Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-04-29
2001-05-29
Russel, Jeffrey E. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S015800, C514S016700, C514S017400, C514S018700, C530S327000, C530S328000, C530S329000, C530S330000, C530S331000, C530S345000
Reexamination Certificate
active
06239107
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns novel conjugates of a lipophilic moiety and a peptide of 3-12 amino acids. The present invention further concerns pharmaceutical compositions comprising as an active ingredient said novel conjugates. The pharmaceutical compositions of the invention are preferably used for the treatment of male impotence or for the treatment of neurodegenerative diseases.
BACKGROUND OF THE INVENTION
Vasoactive intestinal peptide (VIP), a 28 amino acid neuropeptide widely distributed in the mammalian nervous system, has potent neurotrophic actions that influence nerve cell function. In the central nervous system, this role of VIP is translated into developmental effects, display of growth factor activities and maintenance of neuronal survival and function. Neurons, which are capable of releasing VIP, innervate blood vessels throughout the body, as well as the trachea in the lung, and the released VIP serves as a potent vasodilator, inducing smooth muscle relaxation. Radioligand binding assays, pharmacological experiments, molecular cloning and development of superactive novel derivatives have indicated several classes of VIP receptor sites and several potential therapeutical uses.
Two possible therapeutical uses of VIP, modified VIP or lipophilic VIP derivatives were reported in our previous Patents IL 87055, EP 0354992 and U.S. Pat. No. 5,147,855 and published patent applications EP 0540969 and EP 0620008 which are directed to the treatment of male impotence by transdermal administration and to the treatment of neurodegenerative diseases, respectively.
VIP is a hydrophilic peptide of a very short half life in the serum (Said, S. I., Editor,
Vasoactive intestinal peptide
in: Advances in Peptide Hormone Research Series, Raven Press, New York, 1-512 (1982)) having the following sequence:
(SEQ ID NO:1)
1 2 3 4 5 6 7 8 9 10 11 12
His-Ser-Asp-Ala-Val-Phe-Tyr-Asp-Asn-Tyr-Thr-Arg-
13 14 15 16 17 18 19 20 21 22 23 24
Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-
25 25 27 28
Ser-Ile-Leu-Asn-NH
2
To enhance its biological availability and increase its stability the present inventors have resorted to two chemical modifications reported in said patents and applications. The first was lipophilization, namely, the addition of a fatty acid moiety, designed to augment VIP's ability to penetrate biological membranes without loss of activity; thus, stearoyl-VIP, a molecule combining VIP with a stearic acid moiety at its N-terminal was designed (EP 0354992). The second modification was the replacement of native amino acids with unnatural amino acids, namely, a substitution of methionine (amino acid 17 of VIP) by norleucine, aimed at stabilizing the molecule against oxidation as well as at increasing lipophilicity; thus, stearoyl-Nle-VIP was designed (Gozes et al.,
Endocrinology,
134:2121-2125 (1994); Fauchere et al.,
Int. J. Peptide Protein Res.,
32:269-278 (1988); EP 0540969). Unmodified VIP fragments derived from the 17-24 positions of the VIP sequence are described in EP 0225020 as ulcer inhibitors.
A major obstacle in the use of any substance as a medicament is its distribution in the body. The modified VIP or lipophilic VIP used for transdermal treatment of male impotence reported in the abovementioned EP 0354992 and EP 0540969 have to penetrate through the dermis and reach the erectile tissues in a short a time span as possible.
VIP, modified VIP or lipophilic VIP used to treat neurodegenerative diseases described in EP 0620008 have to pass the blood brain barrier in order to exert their therapeutic effect on brain cells.
It would have been desirable, both for the purpose of treatment of male impotence and for the purpose of administration to the CNS for the treatment of neurodegenerative diseases, to use molecules that, while having the physiological activity of the full VIP peptide, are smaller in size and thus are able to improve the bioavailability of the therapeutic compound at the target tissue. Furthermore, smaller molecules are at times more stable to degradation than larger molecules since, as a rule, they have less sites available to degradation.
SUMMARY OF THE INVENTION
The present invention is based on the surprising finding that short fragments of VIP or modified VIP conjugated to a lipophilic moiety, which are 3-12 amino acids long, are physiologically active in the treatment of impotence and/or neurodegenerative diseases. The advantage of using short physiologically active peptides conjugated to a lipophilic moiety versus the usage of the full VIP molecule is better biodistribution and bioavailability in the body, as well as ease of preparation. Furthermore, the invention concerns short cyclic peptides containing said short fragments of VIP or of modified VIP conjugated to a lipophilic moiety which in addition to the above advantages feature the advantage of being relatively degradation resistant.
The present invention is concerned with a conjugate of a peptide coupled to a lipophilic moiety, wherein the peptide has at least 3 and at most 12 amino acid residues, said conjugate being selected from the formulae:
(i) R
1
-X
1
-X
1
′-X
1
″-X
2
-NH—R
2
(SEQ ID NO:2);
(ii) R
1
-X
3
-Ser-X
4
-Leu-Asn-NH—R
2
(SEQ ID NO:3);
wherein
R
1
is H or a lipophilic moiety;
R
2
is H, a lipophililc moiety, a lipophilic moiety substituted by X
3
-Ser-X
4
-Leu-Asn-NHR
1
(SEQ ID NO:79) or a spacer consisting of 1-3 residues of a non-charged amino acid coupled to X
1
-X
1
′-X
1
″-X
2
NHR
1
(SEQ ID NO:80),
with the proviso that at least one of R
1
and R
2
is a lipohilic moiety;
X
1
is a covalent bond, Ala, Val, Ala-Val, Val-Ala, L-Lys, D-Lys, Ala-Lys, Val-Lys, Ala-Val-Lys; Val-Ala-Lys or Orn;
X
1
is L-Lys, D-Lys or Orn;
X
1
″ is L-Tyr, D-Tyr, Phe, Trp or the residue of p-amino phenylalanine;
X
4
is Ile or Tyr;
X
5
is a residue of a hydrophobic aliphatic amino acid;
X
2
is X
5
, X
5
-Asn, X
5
-Ser, X
5
-Ile, X
5
-Tyr, X
5
Leu, X
5
-Nle, X
5
-D-Ala, X
5
-Asn-Ser, X
5
-Asn-Ser-Ile (residue 1-4 of SEQ ID NO:75), X
5
-Asn-Ser-Tyr (residues 1-4 of SEQ ID NO:76); X
5
-Asn-Ser-Ile-Leu (residues 1-5 of SEQ ID NO:75), X
5
-Asn-Ser-Tyr-Leu (residues 1‥5 of SEQ ID NO:76), X
5
-Asn-Ser-Ile-Leu-Asn (SEQ ID NO:75) or X
5
-Asn-Ser-Tyr-Leu-Asn (SEQ ID NO:76);
X
3
is a covalent bond, Asn, X
5
, X
5
-Asn, Tyr-X
5
, Tyr-X
5
-Asn, Lys-X
5
, Lys-X
5
-Asn, Lys-Tyr-X
5
, Lys-Tyr-X
5
-Asn (residues 4-7 of SEQ ID NO:77), Lys-Lys-Tyr-X
5
((residues 3-6 of SEQ ID NO:77), Lys-Lys-Tyr-X
5
-Asn (residues 3-7 of SEQ ID NO:77), Val-Lys-Lys-Tyr-X
5
(residues 2-6 SEQ ID NO:78), Val-Ala-Lys-Lys-Tyr-X
5
-Asn (SEQ ID NO:77), or Ala-Val-Lys-Lys-tyr-X
5
-Asn (SEQ ID NO:78);
X
6
is a covalent bond or Asn, Ser, Ile, Tyr, Leu, Asn-Ser, Asn-Ser-Ile, Asn-Ser-Tyr, Asn-Ser-Ile-Leu (residues 2-5 of SEQ ID NO:75), Asn-Ser-Tyr-Leu (residues 2-5 of SEQ ID NO:76), Asn-Ser-Ile-Leu-Asn (residues 2-6 of SEQ ID NO:75) or Asn-Ser-Tyr-Leu-Asn (residues 2-6 of SEQ ID NO:76);
X
7
is a covalent bond or Asn;
X
8
is a covalent bond, X
5
, Tyr, Lys, Tyr-X
5
, Lys-X
5
, Lys-Tyr-X
5
, Lys-Lys-Tyr-X
5
(residues 3-6 of SEQ ID NO:77), Val-Lys-Lys-Tyr-X
5
(residues 2-6 of SEQ ID NO:78), Ala-Lys-Lys-Tyr-X
5
(residues 2-6 of SEQ ID NO:77), or Ala-Val-Lys-Lys-Tyr-X
5
(residues 1-6 of SEQ ID NO:78);
Z is —CCNH—, —NHCO—, —S—S—, —S(CH
2
)
t
CO—NH— or —NH—CO(CH
2
)
t
S—;
m is 1 or 2 when Z is —CONH—, —S—S— or —S(CH
2
)
t
CO—NH—, or m is 2, 3 or 4 when Z is —NH—CO— or —NH—CO(CH
2
)
t
S—;
n is 1 or 2 when Z is —NH—CO—, —S—S— or —NH—CO(CH
2
)
t
S—, or n is 2, 3 or 4 when Z is —CONH— or —S(CH
2
)
t
CO—CO—NH—, and
t is 1 or 2,
with the proviso that the conjugate stearoyl-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH
2
(SEQ ID
Fridkin Matityahu
Gozes Illana
Browdy and Neimark
Russel Jeffrey E.
Yeda Research & Development Co. Ltd.
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