FSH-RELEASING PEPTIDES

PEPTIDES LIBERATEURS DE F.S.H.

English Abstract

Lamprey LHRH-III is a potent FSH-releasing factor, and may be used to enhance
fertility. Antagonists to lamprey LHRH-III may be used to inhibit fertility.

French Abstract

La LH-RH-III de la lamproie est un puissant facteur libérateur de F.S.H., et il peut être utilisé pour stimuler la fécondité. Des antagonistes de la LH-RH-III de la lamproie peuvent être utilisés pour inhiber la fécondité.

Claims

32
What is claimed:
1. A method of altering the level of secretion of FSH in a vertebrate animal,
comprising administering to the animal an effective amount of a peptide
selected from the
group consisting of an 1-LHRH-III agonist peptide; an 1-LHRH-III superagonist
peptide; an
1-LHRH-III antagonist peptide; a peptide selected from the group consisting of
SEQ. ID NO.
6 through SEQ. ID NO. 41; and 1-LHRH-III (SEQ. ID NO. 1); wherein the amount
of the
peptide administered causes a selective change in the serum FSH level of the
animal, but does
not cause a proportional change in the serum LH level of the animal.
2. A method as recited in Claim 1, wherein the level of secretion of FSH in
the
animal is increased; said method comprising administering to the animal an
effective amount
of I-LHRH-III (SEQ. ID NO. 1).
3. A method as recited in Claim 2, wherein the animal is a mammal.
4. A method as recited in Claim 3, wherein the animal is a human.
5. A method as recited in Claim 4, wherein the human is male.
6. A method as recited in Claim 4, wherein the human is female.
7. A method of increasing the fertility of a vertebrate animal, comprising
increasing the level of FSH secretion in the animal by the method of Claim 2.
8. A method as recited in Claim 7, wherein the animal is a mammal.
9. A method as recited in Claim 8, wherein the animal is a human.
10. A method as recited in Claim 9, wherein the human is male.
11. A method as recited in Claim 9, wherein the human is female.

33
12. A method as recited in Claim 1, wherein the level of secretion of FSH in
the
animal is increased; said method comprising administering to the animal an
effective amount
of an 1-LHRH-III agonist peptide or 1-LHRH-III superagonist peptide; wherein
the amount of
the peptide administered causes a selective increase in the serum FSH level of
the animal, but
does not cause a proportional increase in the serum LH level of the animal.
13. A method as recited in Claim 12, wherein the peptide is selected from the
group consisting of decapeptides whose sequences are identical to the sequence
of 1-LHRH-III
(SEQ. ID NO. 1), except that either or both of the following conditions is
satisfied:
(a) the sixth amino acid residue is a natural or xenobiotic amino acid residue
other than aspartic acid; or
(b) the ninth amino acid residue is prolyl ethyl amide (ProNHEt); and the
tenth amino acid residue of 1-LHRH-III is deleted.
14. A method as recited in Claim 13, wherein the peptide is selected from the
group consisting of SEQ. ID NO. 6 through SEQ. ID NO. 32.
15. A method as recited in Claim 12, wherein the animal is a mammal.
16. A method as recited in Claim 15, wherein the animal is a human.
17. A method as recited in Claim 16, wherein the human is male.
18. A method as recited in Claim 16, wherein the human is female.
19. A method of increasing the fertility of a vertebrate animal, comprising
increasing the level of FSH secretion in the animal by the method of Claim 12.

34
20. A method as recited in Claim 19, wherein the peptide is selected from the
group consisting of decapeptides whose sequences are identical to the sequence
of 1-LHRH-III
(SEQ. ID NO. 1), except that either or both of the following conditions is
satisfied:
(a) the sixth amino acid residue is a natural or xenobiotic amino acid residue
other than aspartic acid; or
(b) the ninth amino acid residue is prolyl ethyl amide (ProNHEt); and the
tenth amino acid residue of I-LHRH-III is deleted.
21. A method as recited in Claim 20, wherein the peptide is selected from the
group consisting of SEQ. ID NO. 6 through SEQ. ID NO. 32.
22. A method as recited in Claim 19, wherein the animal is a mammal.
23. A method as recited in Claim 19, wherein the animal is a human.
24. A method as recited in Claim 23, wherein the human is male.
25. A method as recited in Claim 23, wherein the human is female.
26. A method as recited in Claim 1, wherein the level of secretion of FSH in
the
animal is decreased; said method comprising administering to the animal an
effective amount
of an 1-LHRH-III antagonist peptide; wherein the amount of the peptide
administered causes a
selective decrease in the serum FSH level of the animal, but does not cause a
proportional
decrease in the serum LH level of the animal.
27. A method as recited in Claim 26, wherein the antagonist is selected from
the
group consisting of SEQ. ID NO. 33 through SEQ. ID NO. 41.
28. A method as recited in Claim 26, wherein the animal is a mammal.
29. A method as recited in Claim 28, wherein the animal is a human.
30. A method as recited in Claim 29, wherein the human is male.

35
31. A method as recited in Claim 29, wherein the human is female.
32. A method of decreasing the fertility of a vertebrate animal, comprising
decreasing the level of FSH secretion in the animal by the method of Claim 26.
33. A method as recited in Claim 32, wherein the antagonist is selected from
the
group consisting of SEQ. ID NO. 33 through SEQ. ID NO. 41.
34. A method as recited in Claim 32, wherein the animal is a mammal.
35. A method as recited in Claim 34, wherein the animal is a human.
36. A method as recited in Claim 35, wherein the human is male.
37. A method as recited in Claim 35, wherein the human is female.
38. A composition selected from the group consisting of an I-LHRH-III agonist
peptide; an 1-LHRH-III superagonist peptide; an 1-LHRH-III antagonist peptide;
a peptide
selected from the group consisting of SEQ. ID NO. 6 through SEQ. ID NO. 41;
and an
effective amount of substantially pure 1-LHRH-III (SEQ. ID NO. 1) admixed with
a
pharmaceutically acceptable carrier; wherein said composition has the property
that said
composition will, when administered in an effective amount to a vertebrate
animal, cause a
selective change in the serum FSH level of the animal, but will not cause a
proportional
change in the serum LH level of the animal.
39. A composition as recited in Claim 38, wherein said composition comprises
an
effective amount of substantially pure 1-LHRH-III (SEQ. ID NO. 1) admixed with
a
pharmaceutically acceptable carrier.
40. A composition as recited in Claim 38, wherein said composition comprises a
peptide selected from the group consisting of SEQ. ID NO. 6 through SEQ. ID
NO. 32.
41. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 6.
42. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 7.

36
43. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 8.
44. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 9.
45. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 10.
46. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 11.
47. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 12.
48. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 13.
49. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 14.
50. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 15.
51. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 16.
52. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 17.
53. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 18.
54. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 19.
55. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 20.
56. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 21.
57. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 22.
58. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 23.
59. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 24.
60. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 25.

37
61. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 26.
62. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 27.
63. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 28.
64. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 29.
65. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 30.
66. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 31.
67. A peptide as recited in Claim 40, wherein said peptide is SEQ. ID NO. 32.
68. A composition as recited in Claim 38, wherein said composition comprises a
peptide selected from the group consisting of SEQ. ID NO. 33 through SEQ. ID
NO. 41.
69. A peptide as recited in Claim 68, wherein said peptide is SEQ. ID NO. 33.
70. A peptide as recited in Claim 68, wherein said peptide is SEQ. ID NO. 34.
71. A peptide as recited in Claim 68, wherein said peptide is SEQ. ID NO. 35.
72. A peptide as recited in Claim 68, wherein said peptide is SEQ. ID NO. 36.
73. A peptide as recited in Claim 68, wherein said peptide is SEQ. ID NO. 37.
74. A peptide as recited in Claim 68, wherein said peptide is SEQ. ID NO. 38.
75. A peptide as recited in Claim 68, wherein said peptide is SEQ. ID NO. 39.
76. A peptide as recited in Claim 68, wherein said peptide is SEQ. ID NO. 40.
77. A peptide as recited in Claim 68, wherein said peptide is SEQ. ID NO. 41.

Description

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
1
FSH-RELEASING PEPT)(DES
This invention was made with support from the United States Government under
Grants DK43900 and MH51853 awarded by the National Institutes of Health. The
United
States Government has certain rights in this invention.
The benefit of the 04 June 1997 filing date of provisional application serial
number
60/nnn,nnn (which was a conversion of nonprovisional application serial number
08/869,153)
is claimed under 35 U.S.C. ~ 119(e) in the United States. The benefit of the
04 June 1997
filing date of application serial number 08/869,153 is claimed under
applicable treaties and
conventions outside the United States.
TECHNICAL FIELD
This invention pertains to compositions and methods for selectively
stimulating or
inhibiting the release of follicle-stimulating hormone from the anterior lobe
of the pituitary
gland.
BACKGROUND ART
The brain controls the release of gonadotropin hormones from the anterior
pituitary
gland. Two important gonadotropins are follicle-stimulating hormone (FSH) and
luteinizing
hormone (LH). FSH is critical for spermatogenesis and for ovarian follicle
development,
while LH is critical to androgen secretion in males, and estrogen secretion,
ovulation, and
formation of the corpus luteum in females. A hormone with specific activity
for releasing
FSH but not LH could be used to increase fertility in humans or other animals,
or to correct
fertility problems caused by defective hypothalamic control of FSH secretion.
Conversely,
antisera or other antagonists to an FSH-specific releasing factor will inhibit
FSH secretion,
thereby inhibiting spermatogenesis in males, or inhibiting development of
follicles and ovarian
development in females, providing a new antifertility drug. It is also
possible that very high
doses of an FSH-specific releasing factor will inhibit FSH secretion, rather
than stimulate it.

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
2
Prior indirect evidence has suggested that separate factors could be
responsible for
triggering the release of FSH and for triggering the release of LH in mammals.
However,
this hypothesis could not previously be confirmed, because no previous work
successfully
isolated a potent factor that selectively induces the release of FSH, but not
LH. See McCann,
S. et al., Control of follicle-stimulating hormone and luteinizing hormone
release by
hypothalamic peptides, Annals New York Academy of Sciences, 687:55-59, 1993;
Dees, W.
et al., Ethanol and the pulsatile release of luteinizing hormone, follicle
stimulating hormone
and prolactin in ovariectomized rats, Alcohol, 2:641-646, 1985; Dhariwal, A.
et al.,
Separation of follicle-stimulating hormone-releasing factor from luteinizing
hormone-releasing
factor, Endocrinology, 76:290-294, 1965; Dhariwal, A. et al., Chromatographic
behavior of
follicle stimulating hormone-releasing factor on Sephadex and carboxy methyl
cellulose,
Neuroendocrinology, 2:294-303, 1967; Igarashi, M. et al., A hypothalamic
follicle stimulating
hormone-releasing factor,. Endocrinology, 74:446-452, 1964; Lumpkin, M. et
al., Effect of
destruction of the dorsal anterior hypothalamus on follicle-stimulating
hormone secretion in
the rat, Endocrinology, 115:2473-2480, 1984; Samson, W. et al.,
Chromatographic and
biologic analysis of ME and OVLT LHRH, Peptides, 1:97-102, 1980; Mizunuma, H.,
et al.,
Evidence for an FSH-releasing factor in the posterior portion of the rat
median eminence,
Life Sci., 33:2003-2009, 1983.
Luteinizing hormone releasing hormone (LHRH, also known as gonadotropin-
releasing
hormone, or GnRH) has both LH-releasing activity and FSH-releasing activity.
See Schally,
A. et al., Gonadotropin-releasing hormone: one polypeptide regulates secretion
of luteinizing
and follicle-stimulating hormones, Science, 173:1036-1038, 1971; and D.
Lincoln,
Gonadotropin-releasing hormone (GnRH): basic physiology, pp. 218-229 in L.
DeGroot et
al., Endocrinology, 1995. The latter states at page 218: "There is no
convincing evidence
for the existence of a separate and specific FSH-releasing hormone, although
some
components of the GnRH precursor and some GnRH analogues appear to differ in
the degree
to which they stimulate the secretion of the two gonadotropins."
Sower, S. et al., Primary structure and biological activity of a third
gonadotropin
releasing hormone from lamprey brain, Endocrinology, 132:1125-1131, 1993
reported the
structure of lamprey GnRH-III (referred to as 1-LHRH-III in this
specification), and reported
that it stimulated estradiol and progesterone release from Petromyzon marinus
(lamprey)
ovaries. (Lampreys, jawless fish, are representatives of what is generally
considered to be the
most primitive of the extant classes of vertebrates.)
Lamprey I-LHRH-I has been reported to have relatively low activity in
releasing
either FSH or LH in rats. Yu, W. et al., Selective FSH-releasing activity of
[D-Trp~]GAP,_13:

CA 02293664 1999-12-03
WO 98/SSI36 PCT/US98/11512
3
comparison with gonadotropin-releasing abilities of analogs of GAP and natural
LHRHs,
Brain Res. Bull., 25:867-873, 1990.
Schally, A. et al., Re-examination of porcine and bovine hypothalamic
fractions for
additional luteinizing hormone and follicle stimulating hormone-releasing
activities,
Endocrinology, 98:380-391, 1976 reported that in vivo FSH-releasing activity
could not be
separated from LH-releasing activity from porcine hypothalami by fractionation
on Sephadex,
and concluded that there was only one gonadotropin-releasing hormone (GnRH).
By contrast, Lumpkin, M. et al., Purification of FSH-releasing factor: Its
dissimilarity
from LHRH of mammalian, avian, and piscian origin, Brain Res. Bull., 18:175-
178, 1987
reported that FSH-releasing activity was separated from the LH-releasing
activity in ovine
hypothalami on Sephadex G-25, but did not isolate the factor causing FSH
release.
Neurons that are immunopositive for 1-LHRH-I have been identified in human
hypothalami, projecting from the arcuate region to the median eminence. Stops,
E. et al,
Polygenic expression of gonadotropin-releasing hormone (GnRH) in human?,
Peptides, 9:419-
423, 1988.
Lincoln, D., Luteinizing Hormone-Releasing Hormone, pp. 142-151 in DeGroot et
al.
(ed), Endocrinology, 1989, discloses various agonists and antagonists for
mammalian LHRH.
W. Yu et al., "A hypothalamic follicle-stimulating hormone-releasing
decapeptide in
the rat," Proc. Natl. Acad. Sci. USA, 94:9499-9503, 1997 discloses some of the
work
reported in the present specification, but is not believed to constitute prior
art.
United States patent 4,973,577 discloses a 28,000 dalton protein isolated from
porcine
follicular fluid that stimulates the release of FSH, but not of LH. This
protein has a relatively
slow onset of action, and is relatively difficult to synthesize. The protein
was said to be a
homodimer of two chains of 116 amino acid residues each, or 232 residues
total.
United States patent 3,888,836 discloses a method for synthesizing mammalian
LHRH. Mammalian LHRH causes increased serum levels of both LH and FSH.
United States patent 4,721,775 discloses certain peptides that non-selectively
induce
the secretion of both LH and FSH.
.30 Attempts in our laboratory to purify FSH-releasing factor (FSH-RF} by
fractionation
of lamb hypothalami (discussed in some of the papers cited above) were
successful only at
certain seasons of the year, and even then we found that activity was lost
after samples were
stored at -20°C (unpublished data). Thus our prior work did not
successfully isolate or
identify the putative FSH-releasing factor.

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
4
Other studies in our laboratory confirmed FSH-releasing activity by incubating
stalk
median eminence (SME)-extracts in vitro with hemipituitaries from male rats.
VJe confirmed
the FSH-releasing activity of sheep and rat SME extracts in this assay, and
found that the
FSH-releasing activity emerged from columns of Sephadex G-25 just prior to
emergence of
LHRH, similar to results we had seen in an in vivo assay in ovariectomized,
estrogen- and
progesterone-blocked female rats. Even where we were able to extract crude or
partially
purified fractions showing selective FSH-releasing activity, that activity was
relatively low
compared to the activity of fractions with LH-releasing activity (unpublished
data).
Our laboratory also screened known LHRH's from various species for selective
FSH-
releasing activity; and we also evaluated the activity of 25 analogs of LHRH
in in vivo assays.
(LHRH's from various species are disclosed in Lumpkin et al., 1987.) One
analog was found
to have only FSH-releasing activity, but its potency was very low, and the
slope of its dose-
response curve was flat. Of the known forms of LHRH from other species, we
found that
only chicken (c) LHRH-II had slightly preferential FSH-releasing activity in
vivo (unpublished
data).
DISCLOSURE OF INVENTION
We have unexpectedly discovered that 1-LHRH-III is the long-sought FSH-
releasing
factor. This activity has been confirmed in vitro by incubation with hemi-
anterior pituitaries
of adult male rats. Following intravenous injection at the lowest dose tested
to date (10
picomoles), this peptide produced an increase in FSH in vivo (P < 0.01) within
ten minutes,
but no significant increase in LH. Such a selective effect has not previously
been reported for
any analog of mammalian LHRH.

CA 02293664 1999-12-03
WO 98155136 PCTNS98/11512
MODES FOR CARRYING OUT THE INVENTION _
TABLE 1
Structure of Vertebrate LIiRII's Tested
5 Mammal pGlu-His-Trp-Ser-Tyr-Gly-Leu-ArQ-(SEQ. ID
Pro-Gly-NHZ NO. 2)
(m-LHRH)
Lamprey III pGiu-His-Trp-Ser-His-Asp-Trp-Lys-(SEQ. ID
Pro-Gly-NHz NO. 1)
(1-LHRH-III)
Lamprey I pGlu-His-Tyr-Ser-Leu-Glu-Trp-Lys-(SEQ. ID
Pro-Gly-NHZ NO. 3)
(1-LHRH-I)
Salmon pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-(SEQ. ID
Pro-Gly-NHz NO. 4)
(s-LHRH)
Chicken II pGlu-His=frp-Ser-His-Gly-Trp-Tyr-(SEQ. ID
Pro-Gly-NHS NO. 5)
(c-LHRH-II)
General
Adult male and female Sprague-Dawley rats (Holtzmann, Madison, WI; 200-250 g)
were housed two per cage under controlled conditions of temperature (23-
25°C) and lighting
(on from 0500 to 1700 hr). The animals had free access to a pellet diet and to
tap water.
The 1-LHRH-III used in the experiments reported here was synthesized by
standard
solid-state peptide synthesis methods, and was purified to greater than 97%
purity by
preparative reverse-phase high performance liquid chromatography. All other
peptides used
were purchased from Peninsula Laboratories (Belmont, CA), except as otherwise
noted.
The significance of differences among muitiple groups was determined by
analysis of
variance, with subsequent Newman-Keuls multiple comparisons at each point.
Student's t-test
was used to determine the significance of differences between two groups.
In vitro studies
. 30 . After acclimatization for 5 or more days in the vivarium, male rats
were killed by
decapitation. Following removal of the posterior lobe, the anterior pituitary
(AP) was bisected
longitudinally, and each AP was incubated in a tube containing 0.5 ml Krebs-
Ringer
bicarbonate (5 mM ascorbic acid; pH 7.4) buffer (KRB) in an atmosphere of 95 %
OZ/5 % COZ
in a Dubnoff shaker (50 cycles per min) for a period of b0 min. Following this
pre-
incubation period, the APs were incubated for 3 hr in fresh KRB buffer alone
(control), or in

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
6
KRB containing one of various concentrations of synthetic mammalian (m}-LHRH,
1-LHRH-__
III, 1-LHRH-I, chicken (c)-LHRH-II, or salmon (s}-LHRH. The medium was then
aspirated,
and was stored frozen at -20°C until radio-immunoassays (RIA) for FSH
and LH were
conducted. FSH and LH were measured using kits supplied by the National
Institute of
Arthritis Digestive Diabetes and Kidney Disease; hormone values were expressed
in terms of
NIH-rFSH-RP-2 and NIH-rLH-RP-3 standards. The experiments were repeated twice.
The
inter- and infra-assay coefficients of variation for FSH assays were 7.5 % and
5.2 % ,
respectively; and were 6.5 % and 4.8 % for LH assays.
In vivo studies
Female rats were ovariectomized under anesthesia with isoflurane 4 weeks prior
to the
in vivo experiments. Three days before blood sampling, each ovariectomized rat
was injected
subcutaneously with SO icg estradiol benzoate (Sigma Chemical Co., St. Louis,
MO) dissolved
in 0.1 ml sesame oil, and 25 mg progesterone (Eli Lilly, Indianapolis, IN)
dissolved in 0.5 ml
sesame oil. One day prior to testing, each animal was implanted with a jugular-
atrial catheter.
On the morning of testing, polyethylene tubing (PE-50) was connected to the
distal end of the
jugular-atrial catheter on the rat's dorsum to facilitate blood sampling and
intravenous
injection. Animals were acclimatized one hour before initial blood samples
were taken.
Heparinized blood samples (5 ml) were collected just before, and at 10, 30,
and 60 minutes
after injection of 0.5 ml isotonic saline or 1-LHRH-III (10 or 100 pmole) in
0.5 ml isotonic
saline. After removal of each blood sample, an equal volume of isotonic saline
was
administered to maintain blood volume.
Effects of 1-LHRH 1, 1 LHRH 111, and Mammalian (m)-LIIRH on FSH Release In
Vitro
Lamprey 1-LHRH-III caused FSH release in a dose-related fashion, with a
minimal
effective concentration (MEC) of 10-9 M (the lowest concentration tested) or
lower, and a
maximal effect at about 10'6 M. Thereafter, release of FSH levelled off
through the highest
concentration tested (10'° M).
Lamprey 1-LHRH-I caused a small but statistically significant release of FSH
at a
concentration of 10-s M; the amount of FSH released was significantly less
than that caused by
1-LHRH-III at the two concentrations tested (10~ and 10-5 M). By contrast, m-
LHRH
produced equivalent levels of FSH release at the two concentrations tested {4
x 10-9 and 2 x 10
-g M) as compared to the levels induced by I-LHRH-III. See Table 2.

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
7
Table 2
Group FSH (ng/ml)
KRB (control) 97.7 20.4
~5 m-LHRH, 4 x 109 M ** 218.5 35.7
m-LHRH, 2 x 10-8 M ** 257.5 42.4
- 1-LHRH-III, 10-9 M * 228.3 52.5
1-LHRH-III, 10-$ M ** 255.7 42.4
I-LHRH-III, 10'' M *** 213.0 17.9
1-LHRH-III, 10-6 M ** 278.2 40.9
1-LHRH-III, 10'5 M ** 283.0 60.8
I-LHRH-III, 10~ M **** 368.6 40.9
I-LHRH-I, 10~ M * 164.0 29.3
1-LHRH-I, 10-5 M ** 178.2 17.0
n = 6 in each case; * signifies p < 0.05; ** signifies p < 0.01; *** signifies
p < 0.001; and **** signifies p < 0.0001 versus KRB control
Effect of 1-LFIRH 1, 1-LHRH Ill, and m-LIIRH on LH Release In Vitro
In contrast to its effects on FSH release, I-LHRH-III had a much weaker effect
on LH
release. In fact, I-LHRH-III only caused the release of significant and
comparable
concentrations of LH at the three highest concentrations tested (10'6 -
10'° M). We found that
I-LHRH-I was inactive at the two concentrations tested (10-6 and 10'5 M).
Mammalian LHRH
gave a dose-related stimulatory effect on LH release, and was active at the
lowest
concentration tested (4 x 10-9 M). See Table 3.

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
8
Table 3
Group ~ (~~~)
KRB (control) 139.3 t 16.9
S m-LHRH, 4 x 10'9 M 183.1 t 27.0
m-LHRH, 2 x 10'e M ** 262.0 t 36.1
1-LHRH-III, 10'9 M 171.8 27.3
1-LHRH-III, 10'e M 165.7 t 26.6
I-LHRH-III, 10'' M 179.9 28.4
1-LHRH-III, 10'6 M * 233.6 33.8
1-LHRH-III, 10-5 M * 202.9 31.2
1-LHRH-III, 10~ M ** 236.6 19.3
1-LHRH-I, 10-6 M 134.1 23.2
1-LHRH-I, 10'5 M 120.4 8.1
n = 6 in each case; * signifies p < 0.05; ** signifies p < 0.01; versus KRB
control
?0 Effects of Salmon and Chicken LFIRH II on Gonadotropin Release In Vitro
Salmon (s)-LHRH stimulated the release of both FSH and LH at the two
concentrations tested (10 ~' and 106 M). See Tables 4 and S. Chicken (c) LHRH-
II stimulated
LH release at doses from 10-8 to 10-° M, but the dose effect was not
statistically significant.
The c-LHRH-II had equivalent LH-releasing activity to that of m-LHRH, but 1-
LHRH-III
showed no LH-releasing activity in this experiment. The c-LHRH-II only
significantly
increased FSH release at the highest concentration tested (10 ~ M). In this
experiment, the
MEC for 1-LHRH-III for a statistically significant release of FSH was two
orders of
magnitude lower than that for c-LHRH-II.

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/115I2
9
Table 4
Group ~ FSH (ng/ml)
KRB (control) 157.5 t 13.2
~5 m-LHRH, 2 x 10'8 M ** 320.5 t 42.6
1-LHRH-III, 10'~ M 196.3 t 21.2
1-LHRH-III, 10'8 M * 249.3 31.3
1-LHRH-III, 10~' M * 203.2 16.4
1-LHRH-III, 10-6 M 215.0 38.0
c-LHRH-II, 10-8 M 123.7 18.0
c-LHRH-II, 10'' M 209.3 44.0
c-LHRH-II, 10'6 M *** 263.7 17.8
s-LHRH, 10 M * 233.0 37.4
s-LHRH, 10'6 M 203.2 t 39.2
n = 6 in each case; * signifies p < 0.05; ** signifies p < 0.01; and ***
signifies
p < 0.001 versus KRB control

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
Table 5
Group LH (ng/ml)
KRB (control) 149.3 17.0
5 m-LHRH, 2 x 10-8 M * 214.7 30.3
1-LHRH-III, 10-9 M 158.7 10.2
- 1-LHRH-III, 10-8 M 163.2 6.5
1-LHRH-III, 10-' M 182.7 t 13.7
1-LHRH-III, 10-6 M 176.0 t 9.3
10 c-LHRH-II, 10'8 M * 199.3 13.6
c-LHRH-II, 10-' M * 216.3 17.7
c-LHRH-II, 10-6 M * 239.0 31.7
s-LHRH, 10-' M * 230.8 38.8
s-LHRH, 10~ M 186.2 13.6
n = 6 in each case; * signifies p < 0.05 versus KRB control
Effects of 1-LFIRH 111 on FSII and LH Release In Vivo
Saline-injected control animals experienced a significant decline in plasma
FSH levels
10 minutes after injection, followed by a return to levels that did not differ
significantly from
pre-injection FSH levels by 30- and 60-minutes post-injection (data not
shown).
Compared to the control animals, animals injected with the lowest dose of 1-
LHRH-III
(10 pmole) showed a highly significant increase in plasma FSH 10 minutes after
injection, an
effect that vanished by 30 minutes post-injection. See Table 6. Increasing the
dose of 1-
LHRH-III to 100 pmole produced a slightly greater effect at 10 minutes that
was maintained
minutes after injection. Thus the 100 pmole dose caused a more prolonged
effect than the
10 pmole dose.

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/I 1512
11
Table 6
Group Change in FSH from baseline,
10 minutes post-injection (ng/ml)
saline -3.85 0.82
1-LHItH-III, 10 pmole ** -0.75 t 0.37
1-LHItH-III, 100 pmole ** -0.43 0.61
n = 6 in each case, except n = 7 for 1-LHRH-III, 100 pmole; ** signifies p <
0.01
versus saline control
Injection of saline diluent produced a slight decrease in LH plasma
concentration i0
minutes post-injection, an effect that continued for the duration of the
experiment, but that was
not statistically significant. Neither the 10 pmole nor the 100 pmole dose of
1-LHItH-III
produced a statistically significant difference in LH levels versus saline
control at any of the
times measured. See Table 7.
Table 7
Group Change in LH from baseline,
10 minutes post-injection (ng/tnl)
saline -0.070 0.122
1-LHItH-III, 10 pmole -0.022 0.078
1-LHItH-III, 100 pmole 0.097 t 0.077
n = 6 in each case, except n = 7 for 1-LHRH-III, 100 pmole; the measured
values
were not significantly different from one another.
Thus I-LHItH-III caused the release of FSH in_ vivo, but not LH, at each of
the two
doses tested, 10 pmole and 100 pmole.
As these results have demonstrated, I-LHItH-III is the first highly specific
and potent
FSH-releasing peptide discovered. The 1-LHRH-III behaves completely
differently from the

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
12
other polypeptides tested. We found that 1-LHRH-I had minimal potency to
release either
FSH or LH. Salmon LHRH and chicken LHRH-II had low potency, and lacked
specificity
for FSH release. The in vivo assays using the other known vertebrate LHRH's
showed that
only one of them, c-LHRH-II, possessed even slight selectivity for FSH
release.
We conclude that 1-LHRH-III is a highly conserved peptide in vertebrates, and
in
particular that it or a closely related peptide is the mammalian peptide
hormone responsible
for the potent and specific release of FSH. If I-LHRH-III is not identical to
the mammalian
FSH-RF, the degree of homology between the two is quite high.
Mammalian m-LHRH and 1-LHRH-III were approximately equipotent toward releasing
FSH. The decreased potency of I-LHRH-III toward releasing LH is probably
accounted for
by the fact that 1-LHRH-II1 only has 60% homology with m-LHRH (see Table 1).
The
differences in sequences are accounted for by the differing amino acids in
positions 5-8, which
presumably cause a drastic decrease in LH-releasing activity and an increase
in FSH-releasing
capabilities. It is probable that the tetrapeptide 1-LHRH-III 5-8 binds to the
active site of a
putative specific FSH-RF receptor. Presumably, the FSH-RF receptor confers
this specificity
for FSH release, whereas the LHRH receptors stimulate the release of both
hormones, albeit
with a greater sensitivity for LH than FSH release. FSH-RF receptors may
reside on
gonadotropes that contain only FSH. LHRH receptors may cause release of both
hormones
from gonadotropes that contain both FSH and LH. LHRH receptors may also be
located on
gonadotropes that only contain LH.
Pulsatile gonadotropin release in the rat is characterized by simultaneous
pulses of
FSH and LH, by pulses of LH alone, and by pulses of FSH alone. We hypothesize
that the
first two types of pulses may be accounted for by LHRH, and the third by FSH-
RF.
The discovery of FSH-RF has important implications for veterinary and human
medicine. For example, treatment of farm animals with FSH-RF should lead to
maturation of
increased . numbers of ovarian follicles and subsequent ovulations, leading to
increased litter
sizes. FSH-RF may be used as a drug to increase fertility in humans.
Related peptides are expected to be agonists or "superagonists" of 1-LHRH-III.
These
agonists will be prepared and tested in similar assays. Particular examples
include peptides in
which (a) the Aspb amino acid residue has been replaced with another amino
acid residue
(naturally occurring or xenobiotic); or (b) in which the Pro9 residue has been
replaced with
ProNHEt (prolyl ethyl amide) and the Gly'°-NHZ has been deleted; or (c)
both. Examples of
such agonists include the following:

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
13
pGlu-His-Trp-Ser-His-Asp-Trp-Lys-(ProNHEt) (SEQ. ID NO. 6)
pGlu-His-Trp-Ser-His-Ala-Trp-Lys-Pro-Gly-NHZ (SEQ. ID NO. 7)
pGlu-His-Trp-Ser-His-Ala-Trp-Lys-(ProNHEt) (SEQ. ID NO. 8)
pGlu-His-Trp-Ser-His-(D-Ala)-Trp-Lys-Pro-Gly-NHZ (SEQ. ID NO. 9)
pGlu-His-Trp-Ser-His-(D-Ala)-Trp-Lys-(ProNHEt) (SEQ. ID NO. 10)
pGlu-His-Trp-Ser-His-Leu-Trp-Lys-Pro-Gly-NHZ (SEQ. ID NO. 11)
pGlu-His-Trp-Ser-His-Leu-Trp-Lys-(ProNHEt) (SEQ. ID NO. 12)
pGlu-His-Trp-Ser-His-(D-Leu)-Trp-Lys-Pro-Gly-NHZ (SEQ. ID NO. 13)
pGlu-His-Trp-Ser-His-(D-Leu)-Trp-Lys-(ProNHEt) (SEQ. ID NO. 14)
pGlu-His-Trp-Ser-His-(SerBu~-Trp-Lys-Pro-Gly-NHz (SEQ. ID NO. 15)
pGlu-His-Trp-Ser-His-(SerBu')-Trp-Lys-(ProNHEt) {SEQ. ID NO. 16)
pGlu-His-Trp-Ser-His-(D-SerBu')-Trp-Lys-Pro-Gly-NHZ (SEQ. ID NO. 17)
pGlu-His-Trp-Ser-His-(D-SerBu')-Trp-Lys-(ProNHEt) (SEQ. ID NO. 18)
pGlu-His-Trp-Ser-His-Trp-Trp-Lys-Pro-Gly-NHZ (SEQ. ID NO. 19)
pGlu-His-Trp-Ser-His-Trp-Trp-Lys-(ProNHEt) (SEQ. ID NO. 20)
pGlu-His-Trp-Ser-His-(D-Trp)-Trp-Lys-Pro-Gly-NHZ (SEQ. ID NO. 21)
pGlu-His-Trp-Ser-His-(D-Trp)-Trp-Lys-(ProNHEt) (SEQ. ID NO. 22)
pGlu-His-Trp-Ser-His-(His-Bzl)-Trp-Lys-Pro-Gly-NHZ (SEQ. ID NO. 23)

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
14
pGlu-His-Trp-Ser-His-(His-Bzl)-Trp-Lys-(ProNHEt) (SEQ. ID NO. 24) -
pGlu-His-Trp-Ser-His-(D-His-Bzl)-Trp-Lys-Pro-Gly-NHZ {SEQ. ID NO. 25)
pGlu-His-Trp-Ser-His-(D-His-Bzl)-Trp-Lys-(ProNHEt) (SEQ. ID NO. 26)
pGlu-His-Trp-Ser-His-Nal(2)-Trp-Lys-Pro-Gly-NHZ (SEQ. ID NO. 27)
pGlu-His-Trp-Ser-His-Nal(2)-Trp-Lys-(ProNHEt) (SEQ. ID NO. 28)
pGlu-His-Trp-Ser-His-(D-Nal(2)}-Trp-Lys-Pro-Gly-NHz (SEQ. ID NO. 29)
pGlu-His-Trp-Ser-His-(D-Nal(2))-Trp-Lys-(ProNHEt) (SEQ. ID NO. 30)
pGlu-His-Trp-Ser-His-Nal(2)-Trp-Lys-Pro-(aza-Gly)-NHZ (SEQ. ID NO. 31)
pGlu-His-Trp-Ser-His-(D-Nal(2))-Trp-Lys-Pro-(aza-Gly)-NHZ (SEQ. ID NO. 32)
Note: "(aza-Gly)-NHZ" in SEQ. ID NOs. 31 and 32 denotes -NH-NH-CONHZ.
Converseiy, inhibitory analogs of the peptide, or antibodies against the
peptide, may
be used as potent antifertility drugs. Monoclonal or polyclonal antibodies
against the peptide
may be raised using standard techniques, initially conjugating the peptide to
a carrier such as
bovine serum albumin or keyhole limpet hemocyanin. Particular examples of
antagonists
include peptides in which one or more of residues 1, 2, 3, 6, and 10 have been
replaced with
another amino acid residue (naturally occurring or xenobiotic). Examples of
such antagonists
include the following:
pGlu-(D-Phe)-Trp-Ser-His-(D-Ala)-Trp-Lys-Pro-Gly-NHZ (SEQ. ID NO. 33)
pGlu-(D-Phe)-(D-Trp)-Ser-His-(D-Trp)-Trp-Lys-Pro-Gly-NH2 (SEQ. ID NO. 34)
(D-pyro-Glu)-(D-Phe)-(D-Trp)-Ser-His-(D-Trp)-Trp-Lys-Pro-Gly-NHZ (SEQ. ID NO.
35)

CA 02293664 1999-12-03
WO 98!55136 PCT/US98l11512
(N-Ac-D-Phe)-(D-p-Cl-Phe)-{D-Trp)-Ser-His-(D-Trp)-Trp-Lys-Pro-Gly-NHZ (SEQ.
ID NO. 3b)
(N-Ac-D-p-CI-Phe)-(Ac-D-p-Cl-Phe)-(D-Trp)-Ser-His-(D-Phe)-Trp-Lys-Pro-(D-Ala)-
5 NHZ (SEQ. ID NO. 37)
(N-Ac-D-p-CI-Phe)-(Ac-D-p-CI-Phe)-(D-Trp)-Ser-His-(D-Arg)-Trp-Lys-Pro-(D-Ala)-
NHZ (SEQ. ID NO. 38)
10 (N-Ac-D-Nal(2))-(p-F-D-Phe)-(D-Trp)-Ser-His-(D-Arg)-Trp-Lys-Pro-Gly-NHz
(SEQ.
ID NO. 39)
(N-Ac-D-Nal(2))-(D-p-CI-Phe)-(D-Trp)-Ser-His-(D-L-Arg-Et2)-Trp-Lys-Pro-(D-Ala}-
NHZ (SEQ. ID NO. 40)
(N-Ac-D-Nal(2))-(D-p-Cl-Phe)-(D-3-Pal)-Ser-His-{D-Arg)-(D-Trp)-Lys-Pro-(D-Ala)-
NHz (SEQ. ID NO. 41)
The effect of the peptide, its agonists, and its antagonists will be
characterized in rats,
and then in large mammals, such as sheep and pigs. After successful testing in
large
mammals, in vivo tests in primates will be conducted, in monkeys and in
humans, in
accordance with applicable laws and regulations.
The peptide, an agonist, or an antagonist, combined with a pharmaceutically
acceptable carrier, may be administered to mammals, including humans,
intravenously,
subcutaneously, percutaneously, intramuscularly, or intranasally to increase
fertility. It may
also be administered to other vertebrates to increase fertility, for example
sheep, cattle, pigs,
chickens, turkeys, channel catfish, tilapia, and koi.
The active compound may be administered as a pharmaceutically acceptable salt,
such
. 30 as an acid addition salt; metal complex, e.g. with zinc, iron; or the
like (which are considered
salts for purposes herein). Illustrative of such acid addition salts are
hydrochloride,
hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate,
succinate, malate,
ascorbate, tartrate, and the like. Intravenous or other injections may be
administered in
isotonic saline, phosphate buffers, and the like.

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
16
The dosage will vary depending on the specific purpose for which the peptide
is
administered; appropriate dosages may readily be determined by those of skill
in the art, an
"effective amount" being that which elevates or depresses serum FSH levels by
a statistically
significant amount, or that which enhances or inhibits fertility to a
statistically significant
degree.
For example, a patient experiencing infertility of unknown cause may be tested
for 1-
LHRH-III or FSH deficit through means otherwise known in the art, such as
radioimmunoassay. Abnormally low FSH levels may indicate a deficiency in 1-
LHRIi-III.
Such patients may be tested for a positive response to exogenous 1-LHRH-III.
If
administration of exogenous 1-LHRH-III produces an increase in FSH, that
Ending indicates
that administration of exogenous 1-LHRH-III is a potential treatment for
infertility.
As may prove most economical, the l-LHRH-III may be synthesized through any of
various means known in the art, for example, synthesis on a solid-state
peptide synthesizer, or
expression of a cloned gene in E. coli.
The complete disclosures of all references cited in this specification are
hereby
incorporated by reference. In the event of an otherwise irreconcilable
conflict, however, the
present specification shall control.

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
17
SEQUENCE FISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: McCann, Samuel M.
Yu, Wen H.
(ii) TITLE OF INVENTION: FSH-Releasing Peptides
IO
(iii) NUMBER OF SEQUENCES: 41
{iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: John H. Runnels
15 (B) STREET: P. 0. Box 2471
(C) CITY: Baton Rouge
(D) STATE: LA
(E) COUNTRY: USA
(F) ZIP: 70821-2471
20
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
25 (D) SOFTWARE: PatentIn Release #1.0, Version #1.25;
WordPerfect
5.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
3O {B) FILING DATE:
{C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Runnels, John H.
35 (B) REGISTRATION NUMBER: 33451
(C) REFERENCE/DOCKET NUMBER: 9703P.1
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (504) 387-3221
40 (B) TELEFAX: (504) 346-8049
(2) INFORMATION
FOR SEQ
ID NO:
l:
45 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
50 (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION:/note= "Xaa at 1 is pyro-Glu;
Xaa at
is Gly-NH2"
55
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Xaa His Trp Ser His Asp Trp Lys Pro Xaa
1 5 10
60

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
18
(2) INFORMATION FOR SEQ ID N0:2: _
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION:/note= "Xaa at 1 is gyro-Glu; Xaa
at
10 is Gly-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Xaa His Trp Ser Tyr Gly Leu Arg Pro Xaa
1 5 10
(2) INFORMATION FOR SEQ ID N0:3:
0
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION:/note= "Xaa at 1 is pyro-Glu; Xaa
at
10 is Gly-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Xaa His Tyr Ser Leu Glu Trp Lys Pro Xaa
1 5 to
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION:/note= "Xaa at 1 is gyro-Glu; Xaa
at
10 is Gly-NH2"
SO (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Xaa His Trp Ser Tyr Gly Trp Leu Pro Xaa
1 5 10

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
19
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
S (B) TYPE: amino acid
' (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION:/note= ~~Xaa at 1 is pyro-Glu; Xaa
at
10 is Gly-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Xaa His Trp Ser His Gly Txp Tyr Pro Xaa
1 5 10
{2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
{D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION:/note= ~~Xaa at 1 is pyro-Glu; Xaa
at 9
i6 (PrONHEt)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Xaa His Trp Ser His Asp Trp Lys Xaa
1 5
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION:/note= "Xaa at 1 is pyro-Glu; Xaa
at
10 is Gly-NH2"
SO (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Xaa His Trp Ser His Ala Txp Lys Pro Xaa
1 5 10

CA 02293664 1999-12-03
WO 98/55136 PCTNS98/11512
(2) INFORMATION FOR SEQ ID N0:8: -
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
5 (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa at
9 is (ProNHEt)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Xaa His Trp Ser His Ala Trp Lys Xaa
1 5
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is gyro-Glu; Xaa at
6 is (D-Ala); Xaa at 10 is Gly-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Xaa His Trp Ser His Xaa Trp Lys Pro Xaa
1 5 10
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix) FEATURE
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa at
6 is (D-Ala); Xaa at 9 is (ProNHEt)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
Xaa His Trp Ser His Xaa Trp Lys Xaa
1 5

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
21
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
S (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION:/note= "Xaa at 1 is pyro-Glu; Xaa
at
10 is Gly-NH2"
(xi) .SEQUENCE DESCRIPTION: SEQ ID N0:11:
IS
Xaa His Txp Ser His Leu Trp Lys Pro Xaa
1 5 10
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
2S
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa
at
9 is
(ProNHEt)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Xaa His Trp Ser His Leu Trp Lys Xaa
3S 1 5
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
4S
(ix) .FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa
at
6 is
(D-Leu);
Xaa
at
10
is
Gly-NH2"
SO (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Xaa His Txp Ser His Xaa Trp Lys Pro Xaa
1 5 10

CA 02293664 1999-12-03
WO 98/55136 PCTNS98/11512
22
(2) INFORMATION FOR SEQ ID N0:14: _
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa at
6 is (D-Leu) ; Xaa at 9 is (ProNHEt) "
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Xaa His Trp Ser His Xaa Trp Lys Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa at
6 is (SerBut); Xaa at 10 is Gly-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Xaa His Txp Ser His Xaa Tzp Lys Pro Xaa
1 s to
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa at
6 is (SerBut); Xaa at 9 is (ProNHEt)"
SO (xi) SEQBENCE DESCRIPTION: SEQ ID N0:16:
Xaa His Trp Ser His Xaa Trp Lys Xaa
1 5

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
23
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
S (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
i0 (ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is gyro-Glu; Xaa at
6 is (D-SerBut); Xaa at 10 is Gly-NH2"
IS (xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Xaa His Tzp Ser His Xaa Trp Lys Pro Xaa
1 5 10
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
2S
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
6 is (D-SerBut) ; X~ at 9FORMATP O~~E ; ate= "Xaa at 1 is pyro-Glu; Xaa at
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Xaa His Tzp Ser His Xaa Txp Lys Xaa
3S 1 s
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
4S
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is gyro-Glu; Xaa at
10 is Gly-NH2"
SO (xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Xaa His Trp Ser His Trp Trp Lys Pro Xaa
1 5 10

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
24
(2) INFORMATION FOR SEQ ID N0:20: -
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
S (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
lO (ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at is pyro-Glu;
1 Xaa at
9 is (ProNHEt) "
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
1S
Xaa His Tzp Ser His Trp Trp Lys Xaa
1 5
(2) INFORMATION FOR SEQ ID N0:21:
20
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
2S
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at is pyro-Glu;
1 Xaa at
30 6 is (D-Trp); Xaa at 10 is Gly-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Xaa His Tzp Ser His Xaa Trp Lys Pro Xaa
3S 1 s to
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
40 (A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
4S
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at is pyro-Glu;
1 Xaa at
6 is (D-Trp); Xaa at 9 is (ProNHEt)"
SO (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Xaa His Tzp Ser His Xaa Tzp Lys Xaa
1. 5

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
2S
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa at
6 is (His-Bzl); Xaa at 10 is Gly-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
Xaa His Trp Ser His Xaa Tzp Lys Pro Xaa
1 5 10
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
2S (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa at
6 is (His-Bzl); Xaa at 9 is (ProNHEt)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Xaa His Trp Ser His Xaa Trp Lys Xaa
3S 1 5
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
4S
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa at
6 is (D-His-Bzl); Xaa at 10 is Gly-NH2"
SO (xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
Xaa His Trp Ser His Xaa Trp Lys Pro Xaa
1 5 10

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
26
(2) INFORMATION FOR SEQ ID N0:26: _
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
S (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULfi TYPE: peptide
IO (ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is gyro-Glu; Xaa at
6 is (His-Bzl); Xaa at 9 is (ProNHEt)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
IS
Xaa His Trp Ser His Xaa Trp Lys Xaa
1 5
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
2S
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa at
6 is Nal(2); Xaa at 10 is Gly-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Xaa His Trp Ser His Xaa Trp Lys Pro Xaa
3S 1 5 i o
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
4S
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa at
6 is Nal(2); Xaa at 9 is (ProNHEt)"
SO (xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Xaa His Trp Ser His Xaa Trp Lys Xaa
1 5

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
27
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa at
6 is (D-Nal(2)); Xaa at 10 is Gly-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
IS
Xaa His Trp Ser His Xaa Tzp Lys Pro Xaa
1 5 10
(2} INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa at
6 is (D-Nal(2)); Xaa at 9 is (ProNHEt)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
Xaa His Trp Ser His Xaa Trp Lys Xaa
1 5
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
4S
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu; Xaa at
6 is Nal(2); Xaa at 10 is (aza-Gly)"
SO (xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
Xaa His Trp Ser His Xaa Trp Lys Pro Xaa
1 5 10

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
28
(2) INFORMATION FOR SEQ ID N0:32: -
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
$ (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
lO (ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu;
Xaa at
6 is (D-Nal(2)); Xaa at 10 is (aza-Gly)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
15
Xaa His Trp Ser His Xaa Trp Lys Pro Xaa
1 5 10
(2) INFORMATION FOR SEQ ID N0:33:
..0
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
25
(ii) MOLECULE TYPE: peptide
( ix) FEATURE
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu;
Xaa at
30 2 is (D-Phe); Xaa at 6 is (D-Ala); Xaa at Gly-NH2"
is
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
Xaa Xaa Trp Ser His Xaa Trp Lys Pro Xaa
35 1 s to
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
40 (A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
45
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is pyro-Glu;
Xaa at
2 is (D-Phe) : Xaa at 3 is (D-Trp) ; Xaa is (D-Trp) ; Xaa at
at 6 10 is
Gly-NH2"
50
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
Xaa Xaa Xaa Ser His Xaa Trp Lys Pro Xaa
1 5 10
SS

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/I1512
29
(2) INFORMATION FOR SEQ ID N0:35: _
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
0 (ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is
(D-pyro-Glu); Xaa at 2 is (D-Phe); Xaa at 3 is (D-Trp); Xaa at 6 is
(D-Trp); Xaa at 10 is Gly-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
Xaa Xaa Xaa Ser His Xaa Trp Lys Pro Xaa
1 5 10
(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is (N-Ac-D-Phe); Xaa
at 2 is (D-p-C1-Phe); Xaa at 3 is (D-Trp); Xaa at 6 is (D-Trp); Xaa at
10 is Gly-NH2"
3S (xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
Xaa Xaa Xaa Ser His Xaa Trp Lys Pro Xaa
1 5 10
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is
(N-Ac-D-p-C1-Phe); Xaa at 2 is (Ac-D-p-C1-Phe); Xaa at 3 is (D-Trp); Xaa
at 6 is (D-Phe); Xaa at 10 is (D-Ala-NH2)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
SS Xaa Xaa Xaa Ser His Xaa Trp Lys Pro Xaa
1 5 10

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
(2) INFORMATION FOR SEQ ID N0:38: _
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
$ (B} TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
10 ( ix) FEATURE
(D) OTHER INFORMATION: /note= "Xaa at 1 is
(N-Ac-D-p-C1-Phe); Xaa at 2 is (Ac-D-p-C1-Phe); Xaa at 3 is (D-Trp); Xaa
at 6 is (D-Arg); Xaa at 10 is (D-Ala-NH2)"
IS
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
Xaa Xaa Xaa Ser His Xaa Trp Lys Pro Xaa
1 5 10
20
(2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
25 (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
30 (ix) FEATURE:
(D) OTHER INFORMATION: /note= "Xaa at 1 is
(N-Ac-D-Nal(2)); Xaa at 2 is (p-F-D-Phe); Xaa at 3 is (D-Txp);
Xaa at 6
is (D-Arg); Xaa at 10 is Gly-NH2"
3S (xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
Xaa Xaa Xaa Ser His Xaa Trp Lys Pro Xaa
1 5 10
(2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix) FEATURE
(D) OTHER INFORMATION: /note= "Xaa at 1 is
(N-Ac-D-Nal(2)); Xaa at 2 is (D-p-C1-Phe); Xaa at 3 is (D-Trp);
Xaa at 6
is (D-L-Arg-Et2); Xaa at 10 is (D-Ala-NH2)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
Xaa Xaa Xaa Ser His Xaa Trp Lys Pro Xaa
1 5 10

CA 02293664 1999-12-03
WO 98/55136 PCT/US98/11512
31
(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION:/note= "Xaa at 1 is
(N-Ac-D-Nal(2)); Xaa at 2 is (D-p-C1-Pize); Xaa at 3 is (D-3-Pal); Xaa at
6 is (D-Arg); Xaa at 7 is (D-Trp); Xaa at 10 is (D-Ala-NH2)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
Xaa Xaa Xaa Ser His Xaa Xaa Lys Pro Xaa
1 5 10