U.S. patent application number 09/771770 was filed with the patent office on 2001-09-06 for use of compounds for the regulation of food intake.
Invention is credited to Andersen, Maibritt Bansholm, Hansen, Birgit Sehested, Raun, Kirsten, Thim, Lars, Tullin, Soren.
Application Number | 20010020012 09/771770 |
Document ID | / |
Family ID | 27439798 |
Filed Date | 2001-09-06 |
United States Patent
Application |
20010020012 |
Kind Code |
A1 |
Andersen, Maibritt Bansholm ;
et al. |
September 6, 2001 |
Use of compounds for the regulation of food intake
Abstract
Compounds that are ligands for the growth hormone secretagogue
receptor type 1A (GHS-R 1A), as well as pharmaceutically acceptable
salts of such compounds, are useful for the manufacture of
medicaments for the regulation of food intake.
Inventors: |
Andersen, Maibritt Bansholm;
(Roskilde, DK) ; Hansen, Birgit Sehested;
(Stenlose, DK) ; Raun, Kirsten; (Lyngby, DK)
; Tullin, Soren; (Soborg, DK) ; Thim, Lars;
(Gentofte, DK) |
Correspondence
Address: |
Steve T. Zelson, Esq.
Novo Nordisk of North America, Inc.
Suite 6400
405 Lexington Avenue
New York
NY
10174-6401
US
|
Family ID: |
27439798 |
Appl. No.: |
09/771770 |
Filed: |
January 29, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60181303 |
Feb 9, 2000 |
|
|
|
Current U.S.
Class: |
514/46 ; 514/1.9;
514/15.7; 514/16.4; 514/16.8; 514/21.91; 514/278; 514/323; 514/4.9;
514/5.1; 514/6.9; 514/7.4 |
Current CPC
Class: |
A61K 31/7076 20130101;
A61K 38/17 20130101; A61K 38/25 20130101; A61K 31/44 20130101; G01N
33/74 20130101; A61K 38/06 20130101; A61K 31/00 20130101; G01N
2500/00 20130101; A61K 31/4747 20130101; A61K 49/0008 20130101;
A61K 38/05 20130101; A61K 31/454 20130101 |
Class at
Publication: |
514/46 ; 514/19;
514/323; 514/278 |
International
Class: |
A61K 038/05; A61K
031/7076; A61K 031/4747; A61K 031/454 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 1, 2000 |
DK |
PA 2000 00161 |
Jul 17, 2000 |
DK |
PA 2000 01107 |
Claims
What is claimed is:
1. The use of a compound that is a ligand for the receptor GHS-R
1A, or pharmaceutically acceptable salts thereof, for the
manufacture of a medicament for the regulation of food intake.
2. The use according to claim 1, wherein the compound does not
induce a therapeutic effective growth hormone release at the
therapeutic dose of the compound.
3. The use according to claims 1 or 2, wherein the medicament is
for humans.
4. The use according to any one of claims 1-3, wherein the
medicament is a non-injectable medicament.
5. The use according to any one of claims 1-4, wherein the ligand
is an agonist.
6. The use according to any one of claims 1-4, wherein the ligand
is an antagonist.
7. The use according to any one of claims 1-5, wherein the compound
is selected from the group consisting of adenosine, ghrelin and
ghrelin homologues.
8. A method for the regulation of food intake, which method
comprises administering an effective amount of a compound as
defined in any one of claims 1, 2 or 5-7 to a patient in need of
such a treatment.
9. A method for identifying compounds for the regulation of food
intake, characterized by screening out compounds that act as
ligands for the GHS receptor type 1A (GHS-R 1A).
10. Compounds identified by a method according to claim 9.
11. The use of a compound as defined in any one of claims 1, 2 or
5-7 for the manufacture of a medicament for the regulation of Body
Mass Index (BMI).
12. The use of a compound as defined in any one of claims 1, 2 or
5-7 for the manufacture of a medicament for the treatment of
anorexia.
13. The use of a compound as defined in any one of claims 1, 2 or
5-7 for the manufacture of a medicament for the treatment of lack
of appetite in children with a growth hormone deficiency.
14. The use of a compound as defined in any one of claims 1, 2 or
5-7 for the manufacture of a medicament for the treatment of
obesity.
15. The use of a compound as defined in any one of claims 1, 2 or
5-7 for the manufacture of a medicament for the treatment of Type
II diabetes.
16. The use of a compound according to any one of claims 1,2 or 5-7
for the manufacture of a medicament for the treatment of wasting
associated with AIDS.
17. A pharmaceutical composition comprising, as an active
ingredient, a compound as defined in any one of claims 1, 2 or 5-7
or a pharmaceutically acceptable salt thereof together with a
pharmaceutically acceptable carrier or diluent.
18. A composition according to claim 17 in unit dosage form,
comprising from about 0.05 to about 2000 mg, preferably from about
0.1 to about 500 mg of a compound as defined in any one of claims
1, 2 or 5-7 or a pharmaceutically acceptable salt thereof.
19. A pharmaceutical composition for the regulation of food intake,
the composition comprising, as an active ingredient, a compound as
defined in any one of claims 1, 2 or 5-7 or a pharmaceutically
acceptable salt thereof together with a pharmaceutically acceptable
carrier or diluent.
20. A pharmaceutical composition according to any one of the claims
17-19 for oral, nasal, transdermal, pulmonal or parenteral
administration.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the use of a compound that
is a ligand for the growth hormone secretagogue receptor type 1A
(GHS-R 1A) for the regulation of food intake or food intake. The
present invention also embraces pharmaceutical compositions
comprising these compounds and methods of using the compounds and
their pharmaceutical compositions.
BACKGROUND OF THE INVENTION
[0002] Stimulation of food intake is important in connection with
patients suffering from anorexia due to chronic medical conditions,
eating disorders, and other conditions in which excessive weight
loss has produced a detrimental effect on the patients' health.
[0003] In other situations, suppression of food intake is
important. Obesity is a well-known risk factor for the development
of many very common diseases such as atherosclerosis, hypertension,
Type II diabetes (non-insulin dependent diabetes mellitus (NIDDM)),
dyslipidemia, coronary heart disease, and osteoarthritis and
various malignancies. It also causes considerable problems through
reduced mobility and decreased quality of life. The incidence of
obesity and thereby also these diseases is increasing throughout
the entire industrialized world. Except for exercise, diet and food
restriction, no convincing treatment for reducing body weight
effectively and acceptably currently exist. However, due to the
important effect of obesity as a risk factor in serious and even
fatal and common diseases, it will be important to find
pharmaceutical compounds useful in prevention and treatment of
obesity.
[0004] Even mild obesity increases the risk of premature death,
diabetes, hypertension, atherosclerosis, gallbladder disease and
certain types of cancer. In the industrialized western world the
prevalence of obesity has increased significantly in the past few
decades. Because of the high prevalence of obesity and its health
consequences, its prevention and treatment should be a high public
health priority.
[0005] When energy intake exceeds energy expenditure, the excess
calories are stored in adipose tissue, and if this net positive
balance is prolonged, obesity results, i.e., there are two
components to weight balance, and an abnormality on either side
(intake or expenditure) can lead to obesity.
SUMMARY OF THE INVENTION
[0006] The present invention relates, inter alia, to the use of a
compound that is a ligand for the receptor GHS-R 1A, or
pharmaceutically acceptable salts thereof, for the manufacture of a
medicament for the regulation of food intake.
DESCRIPTION OF THE INVENTION
[0007] Accordingly, the present invention provides the use of a
compound that is a ligand for the receptor GHS-R 1A, or
pharmaceutically acceptable salts thereof, for the manufacture of a
medicament for the regulation of food intake.
[0008] A further aspect of the present invention relates to a
method for the regulation of food in-take, which method comprises
administering an effective amount of a compound that is a ligand
for the receptor GHS-R 1A, or pharmaceutically acceptable salts
thereof, to a patient in need of such a treatment.
[0009] A still further aspect of the present invention relates to
the use of a compound that is a ligand for the receptor GHS-R 1A,
or pharmaceutically acceptable salts thereof, for the manufacture
of a medicament for the regulation of Body Mass Index (BMI).
[0010] A further aspect of the present invention relates to the use
of a compound that is a ligand for the receptor GHS-R 1A, or
pharmaceutically acceptable salts thereof, for the manufacture of a
medicament for the treatment of anorexia.
[0011] A still further aspect of the present invention relates to
the use of a compound that is a ligand for the receptor GHS-R 1A,
or pharmaceutically acceptable salts thereof, for the manufacture
of a medicament for the treatment of lack of appetite in children
with a growth hormone deficiency.
[0012] A further aspect of the present invention relates to the use
of a compound that is a ligand for the receptor GHS-R 1A, or
pharmaceutically acceptable salts thereof, for the manufacture of a
medicament for the treatment of obesity.
[0013] A still further aspect of the present invention relates to
the use of a compound that is a ligand for the receptor GHS-R 1A,
or pharmaceutically acceptable salts thereof, for the manufacture
of a medicament for the treatment of Type II diabetes.
[0014] A further aspect of the present invention relates to the use
of a compound that is a ligand for the receptor GHS-R 1A, or
pharmaceutically acceptable salts thereof, for the manufacture of a
medicament for the treatment of wasting associated with various
diseases or conditions, e.g., wasting associated with AIDS, chronic
liver disease, chronic obstructive pulmonary disease (COPD) or
respiratory insufficiency in general, as well as wasting associated
with bone fractures or with ageing. Wasting, which involves a
progressive loss of body mass, including muscle mass, is normally
attributable to a catabolic state of metabolism, and is frequently
difficult to reverse by purely dietary means.
[0015] In one embodiment of the present invention the receptor
GHS-R 1A is the human GHS-R 1A receptor. In another embodiment the
medicament is for humans.
[0016] In a still further embodiment of the invention the compound
does not induce a therapeutically effective growth hormone release
at the therapeutic dose of the compound.
[0017] In a further embodiment of the invention the medicament is a
non-injectable medicament. In a still further embodiment the
medicament is an oral medicament.
[0018] The receptor GHS-R 1A (the growth hormone secretagogue
receptor type 1A) is described by Howard, A. D. et al. (1996) in
Science 273, 974-977.
[0019] The binding of a compound to the receptor GHS-R 1A can,
e.g., be measured by the use of the assays as described in Example
1 herein.
[0020] In one embodiment of the invention the ligand has a potency
(EC.sub.50) on the GHS-R 1A of less than 500 nM. In another
embodiment the ligand has a potency (EC.sub.50) on the GHS-R 1A of
less than 100 nM.
[0021] In a further embodiment the binding constant (K.sub.D) of
the ligand is less than 500 nM. In a still further embodiment the
binding constant (K.sub.D) of the ligand is less than 100 nM.
[0022] In yet another embodiment of the invention the ligand is an
agonist for the receptor GHS-R 1A. In another embodiment of the
invention the ligand is an antagonist for the receptor GHS-R
1A.
[0023] In a still further embodiment the compound employed in
accordance with the invention is adenosine. In a further embodiment
the compound is ghrelin or a peptide homologous thereto. Ghrelin is
described by Kojima in Nature (1999), vol. 402, 656-660.
[0024] Peptides homologous to ghrelin are peptides which have an
amino acid sequence which has a degree of identity to ghrelin of at
least about 70%, preferably at least about 80%, more preferably at
least about 90%, even more preferably at least about 95%, and most
preferably at least about 97%, and which qualitatively retain the
activity as a ligand for the receptor GHS-R 1A. The degree of
identity between two or more amino acid sequences may be determined
by means of computer programs known in the art, such as GAP
provided in the GCG program package (Needleman and Wunsch, 1970,
Journal of Molecular Biology 48:443-453). For the purposes of
determining the degree of identity between two amino acid sequences
for the present invention, GAP is used with the following settings:
GAP creation penalty of 3.0 and GAP extension penalty of 0.1.
[0025] Within the context of the present invention, "a
therapeutically effective growth hormone release" is to be
understood as a growth hormone release that would have a
therapeutic effect in treatment of the specific indication, e.g.,
regulation of food intake.
[0026] The therapeutic dose of the compound will depend upon the
frequency and mode of administration, the sex, age, weight and
general condition of the subject treated, the nature and severity
of the condition treated and any concomitant diseases to be
treated, and other factors evident to those skilled in the art. In
one embodiment, the effective amount of the compound is in the
range from about 0.05 to about 2000 mg, preferably from about 0.1
mg to about 1000 mg, and especially from about 0.5 to about 500 mg
per day.
[0027] The term obesity implies an excess of adipose tissue. In
this context, obesity is best viewed as any degree of excess
adiposity that imparts a health risk. The distinction between
normal and obese individuals can only be approximated, but the
health risk imparted by obesity is probably a continuum with
increasing adiposity. However, in the context of the present
invention, individuals with a Body Mass Index (BMI, defined as the
body weight of a human individual in kilograms divided by the
square of the height of that individual in meters) above 25 are to
be regarded as obese.
[0028] The present invention also encompasses pharmaceutically
acceptable salts of the present compounds. Such salts include
pharmaceutically acceptable acid addition salts, pharmaceutically
acceptable metal salts, ammonium salts and alkylated ammonium
salts. Acid addition salts include salts of inorganic acids as well
as organic acids. Representative examples of suitable inorganic
acids include hydrochloric, hydrobromic, hydriodic, phosphoric,
sulpfuric and nitric acids and the like. Representative examples of
suitable organic acids include formic, acetic, trichloroacetic,
trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric,
glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric,
pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic,
tartaric, ascorbic, pamoic, bismethylene salicylic,
ethanedisulfonic, gluconic, citraconic, aspartic, stearic,
palmitic, ethylenediaminetetraacetic (EDTA), p-aminobenzoic,
glutamic, benzenesulfonic and p-toluenesulfonic acids and the like.
Further examples of pharmaceutically acceptable inorganic or
organic acid addition salts include the pharmaceutically acceptable
salts listed in J. Pharm. Sci. 1977, 66, 2, which is incorporated
herein by reference. Examples of metal salts include lithium,
sodium, potassium and magnesium salts and the like. Examples of
ammonium and alkylated ammonium salts include ammonium,
methylammonium, dimethylammonium, trimethylammonium, ethylammonium,
hydroxyethylammonium, diethylammonium, butylammonium and
tetramethylammonium salts and the like.
[0029] Also included within the scope of compounds or
pharmaceutically acceptable acid addition salts thereof in the
context of the present invention are any hydrates (hydrated forms)
thereof.
[0030] Within the context of the present invention, a "ligand for
the receptor GHS-R 1A" is understood to refer to any compound that
has affinity to the receptor GHS-R 1A as measured by the method as
described in Example 1 herein.
[0031] Within the context of the present invention, treatment is to
be understood as treatment and/or prevention.
[0032] In a still further aspect, the invention relates to a
pharmaceutical composition comprising, as an active ingredient, a
compound as defined above or a pharmaceutically acceptable salt
thereof together with a pharmaceutically acceptable carrier.
[0033] In a further aspect of the invention the present compounds
may be administered in combination with further pharmacologically
active substances, e.g., an antidiabetic agent or other
pharmacologically active material, including other compounds for
the treatment and/or prevention of insulin resistance and diseases
wherein insulin resistance is the pathophysiological mechanism.
[0034] Furthermore, the compounds according to the invention may be
administered in combination with antiobesity agents or food
intake-regulating agents.
[0035] A still further aspect of the present invention is a method
of identifying candidate compounds that regulate food intake,
characterized by screening out compounds that act as ligands for
the receptor GHS-R 1A. This method for identifying candidate
compounds comprises:
[0036] (a) contacting a growth hormone secretagogue receptor type
1A (GHS-R 1A), or a fragment thereof having GHS-R 1A ligand-binding
activity, in the presence of increasing amounts of a compound of
interest not known to have GHS-R 1A ligand-binding activity;
[0037] (b) measuring the binding of the known GHS-R 1A ligand to
the GHS-R 1A receptor; and
[0038] (c) determining the concentration of the compound of
interest at which the binding of said ligand to said receptor is
reduced to 50% of binding in the absence of said compound,
[0039] wherein, if said concentration is about 500 nm or less, the
compound is a candidate compound that regulates food intake.
[0040] A further aspect of the present invention relates to a
compound identified by or identifiable by this method.
PHARMACEUTICAL COMPOSITION
[0041] The compounds of the invention may be administered alone or
in combination with pharmaceutically acceptable carriers or
excipients, in either single or multiple doses. The pharmaceutical
compositions according to the invention may be formulated with
pharmaceutically acceptable carriers or diluents as well as any
other known adjuvants and excipients in accordance with
conventional techniques such as those disclosed in Remington: The
Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack
Publishing Co., Easton, Pa., 1995.
[0042] The pharmaceutical compositions may be specifically
formulated for administration by any suitable route such as the
oral, rectal, nasal, pulmonary, topical (including buccal and
sublingual), transdermal, intracisternal, intraperitoneal, vaginal
and parenteral (including subcutaneous, intramuscular, intrathecal,
intravenous and intradermal) route, the oral route being preferred.
It will be appreciated that the choice of route will depend on the
general condition and age of the subject to be treated, the nature
of the condition to be treated and the active ingredient
chosen.
[0043] Pharmaceutical compositions for oral administration include
solid dosage forms such as capsules, tablets, dragees, pills,
lozenges, powders and granules. Where appropriate, they can be
prepared with coatings, such as enteric coatings, or they can be
formulated so as to provide controlled release of the active
ingredient, such as sustained or prolonged release according to
methods well known in the art.
[0044] Liquid dosage forms for oral administration include
solutions, emulsions, suspensions, syrups and elixirs.
[0045] Pharmaceutical compositions for parenteral administration
include sterile aqueous and non-aqueous injectable solutions,
dispersions, suspensions or emulsions, as well as sterile powders
to be reconstituted in sterile injectable solutions or dispersions
prior to use. Depot injectable formulations are also contemplated
as being within the scope of the present invention.
[0046] Other suitable administration forms include suppositories,
sprays, ointments, creams, gels, inhalants, dermal patches,
implants, etc.
[0047] A typical oral dosage of a compound employed according to
the invention is in the range of from about 0.0001 to about 100
mg/kg body weight per day, preferably from about 0.001 to about 10
mg/kg body weight per day, and more preferably from about 0.01 to
about 1 mg/kg body weight per day, administered in one or more
doses, such as 1 to 3 doses.
[0048] The formulations may conveniently be presented in unit
dosage form by methods known to those skilled in the art. A typical
unit dosage form for oral administration one or more times per day,
such as 1 to 3 times per day, may contain from about 0.05 to about
2000 mg, preferably from about 0.1 to about 500 mg, and more
preferably from about 0.5 mg to about 200 mg of a compound employed
according to the invention.
[0049] For parenteral routes, such as intravenous, intrathecal,
intramuscular and similar routes of administration, doses are
typically of the order of about half the dose employed for oral
administration.
[0050] The compounds of this invention are generally utilized as
the free substance or as a pharmaceutically acceptable salt
thereof. One example is an acid addition salt of a compound having
a free base functionality. When a compound of the invention
contains a free base functionality, such salts are suitably
prepared in a conventional manner by treating a solution or
suspension of the free base form of the compound with, typically,
one equivalent (chemical equivalent, i.e. acid-base equivalent) of
a pharmaceutically acceptable acid, for example an inorganic or
organic acid chosen among the representative examples thereof
mentioned above. Physiologically acceptable salts of a compound
with a hydroxy group include the anion of said compound in
combination with a suitable cation, such as sodium or ammonium
ion.
[0051] For parenteral administration, solutions of the present
compounds in sterile aqueous solution, aqueous propylene glycol or
sesame or peanut oil may be employed. Such aqueous solutions should
be suitably buffered if necessary, and the liquid diluent first
rendered isotonic with sufficient saline or glucose. The aqueous
solutions are particularly suitable for intravenous, intramuscular,
subcutaneous and intraperitoneal administration. The sterile
aqueous media employed are all readily available by standard
techniques known to those skilled in the art.
[0052] Suitable pharmaceutical carriers include inert solid
diluents or fillers, sterile aqueous solution and various organic
solvents. Examples of solid carriers are lactose, terra alba,
sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia,
magnesium stearate, stearic acid or lower alkyl ethers of
cellulose. Examples of liquid carriers are syrup, peanut oil, olive
oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene
or water. Similarly, the carrier or diluent may include any
sustained-release material known in the art, such as glyceryl
monostearate or glyceryl distearate, alone or mixed with a wax. The
pharmaceutical compositions formed by combining the compounds of
the invention and the pharmaceutically acceptable carriers are then
readily administered in a variety of dosage forms suitable for the
disclosed routes of administration. The formulations may
conveniently be presented in unit dosage form by methods known in
the art of pharmacy.
[0053] Formulations of the present invention suitable for oral
administration may be presented as discrete units such as capsules
or tablets, each containing a predetermined amount of the active
ingredient, and which may include a suitable excipient. These
formulations may be in the form of powder or granules, as a
solution or suspension in an aqueous or non-aqueous liquid, or as
an oil-in-water or water-in-oil liquid emulsion.
[0054] If a solid carrier is used for oral administration, the
preparation may be tabletted, placed in a hard gelatin capsule in
powder or pellet form, or it can be in the form of a troche or
lozenge. The amount of solid carrier will vary widely, but will
usually be from about 25 mg to about 1 g. If a liquid carrier is
used, the preparation may be in the form of a syrup, emulsion, soft
gelatin capsule or sterile injectable liquid, such as an aqueous or
non-aqueous liquid suspension or solution.
[0055] A typical tablet that may be prepared by conventional
tabletting techniques may contain:
1 Core: Active compound (as free compound or salt thereof) 5 mg
Colloidal silicon dioxide (Aerosil .TM.) 1.5 mg Cellulose,
microcryst. (Avicel .TM.) 70 mg Modified cellulose gum (Ac-Di-Sol
.TM.) 7.5 mg Magnesium stearate q.s. Coating:
Hydroxypropylmethylcellulose (HPMC) approx. 9 mg *Mywacett .TM.
9-40 T approx. 0.9 mg *Acylated monoglyceride used as plasticizer
for film coating.
[0056] The compounds of the invention may be administered to a
mammal, especially a human, in need thereof. Such mammals include
also animals, both domestic animals, e.g., household pets, and
non-domestic animals such as wildlife.
[0057] If desired, the pharmaceutical composition of the invention
may comprise the compound of the invention in combination with
further pharmacologically active substances such as those described
in the foregoing.
[0058] The present invention is further illustrated by the
following examples which, however, are not to be construed as
limiting the scope of protection.
EXAMPLES
Example 1
Identification of Ligands for the Receptor GHS-R 1A
[0059] Transfection
[0060] Lipofectamine (Life Technologies, Rockville, Md., U.S.A.)
was used for transfection of BHK cells with a GHS-R 1A expression
vector (Howard, A. D. et al. (1996), Science 273, 974-977).
[0061] Receptor Binding Assay
[0062] Receptor binding was assayed as described in Hansen, B. S.
et al (1999) Eur.J.Endocrinol. 141:180-189. Briefly, crude
membranes from stably transfected BHK/GHS-R 1A cells were suspended
at 0.5 mg protein/ml in homogenization buffer (25 mM Tris-base, 2.5
mM EDTA, 10 mM MgCl.sub.2 and 30 .mu.g/ml bacitracin). In a
microtiter plate 10 .mu.l membrane suspension was combined with
either .sup.35S-labelled MK0677 (see Example 4) (Amersham Pharmacia
Biotech, Essex, UK) or 2-.sup.3H-adenosine (Amersham) as well as
binding buffer (2.5 mM Tris-base, 2.5 mM EDTA and 10 mM MgCl.sub.2)
to a total volume of 250 .mu.l. Non-specific binding was determined
by adding 10 .mu.M MK0677 (see Example 4) or 10 .mu.M adenosine to
the assay. The assay was subsequently incubated at 30.degree. C.
for 60 minutes, followed by application of the samples to GF/B
filters (Whatman, Kent, UK) which had been pre-treated with 0.5%
polyethylenimine for 60 minutes. The filters were subsequently
washed in 0.9% NaCl and counted using an Optiphase.TM. `HiSafe 3`
counter (Wallac, Turku, Finland).
[0063] To test for compounds that can compete with binding of
either .sup.35S-MK0677 or .sup.3H-adenosine to the GHS-R 1A,
different concentrations (0.001 nmol/l to 10 .mu.mol/l) of the
compound were added to the incubation described above.
[0064] Specific binding was determined as the difference between
total binding and non-specific binding (binding in the presence of
10 .mu.M unlabelled ligand). Binding curves were generated using
either the non-linear regression or hyperbolic fit feature of the
GraphPad.TM. Prism software package (GraphPad, San Diego, Calif.,
U.S.A.).
[0065] Calcium Imaging
[0066] To test for agonism or antagonism of the compounds that can
compete with the binding of the radiolabelled ligands in the above
receptor assay, a functional assay based on stimulation of the
release of Ca.sup.++ via the GHS-R was developed.
[0067] GHSR-expressing cells were cultured in Lab-TekTM chambered
coverglasses (Nalge Nunc Int., Naperville, Ill., USA). Prior to the
experiment, cells were loaded with the Ca.sup.2+-sensitive dye,
Fura2-AM (Molecular Probes Inc., Eugene, Oreg., U.S.A.), according
to standard procedures. The chambers were placed on a
temperature-regulated microscope stage and kept at 37.degree. C.
Fluorescence images were acquired using the MetaFluor.TM. software
package (Universal Imaging Corporation, West Chester, Pa., U.S.A.)
together with a Zeiss Axiovert.TM. 100S inverted microscope (Carl
Zeiss, Oberkochen, Germany) and a Princeton.TM. MicroMAX-5-1300Y
CCD camera (Princeton instruments, Trenton, N.J., U.S.A.). The
microscope was also equipped with a 530 nm.+-.15 nm emission
filter, a 500 nm dichroic mirror (Delta Lys & Optik, Lyngby,
Denmark) and a filterwheel (LUDL electronic products, Hawthorne,
N.Y., USA) harbouring 340 nm.+-.10 nm and 380 nm.+-.10 nm
excitation filters (Delta Lys & Optik). Images were acquired
every 3 seconds. After acquisition of 12-14 images, the cells were
stimulated with adenosine, MK0677 or other compounds. To test for
antagonism the compounds were added together with either adenosine
or MK0677. In each experiment the Fura2 ratio (ratio between the
measured intensities of emission at 510 nm following excitation at
340 nm and at 380 nm, respectively) was followed in 50 cells. A
normalized ratio was generated for each cell by dividing the Fura2
ratio at time t with the ratio at time zero. The data represented
the average normalized Fura2 ratio for 50 cells in a typical
experiment. All experiments were repeated at least 4 times (giving
similar results).
Example 2
Identification of Adenosine as a Ligand for the Receptor GHS-R
1A
[0068] The methods of example 1 were used. Adenosine was found to
be a potent ligand for the GHS-R 1A (EC.sub.50.about.50 nM using
the Ca.sup.++ assay described above). Binding studies were
performed to characterize the binding of adenosine to the GHS-R 1A,
and a K.sub.D of 87.+-.10 nM was determined.
[0069] Adenosine was, however, unable to stimulate GH secretion
from rat pituitary cells (assay described e.g. in Hansen, B. S. et
al (1999) Eur.J.Endocrinol. 141:180-189).
Example 3
Adenosine Does Not Stimulate Growth Hormone Release, But Stimulates
Feeding
[0070] The effect on GH release was studied in Halothane
anaesthetized male Wistar rats after intracerebroventricular (icv)
and intravenous (iv) administration of adenosine and also in
pentobarbital anaesthetized catheterized female Sprague Dawley (SD)
rats after iv administration. Vehicle or adenosine was given to
groups of rats (n=4-6) in the following doses: 10 .mu.g/rat and 100
.mu.g/rat icv dissolved in 5 .mu.l saline, 1 mg/kg iv and also 10
mg/kg iv to the SD rats. Blood samples were obtained from
anaesthetized animals before dosing, and either 10 min after dosing
or by frequent blood sampling through a catheter up until 45 min
after dosing. The plasma was analyzed for rat GH.
[0071] The effect on feeding was studied in conscious non-deprived
male Wistar rats (n=7-10) after icv dosing of vehicle or adenosine
(1 .mu.g/rat and 10 .mu.g/rat in 5 .mu.l saline). Food intake was
measured in feeding boxes with standard chow and water placed on
balances connected to a computer. Changes in weight of chow and
water were continuously registered. Food and water intake were
measured for 3 hours after drug injection during early daytime,
when food intake is normally at a very low level.
[0072] The results showed that adenosine did not stimulate GH
release in any of the given doses, neither by the icv nor the iv
route of administration.
[0073] With respect to the orexigenic effect of adenosine, there
was no effect after 1 .mu.g/rat, but food intake was significantly
increased compared to the vehicle control group after injection of
10 .mu.g/rat adenosine icv.
Example 4
Correlation Between GHS-R 1A Binding Affinity and the Effect on
Food Intake
[0074] The compounds used were characterized by showing equipotency
with respect to in vitro GH release from primary rat somatotrophs,
although they displayed significantly different binding affinity to
the GHS-R 1A.
[0075] The following compounds (structures shown below) were
employed: NNC 26-1291, with a high binding affinity to the receptor
similar to that of MK0677 (K.sub.l=0.4.+-.0.06 nM), NNC 26-1187
with an intermediate binding affinity (K.sub.l=22.2.+-.7 nM), and
NN703 displaying a weak binding affinity (K.sub.l=112.+-.29
nM).
[0076] NN703 is described in WO 97/23508, and this compound,
together with MK0677 (the latter also being denoted MK677), is also
described in, e.g., Drug Discovery Today, vol. 4, No.11, November
1999, pp. 497-506. NNC 26-1291 and NNC 26-1187 are growth hormone
secretagogues of a non-peptidyl nature and are described in WO
99/58501 and WO 00/26252, respectively.
[0077] The effect on feeding was tested after icv injections in
non-deprived male Wistar rats. The compounds were dissolved in 5
.mu.l saline and injected in the following doses: 10.0 .mu.g per
rat (NNC 26-1291) and 30.0 .mu.g per rat (NN703 and NNC
26-1187).
[0078] Food intake was measured in feeding boxes, where standard
chow and water were placed on balances and changes in weight
continuously registered. Food and water intake was measured for 3
hours after drug injection in the beginning of the light part of
the diurnal cycle, when food intake is normally at a very low
level. All three compounds increased food intake. The effect of
NN703 was moderate, with a food intake at about 3 grams, while NNC
26-1187 and particularly NNC 26-1291 showed stronger effects with
food intakes at about 4-5 grams. For comparison, food intake in
control animals is normally about 1 gram, and icv administration of
10 .mu.g porcine neuropeptide Y (NPY), which has a strong effect on
food intake, increases food consumption to about 8 grams in this
model.
[0079] The compounds employed were as follows: 1
Example 5
Ghrelin Stimulates Feeding, But Not GH Release, After icv
Administration
[0080] Non-deprived male Wistar rats (n=6-10) were used for the
study, dosed with vehicle or with ghrelin at 10 .mu.g/rat icv
dissolved in 5 .mu.l saline. Food intake was measured in conscious
animals, using feeding boxes. Food and water intake were measured
for 3 hours after drug injection during early daytime, when food
intake is normally at a very low level. GH release was measured in
Halothane-anaesthetized animals from which blood samples were
obtained, before dosing and 10 min after dosing, via the orbital
plexus. The plasma was analyzed for rat GH.
[0081] Ghrelin administered in this manner (icv administration)
produced a significant effect on feeding, compared to vehicle, but
did not stimulate GH release. In contrast, it is known that ghrelin
administration by the iv route causes stimulation of GH
release.
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