U.S. patent application number 10/541526 was filed with the patent office on 2006-08-24 for modification of feeding behaviour.
Invention is credited to Stephen Robert Bloom, Catherine Louise Dakin, Mohammad Ali Ghatei, Caroline Jane Small.
Application Number | 20060189522 10/541526 |
Document ID | / |
Family ID | 9950947 |
Filed Date | 2006-08-24 |
United States Patent
Application |
20060189522 |
Kind Code |
A1 |
Bloom; Stephen Robert ; et
al. |
August 24, 2006 |
Modification of feeding behaviour
Abstract
The present invention relates to compositions and methods for
use in the prevention or treatment of excess weight in a mammal.
The compositions comprise oxyntomodulin which is shown to reduce
food intake and/or increase energy expenditure.
Inventors: |
Bloom; Stephen Robert;
(London, GB) ; Ghatei; Mohammad Ali; (London,
GB) ; Small; Caroline Jane; (London, GB) ;
Dakin; Catherine Louise; (London, GB) |
Correspondence
Address: |
PATREA L. PABST;PABST PATENT GROUP LLP
400 COLONY SQUARE
SUITE 1200
ATLANTA
GA
30361
US
|
Family ID: |
9950947 |
Appl. No.: |
10/541526 |
Filed: |
January 12, 2004 |
PCT Filed: |
January 12, 2004 |
PCT NO: |
PCT/GB04/00017 |
371 Date: |
July 7, 2005 |
Current U.S.
Class: |
514/4.9 ;
514/11.7; 514/17.7; 514/5.2 |
Current CPC
Class: |
A61P 3/04 20180101; A61K
38/2271 20130101; A61K 38/26 20130101; A61P 3/06 20180101; A61P
3/00 20180101; A61K 38/22 20130101; A61K 38/22 20130101; A61K
2300/00 20130101; A61K 38/2271 20130101; A61K 2300/00 20130101;
A61K 38/26 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 38/22 20060101
A61K038/22 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 10, 2003 |
GB |
030051.7 |
Claims
1-37. (canceled)
38. A pharmaceutical composition comprising oxyntomodulin and one
or more additional agents which influence weight and/or food
intake.
39. The composition of claim 38, wherein the composition is in a
form suitable for administration via a route peripheral to the
brain.
40. The composition of claim 39, wherein the composition is in a
form suitable for administration by a route selected from the group
consisting of oral, rectal, intravenous, intramuscular,
intraperitoneal, buccal, sublingual, nasal, subcutaneous and
transdermal administration.
41. The composition of claim 38, wherein the one or more additional
agents have one of more effects selected from the group consisting
of reduces food intake, reduces hunger, reduces weight, reduces or
prevents obesity, increases energy expenditure and reduces nutrient
availability in a mammal.
42. The composition of claim 38, wherein the one or more additional
agents are selected from the group consisting of GLP-1 or an
agonist thereof; PYY or an agonist thereof; and a combination of
PYY or an agonist thereof and GLP-1 or an agonist thereof.
43. The composition of claim 42, wherein the composition is in a
form suitable for peripheral administration and wherein the one or
more additional agents are in a dose of 0.1 nmoles per kg body
weight of the subject or more, for example, 0.2 nmoles or more, for
example, 0.4 nmoles or more, for example, 0.6 nmoles or more, for
example, 0.8 nmoles or more, for example, 1.0 nmole or more, for
example, 1.2 nmoles or more, for example, 1.4 nmoles or more, for
example, 1.6 nmoles or more, for example, 1.8 nmoles or more, for
example, 2.0 nmoles or more, for example, 2.2 nmoles or more, for
example, 2.4 nmoles or more, for example, 2.6 nmoles or more, for
example, 2.8 nmoles or more, for example, 3.0 nmoles or more, for
example, up to 3.2 nmoles per kg body weight.
44. The composition of claim 42, wherein the composition is in a
form suitable for peripheral administration, and wherein the one or
more additional agents are in a dose of up to 3.0 nmoles per kg
body weight, for example, up to 2.8 nmoles, for example, up to 2.6
nmoles, for example, up to 2.4 nmoles, for example, up to 2.2
nmoles, for example, up to 2.0 nmoles, for example, up to 1.8
nmoles, for example, up to 1.4 nmoles, for example, up to 1.2
nmoles, for example, up to 1.0 nmoles, for example, up to 0.8
nmoles, for example, up to 0.6 nmoles, for example, up to 0.4
nmoles, for example, up to 0.2 nmoles per kg body weight.
45. The composition of claim 38, wherein the composition is in a
form suitable for peripheral administration, and wherein the
oxyntomodulin is in a dose of for example, 0.1 nmoles or more per
kg body weight of the subject, for example, 0.2 nmoles or more, for
example, 0.5 nmoles or more, for example, 1 nmole or more, for
example, 1.5 nmoles or more, for example, 2 nmole or more, for
example, 2.5 nmoles or more, for example, 3 nmoles or more, for
example, 4 nmoles or more, for example, 5 nmoles or more, for
example, 6 nmoles or more, for example, 7 nmoles or more, for
example, 8 nmoles or more, for example, 9 nmoles or more, for
example, 10 nmoles, for example, 11 nmoles or more, for example, up
to 12 nmoles per kg body weight.
46. The composition of claim 38 in unit dosage form wherein the
oxyntomodulin is in a dose of up to 11 nmoles per kg body weight,
for example, up to 10 nmoles, for example, up to 9 nmoles, for
example, up to 8 nmoles, for example, up to 7 nmoles, for example,
up to 6 nmoles, for example, up to 5 nmoles, for example, up to 4
nmoles, for example, up to 3 nmoles, for example, up to 2 nmoles,
for example, up to 1 nmole, for example, up to 0.5 nmoles, for
example, up to 0.4 nmoles, for example, up to 0.2 nmoles per kg
body weight.
47. The composition of claim 38, wherein the composition is in a
form suitable for subcutaneous administration and wherein the dose
of oxyntomodulin is from 0.5 mg to 2 mg.
48. A method for decreasing calorie intake in a subject, for
decreasing appetite in a subject, for decreasing food intake in a
subject, for increasing energy expenditure in a subject, for weight
control or treatment in a subject, for reduction or prevention of
obesity in a subject; for preventing and reducing weight gain in a
subject; for inducing and promoting weight loss in a subject; a
method for reducing obesity as measured by the Body Mass Index; a
method for controlling of any one or more of appetite, satiety and
hunger in a subject; a method for maintaining desired body weight,
a desired Body Mass Index, and/or a desired appearance and good
health in a subject; a method for improving lipid profile in a
subject; a method for alleviating a condition or disorder in a
subject, which condition or disorder can be alleviated by reducing
nutrient availability and/or by increasing energy expenditure; or a
method for reducing levels of circulating ghrelin in a subject,
which comprises administering oxyntomodulin and one or more
additional agents, which influence weight and/or food intake to the
subject
49. The method of claim 48 for controlling any one or more of
appetite, satiety and hunger in a subject which comprises inducing,
increasing, enhancing or promoting satiety and/or sensations of
satiety in a subject.
50. The method of claim 48 for controlling any one or more of
appetite, satiety and hunger in a subject which comprises reducing,
inhibiting or suppressing hunger or sensations of hunger in a
subject.
51. The method of claim 48, wherein the effect is achieved by
reducing levels of circulating ghrelin.
52. The method of claim 48, wherein the oxyntomodulin is
administered via a route peripheral to the brain.
53. The method of claim 52, wherein the oxyntomodulin is
administered by a route selected from the group consisting of oral,
rectal, intravenous, intramuscular, intraperitoneal, buccal,
sublingual, nasal, subcutaneous and transdermal administration.
54. The method of claim 48, wherein the one or more additional
agents have an effect selected from the group consisting of reduces
food intake and/or reduces hunger, reduces weight, reduces or
prevents obesity, increases energy expenditure and reduces nutrient
availability in a mammal.
55. The method of claim 54, wherein the one or more additional
agents are selected from the group consisting of GLP-1 or an
agonist thereof; PYY or an agonist thereof; and a combination of
PYY or an agonist thereof and GLP-1 or an agonist thereof.
56. The method of claim 55, wherein the one or more additional
agents are administered peripherally at a dose of 0.1 nmoles per kg
body weight of the subject or more, for example, 0.2 nmoles or
more, for example, 0.4 nmoles or more, for example, 0.6 nmoles or
more, for example, 0.8 nmoles or more, for example, 1.0 nmole or
more, for example, 1.2 nmoles or more, for example, 1.4 nmoles or
more, for example, 1.6 nmoles or more, for example, 1.8 nmoles or
more, for example, 2.0 nmoles or more, for example, 2.2 nmoles or
more, for example, 2.4 nmoles or more, for example, 2.6 nmoles or
more, for example, 2.8 nmoles, for example, 3.0 nmoles or more, for
example, up to 3.2 nmoles per kg body weight.
57. The method of claim 55, wherein the one or more additional
agents are administered peripherally in an amount of up to 3.0
nmoles per kg body weight, for example, up to 2.8 nmoles, for
example, up to 2.6 nmoles, for example, up to 2.4 nmoles, for
example, up to 2.2 nmoles, for example, up to 2.0 nmoles, for
example, up to 1.8 nmoles, for example, up to 1.4 nmoles, for
example, up to 1.2 nmoles, for example, up to 1.0 nmole, for
example, up to 0.8 nmoles, for example, up to 0.6 nmoles, for
example, up to 0.4 nmoles, for example, up to 0.2 nmoles per kg
body weight.
58. The method of claim 48, wherein the oxyntomodulin is
administered peripherally at a dose of, for example, 0.1 nmoles or
more per kg body weight of the subject, for example, 0.2 nmoles or
more, for example, 0.5 nmoles or more, for example, 1 nmole or
more, for example, 1.5 nmoles or more, for example, 2 nmole or
more, for example, 2.5 nmoles or more, for example, 3 nmoles or
more, for example, 4 nmoles or more, for example, 5 nmoles or more,
for example, 6 nmoles or more, for example, 7 nmoles or more, for
example, 8 nmoles or more, for example, 9 nmoles or more, for
example, 10 nmoles or more, for example, 11 nmoles or more, for
example, up to 12 nmoles per kg body weight.
59. The method of claim 48, wherein the oxyntomodulin is
administered at a dose of up to 11 nmoles per kg body weight, for
example, up to 10 nmoles, for example, up to 9 nmoles, for example,
up to 8 nmoles, for example, up to 7 nmoles, for example, up to 6
nmoles, for example, up to 5 nmoles, for example, up to 4 nmoles,
for example, up to 3 nmoles, for example, up to 2 nmoles, for
example, up to 1 nmoles, for example, up to 0.5 nmoles, for
example, up to 0.4 nmoles, for example, up to 0.2 nmoles per kg
body weight.
60. The method of claim 50, wherein the oxyntomodulin is
administered at a dose of 0.5 mg to 2 mg before meals.
61. The method of claim 50, wherein the oxyntomodulin and the one
or more additional agents are administered simultaneously, or
sequentially in any order.
Description
INTRODUCTION
[0001] The present invention relates to compositions and methods
for use in weight loss in mammalian animals.
BACKGROUND OF THE INVENTION
[0002] One of the diseases with the highest incidence but which
lacks effective treatment is obesity. It is a debilitating
condition which reduces quality of life and substantially increases
the risk of other diseases.
[0003] In the USA 25% of the adult population is now considered to
be clinically obese. It has been estimated that $45 billion of US
healthcare costs, or 8% per annum of total healthcare spend, is a
direct result of obesity. In Europe the problem is increasing. It
has been predicted that without new approaches over 20% of the UK
population will be clinically obese by 2005. The fact that obesity
is a metabolic disease is being increasingly recognised by the
medical profession and the health authorities. There is, however, a
shortage of effective and safe drugs which can be used in
conjunction with diet and exercise for the long-term management of
obesity.
[0004] It is an object of the present invention to provide such
drugs and also to provide means to identify and develop further
such drugs.
[0005] Preproglucagon is a 160 amino acid polypeptide which is
cleaved in a tissue specific manner by prohormone convertase-1 and
-2 giving rise to a number of products with a variety of functions
in both the central nervous system (CNS) and peripheral tissues. In
the intestine and in the CNS, the major post-translational products
of preproglucagon cleavage are glucagon-like peptide-1 (GLP-1),
glucagon-like peptide-2 (GLP-2), glicentin and oxyntomodulin (OXM),
as shown in Figure A. While GLP-1 and GLP-2 have been shown to
inhibit food intake, no such role has been demonstrated in humans
for the distinct peptide OXM. The importance of OXM as a
biologically active peptide in humans has not been
demonstrated.
SUMMARY OF THE INVENTION
[0006] The present invention is based on our surprising
observations that the OXM peptide can inhibit food intake, reduce
weight and increase energy expenditure in humans, and also that OXM
infusion suppresses fasting plasma ghrelin.
[0007] The present invention provides a method for the prevention
or treatment of excess weight in a mammal, the method comprising
administering a composition comprising OXM to a mammal. The mammal
is likely to be in need of prevention or treatment of excess
weight. The weight loss may be cosmetic. The composition comprising
OXM will be administered in an effective concentration.
[0008] The present invention also provides the following methods of
treatment of a subject: a method for decreasing calorie intake in a
subject, a method for decreasing appetite in a subject, a method
for decreasing food intake in a subject, a method for weight
control or treatment in a subject, a method for reduction or
prevention of obesity, and a method for increasing energy
expenditure; in particular any one or more of the following:
preventing and reducing weight gain; inducing and promoting weight
loss; and reducing obesity as measured by the Body Mass Index. The
methods include control of any one or more of appetite, satiety,
hunger and energy expenditure, in particular any one or more of the
following: reducing, suppressing and inhibiting appetite; inducing,
increasing, enhancing and promoting satiety and sensations of
satiety; and reducing, inhibiting and suppressing hunger and
sensations of hunger; and increasing energy expenditure. The
methods further include maintaining any one or more of a desired
body weight, a desired Body Mass Index, a desired appearance and
good health. In all the above methods OXM is administered to a
subject, generally by a peripheral route of administration.
[0009] The present invention also provides a method for improving
lipid profile in a subject. The method includes administering to
the subject an effective amount of OXM. An improvement in lipid
profile includes, but is not limited to, at least one method of
reducing cholesterol levels, reducing triglyceride levels and
increasing HDL cholesterol levels. OXM can be administered
peripherally, such as in a single or divided dose.
[0010] In another embodiment, a method is disclosed herein for
alleviating a condition or disorder which can be alleviated by
reducing nutrient availability and/or increasing energy
expenditure. The method includes administering to a subject a
therapeutically effective amount of OXM.
[0011] The present invention provides a pharmaceutical composition
comprising OXM and a pharmaceutically suitable carrier, in a form
suitable for oral, rectal, parenteral eg intravenous,
intramuscular, or intraperitoneal, mucosal e.g. buccal, sublingual,
nasal, subcutaneous or transdermal administration, including
administration by inhalation. If in unit dosage form, the dose per
unit may be, for example, as described below or as calculated on
the basis of the per kg doses given below.
[0012] The present invention also includes OXM or an agonist
thereof for use in the manufacture of a medicament for
administration by a route peripheral to the brain for any of the
methods of treatment described above. Examples of peripheral routes
include oral, rectal, parenteral eg intravenous, intramuscular, or
intraperitoneal, mucosal e.g. buccal, sublingual, nasal,
subcutaneous or transdermal administration, including
administration by inhalation. Preferred dose amounts of OXM for the
medicaments are given below.
[0013] The present invention provides a method for cosmetic weight
loss in a mammal, the method comprising administering a composition
comprising OXM to a mammal. In this circumstance, the weight loss
is purely for the purposes of cosmetic appearance.
[0014] The present invention further provides the use, in
combination, of OXM and another agent that has an influence in any
way on weight and/or food intake, for example, an agent that has
any one of more of the following effects: reduces food intake
and/or reduces hunger, reduces weight, reduces or prevents obesity,
increases energy expenditure or reduces nutrient availability in a
mammal, especially a human. The other agent is, for example, GLP-1
or an agonist thereof receptor, or PYY or an agonist thereof, or
another substance that is or is derived from a naturally food
influence substance, for example, amylin, leptin, exendin-4 or
agonists thereof. If desired, more than one other agent may be used
in combination with OXM, for example, GLP-1 or an agonist thereof
and PYY or an agonist thereof may be used. (It will be understood
that a reference to a substance "or an agonist thereof" includes
mixtures of the substances and one or more agonists thereof, and
also mixtures of two or more agonists.)
[0015] In the methods of the invention, OXM is administered in an
amount effective to achieve the desired result, as is any other
agent used in combination with OXM. In each case, the subject,
generally a human, may be overweight and/or may be diabetic.
BRIEF DESCRIPTION OF THE FIGURES
[0016] Figure A is a graphical representation of preproglucagon and
its component parts.
[0017] FIG. 1 is a comparison of the effects of ICV and iPVN
proglucagon-derived and related products on food intake in fasted
rats. FIG. 1A illustrates the cumulative food intake (g) up to 8 h
after ICV injection of GLP-1, OXM, glucagon, or glicentin (all 3
nmol) into fasted animals. *, P<0.05 vs. saline control. FIG. 1B
illustrates cumulative food intake (g) up to 24 h after an acute
iPVN injection of GLP-1, OXM (both 1 nmol), or exendin-4 (0.03
nmol) into fasted animals. *, P<0.01 vs. saline control for all
groups at 1, 2, and 4 h. *, P<0.05 vs. saline control for
exendin-4 only at 8 h.
[0018] FIG. 2 shows two graphs of the effects of ICV and iPVN OXM
on food intake in fasted rats. FIG. 2A, cumulative food intake (g)
up to 8 h after an acute ICV injection of OXM (0.3, 1, 3, or 10
nmol). FIG. 2B, cumulative food intake (g) up to 8 h after an acute
iPVN injection of OXM (0.1, 0.3, or 1.0 nmol) into fasted animals.
*, P<0.05 vs. saline control.
[0019] FIG. 3 shows two bar graphs of the effect of ICV OXM at the
onset of the dark phase. Sated rats received an ICV injection of
OXM, GLP-1 (3 nmol), or saline at the onset of the dark phase. Food
intake (grams; A) and behaviors (3) at 1 h postinjection were
determined. *, P<0/05 vs. saline control.
[0020] FIG. 4 shows two bar graphs of the inhibition of OXM and
GLP-1 effects on food intake by exendin-(9-39). FIG. 4A, food
intake 1 h after an acute ICV injection of GLP-1 (3 nmol), GLP-1
plus exendin-(9-39) (30 nmol), OXM (3 nmol), OXM and exendin-(9-39)
(30 nmol), or exendin-(9-39) alone (30 nmol). FIG. 4B, food intake
after an acute iPVN injection of GLP-1 (1 nmol), GLP-1 and
exendin-(9-39) (10 nmol), OXM (1 nmol), OXM and exendin-(9-39) (10
nmol), or exendin-(9-39) alone (10 nmol) into fasted animals. **,
P<0.005 vs. saline control.
[0021] FIG. 5 is a graph of the competition of [.sup.125I] GLP-1
binding in rat hypothalamic membranes by GLP-1 and OXM.
[0022] FIG. 6 illustrates the effect of a) IP OXM (30, 100 and 300
nmol/kg in 500 .mu.l saline) or saline on cumulative food intake
(g) in 24-hour fasted rats injected during the early dark phase
(closed squares=saline, open circles=OXM 30 nmol/kg, closed
triangles=OXM 100 nmol/kg, open triangles=OXM 300 nmol/kg); and b)
IP OXM (30 and 100 nmol/kg in 500 .mu.l saline) or saline on
cumulative food intake in non-fasted rats injected prior to the
onset of the dark phase (closed squares=saline, open circles=OXM 30
nmol/kg, closed triangles=OXM 100 nmol/kg). *P<0.05 vs.
saline.
[0023] FIG. 7 illustrates the effect of twice daily IP injections
of OXM (50 nmol/kg) or saline for seven days on a) cumulative food
intake (g); and b) body weight gain (g). *P<0.05, **P<0.01,
***P<0.005 vs. saline.
[0024] FIG. 8 illustrates the effect of IP OXM (50 nmol/kg), saline
or a positive control (1 hour=GLP-1 (50 nmol/kg); 2 hours=CCK (15
nmol/kg)) on gastric emptying in 36hour fasted rats. Contents (dry
weight) of the stomach were expressed as a percentage of the food
intake during the 30-minute feeding period. **P<0.01 vs.
saline.
[0025] FIG. 9 illustrates the effect of increasing doses of OXM
(0.01-1.0 nmole) on 1 hour food intake when administered into the
arcuate nucleus of 24-hour fasted rats. *P<0.05, **P<0.01,
***P<0.05 vs. saline.
[0026] FIG. 10 illustrates the effect of iARC administration of
exendin 9-39 (5 nmoles) or saline injected 15 minutes prior to IP
administration of OXM (30 nmol/kg), GLP-1 (30 nmol/kg) or saline on
1 hour food intake (g). (S=saline, G=GLP-1 (30 nmol/kg), Ox=OXM (30
nmol/kg), Ex=exendin 9-39 (5 nmoles)).
[0027] FIG. 11a illustrates the expression of fos-like
immunoreactivity in response to A) IP saline or B) IP OXM (50
nmol/kg) in the arcuate nucleus of the hypothalamus (x40
magnification). ***P<0.005 vs. saline; and
[0028] FIG. 11b illustrates the expression of fos-like
immunoreactivity in response to A) IP saline, B) IP OXM (50
nmol/kg) or C) IP CCK (15 nmol/kg) in the NTS and AP of the
brainstem.
[0029] FIG. 12 shows the protocol of the study of the effect of
intravenous infusion of OXM on food intake in human subject. The
scale represents time (min). Infusion of OXM (3.0 pmol/kg/min) and
saline was from 0-90 minutes. The buffet meal was presented at 75
minutes.
[0030] FIG. 13 shows the calories consumed by the human subject at
the buffet meal. Each line represents the calories consumed by an
individual subject with saline and OXM infusion. The bold line
shows the mean calorie intake for all volunteers. The mean fall in
calories with OXM infusion is 17.6.+-.5.7%.
[0031] FIG. 14 is a visual analogue scale showing the response of
the human subjects to the question `How hungry are you right now?`
There was a significant fall in subjective hunger during OXM
infusion. Hunger scores diminished considerably following the
buffet meal.
[0032] FIG. 15 shows the effect of IP administration of OXM (30
nmoles/kg and 100 nmoles/kg) on fasting plasma ghrelin-IR 30 and 90
minutes post-injection in rats. The solid blocks show the results
with the saline control, the hatched block the results with
OXM.
[0033] FIG. 16 shows energy intake in kJ calories consumed by human
subjects at a buffet meal. Each line represents the energy intake
of an individual subject with saline and with OXM infusion. The
bold line shows the mean calorie intake for all volunteers.
[0034] FIG. 17 shows the energy intake at the buffet meal, and the
cumulative 12 and 24 hour energy intake of human subjects. The
solid blocks show the results with the saline control, the hatched
block the results with OXM.
[0035] FIG. 18 shows the relative hunger scores of the human
subjects during a fasting period and after a meal, with infusion of
OXM or a saline control for the period shown.
[0036] FIG. 19 shows the OXM-like immunoreactivity (OLI) in pmol/L
determined by an RIA during a fasting period and after a meal, with
infusion of OXM or a saline control for the period shown.
[0037] FIG. 20 shows gel permeation analysis of plasma samples
during OXM infusion. The single immunoreactive peak elutes at the
same position as synthetic OXM.
[0038] FIG. 21 shows the change in plasma ghrelin levels during a
fasting period and after a meal, with infusion of OXM or a saline
control for the period shown.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The present invention is based on the surprising observation
that, found that contrary to expectations, the OXM peptide can
inhibit food intake and reduce weight.
[0040] In this text, the term "oxyntomodulin" is the same as "OXM"
and relates to any composition which includes an OXM peptide
sequence or an analogue thereof as follows:
[0041] OXM sequences are well known and documented in the art. The
present invention relates to all of the sequences recited herein
including, in particular, TABLE-US-00001 the OXM human sequence SEQ
ID NO: 1 (which is the same as the reat, hamster and bovine OXM
sequence), as follows: SEQ ID NO: 1 His Ser Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val Gln Trp
Leu Met Asn Thr Lys Arg Asn Lys Asn Asn Ile Ala the OXM angler fish
sequence SEQ ID NO: 2 as follows: SEQ ID NO: 2 His Ser Glu Gly Thr
Phe Ser Asn Asp Tyr Ser Lys Tyr Leu Glu Asp Arg Lys Ala Gln Glu Phe
Val Arg Trp Leu Met Asn Asn Lys Arg Ser Gly Val Ala Glu and the eel
OXM sequence SEQ ID NO: 3 as follows: SEQ ID NO: 3 His Ser Gln Gly
Thr Phe Thr Asn Asp Tyr Ser Lys Tyr Leu Glu Thr Arg Arg Ala Gln Asp
Phe Val Gln Trp Leu Met Asn Ser Lys Arg Ser Gly Gly Pro Thr
[0042] The term OXM used in this text also covers any analogue of
the above OXM sequence, wherein the histidine residue at position 1
is maintained or replaced by an aromatic moiety carrying a positive
charge or a derivative thereof, preferably wherein the moiety is an
amino acid, more preferably wherein it is a histidine derivative,
while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21 or 22 of the other amino acids in the above OXM
sequence can be independently replaced by any other independently
chosen amino acid, with the exception of histidine in position
1.
[0043] Any one or more (to 22) other alpha-amino acid residue in
the sequence can be independently replaced by any other one
alpha-amino acid residue. Preferably, any amino acid residue other
than histidine is replaced with a conservative replacement as well
known in the art i.e. replacing an amino acid with one of a similar
chemical type such as replacing one hydrophobic amino acid with
another.
[0044] As discussed above, 1 to 22 of the amino acids can be
replaced. In addition to the replacement option above, this may be
by a non-essential or modified or isomeric form of an amino acid.
For example, 1 to 22 amino acids can be replaced by an isomeric
form (for example a D-amino acid), or a modified amino acid, for
example a nor-amino acid (such as norleucine or norvaline) or a
non-essential amino acid (such as taurine). Furthermore, 1 to 22
amino acids may be replaced by a corresponding or different amino
acid linked via its side chain (for example gamma-linked glutamic
acid). For each of the replacements discussed above, the histidine
residue at position 1 is unaltered or defined above.
[0045] In addition, 1, 2, 3, 4 or 5 of the amino acid residues can
be removed from the OXM sequence with the exception of histidine at
the 1 position (or as defined above). The deleted residues may be
any 2, 3, 4 or 5 contiguous residues or entirely separate
residues.
[0046] The C-terminus of the OXM sequence may be modified to add
further amino acid residues or other moieties. The OXM above may be
provided as the corresponding salt thereof. Examples of
pharmaceutically acceptable salts of OXM and its analogues include
those derived from organic acids such as methanesulphonic acid,
benzenesulphonic acid and p-toluenesulphonic acid, mineral acids
such as hydrochloric and sulphuric acid and the like, giving
methanesulphonate, benzenesulphonate, p-toluenesulphonate,
hydrochloride and sulphate, and the like, respectively or those
derived from bases such as organic and inorganic bases. Examples of
suitable inorganic bases for the formation of salts of compounds
for this invention include the hydroxides, carbonates, and
bicarbonates of ammonia, lithium, sodium, calcium, potassium,
aluminium, iron, magnesium, zinc and the like. Salts can also be
formed with suitable organic bases. Such bases suitable for the
formation of pharmaceutically acceptable base addition salts with
compounds of the present invention include organic bases which are
nontoxic and strong enough to form salts. Such organic bases are
already well known in the art and may include amino acids such as
arginine and lysine, mono-, di-, or trihydroxyalkylamines such as
mono-, di-, and triethanolamine, choline, mono-, di-, and
trialkylamines, such as methylamine, dimethylamine, and
trimethylamine, guanidine; N-methylglucosamine; N-methylpiperazine;
morpholine; ethylenediamine; N-benzylphenethylamine;
tris(hydroxymethyl) aminomethane; and the like.
[0047] Salts may be prepared in a conventional manner using methods
well known in the art. Acid addition salts of said basic compounds
may be prepared by dissolving the free base compounds in aqueous or
aqueous alcohol solution or other suitable solvents containing the
required acid. Where OXM contains an acidic function a base salt of
said compound may be prepared by reacting said compound with a
suitable base. The acid or base salt may separate directly or can
be obtained by concentrating the solution eg. by evaporation. OXM
may also exist in solvated or hydrated forms.
[0048] The OXM of the present invention may be conjugated to one or
more groups such as a lipid, sugar, protein or polypeptide. The OXM
can be conjugated by being attached to the group (for example via a
covalent or ionic bond) or can be associated therewith. The
conjugated link is preferably not through the C or N terminus amino
acid, when the OXM is attached to the group. The OXM can be
conjugated to a polymer such as polyethylene glycol,
polyvinylpyrrolidone, polyvinylalcohol,
polyoxyethylene-polyoxypropylene copolymers, polysaccharides such
as cellulose, cellulose derivatives, chitosan, acacia gum, karaya
gum, guar gum, xanthan gum, tragacanth, alginic acid, carrageenan,
agarose, and furcellarans, dextran, starch, starch derivatives,
hyaluronic acid, polyesters, polyamides, polyanhydrides, and
polyortho esters.
[0049] The OXM can be chemically modified. In particular, the amino
acid side chains, the N terminus and/or the C acid terminus of OXM
can be modified. For example, the OXM can undergo one or more of
alkylation, disulphide formation, metal complexation, acylation,
esterification, amidation, nitration, treatment with acid,
treatment with base, oxidation or reduction. Methods for carrying
out these processes are well known in the art. In particular the
OXM is provided as a lower alkyl ester, a lower alkyl amide, a
lower dialkyl amide, an acid addition salt, a carboxylate salt or
an alkali addition salt thereof. In particular, the amino or
carboxylic termini of the OXM may be derivatised by for example,
esterification, amidation, acylation, oxidation or reduction. In
particular, the carboxylic terminus of the OXM can be derivatised
to form an amide moiety.
[0050] The OXM can be treated with metals, in particular with
divalent metals. For the purposes of this invention the OXM can
therefore be provided in the presence of one or more of the
following metals, zinc, calcium, magnesium, copper, manganese,
cobalt, molybdenum or iron.
[0051] The OXM can be provided in the form of a pharmaceutical
composition in combination with a pharmaceutically acceptable
carrier or diluent. Suitable carriers and/or diluents are well
known in the art and include pharmaceutical grade starch, mannitol,
lactose, magnesium stearate, sodium saccharin, talcum, cellulose,
glucose, sucrose, (or other sugar), magnesium carbonate, gelatin,
oil, alcohol, detergents, emulsifiers or water (preferably
sterile). The composition may be a mixed preparation of a
composition or may be a combined preparation for simultaneous,
separate or sequential use (including administration). The OXM can
be provided as a crystalline solid, a powder, an aqueous solution,
a suspension or in oil.
[0052] The compositions according to the invention for use in the
aforementioned indications may be administered by any convenient
method, for example by oral, rectal, parenteral eg intravenous,
intramuscular, or intraperitoneal, mucosal e.g. buccal, sublingual,
nasal, subcutaneous or transdermal administration, including
administration by inhalation, and the compositions adapted
accordingly.
[0053] For oral administration, the composition can be formulated
as liquids or solids, for example solutions, syrups, suspensions or
emulsions, tablets, capsules and lozenges.
[0054] A liquid formulation will generally consist of a suspension
or solution of the compound or physiologically acceptable salt in a
suitable aqueous or non-aqueous liquid carrier(s) for example
water, ethanol, glycerine, polyethylene glycol or an oil. The
formulation may also contain a suspending agent, preservative,
flavouring or colouring agent.
[0055] A composition in the form of a tablet can be prepared using
any suitable pharmaceutical carrier(s) routinely used for preparing
solid formulations. Examples of such carriers include magnesium
stearate, starch, lactose, sucrose and microcrystalline
cellulose.
[0056] A composition in the form of a capsule can be prepared using
routine encapsulation procedures. For example, powders, granules or
pellets containing the active ingredient can be prepared using
standard carriers and then filled into a hard gelatin capsule;
alternatively, a dispersion or suspension can be prepared using any
suitable pharmaceutical carrier(s), for example aqueous gums,
celluloses, silicates or oils and the dispersion or suspension then
filled into a soft gelatin capsule.
[0057] Compositions for oral administration may be designed to
protect the active ingredient against degradation as it passes
through the alimentary tract, for example by an outer coating of
the formulation on a tablet or capsule.
[0058] Typical parenteral compositions, including compositions for
subcutaneous administration, comprise a solution or suspension of
the compound or physiologically acceptable salt in a sterile
aqueous or non-aqueous carrier or parenterally acceptable oil, for
example polyethylene glycol, polyvinyl pyrrolidone, lecithin,
arachis oil or sesame oil. Alternatively, the solution can be
lyophilised and then reconstituted with a suitable solvent just
prior to administration.
[0059] Compositions for nasal or oral administration may
conveniently be formulated as aerosols, drops, gels and powders.
Aerosol formulations typically comprise a solution or fine
suspension of the active substance in a physiologically acceptable
aqueous or non-aqueous solvent and are usually presented in single
or multidose quantities in sterile form in a sealed container,
which can take the form of a cartridge or refill for use with an
atomising device. Alternatively the sealed container may be a
unitary dispensing device such as a single dose nasal inhaler or an
aerosol dispenser fitted with a metering valve which is intended
for disposal once the contents of the container have been
exhausted. Where the dosage form comprises an aerosol dispenser, it
will contain a pharmaceutically acceptable propellant. The aerosol
dosage forms can also take the form of a pump-atomiser.
[0060] Compositions suitable for buccal or sublingual
administration include tablets, lozenges and pastilles, wherein the
active ingredient is formulated with a carrier such as sugar and
acacia, tragacanth, or gelatin and glycerin.
[0061] Compositions for rectal or vaginal administration are
conveniently in the form of suppositories (containing a
conventional suppository base such as cocoa butter), pessaries,
vaginal tabs, foams or enemas.
[0062] Compositions suitable for transdermal administration include
ointments, gels, patches and injections including powder
injections.
[0063] Conveniently the composition is in unit dose form such as a
tablet, capsule or ampoule.
[0064] OXM may be administered peripherally at a dose of, for
example, 0.1 nmoles or more per kg body weight of the subject, for
example, 0.2 nmoles or more, for example, 0.5 nmoles or more, for
example, 1 nmole or more, for example, 1.5 nmoles or more, for
example, 2 nmole or more, for example, 2.5 nmoles or more, for
example, 3 nmoles or more, for example, 4 nmoles or more, for
example, 5 nmoles or more, for example, 6 nmoles or more, for
example, 7 nmoles or more, for example, 8 nmoles or more, for
example, 9 nmoles or more, for example, 10 nmoles, for example, 11
nmoles or more, for example, up to 12 nmoles per kg body weight.
The amount used may be up to 11 nmoles per kg body weight, for
example, up to 10 nmoles, for example, up to 9 nmoles, for example,
up to 8 nmoles, for example, up to 7 nmoles, for example, up to 6
nmoles, for example, up to 5 nmoles, for example, up to 4 nmoles,
for example, up to 3 nmoles, for example, up to 2 nmoles, for
example, up to 1 nmoles, for example, up to 0.5 nmoles, for
example, up to 0.4 nmoles, for example, up to 0.2 nmoles per kg
body weight. The dose is generally in the range of from 0.1 to 12
nmoles per kg body weight, for example, within any combination of
upper and lower ranges given above. A dose may be calculated on an
individual basis or on the basis of a typical subject, often a 70
or 75 kg subject. The dose may be administered before each
meal.
[0065] For subcutaneous administration, a dose of OXM within the
range of from 100 nmol to 500 nmol i.e. about 0.5 mg to about 2 mg,
which dose is calculated on the basis of a 75 kg subject, may be
administered, generally before meals.
[0066] A pharmaceutical preparation in unit dosage form for
peripheral administration preferably comprises an amount of OXM
calculated on the basis of the per kg doses given above. Typically,
the dose may be calculated on the basis of a 70 or 75 kg subject. A
composition for subcutaneous administration, for example, may
comprise a unit dose of OXM within the range of from 100 nmol to
500 nmol i.e. about 0.5 mg to about 2 mg, calculated on the basis
of a 75 kg subject.
[0067] The OXM can be used as a prophylaxis to prevent excess
weight gain or can be used as a therapeutic to lose excess
weight.
[0068] The excess weight is typically obesity, although the mammal
will not be certified as clinically obese in order to be suffering
from excess weight. The OXM may be in liquid, solid or semi-solid
form.
[0069] In today's society, the prevention or treatment of excess
weight in a mammal is a real need. Preferably the mammal is a
human, although it may also include other mammalian animals, such
as horses, canine animals (in particular domestic canine animals),
feline animals (in particular domestic feline animals) as well as
mammals which are produced for meat, such as porcine, bovine and
ovine animals. The present invention can be used to prevent excess
weight in such animals in order to maximise lean meat
production.
[0070] Throughout this text, the term "prevention" means any effect
which mitigates any excess weight, to any extent. Throughout this
text, the term "treatment" means amelioration of excess weight, to
any extent.
[0071] Suitable doses of OXM include those that raise the
concentration of OXM significantly above the basal concentration of
OXM, such as, but not limited to, a dose that that mimic
postprandial serum concentrations of OXM. Thus, in one embodiment,
OXM is administered to a reduction in calorie intake, food intake,
or appetite equivalent to the reduction in calorie intake, food
intake, or appetite, or to increase the energy expenditure, caused
by the postprandial level of OXM.
[0072] For all methods disclosed herein, the dose of OXM can be
based on the physiological levels observed post-prandially. A
single dose may be administered per day, or divided doses can be
used (see above).
[0073] It is preferable to administer OXM via a peripheral route of
administration, that is to say, via a route other than directly to
the brain. Examples of such routes include oral, rectal, parenteral
eg intravenous, intramuscular, or intraperitoneal, mucosal e.g.
buccal, sublingual, nasal, subcutaneous or transdermal
administration, including administration by inhalation.
[0074] The present invention provides a pharmaceutical composition
comprising OXM and a pharmaceutically suitable carrier, in a form
suitable for oral, rectal, parenteral eg intravenous,
intramuscular, or intraperitoneal, mucosal e.g. buccal, sublingual,
nasal, subcutaneous or transdermal administration, including
administration by inhalation. If in unit dosage form, the dose may
per unit may be calculated on the basis of the per kg doses given
above.
[0075] The present invention also includes OXM or an agonist
thereof for use in the manufacture of a medicament for
administration by a peripheral route for any of the methods of
treatment described above. Examples of peripheral routes include
oral, rectal, parenteral eg intravenous, intramuscular, or
intraperitoneal, mucosal e.g. buccal, sublingual, nasal,
subcutaneous or transdermal administration, including
administration by inhalation. Preferred dose amounts of OXM for the
medicaments are given above.
[0076] The present invention provides a method for cosmetic weight
loss in a mammal, the method comprising administering a composition
comprising OXM to a mammal. In this circumstance, the weight loss
is purely for the purposes of cosmetic appearance.
[0077] All preferred features given above apply to this aspect of
the invention.
[0078] Without being bound to this theory, it is understood that
the present invention provides the prevention or treatment of
excess weight by the administration of OXM which acts as an
inhibitor to food intake to the mammalian body and/or increases
energy expenditure. Such reduced food intake and/or increased
energy expenditure results in the prevention or treatment of excess
weight in a mammal. In this text the term "food" includes a
substance which is ingested and which has calorific value.
Furthermore, we have found that OXM infusion suppresses fasting
plasma ghrelin. This is an important finding because ghrelin is a
powerfill stimulant of appetite in man and preprandial rises in
plasma ghrelin have been suggested to be a trigger for meal
initiation. Without being bound by the hypothesis, we consider that
inhibition of the normal preprandial rise in ghrelin by OXM is
likely to be one mechanism by which OXM infusion reduces
appetite.
[0079] The present invention further provides the use, in
combination, of OXM and another agent that has an influence in any
way on weight and/or food intake, for example, an agent that has
any one of more of the following effects: reduces food intake
and/or reduces hunger, reduces weight, reduces or prevents obesity,
increases energy expenditure or reduces nutrient availability in a
mammal, especially a human. The other agent is, for example, GLP-1
or an agonist thereof receptor, or PYY or an agonist thereof, or
another substance that is or is derived from a naturally food
influence substance, for example, amylin, leptin, exendin-4 or
agonists thereof. If desired, more than one other agent may be used
in combination with OXM, for example, GLP-1 or an agonist thereof
and PYY or an agonist thereof may be used. (It will be understood
that a reference to a substance "or an agonist thereof" includes
mixtures of the substances and one or more agonists thereof, and
also mixtures of two or more agonists.)
[0080] In one embodiment OXM may be used with GLP-1 or an agonist
thereof. OXM appears to have an arcuate site of action, whereas
GLP-1 acts via the brain stem. The use of the two agents in
combination may give a synergistic effect.
[0081] GLP-1, like OXM, is a post-translational product of
preproglucagon, see Figure A. The initial post-translational
product is GLP-1 (1-37). Human GLP-1 (1-37) has the following amino
acid sequence, SEQ ID NO: 4:
His Asp Glu Phe Glu Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser
Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg Gly
SEQ ID NO: 4
[0082] Further modifications give GLP-1 (1-36) SEQ.ID.NO: 5, and
the amide thereof GLP-1 (1-36) NH.sub.2; GLP-1 (7-37) SEQ.ID.NO:6;
and GLP-1 (7-36) SEQ.ID.NO:7 and the amine thereof, GLP-1 (7-36)
NH.sub.2, which is the most biologically active of the GLP-1
peptides. The term "GLP-1" is used herein to denote any of the
GLP-1 peptides defined above, especially GLP-1 (7-36) NH.sub.2,
also known as GLP-1 (7-36) amide. The terms encompasses GLP-1
peptides of any animal origin, especially the human peptides.
[0083] A GLP-1 agonist is a peptide, small molecule, or chemical
compound that preferentially binds to the GLP-1 receptor and
stimulates the same biological activity as does GLP-1. In one
embodiment, an agonist for the GLP-1 receptor binds to the receptor
with an equal or greater affinity than GLP-1. In another
embodiment, an agonist selectively binds the GLP-1 receptor, as
compared to binding to another receptor. Exendin-4, which is a
39-amino acid peptide isolated from the salivary glands of the Gila
monster (Heloderma suspectum) (Eng J et al J Biol Chem
267:7402-7405, 1992) is an example of an agonist at the GLP-1
receptor. Molecules derived from exendin-4 and that also have GLP-1
agonist activity are further examples of GLP-1 agonists. GLP-1
agonists include GLP-1 related peptides and peptides that result
from natural or synthetic enzymatic or chemical processing of
preproglucagon or of a GLP-1 peptide or a related peptide.
[0084] Any compound that is described as being a GLP-1 agonist may
be used in the present invention, as may any compound that is
tested for GLP-1 agonist activity, for example, as described above,
and found to function as a GLP-1 agonist. A recombinant GLP-1
receptor suitable for use in screening is disclosed in WO93/19175.
Many GLP-1 agonists are known and are described in the art.
Examples of published patent specifications that disclose GLP-1
agonists are the following: WO2002/67918, WO2002/66479,
WO2002/03978, WO2001/89554, WO2001/14386, WO2001/66135,
WO2001/35988, WO2001/14368, WO2001/04156, WO2000/78333,
WO2000/59887, WO2000/42026, EP 0955314, and WO99/43707. Examples of
GLP-1 agonists are Arg34,
Lys26(N-epsilon-(gamma-Glu(N-alpha-hexadecanoyl)))-GLP-1 (7-37),
IP7-GLP-1 (7-37)OH.
[0085] It may be advantageous to use PYY or an agonist thereof with
OXM. PYY has a sustained duration of action, for example, when
administered peripherally, it continues to act after it has been
cleared from the circulating blood, for example, for up to 24 hours
after administration. Accordingly, PYY is effective when two or
even one dose per day is administered. Without being limited by the
following, OXM appears to have an immediate effect, which may not
be sustained for a prolonged period. OXM may be administered
several times per day, for example, before a meal. The use of long
acting PYY with short acting OXM enables "fine tuning" of
administration regimes to the needs of the user.
[0086] PYY is a 36-residue peptide amide isolated originally from
porcoine intestine (Tatemoto et al. Proc. Natl. Acad. Sci. 79:2514,
1982). The term as used herein includes PYY obtained or derived
from any species. Thus, PYY includes the human full length
polypeptide, which has the following sequence, SEQ ID NO: 8:
Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu
Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln
Arg Tyr SEQ ID NO: 8
[0087] and species variations of PYY, including e.g. murine,
hamster, chicken, bovine, rat, and dog. In one embodiment, PYY
agonists do not include NPY. The term PYY as used herein also
includes PYY.sub.3-36. It may be advantageous to use PYY.sub.3-36.
A PYY agonist is any compound which binds to a receptor that
specifically binds PYY, and elicits an effect of PYY. In one
embodiment, a PYY agonist is a compound that affects food intake,
caloric intake, or appetite, and/or which binds specifically in a Y
receptor assay or competes for binding with PYY, such as in a
competitive binding assay with labeled PYY. PYY agonists include,
but are not limited to, compounds that bind to the Y2 receptor.
[0088] PYY agonists and compounds that may be used as PYY agonists
are disclosed in the art. For example, contemplated as useful PYY
agonists are Y2 specific NPY peptide agonists as described in U.S.
Pat. No. 5,026,685; U.S. Pat. No. 5,574,010; U.S. Pat. No.
5,604,203; U.S. Pat. No. 5,696,093; U.S. Pat. No. 6,046,167. There
may also be used variants of PYY and of neuropeptide Y that are
analogous to the variants and modifications of OXM described
above.
[0089] If desired, OXM may be used in with both GLP-1 or an agonist
thereof and PYY or an agonist thereof.
[0090] The use of a combination of any of OXM and GLP-1 or an
agonist thereof and PYY or an agonist thereof may serve to increase
the effectiveness of any of the agents compared with its use alone,
for example, as described above. Alternatively or in addition, use
of the two or three agents in combination may reduce any tendency
for "escape" when using an agent alone. The term "escape" is used
to denote a reduction in effect of an agent with time. For example,
if any one of the agents above has been used alone, its effect may
reduce with time. Use of one or both of the other agents in
addition may reduce or prevent the tendency for that reduction in
effectiveness. For example, PYY has a sustained effect and may be
used for prolonged periods. If the effect of PYY should appear to
reduce, or to reduce or prevent any such reduction in effect, OXM
may be administered in addition to the PYY. GLP-1 may also be used
for the same purpose, with OXM or with OXM and PYY.
[0091] If desired, one or more other agents, such as, but not
limited to, an additional appetite suppressant, may also be
administered. Specific, non-limiting example of an additional
appetite suppressant include amfepramone (diethylpropion),
phentermine, mazindol and phenylpropanolamine, fenfluramine,
dexfenfluramine, and fluoxetine.
[0092] When used in combination with another agent, OXM may be
administered simultaneously or substantially simultaneously as the
other agent, or sequentially, in either order. OXM and the other
agent may be administered in a single pharmaceutical composition or
in separate compositions, and they may be administered by the same
route or by different routes. It is generally more convenient to
administer all the active agents in a single composition. However,
in some cases it may be necessary or appropriate to administer the
active agents by different routes. For example, peptides are
generally not stable on oral administration unless modified or
formulated in a special way, so must generally be administered via
a non-oral route. Some agonists, for example, GLP-1 agonists, are
chemical compounds that are stable when administered orally. It may
be appropriate to administer OXM non-orally and the other component
by a non-oral route.
[0093] According to a preferred aspect of the invention, a
therapeutically effective amount of OXM or an agonist thereof is
administered with a therapeutically effective amount of GLP-1 or an
agonist thereof and/or PYY or an agonist thereof. The term
"GLP-1/PYY" is used herein to denote GLP-1 or an agonist thereof
and/or PYY or an agonist thereof.
[0094] The OXM or agonist thereof and the GLP-1/PYY may be
administered simultaneously or substantially simultaneously, or
sequentially, in any order. The OXM or agonist thereof and the
GLP-1/PYY may be administered in a single pharmaceutical
composition or in separate compositions, and they may be
administered by the same route or my different routes.
[0095] If the OXM and the GLP-1/PYY are to be administered in a
single pharmaceutical composition, that composition may be any of
those described above for OXM or an agonist thereof. The
composition may enable simultaneous or substantially simultaneous
administration of the OXM or agonist thereof and the GLP-1/PYY. If
desired, the OXM or agonist thereof and the GLP-1/PYY may be
compartmentalized in the composition, for example, in different
layers of a tablet, or in different granules in a capsule. If
desired, such compartmentalization may be designed to give
different release properties to the components to enable delivery
of the OXM or agonist component and the GLP-1/PYY at different
times, for example, sequentially.
[0096] Alternatively, the OXM or agonist thereof and the GLP-1/PYY
may be formulated in separate pharmaceutical compositions, for
example, any of the pharmaceutical compositions described above for
OXM and agonists thereof. Such separate compositions may be
administered simultaneously or substantially simultaneously, or
they may be administered sequentially, in any order. For example,
PYY may be administered two times or even once per day, with OXM
being administered up to several times per day, for example, before
meals.
[0097] If administered separately, whether sequentially or
simultaneously (or substantially simultaneously), the OXM or
agonist thereof and the GLP-1/PYY may be administered by the same
route or by different routes, for example, as described above.
[0098] When used in combination therapy as described above, OXM may
be used in a dose as disclosed above in relation to peripheral
administration when used alone, that is to say, OXM may be
administered peripherally at a dose of, for example, 0.1 nmoles or
more per kg body weight of the subject, for example, 0.2 nmoles or
more, for example, 0.5 nmoles or more, for example, 1 nmole or
more, for example, 1.5 nmoles or more, for example, 2 nmole or
more, for example, 2.5 nmoles or more, for example, 3 nmoles or
more, for example, 4 nmoles or more, for example, 5 nmoles or more,
for example, 6 nmoles or more, for example, 7 nmoles or more, for
example, 8 nmoles or more, for example, 9 nmoles or more, for
example, 10 nmoles, for example, 11 nmoles or more, for example, up
to 12 nmoles per kg body weight. The amount used may be up to 11
nmoles per kg body weight, for example, up to 10 nmoles, for
example, up to 9 nmoles, for example, up to 8 nmoles, for example,
up to 7 nmoles, for example, up to 6 nmoles, for example, up to 5
nmoles, for example, up to 4 nmoles, for example, up to 3 nmoles,
for example, up to 2 nmoles, for example, up to 1 nmoles, for
example, up to 0.5 nmoles, for example, up to 0.4 nmoles, for
example, up to 0.2 nmoles per kg body weight. The dose is generally
in the range of from 0.1 to 12 nmoles per kg body weight, for
example, within any combination of upper and lower ranges given
above.
[0099] GLP-1 or an agonist thereof may be administered peripherally
at a dose of, for example, 0.1 nmoles or more per kg body weight of
the subject, for example, 0.2 nmoles or more, for example, 0.4
nmoles or more, for example, 0.6 nmoles or more, for example, 0.8
nmoles or more, for example, 1.0 nmole or more, for example, 1.2
nmoles or more, for example, 1.4 nmoles or more, for example, 1.6
nmoles or more, for example, 1.8 nmoles or more, for example, 2.0
nmoles or more, for example, 2.2 nmoles or more, for example, 2.4
nmoles or more, for example, 2.6 nmoles or more, for example, 2.8
nmoles, for example, 3.0 nmoles or more, for example, up to 3.2
nmoles per kg body weight. The amount used may be up to 3.0 nmoles
per kg body weight, for example, up to 2.8 nmoles, for example, up
to 2.6 nmoles, for example, up to 2.4 nmoles, for example, up to
2.2 nmoles, for example, up to 2.0 nmoles, for example, up to 1.8
nmoles, for example, up to 1.4 nmoles, for example, up to 1.2
nmoles, for example, up to 1.0 nmoles, for example, up to 0.8
nmoles, for example, up to 0.6 nmoles, for example, up to 0.4
nmoles, for example, up to 0.2 nmoles per kg body weight. The dose
is generally in the range of from 0.1 to 3.2 nmoles per kg body
weight, for example, within any combination of upper and lower
ranges given above.
[0100] PYY or an agonist thereof may be used at a dose within the
ranges disclosed above for GLP-1. The doses of the various agent
may be independent of each other or, for example, equimolar doses
may be used, for example, equimolar doses of GLP-1 or an agonist
thereof and PYY or an agonist thereof. A dose may be calculated on
an individual basis or on the basis of a typical subject, often a
70 or 75 kg subject.
[0101] A further embodiment of the present invention is a
pharmaceutical composition comprising oxyntomodulin and one or more
other agents having an influence in any way on weight and/or food
intake, for example, an agent that has any one of more of the
following effects: reduces food intake and/or reduces hunger,
reduces weight, reduces or prevents obesity, increases energy
expenditure or reduces nutrient availability in a mammal,
especially a human, in admixture or conjunction with a
pharmaceutically suitable carrier. The agents are as defined above
and are, for example, GLP-1 or an agonist and/or PYY agonist
thereof. The compositions may be, for example, as described above
for OXM pharmaceutical compositions. Doses of the OXM and other
agents are, for example, as described above.
[0102] A pharmaceutical preparation in unit dosage form for
peripheral administration preferably comprises an amount of OXM
calculated on the basis of the per kg doses given above. Typically,
the dose may be calculated on the basis of a 75 kg subject. A
composition for subcutaneous administration, for example, may
comprise a unit dose of OXM within the range of from 100 nmol to
500 nmol i.e. about 0.5 mg to about 2 mg, calculated on the basis
of a 75 kg subject.
[0103] The present invention also provides the use of OXM in the
manufacture of a medicament for the treatment of a subject
according to any of the methods disclosed above.
[0104] When OXM and another agent that reduces food intake, for
example, PYY or an agonist thereof and/or GLP-1 or an agonist
thereof are used in the manufacture of a medicament for use in a
treatment as described herein, the medicament may be a single
pharmaceutical composition comprising all the components, as
described above, or may be a two or more component medicament, one
component being a pharmaceutical composition comprising OXM, the
other component(s) each being a pharmaceutical composition
comprising the other agent(s) that reduce food intake, see
above.
[0105] The medicament, whether a one component medicament or a two
or more component medicament as described above, will generally be
packaged with instructions relating to its use. Such instructions
will refer to the timing, dose and route of administration of the
component(s).
[0106] The preferred features above relating to methods and
compositions relating to OXM when used in combination with other
agent also applies to its use in the manufacture of a medicament as
described above.
[0107] In all embodiments of the invention, the particular dosage
regime for which will ultimately be determined by the attending
physician and will take into consideration such factors as the OXM
being used, animal type, age, weight, severity of symptoms and/or
severity of treatment to be applied, method of administration of
the medicament, adverse reaction and/or contra indications.
Specific defined dosage ranges can be determined by standard
designed clinical trials with patient progress and recovery being
fully monitored.
[0108] Such trials may use an escalating dose design using a low
percentage of the maximum tolerated dose in animals as the starting
dose in man. Examples of suitable doses are given above.
[0109] Preferred features of each aspect of the invention are as
for each of the other aspects mutatis mutandis.
[0110] The present invention is now described by way of example
only in the following non-limiting Examples.
EXAMPLES
Example 1
OXM Causes a Potent Decrease in Fasting-Induced Refeeding when
Injected Both ICV and iPVN
Peptides and Chemicals
[0111] GLP-1, glicentin, glucagon, and SP-1 were purchased from
Peninsula Laboratories, Inc. (St. Helens, UK). OXM was purchased
from IAF BioChem Pharma (Laval, Canada). Exendin-4 and
exendin-(9-39) were synthesised at Medical Research Council,
Hemostasis Unit, Clinical Sciences Center, Hammersmith Hospital,
London, UK using F-moc chemistry on an 396 MPS peptide synthesiser
(Advanced ChemTech, Inc.) and purified by reverse phase HPLC on a
C.sub.8 column (Phenomex, Macclesfield, UK). The correct molecular
weight was confirmed by mass spectrometry. All chemicals were
purchases from Merck & Co. (Lutterworth, Leicester, UK) unless
otherwise stated.
Animals
[0112] Adult male Wistar rats (ICSM, Hammersmith Hospital) were
maintained in individual cages under controlled conditions of
temperature (21-23.degree. C.) and light (12 h of light, 12 h of
darkness) with ad libitum access to food (RM1 diet, Special Diet
Services UK Ltd., Witham, UK) and tap water. Animals were handled
daily after recovery from surgery until completion of the studies.
All animal procedures undertaken were approved by the British Home
Office Animals (Scientific Procedures Act 1986 (Project License PIL
90/1077).
ICV and iPVN Cannulation and Infusions of Test Compounds
[0113] Animals had permanent stainless steel guide cannulas
(Plastics One, Roanoke, Va.) stereotactically implanted ICV
(intracerebraventricularly) or iPVN (into the hypothalamic
paraventricular nucleus). All studies were carried out in the early
light phase, between 0900-1100 h, after a 24-h fast, and food
intake was measured 1, 2, 4, 8, and 24 h postinjection.
Feeding Study Protocols
[0114] Comparison of the effect of proglucagon-derived products and
related peptides on food intake.
[0115] In study 1a, rats were injected ICV with 10 .mu.l saline,
GLP-1 (13 nmol), OXM (3 nmol), glucagon (3 nmol), or glicentin (3
nmol; n=8/group).
[0116] In all studies, TABLE-US-00002 the human OXM with the
following sequence, SEQ ID NO: 1, was used: SEQ ID NO: 1 His Ser
Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser Arg Arg Ala
Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn Lys Asn Asn Ile
Ala Human GLP-1 with the following sequence, SEQ ID NO: 7, was
used: SEQ ID NO:7 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly
Arg,
[0117] In study 1b, rats were injected iPVN with 1.mu. saline,
GLP-1 (1.0 nmol), OXM (1.0 nmol), glicentin (1.0 nmol), glucagon
(1.0 nmol), or SP-1 (3.0 nmol; n=12-15/group). Exendin-4, when
injected ICV, inhibits food intake more potently than GLP-1.
Therefore, exendin-4 was injected iPVN at a dose of 0.03 nmol.
Investigation of the Effect of Increasing Doses of OXM on Food
Intake
[0118] In study 2a, rats were injected ICV with saline, GLP-1 (3
nmol), or OXM (0.3, 1, 3 or 10 nmol; n=8/group). In study 2b, rats
were injected iPVN with saline, GLP-1 (1.0 nmol), or OXM (0.1, 0.3,
or 1.0 nmol; n-12-15/group). To assess whether OXM acts via the
GLP-1 receptor, a study using the GLP-1 receptor antagonist
exendin-(9-39) was performed.
Night Time Feeding and Behavioural Analysis.
[0119] Study 3. It is possible that OXM inhibits food intake via
nonspecific taste aversion, and that it is not a true satiety
factor. Therefore, ICV cannulated rats were administered GLP-1 (3
nmol), OXM (3 nmol), or saline (n=6/group) at the onset of the dark
phase. Food intake was measured 1 h postinjection (study 3a), and
behaviour was assessed (study 3b). Rats were observed for 1 h
postinjection using a behavioural score sheet.
[0120] In study 4a, rats were injected with ICV with saline, GLP-1
(3 nmol), GLP-1 (3 nmol) plus exendin-(9-39) (30 nmol), OXM (3
nmol), OXM (3 nmol) plus exendin-(9-39) (30 nmol), or
exendin-(9-39) alone (30 nmol). In study 4b, rats were iPVN
injected with saline, GLP-1 (1 nmol), GLP-1 (1 nmol) plus
exendin-(9-39) (10 nmol), OXM (1 nmol), OXM (1 nmol) plus
exendin-(9-39) (10 nmol), or exendin-(9-39) alone (10 nmol;
n=10-12/group).
Receptor Binding Assays. Study 5.
[0121] Receptor binding assays were performed in a final volume of
0.5 ml rat hypothalamic membranes (200 .mu.g protein), 500 Bq (100
pM) [.sup.125I]GLP-1, and unlabeled competing peptides (GLP-1 and
OXM) as specified. Membranes were incubated at room temperature for
90 min. Bound and free radioactivity were separated by
centrifugation (2 min, 4.degree. C.). Pelleted membranes were
washed with assay buffer (0.5 ml, ice-cold), and the membranes were
centrifuged as described above. The supernatant was removed, and
the radioactivity in the pellet was counted using a
.gamma.-counter. Specific (saturable) binding was calculated as the
difference between the amount of [.sup.125I]GLP-1 bound in the
absence (total binding) and presence of 1 .mu.m GLP-1 or OXM
(nonsaturable binding). All curves were constructed with points in
triplicate. IC.sub.50 values were calculated using the Prism 3
program (GraphPad Software, Inc., San Diego, Calif.).
Statistics
[0122] For food intake analyses, data are presented as the
mean.+-.SEM. Statistical differences between experimental groups
were determined by ANOVA, followed by a post-hoc least significant
difference test (Systat 8.0, Evanston, Ill.). For behavioural
analyses, data are expressed as the median number of occurrences of
each behaviour and the range. Comparisons between groups were made
using the Mann-Whitney U test (Systat 8.0). In all cases, P<0.05
was considered statistically significant.
Results
[0123] Comparison of the effects of proglucagon-derived products
and related peptides on food intake
ICV Administration.
[0124] In study 1a, OXM and GLP-1 (3 nmol) significantly reduced
refeeding. This inhibition of food intake lasted until 4 h
postinjection (FIG. 1A). Glucagon and glicentin (3 nmol) failed to
affect food intake at any time point (FIG. 1A).
iPVN Administration.
[0125] In study 1b, OXM, GLP-1 (3 nmol) and exendin-4 (0.03 nmol)
also inhibited refeeding when injected iPVN. This inhibition lasted
at least 8 h postinjection, longer than when injected ICV (FIG.
1B). Glicentin, glucagon (1 nmol), and SP-1 (3 nmol) failed to
affect food intake at any time point when injected iPVN.
Effects of Increasing Doses of OXM on Food Intake
ICV Administration.
[0126] In study 2a, when injected ICV, OXM reduced refeeding in a
dose-dependent manner, reaching a maximal effect at a dose of 3
nmol 1, 2, and 4 h postinjection (FIG. 2A).
iPVN Administration.
[0127] In study 2b, food intake was significantly reduced by
iPVN-injected GLP-1 and OXM (both 1 nmol) until 8 h postinjection
(FIG. 2B).
Effect of OXM in ICV-Cannulated Sated Rats at the Onset of the Dark
Phase.
[0128] The dark phase is the rats' natural feeding time. Therefore,
assessing the effect of a putative satiety factor in non-fasted
animals at this time would represent a more physiological
effect.
Effect of OXM on Food Intake.
[0129] In study 3a, when injected in the early dark phase, both
GLP-1 and OXM (3 nmol) significantly reduced food intake compared
with that of saline-treated animals 1 h postinjection [FIG.
3A].
Observation of Behaviour After ICV Injection of OXM.
[0130] ICV administration of OXM (3 nmol) in the early dark phase
led to a significant decrease in feeding episodes (study 3a) and an
increase in rearing behaviour (study 3b) [FIG. 3B]. There was no
change in grooming, still, head down, burrowing, or locomotion
episodes.
[0131] To assess whether OXM acts via the GLP-1R, a study using the
GLP-1R antagonist, exendin-(9-39) was performed.
ICV Administration. Study 4.
[0132] ICV coadministration of the GLP-1 receptor antagonist
exendin-(9-39) with GLP-1 at a ratio of 10:1 (antagonist/agonist)
blocked the anorectic effects of GLP-1 [FIG. 4A]. Furthermore,
coadministration of exendin-(9-39) with OXM resulted in attenuation
of the anorectic effect of OXM [FIG. 4A].
iPVN Administration.
[0133] Similarly, when injected iPVN, the anorectic effects of both
GLP-1 and OXM were blocked when coinjected with exendin-(9-39)
[FIG. 4B].
Receptor Binding Assays. Study 5.
[0134] The affinity (IC.sub.50) of GLP-1 for the GLP-receptor in
rat hypothalamic membrane preparations was 0.16 nM (FIG. 5). The
affinity of OXM for the GLP-1 receptor in the same membrane
preparations was 8.2 nM (FIG. 5), which is approximately 2 orders
of magnitude weaker than that of GLP-1.
Discussion.
[0135] OXM causes a potent decrease in fasting-induced refeeding
when injected both ICV and iPVN. The effect was sustained until 8 h
(iPVN) or 4 h (ICV) postinjection. The effect of OXM is
approximately of the same magnitude and time course as that of
GLP-1 when administered ICV and iPVN at equimolar doses. In
addition, OXM inhibits food intake in nonfasted rats at the onset
of the dark phase, and at that time they showed no signs of
aversive behaviour.
[0136] It has been suggested that there is an OXM-specific binding
site in gastric mucosa. However, no such binding site has been
identified in the CNS. Therefore, it was proposed that OXM mediated
its effects via the hypothalamic GLP-IR, as GLP-1 and OXM have
similar potency in feeding studies. It has been shown that OXM has
a nanomolar affinity for the GLP-IR (IC.sub.50=8.2 nM). This
affinity is approximately 2 orders of magnitude weaker than that of
GLP-1 (IC.sub.50=0.16 nM). Yet despite this reduced affinity for
the GLP-1R, OXM reduces food intake to the same magnitude. One
explanation for this is that OXM might act through both the GLP-1R
and its own receptor in the hypothalamus. Thus, OXM could elicit a
response comparable to that of GLP-1 despite its lower affinity for
the GLP-IR.
[0137] Exendin-(9-39), a fragment of the GLP-1R agonist exendin-4,
is a potent and selective antagonist at the GLP-1R. When GLP-1 and
exendin-(9-39) are coinjected, the anorectic actions of GLP-1 are
blocked. When OXM is coinjected with exendin-(9-39), the anorectic
effects of OXM are also completely blocked. This would strengthen
the argument that OXM is mediating its effects via the GLP-1R.
[0138] We investigated the effects of glicentin, and glucagon after
an acute ICV injection in fasted rats. No effect on fasting-induced
food intake was seen after the administration of these peptides. In
addition, there was no effect of these peptides when they were
administered iPVN. When SP-1, the putative minimal active structure
of OXM, was injected iPVN, no inhibition of food intake was
observed. Therefore the effect seen by OXM is specific.
Example 2
Peripheral Administration of OXM Also Reduces Food Intake and Body
Weight Gain.
Peptides and Chemicals
[0139] OXM was purchased from IAF BioChem Pharma (Laval, Canada).
GLP-1 was purchased from Peninsula Laboratories Inc. (St. Helens,
UK). Exendin 9-39 was synthesised at Medical Research Council,
Hemostasis Unit, Clinical Sciences Centre, Hammersmith Hospital,
London, UK using F-moc chemistry on a 396 MPS peptide synthesizer
(Advanced ChemTech Inc., Louisville, Ky.) and purified by reverse
phase HPLC on a C.sub.8 column (Phenomex, Macclesfield, UK), using
a gradient of acetonitrile on 0.1% trifluoroacetic acid. Correct
molecular weight was confirmed by mass spectrometry. All chemicals
were purchases from Merck Eurolab Ltd. (Lutterworth,
Leicestershire, UK), unless otherwise stated.
Animals
[0140] Adult male Wistar rats (180-200 g) were maintained in
individual cages under controlled conditions of temperature
(21-23.degree. C.) and light (12 hours light, 12 hours dark) with
ad libitum access to standard rat chow (RM1 diet, Special Diet
Services UK Ltd., Witham, Essex, UK) and water. All procedures
undertaken were approved by the British Home Office Animals
(Scientific Procedures) Act 1986 (Project Licenses PPL: 90/1077,
70/5281 and 70/5516).
Intra-Arcuate Nucleus Cannulation
[0141] Animals had permanent indwelling, unilateral, stainless
steel guide cannulae (Plastics One, Roanoke, Va.) stereotactically
implanted into the arcuate nucleus of the hypothalamus, using a
cannulation protocol using cannulae positioned 3.3 mm posterior to
and 0.3 mm lateral to bregma and 9.0 mm below the outer surface of
the skull.
Intra-Peritoneal (IP) Injections
[0142] All IP injections were delivered using a 1 ml syringe and a
25 gauge needle. The maximum volume of injection was 500 .mu.l, and
was adjusted according the weight of the individual animal. All
peptides were dissolved in saline.
[0143] In these studies, the human OXM and human GLP-1 were used
with the sequences provided on pages 15 and 16 above.
In Vivo Protocols
1. Investigating the Dose-Response Effect of Peripheral
Administration of OXM on Food Intake in Fasted Animals:
[0144] Animals were fasted for 24 hours prior to the study. During
the early light phase (09.00-10.00 hr), rats were given a single IP
injection of saline, GLP-1 (30 nmol/kg body weight as a positive
control) or OXM (10-300 nmol/kg body weight) (n=12 per group) in a
volume of 500 .mu.l. Following the injection, the animals were
returned to their home cages and provided with a pre-weighed amount
of chow. Food intake was measured 1, 2, 4, 8 and 24 hours
post-injection.
2. Investigating the Effect of Peripheral Administration of OXM on
Food Intake in Non-Fasted Animals During the Dark Phase:
[0145] The dark phase is the "normal" feeding time for rats.
Therefore, any inhibition of food intake at this time could be
considered to be more physiological than alterations to refeeding
following a fast. Animals received a single IP injection of saline
or OXM (3-100 nmol/kg body weight) (n=12 per group) prior to lights
out (18.00-19.00 hr). Food intake was measured 1, 2, 4, 8 and 12
hours post-lights-out.
3. The Effect of Repeated IP Injections of OXM
[0146] 45 Animals were randomised by weight into three groups (n=15
per group): 1) Saline-treated with ad libitum access to food, 2)
OXM-treated (50 nmol/kg body weight per injection--a dose based on
the previous dose-response experiment) with ad libitum access to
food, 3) Saline-treated, but food restricted to the mean light and
dark phase food intake of the OXM-treated group. Animals were
injected twice daily (07.00 and 18.00 hr) for seven days. Food
intake (g), body weight (g) and water intake (ml) were measured
daily. On the eighth day, the animals were killed by decapitation.
Epididymal white adipose tissue (WAT) and interscapular brown
adipose tissue (BAT) were removed and weighed as an assessment of
body adiposity.
4. Investigating the Effect of Peripheral Administration of OXM on
Gastric Emptying
[0147] Animals were fasted for 36 hours to ensure that the stomach
was empty. During the early light phase (09:00-10:00) were allowed
ad libitum access to a pre-weighed amount of standard rat chow for
thirty minutes. After that time, the food was removed and
reweighed. The animals were then IP injected with saline, OXM (50
nmol/kg body weight) or CCK-8 (15 nmol/kg body weight). Rats were
then killed at the same times as those used in the previous feeding
studies: 1, 2, 4 or 8 hours post-feeding (n=12 per group per
time-point). The CCK-8 group was used as a positive control for the
experiment at the two-hour time-point only. Animals were killed by
carbon dioxide asphyxiation. A laparotomy was rapidly performed and
the stomach exposed. The pyloric junction was ligated (2.0 Mersilk,
Johnson & Johnson, Belgium), followed by ligation of the
gastro-oesophogeal junction and the stomach was removed. The
gastric contents were then removed, placed in a pre-weighed
weighing boat and left to air-dry for 48 hours. Once dry, the
contents were weighed and the percentage of the chow ingested
during the half-hour re-feeding period remaining in the stomach per
rat was then calculated using the following formula: % .times.
.times. food .times. .times. remaining .times. .times. in .times.
.times. the .times. .times. stomach = dry .times. .times. weight
.times. .times. of .times. .times. stomach .times. .times. content
weight .times. .times. of .times. .times. food .times. .times.
ingested .times. 100 ##EQU1## 5. Investigating the Effect of
Increasing Doses of Intra-Arcuate OXM
[0148] Intra-arcuate (Intra-ARC (iARC)) cannulated rats (n=12-15
per group) were randomised by weight into 6 groups. During the
early light phase (0900-1000), 24-hour fasted rats received an iARC
injection of saline, OXM (0.01, 0.03, 0.1, 0.3 or 1.0 nmoles). Food
intake was measured 1, 2, 4, 8 and 24 hours post-injection.
6. Investigating Whether Peripherally Administered OXM is Acting
Directly Via Arcuate Nucleus GLP-1 Receptors.
[0149] Rats cannulated into the arcuate nucleus were randomised
into 6 groups (n=10-12 per group). During the early light phase
(0900-1000) 24-hour fasted rats received an iARC injection of
saline or exending.sub.9-39 (5 nmoles) followed by an IP injection
of saline, OXM (30 nmoles/kg body weight) or GLP-1 (30 nmoles/kg
body weight) 15 minutes later. The injection details are described
in Table 1 below. TABLE-US-00003 TABLE 1 Group Intra-ARC injection
IP injection 1 Saline Saline 2 Saline OXM (30 nmoles/kg) 3 Saline
GLP-1 (30 nmoles/kg) 4 Exendin 9-39 Saline (5 nmoles) 5 Exendin
9-39 OXM (30 nmoles/kg) (5 nmoles) 6 Exendin 9-39 GLP-1 (30
nmoles/kg) (5 nmoles)
Immunohistochemistry
[0150] 90 minutes after an IP injection of OXM (50 nmol/kg), CCK
(15 nmol/kg) or saline, rats were terminally anaesthetized was
transcardially perfused with 0.1 M phosphate buffered saline (PBS)
following by 4% PB-formalin (PBF). The brains were removed and
post-fixed overnight in PBF and then transferred to PB-sucrose (20%
w/v) overnight. 40 .mu.m coronal sections of brain and brainstem
were cut on a freezing microtome and stained for fos-like
immunoreactivity (FLI) by the avitin-biotin-peroxidase method. The
sections were then mounted on poly-L-lysine-coated slides,
dehydrated in increasing concentrations of ethanol (50-100%),
delipidated in xylene and coverslipped using DPX mountant. Slides
were examined for FLI-positive nuclei using a light microscope
(Nikon Eclipse E-800) and images captured using a microimager
(Xillix MicroImager). The numbers of FLI-positive nuclei in the
hypothalamus and brainstem were counted by an independent member of
the research team who was blinded to the experimental groups. The
average number of FLI-positive nuclei per section was calculated
and expressed as an integer for each animal.
Hypothalamic Explant Static Incubation
[0151] A static incubation system was used. Male Wistar rats were
killed by decapitation and the whole brain removed immediately. The
brain was mounted, ventral surface uppermost, and placed in a
vibrating microtome (Microfield Scientific Ltd., Dartmouth, UK). A
1.7 mm slice was taken from the basal hypothalamus, blocked lateral
to the Circle of Willis and incubated in chambers containing 1 ml
of artificial cerebrospinal fluid which was equilibrated with 95%
O.sub.2 and 5% CO.sub.2. The hypothalamic slice encompassed the
medial pre-optic area, PVN (paraventricular hypothalamic nucleus),
dorsomedial nucleus, ventromedial nucleus, lateral hypothalamus and
ARC. The tubes were placed on a platform in a water bath maintained
at 37.degree. C. After an initial 2-hour equilibration period, each
explant was incubated for 45 minutes in 600 .mu.l aCSF (basal
period) before being challenged with a test period. OXM, 100 nM was
used as a dose representing a concentration ten times that of its
IC.sub.50 for the GLP-1 receptor. The viability of the tissue was
confirmed by a final 45-minute exposure to aCSF containing 56 mM
KCl. At the end of each experimental period, the aCSF was removed
and stored at -20.degree. C. until measurement of
.alpha.MSH-immunoreactivity by radioimmunoassay.
Radioimmunassay to Measure .alpha.MSH-IR
[0152] Alpha-MSH was measured using an in-house radioimmunoassay,
developed using an antibody from Chemicon International Inc.
Statistical Analysis
[0153] Data from IP and iARC feeding studies were analyzed by ANOVA
with post-hoc LSD (least significant difference) test. Fat pad
weights from different treatment groups were analyzed using an
unpaired t test. Data from the hypothalamic explant incubation
study, in which each explant was compared with its own basal
period, were analyzed by paired t test. In all cases P<0.05 was
considered to be statistically significant.
Results
1. The Effect of Peripheral Administration of OXM in Fasted
Animals:
[0154] Intraperitoneal administration of OXM (100 nmol/kg and 300
nmol/kg) caused a significant inhibition in refeeding in 24-hour
fasted animals one hour post-injection, compared with saline
controls (1 hour: OXM 100 nmol/kg, 5.4.+-.0.2 g (P<0.05), 300
nmol/kg, 4.5.+-.0.2 g (P<0.05) vs. saline, 6.3.+-.0.2 g). The
reduction in food intake caused by 100 nmol/kg was sustained until
8 hours post-injection. However, the highest dose of OXM (300
nmol/kg) continued to significantly inhibited food intake 24 hours
post-injection (8 hours: OXM, 300 nmol/kg, 9.5.+-.0.6 g vs. saline,
17.5.+-.0.7 g; P<0.05) (FIG. 6a). The 30 nmol/kg and 10 nmol/kg
failed to alter food intake at any time-point investigated.
2. The Effect of Peripheral Administration of OXM in Non-Fasted
Animals on Dark Phase Food Intake:
[0155] OXM, 3 and 10 nmol/kg, failed to affect food intake at any
time-point investigated in nocturnally feeding rats injected
immediately prior to the dark phase. However, OXM, 30 nmol/kg,
significantly inhibited food intake until 2 hours post-injection (2
hours: OXM, 30 nmol/kg, 4.5.+-.0.4 g vs. saline, 5.8.+-.0.4 g;
P<0.05). Food intake was reduced 4 hours post-injection, but
this was not significant. OXM, 100 nmol/kg, significantly inhibited
food intake throughout the dark phase (8 hours: OXM, 100 nmol/kg,
14.1.+-.0.8 g vs. saline, 16.9.+-.0.5 g; P<0.05) (FIG. 6b).
3. The Effect of Repeated IP Administration of OXM
[0156] Twice-daily IP injections of OXM (50 nmol/kg) for seven days
caused a significant decrease in cumulative daily food intake,
compare with saline-treated control animals (Cumulative food intake
day 7: OXM, 50 nmol/kg, 168.+-.4.6 g vs. saline, 180.+-.4.3 g;
P<0.01) (FIG. 7a). Furthermore, OXM-treated animals gained
weight significantly more slowly than saline controls (cumulative
weight gain day 7: OXM, 50 nmol/kg, 21.0.+-.1.5 g vs. saline,
37.6.+-.1.9 g; P<0.005). Moreover, the food restricted "pair
fed" animals did not gain weight as slowly as OXM-treated animals,
despite receiving the same food intake (Day 7: pair fed,
33.5.+-.2.0 g; P=NS vs. saline (ad libitum fed), P<0.05 vs. OXM)
(FIG. 7b). In addition, chronic OXM caused a decrease in adiposity
that was not seen in saline-injected pair fed animals (Table 2).
Water intake was significantly reduced in OXM-treated animals on
days 1 and 2 of the experiment (Day 1: OXM, 24.1.+-.1.28 ml vs.
saline, 28.1.+-.1.33 ml; P<0.05). On subsequent days, there was
an increase in daily water intake compared with saline-treated
animals (days 3-6). However, by day 7, there was no difference in
water intake between saline and OXM-treated groups (not shown). The
body weight difference between the "pair fed" rats and the OXM
treated rats is due to increased energy expenditure since the two
groups ate the same amount of food. TABLE-US-00004 TABLE 2 The
effect of twice-daily IP administration of saline or OXM (50
nmol/kg) for seven days on the weight of epididymal WAT and
interscapular BAT in food restricted and ad libitum fed rats.
Tissue/ hormone Saline OXM Pairfed WAT 0.69 .+-. 0.02 0.51 .+-.
0.01.sup.a 0.61 .+-. 0.02.sup.b BAT 0.16 .+-. 0.01 0.12 .+-.
0.01.sup.a 0.15 .+-. 0.01.sup.b
4. The Role of Delayed Gastric Emptying on the Anorectic Effect of
OXM:
[0157] One hour after food was presented to the 36-hour fasted
rats, the dry weight of the contents of the stomachs (as a
percentage of the food consumed during the 30 minute feeding
period) of GLP-1-treated animals were significantly greater than
that of saline-treated animals (1 hour: GLP-1, 50 nmol/kg,
76.9.+-.2.7 g vs. saline, 65.8.+-.1.6 g; P<0.01), suggesting
that GLP-1 caused a significant decrease in gastric emptying. The
contents of the stomachs of OXM-treated animals were greater than
those of the saline treated controls, although this was not
statistically significant (1 hour: OXM, 50 nmol/kg, 72.0.+-.1.4 g
vs. saline 65.8.+-.1.6 g; P=0.07). Two hours post-feed, OXM did not
affect the contents of the stomach, compared with saline-treated
animals. However, animals injected with the positive control for
this time-point, CCK (15 nmol/kg), had significantly greater
stomach content (2 hours: CCK, 15 nmol/kg, 64.7.+-.6.4 g vs.
saline, 38.5 g; P<0.01), suggesting that CCK caused a
significant decrease in the rate of gastric emptying. There was no
effect of OXM on the contents of the stomach, compared with
saline-treated animals, at 4 or 8 hours post-feed (FIG. 8).
5. Investigating the Effect of Increasing Doses of OXM Injected
Intra-Arcuate Nucleus
[0158] Food intake was significantly inhibited by all doses (except
0.01 nmoles) of OXM administered iARC during the 1.sup.st hour of
re-feeding following a 24-hour fast (1 hour: OXM 0.03 nmoles,
6.1.+-.0.5 g (P<0.05); 0.1 nmoles, 5.6.+-.0.4 g (P<0.05); 0.3
nmoles, 5.1.+-.0.6 g (P<0.01); 1.0 nmole, 3.6.+-.0.5 g
(P<0.005) all vs. saline, 7.7.+-.0.2 g) (FIG. 9). OXM 0.3 and
1.0 nmoles continued to significantly inhibit food intake until 8
hours post-injection. Twenty-four hours post-injection, food intake
was inhibited by OXM 1.0 nmoles, although this was not significant
(24 hours: OXM, 1.0 nmole, 37.8.+-.3.0 g vs. saline, 40.8.+-.1.6 g;
P=NS).
6. Investigating Whether Peripherally Administered OXM is Acting
Via Arcuate Nucleus GLP-1 Receptors
[0159] Intraperitoneal administration of both GLP-1 (30 nmol/kg)
and OXM (30 nmol/kg) caused a significant inhibition of food intake
one hour into the dark phase (1 hour: GLP-1, 5.0.+-.0.6 g, OXM,
5.1.+-.0.4 g vs. saline, 9.2.+-.0.3 g). However, the anorexia
caused by IP administration of OXM was blocked by prior
administration of the GLP-1 receptor antagonist, exendin 9-39 (300
nmol/kg), injected directly into the ARC (Table 3 & FIG. 10).
Inhibition of food intake by IP GLP-1 was not affected by prior
iARC administration of exendin 9-39. TABLE-US-00005 TABLE 3 The
effect of iARC administration of exendin 9-39 (5 nmoles) or saline
injected 15 minutes prior to IP administration of OXM (30 nmol/kg),
GLP-1 (30 nmol/kg) or saline on 1 hour food intake (g). Peptide
Food intake (g) S.E.M. Saline/saline 9.2 0.3 Saline/GLP-1 5.0 0.6
Exendin 9-39/GLP-1 5.0 0.3 Saline/OXM 5.1 0.4 Exendin 9-39/OXM 9.4
0.4 Exendin 9-39/saline 9.0 0.3 (S = saline, G = GLP-1 (30
nmol/kg), Ox = OXM (30 nmol/kg), Ex = exendin 9-39 (5 nmoles)).
7. Mapping the Expression of FLI in the Hypothalamus in Response IP
OXM:
[0160] After IP OXM administration (50 nmol/kg) dense staining of
FLI was found almost exclusively in the hypothalamic arcuate
nucleus (FIG. 11a). No other hypothalamic nuclei (PVN
(paraventricular hypothalamic nucleus), DMH (dorsomedial
hypothalamic nucleus), VMH (ventromedial hypothalamic nucleus))
demonstrated specific c-fos staining.
[0161] In the brainstem, IP CCK (15 nmol/kg) caused dense staining
of FLI, most notably in the NTS (nucleus tractus solitarius) and
the area postrema (FIG. 6b). However, neither IP saline nor IP OXM
(50 nmol/kg) caused a specific increase in c-fos expression in the
same brainstem nuclei investigated (FIG. 11b).
8. Changes in Alpha-MSH Release from Hypothalamic Explants when
Incubated with OXM
[0162] Incubating OXM (100 nM) was hypothalamic explants caused a
significant increase in the release of .alpha.-MSH, compared with
basal release (.alpha.-MSH: OXM, 100 nM, 4.1.+-.0.6 fmol/explant
vs. 2.6.+-.0.5 fmol/explant; P<0.005). Explant viability was
assessed by incubation with 56 mM KCl, and viability was confirmed
in >80% of explants. Those explants that were not viable were
excluded from the analysis.
Discussion
[0163] Peripheral administration of OXM causes a reduction in food
intake in rats. This was seen following a fast in the light phase
and during the nocturnal feeding phase. The anorectic effect was
potent and sustained for periods up to 24 hours. Twice-daily IP
administration of OXM for seven days caused a reduction in daily
food intake compared with those treated with saline, with no
tachyphylaxis. Animals treated with OXM gained significantly less
weight than pair fed animals, despite the two groups receiving
identical daily caloric intake. Intraperitoneal administration of
OXM did transiently reduce water intake although this was not
sustained, suggesting that the reduction in the rate of body weight
gain was not due to dehydration.
[0164] On conclusion of the chronic study, epididymal WAT and
interscapular BAT were removed and weighed. It was found that there
was a reduction in the weights of all fat pads in OXM-treated
animals compared with pair-fed animals, despite identical food
intake. Therefore it appears that peripheral OXM administration is
also affecting other metabolic parameters.
[0165] A major contributor to satiety is delayed gastric emptying
via vagally-mediated mechanism that leads to brainstem activation.
Both GLP-1 and OXM are potent inhibitors of gastric emptying in
rodents and humans and in the case of GLP-1, this is thought to be
the dominant mechanism through which it promotes satiety. We
hypothesized that OXM was acting in the same way, and that its
effects on gastric emptying were the cause of sustained anorexia.
However, although peripheral administration of OXM led to a slight
delay in gastric emptying in the first hour after the
re-introduction of food, this was non-significant and the effect
was short-lived. This suggested that OXM does slow gastric
emptying, but it is not likely to be responsible for the robust and
sustained inhibition of food intake.
[0166] We report here that peripheral administration of OXM
increases FLI in almost exclusively in the ARC. Furthermore, we
found that incubating hypothalamic explant with OXM caused a
significant increase in the release of the POMC
(pro-opiomelanocortin)-derived product, .alpha.MSH from
hypothalamic explants. IP OXM did not affect the expression of FLI
in the NTS and AP--areas known to be important in integrating
vagally mediated information, further strengthening the notion that
OXM is not acting via these pathways.
[0167] It is thought that nuclei in the brainstem are the primary
site of GLP-1 action, and information is subsequently relayed to
the hypothalamic PVN, where its anorectic effects are mediated.
Direct injection of OXM into the ARC, even at very low doses caused
a robust and sustained inhibition of food intake, further
supporting the hypothesis that that the ARC is the site of the
actions of OXM. Anorectic effects caused by peripheral
administration of OXM were blocked by prior administration of
exendin 9-39 into the ARC. Interestingly, however, the anorectic
actions of peripherally administered GLP-1 were not. This finding
strongly indicates that OXM is acting via GLP-1 receptors in the
ARC. In addition, it has identified distinct pathways which mediate
the actions of GLP-1 and OXM.
[0168] Taken together, these data demonstrate that OXM is
potentially important in both long and short-term regulation of
food intake and body weight maintenance. Rather than reducing
appetite via "traditional" satiety pathways, involving slowing of
gastric emptying and activation of brainstem nuclei, circulating
OXM is mediating its anorectic effects via direct interaction with
the ARC, potentially by activating POMC (pro-opiomelanocortin)
neurons within the nucleus. Therefore, OXM may be useful in the
treatment or prevention of excess weight such as obesity in
mammals, and further represents a novel target for the development
of therapeutic agents in the treatment of excess weight such as
obesity in mammals.
Example 3
Investigation of the Effect of OXM Infusion on Food Intake in Human
Subjects
Methods
Study 1
[0169] The study design was a double-blind placebo-controlled
crossover, see FIG. 12. 13 healthy volunteers (age 27.+-.2 yrs; BMI
25.3.+-.0.7 kg.sup.-2) received a 90 minute intravenous infusion of
OXM (3.0 pmol/kg/min) and an infusion of saline .gtoreq.1 week
apart, in random order. OXM was dissolved in saline containing
haemaccel (5% by volume) to reduce adsorption to the syringe and
tubing. Volunteers completed a food diary for three days prior to
each infusion and for the subsequent 24 hours. Subjects were
instructed to follow a similar diet on the days preceding each
infusion. They consumed an identical meal (of their choice) on the
night before each infusion and fasted from 9 pm.
[0170] On each study day intravenous cannulae were inserted
bilaterally into arm veins, one for administration of the infusion,
while the other was used for blood-sampling. Subjects were attached
to a cardiac monitor and blood pressure was measured every 15 min.
Blood samples were collected every 30 minutes into Lithium-Heparin
tubes (LIP LTD, UK) containing 5,000 Kallikrein Inhibitor Units
(0.2 ml) of aprotinin (Trasylol, Bayer) and stored on ice.
Following centrifugation plasma was immediately separated and
stored at -70.degree. C. until analysis.
[0171] 15 min before termination of the infusion, subjects were
offered a buffet meal which was provided in excess so that all
appetites could be satisfied and subjects would be unable to assess
their own food intake. The choices consisted of chicken curry,
plain boiled rice, fruit salad, and a variety of mini chocolate
bars and fruit-flavoured sweets. Water was freely available.
Dietary intake was calculated by weighing food and water pre and
postprandially.
[0172] Food intake for 24 hours following the buffet meal was
recorded in food diaries and energy intake was calculated with the
aid of the Dietplan program (Forestfield Software LTD, West Sussex,
UK).
[0173] Every 30 min subjects completed visual analogue scales (VAS)
rating hunger, satiety, fullness, prospective food consumption and
nausea. These consisted of 100 mm scales with the text expressing
the most positive and the negative rating anchored at each end.
Study 2
[0174] The same protocol was followed as for Study 1, except that
the eight healthy fasting volunteers were administered OXM
subcutaneously at doses from 100 nmol to 250 nmol (in normal
saline) thirty minutes before the buffet.
Results
Study 1
[0175] OXM infusion led to a significant fall in calories consumed
at the buffet meal (192.+-.59 kcal; 17.6.+-.5.7%). 12/13 subjects
showed a decrease in calories consumed with OXM infusion, see FIG.
13. OXM infusion was associated with a significant fall in
subjective hunger scores, see FIG. 14 (VAS `How hungry are you
right now?` 60 min P<0.05). There were no adverse effects of OXM
infusion. In particular there was no effect of OXM on feelings of
sickness (nausea) (VAS `How sick do you feel right now?` 75 min
P=0.8). The effect appears to be rapid.
[0176] Study 2 TABLE-US-00006 The results obtained are shown in
Table 4. wt nmol nmol Energy (Kcal) Kcal Dif Name KG BMI dose
dose/kg saline sc oxm Diff % 01 89 27 100 1.12 1344 283 -1061 -79
02 95 28 100 1.05 1059 840 -219 -21 03 89 28 150 1.69 917 731 -186
-20 04 91 30 150 1.65 530 467 -63 -12 05 92 31 150 1.63 381 283 -98
-26 06 87 29 200 2.29 922 667 -255 -28 07 113 36 250 2.21 966 875
-91 -9 08 106 36 250 2.36 910 742 -168 -18 MEAN 879 611 -268 -32
SEM 106 84 116 12
Discussion
[0177] The demonstration that parenteral administration of OXM to
human subjects results in a decrease in calories consumed and a
significant reduction in subjective sensations of hunger without
undesirable side effects, in particular, feelings of sickness
(nausea) is confirmation the utility of OXM in the treatment or
prevention of excess weight such as obesity in mammals, and as a
novel target for the development of therapeutic agents in the
treatment of excess weight such as obesity in mammals.
Example 4
Investigation of the Plasma OXM-Immunoreactivity (IR) and
Ghrelin-Immunoreactivity IR (Ghrelin-IR) Levels Following IP
Administration of OXM.
Methods
[0178] OXM or saline were administered to fasted rats to
investigate the plasma OXM-IR and ghrelin-IR levels following IP
OXM. Plasma OXM-IR levels were measured, using a previously
described assay, which also measures enteroglucagon (i.e.,
N-terminally elongated OXM) (Ghatei M A, Uttenthal L O,
Christofides N D, Bryant M G, Bloom S R 1983 J Clin Endocrinol
Metab 57:488-495). The OXM-IR assay could detect changes of 10
pmol/L (95% confidence limit) with an intra-assay variation of
5.7%. The ghrelin radioimmunoassay (English P J, Ghatei M A, Malik
I A, Bloom S R, Wilding J P 2002 J Clin Endocrinol Metab 87:2984)
measured both octanoyl and des-octanoyl ghrelin (Total ghrelin). It
did not cross-react with any known gastrointestinal or pancreatic
peptide hormones and could detect changes of 10 pmol/L (95%
confidence limit) with an intra-assay variation of 9.5%.
[0179] Rats (n=10 per group) were IP injected with OXM (30
nmoles/kg and 100 nmoles/kg) or saline at the beginning of the
light phase. The rats were decapitated 30 and 90 minutes following
the IP injection, trunk blood collected. All plasma was collected
and frozen at -20 C until assayed for OXM-IR and ghrelin-IR. During
the entire post-injection period the rats remained fasted. The time
points and the doses of OXM were chosen by reference to previous
feeding studies.
[0180] The release of gut hormones has been found to be influenced
by the content of the diet, in particular the fat content. For this
reason, a further three groups (n=10) were investigated: a) Rats
fasted overnight and killed at the beginning of the light phase, b)
Rats fed high fat rat chow (45% fat, Research Diets Inc.) overnight
and decapitated at the beginning of the light phase, c) Rats fasted
overnight and at lights-on, they were given ad libitum access to
45% high fat chow for 2 h. The rats were decapitated at the end of
this 2-hour high fat meal (i.e., two hours into the light phase).
All plasma was collected and frozen at -20.degree. C. until assayed
for OXM-IR and ghrelin-IR.
Results
[0181] IP administration of OXM (30 nmoles/kg and 100 nmoles/kg)
increased plasma OXM-IR 30 and 90 minutes post-injection (30 min
plasma OXM-IR pmol/L: saline 61.8.+-.8.9, OXM 30 nmoles/kg
448.9.+-.184.4, OXM 100 nmoles/kg 997.1.+-.235.4. 90-min plasma
OXM-IR pmol/L: saline 47.5.+-.4.5, OXM 30 nmoles/kg 150.6.+-.52.5,
OXM 100 nmoles/kg 107.8.+-.25.0).
[0182] The plasma OXM-IR levels were determined in three additional
groups: a) Rats fasted overnight and killed at the beginning of the
light phase (plasma OXM-IR pmol/L: 51.9.+-.5.8), b) Rats fed high
fat rat chow overnight and decapitated at the beginning of the
light phase (plasma OXM-IR pmol/L: 220.2.+-.22.2), c) Rats fasted
overnight, then given ad libitum access to high fat chow for 2
hours at lights-on, were decapitated at the end of the 2-hour high
fat meal (plasma OXM-IR pmol/L: 254.0.+-.32.7).
[0183] IP administration of OXM (30 nmoles/kg and 100 nmoles/kg)
significantly decreased fasting plasma ghrelin-IR 30 and 90 minutes
post-injection (30 min plasma ghrelin pmol/L: saline,
1056.9.+-.64.0, OXM, 30 nmoles/kg 867.4.+-.42.0 (p<0.01), OXM,
100 nmoles/kg 860.0.+-.47.5 (p<0.02). Ninety-minute plasma
ghrelin-IR pmol/L: saline, 1055.2.+-.52.5, OXM, 30 nmoles/kg
886.9.+-.36.3 (p<0.01), OXM, 100 nmoles/kg 900.0.+-.52.9
(P<0.05), see FIG. 15.
[0184] Plasma ghrelin-IR levels were determined in 3 additional
groups: a) Rats fasted overnight and killed at the beginning of the
light phase (plasma ghrelin-IR pmol/L: 1066.1.+-.80.9), b) Rats fed
high fat rat chow overnight and decapitated at the beginning of the
light phase (plasma ghrelin-IR pmol/L: 611.3.+-.16.9), c) Rats
fasted overnight, at lights-on they were given ad libitum access to
high fat chow for 2 h, were decapitated at the end of the 2-hour
high fat meal (plasma ghrelin pmol/L: 648.9.+-.57.3).
Example 5
Investigation of the Effect of OXM Infusion on Human Subjects
Methods
Study Design
[0185] The study design was as in Example 3. Subjects remained in
the study room until t.sub.225. They continued to complete VAS
until 09:00 the following morning and recorded food intake in
diaries for 24 hours following the buffet meal (until 13:00 the
following day). Food diaries were analysed by a dietician blinded
to the study and energy intake was calculated with the aid of the
Dietplan program (Forestfield Software LTD, West Sussex, UK).
Plasma Hormone Measurements
[0186] All samples were assayed in duplicate and within one assay
to eliminate inter-assay variation. Plasma OLI, pancreatic
glucagon, peptide YY (PYY), insulin, glucagon-like peptide-1
(GLP-1) and ghrelin were measured using established in-house RIAs.
The OLI assay (Ghatei M A, Uttenthal L O, Christofides N D, Bryant
M G, Bloom S R 1983 J Clin Endocrinol Metab 57:488-495) could
detect changes of 10 pmol/L (95% confidence limit) with an
intra-assay variation of 5.7%. The PYY assay (Adrian T E, Savage A
P, Sagor G R, Allen J M, Bacarese-Hamilton A J, Tatemoto K, Polak J
M, Bloom S R 1985 Gastroenterology 89:494-499) could detect changes
of 2 pmol/L (95% confidence limit) with an intra-assay variation of
5.8%. The PYY antibody was specific for the C-terminus of PYY and
cross-reacts fully with human PYY 3-36. The insulin assay (Kreymann
B, Williams G, Ghatei M A, Bloom S R 1987 Lancet 2:1300-1304) could
detect changes of 6 pmol/L (95% confidence limit) with an intra-say
variation of 5.4%. The GLP-1 assay (Kreymann B, Williams G, Ghatei
M A, Bloom S R 1987 Lancet 2:1300-1304) could detect changes of 8
pmol/L (95% confidence limit) with an intra-assay variation of
6.1%. The GLP-1 antibody was specific for amidated GLP-1 and does
not cross-react with GLP-1 (1-37), GLP-1 (1-36) or GLP-1 (7-37).
The ghrelin assay (English P J, Ghatei M A, Malik I A, Bloom S R,
Wilding J P 2002 J Clin Endocrinol Metab 87:298) could detect
changes of 10 pmol/L (95% confidence limit) with an intra-assay
variation of 9.5%. Plasma leptin was measured using the Linco
Research (Missouri, USA) human leptin RIA kit.
Results
1. Effects of OXM Infusion on Energy Intake
[0187] OXM infusion significantly reduced energy intake at the
buffet meal by 19.3.+-.5.6% (reduction vs saline: 220.+-.60 kcal,
P<0.01). 12 out of the 13 subjects studied showed a decrease in
energy intake with OXM infusion (FIG. 17). There was no obvious
cause for the failure of response in one subject. OXM infusion
significantly reduced cumulative 12 hour energy intake by
11.3.+-.6.2% (reduction vs saline: 365.+-.159 kcal, P<0.05)
(FIG. 3). Cumulative 24 hour energy intake was not significantly
altered (saline 3043.+-.366 kcal, OXM 2768.+-.297 kcal). OXM did
not change water consumption or the proportion of calories obtained
from different macronutrients at the buffet meal or in the
subsequent cumulative 12 and 24 hr food intake.
2. Effects of OXM Infusion on Appetite and Palatability
[0188] During infusion of saline visual analogue scores for hunger
did not change significantly throughout the fasting period (FIG.
18) whereas OXM infusion caused a significant fall in hunger
(incremental AUC t.sub.0 to t.sub.75: saline+273.+-.128 mmmin, OXM
minus 374.+-.185 mmmin, P<0.05). The decrease in hunger
following the buffet meal was similar on saline and OXM infusion
days and hunger scores remained similar thereafter. The duration of
the meal was significantly reduced by OXM (saline 19.2.+-.1.3 min,
OXM 15.1.+-.1.8 min, P<0.05). There was no significant effect of
OXM on visual analogue scores for satiety, prospective food
consumption, nausea and meal palatability (data not shown).
3. Plasma Levels of OXM-Like Immunoreactivity (OLI)
[0189] Infusion of OXM elevated plasma OLI from 62.+-.5 pmol/L to a
peak of 907.+-.32 pmol/L at t.sub.60 (FIG. 19). In comparison, on
the saline infusion day, consumption of the buffet meal led to a
peak postprandial OLI level of 151.+-.18 pmol/L at 195 min. Gel
permeation analysis of plasma samples during OXM infusion (FIG. 20)
demonstrated a single immunoreactive peak eluting in the same
position as synthetic OXM (K.sub.av=0.6). Thus intact full-length
OXM was the principle circulating form.
4. Effects of OXM Infusion on Plasma Ghrelin
[0190] During the saline infusion, plasma ghrelin levels increased
throughout the fasting period (t.sub.0 461.+-.32 pmol/L, t.sub.75
484.+-.35 pmol/L) and decreased postprandially (t.sub.225 357.+-.28
pmol/L). However, during infusion of OXM fasting levels of ghrelin
decreased before the meal (t.sub.0 482.+-.33 pmol/L, t.sub.75
435.+-.35 pmol/L) and there was a further postprandial reduction in
ghrelin (t.sub.225 356.+-.31 pmol/L). Hence plasma ghrelin prior to
the buffet meal was significantly reduced by OXM infusion compared
to saline (mean change in ghrelin from t.sub.0 to t.sub.75:
saline+24.+-.10 pmol/L, OXM minus 47.+-.11 pmol/L, P<0.0001)
(FIG. 21). The suppression in plasma ghrelin due to OXM infusion
represents 44.+-.10% of the postprandial decrease in ghrelin on the
corresponding saline infusion day (mean postprandial decrease
155.+-.19 pmol/L).
5. Effects of OXM Infusion on Plasma Hormones Levels
[0191] There was no significant effect of OXM infusion on fasting
plasma levels of PYY, insulin, pancreatic glucagon, GLP-1 or leptin
(table 1). Plasma concentrations of leptin in female subjects were
higher than in males as has previously been reported.
Discussion
[0192] We have demonstrated that systemic administration of OXM
significantly reduces food intake in healthy human subjects.
Intravenous infusion of OXM reduced calorie intake by 19% at the
buffet meal and cumulative energy intake was decreased in the 12
hours post-infusion. Much smaller alterations in food consumption
would lead to weight loss if sustained in the long term. However
there was no significant effect of OXM on cumulative 24 hour energy
intake. Our work indicates that OXM may increase energy
expenditure. OXM did not affect enjoyment of the meal, which is
important in view of its potential therapeutic use.
[0193] Ghrelin is a powerful stimulant of appetite in man (Wren A
M, Seal L J, Cohen M A, Brynes A E, Frost G S, Murphy K G, Dhillo W
S, Ghatei M A, Bloom S R 2001 Ghrelin enhances appetite and
increases food intake in humans. J Clin Endocrinol Metab 86:5992)
and preprandial rises in plasma ghrelin have been suggested to be a
trigger for meal initiation (Cummings D E, Purnell J Q, Frayo R S,
Schmidova K, Wisse B E, Weigle D S 2001 A preprandial rise in
plasma ghrelin levels suggests a role in meal initiation in humans.
Diabetes 50:1714-1719). Hence our novel finding that OXM infusion
suppresses fasting plasma ghrelin is potentially important.
Inhibition of the normal preprandial rise in ghrelin by OXM is
likely to be one mechanism by which OXM infusion reduces appetite.
This finding may also shed light on the poorly understood mechanism
by which ghrelin levels are reduced postprandially. In rodents,
fasting increases plasma ghrelin, while oral intake of glucose, but
not water, decreases ghrelin secretion, suggesting that suppression
of plasma ghrelin is related to ingestion of nutrients rather than
stomach distension. Hence OXM released in response to nutrient
ingestion may contribute to the normal postprandial inhibition of
plasma ghrelin. It is believed that only a proportion of total
circulating ghrelin is the biologically-active, octanoylated form.
The effect of food consumption and OXM infusion may be to primarily
reduce levels of this active ghrelin.
[0194] Intravenous infusion of OXM has been shown to inhibit
gastric emptying in humans. Suppression of gastric-emptying may
lead to increased gastric distension which may contribute to
satiety by causing a sensation of fullness. In the current study
hunger scores were significantly reduced by OXM in the fasting
state when gastric distension is unlikely to be important. Hence
the reduction in appetite in the pre-meal period is unlikely to
result from effects of OXM on gastric emptying. The anorectic
effect of OXM does not appear to be mediated by stimulation of the
release of PYY or leptin as concentrations of these hormones were
unaffected by OXM infusion.
[0195] We have demonstrated in humans the anorectic effect of
elevated circulating levels of OXM. Infusion of OXM produced
circulating levels of OLI which were comparable to the elevated
concentrations seen in tropical sprue and following jejuno-ileal
bypass surgery for morbid obesity. Therefore OXM may contribute to
the loss of appetite and weight loss observed in these conditions.
We consider that lower postprandial concentrations of OXM
contribute to the physiological reduction of appetite in normal
individuals and that exogenous administration of OXM has potential
to reduce food intake and/or increase energy expenditure in the
obese.
[0196] Taken together, these data demonstrate that OXM is
potentially important in both long and short-term regulation of
food intake, energy expenditure and body weight maintenance.
Therefore, OXM may be useful in the treatment or prevention of
excess weight such as obesity in mammals, and further represents a
target for the development of therapeutic agents in the treatment
of excess weight such as obesity in mammals, especially humans.
Sequence CWU 1
1
9 1 37 PRT Homo sapiens 1 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr
Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln
Trp Leu Met Asn Thr Lys Arg Asn 20 25 30 Lys Asn Asn Ile Ala 35 2
36 PRT Lophius piscatorius 2 His Ser Glu Gly Thr Phe Ser Asn Asp
Tyr Ser Lys Tyr Leu Glu Asp 1 5 10 15 Arg Lys Ala Gln Glu Phe Val
Arg Trp Leu Met Asn Asn Lys Arg Ser 20 25 30 Gly Val Ala Glu 35 3
36 PRT Anguilla japonica 3 His Ser Gln Gly Thr Phe Thr Asn Asp Tyr
Ser Lys Tyr Leu Glu Thr 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln
Trp Leu Met Asn Ser Lys Arg Ser 20 25 30 Gly Gly Pro Thr 35 4 37
PRT Homo sapiens 4 His Asp Glu Phe Glu Arg His Ala Glu Gly Thr Phe
Thr Ser Asp Val 1 5 10 15 Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
Glu Phe Ile Ala Trp Leu 20 25 30 Val Lys Gly Arg Gly 35 5 36 PRT
Homo sapiens 5 His Asp Glu Phe Glu Arg His Ala Glu Gly Thr Phe Thr
Ser Asp Val 1 5 10 15 Ser Ser Tyr Leu Glu Gly Gly Ala Ala Lys Glu
Phe Ile Ala Trp Leu 20 25 30 Val Lys Gly Arg 35 6 31 PRT Homo
sapiens 6 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15 Gly Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg Gly 20 25 30 7 30 PRT Homo sapiens 7 His Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gly Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 8 36 PRT Homo
sapiens 8 Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro
Glu Glu 1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu
Asn Leu Val Thr 20 25 30 Arg Gln Arg Tyr 35 9 37 PRT Homo sapiens 9
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5
10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg
Asn 20 25 30 Arg Asn Asn Ile Ala 35
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