Note: Descriptions are shown in the official language in which they were submitted.
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USE OF LEPTIN ANTAGOMSTS FOR TIC TREATMIaVVT OF DIABETES
The invention relates to a novel use, in particular a use for the treatment of
diabetes and complications thereof.
Non-insulin-dependent diabetes (NIDDM) is known to be caused by insulin
resistance (particularly in skeletal muscle, adipose tissue and liver) and an
inadequate
insulin secretion from the beta-cells of the Islets of Langerhans in the
pancreas. Thus,
despite hyperinsulinaemia there is insufficient insulin to compensate for the
insulin
resistance and to maintain blood glucose in the desirable range.
Pelleymounter et al (Science, 1995, 269, 540-543) have reported that the ob
polypeptide or "leptin" lowers both plasma insulin and glucose levels in the
genetically
obese oblob mouse.
United Kingdom patent application, Publication Number 2292382 relates inter
alia to polypeptides, OB polypeptides, and antagonists thereof and their use
for
modulating bodyweight. The disclosures of GB 2292382 are incorporated herein
by
reference.
We have now shown that recombinant leptin directly inhibits insulin release
from
both isolated islets and the perfused pancreas of the oblob mouse. An
antagonist of leptin
is therefore indicated to be of value in enhancing insulin secretion and
thereby assisting in
the control of blood glucose levels. We have further shown that leptin
inhibits basal and
insulin-stimulated glycogen synthesis in isolated soleus muscle of oblob mice.
An
antagonist of leptin is therefore also indicated to be of value in enhancing
glucose
utilisation and the action of insulin to enhance glucose utilisation.
Antagonists are
therefore indicated to be of direct use in the treatment of disorders
resulting from
deficiencies in insulin secretion and action and of hyperglycaemia, such as
non-insulin-
dependent diabetes. Moreover, since hyperglycaemia is believed to lead to many
of the
long term complications of diabetes, an antagonist which enhances insulin
secretion and
assists in blood glucose control, may be useful in the treatment of diabetic
complications,
such as retinopathy, nephropathy and angiopathy.
Accordingly, the invention provides the use of an antagonist of leptin for the
treatment of disorders resulting from deficiencies in insulin secretion and of
hyperglycaemia, such as non-insulin-dependent diabetes (NIDDM).
In a further aspect, there is provided the use of an antagonist of leptin for
the
maunfacture of a medicament for the treatment of disorders resulting from
deficiencies in
insulin secretion and of hyperglycaemia, such as non-insulin-dependent
diabetes
(NIDDM).
Suitable antagonists of leptin are as disclosed in GB2292382 and may be
prepared
according to methods disclosed therein.
Particular antagonists include protein antagonists.
Particular antagonists include non-protein antagonists, especially small
organic
molecule antagonists.
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The present invention also extends to a method for the treatment of disorders
resulting from deficiencies in insulin secretion and of hyperglycaemia, such
as non-
insulin-dependent diabetes (NIDDM), in a human or non-human mammal, which
method
45 comprises the administration to human or non-human mammal in need of such
treatment,
an effective, pharmaceutically acceptable, non-toxic amount of an antagonist
of leptin.
The present invention also extends to a pharmaceutical composition comprising
an antagonist of leptin, or a pharmaceutically acceptable derivative thereof,
and a
pharmaceutically acceptable carrier.
50 Particular compositions of the invention are those used for the treatment
of
disorders resulting from deficiencies in insulin secretion and of
hyperglycaemia, such as
non-insulin-dependent diabetes (NIDDM).
Suitable pharmaceutically acceptable Garners are as dictated by conventional
practice such as those disclosed in GB2292382 or in International Patent
Application,
55 Publication Number WO 94/01420.
The compositions of the invention are prepared according to conventional
practice, such as described in the above mentioned patent applications
The dosages of the antagonists may be determined according to conventional
methodology such as those described in the above mentioned patent
applications.
60 The following is a brief description of the figures (Figures 1-S and Table
1) of the
application:
Figure 1 and Table 1 show the effect of Ieptin (100nM) on basal insulin
secretion from
the perfused pancreas of ob mice;
Figure 2 shows the effect of ieptin (100 nmol/1) on glucose stimulated (16.7
mmoUl)
65 insulin secretion from ob/ob islets;
Figure 3 shows the dose - dependency of inhibitory effects of recombinant
leptin on
glucose-stimulated (16.7 mmol/1) insulin secretion from isolated pancreatic
islets of ob/ob
mice;
Figure 4 shows the effect of leptin on glycogen synthesis in isolated soleus
muscle of
70 ob/ob mice; and
Figure 5 shows the effect of leptin on the glycogen synthesis in isolated
soleus muscle of
ob/ob mice.
Lee et al (Nature, 1995, 379, 632-635) have shown that there are a number of
forms of the leptin receptor. If the islet receptors) differs in nature or
proportions to that
75 in other tissues, an especially useful antagonist would be one that
antagonizes the action
of leptin at the islet and/or skeletal muscle receptor, but does not
antagonize other leptin
receptors (e.g. in the hypothalamus) and thereby exacerbate insulin
resistance.
Mutations in the mouse ob gene (J. Hered. 41, 317-318 ) 1950)) and the db gene
(Science 153, 1127-1128 (1966)) result in obesity and non-insulin dependent
diabetes.
80 The ob gene product, leptin, is expressed exclusively in adipose tissue
(Nature (Lond.),
372, 425-432 (1994)) and it has been demonstrated that daily injections of
recombinant
leptin inhibits food intake and reduces body weight and fat-mass in ob/ob mice
(Science
269, 543-546 (1995); Science 269, 540-543 (1995); Science 269, 546-549
(1995)). Such
treatment also results in a reduction in the hyperinsulinaemia (Science 269,
540-543
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85 (1995)). Recently, the gene that codes for the receptor for the ob-gene has
been identified
- (Cell 83, 1263-1271 (1995)). The leptin receptor has several alternatively
spliced
variants. One of these spliced variants is expressed at a high level in
hypothalamus, and
is believed to be the functional receptor in the regulation of energy balance.
It is
abnormally spliced in C57B1/KSJ db/db mice (Nature (Loud.) 379, 622-635
(1996); Cell
90 84, 491-495 (1996); Science 271, 994-996 (1996)) so that the cytoplasmic
region is
missing, leading to defective signal transduction. We now report that, in
addition to
hypothalamus, the predicted functional leptin receptor ob-Rb, is expressed in
several
tissues, including pancreatic islets, in ob/ob mice. Furthermore, recombinant
leptin
inhibits directly insulin release from both isolated islets and the perfused
pancreas of the
95 ob/ob mouse, and it inhibits basal and insulin-stimulated glycogen
synthesis in isolated
soleus muscle of oblob mice, demonstrating that leptin has both central and
peripheral
actions.
The diabetic (db) gene product in mice has been identified as the receptor for
leptin. At least six alternatively spliced forms of the leptin message have
been identified
100 (Nature (Loncl ) 379, 622-635 (1996)). One of these, Ob-Rb, has an
extensive intracellular
region containing a Box 2 sequence motif, which is required for the binding of
JAK
protein kinases and is believed to encode the functional receptor. Short
antisense
oligodeoxynucleotide probes (Trayhurn et al, Biochem. Soc. Traps. Vol 23 page
4945,
1995) 30-34 mers, were designed to hybridise with different domains in the
mouse leptin
i OS receptor mRNA sequence and used to detect either expression of all
transcripts of the
leptin-receptor or to detect solely the leptin receptor long-form, Ob-Rb. Each
oligonucleotide probe sequence was unique to the leptin receptor and had no
significant
homology to any other known sequence.
Expression in lean +/+ mice of total leptin receptor mRNA was detected in
110 hypothalamus, kidneys, lung, liver, whole pancreas, brain, soleus muscle
and spleen but
not in white adipose tissue, pituitary or heart. Total leptin receptor mRNA,
standardised
to poly(A) mRNA levels was over-expressed in ob/ob mice relative to the lean
littermates. However, there were tissue specific differences in
overexpression. Thus,
hypothalamus which shows the highest level of expression in lean mice was only
2-fold
115 over-expressed in ob/ob mice. In kidney, total leptin mRNA was over-
expressed 4-fold
but in some other tissues that had low expression of total leptin mRNA in lean
mice, total
leptin mRNA was over-expressed by up to 10-fold in ob/ob mice.
Northern blot analysis of hypothalamus probed with a 34-mer corresponding to
bases 3329-3363, which are part of the intracellular loop present only in the
long-form of
120 this leptin receptor Ob-Rb, gave a single sharp band. This contrasted with
the multiple
bands when the blot was probed with the 33-mer corresponding to bases 1877-
1910,
which is a sequence common to all the known splice variants of the leptin
receptor.
Molecular weight markers indicated that the 34-mer hybridised to a mRNA
fragment of
approximately 3400 b.p. consistent with the putative functional leptin
receptor. This was
125 found to be expressed at a high level in hypothalamus, in
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agreement with the recent studies of Lee et al (Nature (Loud.) 379, 622-635
(1996)).
- Furthermore, the hypothalami from ob/ob mice showed 2-fold over-expression
relative to
the +/+ mice. The Ob-Rb leptin receptor is also present (and over-expressed
relative to
lean +/+ mice) in liver, kidney and lung but not in whole brain, heart, soleus
muscle or
130 pituitary. The finding of relative over-expression of the Ob-Rb leptin in
ob/ob mice
relative to lean +/+ mice is consistent with the findings of several workers
(Science 269,
540-543 (1995); Proc. Natl, Acad. Sci., USA 93, 1726-1730 (1996)) that
recombinant
leptin is more effective in reducing food intake and body weight in the ob/ob
mice. It
also raises the possibility that leptin regulates the expression of its
receptor. This could
135 result in leptin resistant states and could explain the relative lack of
activity of
recombinant leptin in dietary induced obesity (Science 269, 540-543 (1995)).
Using the slot-blot technique, no expression of the long-form of the leptin
receptor
was detected in whole pancreata from either lean +/+ or ob/ob mice. However, a
strong
signal was obtained using mRNA from ob/ob mouse pancreatic islets.
140 Previous studies have demonstrated that daily intraperitoneal injections
of
recombinant leptin for 28 days produced a dose-dependent significant reduction
in serum
insulin and blood glucose in ob/ob mice but not in lean mice (Science 269, 540-
543
(1995)). Given the high expression of the long-form of the leptin receptor,
which is the
putative functional receptor, in pancreatic islets from ob/ob mice, we decided
to examine
145 the functional response on insulin secretion using the ob/ob mouse
perfused pancreas
(Fig. l , Table 1 ).
Leptin ( 1 OOnM) produced an immediate reduction in the insulin release from
the
isolated pancreas.
The action of leptin in the perfused pancreas preparation could be either
direct on
1 SO islets or via the release of a further mediator from the vasculature. To
clarify this, the
effect of leptin on insulin release from ob/ob mouse isolated pancreatic
islets was
determined. Leptin (100nM) completely inhibited the stimulatory effect of
16.7mM
glucose on insulin release in islets isolated from overnight-fasted mice
(Fig.2). The
inhibitory effect of leptin was dose-related over the range 1-100nM (Fig. 3).
Also leptin
155 (10 nm) inhibited glucose-stimulated insulin secretion by islets from wild-
type mice, but
leptin ( 100 nM) had no effect on insulin secretion by islets from ob/ob mice.
To evaluate the possibility that leptin might directly inhibit glucose uptake
and
insulin action, [14C]-glucose incorporation into glycogen was measured in
isolated intact
soleus muscles, from ob/ob mice, weighing 4-6mg using the method of Challiss
et al.
160 (Biochemical Pharmacology, 1988, 37, 947-950). Recombinant marine leptin
at 100nM
inhibited glycogen synthesis in soleus muscle (Fig 4), with 35% inhibition at
basal (P<
0.01 ), and 28%, 30% and 45% at low insulin concentrations ( 10, 50 and 1
OOuU/ml
respectively, P< 0.05). The maximal response to insulin (10,000uU/ml) was not
significantly affected by leptin. The effects of lower concentrations of
leptin ( 1 and
165 lOnM) were examined in the absence of insulin and in the presence of
100uU/ml insulin.
l OnM leptin caused significant inhibition of both basal and insulin-
stimulated glycogen
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synthesis (32% and 35% respectively; P< 0.05), whereas 1nM leptin did not have
a
significant effect (Fig 5).
Obesity is the commonest nutritional disorder in Western Society and in many
170 developing countries. It is strongly associated with non-insulin dependent
diabetes. The
basis of this association has largely been assumed to relate to the increase
in insulin
resistance that occurs with developing adiposity. Insulin resistance might be
expected to
result in glucose intolerance but it is commonly believed that the development
of non-
insulin dependent diabetes requires the additional independent development of
a
175 pancreatic lesion.
Initial studies using infusion of recombinant leptin to ob/ob mice and lean
littermates suggested that the primary action of leptin was to control
appetite possibly
through a suppression of central NPY release (Nature (Loud.) 337, 530-532
(1995)).
However, a more recent study which included a group of ob/ob mice that
consumed the
180 same amount of food as leptin infused mice, demonstrated that leptin had
significant
metabolic actions (Proc. Natl, Acad. Sci., USA 93, 1726-1730 (1996)). The
infusion of
leptin to ob/ob mice resulted in a significantly lower body weight and fat pad
weight
relative to pair-fed mice. However, the most dramatic difference between the
mice
infused with leptin and the pair-fed animals was in the serum insulin
concentration. Pair-
185 feeding reduced the insulin concentration from 30.6 ~ 6.2 mg/ml to 14.2 t
4.2 mg/ml. In
the mice infused with leptin, the insulin concentration 0.09 ~ 0.08 ng/ml was
not
significantly different from lean animals (Proc. Natl, Acad. Sci., USA 93,
1726-1730
(1996)). The present study demonstrates that the leptin receptor spliced
variant that
encodes the functional receptor is present in pancreatic islets of ob/ob mice,
and that
190 leptin will directly inhibit basal insulin secretion in the perfused
pancreas and glucose
stimulated insulin release by isolated islets of the ob/ob mice. These data
suggest for the
first time that leptin over-production following excess adiposity may directly
modify
insulin secretion and could be involved in the development of the diabetic
syndrome.
These data further suggest that leptin overproduction associated with obesity
may be one
195 of a number of factors responsible for inducing insulin resistance in
obesity.
Accordingly, in a further particular aspect the present invention provides the
use
of an antagonist of leptin for the treatment of insulin resistance, especially
that associated
with obesity.
Further provided is the use of an antagonist of leptin for the maunfacture of
a
200 medicament for the treatment of insulin resistance, especially that
associated with obesity.
Also provided is a method for the treatment of of insulin resistance,
especially that
associated with obesity, in a human or non-human mammal, which method
comprises the
administration to human or non-human mammal in need of such treatment, an
effective,
pharmaceutically acceptable, non-toxic amount of an antagonist of leptin.
205 A further particular pharmaceutical composition of the invention is
therefore a
pharmaceutical composition useful for the treatment of of insulin resistance,
especially
that associated with obesity.
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In addition to pancreatic islets, we have also detected the long-form of the
leptin
receptor in liver, kidney and lung. The functional effects of leptin in these
tissues are at
210 present unknown. However, Levin et al (Proc. Natl, Acad. Sci., USA 93,
1726-1730
(1996)) noted that hepatic glycogen content of liver was significantly reduced
in leptin-
infused ob/ob mice but not in pair-fed animals. Together with the current data
these
findings are suggestive that leptin might directly affect hepatic glycogen
metabolism.
In summary, the present results present both molecular biology and functional
215 evidence for leptin having widespread peripheral metabolic activity as
well as a central
action on food intake.
The disclosures of the above mentioned references including patent applicatons
GB2292382 and WO 94/01420 are incorporated herein by reference.
The following Figures and Table illustrate the invention but do not limit it
in any
220 way.
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Table 1
Control Leptin
AUC 0-l5min 0.20810.022 (4) 0.19010.028 (5)
AUC 16-30min 0.19610.019 (4) 0.12810.019 (5)*
change 96.5111.3 (4) J 69.20.45 (5)*
0-l5min: Stabilization period
I6-30min: treated period (PBS or Leptin)
AUC: area under the curve
Change: the means of 16-30min vs the means of 0-l~min
Results are meanstSEM (n=4.5). *P<0.05 (un-paired test)
_7_
