U.S. patent application number 13/618265 was filed with the patent office on 2013-01-24 for peptide yy and peptide yy agonists for treatment of metabolic disorders.
This patent application is currently assigned to Amylin Pharmaceuticals, LLC. The applicant listed for this patent is James R. Paterniti, JR., Richard A. PITTNER, Andrew A. Young. Invention is credited to James R. Paterniti, JR., Richard A. PITTNER, Andrew A. Young.
Application Number | 20130023466 13/618265 |
Document ID | / |
Family ID | 22971453 |
Filed Date | 2013-01-24 |
United States Patent
Application |
20130023466 |
Kind Code |
A1 |
PITTNER; Richard A. ; et
al. |
January 24, 2013 |
Peptide YY and Peptide YY Agonists for Treatment of Metabolic
Disorders
Abstract
Methods and compositions are disclosed to treat metabolic
disorders such as obesity, diabetes, and increased cardiovascular
risk comprising administering a therapeutically effective amount of
a PYY or a PYY agonist.
Inventors: |
PITTNER; Richard A.; (Menlo
Park, CA) ; Young; Andrew A.; (Chapel Hill, NC)
; Paterniti, JR.; James R.; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PITTNER; Richard A.
Young; Andrew A.
Paterniti, JR.; James R. |
Menlo Park
Chapel Hill
San Diego |
CA
NC
CA |
US
US
US |
|
|
Assignee: |
Amylin Pharmaceuticals, LLC
San Diego
CA
|
Family ID: |
22971453 |
Appl. No.: |
13/618265 |
Filed: |
September 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10016969 |
Dec 14, 2001 |
8273713 |
|
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13618265 |
|
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60256216 |
Dec 14, 2000 |
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Current U.S.
Class: |
514/5.2 ;
514/6.9; 514/7.4; 514/9.7 |
Current CPC
Class: |
A61K 38/2271 20130101;
A61P 3/06 20180101; A61P 3/10 20180101; A61P 9/12 20180101; A61P
3/08 20180101; A61K 38/22 20130101; A61P 3/12 20180101; A61P 9/00
20180101; A61P 3/04 20180101 |
Class at
Publication: |
514/5.2 ;
514/6.9; 514/7.4; 514/9.7 |
International
Class: |
A61K 38/22 20060101
A61K038/22; A61P 3/04 20060101 A61P003/04; A61P 9/12 20060101
A61P009/12; A61P 9/00 20060101 A61P009/00; A61P 3/10 20060101
A61P003/10; A61P 3/06 20060101 A61P003/06 |
Claims
1. A method of treating obesity comprising administering to an
obese subject a therapeutically effective amount of a PYY or a PYY
agonist.
2. The method of claim 1 wherein the subject is at least one of
insulin resistant or glucose intolerant.
3. The method of claim 1 wherein the subject has diabetes
mellitus.
4. The method of claim 1 wherein the PYY agonist is PYY[3-36].
5. The method of claim 1 wherein the PYY or PYY agonist is
administered peripherally.
6. The method of claim 1 wherein about 1 .mu.g to about 5 mg of the
PYY or PYY agonist is administered per day in single or divided
doses.
7. The method of claim 1 wherein about 0.01 .mu.g/kg to about 500
.mu.g/kg of the PYY or PYY agonist is administered per dose.
8. A method of reducing food intake comprising administering to a
subject a therapeutically effective amount of a PYY or a PYY
agonist.
9. The method of claim 8 wherein the PYY agonist is PYY[3-36].
10. The method of claim 8 wherein the PYY or PYY agonist is
administered peripherally.
11. The method of claim 8 wherein about 1 .mu.g to about 5 mg of
the PYY or PYY agonist is administered per day in single or divided
doses.
12. The method of claim 8 wherein about 0.01 .mu.g/kg to about 500
.mu.g/kg of the PYY or PYY agonist is administered per dose.
13. A method of treating diabetes mellitus in a subject comprising
administering an effective amount of a PYY or a PYY agonist.
14. The method of claim 13 wherein the PYY or PYY agonist is
administered peripherally.
15. The method of claim 13 wherein the subject has Type II
diabetes.
16. The method of claim 13 wherein the subject is overweight.
17. The method of claim 13 wherein the PYY agonist is
PYY[3-36].
18. The method of claim 13 wherein about 1 .mu.g to about 5 mg of
the PYY or PYY agonist is administered per day in single or divided
doses.
19. The method of claim 13 wherein about 0.01 .mu.g/kg to about 500
.mu.g/kg of the PYY or PYY agonist is administered per dose.
20. A method of improving lipid profile in a subject comprising
administering to the subject an effective amount of a PYY or a PYY
agonist.
21. The method of claim 20 wherein the improvement in lipid profile
comprises at least one of reducing LDL cholesterol levels, reducing
triglyceride levels and increasing HDL cholesterol levels.
22. The method of claim 20 wherein the PYY or PYY agonist is
administered peripherally.
23. A method for treating conditions or disorders which can be
alleviated by reducing nutrient availability in a subject
comprising administering to said subject a therapeutically
effective amount of a PYY or a PYY agonist.
24. The method of claim 23 wherein the condition or disorder is
hypertension.
25. The method of claim 23 wherein the condition or disorder is
dyslipidemia.
26. The method of claim 23 wherein the condition is cardiovascular
risk.
27. The method of claim 23 wherein the disorder is an eating
disorder.
28. The method of claim 23 wherein the condition or disorder is
insulin-resistance.
29. The method of claim 23 wherein the condition is obesity.
30. The method of claim 23 wherein the condition is diabetes
mellitus.
31. The method of claim 23 wherein about 1 .mu.g to about 5 mg of
the PYY or PYY agonist is administered per day in single or divided
doses.
32. The method of claim 23 wherein about 0.01 .mu.g/kg to about 500
.mu.g/kg of the PYY or PYY agonist is administered per dose.
33. The method of any of claims 1, 8, 13, 20, and 23 wherein the
PYY agonist has a potency in at least one of a food intake or
gastric emptying assay greater than NPY.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of and claims priority to
pending U.S. patent application Ser. No. 10/016,969, entitled
"Peptide YY and Peptide YY Agonists for Treatment of Metabolic
Disorders," filed Dec. 14, 2001, and claims priority to U.S.
Provisional Application Ser. No. 60/256,216 entitled "Peptide YY
and Peptide YY Agonists for Treatment of Obesity, Diabetes, and
other Metabolic Disorders," filed Dec. 15, 2000, each of which are
hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to methods and compositions
for treating metabolic conditions or disorders, particularly those
which can be alleviated by reducing caloric availability, for
example diabetes, obesity, eating disorders, insulin-resistance
syndrome (Syndrome X), glucose intolerance, dyslipidemia, and
cardiovascular disorders.
BACKGROUND
[0003] A number of related hormones make up the pancreatic
polypeptide (PP) family. Pancreatic polypeptide was discovered as a
contaminant of insulin extracts and was named more by its organ of
origin, rather than functional importance (Kimmel, Pollock et al.
Endocrinology 83: 1323-30, 1968). It is a 36-amino acid peptide
[SEQ ID NO.: 1] containing distinctive structural motifs. A related
peptide was subsequently discovered in extracts of intestine and
named Peptide YY (PYY) because of the N- and C-terminal tyrosines
(Tatemoto. Proc Natl Acad Sci USA 79: 2514-8, 1982) [SEQ ID NO.:
2]. A third related peptide was later found in extracts of brain
and named Neuropeptide Y (NPY) (Tatemoto. Proc Natl Acad Sci USA
79: 5485-9, 1982; Tatemoto, Carlquist et al. Nature 296: 659-60,
1982) [SEQ ID NO.: 4].
[0004] These three related peptides have been reported to exert
various biological effects. Effects of PP include inhibition of
pancreatic secretion and relaxation of the gallbladder. Centrally
administered PP produces modest increases in feeding that may be
mediated by receptors localized to the hypothalamus and brainstem
(reviewed by (Gehlert. Proc Soc Exp Biol Med 218: 7-22, 1998)).
[0005] Release of PYY [SEQ ID NO.: 2] occurs following a meal. An
alternate molecular form of PYY is PYY[3-36] [SEQ ID NO.: 3]
(Eberlein, Eysselein et al. Peptides 10: 797-803, 1989) (Grandt,
Schimiczek et al. Regul Pept 51: 151-9, 1994). This fragment
constitutes approximately 40% of total PYY-like immunoreactivity in
human and canine intestinal extracts and about 36% of total plasma
PYY immunoreactivity in a fasting state to slightly over 50%
following a meal. It is apparently a dipeptidyl peptidase-IV (DPP4)
cleavage product of PYY. PYY[3-36] is reportedly a selective ligand
at the Y2 and Y5 receptors, which appear pharmacologically unique
in preferring N-terminally truncated (i.e. C-terminal fragments of)
NPY analogs. Peripheral administration of PYY reportedly reduces
gastric acid secretion, gastric motility, exocrine pancreatic
secretion (Yoshinaga, Mochizuki et al. Am J Physiol 263: G695-701,
1992) (Guan, Maouyo et al. Endocrinology 128: 911-6, 1991) (Pappas,
Debas et al. Gastroenterology 91: 1386-9, 1986), gallbladder
contraction and intestinal motility (Savage, Adrian et al. Gut 28:
166-70, 1987). The effects of central injection of PYY on gastric
emptying, gastric motility and gastric acid secretion, as seen
after direct injection in or around the hindbrain/brainstem (Chen
and Rogers. Am J Physiol 269: R787-R792, 1995) (Chen, Rogers et al.
Regul Pept 61: 95-98, 1996) (Yang and Tache. Am J Physiol 268:
G943-8, 1995) (Chen, Stephens et al. Neurogastroenterol Motil 9:
109-116, 1997), may differ from those effects observed after
peripheral injection. For example, centrally administered PYY had
some effects opposite to those described herein for peripherally
injected PYY[3-36] in that gastric acid secretion was stimulated,
not inhibited. Gastric motility was suppressed only in conjunction
with TRH stimulation, but not when administered alone, and was
indeed stimulatory at higher doses through presumed interaction
with PP receptors. PYY has been shown to stimulate food and water
intake after central administration (Morley, Levine et al. Brain
Res 341: 200-203, 1985) (Corp, Melville et al. Am J Physiol 259:
R317-23, 1990).
[0006] Likewise, one of the earliest reported central effects of
NPY [SEQ ID NO.: 4] was to increase food intake, particularly in
the hypothalamus (Stanley, Daniel et al. Peptides 6: 1205-11,
1985). PYY and PP are reported to mimic these effects, and PYY is
more potent or as potent as NPY (Morley, J. E., Levine, A. S.,
Grace, M., and Kneip, J. Brain Res 341: 200-203, 1985) (Kanatani,
Mashiko et al. Endocrinology 141: 1011-6, 2000) (Nakajima, Inui et
al. J Pharmacol Exp Ther 268: 1010-4, 1994). Several groups found
the magnitude of NPY-induced feeding to be higher than that induced
by any pharmacological agent previously tested, and also extremely
long-lasting. NPY-induced stimulation of feeding has been
reproduced in a number of species. Among the three basic
macronutrients (fat, protein, and carbohydrate), the intake of
carbohydrates was preferentially stimulated. No tolerance was seen
towards the orexigenic effect of NPY, and when administration of
the peptide was repeated over 10 days, a marked increase in the
rate of weight gain was observed. Following starvation, the
concentration of NPY in the hypothalamic PVN increased with time,
and returned rapidly to control levels following food
ingestion.
[0007] Pharmacological studies and cloning efforts have revealed a
number of seven transmembrane receptors for the PP family of
peptides, and these receptors have been assigned the names Y1
through Y6 (and a putative PYY-preferring receptor or Y7). Typical
signaling responses of these receptors are similar to those of
other G.sub.i/G.sub.o-coupled receptors, namely inhibition of
adenylate cyclase. Even with fairly low sequence homology among
receptors, it is apparent that there is a clustering of amino acid
sequence similarity between Y1, Y4 and Y6 receptors, while Y2 and
Y5 define other families. Other binding sites have been identified
by the rank order of potency of various peptides. The
NPY-preferring receptor, which has not been cloned, has been termed
Y3, and PYY-preferring receptors have also been shown to exist
(putative Y7) (Reviewed in (Michel, Beck-Sickinger et al. Pharmacol
Rev 50: 143-50, 1998) (Gehlert, D. R. Proc Soc Exp Biol Med 218:
7-22, 1998)).
[0008] The Y5 and Y1 receptors have been suggested as the primary
mediators of the food intake response (Marsh, Hollopeter et al. Nat
Med 4: 718-21, 1998) (Kanatani, A., Mashiko, S., Murai, N.,
Sugimoto, N., Ito, J., Fukuroda, T., Fukami, T., Morin, N.,
MacNeil, D. J., Van der Ploeg, L. H., Saga, Y., Nishimura, S., and
Ihara, M. Endocrinology 141: 1011-6, 2000). The prevalent idea has
been that endogenous NPY, via these receptors, increases feeding
behavior. Proposed therapies for obesity have invariably been
directed toward antagonism of NPY receptors, while therapies for
treating anorexia have been directed toward agonists of this ligand
family (see, e.g., U.S. Pat. Nos. 5,939,462; 6,013,622; and
4,891,357). In general, PYY and NPY are reported to be equipotent
and equally effective in all Y1, Y5 (and Y2) receptor assays
studied (Gehlert, D. R. Proc Soc Exp Biol Med 218: 7-22, 1998).
[0009] The main characteristic of putative Y3 receptors is that
they recognize NPY, while PYY is at least an order of magnitude
less potent. The Y3 receptor represents the only binding
site/receptor that shows a preference for NPY.
[0010] There is an additional binding site/receptor which shows
preference for PYYs, termed PYY-preferring receptor, which is
referred to herein as the Y7 receptor (s). Different rank orders of
binding to this receptor, or class of receptors, have been
reported, suggesting that there may be more than one receptor in
this family. In most cases it has been applied to describe a
receptor where PYY was three to five times more potent than NPY.
The International Union of Pharmacology recommendations for the
nomenclature of NPY, PYY and PP receptors are that the term
PYY-preferring receptor is not used unless a potency difference of
at least twenty fold between PYY and NPY is observed (Michel, M.
C., Beck-Sickinger, A., Cox, H., Doods, H. N., Herzog, H.,
Larhammar, D., Quirion, R., Schwartz, T., and Westfall, T.
Pharmacol Rev 50: 143-50, 1998). However, for purposes of this
disclosure, reference to the Y7 receptor or pharmacology of a
PYY-preferring receptor means a receptor having any degree of
preference for PYY over NPY.
[0011] Obesity and its associated disorders are common and very
serious public health problems in the United States and throughout
the world. Upper body obesity is the strongest risk factor known
for type 2 diabetes mellitus, and is a strong risk factor for
cardiovascular disease. Obesity is a recognized risk factor for
hypertension, atherosclerosis, congestive heart failure, stroke,
gallbladder disease, osteoarthritis, sleep apnea, reproductive
disorders such as polycystic ovarian syndrome, cancers of the
breast, prostate, and colon, and increased incidence of
complications of general anesthesia. (see, e.g., (Kopelman. Nature
404: 635-43, 2000)). It reduces life-span and carries a serious
risk of co-morbidities above, as well disorders such as infections,
varicose veins, acanthosis nigricans, eczema, exercise intolerance,
insulin resistance, hypertension hypercholesterolemia,
cholelithiasis, orthopedic injury, and thromboembolic disease
(Rissanen, Heliovaara et al. BMJ 301: 835-7, 1990). Obesity is also
a risk factor for the group of conditions called insulin resistance
syndrome, or "Syndrome X." Recent estimates for the medical cost of
obesity and associated disorders are $150 billion worldwide. The
pathogenesis of obesity is believed to be multifactoral but the
basic problem is that in obese subjects nutrient availability and
energy expenditure do not come into balance until there is excess
adipose tissue. Obesity is currently a poorly treatable, chronic,
essentially intractable metabolic disorder. A therapeutic drug
useful in weight reduction of obese persons could have a profound
beneficial effect on their health.
[0012] All documents referred to herein are incorporated by
reference into the present application as though fully set forth
herein.
SUMMARY OF THE INVENTION
[0013] It has been discovered that, contrary to reported activities
of central administration of members of the pancreatic polypeptide
family, peripheral administration of PYY and PYY agonists reduces
nutrient availability and is useful in the treatment of obesity and
related disorders. PYY and PYY agonist compositions and uses
thereof are disclosed herein to modulate nutrient availability in a
patient for treating metabolic disorders which may be benefited by
a reduction in nutrient availability. These methods will be useful
in the treatment of, for example, obesity, diabetes, including but
not limited to type 2 or non-insulin dependent diabetes, eating
disorders, insulin-resistance syndrome, and cardiovascular
disease.
[0014] By "PYY" is meant a Peptide YY polypeptide obtained or
derived from any species. Thus, the term "PYY" includes both the
human full length, 36 amino acid peptide as set forth in SEQ ID NO:
2, and species variations of PYY, including e.g., murine, hamster,
chicken, bovine, rat, and dog PYY, for example. By "PYY agonist" is
meant any compound which elicits an effect of PYY to reduce
nutrient availability, for example a compound (1) having activity
in the food intake, gastric emptying, pancreatic secretion, or
weight loss assays described herein in Examples 1, 2, 5, or 6, and
(2) which binds specifically in a Y receptor assay (Example 10) or
in a competitive binding assay with labeled PYY or PYY[3-36] from
certain tissues having an abundance of Y receptors, including e.g.,
area postrema (Example 9), wherein the PYY agonist is not
pancreatic polypeptide. Preferably, PYY agonists would bind in such
assays with an affinity of greater than 1 .mu.M, and more
preferably with an affinity of greater than 1-5 nM.
[0015] Such agonists can comprise a polypeptide having a functional
PYY domain, an active fragment of PYY, or a chemical or small
molecule. PYY agonists may be peptide or non-peptide compounds, and
include "PYY agonist analogs," which refer to any compound
structurally similar to a PYY that have PYY activity typically by
virtue of binding to or otherwise directly or indirectly
interacting with a PYY receptor or other receptor or receptors with
which PYY itself may interact to elicit a biological response. Such
compounds include derivatives of PYY, extended PYY molecules having
more than 36 amino acids, truncated PYY molecules having less than
36 amino acids, and substituted PYY molecules having one or more
different amino acids, or any combination of the above. Such
compounds may also be modified by processes such as amidation,
glycosylation, acylation, sulfation, phosphorylation, acetylation
and cyclization.
[0016] One such PYY agonist analog is PYY[3-36], identified herein
as SEQ ID NO: 3. Polypeptides with numbers in brackets refer to
truncated polypeptides having the sequence of the full length
peptide over the amino acid positions in the brackets. Thus,
PYY[3-36] has a sequence identical to PYY over amino acids 3 to 36.
A PYY agonist may bind to a PYY receptor with higher or lower
affinity, demonstrate a longer or shorter half-life in vivo or in
vitro, or be more or less effective than native PYY.
[0017] By "condition or disorder which can be alleviated by
reducing caloric (or nutrient) availability" is meant any condition
or disorder in a subject that is either caused by, complicated by,
or aggravated by a relatively high nutrient availability, or that
can be alleviated by reducing nutrient availability, for example by
decreasing food intake. Such conditions or disorders include, but
are not limited to, obesity, diabetes, including type 2 diabetes,
eating disorders, and insulin-resistance syndromes.
[0018] In one aspect, the invention provides a method of treating
obesity in an obese or overweight subject by administering a
therapeutically effective amount of a PYY or a PYY agonist. While
"obesity" is generally defined as a body mass index over 30, for
purposes of this disclosure, any subject, including those with a
body mass index of less than 30, who needs or wishes to reduce body
weight is included in the scope of "obese." Subjects who are
insulin resistant, glucose intolerant, or have any form of diabetes
mellitus (e.g., type 1, 2 or gestational diabetes) can benefit from
this method.
[0019] In other aspects, the invention features methods of reducing
food intake, treating diabetes mellitus, and improving lipid
profile (including reducing LDL cholesterol and triglyceride levels
and/or changing HDL cholesterol levels) comprising administering to
a subject a therapeutically effective amount of a PYY or a PYY
agonist. In a preferred embodiment, the methods of the invention
are used to treat conditions or disorders which can be alleviated
by reducing nutrient availability in a subject in need thereof,
comprising administering to said subject a therapeutically
effective amount of a PYY or a PYY agonist. Such conditions and
disorders include, but are not limited to, hypertension,
dyslipidemia, cardiovascular disease, eating disorders,
insulin-resistance, obesity, and diabetes mellitus of any kind.
[0020] In the methods of the invention, preferred PYY agonists are
those having a potency in one of the assays described herein
(preferably food intake, gastric emptying, pancreatic secretion, or
weight reduction assays) which is greater than the potency of NPY
in that same assay.
[0021] For all indications, in preferred embodiments, a preferred
PYY agonist is PYY[3-36], and is preferably administered
peripherally at a dose of about 1 .mu.g to about 5 mg per day in
single or divided doses, or at about 0.01 .mu.g/kg to about 500
.mu.g/kg per dose, more preferably about 0.05 .mu.g/kg to about 250
.mu.g/kg, most preferably below about 50 .mu.g/kg. Dosages in these
ranges will vary with the potency of each agonist, of course, and
are readily determined by one of skill in the art.
[0022] In the methods of the present invention, PYY's and PYY
agonists may be administered separately or together with one or
more other compounds and compositions that exhibit a long term or
short-term action to reduce nutrient availability, including, but
not limited to other compounds and compositions that comprise an
amylin or amylin agonist, a cholecystokinin (CCK) or CCK agonist, a
leptin (OB protein) or leptin agonist, an exendin or exendin
agonist, or a GLP-1 or GLP-1 agonist. Suitable amylin agonists
include, for example, [25,28,29Pro-]-human amylin (also known as
"pramlintide," and described in U.S. Pat. Nos. 5,686,511 and
5,998,367) and salmon calcitonin. The CCK used is preferably CCK
octopeptide (CCK-8). Leptin is discussed in, for example,
(Pelleymounter, Cullen et al. Science 269: 540-543, 1995) (Halaas,
Gajiwala et al. Science 269: 543-6, 1995) and (Campfield, Smith et
al. Science 269: 546-549, 1995). Suitable exendins include
exendin-3 and exendin-4, and exendin agonist compounds include, for
example, those described in PCT Publications WO 99/07404, WO
99/25727, and WO 99/25728.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a plot of the activity of Y receptor ligands in a
food intake assay in overnight-fasted NIH/SW mice.
[0024] FIG. 2 is a plot of activity of various Y receptor ligands
on gastric emptying in HSD rats.
[0025] FIG. 3 demonstrates inhibition of gastric acid secretion in
rats upon acute peripheral administration of PYY[3-36]. Data are
expressed as .mu.mol of acid secreted/10 min.
[0026] FIG. 4 is a bar graph demonstrating that acute peripheral
administration of PYY[3-36] prevents gallbladder emptying in mice.
This effect could be reversed by administration of CCK-8.
[0027] FIG. 5 shows an acute, dose-dependent effect of
subcutaneously administered PYY[3-36] to inhibit CCK-8-stimulated
exocrine pancreatic secretion (assayed by amylase activity) in
rats.
[0028] FIG. 6 illustrates a decreased body weight gain in fattened
C57BI/6 (diet-induced obese, or DIO) mice with continuous
peripheral PYY[3-36] infusion over a period of four weeks.
[0029] FIG. 7 shows the effect of continuous peripheral infusion of
PYY[3-36] to decrease caloric efficiency in fattened C57BI/6
(diet-induced obese, or DIO) mice over a period of four weeks.
[0030] FIG. 8 demonstrates the improvement in glycemic control as
measured by percent change in HbA1c over a period of 28 days in
obese diabetic (ZDF) rats upon continuous peripheral infusion of
PYY[3-36].
DETAILED DESCRIPTION OF THE INVENTION
[0031] It has been generally accepted that endogenous NPY (reviewed
in (Schwartz, Woods et al. Nature 404: 661-71, 2000)), and PYY
(Morley, J. E., Levine, A. S., Grace, M., and Kneip, J. Brain Res
341: 200-203, 1985)), via their receptors, increase feeding
behavior. Methods directed at therapies for obesity have invariably
attempted to antagonize Y receptors, while claims for treating
anorexia have been directed at agonists of this ligand family.
However, as described and claimed herein, it has been surprisingly
discovered that peripheral administration of PYY and agonists
thereof has a potent effect to reduce nutrient availability, rather
than increase it as suggested by reports in the patent and
scientific literature (see e.g. U.S. Pat. Nos. 5,912,227 and
6,315,203 which disclose the use of PYY receptor agonists to
increase weight gain). The spectrum of actions of inhibition of
food intake, slowing of gastric emptying, inhibition of gastric
acid secretion, and inhibition of pancreatic enzyme secretion, are
useful to exert clinical benefit in metabolic diseases such as type
1, type 2, or gestational diabetes mellitus, obesity and other
manifestations of insulin-resistance syndrome (Syndrome X), and in
any other use for reducing nutrient availability.
[0032] Use of PYY and PYY agonists to reduce food intake and
nutrient availability, and in treating disorders such as diabetes
and obesity, has not been previously asserted. In fact, such
utility in diabetes would not be predicted from the absence of
acute effect on blood glucose and from reports of inhibition of
insulin secretion. However, it is demonstrated herein that this
group of ligands and agonist ligands will be useful in these
condition and related conditions.
[0033] The Applicant's data demonstrate that the effects of
peripherally-administered PYY or PYY[3-36] to reduce food intake
and to delay gastric emptying are determined by interactions with
one or more unique receptor classes in, or similar to, those in the
Y-fold family. The data are best explained by interactions with a
receptor or receptors similar to the PYY-preferring (or Y7)
receptors, and are less well explained by interactions with the
other known Y receptors such as Y1-Y6. Table 1 (below) shows
published PP family ligand potencies reported at the known
receptors, as well as certain unpublished data of Applicants, and
the rank order of potencies of various ligands. The rank order of
potency in the Examples herein does not correspond to any single
published receptor pharmacology, and indicates a novel mechanism of
PYY action in reducing caloric availability.
TABLE-US-00001 TABLE 1 Summary of receptor pharmacology for the PP
family of receptors derived from published data and patents. The
peripheral appetite and gastric emptying data are unpublished data
of the Applicant. RECEPTORS PHARMACOLOGY REFERENCE Food Intake
PYY[3-36] .gtoreq. PYY >> NPY = NPY[3-36] = PP = Applicant
data Inhibition Ac-PYY[22-36] (See FIG. 1) (peripheral) Gastric
PYY[3-36] .gtoreq. PYY >> NPY = NPY[3-36] = PP = Applicant
data Emptying Ac-PYY[22-36] (See FIG. 2) Food Intake PYY .gtoreq.
PYY[3-36] = NPY = NPY[3-36] > PP (Iyengar, Li et al. J Pharmacol
Exp Stimulation Ther 289: 1031-40, 1999) (central) Y1 NPY = PYY
> NPY[3-36] = PYY[3-36] = PP (Iyengar, S., Li, D. L., and
Simmons, R. M. J Pharmacol Exp Ther 289: 1031-40, 1999) (Gehlert,
D. R. Proc Soc Exp Biol Med 218: 7-22, 1998; Michel, M. C.,
Beck-Sickinger, A., Cox, H., Doods, H. N., Herzog, H., Larhammar,
D., Quirion, R., Schwartz, T., and Westfall, T. Pharmacol Rev 50:
143-50, 1998) U.S. Pat. No. 5,968,819 Y2 NPY = PYY = PYY[3-36] =
NPY[3-36] >> PP (Gehlert, D. R. Proc Soc Exp Biol Med 218:
7-22, 1998; Michel, M. C., Beck- Sickinger, A., Cox, H., Doods, H.
N., Herzog, H., Larhammar, D., Quirion, R., Schwartz, T., and
Westfall, T. Pharmacol Rev 50: 143-50, 1998; Iyengar, S., Li, D.
L., and Simmons, R. M. J Pharmacol Exp Ther 289: 1031-40, 1999)U.S.
Pat. No. 5,968,819 Y3 NPY > PP > PYY (Gehlert, D. R. Proc Soc
Exp Biol Med 218: 7-22, 1998; Michel, M. C., Beck- Sickinger, A.,
Cox, H., Doods, H. N., Herzog, H., Larhammar, D., Quirion, R.,
Schwartz, T., and Westfall, T. Pharmacol Rev 50: 143-50, 1998) Y4
PP > PYY > NPY > PYY[3-36] = NPY[3-36] (Gehlert, D. R.
Proc Soc Exp Biol Med 218: 7-22, 1998; Michel, M. C., Beck-
Sickinger, A., Cox, H., Doods, H. N., Herzog, H., Larhammar, D.,
Quirion, R., Schwartz, T., and Westfall, T. Pharmacol Rev 50:
143-50, 1998; Iyengar, S., Li, D. L., and Simmons, R. M. J
Pharmacol Exp Ther 289: 1031-40, 1999)U.S. Pat. No. 5,968,819 Y5
NPY = PYY .gtoreq. PP .gtoreq. PYY[3-36] = NPY[3-36] (Gehlert, D.
R. Proc Soc Exp Biol Med 218: 7-22, 1998; Michel, M. C., Beck-
Sickinger, A., Cox, H., Doods, H. N., Herzog, H., Larhammar, D.,
Quirion, R., Schwartz, T., and Westfall, T. Pharmacol Rev 50:
143-50, 1998; Iyengar, S., Li, D. L., and Simmons, R. M. J
Pharmacol Exp Ther 289: 1031-40, 1999)U.S. Pat. No. 5,968,819 Y6
NPY = PYY .gtoreq. NPY[3-36] > PP (Gehlert, D. R. Proc Soc Exp
Biol Med 218: 7-22, 1998; Michel, M. C., Beck- Sickinger, A., Cox,
H., Doods, H. N., Herzog, H., Larhammar, D., Quirion, R., Schwartz,
T., and Westfall, T. Pharmacol Rev 50: 143-50, 1998; Iyengar, S.,
Li, D. L., and Simmons, R. M. J Pharmacol Exp Ther 289: 1031-40,
1999)U.S. Pat. No. 5,968,819 (Y7) PYY > NPY >> PYY[3-36] =
PP (Yang, Li et al. Br J Pharmacol 123: 1549-54, 1998) (Y7)
PYY[3-36] .gtoreq. PYY > NPY >> PP (Haynes, Hill et al. Br
J Pharmacol 122: 1530-6, 1997) (Y7) PYY >> NPY = PYY[3-36] =
PP (Kawakubo, Yang et al. Brain Res 854: 30-4, 2000)
[0034] Any PYY or PYY agonist may be useful in the invention.
Preferred PYY agonists include peptide agonists, particularly PYY
agonist analogs such as PYY[3-36]. Analogs may be made by, e.g.,
conservative amino acid substitution of the sequence of PYY or
portions thereof, and can be tested in the assays provided in the
Examples or other suitable assays that distinguish the actions of
PYY from those of NPY or PP. Non-peptide agonists are also
contemplated.
[0035] The spectrum of actions exhibited by PYY, e.g., inhibition
of food intake, slowing of gastric emptying, inhibition of gastric
acid secretion, inhibition of pancreatic enzyme secretion, etc.,
act in a coordinated way to restrict nutrient assimilation and
thereby exert clinical benefit in metabolic diseases such diabetes
mellitus, obesity, cardiovascular disease (atherosclerosis,
hypertension, dyslipidemia, etc.), and manifestations of
insulin-resistance syndromes (e.g., Syndrome X).
[0036] The human sequences of peptides in the PP ligand family
referred to herein are as follows (in conventional one-letter amino
acid code):
TABLE-US-00002 PP: (SEQ ID NO: 1)
APLEPVYPGDNATPEQMAQYAADLRRYINMLTRPRY PYY: (SEQ ID NO: 2)
YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY PYY[3-36]: (SEQ ID NO: 3)
IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY NPY: (SEQ ID NO: 4)
YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY
[0037] These peptides are C-terminally amidated when expressed
physiologically, but need not be for the purposes of the instant
invention. These peptides may also have other post-translational
modifications.
[0038] PYY and peptide-based PYY agonists described herein may be
prepared using standard recombinant expression or chemical peptide
synthesis techniques known in the art, e.g., using an automated or
semiautomated peptide synthesizer.
[0039] Solid phase peptide synthesis may be carried out with an
automatic peptide synthesizer (e.g., Model 430A, Applied Biosystems
Inc., Foster City, Calif.) using the NMP/HOBt (Option 1) system and
tBoc or Fmoc chemistry (see, Applied Biosystems User's Manual for
the ABI 430A Peptide Synthesizer, Version 1.3B Jul. 1, 1988,
section 6, pp. 49-70, Applied Biosystems, Inc., Foster City,
Calif.) with capping. Peptides may be also be assembled using an
Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Ky.).
Peptides may be purified by RP-HPLC (preparative and analytical)
using, e.g., a Waters Delta Prep 3000 system and a C4, C8 or C18
preparative column (10.mu., 2.2.times.25 cm; Vydac, Hesperia,
Calif.).
[0040] Peptide compounds useful in the invention may also be
prepared using recombinant DNA techniques, using methods now known
in the art. See, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2d Ed., Cold Spring Harbor (1989). Non-peptide
compounds useful in the present invention may be prepared by
art-known methods. For example, phosphate-containing amino acids
and peptides containing such amino acids, may be prepared using
methods known in the art. See, e.g., Bartlett and Landen, Biorg.
Chem. 14:356-377 (1986).
[0041] The compounds described above are useful in view of their
pharmacological properties. In particular, the compounds of the
invention possess activity as agents to reduce nutrient
availability, including reduction of food intake.
[0042] The compositions or pharmaceutical composition can be
administered by any route, including intravenously,
intraperitoneal, subcutaneous, and intramuscular, orally,
topically, transmucosally, or by pulmonary inhalation. Compositions
useful in the invention may conveniently be provided in the form of
formulations suitable for parenteral (including intravenous,
intramuscular and subcutaneous), nasal or oral administration. In
some cases, it will be convenient to provide a PYY or a PYY agonist
and another food-intake-reducing, plasma glucose-lowering or plasma
lipid-altering agent, such as an amylin, an amylin agonist, a CCK
or CCK agonist, or a leptin or leptin agonist, or an exendin or
exendin agonist, in a single composition or solution for
administration together. In other cases, it may be more
advantageous to administer the additional agent separately from
said PYY or PYY agonist.
[0043] A suitable administration format may best be determined by a
medical practitioner for each patient individually. Various
pharmaceutically acceptable carriers and their formulation are
described in standard formulation treatises, e.g., Remington's
Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and
Hanson, M. A. "Parenteral Formulations of Proteins and Peptides:
Stability and Stabilizers," Journal of Parenteral Science and
Technology, Technical Report No. 10, Supp. 42:2 S (1988).
[0044] Compounds useful in the invention can be provided as
parenteral compositions for e.g., injection or infusion.
Preferably, they are suspended in an aqueous carrier, for example,
in an isotonic buffer solution at a pH of about 3.0 to about 8.0,
preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to
about 5.0. Useful buffers include sodium citrate-citric acid and
sodium phosphate-phosphoric acid, and sodium acetate/acetic acid
buffers. A form of repository or "depot" slow release preparation
may be used so that therapeutically effective amounts of the
preparation are delivered into the bloodstream over many hours or
days following transdermal injection or delivery.
[0045] Since the products of the invention are amphoteric, they may
be utilized as free bases, as acid addition salts or as metal
salts. The salts must, of course, be pharmaceutically acceptable,
and these will include metal salts, particularly alkali and
alkaline earth metal salts, e.g., potassium or sodium salts. A wide
variety of pharmaceutically acceptable acid addition salts are
available. Such products are readily prepared by procedures well
known to those skilled in the art.
[0046] For use by the physician, the compositions will be provided
in dosage unit form containing an amount of a PYY or a PYY agonist
with or without another active ingredient, e.g., a food
intake-reducing, plasma glucose-lowering or plasma lipid-altering
agent. Therapeutically effective amounts of a PYY or a PYY agonist
for use in reducing nutrient availability are those that suppress
appetite at a desired level. As will be recognized by those in the
field, an effective amount of therapeutic agent will vary with many
factors including the age and weight of the patient, the patient's
physical condition, the blood sugar level, the weight level to be
obtained, and other factors
[0047] The effective daily appetite-suppressing dose of the
compounds will typically be in the range of about 1 to 30 .mu.g to
about 5 mg/day, preferably about 10 to 30 .mu.g to about 2 mg/day
and more preferably about 5 to 100 .mu.g to about 1 mg/day, most
preferably about 5 .mu.g to about 500 .mu.g/day, for a 50 kg
patient, administered in a single or divided doses. Preferably,
dosages are between about 0.01 to about 100 .mu.g/kg/dose. The
exact dose to be administered is readily determined by one of skill
in the art and is dependent upon the potency of the particular
compound, as well as upon the age, weight and condition of the
individual. Administration should begin whenever the suppression of
nutrient availability, food intake, weight, blood glucose or plasma
lipid lowering is desired, for example, at the first sign of
symptoms or shortly after diagnosis of obesity, diabetes mellitus,
or insulin-resistance syndrome. Administration may be by any route,
e.g., injection, preferably subcutaneous or intramuscular, oral,
nasal, transdermal, etc. Dosages for certain routes, for example
oral administration, should be increased to account for decreased
bioavailablity, for example, by about 5-100 fold.
[0048] The optimal formulation and mode of administration of
compounds of the present application to a patient depend on factors
known in the art such as the particular disease or disorder, the
desired effect, and the type of patient. While the compounds will
typically be used to treat human subjects they may also be used to
treat similar or identical diseases in other vertebrates such as
other primates, farm animals such as swine, cattle and poultry, and
sports animals and pets such as horses, dogs and cats.
Screening for Additional PYY Agonists
[0049] Other PYY agonists can be identified by using the receptor
binding assays described below (e.g., in Examples 9 and 10) or
known in the art in combination with the physiological screens
described in the Examples below. Potential PYY agonists can be
compared with the activity of PYY or PYY[3-36].
[0050] Alternatively, once one or more PYY-preferring (Y7)
receptors have been characterized and cloned, alternative assays
and high throughput screens can be implemented as discussed below
or known in the art. Y7 receptors are those with an affinity for
PYY or PYY[3-36] greater than their affinity for NPY. Methods of
screening for compounds which modulate PYY receptor activity
comprise contacting test compounds with PYY receptors and assaying
for the presence of a complex between the compound and the PYY
receptors. In such assays, the test ligand is typically labelled.
After suitable incubation, free ligand is separated from that
present in bound form, and the amount of free or uncomplexed label
is a measure of the ability of the particular compound to bind to
the PYY receptors. Alternatively, bound labelled ligand may be
measured (e.g., using expressed membrane-bound Y7 receptors).
[0051] In another embodiment of the invention, high throughput
screening for compounds having suitable binding affinity to PYY
receptors is employed. For example, large numbers of different
small peptide test compounds are synthesised on a solid substrate.
The peptide test compounds are contacted with the PYY receptor and
washed. Bound PYY receptor is then detected by methods well known
in the art. Purified test compounds can also be coated directly
onto plates for use in the aforementioned drug screening
techniques. In addition, if the test compounds are proteins,
antibodies can be used to capture the protein and immobilize it on
the solid support by any means known in the art.
[0052] Other embodiments of the invention comprise using
competitive screening assays in which neutralizing antibodies
capable of binding a polypeptide of the invention specifically
compete with a test compound for binding to the polypeptide. In
this manner, the antibodies can be used to detect the presence of
any peptide that shares one or more antigenic determinants with a
PYY agonist. Radiolabeled competitive binding studies are described
in A. H. Lin et al. Antimicrobial Agents and Chemotherapy, 1997,
vol. 41, no. 10. pp. 2127-2131, the disclosure of which is
incorporated herein by reference in its entirety.
[0053] To assist in understanding the present invention, the
following Examples are included. The experiments relating to this
invention should not, of course, be construed as specifically
limiting the invention and such variations of the invention, now
known or later developed, which would be within the purview of one
skilled in the art are considered to fall within the scope of the
invention as described herein and hereinafter claimed.
EXAMPLES
[0054] In experiments described below, members of the PP ligand
family were used in various assays. Unless otherwise stated, all
peptide test compounds were dissolved in saline to a concentration
of between 1-5 mg/ml without measurement of pH. In all cases,
preparations were clear to the eye prior to administration.
Example 1
Activity of Y Receptor Ligands on Food Intake in Overnight-Fasted
NIH/SW Mice
[0055] Female NIH/Swiss mice (8-12 weeks old) were group housed
with a 12:12 hour light:dark cycle with lights on at 0600. Water
and a standard pelleted mouse chow diet were available ad libitum,
except as noted. Animals were fasted and housed individually
starting at approximately 1500 hrs, 1 day prior to experiment. The
morning of the experiment (approx. 0630 hrs), all animals were
weighed and divided into experimental groups so as to give the most
similar weight distribution between groups. In a typical study,
n=10 for the control group and at least 5 for each treatment
group.
[0056] At time=0 min, all animals were given an intraperitoneal
injection of vehicle or compound in a volume of 5 ml/kg and
immediately given a pre-weighed amount (10-15 g) of the standard
chow. Increasing dosages of PYY[3-36], or PYY (0.1 .mu.g/kg to 500
.mu.g/kg and NPY (100 and 500 .mu.g/kg), and single high doses of
NPY[3-36] (100 .mu.g/kg), N-terminal acetylated Ac-PYY[22-36] (200
.mu.g/kg) and PP (500 .mu.g/kg) were provided, as indicated on FIG.
1. Food was removed and weighed at 1 hr to determine the amount of
food consumed (Morley, Flood et al. Am J Physiol 267: R178-R184,
1994).
Analysis:
[0057] Food intake was calculated by subtracting the weight of the
food remaining after one hour from the weight of the food provided
initially at time=0. The effects of treatment on food intake are
expressed as % change relative to control.
[0058] Significant treatment effects were identified by ANOVA
(p<0.05). Where a significant difference existed, test means
were compared to the control mean using Dunnett's test (Prism
v2.01, GraphPad Software Inc., San Diego, Calif.).
Results:
[0059] As seen in FIG. 1, PYY administered peripherally
(intraperitoneal injection) at doses of 10, 100 and 500 .mu.g/kg
significantly reduced food intake measured over 60 min in
overnight-fasted female NIH/SW mice. These doses of PYY[3-36] had
approximately equal efficacy. PP and NPY showed a trend toward
activity at 500 .mu.g/kg. But NPY and NPY[3-36] [SEQ ID NO.: 5]
were inactive at 100 .mu.g/kg. Ac-PYY[22-36] [SEQ ID NO.: 6] at 200
.mu.g/kg and was also inactive. The rank order of potency was:
PYY[3-36].gtoreq.PYY>>NPY=NPY[3-36]=PP=Ac-PYY[22-36]. The
rank order of potency, and in particular the lack of effect of NPY,
does not reflect the pharmacology of any of the known cloned
receptors.
[0060] PP given peripherally has been reported to reduce feeding
(Asakawa, Inui et al. Peptides 20: 1445-8, 1999). Additionally, PP
given peripherally to obese mice reportedly reduced food intake and
body weight gain (Malaisse-Lagae, Carpentier et al. Experientia 33:
915-7, 1977). The ob/ob mouse is reported to be hypersensitive to
several anorexigens (Young and Bhaysar. Program and Abstracts, 10th
International Congress of Endocrinology 419 (poster P2-58), 1996).
Mice over expressing PP were reported to decrease body weight and
food intake (Ueno, Inui et al. Gastroenterology 117: 1427-32,
1999). Applicants were unable to reduce food intake with PP in the
test system indicated in FIG. 1. The Asakawa et al. studies were
acute single-injection studies and no data on body weight change
were provided. Although the PP transgenic mouse study (Ueno et al.,
Gastroenterology 117: 1427-32, 1999) claims to show decreased body
weight and food intake in overexpressing animals, half of the
animals died in the perinatal period, which could signal
pathophysiology apart from a straightforward explanation of
decreased milk intake leading to starvation. In addition, the gene
expression system is not pancreas-specific, and peptide is
expressed in the brain, which confuses any interpretation of the
over-expression data. Ueno et al. conclude from their data that PP
could be involved in feeding and body weight regulation partly
through regulation of GE, but the data in Examples 1 and 2 (below)
show PP to have little or no effect on food consumption, and to be
essentially inactive in slowing gastric emptying. Importantly, PP
is only 50% homologous to PYY (or NPY), has a different primary
tissue localization (pancreas vs. intestinal L-cells vs. neurons),
and an apparent preference for the Y4 receptor over Y1 and Y2. NPY,
which is 70% homologous to PYY, is a powerful orexigen when
administered centrally. It produces only a modest decrease in food
intake, and is completely inactive in the gastric emptying assay of
Example 2 (below) when given peripherally.
Example 2
Activity of Y Peptide Ligands on Gastric Emptying in HSD Rats
[0061] Male HSD rats, 180-215 g, were housed with a 12:12 hour
light:dark cycle and fasted for 20 hrs (overnight). At time=0 min,
test peptide (PYY[3-36], PYY, Ac-PYY[22-36], NPY, NPY[3-36], or PP)
or saline vehicle was injected (intraperitoneal) into conscious
rats (n=6/group). At t=1 min, a solution of 1 mL sterile water
containing 5 .mu.Ci of .sup.3H-3-O-methyl-glucose was gavaged by
oropharyngeal tube to conscious rats. Blood samples (10 .mu.l) were
collected 40 min after gavage and assayed for counts per minute
(CPM) in plasma. To eliminate pain during tail vein sampling, 2%
Lidocaine (0.1 ml) was injected 3-4 cm from the end of the tail
(Gedulin, Jodka et al. Gastroenterology 108: A604, 1995).
Data Analysis:
[0062] Effects of the test compound were expressed as percent
change relative to control, which was calculated as -100*(1-(mean
value test rats/mean value controls)).
[0063] Relative activity was defined as significant if p<0.05 as
determined by ANOVA. Where a significant difference existed, test
means were compared to the control mean using Dunnett's test (Prism
v2.01, GraphPad Software Inc., San Diego, Calif.).
Results:
[0064] As seen in FIG. 2, PYY[3-36] administered peripherally
(intraperitoneal injection) at doses greater or equal to 10
.mu.g/kg significantly and dose-dependently reduced gastric
emptying measured at 40 minutes in HSD rats. PYY at 100 and 500
.mu.g/kg was also efficacious. By contrast, NPY, NPY[3-36], or PP
injected at 500 .mu.g/kg and Ac-PYY[22-36] at 200 .mu.g/kg were
inactive. The order of potency of the test compounds is as follows:
PYY[3-36].gtoreq.PYY>>NPY=NPY[3-36]=PP=Ac-PYY[22-36]. This
potency profile is similar to that seen for food intake (FIG. 1).
The lack of effect of NPY does not reflect the pharmacology of any
known cloned receptors. It is significant that Ac-PYY[22-36] was
inactive in both assays, since Balasubramanian et al., U.S. Pat.
No. 5,604,203 reported that this subpeptide is a ligand for both
rat intestinal PYY receptors, and the Y2 receptor.
Example 3
Acute Peripheral Administration of PYY[3-36] Inhibits Gastric Acid
Secretion in Rats
[0065] Male Harlan Sprague Dawley rats were housed in a 12:12 hour
light:dark cycle. All experiments were performed during the light
cycle. Animals were fasted for approximately 20 hours before
experimentation but were given free access to water until the start
of the experiment.
[0066] Rats (age 11-16 weeks, body mass 291-365 g) were surgically
fitted with gastric fistulae (Kato, Martinez et al. Peptides 16:
1257-1262, 1995). Overnight fasted rats were weighed and their
gastric fistulae were uncapped and attached to flexible Tygon
tubing (3/8.times. 1/16) into which was fitted a piece of PE205
tubing that would extend up into the stomach. Saline was injected
through the narrower PE205 tubing and the effluent collected from
the Tygon tubing. To ensure proper flow through the fistulae and an
empty stomach, the stomach was flushed several times with .about.5
mL of room temperature saline solution until flow was easy and the
effluent was clean. Gastric acid secretion was measured at 10 min
intervals by injecting 5 mL of saline followed by 3 mL of air and
collecting the effluent. Three mL of each gastric aspirate were
titrated to pH 7.0 with 0.01 N sodium hydroxide using a pH meter.
The amount of base required for each titration, corrected to the
total volume collected, was used to calculate the moles of acid in
each sample.
[0067] After a baseline sample was collected, and the recovered
volume recorded, the animal was given a subcutaneous injection of
125 .mu.g/kg pentagastrin to stimulate gastric secretion. Gastric
acid secretion was sampled every 10 minutes. Forty minutes after
pentagastrin injection, the animal was given a subcutaneous
injection of 100 .mu.g/kg PYY[3-36] or saline and sampling of
gastric secretion was continued every 10 minutes for a total of 2
hrs. Data are expressed as .mu.mol of acid secreted per 10 minute
sampling interval (mean.+-.SEM n=4/group).
Results:
[0068] FIG. 3 demonstrates that PYY[3-36] administered acutely by
peripheral (intraperitoneal) injection (100 .mu.g/kg) inhibited
pentagastrin-stimulated gastric acid secretion in rats. The
ED.sub.50 for this effect was .apprxeq.20 .mu.g/kg.
Example 4
Acute Peripheral Administration of PYY[3-36] Prevents Gallbladder
Emptying in Mice--Reversible by CCK-8
[0069] Mice were housed in a 12:12 hour light:dark cycle room with
free access to water and mouse chow until the start of the
experiment. At t=0, mice were given a subcutaneous injection of 1,
10, 100, or 1000 .mu.g/kg PYY[3-36], 1 or 10 .mu.g/kg CCK-8, both
or saline (treatment and n/group as indicated in FIG. 4). Thirty
minutes later, animals were anesthetized, and their intact
gallbladders removed and weighed.
Analysis:
[0070] Data are expressed as organ weight in mg. Activity was
defined as change from the mean of the control group. Statistical
significance was defined as p<0.05 by ANOVA and/or Dunnett's
test.
Results:
[0071] As seen in FIG. 4, PYY[3-36] administered by acute
peripheral injection at doses greater or equal to 10 .mu.g/kg
prevented gallbladder emptying in mice. This inhibition of emptying
had an ED.sub.50.apprxeq.31 .mu.g/kg and could be overridden by
CCK-8, even at the highest doses of PYY[3-36] tested.
Example 5
Acute Peripheral Administration of PYY[3-36] Inhibits
CCK-8-Stimulated Exocrine Pancreatic Secretion (Amylase) in
Rats
[0072] Male Harlan Sprague Dawley rats were housed in a 12:12 hour
light:dark cycle. All experiments were performed during the light
cycle. Animals were fasted for approximately 20 hours before
experimentation but were given free access to water until the start
of the experiment.
[0073] Rats were anesthetized with 5% halothane, maintained with 2%
halothane during surgery and with 1% halothane thereafter.
Tracheotomy and cannulation of the right femoral artery were
performed and body temperature was controlled with a
thermoregulator that switched a heated operating table. The femoral
arterial line, used for blood sampling, was perfused with
heparinized saline (2 U/ml) and connected to a pressure transducer
for blood pressure recording. Through a midline incision, two
polyethylene cannulae were inserted into the common bile-pancreatic
duct at a point about 0.5 cm above where the duct enters the
pancreas. The first cannula was inserted up toward the liver to
collect bile. The other end of this cannula was placed into the
duodenum through a small incision in the duodenum. Thus, bile
flowed directly from the liver to the small intestine, being
shunted away from the pancreas completely. A second polyethylene
cannula inserted into the common bile-pancreatic duct near the
first was directed toward the pancreas to collect pancreatic juice.
The pancreatic duct was ligated at its entry into the duodenum,
forcing secreted pancreatic juice into the collection cannula.
[0074] Pancreatic juice was collected over 15 min intervals between
t=-15 to +60 min. The volume of pancreatic juice (measured by
weight) and activities of amylase were determined for each
15-minute aliquot (Taniguchi, Yazaki et al. Eur J Pharmacol 312:
227-33, 1996). Pancreatic juice was diluted 1:2000 before assay.
Enzyme secretion was expressed in units per 15 min obtained by
multiplying activity by volume collected (Taniguchi, H., Yazaki,
N., Yomota, E., Shikano, T., Endo, T., and Nagasaki, M. Eur J
Pharmacol 312: 227-33, 1996).
Statistical Analysis.
[0075] Pairwise statistical analyses were performed using Student's
t-test; multiple comparisons to a control used Dunnett's test;
general effects were tested by one-way ANOVA. Results are reported
as mean.+-.standard error of the mean. P<0.05 is used as the
level of significance.
Results:
[0076] FIG. 5 shows that PYY[3-36] administered by acute peripheral
(subcutaneous) injection at 30 .mu.g/kg blocked CCK-8-stimulated
pancreatic secretion in rats as measured by amylase activity in
pancreatic juice. In the absence of CCK-8, PYY[3-36] at 300
.mu.g/kg had no effect on basal amylase activity when compared to
saline-injected controls.
Example 6
Continuous Peripheral PYY[3-36] Infusion Decreases Body Weight Gain
in Fattened C57B1/6 (DIO) Mice
[0077] Male C57B1/6 mice (4 weeks-old at start of study) were fed
high fat (HF; 58% of dietary kcal as fat) or low fat (LF; 11% of
dietary kcal as fat) chow. After 7 weeks on chow, each mouse was
implanted with an osmotic pump (Alzet #2004) that delivered the
dose indicated in FIG. 6 of PYY[3-36] (30, 100, 300, or 1000
.mu.g/kg/day) continuously for 4 weeks. Body weight and food intake
were measured weekly (Surwit, Feinglos et al. Metabolism--Clinical
and Experimental 44: 645-651, 1995).
Data Analysis:
[0078] Effects of the test compound are expressed as the mean.+-.sd
of change in grams from starting weight of at least 14 mice per
treatment group ((p<0.05 ANOVA, Dunnett's test (Prism v2.01,
GraphPad Software Inc., San Diego, Calif.).
Results:
[0079] FIG. 6 demonstrates that PYY[3-36] administered by
continuous peripheral infusion produced a dose-related decrease in
body weight gain in diet-induced obese (DIO) mice. The effects were
significant at 300 .mu.g/kg/day for the first 3 weeks and at all
time points for the 1000 .mu.g/kg/day dose.
Example 7
Continuous Peripheral Infusion of PYY[3-36] Decreases Caloric
Efficiency in Fattened C57B1/6 (DIO) Mice
[0080] Male C57B1/6 mice (4 weeks-old at start of study) were fed
high fat (HF; 58% of dietary kcal as fat) or low fat (LF; 11% of
dietary kcal as fat) chow. After 7 weeks on chow, each mouse was
implanted with an osmotic pump (Alzet #2004) that delivered the
dose indicated in FIG. 6 of PYY[3-36] (30, 100, 300, or 1000
.mu.g/kg/day) continuously for 4 weeks. Body weight and food intake
were measured weekly (Surwit, Feinglos, et al Metabolism--Clinical
and Experimental 44: 645-651, 1995).
Analysis:
[0081] Effects of the test compound are expressed as change in body
weight (g) from the starting weight per kcal consumed. Kcal
consumed was computed by multiplying the weight of food consumed
(g) by the caloric density (kcal/g) specified by the manufacturer.
Note that these data are derived from the animals used in Example
3.
[0082] Activity was defined as the change of the mean.+-.sd of n=at
least 14 mice/group. Significance was defined as p<0.05 in an
ANOVA or Dunnett's test.
Results:
[0083] FIG. 7 shows that PYY[3-36] administered subchronically by
continuous peripheral infusion produced a dose-related decrease in
caloric efficiency (measured as body weight gained/kcal consumed)
in diet-induced obese (DIO) mice. The effects were significant at
all time points for 1000 .mu.g/kg/day and at some time points for
300 .mu.g/kg/day.
Example 8
Continuous Peripheral Infusion of PYY[3-36] for 28-Days Improves
Glycemic Control in Obese Diabetic (ZDF) Rats
[0084] Diabetic fatty Zucker rats (ZDF) (7 weeks of age) were
housed in a 12:12 hour light:dark cycle room and given ad libitum
access to high fat rodent chow and water. After 1 week of
acclimatization, blood samples were drawn, and animals were sorted
by starting HbA1c to provide a similar range in each treatment
group.
Animals were implanted with osmotic pumps which delivered the doses
indicated in FIG. 8 of PYY[3-36] or saline continuously to the
periphery for 28 days. HbA1c was measured at weekly intervals.
HbA1c levels (%) were plotted against time (Brown, Henke et al.
Diabetes 48: 1415-24, 1999).
Results:
[0085] As indicated in FIG. 8, PYY[3-36] administered continuously
by peripheral infusion to Diabetic Zucker Fatty (ZDF) rats produced
a dose-dependent improvement in long term blood glucose control as
measured by the level of HbA1c. The improvement in glycemic control
increased throughout the treatment period, and was significant at
all doses of PYY[3-36] at 28 days.
[0086] The above Examples directly indicate that PYY agonists have
utility in reducing caloric availability, and can be used as
therapeutics to treat conditions that benefit from reduced caloric
availability, such as obesity and type 2 diabetes. Further, the
Examples indicate that the effects of PYY agonists to reduce
caloric availability could occur through several mechanisms, and
provide a framework to identify a PYY agonist. Since PYY and NPY
are reported to be equipotent and equally effective in all Y1 and
Y2 receptor assays studied, the data in Examples 1 and 2 above
indicate that the effects of PYY and agonists on reducing food
intake (FIG. 1) and delaying gastric emptying (FIG. 2) are not
mediated through Y1 or Y2 receptors. Those data show that PYY's
effects on food intake and gastric emptying are not comparable to
reported effects on Y1 and Y2 receptors, since NPY showed little or
no activity in these assays.
[0087] FIG. 1 illustrates one aspect of the invention. It is known
that central administration of a PYY or NPY agonist increases food
intake (Clark, Kalra et al. Endocrinology 115: 427-9, 1984; Clark,
Sahu et al. Regul Pept 17: 31-9, 1987). Surprisingly, we have found
that peripheral administration of PYY or PYY[3-36] effectively
decreases food intake. Not presented here, we have documented that
PYY[3-36] reduced food intake in long term studies in other rodent
models including the ob/ob mouse, and fa/fa rat. Administered
peripherally, other members of the PP family have little or no
effect on food intake. The order of potency, and in particular, the
lack of effect of NPY, does not reflect the pharmacology of any of
the known cloned Y receptors. The unique pharmacology of PYY
agonists is further established by their potent effect to delay
gastric emptying compared with other PP family members which are
inactive in this assay (see, e.g., data in FIG. 2).
[0088] Characterization of the PYY agonist PYY[3-36] illustrates
additional mechanisms that could reduce caloric availability. These
include decreased gastric acid secretion (FIG. 3), decreased
exocrine pancreatic secretion (FIG. 5), and delayed gallbladder
emptying (FIG. 4). Without being bound by any theory, we
hypothesize that this entire spectrum of effects on food intake and
gastrointestinal function contributes to the utility of PYY
agonists to decrease caloric availability. For example, PYY and
PYY[3-36] reportedly inhibited vagally stimulated gastric acid
output in rabbits (Lloyd, Grandt et al. Am J Physiol 270:
G123-G127, 1996). PYY also inhibited pentagstrin-stimulated gastric
acid secretion in humans (Adrian, Ferri et al. Gastroenterology 89:
1070-7, 1985) and rats (Greeley, Guo et al. Proc Soc Exp Biol Med
189: 325-8, 1988), and CRF-induced gastric-acid secretion in rats
(Gue, Junien et al. Br J Pharmacol 118: 237-42, 1996). PYY
(Yoshinaga, Mochizuki et al Am J Physiol 263: G695-701, 1992)
(Guan, Maouyo, et al. Endocrinology 128: 911-6, 1991) (Pappas,
Debas et al. Am J Physiol 248: G118-23, 1985) and PYY[3-36] (Deng,
Guarita et al. Dig Dis Sci 46: 156-65, 2001) have also been
reported to inhibit pancreatic enzyme secretion. In normal humans,
PPY was recently reported to reduce the cephalic, but not the
CCK-dependent phase of gallbladder emptying (Hoentjen, Hopman et
al. Scand J Gastroenterol 35: 166-71, 2000).
[0089] We propose that treatment with a PYY agonist via mechanisms
identified herein will decrease body weight. We have established,
in several obese rodent models, that peripheral administration of
PYY[3-36] produces a dose-dependent decrease in body weight and/or
rate of weight gain. We demonstrate the effect herein in a
diet-induced obese (DIO) mouse model (FIG. 6).
[0090] Additionally, as summarized in FIG. 1, peripheral
administration of a PYY agonist decreases food intake. From a
calculation of the body weight gained per kcal consumed in the DIO
mouse study, it is clear that peripheral administration of a PYY
agonist decreases the efficiency with which calories are converted
into body mass (FIG. 7). Thus, the present Examples support an
effect of PYY agonists to reduce body weight gain in association
with reduced caloric availability.
[0091] PYY and PYY agonists in particular have utility in the
treatment of diseases that will benefit from reduced caloric
availability such as obesity, type 2 diabetes and cardiovascular
disease. We have examined the antidiabetic activities of PYY[3-36]
in an obese diabetic rodent, the ZDF rat. Peripheral administration
of the PYY agonist produces a significant, robust and dose-related
improvement in glycemic control as measured by hemoglobin A1c
levels (FIG. 8). While not presented in the Example, food intake
was also reduced by PYY[3-36] administration.
Example 9
Area Postrema Assay
[0092] Peripherally administered PYY has been reported to activate
neurons in the area postrema (Bonaz, Taylor et al. Neurosci Lett
163: 77-80, 1993). Evaluation of the PYY agonist activity of
potential compounds of the invention can be carried out using the
area postrema assay as follows, in combination with an assay of PYY
effect, for example those of Examples 1 and 2.
Membrane Preparation
[0093] In this assay, area postrema membranes were prepared from
tissue dissected from the pig or bovine brain stem. Area postrema
membrane preparations are initiated by brief (4-10 seconds)
homogenization of tissues using, e.g., a Polytron tissue
homogonizer (Brinkman Instruments, NY) at ice-cold temperatures in
a buffered solution such as phosphate buffered saline (138 mM NaCl,
8.1 mM Na.sub.2PO.sub.4, 2.5 mM KCl, 1.2 mM KH.sub.2PO.sub.4, 0.9
mM CaCl.sub.2, 0.5 mM MgCl.sub.2, pH 7.4). Following tissue
disruption, large particles and debris were cleared by
centrifugation (200.times.g, 5 minutes, 4.degree. C.) and the
supernatant fraction is reserved on ice. Membranes are isolated
from the supernatant fraction by high-speed centrifugation (at
least 40,000.times.g, for at least 10 minutes, 4.degree. C.).
Membranes are normally washed at least twice by re-homogenization
in fresh buffer and recentrifugation, in order to remove endogenous
interfering substances. Washed membranes are resuspended in buffer
containing a proteolytic enzyme inhibitors such as
phenylmethylsulfonyl fluoride (PMSF) or bacitracin. Volumes of
buffer may be added sufficient to adjust the final tissue
concentration to a level suitable for the particular screening
method employed.
Binding Reactions
[0094] In one embodiment, incubation mixtures for the screening
method are set up as follows. To glass or polymeric tubes are added
a small volume of Buffer Mixture ("HBBM") composed of a buffer
solution such as HEPES containing a protease inhibitor such as
bacitracin or PMSF, protease-free serum albumin (preferable
fraction V BSA, protease-free) and, optionally, a Mg.sup.2+ or
Ca.sup.2+ salt, and EDTA. To the Buffer Mixture is added a small
volume of buffer containing the unlabeled molecules to be tested
for agonist activity at concentrations of about from 10.sup.-11 to
10.sup.-6 M. Control tubes contain buffer alone. To this mixture is
added amounts of labeled area postrema preparation ligand (here,
PYY), in buffer so as to produce final concentrations of from about
10 to about 100 pM. Because of the high specific activities
obtainable and ease of chemical labeling, .sup.125I is preferred to
label the area postrema ligands. Ligands may be isolated from human
tissues, from animal tissues, or produced by chemical, synthetic,
or recombinant means. Labeled area postrema preparation ligands are
dissolved in sterile water containing protease-free Fraction V BSA,
aliquoted, and stored frozen until use.
[0095] Reactions are begun by adding, for example, membranes to
each incubation tube. The amount of membrane protein required per
tube is varied so that the amount of labeled ligand bound by the
membranes in the assay is less than, for example, 10% of the total
concentration of ligand in the assay (typically about 100
.mu.g).
[0096] Reaction mixtures are incubated for a period of time and at
a temperature sufficient to reach steady-state conditions within
the period. The term "steady state" as used herein is intended to
encompass the sum total of all reactions and processes that
influence the net amount of bound hormone. It may or may not be
synonymous with "equilibrium." Typically, tubes are incubated for
about 60 minutes at room temperature.
Detection
[0097] When membranes are used, they are isolated following binding
in order to determine the amount of labeled ligand bound after
competition between labeled and unlabeled ligands. It is convenient
to collect membranes by filtration with a vacuum-powered Brandel
Cell Harvester (Brandel Instruments, Gaithersburg, Md., Model M-24)
through glass fiber filters (e.g., GF/B, Whatman) that have been
presoaked with a regent in order to reduce nonspecific binding
(NSB). Preferred is presoaking filters for about 5 hours in about
0.3% polyethyleneimine. The skilled artisan will know of other
membrane collecting devices, such as the Millipore Filtration
Assembly ((Model 1225) or the Sandbeck filter box (Bennett, J. P.,
in Neurotransmitter Receptor Binding, H. I. Yamura, et al., Raven,
New York 1978, Pages 57-90), collecting filters, and NSB-reducing
reagents that can be used in receptor binding assays. Both
immediately before and immediately after filtration, filters are
washed with large (milliliter) volumes of ice cold buffer to remove
contaminating materials, e.g., unbound labeled ligand. Filters are
removed and the amount of labeled ligand bound to membranes is
quantified. Where .sup.125I is the label, radioactivity may be
assessed in a gamma ray counter. Where a chemiluminescent reporter
molecule (e.g., AMPPD, Tropix, Inc., Bedford, Mass.) is used, the
light produced may be quantified in a luminometer. Enzymatic and
fluorescent labels may also be used.
[0098] Instead of by filtration, membranes may be isolated
following incubation by centrifugation (e.g., Beckman--2-21-M
refrigerated centrifuge at 21,000 rpm or a Beckman 12 or Eppendorf
microfuge), washed with ice cold buffer, then counted as such or
following solubilization of membranes by detergent or alkali.
Data Analysis
[0099] Scatchard plot saturation analyses of binding data, wherein
bound/free (B/F) labeled ligand is plotted as a function of the
amount bound, are performed by standard methods. See, e.g.,
(Scatchard. Ann NY Acad Sci 51: 660, 1949).
[0100] Competition curves, wherein the amount bound (B) is plotted
as a function of the log of the concentration of ligand may be
analyzed by computer, e.g., analyses by nonlinear regression to a
4-parameter logistic equation (Prism Program; GraphPAD Software,
San Diego, Calif.) or the ALLFIT program (Version 2.7 (NIH,
Bethesda, Md. 20892)) (Munson and Rodbard. Anal Biochem 107:
220-39, 1980; de Lean, A., Munson, P. J. et al. 1988).
[0101] To determine binding constants, Scatchard saturation curves
may be generated and analyzed according to a modification of the
method of Scatchard, as described by Bylund, D. B., et al.,
"Methods for Receptor Binding," In H. I. Yamamura et al., eds.,
Methods in Neurotransmitter Analysis, Raven Press, New York, 1990
pp. 1-35.
[0102] In order to obtain specific binding values experimentally, a
broad range of tracer concentrations of labeled ligand (typically,
1-150 pM) is used to obtain total binding and duplicate tubes
reassessed, in the presence of a very high concentration, e.g., 100
nM, of unlabeled ligand, to obtain nonspecific binding (NSM). The
latter value is subtracted from each total binding value in order
to obtain specific binding at every concentration of labeled
ligand.
Example 10
Y Receptor Binding Assay
[0103] Evaluation of the PYY agonist activity of potential
compounds of the invention can be carried out by investigating
their interaction with any of the known Y receptors, such as Y1-Y6,
or with one or more unique receptor classes similar to the
PYY-preferring receptors (such as Y7) expressed in cells, in
combination with an assay of PYY effect, for example those of
Examples 1 and 2. These cells may endogenously express the Y
receptor of interest (such as SK-N-MC cells that express Y1
receptors or SK-N-BE2 cells which express Y2 receptors) or may be
other cells (such as COS-7 or HEK293 cells) that are transfected
with the clone of the Y receptor of interest. Binding to SK-N-BE2
cells is used as an example.
Cell Culture
[0104] SK-N-BE2 cells are grown on, for example 150 mm plates in
tissue culture medium with supplements (Dulbecco's Modified Eagle
Medium with 10% fetal calf serum, 4 mM glutamine, 100 units/mL
penicillin and 100 .mu.g/mL streptomycin) at 37.degree. C. in 5%
CO.sub.2 humidified atmosphere. Stock plates are trypsinized and
split 1:6 every 3-4 days.
Membrane Preparation
[0105] Cells are scraped from the plates in a small volume of a
buffered solution such as phosphate buffered saline (138 mM NaCl,
8.1 mM Na.sub.2PO.sub.4, 2.5 mM KCl, 1.2 mM KH.sub.2PO.sub.4, 0.9
mM CaCl.sub.2, 0.5 mM MgCl.sub.2, pH 7.4), or are trypsinized,
washed and resuspended in buffered solution. Membrane preparations
are initiated by brief (10 seconds) homogenization of cells using,
e.g., a Polytron tissue homogonizer (Brinkman Instruments, NY) at
ice-cold temperatures. Membranes are further prepared by
centrifugation as described above in Example 9. Binding reactions,
detection and data analysis are as described in Example 9.
[0106] Various modifications of the invention in addition to those
shown and described herein will be apparent to those skilled in the
art from the foregoing description and fall within the scope of the
following claims.
Sequence CWU 1
1
6136PRTHomo sapiens 1Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asn
Ala Thr Pro Glu Gln 1 5 10 15 Met Ala Gln Tyr Ala Ala Asp Leu Arg
Arg Tyr Ile Asn Met Leu Thr 20 25 30 Arg Pro Arg Tyr 35 236PRTHomo
sapiens 2Tyr 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 334PRTHomo sapiens 3Ile
Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn 1 5 10
15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln
20 25 30 Arg Tyr 436PRTHomo sapiens 4Tyr Pro Ser Lys Pro Asp Asn
Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met Ala Arg Tyr Tyr
Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr 20 25 30 Arg Gln Arg
Tyr 35 534PRTHomo sapiens 5Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala
Pro Ala Glu Asp Met Ala 1 5 10 15 Arg Tyr Tyr Ser Ala Leu Arg His
Tyr Ile Asn Leu Ile Thr Arg Gln 20 25 30 Arg Tyr 615PRTHomo sapiens
6Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr 1 5 10
15
* * * * *