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Patent 1186302 Summary

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(12) Patent: (11) CA 1186302
(21) Application Number: 375536
(54) English Title: LRF ANTAGONISTS
(54) French Title: ANTAGONISTES DU LRF
Status: Expired
Bibliographic Data
(52) Canadian Patent Classification (CPC):
  • 530/7.06
(51) International Patent Classification (IPC):
  • C07K 7/06 (2006.01)
  • A61K 38/08 (2006.01)
  • A61P 15/00 (2006.01)
  • C07K 1/00 (2006.01)
(72) Inventors :
  • VALE, WYLIE, W., JR. (United States of America)
  • RIVIER, JEAN E.F. (United States of America)
(73) Owners :
  • SALK INSTITUTE FOR BIOLOGICAL STUDIES (THE) (Not Available)
(71) Applicants :
(74) Agent: MACRAE & CO.
(74) Associate agent:
(45) Issued: 1985-04-30
(22) Filed Date: 1981-04-15
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): No

(30) Application Priority Data:
Application No. Country/Territory Date
182,594 United States of America 1980-08-29
140,487 United States of America 1980-04-15

Abstracts

English Abstract




LRF ANTAGONISTS



ABSTRACT

Peptides which inhibit the secretion of
gonadotropins by the pituitary gland and inhibit the
release of steroids by the gonads. Administration of an
effective amount prevents ovulation of female mammalian
eggs and/or the release of steroids by the gonads. The
peptides have the structure:

X-R1-R2-R3-Ser-Tyr-R4-R5-Arg-Pro-R6

wherein X is hydrogen or an acyl group having 7 or less
carbon atoms; R1 is dehydro Pro, dehydro D-Pro, Thz or
D-Thz; R2 is D-Phe, D-His, D-Trp, Trp, Cl-D-Phe,
dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe,
AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe, Br-D-Phe,
dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or
CH3-D-Phe; R3 is D-Trp, Trp, D-Phe or D-His; R4 is
Gly or a D-isomer amino acid; R5 is Leu or N.alpha.Me-Leu;
and R6 is Gly-NH2 or NHCH2CH3.


Claims

Note: Claims are shown in the official language in which they were submitted.




The embodiments of the invention in which an
exclusive property or privilege is claimed are defined
as follows:
1. A method for the manufacture of a compound
having the formula:

X-R1-R2-R3-Ser-Tyr-R4-R5-Arg-Pro-R6
wherein X is hydrogen or an acyl group having 7 or less
carbon atoms; R1 is dehydro Pro, dehydro D-Pro, Thz or
D-Thz; R2 is D-Phe, D-His, D-Trp, Trp, Cl-D-Phe,
dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe,
AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe, Br-D-Phe,
dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or
CH3-D-Phe; R3 is D-Trp, Trp, D-Phe or D-His; R4 is
Gly or a D-isomer amino acid; R5 is Leu or N.alpha.Me-Leu;
and R6 is Gly-NH2 or NHCH2CH3.
comprising (a) forming an intermediate compound having
the formula:
X1-R1-R2-R3-Ser(X2)-Tyr-(X3)-R4-
R5-Arg(X4)-Pro-X5 wherein X1 is an .alpha.-amino
protecting group; X2 is a protecting group for the
alcoholic hydroxyl group of Ser; X3 is a protecting
group for the phenolic hydroxyl group of Tyr; X4 is a
protecting group for the nitrogen atoms of Arg; and X5
is selected from Gly-O-CH2[resin support],
O-CH2-[resin support], Gly-NH-[resin support],
Gly-NH2 and NHCH2CH3;
(b) splitting off one or more of the groups
X1 to X5 and, if desired, converting a resulting
peptide into a nontoxic salt thereof.
2. A method in accordance with Claim 1
wherein, in step (a), the intermediate compound is formed
with R2 representing dichloro-D-Phe, CF3-D-Phe,
F-D-Phe, difluoro-D-Phe, AcNH-D-Phe, NO2-D-Phe,
dinitro-D-Phe, Br-D-phe, dibromo-D-Phe, CH3S-D-Phe,
OCH3-D-Phe or CH3-D-Phe.
3. A method in accordance with Claim 2 wherein
R1 is dehydro-Pro.



18


4. A method in accordance with Claim 3 wherein
R2 is dichloro-D-Phe.
5. A method in accordance with Claim 3 wherein
R2 is 4 CF3-D-Phe.
6. A method in accordance with Claim 3 wherein
R2 is 4 AcNH-D-Phe.
7. A method in accordance with Claim 3 wherein
R2 is 4 NO2-D-Phe.
8. A method in accordance with Claim 3 wherein
R2 is 4 Br-D-Phe.
9. A method in accordance with Claim 3 wherein
R2 is 4 CH3S-D-Phe.
10. A method in accordance with Claim 3 wherein
R2 is 4 F-D-Phe.
11. A method in accordance with Claim 3 wherein
R2 is 4 OCH3-D-Phe.
12. A method in accordance with Claim 3 wherein
X is acrylyl.
13. A method in accordance with Claim 3 wherein
X is acetyl.
14. A method in accordance with Claim 3 wherein
R2 is 2,4 Cl-D-Phe, 3,4 Cl-D-Phe, 4F-D-Phe,
4NO2-D-Phe, 4Br-D-Phe, or 4CH3S-D-Phe.
15. A method in accordance with Claim 14
wherein R3 is D-Trp.
16. A method in accordance with Claim 14
wherein R4 is D-Trp.
17. A method in accordance with Claim 14
wherein R4 is a lipophilic, aromatic D-isomer amino
acid.
18. A method in accordance with Claim 14
wherein R5 is Leu.
19. A method in accordance with Claim 14
wherein R6 is Gly-NH2.
20. A method in accordance with Claim 2
wherein R5 is Leu.


19



21. A method in accordance with Claim 20
wherein X is acrylyl, R3 is D-Trp, R4 is D-Trp and
R6 is Gly-NH2.
22. A method in accordance with Claim 20
wherein X is acetyl, R3 is D-Trp, R4 is D-Trp and
R6 is Gly-NH2.
23. A method in accordance with Claim 1
wherein, in step (a), the intermediate compound is formed
with R2 representing 4 Cl-D-Phe.
24. A method in accordance with Claim 23
wherein R4 is D-Trp.
25. A method in accordance with Claim 23
wherein R4 is imBzl-D-His.
26. A method in accordance with Claim 23
wherein R4 is a lipophilic aromatic D-isomer amino
acid.
27. A method in accordance with Claim 26
wherein R5 is Leu.
28. A method in accordance with Claim 26
wherein R6 is Gly-NH2.
29. A method in accordance with Claim 26
wherein X is acetyl.
30. A method in accordance with Claim 26
wherein X is acrylyl.
31. A method in accordance with Claim 26
wherein R1 is dehydro-Pro.
32. A method in accordance with Claim 26
wherein R1 is dehydro-D-Pro.
33. A method in accordance with Claim 26
wherein R1 is Thz.
34. A method in accordance with Claim 26
wherein R1 is D-Thz.
35. A method in accordance with Claim 1
wherein, in step (a), the intermediate compound is formed
with R2 representing D-Phe and R3 representing D-Trp.
36. A method in accordance with Claim 35
wherein X is hydrogen.






37. A method in accordance with Claim 35
wherein X is acetyl.
38. A method in accordance with Claim 35
wherein X is acrylyl.
39. A method in accordance with Claim 37
wherein R5 is Leu.
40. A method in accordance with Claim 37
wherein R5 is N MeLeu.
41. A method in accordance with Claim 37
wherein R6 is Gly-NH2.
42. A method in accordance with Claim 37
wherein R6 is NHCH2CH3.
43. A peptide, or a nontoxic salt thereof,
having the formula:

X-R1-R2-R3-Ser-Tyr-R4-R5-Arg-Pro-R6

wherein X, R1, R2, R3, R4, R5 and R6 are
defined as in Claim 1 when prepared by the method of
Claim 1 or by an obvious chemical equivalent thereof.
44. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro and R2 is dichloro-D-Phe,
CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe,
NO2-D-Phe, dinitro-D-Phe, Br-D-Phe, dibromo-D-Phe,
CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe when prepared
by the method of Claim 2 or by an obvious chemical
equivalent thereof.
45. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro and R2 is dichloro-D-Phe,
CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe,
NO2-D-Phe, dinitro-D-Phe, Br-D-Phe, dibromo-D-Phe,
CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe when prepared
by the method of Claim 3 or by an obvious chemical
equivalent thereof.
46. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro and R2 is dichloro-D-Phe
when prepared by the method of Claim 4 or by an obvious
chemical equivalent thereof.



21


47. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro and R2 is 4 CF3-D-Phe
when prepared by the method of Claim 5 or by an obvious
chemical equivalent thereof.
48. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro and R2 is 4 AcNH-D-Phe
when prepared by the method of Claim 6 or by an obvious
chemical equivalent thereof.
49. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro and R2 is 4 NO2-D-Phe
when prepared by the method of Claim 7 or by an obvious
chemical equivalent thereof.
50. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro and R2 is 4 Br-D-Phe when
prepared by the method of Claim 8 or by an obvious
chemical equivalent thereof.
51. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro and R2 is 4 CH3S-D-Phe
when prepared by the method of Claim 9 or by an obvious
chemical equivalent thereof.
52. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro and R2 is 4 F-D-Phe when
prepared by the method of Claim 10 or by an obvious
chemical equivalent thereof.
53. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro and R2 is 4 CH3-D-Phe
when prepared by the method of Claim 11 or by an obvious
chemical equivalent thereof.
54. A peptide in accordance with Claim 43
wherein X is acrylyl, R1 is dehydro-Pro and R2 is
dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe,
AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe, Br-D-Phe,
dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe
when prepared by the method of Claim 12 or by an obvious
chemical equivalent thereof.


22



55. A peptide in accordance with Claim 43
wherein X is acetyl, R1 is dehydro-Pro and R2 is
dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe,
AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe, Br-D-Phe,
dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe
when prepared by the method of Claim 13 or by an obvious
chemical equivalent thereof.
56. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro and R2 is 2,4 Cl-D-Phe,
3,4 Cl-D-Phe, 4 F-D-Phe, 4NO2-D-Phe, 4Br-D-Phe or
4CH3S-D-Phe when prepared by the method of Claim 14 or
by an obvious chemical equivalent thereof.
57. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro, R2 is 2,4 Cl-D-Phe, 3,4
Cl-D-Phe, 4 F-D-Phe, 4NO2-D-Phe, 4Br-D-Phe or
4CH3S-D-Phe and R3 is D-Trp when prepared by the
method of Claim 15 or by an obvious chemical equivalent
thereof.
58. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro, R2 is 2,4 Cl-D-Phe, 3,4
Cl-D-Phe, 4 F-D-Phe, 4NO2-D-Phe, 4Br-D-Phe or
4CH3S-D-Phe and R4 is D-Trp when prepared by the
method of Claim 16 or by an obvious chemical equivalent
thereof.
59. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro, R2 is 2,4 Cl-D-Phe, 3,4
Cl-D-Phe, 4 F-D-Phe, 4NO2-D-Phe, 4Br-D-Phe or
4CH3S-D-Phe and R4 is a lipophilic, aromatic
D-isomer amino acid when prepared by the method of Claim
17 or by an obvious chemical equivalent thereof.
60. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro, R2 is 2,4 Cl-D-Phe, 3,4
Cl-D-Phe, 4 F-D-Phe, 4NO2-D-Phe, 4Br-D-Phe or
4CH3S-D-Phe and R5 is Leu when prepared by the
method of Claim 18 or by an obvious chemical equivalent
thereof.

23



61. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro, R2 is 2,4 Cl-D-Phe, 3,4
Cl-D-Phe, 4 F-D-Phe, 4NO2-D-Phe, 4Br-D-Phe or
4CH3S-D-Phe and R6 is Gly-NH2 when prepared by the
method of Claim 19 or by an obvious chemical equivalent
thereof.
62. A peptide in accordance with Claim 43
wherein R2 is dichloro-D-Phe, CF3-D-Phe, F-D-Phe,
difluoro-D-Phe, AcNH-D-Phe, NO2-D-Phe, dinitro-D-Phe,
Br-D-phe, dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or
CH3-D-Phe and R5 is Leu when prepared by the method
of Claim 20 or by an obvious chemical equivalent thereof.
63. A peptide in accordance with Claim 43
wherein X is acrylyl, R2 is dichloro-D-Phe,
CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe,
NO2-D-Phe, dinitro-D-Phe, Br-D-phe, dibromo-D-Phe,
CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe, R3 is
D-Trp, R4 is D-Trp and R6 is Gly-NH2 when prepared
by the method of Claim 21 or by an obvious chemical
equivalent thereof.
64. A peptide in accordance with Claim 43
wherein X is acrylyl, R2 is dichloro-D-Phe,
CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe,
NO2-D-Phe, dinitro-D-Phe, Br-D-phe, dibromo-D-Phe,
CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe, R3 is
D-Trp, R4 is D-Trp and R6 is Gly-NH2 when prepared
by the method of Claim 22 or by an obvious chemical
equivalent thereof.
65. A peptide in accordance with Claim 43
wherein R2 is 4 Cl-D-Phe when prepared by the method
of Claim 23 or by an obvious chemical equivalent thereof.
66. A peptide in accordance with Claim 43
wherein R2 is 4 Cl-D-Phe and R4 is D-Trp when
prepared by the method of Claim 24 or by an obvious
chemical equivalent thereof.
67. A peptide in accordance with Claim 43
wherein R2 is 4 Cl-D-Phe and R4 is imBzl-D-His when
prepared by the method of Claim 25 or by an obvious
chemical equivalent thereof.


24



68. A peptide in accordance with Claim 43
wherein R2 is 4 Cl-D-Phe and R4 is a lipophilic
aromatic D-isomer amino acid when prepared by the method
of Claim 26 or by an obvious chemical equivalent thereof.
69. A peptide in accordance with Claim 43
wherein R2 is 4 Cl-D-Phe, R4 is a lipophilic
aromatic D-isomer amino acid and R5 is Leu when
prepared by the method of Claim 27 or by an obvious
chemical equivalent thereof.
70. A peptide in accordance with Claim 43
wherein R2 is 4 Cl-D-Phe, R4 is a lipophilic
aromatic D-isomer amino acid and R6 is Gly-NH2 when
prepared by the method of Claim 28 or by an obvious
chemical equivalent thereof.
71. A peptide in accordance with Claim 43
wherein X is acetyl, R2 is 4 Cl-D-Phe and R4 is a
lipophilic aromatic D-isomer amino acid when prepared by
the method of Claim 29 or by an obvious chemical
equivalent thereof.
72. A peptide in accordance with Claim 43
wherein X is acrylyl, R2 is 4 Cl-D-Phe and R4 is a
lipophilic aromatic D-isomer amino acid when prepared by
the method of Claim 30 or by an obvious chemical
equivalent thereof.
73. A peptide in accordance with Claim 43
wherein R1 is dehydro-Pro, R2 is 4 Cl-D-Phe and R4
is a lipophilic aromatic D-isomer amino acid when
prepared by the method of Claim 31 or by an obvious
chemical equivalent thereof.
74. A peptide in accordance with Claim 43
wherein R1 is dehydro-D-Pro, R2 is 4 Cl-D-Phe and
R4 is a lipophilic aromatic D-isomer amino acid when
prepared by the method of Claim 32 or by an obvious
chemical equivalent thereof.






75. A peptide in accordance with Claim 43
wherein R1 is Thz, R2 is 4 Cl-D-Phe and R4 is a
lipophilic aromatic D-isomer amino acid when prepared by
the method of Claim 33 or by an obvious chemical
equivalent thereof.
76. A peptide in accordance with Claim 43
wherein R1 is D-Thz, R2 is 4 Cl-D-Phe and R4 is a
lipophilic aromatic D-isomer amino acid when prepared by
the method of Claim 34 or by an obvious chemical
equivalent thereof.
77. A peptide in accordance with Claim 43
wherein R2 is D-Phe and R3 is D-Trp when prepared by
the method of Claim 35 or by an obvious chemical
equivalent thereof.
78. A peptide in accordance with Claim 43
wherein X is hydrogen, R2 is D-Phe and R3 is D-Trp
when prepared by the method of Claim 36 or by an obvious
chemical equivalent thereof.
79. A peptide in accordance with Claim 43
wherein X is acetyl, R2 is D-Phe and R3 is D-Trp
when prepared by the method of Claim 37 or by an obvious
chemical equivalent thereof.
80. A peptide in accordance with Claim 43
wherein X is acrylyl, R2 is D-Phe and R3 is D-Trp
when prepared by the method of Claim 38 or by an obvious
chemical equivalent thereof.
81. A peptide in accordance with Claim 43
wherein R2 is D-Phe, R3 is D-Trp and R5 is Leu
when prepared by the method of Claim 39 or by an obvious
chemical equivalent thereof.
82. A peptide in accordance with Claim 43
wherein R2 is D-Phe, R3 is D-Trp and R5 is N.alpha.MeLeu
when prepared by the method of Claim 40 or by an obvious
chemical equivalent thereof.
83. A peptide in accordance with Claim 43
wherein R2 is D-Phe, R3 is D-Trp and R6 is
Gly-NH2 when prepared by the method of Claim 41 or by
an obvious chemical equivalent thereof.


26




84. A peptide in accordance with Claim 43
wherein R2 is D-Phe, R3 is D-Trp and R6 is
NHCH2CH3 when prepared by the method of Claim 42 or
by an obvious chemical equivalent thereof.




27

Description

Note: Descriptions are shown in the official language in which they were submitted.


3~Z



LRF ANTAGON I STS
The present invention relates to peptides which
inhihit the release of gona~otropins by the pituitary
gland in mammalians, including humans and to methods of
preventing ovulation and/or inhibitin~ the release of
steroids. More particularly, the present invention is
directed to pep~ides which inhibit gonadal function and
the release of the steroidal hormones, progesterone and
testosterone.
The pituitary gland is attached by a stalk to
the region in the base of the brain known as the
hypothalamus. The pituitary gland has two lobes, the
anterior and the posterior lohes. The posterior lobe of
the pituitary gland stores and passes onto the general
circulation two hormones manufactured in the
hypothalamus, these being vasopre~sin and oxytocin. The
anterior lobe of the pituitary gland secretes a number
of hormones, which are complex protein or glyco-protein
molecules that travel through the bloodstream to various
organs and which, in turn, stimulate the secretion into
the blood stream of other hormones from the peripheral
organs. In part:icular, follicle stlmulatin~ hormon~
~FSH) and lut~iniæing hormone (JJ~I), 30mel-ime~ re~e~red
to as gonadotroE~ins or gonaclotropic hoxmones, are
2S released by the pituitary gland. These hormones, in
combination, regulate the functioning of the gonads to
produce testo~terone in the testes and progesterone and
e~trogen in the ovaries, and also regulate the
production and maturation o~ yametes.
The release o~ a horrnolle by the anterior lobe
of the pituitary gland usua]ly requires a prior release
of another clas~ of hormones produced by the
hypothalamus. One o~ the hypot}latamic hormones acts as
a faator tha~ tri-3ger~ the release of the gonadotropic
hormon~3, par~:lcu]arly r.H. ~the hypothalamic hormone

` 1~L8~i302



which acts as a releasing factor for LH is referxed to
herein as LRF al~hough it has also been referred to as
~JH-RH and as GnRH. LRF has been isolated and
characterized as a decapeptide having the following
structure:
p-Glu-His-Trp-Ser-Tyr-Gly~Leu-Arg-Pro-Gly-NH2
Peptides are compollnds which contain two or
more amino acids in which the carboxyl group of one acid
is linked to the amino group of the other acid7 The
~vrmula for LRF, as represented above, is in accordance
with conventional representation of peptides where the
amino group appears to the left and the carboxyl group
to the right. The position o~ the amino acid r~sidue is
identified by numbering the amino acid residues Erom
left to right. In the case of LRF, the hydroxyl portion
of the carboxyl group of glycine has been replaced with
an amino group (NH2). The abbreviations for the
individual amino acid residues above are conventional
and are based on the trivial name of the amino acid:
where p-Glu is pyroglutamic acid, His is histidine, Trp
is tryptophan, Ser is serine, Tyr is tyrosine, Gly is
glycine, Leu is Leucine, Arg is argi.nin~ and Pro i~
proline. ~xcept or glycine, amino aaids o~
~eptides o~ the invention are o~ the L-conEiguration
unless noted otherwise.
It is known that the substitution of D-amino
aclds ~or Gly in the 6 position o~ the LRF Aecape~tide
provides a peptifle material having rom about 1 to 35
times greater potency than does LRF to eEect the
release o~ LH and other gona~otropin.s by the pituitary
gland oE mammalians. The relea~,ing eEEect is obtai.ned
when the LRF analog is administered to a mammalian
i.ntravenously, subcutaneously, intramuscularly, orally,
intrana.sally or intravaginalJ.y.
Tt i~ also known that substitut.ion of various

118~i3~2


--3--
amino acids for His (or the deletion of His) at the
2-position of the LRF decapeptide produces analogs
having an inhibitory effect on the release of LH and
other gonadotropins by the pituitary gland of
mammalians. In particular, varying degrees of
inhibition of the release o LH are obtained when ~is is
deleted (des ~is) or replaced by D-Alar D-Phe or Gly.
rrhe inhibitory effect of such peptides modified at the
2-position can be f~rther enhanced when a D-amino acid
is substituted for Gly in the 6-position of the
decapeptides. For example, the peptide:
- pGlu-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH2 is 4 ~imes
more potent as an inhibitor ~or the release of
gonadotropins than is the same peptide where Gly is
present in the 6-position rather than D-Ala.
Some female mammalians who have no ovulator~
cycle and who show no pituitary or ovarian de~ec~ begin
to secrete normal amounts of the gonadotropins LH and
FSH after the appropriate administration of LRF. Thus,
the administration of LRF is considered suitable for the
treatment o~ those cases of in~ertility where a
~unctional defect resides in the hypothalamus.
There are al~o reason~ ~or d~sirlng ~o prevent
ovula~ion i.n ~emal~ mammalians, ~nd the administration
of LRF analogs that are antagonistic to the normal
~unction o~ LRF have been used to prevent ovulation.
For this,rea.sont analogs of LRF which are antagoni~tic
, tc> L~F are being investigated ~or their potential use as
a contraceptive or ~or regulating conception periods.
It is desirect to provide peptides which are strongly
antagonistic to endogenous I.RF and which prevent
secretion o~ L~ and the release of steroids by the
gonads oE mammals.
The present invention provides peptides which
inhibit the release o~ gonactotropins ln mammalians,

11i5 ~3~!2



including humans, and also provides methods for
inhibiting the release of steroids by the gonads of male
and female mammalians The improved LRF analogs are
antagonistic to LR~ and have an inhibitory effect on the
reproduction processes of mammalians. These analogs may
be used to inhibit the production of gonadotropins and
sex hormones under various circumstances including
precocious puberty, hormone dependen~ neoplasia,
dysmenorrhea and endometriosis.
Generally, in accordance with the present
invention, peptides have been synthesized which strongly
inhibi.t the secretion of gonadotropins by the p;tuit~ry
gland of mammalians, including humans, and/or inhibit
the release of steroids by the gonads. TheSe peptides
are analogs of LKF wherein there is a 1-position
substitution in the form of either dehydroproline or
meta-thiazolidine-2-carboxylic acid, and preEerably
substituents are also present 2-, 3- and 6-positions.
The l-position substituent may be modified so that its
alpha amino group contain.s an acyl group, such as
formyl, acetyl, acrylyl, vinylacetyl or benæoyl.
Dehydro L-Pro iS preferred in the l~posltion. Modi~ied
D-Phe is pre~erably present in the 2-position and
provides incr~ased antagonistic activity as a result o~
the specific modi~ications present in the benzene ring.
Single substitutions or hydro~en are pre~erably made in
the para- or 4-~osition, and double substitutions are
mad~ preferably in the 2,~- or the 3,4-positions. The
substitutions are most preferably selected ~rom
dichloro, methyl, eluoro, di~luoro, trifluoromethyl,
metho~y, bromo, dibromo, nitro, dinitro, acetylamino and
methyl mercapto. D-Trp is preerred in the 3-position,
and imBzl D-~is or D--Trp or some other lipophilic
aromatic D-amino acid is preferred in the 6-position,
although Gl~ or any D-isomer am~no acid, e.g. D-Leu and

1~8~i3C~


--5--
D-Ser(O-t But), may be used. The substitutions in the
- 7- and 10-positions are op1:ional.
Because these peptides are highly potent to
inhibit release of LH, they are often referred to as LRF
antagonists. The peptides inhibit ovulation o~ female
mammals when administered at very low levels at
proestrous and are also effective to cause resorption of
ertilized eggs if administered shortly after conception.
More specifically, the peptides of the precent
invention are represented by the following formula:
1 2 R3 ser-Tyr-R4-R5-Ar9-pro-R
wherein X is hydrogen or an acyl group having 7 or less
carbon atoms Rl is dehydro Pro, dehydro D-Pro, Thz or
D-Thz; R2 is D-Phe, D-His, D-Trp, Trp, Cl-D-Phe,
dichloro D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe,
AcNH-D-Phe, N02-D-Phe, dinitro-D-Phe, Br-D-Phe,
dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or
CH3-D-Phe; R3 is D-Trp, Trp, D-Phe or D-His; R4 is
Gly or a D-isomer amino acid; R5 is Leu or N Me-Leu;
and R6 is Gly-N~I2 or NHCH2CH3.
By dehydro Pro is meant 3,4 dehydroproline,
C5I~702N, and when X is an acyl radical, it i~
Attached to the ni~ro~en. By Thz i~ meant
meta-thiazolidine-2-carboxylic acid, C4H702NS,
which can be prepared by the treatment of cysteine
hydrochloride with ~ormaldehyde, and for example, Ac-Thz
may be prepared by the reaction o Thz with acetic
anhydride.
The peptides of the present invention can be
synthesized by a solid phase technique using a
chloromethylated resin, a methylbenæhydrylamine resin
(MBTIA) or a benzhydrylamine (B~IA) resin. The synthesis
is conducted in a manner to step~ise add the amino acids
in the chain in the manner set eorth in detail in the
3$ U..S. Patent No. ~,211,G93. Sic~e-chain protecting

3~32


--6--
groups, as are well known in the art, are preferably
added to Ser, Tyr, Arg and ~is before these amino acids
are coupled to the chain being built upon the resin.
Such a method provides the ~ully protected
intermediate peptidoresin. The intermediates o the
invention may be represented as:
X -R1-R2-R3-Ser(X )-Tyr(X )-R4-R5- Arg(X
)-Pro-X wherein: X is an ~-amino protectiny
group of the kype known to be useul in the art in the
stepwise synthesis of polypeptides and when X in the
desired peptide composition is a particular acyl group,
that group may be used as the protecting group. ~mon~
the classes of ~-amino protecting groups covered by X
are (1) acyl-type protecting groups, such as formyl
(For), trifluoroacetyl, phthalyl, p-toluenesulfonyl
(Tos), benzoyl (Bz), henzensulfonyl,
o-nltrophenylsulfen~l (Nps), tritylsulfenyl,
o-ni~rophenoxyacetyl, acrylyl (Acr) r chloroacetyl,
acetyl ~Ac) and ~-chlorobutyryl; (2) aromatic
urethan-type protecting groups, e.g., benzyloxycarbonyl
(Z) and substituted benzyloxycarbonyl, such as
p-chloro-benzyloxycarbonyl, p-nitrobenæyloxycarhonyl,
~-bromohen%yloxycarbonyl and ~-m~ho~yh~rlzy~ox~c~rbonyl;
(3) aliphatlc ure~han prote~inc3 ~roups, such ~s
ter~butyloxycarbonyl (Boc), diisopropylmethoxycarbon~l,
isopropyloxyaarbonyl, ethoxYcarbonyl and
allyloxycarbonyl; (4) cycloalkyl urethan~type protecting
groups, such as cyclopentyloxycarbonyl,
adamantyloxycarbonyl and cyclohexyloxycarbonyl; (5)
thiourethan-type protectinq groups, such as
phenylthiocarbonyl; (~) alkyl-type protecting groups r
such as allyl (Aly), triphenylmethyl(trityl) and benzyl
~Bzl); (7) trialkylsilane groups, such as
trimethylsilane, The preerred a-amino protecting group
is ~oc when X is hydrogen.

~863~2


X is a protecting group for the alcoholic
- hydroxyl group of Ser and is selected from the group
consisting of acetyl, benzoyl, tetrahydropyrany~,
tert-butyl, trityl, benzyl and 2,6-dichlorobenzyl.
Benzyl is preferred.
X3 is a protecting group for the phenolic
hydroxyl group of Tyr selected from the group consisting
of tetrahydropyranyl, tert-butyl, trityl, benzyl,
benzyloxycarbonyl, 4-bromobenzyloxycarbonyl and
2,6-dichlorobenzyl.
X4 is a protecting group for the nitrogen
atoms of Arg and is selected from the group consisting
of nitro, Tos, benzyloxycarbonyl, adamantyloxycarbonyl,
and Boc; alternatively X4 may be hydrogen, which means
there are no protecting groups on the side chain
nitrogen atoms of arginine.
X is selected ~rom Gly-O-CH2-[resin
support]; O-CH2-[resin support] and Gly-NH-~resin
support].
The criterion for selecting side chain
protecting groups or X2-X4 is that the protecting
group must be stable to the reagen~ unc~er ~h~ reaakion
condi.tions selected for removing the a-~mino pro~ectillg
group at each st:ep o~ the synth~sis. The protec~ing
~roup must not be split of under coupllng conditions,
and the protecting group must be removable upon
completi~n of the synthesis o the desired amino acid
~equence under reaction condition~ that will not alter
the peptide chain.
When the X~ group is Gly-O-C~2-[re3in
support], the ester moiety of one of the many fur,ctional
groups o~ the polystyrene resin support is being
representefl. When the X5 ~roup is Gly-NH-[resin
supportl, an amide bond connects Gly to ~E~A resin or to
a MBllA resin.

~81~3~2


-8-
When X is acetyl, formyl, acrylyl, vinylacetyl,
benzoyl or some other acyl group having 7 carbon atoms
or ]ess, it may be employed as the X protecting group
for the a-amino group of Rl in which case it can be
added before coupling of the last amino acid to the
peptide chain. Alternatively, a reaction may be carried
out with the peptide on the resin, e.g. reacting with
acetic acid in the presence of dicyclohexyl carbodiimide
(DCC) or preferably with acetic anhydride.
The fully protected peptide can be cleaved from
the chloromet~ylated resin support by ammonolysis, as ;s
well known in the art, to yield the ully protected
amide intermediate. Deprotection of the peptide as well
as cleavage of the peptide from the benzhydrylamine
resin takes place at 0C with hydrofluoric acid (HF~.
Anisole is added to the peptide prior to treatment with
HF. After the removal of HF, under vacuum, the cleaved,
deprotected peptide is treated with ether, decanted,
taken in dilute acetic acid and lyophilized.
Purification of the peptide i5 effected by ion
exchange chroMotography on a CMC column, followed by
partition chromo~ography using the elution system:
n-butanol; O.].N acetic aci~ (1:1 v-~ume ra~io) on a
column packed with Sephadex G-25 or by using ~PLC as
known in the arts. The peptides o the invention are
effective at levels of less than 200 micrograms per
kilogram o~ body ~eight, when administered at about noon
on the day of proestrous, to prevent ovulation in female
rats. For prolonged suppres~ion of ovulation, it mav be
neceæsary to use dosage levels in the range oE from
about 0.1 to about 5 milligrams per kilo~ram of body
weight. These antagonists are also efective as
contraceptives when administered to male mammals on a
regular basi~.
,Sinc~ these compounds will reduce testosterone

.

~B6302



levels (an undesired consequence in the normal, sexually
active male), it may be reasonable to administer
replacement dosages of testosterone along with the LRF
antagonist.
The following examples further illustrate
various features of the invention b~t are intended to in
no way liMit the scope o~ the invention which is defined
in the appended claims.
EXAMPLE I
The following peptides having the formula
X-dehydro Pro-R2-D-Trp-5er-Tyr-D-Trp-R5-Arg-Pro-Gly-NH
are prepared by the sol;d pha~e procedure referred
to above.
TABLE I
15 PEPTIDE X R2 R5
1 Ac 3,4 C12-D-Phe Leu
2 " 4 CF3-D-Phe "
3 " 4F-D-Phe "
4 " 4 AcNH-D-Phe "
" 4 NO2-D-Phe "
6 " 4 Br-D-Phe "
7 " 4 CH3s_D_phe
8 " 4 OCH3-D~Phe "
9 " 4 CM3-D-Ph~ "
ll 2,4 C12-D-Ph~ "
11 ~cr 3,4 C1~-D-Phe
12 Ac 4 ~CH3-D-Phe N MeLeu
13 " 4 CM3-D-Phe "
14 " 3,4 C12-D-Phe "
For purposes o~ an example, a representative
solid phase synthesis of Peptide No. 1 above, which is
re~erred to as [~c-dehydro Prol, 3,4
C12-D-Phe2,D-Trp3~6~-L~F i3 set forth
hereina~ter. This pept~de has the ~ollowlng formula:

~863~Z


--10--
Ac-dehydro Pro-3,4 C12-D-Phe-D-Trp-Se~-Tyr-D-Trp
-Leu-Arg-Pro-Gly-NH2
A BHA resin is used, and Boc-protected ~ly i~
coupled to the resin over a 2-hour period in CH2C12
using a 3-fold excess of Boc derivative and DCC as an
activating reagent. The glycine residue attaches to the
BHA residue by an amide bond.
Following the coupling of each amino acid
~esidue, washing, deblocking and coupling of the next
amino acid residue is carried out in accordance with the
~ollowing schedule using an automated machine and
beginning with about 5 grams of resin:
STEP RE~GENTS AND O ERATIOI~S MIX TIMES MIN.
1 CH2C12 wash-80 ml. (2 times) 3
lS 2 MethanoltMeOH) wash-30 ml. (2 times) 3
3 CH2C12 wash-~0 ml. (3 times) 3
4 50 percent TFA plus 5 percent 1,2-eth-
anedithiol in CH2CL2-70 ml. (2 times) 10
CH2C12 wash-80 ml. (2 times) 3
6 TEA 12.5 percent in CH2C12 70 ml.
(2 times) 5
7 MeOH wash-4n ml. ~2 times) 2
8 CH2C12 wash-8n ml. (3 times) 3
9 Boc-amino acid (10 mmoles) in 30 ml. o~ either
DME` or CM2C12, depending upon the solubility
o the particular protected amino acid, (1 time)
plus DCC (10 mmoles) in CH2Cl? 30-300
10 MeOH wash-40 ml. (2 times) 3
11 T~A 12.5 percent in CM2C12-70 ml.
(1 time) 3
12 MeO~ wash-30 ml. (2 times) 3
13 CH2Cla wash-80 ml. (2 times) 3
A~ter step 13, an aliquot is taken ~or a
ninhydrin test: i~ the test is negative, go back to step
1 or coupling o~ the next amino acid; if the test is

~L~8~3~


-3].-
posi~ive or slightly posi.tive, go back an~ repeat steps
g ~hrough l?
The above scheclule is used for coupling of each
of the ami.no acids of the peptide of ~he invention a.ter
the first amino acid has been attached, N Boc
protection is used for each o~ the remaining amino acids
throughout the syn'chesis. The side chain o Arg is
protecte~ with Tos. OBzl is used as a side chain
protecting group ~or the hydroxyl group of Ser, and 2-6
1~ dichlorobenzyl is llsed as the side chain protecting
group for the hydroxyl ~roup of 'ryr. N-acetyl dehydro
Pro is introduced as t:he final amino acid. Boc-Arc3(Tos)
and ~oc-D~Trp, ~hich have low solubility in C~2Cl.2,
are coupled using DMF CH2Cl2 mixtures.
Tlle cleavaye o, khe peptide from the resin and
complete deprotection o the side chains'takes place
very readily at 0C. with HF. Anisole is added as a
scavenger prior to HF treatment. After the rernoval of
HF under vacuum, the resin is e~tracted with 50~ ace~ic
acic~, and the wa.shi.ngs are lyophilized to provide a
crude pepticle powder.
Puri~ication of the pepti~e is ~,hen eE~cted b~
ion exchancJe chrom,-ltography on CMC (Whatm~rl CM 3~, U5il1g
~.t ~radi.ent o~ 0,.05 to 0.3M NE~OAc in 50/50
2S Tnethanol/~ater) ~olloweci by partition chromatography in
gel ~iltration column using th~ elution system:
n E3~ttanol.; O.lN ~cet:.ic acid (1:1 volurne ratio).
The peptides set forth in the foregoing table
are assaye~ n vitro and ln vlvo. 'rhe ln vltro ~est is
made using dlssociat,e(l rat pituitary cells rnaintained in
culture Eor 4 days ~rior to the assay. The levels of LH
mediated in response to the applicatioll of pept:icles i.s
assayed by speci~i.c radioimmuno~ssay For rat LH,
Con~rol di~hes o~ cells only receit~e a measure which is
3S 3 nanomol~r i.n L~.E': e~perimental dishes receive a
*trade mark

~ ~8~'3~2


-12-
measure 3 nanomolar in L~F plus a measure having a
concentration of test peptide ranging from 0.01 to 3
nanomolar. The amount of LH secreted in the samples
treated only with LRF is compared with that secreted by
the samples treated with the peptide plus LRF. Results
are calculated and expressed in Table IA ~In Vitro
column) as the molar concentration ratio of test peptide
to LRF (antagonist/LRF) required to reduce the amount of
LH released by 3 nanomolar LRF to 50 percent of the
c~ntrol value (ICR50~.
The peptides described hereinabove are used to
deterrnine effectiveness to prevent ovulation in female
rats. In this test, a specified number of mature female
Sprague-Dawley rats, each having a body weight from 225
to 250 grams, are injected with either 5 or 10
micrograms of peptide in corn oil a~ about noon on the
day of proestrous. Proestrous is the afternoon before
estrous (ovulation)~ A separate female rat group is
used as a control to which the peptide is not
administered. Each of the control rat females has
ovulation a~ estrous. As indicated in the In Vivo
column, the peptides are significantly e~ective to
pr~vent ovulation o~ ~emale rats a~ A very low dosa~e,
and all of the peptide compositions are considered to be5 ~otally e~fec~ive at a dose of one milligram.
TABLE IA
Jn Vitro In Vivo_(10~) In ViVo ~51ls?
Peptide ICR50 Rats Ovulating Rats Ovulating
1 0.039 0/10 ~/7
2 0.070 7/9
3 0.021 0/10 0/10
4 9/10
0.011 0/10 2/10
6 0.010 5/7
7 0.030 1/10 7/10

63~2
; .,.

-13-
8 0.12~ 3/10
9 0.044 1/7 9/9
5/10 4/7
11 0.048 1/10 . 4/10
12 0.29 ~/10
13 0.11 3/4
14 0.05 1/3 8/10
EXAMPLE II
The following peptides having the formula
10 x-Rl-pcl-D-phe-D-Trp-ser-Tyr-R4-R5-Arg-pro-~6
are prepared by the solid phase procedure as generally
described in Bxample I except ~or No. 20 which is
prepared on a chloromethylated resin.
, TABI,E II
15 PEPTIDE X Rl R4 R5 R6
Ac Aehydro Pro D-Trp Leu Gly-NH2
16 " ' " N MeLeu "
17 " dehydro D-Pro " Leu "
18 " N MeLeu n
20 19 Acr dehydro Pro 1l . " ,.
Ac dehydro D-Pro " Leu NHCH2CH3
21 AcThz D~Trp Leu Gly-NH2
2 2 nn n N Me I~ u n
23 "D-Thz " " "
24 ~1n 1l ~eu "
" dehydro Pro (imB~l)D-His n
26 n dehydro D-Pro " " "
27 n ThZ n n n
28 " D-Thz
rhe peptides set forth in the oregoing table
are assayed in vitro and in vivo. The in vitro test is
made using dissociated rat pituitary cells maintained in
culture for 4 days prior to the assay. The levels o~ LH
mediated in response to the application o~ peptides is
as~Ayed by speci~ic radioimmunoassay for rat LH.

363~2
. ~ . ...


Control dishes of cells only receive a measure which is
3 nanomolar in LRF: experimental dishes receive a
measure 3 nanomolar in LRF plus a measure having a
concentration of test peptide ranging rom 1 to 100
nanomolar. The amount of LH secreted in the samples
treated only with LRF is compared with that secreted by
the samples treated with the peptide plus LRF. Results
are calculated and expressed in Table IIA(In Vitro
column) as the molar concentration ratio of test peptide
to LRF (antagonist/LRF) required to reduce the amount Gf
LH released by 3 nanomolar I,RF to 50 percent of the
control value (ICR50).
Several o~ the peptides described hereinabove
are used to determine effectiveness ~o prevent ovulation
in female rats. In this test, four, seven, nine or ten
mature ~emale Sprague-Dawley rats r each having a body
weight from 225 to 250 grams, are injected with 0.02
milligram of peptide (unless otherwise indicated) in
corn oil at about noon on the day of proestrous.
Proestrous is the afternoon before estrous (ovulation).
A separate ten female rat group is used as a con~rol to
which the peptide is not admlnistered. ~ach o~ the ~en
control rat female~ has ovulation a~ estrous. ~8
indicated in the In Vivo column, the peptides are
significantly effective to prevent ovulation o~ female
rat~ at a very low dosage, and all of the peptide
compositions are considered to be totally effective at a
~ose o one milligram.
TABLE IIA
In Vitro In Vivo
Peptide ICR50 ~ats Ovulating
0.043 0/10*
16 0.05~ 0/7
17 0.19 3/8
-~' 35 1~ 0.27 5/10**


,

302


-15-
19 0.03 0/g
- 20 0.2
21 0.14 ~/8
22 0.13 9/10**
23 0.1
24 0.12
0.042 4/10
26 0.2 10/10
27 0.15 7/10**
28 0.1 7/10**
*~0.025 mq. *0.010 mg.
EXAMPLE III
rrhe following peptides having the formula
1 2 R3 Ser-Tyr-D-Trp-R5-Arg-pro R
lS are prepared by a solid phase procedure generally as
described in Example I, except for No. 37 where a
chloromethylated resin is used.
TABLE III
Pep-
tide X Rl R2 R3 R5 R6
2g H dehydro-D,L-Pro D-Phe D-Trp N MeLeu Gly-NH2
30 H " " " I,eu "
31 Ac dehydro-D,L-Pro
32 H " " " ~ MeLeu "
25 33 Ac dehydro-Pro n 1l Leu "
34 Ac dehydro-D-Pro
3S H dehydro-Pro " " " "
36 H dehydro-D-Pro " ~i .. ..
37 ~ dehydro-Pro " " " NHCH2CH3
30 38 Bz dehydrQ-pro D-Trp n 1l Gly-NH2
39 For dehydro-Pro " D-Phe " "
40 For dehydro-Pro D-His Trp " "
41 Acr dehydro-Pro n
42 Bz dehydro-Pro Trp D-His
35 43 Acr dehydro-Pro " D-Trp " "

3C! 2


-16-
44 For dehydro-Pro " D-~is " "
Bæ dehydro-Pro D-His " " "
The peptides set rorth in the fore~oing table
are assayed in vitro and in vivo. The in vitro assay is
made using four day old primary culture of dispersed rat
pituitary cells. The levels of L,H mediated in response
to the application of peptides is assayed by specific
radioimmunoassay for rat LH. Control dishes of cells
only receive a measure which is 3 n~nomolar in LR~^
experimental dishes receive a measure 3 nanamoles of LRF
and a concentration of test peptide ranging from 1 to
100 nanomolar. The amount of LH secreted in the samples
treated only with LRF is compared with that secreted by
the samples treated with t~le peptide plus LRF. Results
are calculated and expressed in Table IIIA (In Vitro
column) as the molar concentration ratio of test peptide
required to reduce the amount of LH released by the 3
nanomolar LRF to 50 percent of the control value
( ICR50 ) -
Several of the peptides described hereinabove
are used to determine effectiveness to prevent ovulation
in female rats. In this test, a number o~ mature female
Sprague-Dawley rats, each having a hody wel~ht from 22$
to 250 grams, are injected with 0.02 milli~ram o~
peptide in corn oil at about noon on the day of
proestrous. Proestrous is the a~ternoon beore e~trous
(ovulation). A separate female rat group is used as a
control to which the peptide is not admini~tered. Each
o~ the control rat females has ovulation at estrous. As
indicated in the In Vivo column, l:he peptides are
~ignificantly effective to prevent ovulation of female
rats at a very low dosage, and all of the peptide
compositions are considered to be totally ef~ective at a
dose of one milligram.

~8~3~2



TA~LE IIIA
In Vitro In Vivo
Peptide ICR50Rats Ovulating
~9 0.5:1 ~/10
0.8:1 7/10
31 0.3:1 1/10
3~ 0.4:1 2/10
33 0.6:1 0/4*
*dose of 0.025 mg.
These peptides can be administered to mammals
intravenously, subcutaneously, intramuscularly,orally,
intranasally or intravaginally to achieve fertility
inhibition and/ox control. Effective dosages will vary
with the form of administration and the particular
species of mammal being treated. An example of one
typical dosage form is a physiological saline solution
containing the peptide which solution is administered to
provide a dose in the range of about 0.1 to S mg/kg of
body weight. Oral administration of the peptide may be
given in either solid form or liquid form.
Although the invention has been described with
regard to its preferred embodimen~s, i~ ~hould ~0
unders~ood that change~ and modlications as would b~
obvious to one having ~he oriAinary skill in ~hi~ art
may be made without departing ~rom the scope of the
invention which is set forth in the claims which are
appended hereto. For example/ other substitutions known
in the art which do not signi~lcantly detract from the
effectiveness of the peptides may be employed in the
peptides oE the invention.

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Administrative Status

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Administrative Status

Title Date
Forecasted Issue Date 1985-04-30
(22) Filed 1981-04-15
(45) Issued 1985-04-30
Expired 2002-04-30

Abandonment History

There is no abandonment history.

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $0.00 1981-04-15
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
SALK INSTITUTE FOR BIOLOGICAL STUDIES (THE)
Past Owners on Record
None
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Description 1993-06-09 17 690
Drawings 1993-06-09 1 8
Claims 1993-06-09 10 349
Abstract 1993-06-09 1 22
Cover Page 1993-06-09 1 16