-proteinaceous molecule such that the complex
is prevented or retarded from crossing the blood brain barrier.
[0152] Still another aspect provides a pharmaceutical composition
comprising a ligand as hereinbefore defined together with a
pharmaceutically acceptable carrier.
[0153] In yet another aspect there is provided a ligand as
hereinbefore defined for use in therapy.
[0154] The present invention is further described by reference to
the following non-limiting examples.
Example 1
[0155] IC-7 was made in accordance with Kallen et. al. (1999)
supra. More specifically IC-7 was developed by substituting the
site III loop of IL-6 with the site III loop of CNTF (Kallen et.
al., (1999) supra.). The site loop is situated on the C-terminal
end of the protein and is the region which binds the either one
gp130R.beta. or the LIFR.beta. (FIG. 3).
Example 2
IC-7 Stimulates Glucose Uptake in Soleus Muscle
[0156] The soleus muscle was dissected tendon to tendon from
anaesthetised C57Bl/6 mice and placed immediately into 2 mL of
pre-gassed (95% O.sub.2, 5% CO.sub.2) Krebs-Henseleit buffer and
incubated for 10 minutes in a 30.degree. C. water bath with
agitation. After 30 minutes of pre-incubation, this buffer was
stimulated with insulin, cytokines or a co-treatment of both and
incubated for a further 30 minutes. Muscles were then placed into 2
mL of Krebs-Henseleit buffer containing .sup.3H-deoxyglucose,
.sup.14C-mannitol and the appropriate stimulus (insulin, cytokine
or both) and incubated for 15 mins. Muscles were washed with
saline, weighed and processed for determination of the uptake of
labelled glucose.
[0157] The soleus muscles of C57Bl/6 mice were assayed for the
effect of insulin, IC-7 and co-treatment of insulin and IC-7 on
glucose uptake. As expected, insulin led to 159% increase in
skeletal muscle glucose uptake (FIG. 3). IC-7 (100 ng/mL)
stimulation led to an equivalent increase in glucose uptake (168%),
while co-treatment had a slight synergistic effect (194%).
IC-7 Increases Fatty Acid Oxidation in Skeletal Muscle
[0158] The soleus and EDL (extensor digitorum longus) muscles were
dissected tendon to tendon from anaesthetised C57Bl/6 mice and
immediately placed into 2 mL of pre-gassed (95% O.sub.2, 5%
CO.sub.2) Krebs-Henseleit buffer and incubated for 30 minutes in a
30.degree. water bath with bioactive and capable of activating the
gp130 receptor in vivo.
Example 3
Acute Effects of IC-7 on Food Intake, Body Mass and Insulin
Sensitivity In Vivo
[0159] The data in Example 2 indicates that IC-7 would be a more
potent gp130 receptor ligand compared with CNTF. This is most
significant because patients in the human trial for Axokine.RTM.
only developed antibodies on high doses of the peptide (Ettinger
et. al. (2003) supra). In previous studies using CNTF, it has been
shown that a subcutaneous injection of 0.3 mg/kg was effective in
activating AMPK and phosphorylating ACC.beta., enhancing fat
oxidation and increasing insulin action. Accordingly, these
experiments are repeated with the addition of 3 doses of IC-7
(0.05, 0.1 and 0.3 mg/kg). Briefly, male C57/Bl6 mice (4 weeks of
age) mice are placed on a high fat diet for 12 weeks. After this
time, conscious mice are injected with the ligands at the
aforementioned doses and skeletal muscle and liver are harvested
after 45 min. Samples are analysed for activation of AMPK, and
phosphorylation of ACC.beta., fat oxidation and insulin signalling
as outlined in our previous paper. This method will enable the
selection of doses of IC-7 for use.
Example 4
Chronic Effects of IC-7 on Food Intake, Body Mass and Insulin
Sensitivity In Vivo
[0160] Male C57/BL6 mice (4 weeks of age) are maintained on either
a chow or high fat diet. Mice are fed ad libitum for 12 weeks so as
to induce obesity and insulin resistance (in the high fat fed
animals). After 11 weeks, animals are injected with CNTF (0.3
mg/kg) or IC-7 or calorically matched and injected with saline
every day for the remaining 1 week of the diet. In addition,
genetically obese mice (ob/ob) (4 weeks of age) are fed a chow diet
for 12 weeks then administered CNTF (0.3 mg/kg), IC-7 or pair fed
and given saline via IP injection every day for 1 week. During the
7 d treatment, body weight, energy turnover and activity patterns
are monitored. After 7 d of treatment with CNTF or IC-7, animals
are agitation. Following this pre-incubation, muscles were
transferred into 2 mL of pre-gassed (95% O.sub.2, 5% CO.sub.2)
Krebs-Henseleit buffer containing .sup.14C-palmitic acid and either
IC-7 or vehicle control and incubated for a further 2 hours.
Muscles were washed in saline, weighed and processed to quantify
any partially oxidized palmitate while the buffer was acidified to
release trapped .sup.14CO.sub.2.
[0161] The soleus and EDL muscles of C57Bl/6 mice were assayed for
the effect of IC-7 on palmitate oxidation. In both soleus and EDL,
100 ng/mL IC-7 led to a significant increase in the total oxidation
of exogenous palmitate (FIG. 5).
[0162] The soleus and EDL muscles of C57Bl/6 mice were assayed to
compare the effects of IC-7 and CNTF on palmitate oxidation. IC-7
(100 ng/mL) significantly increased exogenous palmitate oxidation
in both muscles, while CNTF (100 ng/mL) increased oxidation only in
EDL muscles (n=4) (FIG. 6).
Acute Treatment of IC-7 in C57Bl/6 Mice
[0163] To establish the bioactivity and acute effects of IC-7 in
comparison to CNTF on wildtype, chow fed C57Bl/6 mice, animals were
injected intraperitoneally with vehicle, CNTF (0.3, 0.9 mg/kg), or
IC-7 (0.1, 0.3, 0.9 mg/kg). 45 minutes after administration mice
were anaesthetised and tissues removed. the phosphorylation of
STAT3 was analysed to verify receptor activation.
[0164] C57Bl/6 mice were i.p injected with vehicle, CNTF (0.3, 0.9
mg/kg) or IC-7 (0.2, 0.3, 0.9 mg/kg) and tissues removed after 45
minutes. The phosphorylation of STAT3, a downstream target of gp130
receptor signalling, was determined as a measure of bioactivity of
IC-7 compared to CNTF. In both red gastrocnemius and a soleus
muscle CNTF administration led to a robust increase in pSTAT3 (FIG.
7). IC-7 treatment led to a marked, dose-dependent increase in
pSTAT3 in skeletal muscle. Both CNTF and IC-7 induced a significant
increase in pSTAT3 in liver. In adipose tissue, IC-7 led to a more
robust increase in pSTAT3 in comparison to CNTF. These results
verify dy that IC-7 is sacrificed and fat pads are weighed. Fat
oxidation is measured in skeletal muscle and liver as are
triacylglycerol, diacylglycerol and ceramide in these tissues.
Glucose uptake and insulin signalling are also examined in skeletal
muscle.
[0165] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
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