METHODES D'INHIBITION DE L'OSTEOCLASTOGENESE

METHODS OF INHIBITING OSTEOCLASTOGENESIS

Abrégé français

L'invention concerne des méthodes destinées à inhiber l'ostéoclastogenèse et l'activité des ostéoclastes. L'invention concerne également des méthodes de traitement de patients atteints de maladies caractérisées par une perte osseuse. La présente invention concerne en outre des peptides et des analogues de peptides mis au point à partir d'une boucle de fixation d'un élément de la superfamille de récepteurs de facteur de nécrose tumorale (TNF-R). Selon ces méthodes, on administre au patient une quantité d'inhibiteur efficace pour inhiber l'ostéoclastogenèse. L'invention concerne également des méthodes de modulation de la maturation des cellules dendritiques, de la prolifération des lymphocytes T, et/ou des systèmes récepteurs CD40 chez un individu. Ces méthodes consistent à administrer à l'individu une quantité d'inhibiteur efficace pour moduler la maturation des cellules dendritiques, la prolifération des lymphocytes T, et/ou les systèmes récepteurs CD40.

Abrégé anglais

Methods of inhibiting osteoclastogenesis and the activity of osteoclasts are
disclosed. Methods of treating patients who have diseases characterized bone
loss are disclosed. The present invention also provides peptides and peptide
analogues designed from a binding loop of a member of the tumor necrosis
factor receptor (TNF-R) superfamily. According to the methods, an amount of an
inhibitor effective to inhibit osteoclastogenesis is administered to the
patient. Methods of modulating dendritic cell maturation, T cell
proliferation, and/or CD40 receptor systems in an individual are disclosed.
The methods comprise the step of administering to the individual an amount of
an inhibitor effective to modulating dendriticall maturation, T cell
proliferation, and/or CD40 receptor systems.

Revendications

CLAIMS
1. A method of inhibiting osteoclastogenesis comprising the step of
administering to a
patient an amount of an inhibitor effective to inhibit osteoclastogenesis.
2. The method of claim 1 wherein the inhibitor has the formula:
<IMG>
wherein:
AC is a peptide of 3-18 amino acid residues which corresponds in primary
sequence to a binding loop of a TNF-R superfamily member, and which may
optionally
contain one or more amino acid substitutions, or an analogue thereof wherein
at least one
amide linkage is replaced with a substituted amide or an isostere of amide;
AB1 is a moiety having a first functional group capable of forming a covalent
linkage with one terminus of AC, a second functional group capable of forming
a covalent
linkage with AB2 and a third functional group capable of forming a covalent
linkage with
AA1;
AB2 is a moiety having a first functional group capable of forming a covalent
linkage with the second terminus of AC, a second functional group capable of
forming a
covalent linkage with AB1 and a third functional group capable of forming a
covalent
linkage with AA2;
AA1 is a moiety having hydrophobic properties and a functional group capable
of
forming a covalent linkage with the third functional group of AB2;
-44-

AA2 is a moiety having hydrophobic properties and a functional group capable
of forming
a covalent linkage with the third functional group of AB2;
"=" is a covalent linkage; and
".ident." is a covalent linkage.
3. The method of Claim 2 in which the amino acid substitutions are
conservative.
4. The method of Claim 3 in which the member of TNF-R superfamily is TNF-R p55
5. The method of Claim 4 wherein the inhibitor has the formula:
<IMG>
wherein:
B1 and B10 are each independently a peptide of 1-6 amino acids at least one of
which os a hydrophobic amino acid, an aromatic moiety or a heteroaromatic
moiety;
Z2 is a moiety that is capable of forming a covalent linkage with B1, X3 and
Z9;
Z9 is a moiety that is capable of forming a covalent linkage with B10, X8 and
Z2;
X3 is absent or a hydrophilic amino acid;
X4 is a hydrophobic amino acid;
X5 is a hydrophobic amino acid;
X6 is a hydrophobic amino acid;
X7 is a hydrophobic or hydrophilic amino acid;
X8 is a hydrophobic or hydrophilic amino acid;
-45-

"-" is an amide, substituted amide or an isostere of amide thereof;
"=" is a covalent linkage; and
".ident." is a covalent linkage.
6. The method of Claim 5, wherein:
B1 and B10 are each independently a peptide of 1-2 amino acids, at least one
of
which is an aromatic amino acid;
Z2 and Z9 are each independently a Cys-like amino acid;
X3 is absent or an acidic amino acid;
X4 is an aromatic or apolar amino acid;
X5 is a polar amino acid;
X6 is a polar amino acid;
X7 is an aromatic or polar amino acid;
X8 is an aromatic, apolar or polar amino acid;
"-" is an amide linkage;
"=" is a disulfide linkage; and
".ident." is an amide linkage.
7. The method of Claim 5, wherein:
B1 and B10 are each independently Tyr or Phe;
Z2 and Z9 are each Cys;
X3 is absent or Glu;
X4 is Trp or Leu;
X5 is Ser;
X6 is Gln;
X7 is Tyr or Asn;
X8 is Tyr or Leu;
"-" is an amide linkage;
-46-

"=" is a disulfide linkage; and
".ident." is an amide linkage.
8. The method of Claim 7, wherein said inhibitor is selected from the group
consisting of WP9Q - SEQ ID NO:13, WP9ELY - SEQ ID NO:12, WP9Y - SEQ ID
NO:14, and WP9QY - SEQ ID NO:15.
9. The method of Claim 4, wherein the inhibitor has the formula:
<IMG>
wherein:
B11 and B22 are each independently a peptide of 1-6 amino acids, at least one
of
0 which is a hydrophobic amino acid, an aromatic moiety or a heteroaromatic
moiety;
Z12 is a moiety that is capable of forming a covalent linkage with B11, X13
and Z21;
Z21 is a moiety that is capable of forming a covalent linkage with B22, X20
and Z12;
X13 is absent or hydrophobic amino acid;
X~4 is absent or hydrophilic amino acid;
X15 is a hydrophilic or hydrophobic amino acid;
X16 is a hydrophilic amino acid;
X17 is absent or a hydrophobic amino acid;
-47-

X18 is a hydrophilic amino acid;
X19 is a hydrophilic amino acid;
X20 is a hydrophilic amino acid;
"-" is an amide, a substituted amide or an isostere of amide thereof;
"=" is a covalent linkage; and
".ident." is a covalent linkage.
10. The method of Claim 9, wherein:
B11 and B22 are each independently a peptide of 1-3 amino acids, at least one
of
which is an aromatic amino acid;
Z12 and Z21 are each independently a Cys-like amino acid;
X13 is absent or an aromatic amino acid;
X14 is absent or a polar amino acid;
X15 is a basic, polar or apolar amino acid;
X16 is a polar amino acid;
X17 is absent or an apolar amino acid;
X18 is an acidic amino acid;
X19 is a polar amino acid;
X20 is a basic amino acid;
"-" is an amide linkage;
"=" is a disulfide linkage; and
".ident." is an amide linkage.
11. The method of Claim 10, wherein:
B11 and B22 are each independently Tyr or Phe;
Z12 and Z21 are each Cys;
X13 is absent or Phe;
X14 is absent or Thr;
-48-

X15 is Ala, Asn or Arg;
X16 is Ser;
X17 is absent or Val;
X18 is Glu;
X19 is Asn;
X20 is Arg or His;
"-" is an amide linkage;
"=" is a disulfide linkage; and
".ident." is an amide linkage.
12. The method of Claim 11, wherein said inhibitor is selected from the group
consisting of WP5 - SEQ ID NO:16, WP5N - SEQ ID NO:17, WP5R - SEQ ID NO:18,
WP5J - SEQ ID NO:19, WP5JY - SEQ ID NO:20, WP5JN - SEQ ID NO:21, WP5JR -
SEQ ID NO:22, and WP5VR - SEQ ID NO:23.
13. The method of Claim 4, wherein the inhibitor has the formula:
<IMG>
wherein:
B23 and B33 are each independently a peptide of 1-6 amino acids, at least one
of
which is a hydrophobic amino acid, an aromatic moiety or a heteroaromatic
moiety;
-49-

Z24 is a moiety that is capable of forming a covalent linkage with B23, X25
and Z32;
Z32 is a moiety that is capable of forming a covalent linkage with B33, X31
and Z24;
X25 is absent or a hydrophilic amino acid;
X26 is a hydrophilic amino acid;
X27 is a hydrophilic amino acid;
X28 is a hydrophilic amino acid;
X29 is a hydrophilic amino acid;
X30 is absent or a hydrophilic amino acid;
X31 is absent or a hydrophilic amino acid;
"-" is an amide, a substituted amide or an isostere of amide;
"=" is a covalent linkage; and
".ident." is a covalent linkage.
14. The method of Claim 13, wherein:
B23 and B33 are each independently a peptide of 1-3 amino acids, at least one
of
which is an aromatic amino acid;
Z24 and Z32 are each independently a Cys-like amino acid;
X25 is absent or a basic amino acid;
X26 is a basic amino acid;
X27 is an acidic amino acid;
X28 is an apolar amino acid;
X29 is an apolar amino acid;
X30 is absent or a polar amino acid;
X31 is absent or an apolar amino acid;
"-" is an amide linkage'
"=" is a disulfide linkage; and
".ident." is an amide linkage.
-50-

15. The method of Claim 14, wherein:
B23 and B33 are each independently Tyr or Phe;
Z24 and Z32 are each Cys;
X25 is absent or Arg;
X26 is Lys;
X27 is Glu;
X28 is leu, Pro or Met;
X29 is Gly;
X30 is absent or Gln;
X31 is absent or Val;
"-" is an amide linkage;
"=" is a disulfide linkage; and
".ident." is an amide linkage.
16. The method of Claim 15, wherein said inhibitor is selected from the group
consisting of WP8L - SEQ ID NO:24, WP8JP - SEQ ID NO:25, WP8J - SEQ ID NO:26,
and WP8JF - SEQ ID NO:27.
17. A method of treating patients who have diseases characterized by bone loss
comprising the step of administering to said patient an amount of an inhibitor
effective to
inhibit such bone loss.
18. The method of claim 17 wherein said inhibitor is a compound having the
formula:
<IMG>
-51-

wherein:
AC is a peptide of 3-18 amino acid residues which corresponds in primary
sequence to a binding loop of a TNF-R superfamily member, and which may
optionally
contain one or more amino acid substitutions, or an analogue thereof wherein
at least one
amide linkage is replaced with a substituted amide or an isostere of amide;
AB1 is a moiety having a first functional group capable of forming a covalent
linkage with one terminus of AC, a second functional group capable of forming
a covalent
linkage with AB2 and a third functional group capable of forming a covalent
linkage with
AA1;
AB2 is a moiety having a first functional group capable of forming a covalent
linkage with the second terminus of AC, a second functional group capable of
forming a
covalent linkage with AB1 and a third functional group capable of forming a
covalent
linkage with AA2;
AA1 is a moiety having hydrophobic properties and a functional group capable
of
forming a covalent linkage with the third functional group of AB1;
AA2 is a moiety having hydrophobic properties and a functional group capable
of
forming a covalent linkage with the third functional group of AB2;
"=" is a covalent linkage; and
".ident." is a covalent linkage.
19. The method of claim 18 wherein the inhibitor has the formula:
<IMG>
-52-

wherein:
B1 and B10 are each independently a peptide of 1-6 amino acids, at least one
of
which is a hydrophobic amino acid, an aromatic moiety or a heteroaromatic
moiety;
Z2 is a moiety that is capable of forming a covalent linkage with B1, X3 and
Z9;
Z9 is a moiety that is capable of forming a covalent linkage with B10, X8 and
Z2;
X3 is absent or a hydrophilic amino acid;
X4 is a hydrophobic amino acid;
X5 is a hydrophilic amino acid;
X6 is a hydrophilic amino acid;
X7 is a hydrophobic or hydrophilic amino acid;
X8 is a hydrophobic or hydrophilic amino acid;
"-" is an amide, substituted amide or an isostere of amide thereof;
"=" is a covalent linkage; and
".ident." is a covalent linkage.
20. The method of claim 19 wherein:
B1 and B10 are each independently a peptide of 1-3 amino acids, at least one
of
which is an aromatic amino acid;
Z2 and Z9 are each independently a Cys-like amino acid;
X3 is absent or an acidic amino acid;
X4 is an aromatic or apolar amino acid;
X5 is a polar amino acid;
X6 is a polar amino acid;
X7 is an aromatic or polar amino acid;
X8 is an aromatic, apolar or polar amino acid;
"-" is an amide linkage;
"=" is a disulfide linkage; and
".ident." is an amide linkage.
-53-

21. The method of claim 20 wherein:
B1 and B10 are each independently Tyr or Phe;
Z2 and Z9 are each Cys;
X3 is absent or Glu;
X4 is Trp or Leu;
X5 is Ser;
X6 is Gln;
X7 is Tyr or Asn;
X8 is Tyr or Leu;
"-" is an amide linkage;
"=" is a disulfide linkage; and
".ident." is an amide linkage.
22. The method of claim 18 wherein the compound is selected from the group
consisting of WP9Q - SEQ ID NO: 13, WP9ELY - SEQ ID NO: 12, WP9Y - SEQ ID NO:
14, and WP9QY - SEQ ID NO: 15.
23. The method of claim 18 wherein the inhibitor has the formula:
<IMG>
-54-

wherein:
B11 and B22 are each independently a peptide of 1-6 amino acids, at least one
of
which is a hydrophobic amino acid, an aromatic moiety or a heteroaromatic
moiety;
Z12 is a moiety that is capable of forming a covalent linkage with B11, X13
and Z21;
Z21 is a moiety that is capable of forming a covalent linkage with B22, X20
and Z12
X13 is absent or hydrophobic amino acid;
X14 is absent or a hydrophilic amino acid;
X15 is a hydrophilic or hydrophobic amino acid;
X16 is a hydrophilic amino acid;
X17 is absent or a hydrophobic amino acid;
X18 is a hydrophilic amino acid;
X19 is a hydrophilic amino acid;
X20 is a hydrophilic amino acid;
"-" is an amide, a substituted amide or an isostere of amide thereof;
"=" is a covalent linkage; and
".ident." is a covalent linkage.
24. The method of claim 23 wherein:
B11 and B22 are each independently a peptide of 1-3 amino acids, at least one
of
which is an aromatic amino acid;
Z12 and Z21 are each independently a Cys-like amino acid;
X13 is absent or an aromatic amino acid;
X14 is absent or a polar amino acid;
X15 is a basic, polar or apolar amino acid;
X16 is a polar amino acid;
X17 is absent or an apolar amino acid;
X18 is an acidic amino acid;
X19 is a polar amino acid;
X20 is a basic amino acid;
-55-

"-" is an amide linkage;
"=" is a disulfide linkage; and
".ident." is an amide linkage.
25. The method of claim 24 wherein:
B11 and B22 are each independently Tyr or Phe;
Z12 and Z21 are each Cys;
X13 is absent or Phe;
X14 is absent or Thr;
X15 is Ala, Asn or Arg;
X16 is Ser;
X17 is absent or Val;
X18 is Glu;
X19 is Asn;
X20 is Arg or His;
"-" is an amide linkage;
"=" is a disulfide linkage; and
".ident." is an amide linkage.
26. The method of claim 25 wherein the inhibitor is selected from the group
consisting
of WP5 - SEQ ID NO: 16, WP5N - SEQ ID NO: 17, WP5R - SEQ ID NO: 18, WP5J -
SEQ ID NO: 19, WP5JY - SEQ ID NO: 20, WP5JN - SEQ ID NO: 21, WP5JR - SEQ ID
NO: 22, and WP5VR - SEQ ID NO: 23.
27. The method of claim 18 wherein the inhibitor has the formula:
-56-

<IMG>
wherein:
B23 and B33 are each independently a peptide of 1-6 amino acids, at least one
of
which is a hydrophobic amino acid, an aromatic moiety or a heteroaromatic
moiety;
Z24 is a moiety that is capable of forming a covalent linkage with B23, X25
and Z32;
Z32 is a moiety that is capable of forming a covalent linkage with B33, X31
and Z24;
X25 is absent or a hydrophilic amino acid;
X26 is a hydrophilic amino acid;
X27 is a hydrophilic amino acid;
X28 is a hydrophobic amino acid;
X29 is a hydrophobic amino acid;
X30 is absent or a hydrophobic amino acid;
X31 is absent or a hydrophobic amino acid;
"-" is an amide, a substituted amide or an isostere of amide;
"=" is a covalent linkage; and
".ident." is a covalent linkage.
28. The method of claim 27 wherein:
B23 and B33 are each independently a peptide of 1-3 amino acids, at least one
of
which is an aromatic amino acid;
Z24 and Z32 are each independently a Cys-like amino acid;
-57-

X25 is absent or a basic amino acid;
X26 is a basic amino acid;
X27 is an acidic amino acid'
X28 is an apolar amino acid;
X29 is an apolar amino acid;
X30 is absent or a polar amino acid;
X31 is absent or an apolar amino acid;
"-" is an amide linkage;
"=" is a disulfide linkage; and
".ident." is an amide linkage.
29. The method of claim 28 wherein:
B23 and B33 are each independently Tyr or Phe;
Z24 and Z32 are each Cys;
X25 is absent or Arg;
X26 is Lys;
X27 is Glu;
X28 is Leu, Pro or Met;
X29 is Gly;
X30 is absent or Gln;
X31 is absent or Val;
"-" is an amide linkage;
"=" is a disulfide linkage; and
".ident." is an amide linkage.
30. The method of claim 29 wherein the inhibitor is selected from the group
consisting
of WP8L - SEQ ID NO:24.
-58-

31. The method of claim 17 wherein the disease characterized by bone loss is
selected
from the group consisting of osteoporosis, Paget's disease, metastatic bone
disease,
rheumatoid arthritis, and periodontal disease.
32. The method of claim 31 wherein the disease characterized by bone loss is
osteoporosis.
33. A method of inhibiting bone resorption, comprising the step of
administering to a
patient an amount of an inhibitor effective to inhibit bone resorption.
34. The method of claim 33 wherein said inhibitor has the formula:
<IMG>
wherein:
AC is a peptide of 3-18 amino acid residues which corresponds in primary
sequence to a binding loop of a TNF-R superfamily member, and which may
optionally
contain one or more amino acid substitutions, or an analogue thereof wherein
at least one
amide linkage is replaced with a substituted amide or an isostere of amide;
AB1 is a moiety having a first functional group capable of forming a covalent
linkage with one terminus of AC, a second functional group capable of forming
a covalent
linkage with AB2 and a third functional group capable of forming a covalent
linkage with
AA1;
AB2 is a moiety having a first functional group capable of forming a covalent
-59-

linkage with the second terminus of AC, a second functional group capable of
forming a
covalent linkage with AB1 and a third functional group capable of forming a
covalent
linkage with AA2;
AA1 is a moiety having hydrophobic properties and a functional group capable
of
forming a covalent linkage with the third functional group of AB2;
AA2 is a moiety having hydrophobic properties and a functional group capable
of forming
a covalent linkage with the third functional group of AB2;
"=" is a covalent linkage; and
".ident." is a covalent linkage.
35. The method of Claim 34 in which the amino acid substitutions are
conservative.
36. The method of Claim 35 in which the member of TNF-R superfamily is TNF-R
p55.
37. The method of Claim 36 wherein the inhibitor has the formula:
<IMG>
wherein:
B1 and B10 are each independently a peptide of 1-6 amino acids at least one of
which os a hydrophobic amino acid, an aromatic moiety or a heteroaromatic
moiety;
Z2 is a moiety that is capable of forming a covalent linkage with B1 , X3 and
Z9;
Z9 is a moiety that is capable of forming a covalent linkage with B10, X8 and
Z2;
-60-

X3 is absent or a hydrophilic amino acid;
X4 is a hydrophobic amino acid;
X5 is a hydrophobic amino acid;
X6 is a hydrophobic amino acid;
X7 is a hydrophobic or hydrophilic amino acid;
X8 is a hydrophobic or hydrophilic amino acid;
"-" is an amide, substituted amide or an isostere of amide thereof;
"=" is a covalent linkage; and
".ident." is a covalent linkage.
38. The method of Claim 37, wherein:
B1 and B10 are each independently a peptide of 1-2 amino acids, at least one
of
which is an aromatic amino acid;
Z2 and Z9 are each independently a Cys-like amino acid;
X3 is absent or an acidic amino acid;
X4 is an aromatic or apolar amino acid;
X5 is a polar amino acid;
X6 is a polar amino acid;
X7 is an aromatic or polar amino acid;
X8 is an aromatic, apolar or polar amino acid;
"-" is an amide linkage;
"=" is a disulfide linkage; and
".ident." is an amide linkage.
39. The method of Claim 38, wherein:
B1 and B10 are each independently Tyr or Phe;
Z2 and Z9 are each Cys;
X3 is absent or Glu;
-61-

X4 is Trp or Leu;
X5 is Ser;
X6 is Gln;
X7 is Tyr or Asn;
X8 is Tyr or Leu;
"-" is an amide linkage;
"=" is a disulfide linkage; and
".ident." is an amide linkage.
40. The method of Claim 39, wherein said inhibitor is selected from the group
consisting of WP9Q - SEQ ID NO:13, WP9ELY - SEQ ID NO:12, WP9Y - SEQ ID
NO:14, and WP9QY - SEQ ID NO:15.
41. The method of Claim 36, wherein the inhibitor has the formula:
<IMG>
wherein:
B11 and B22 are each independently a peptide of 1-6 amino acids, at least one
of
which is a hydrophobic amino acid, an aromatic moiety or a heteroaromatic
moiety;
-62-

Z12 is a moiety that is capable of forming a covalent linkage with B11, X13
and Z21;
Z21 is a moiety that is capable of forming a covalent linkage with B22, X20
and Z12;
X13 is absent or hydrophobic amino acid;
X.4 is absent or hydrophilic amino acid;
X15 is a hydrophilic or hydrophobic amino acid;
X16 is a hydrophilic amino acid;
X17 is absent or a hydrophobic amino acid;
X18 is a hydrophilic amino acid;
X19 is a hydrophilic amino acid;
X20 is a hydrophilic amino acid;
"-" is an amide, a substituted amide or an isostere of amide thereof;
"=" is a covalent linkage; and
".ident." is a covalent linkage.
42. The method of Claim 41, wherein:
B11 and B22 are each independently a peptide of 1-3 amino acids, at least one
of
which is an aromatic amino acid;
Z12 and Z21, are each independently a Cys-like amino acid;
X13 is absent or an aromatic amino acid;
X14 is absent or a polar amino acid;
X15 is a basic, polar or apolar amino acid;
X16 is a polar amino acid;
X17 is absent or an apolar amino acid;
X18 is an acidic amino acid;
X19 is a polar amino acid;
X20 is a basic amino acid;
"-" is an amide linkage;
"=" is a disulfide linkage; and
-63-

".ident." is an amide linkage.
43. The method of Claim 42, wherein:
B11 and B22 are each independently Tyr or Phe;
Z12 and Z21 are each Cys;
X13 is absent or Phe;
X14 is absent or Thr;
X15 is Ala, Asn or Arg;
X16 is Ser;
X17 is absent or Val;
X18 is Glu;
X19 is Asn;
X20 is Arg or His;
"-" is an amide linkage;
"=" is a disulfide linkage; and
".ident." is an amide linkage.
44. The method of Claim 43, wherein said inhibitor is selected from the group
consisting of WP5 - SEQ ID NO:16, WP5N - SEQ ID NO:17, WP5R - SEQ ID NO:18,
WP5J - SEQ ID NO:19, WP5JY - SEQ ID NO:20, WP5JN - SEQ ID NO:21, WP5JR -
SEQ ID NO:22, and WP5VR - SEQ ID NO:23.
45. The method of Claim 36, wherein the inhibitor has the formula:
-64-

<IMG>
wherein:
B23 and B33 are each independently a peptide of 1-6 amino acids, at least one
of
which is a hydrophobic amino acid, an aromatic moiety or a heteroaromatic
moiety;
Z24 is a moiety that is capable of forming a covalent linkage with B23, X25
and Z32;
Z32 is a moiety that is capable of forming a covalent linkage with B33, X31
and Z24;
X25 is absent or a hydrophilic amino acid;
X26 is a hydrophilic amino acid;
X27 is a hydrophilic amino acid;
X28 is a hydrophilic amino acid;
X29 is a hydrophilic amino acid;
X30 is absent or a hydrophilic amino acid;
X31 is absent or a hydrophilic amino acid;
"-" is an amide, a substituted amide or an isostere of amide;
"=" is a covalent linkage; and
".ident." is a covalent linkage.
46. The method of Claim 45, wherein:
B23 and B33 are each independently a peptide of 1-3 amino acids, at least one
of
which is an aromatic amino acid;
Z24 and Z32 are each independently a Cys-like amino acid;
-65-

X25 is absent or a basic amino acid;
X26 is a basic amino acid;
X27 is an acidic amino acid;
X28 is an apolar amino acid;
X29 is an apolar amino acid;
X30 is absent or a polar amino acid;
X31 is absent or an apolar amino acid;
"-" is an amide linkage'
"=" is a disulfide linkage; and
".ident." is an amide linkage.
47. The method of Claim 46, wherein:
B23 and B33 are each independently Tyr or Phe;
Z24 and Z32 are each Cys;
X25 is absent or Arg;
X26 is Lys;
X27 is Glu;
X28 is leu, Pro or Met;
X29 is Gly;
X30 is absent or Gln;
X31 is absent or Val;
"-" is an amide linkage;
"=" is a disulfide linkage; and
".ident." is an amide linkage.
48. The method of Claim 47, wherein said inhibitor is selected from the group
consisting of WP8L - SEQ ID NO:24, WP8JP - SEQ ID NO:25, WP8J - SEQ ID NO:26,
and WP8JF - SEQ ID NO:27.
-66-

Description

CA 02380009 2002-O1-25
WO 01/08699 PCT/US00/20510
METHODS OF INHIBITING OSTEOCLASTOGENESIS
FIELD OF THE INVENTION
The present invention relates to the methods of down-modulating
osteoclastogenesis activity, thereby inhibiting bone matrix erosion and thus
preventing
bone loss and treating bone diseases. The present invention also relates to
peptides and
peptide analogues which inhibit TNF binding to its cellular receptors, methods
of
designing similar peptides and peptide analogues, and methods of using such
compounds
to inhibit the biological activities of TNF particularly those activities
relating to bone
resorption, thereby antagonizing its undesirable clinical effects.
BACKGROUND OF THE INVENTION
Osteoclasts are large multinuclear cells which function to erode bone
matrix. They are related to macrophage and other cells that develop from
monocyte cells.
Like macrophage, osteoclasts are derived from haematopoietic progenitor cells.
Bone matrix erosion is a normal process which occurs in coordination with
bone matrix formation, a process in which osteoblasts are involved.
Essentially,
osteoblasts erode bone matrix and tunnel into bone while osteoblasts follow,
line the walls
-1-

CA 02380009 2002-O1-25
WO 01/08699 PCT/US00/20510
of the tunnel and form new bone matrix. Typically, in a normal adult, about 5-
10% of
bone is replaced by these processes annually.
Bone diseases such as osteoporosis and Paget's disease are characterized by
a loss of bone. Similarly, metastatic bone disease, rheumatoid arthritis, and
periodontal
bone disease are also characterized by bone loss. In many cases, bone loss
leads to
fractures in patients. In addition to pain and suffering, patients become
physically
impaired which often leads to complications having negative consequences on
patient
health and quality of life. Moreover, the economic costs attributable to these
diseases are
tremendous.
Receptors and ligands of the Tumor Necrosis Factor (TNF) family have
recently been shown to play an essential part in the differentiation and
activity of
osteoclasts and therefore play a role in bone resorption. On the one hand, TNF-
a is known
to promote osteoclastogenesis, the generation of osteoclasts. On the other
hand, a TNF-
like molecule present on and/or secreted by osteoclasts and stromal cells,
referred to
interchangeably in the field and herein as "Receptor activator of NF-nB
ligand",
(RANKL), "Osteoclast differentiation factor" (ODF), "Osteoprotegerin ligand"
(OPGL),
and "TNF-related activation-induced cytokine" (TRANCE), interacts with a TNF-
receptor-
like molecule, referred to in the field and herein as "Receptor activator of
NF-xB ligand",
(RANK), which, present in the membranes of osteoclast precursors and mature
osteoclasts,
regulates osteoclastogenesis and the resorbing activity of mature osteoclasts.
The
utilization of TNF-a antagonists, such as a monoclonal antibodies, for
therapeutic
purposes, has proven difficult, however, because of immunity to the large
molecule, and
limited entry into some specialized compartments of the body. Suda, et al.
(Endocrine
Reviews 20(3):345-357, 1999), which is incorporated herein by reference,
describe
osteoclast differentiation and function. Filvaroff, E and R. Derynck (Curr.
Biol. 8:R679-
8682, 1998) which is incorporated herein by reference, refer to bone
remodeling and a
signaling system for osteoclast regulation.
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Thus, there is a need for methods of regulating osteoclastogenesis and the
resorting activity of mature osteoclasts. There is also a need for methods of
preventing
bone loss and treating bone diseases.
SUMMARY OF THE INVENTION
The present invention relates to methods of inhibiting osteoclastogenesis
and the resorting activity of mature osteoclasts. According to the present
invention, an
amount of a TRANCE/RANK inhibitor effective to inhibit osteoclast bone erosion
activity
is administered to a patient.
The present invention relates to methods of treating patients who have
diseases characterized by bone loss. According to the present invention, an
amount of a
TRANCE/RANK inhibitor effective to inhibit osteoclastogenesis is administered
to a
patient.
The present invention relates to pharmaceutical compositions which
comprise a TRANCE/RANK inhibitor in an amount effective to inhibit
osteoclastogenesis.
The present invention relates to methods of modulating dendritic cell
maturation, T cell proliferation, and/or CD40 receptor systems in an
individual comprising
the step of administering to the individual an amount of a TRANCE/RANK
inhibitor
effective to modulate dendritic cell maturation, T cell proliferation, and/or
CD40 receptor
systems.
The present invention relates to the use of peptides and peptide analogues
designed from a binding loop of a TNF-R superfamily member. In particular, it
relates to
the use of peptides and peptide analogues designed from three binding loops of
TNF-R.
More specifically, the invention relates to peptides and peptide analogues
which inhibit
activities relating to bone resorption.
Generally, compounds used in the present invention are cyclic peptides or
peptide analogues which are modified at their termini with hydrophobic
moieties. In
embodiments wherein the compound is a peptide, the peptide corresponds in
primary
sequence to a binding loop of a member of the TNF-R superfamily or a portion
thereof. In
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a preferred embodiment, the peptide used in the invention corresponds in
primary
sequence to a binding loop of TNF-R p55 or a portion thereof. In certain
embodiments,
one or more amino acid residues within the peptide are substituted with other
amino acid
residues. Typically, such amino acid substitutions are conservative, i.e.,the
amino acid
residues are replaced with other amino acid residues having similar physical
and/or
chemical properties. In embodiments wherein the compound is a peptide
analogues, the
analogues is obtained by replacing at least one amide linkage in the peptide
with a
substituted amide or an isostere of amide.
In an illustrative embodiment, a compound used in the invention has the
following formula:
AAy
~~~ C
~2= ~2
wherein:
AC is a peptide of 3-18 amino acid residues which corresponds in primary
sequence to a binding loop of a TNF-R superfamily member, and which may
optionally
contain one or more conservative amino acid substitutions, or an analogue
thereof wherein
at least one amide linkage is replaced with a substituted amide or an isostere
of amide;
AB, is a moiety having a first functional group capable of forming a
covalent linkage with one terminus of AC, a second functional group capable of
forming a
covalent linkage with ABZ and a third functional group capable of forming a
covalent
linkage with AA,;
ABZ is a moiety having a first functional group capable of forming a
covalent linkage with the second terminus of AC, a second functional group
capable of
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forming a covalent linkage with ABA and a third functional group capable of
forming a
covalent linkage with AAZ;
AA, is a moiety having hydrophobic properties and a functional group
capable of forming a covalent linkage with the third functional group of AB,;
AAZ is a moiety having hydrophobic properties and a functional group
capable of forming a covalent linkage with the third functional group of AB2;
"--" is a covalent linkage; and
"_" is a covalent linkage.
In a preferred embodiment of the compounds of formula (I), AC is a
peptide which corresponds in primary sequence to a binding loop of TNF-R p55
and which
optionally may contain one or more conservative amino acid substitutions, or
an analogue
thereof. In a particularly preferred embodiment, the peptides and peptide
analogues
specifically inhibit osteoclastogenesis.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the sequence alignment of amino acids in certain
extracellular Cys-rich domains of TNF-R superfamily members: TNF-R p55 (SEQ ID
NO:1), TNF-R p 75 (SEQ ID NO:2), TNF-R-rp (SEQ ID NO: 3), NGF-R p75 (SEQ ID
NO: 4), CD27 (SEQ ID NO:S), CD30 (proximal) SEQ ID N0:6), CD30 (distal) (SEQ
ID
N0:7), CD40 (SEQ ID NO:10), and 4-IBB (SEQ ID NO:11).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The invention provides methods for treating individuals that have diseases
characterized by bone loss. TRANCE/RANK inhibitors are administered to the
individual
in an amount effective to inhibit osteoclastogenesis and/or osteoclast
function and thereby
reduce bone loss, i.e. a therapeutically effective amount.
The invention also provides novel therapeutic pharmaceutical compositions
for treating diseases characterized by bone loss. The pharmaceutical
compositions
comprise a therapeutically effective amount of TRANCE/RANK inhibitors and a
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pharmaceutically acceptable Garner or diluent. In preferred embodiments, the
pharmaceutical compositions are injectable pharmaceutical compositions, i.e.
they are
sterile, pyrogen-free, free of particulate matter, essentially isotonic, and
are otherwise
suitable for injection into humans.
As used herein, the term "TRANCE/RANK inhibitors" refers to peptides
and peptide analogues which inhibit osteoclastogenesis and/or osteoclast
function.
TRANCE/RANK inhibitors can function as an antagonist of the cellular receptor
RANK
by inhibiting TRANCE/RANK.
As used herein, the term "diseases characterized by bone loss" is meant to
refer to diseases, conditions, disorders and syndromes which have as a symptom
or
pathology a decrease in bone mass or density. Examples of diseases
characterized by bone
loss include, but are not limited to, osteoporosis, Paget's disease,
metastatic bone disease,
rheumatoid arthritis and periodontal bone disease.
As used herein, the term "bone resorption" refers to the undesired loss of
bone caused at least in part by osteoclast activity.
As used herein, the term "therapeutically effective amount" is meant to
refer to an amount of a compound which produces a medicinal effect observed as
reduction
in the rate of bone loss in an individual when a therapeutically effective
amount of a
compound is administered to an individual who is susceptible to or suffering
from a
disease characterized by bone loss. Therapeutically effective amounts are
typically
determined by the effect they have compared to the effect observed when a
composition
which includes no active ingredient (i.e. a control) is administered to a
similarly situated
individual.
As used herein, the term "inhibit" means to decrease the amount, quality, or
effect of a particular activity and is used interchangeably with the terms
"reduce",
"minimize", and "lessen" and refers to, for example, the reduction of
osteoclast bone
erosion activity caused by the administration of a therapeutically effective
amount of the
compounds of the present invention to a patient.
As used herein, the term "alkyl" refers to a saturated branched, straight
chain or
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cyclic hydrocarbon group. Typical alkyl groups include, but are not limited
to, methyl,
ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl,
and the like. In
preferred embodiments, the alkyl groups are (C,_CS) alkyl, with (C,-C3) being
particularly
preferred.
As used herein, the term "substituted alkyl" refers to an alkyl group wherein
one or
more hydrogen atoms are each independently replaced with other substituents.
As used herein, the term "alkenyl" refers to an unsaturated branched, straight
chain
or cyclic hydrocarbon group having at least one carbon-carbon double bond. The
group
may be in either the cis or traps conformation about the double bond(s).
Typical alkenyl
groups include, but are not limited to, ethenyl, propenyl, isopropenyl,
butenyl, isobutenyl,
tert-butenyl, pentenyl, hexenyl and the like. In preferred embodiments, the
alkenyl group
is (C,-C~) alkenyl, with (C,-C3) being particularly preferred.
As used herein, the term "alkynyl" refers to an unsaturated branched, straight
chain or cyclic hydrocarbon group having at least one carbon-carbon triple
bond. Typical
alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl,
isobutynyl,
pentynyl, hexynyl and the like. In preferred embodiments, the alkynyl group is
(CI-C~)
alkynyl, with (C,-C3) being particularly preferred.
As used herein, the term "substituted alkynyl" refers to an alkynyl group
wherein
one or more hydrogen atoms are each independently replaced with other
substituents.
As used herein, the term "alkoxy" refers to an -OR group, where R is alkyl,
alenyl
or alkynyl, as defined above.
As used herein, the term "aromatic moiety" refers to a moiety having an
unsaturated cyclic hydrocarbon group which has a conjugated (4n=2) ~ electron
system.
typical aromatic moieties include, but are not limited to, benzene,
naphthalene, anthracene,
azulene, indacene, and the like. In preferred embodiments, the aromatic moiety
contains
5-20 carbons in the ring system, with 5-10 carbon atoms being particularly
preferred.
As used herein, the term "substituted aromatic moiety" refers to an aromatic
moiety wherein one or more hydrogen atoms are each independently replaced with
other
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As used herein, the term "heteroaromatic moiety" refers to an aromatic moiety
wherein one or more of the ring carbon atoms is replaced with another atom
such as N, O
or S. Typical heteroaromatic moieties include, but are not limited to, pyran,
pyrazole,
pyridine, pyrrolke, pyrazine, pyridazine, pyrimidine, pyrrolizine,
quinazoline, quinoline,
quinolizine, quinoxaline, selenophene, thiophere, tellurophene, xanthene and
the like.
As used herein, the term "substituted heteroaromatic moiety" refers to a
heteroaromatic moiety wherein one or more hydrogen atoms are each
independently
replaced with other substituents.
Applicants have discovered that the peptides described infra are useful to
inhibit
osteoclastogenesis and/or osteoclast function. By inhibiting
osteoclastogenesis and/or
osteoclast function, bone erosion can be minimized or even prevented, and bone
loss can
be reduced. Patients suffering from diseases characterized by bone loss can be
treated by
administering an amount of compound effective to inhibit osteoclastogenesis
and/or
osteoclast function. In addition, patients identified as being susceptible to
diseases
characterized by bone loss can be prophylactically treated by administering an
amount of
compound effective to inhibit osteoclastogenesis and/or osteoclast function.
Individuals who have a disease characterized by bone loss can be identified by
those having ordinary skill in the art by well known diagnostic means and
criteria.
Individuals who are susceptible to a disease characterized by bone loss can be
identified by
those having ordinary skill in the art based upon family medical history
and/or the
presence of genetic markers or genes associated with a disease characterized
by bone loss.
According to the invention, TRANCE/RANK inhibitors useful in the invention are
compounds described infra such as peptides and peptide analogues designed from
a
binding loop of a TNF-R superfamily member which function to inhibit
osteoclastogenesis
and/or osteoclast function. Such compounds may be produced by the methods
described
infra or by other techniques well known to those skilled in the art.
According to the invention, TRANCE/RANK inhibitors useful in the invention to
treat diseases characterized by bone loss may be formulated and administered
in the
manner taught infra, or by other techniques well known to those skilled in the
art.
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According to the invention, TRANCE/RANK inhibitors useful in the invention to
treat diseases characterized by bone loss may be formulated and administered
in the
manner taught infra, or by other techniques well known to those skilled in the
art. Some
preferred dosages range from 1nM to 500mM. Some preferred dosages range from
1mM
to 500mM. Some preferred dosages range from lmg to 500 mg. Some preferred
dosages
range from 1000mg to 3000 mg. Some preferred dosages range from 1500 mg to
2500
mg. According to the invention, TRANCE/RANK inhibitors are administered one to
four
times per day.
Pharmaceutical compositions according to the present invention comprise
TRANCE/RANK inhibitors formulated in therapeutically effective doses. In some
embodiments, the pharmaceutical composition is sterile and pyrogen free.
Other aspects of the present invention include the use of TRANCE/RANK-
inhibitors in methods involving other cell types in which TRANCE/RANK-mediated
signaling is involved in cell development and/or activity. Such cell types
include antigen
presenting cells such as dendritic cells and lymphocytes. Anderson et al.
(Nature
390:175-179, 1997) refer to the RANK/RANKL in T cells and dendritic cells.
Similarly,
Kong et al. (Immunol. and Cell Biology 77:188-193, 1999) refer to
osteoprotegerin ligand
as a common link between osteoclastogenesis, lymph node formation and
lymphocyte
development. In addition, Wong et al. (J. Leukocyte Biology 65:715-724, 1999)
refer to
TRANCE as regulating dendritic cell and osteoclast function. TRANCE/RANK
inhibitors
formulated in effective doses as described supra can be used to modulate
dendritic cell
maturation and function, T cell proliferation and CD40 receptor systems.
TNF exerts its biological activities by binding to two TNF-R: p55 and p75. A
comparison of these receptors with several other cell surface receptors
revealed certain
shared structural features that led to their classification as a superfamily
(Beutler et al.,
Science 264:667, 1994). The TNF-R superfamily members possess characteristic
extracellular Cys-rich domains, yet share only about 25% sequence homology.
There are
at least ten members in this superfamily, including: TNF-R p55 and p75, TNF-R
related
protein (rp), CD40, Fas antigen (CD95), low-affinity nerve growth factor
receptor (p74),
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CD27, CD30, 4-1BB and OX40 (Beutler et al., Ann. NY Acad. Sci. pp. 118-133,
1994;
Gruss and Dower, Cytokines and Mol. Ther. 1:75-105, 1995).
Loops and turns in many proteins have been shown to play functionally
important
roles in protein-protein interactions. In a specific embodiment illustrated by
way of
examples, infra, cyclic peptides were designed from three binding loops of TNF-
R p55
which inhibited the binding of TNF to its cellular receptors. In particular,
peptides
designed from loop 1 of domain 3 exhibited the strongest inhibitory
activities. When a
peptide designed from this binding loop was used in combination with peptides
designed
from two other loops, no further increase in inhibitory effects were observed,
indicating
that loop 1 of domain 3 is a dominate ligand binding site in TNF-R.
Based on this finding, corresponding regions of other TNF-R superfamily
members
from which inhibitory peptides and peptide analogues can be designed are
readily
identified by amino acid sequence alignment with the three specific binding
sites of TNF-
R p55 (Fig. 1). Since the dominate binding site of TNF-R p55 falls within
amino acid
residues #119 to 136, which sequence starts and ends with Cys, the same region
in each
TNF-R superfamily member may be used to design peptides and peptide analogues
that
are within the scope of the present invention. In cases where the regions do
not start or
end with Cys, the region may extend to then next Cys. For example, the
corresponding
region in Fas is deleted, and thus this region in starts at residue #97 and
ends with #143.
In the case of NGF-R, the region ends at the Cys at position at 135.
Additionally, residues
74-81 and 97-110 may also be used to design additional peptides and peptide
analogues
within the scope of the present invention. Such compounds are then cyclized
and modified
at their termini with hydrophobic moieties as described in greater detail
below.
Peptides and peptide analogues designed from binding loops of a TNF-R
superfamily
member
Generally, a compound used in the present invention is a cyclic peptide or
peptide
analogue, such as those disclosed in U.S. Application Serial No. 60/146,090,
filed July 28,
1999, which is hereby incorporated by reference in its entirety. The peptide
or peptide
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analogue is modified at its termini with hydrophobic moieties. In embodiments
wherein
the compound is a peptide, the peptide corresponds in primary sequence to a
binding loop
of a member of the TNF-R superfamily of a portion thereof. In a preferred
embodiment,
the peptide corresponds in primary sequence to a binding loop of TNF-R p55 or
a portion
thereof. In certain embodiments, one or more amino acid residues within the
peptide are
substituted with other amino acid residues. Typically, such amino acid
substitutions are
conservative, i. e., the amino acid residues are replaced with other amino
acid residues
having physical and/or chemical properties similar to the residues they are
replacing.
Preferably, conservative amino acid substitutions are those wherein an amino
acid is
replaced with another amino acid encompassed within the same designated class,
as will
be described more thoroughly below. In embodiments wherein the compound is a
peptide
analogue, the analogue is obtained by replacing at least one amide linkage in
the peptide
with a substituted amide or an isostere of amide.
In an illustrative embodiment, a compound used in the invention has the
following
formula:
AA1-
/// C
~2= ~2
(I)
wherein:
AC is a peptide of 3-18 amino acid residues, preferably 5-8 amino acid
residues,
which corresponds in primary sequence to a binding loop of a TNF-R and which
may
optionally contain conservative amino acid substitutions, or an analogue
thereof wherein at
least one amide linkage is replaced with a substituted amide or an isostere of
amide;
AB, is a moiety having a first functional group capable of forming a covalent
linkage with one terminus of AC, a second functional group capable of forming
a covalent
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linkage with ABZ and a third functional group capable of forming a covalent
linkage with
W
ABZ is a moiety having a first functional group capable of forming a covalent
linkage with the second terminus of AC, a second functional group capable of
forming a
covalent linkage with AB, and a third functional group capable of forming a
covalent
linkage with AA2;
AA, is a moiety having hydrophobic properties and a functional group capable
of
forming a covalent linkage with the third functional group of AB2;
AAZ is a moiety having hydrophobic properties and a functional group capable
of forming
a covalent linkage with the third functional group of AB2:
"--" is a covalent linkage; and
"_" is a covalent linkage.
More specifically, the compounds of the invention are illustrated by three
specific
embodiments having the following formulae:
B~-Z =X3-X4
Xs
(11) B~o-Z,~Xs-X~
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B 11zi 2= x1 3- X14
X16
(III) xt7
% 18
B22= 221X20- x1 9
B23z24= X25- x26
x27
28
(IV) j 2~
B33 z32=x31 x30
The designation X" in each case represents an amino acid at the specified
position in the
compound. Similarly, the designation Zn represents an amino acid or other
moiety which
5 is capable of forming covalent linkages with other Z~, such as disulfide
bridges.
The amino acid residues denoted by X" or Zn may be the genetically encoded L-
amino
acids, naturally occurring non-genetically encoded L-amino acids, synthetic L-
amino acids
or D-enantiomers of all of the above. The amino acid notations used herein for
the twenty
genetically encoded L-amino acids and common non-encoded amino acids are
10 conventional and are as follows:
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Amino Acid One-Letter Symbol Common Abbreviation
Alanine A Ala
Arginine R ~'g
Asparagine N Asn
Aspartic acid D Asp
Cysteine C Cys
Glutamine Q Gln
Glutamic acid E Glu
Glycine G Gly
Histidine H His
Isoleucine I Ile
Leucine L Leu
Lysine K Lys
Methionine M Met
Phenylalanine F Phe
Proline P Pro
S Brine S S er
Threonine T Thr
Tryptophan W T~
Tyrosine Y Tyr
V cline V V al
(3-alanine bAla
2,3-diaminopropionic Dpr
acid
-a-aminoisobutyric Aib
acid
N-Methylglycine MeGly
(sarcosine)
Ornithine Orn
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Citrulline Cit
t-buytlalanine t-Bua
t-butylglycine t-Bug
N-methylisoleucine MeIle
phenylglycine Phg
cyclohexylalanine Cha
Norleucine Nle
naphthylalanine Nal
Pyridylananine
3-benzothienyl alanine
4-chlorophenylalanine Phe(4-Cl)
2-fluorophenylalanine Phe(2-F)
3-fluorophenylalanine Phe(3-F)
4-fluorophenylalanine
Penicillamine Pen
1,2,3,4- Tic
tetrahydroisoquinoline-3-
carboxylic acid
(3-thienylalanine Thi
Methionine sulfoxide MSO
Homoarginine hArg
N-acetyl lysine AcLya
2,4-diamino butyric Dbu
acid
p-aminophenylalanine Phe(pHNz)
N-methylvaline MeVal
Homocysteine hCys
Homoserine hSer
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~ s-amino hexanoic acid ~ ~ Aha
The compounds used in the invention are partially defined in terms of amino
acid
residues of designated classes. The amino acids may be generally categorized
into three
main classes: hydrophilic amino acids, hydrophobic amino acids and Cysteine-
like amino
acids, depending primarily on the characteristics of the amino acid side
chain. These
amino classes may be further divided into subclasses. Hydrophilic amino acids
include
amino acids having acidic, basic or polar side chains and hydrophobic amino
acids
including amino acids having aromatic or apolar side chains. Apolar amino
acids ma be
further subdivided to include, among others, aliphatic amino acids. The
definitions of the
classes of amino acids as used herein are as follows:
"Hydrophobic Amino Acid" refers to an amino acid having a side chain that is
uncharged at physiological pH and that is repelled by aqueous solution.
Examples of
genetically encoded hydrophobic amino acids include Ile, Leu and Val. Examples
of non-
genetically encoded hydrophobic amino acids include t-BuA.
"Aromatic Amino Acid" refers to a hydrophobic amino acid having a side chain
containing at least one ring having a conjugated ~-electron system (aromatic
group). The
aromatic group may be further substituted with substituent groups such as
alkyl, alkenyl,
alkynyl, hydroxyl, sulfanyl, nitro and amino groups, as well as others.
Examples of
genetically encoded aromatic amino acids include phenylalanine, tyrosine and
tryptophan.
Commonly encountered non-genetically encoded aromatic amino acids include
phenylglycine, 2-naphthylalanine, (3-2-thienylalanine, 1,2,3,4-
tetrahydroisoquinoline-3-
carboxylic acid, 4-chloro-phenylalanine, 2-fluorophenylalanine, 3-
fluorophenylalanine and
4-fluorophyenylalanine.
"Apolar Amino Acid" refers to a hydrophobic amino acid having a side chain
that
is generlaly uncharged at physiological pH and that is not polar. Examples of
genetically
encoded apolar amino acids include glycine, proline and methionine. Examples
of non-
encoded apolar amino acids include Cha.
"Aliphatic Amino Acid" refers to an apolar amino acid having a saturated or
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unsaturated straight chain, branched or cyclic hydrocarbon side chain.
Examples of
genetically encoded aliphatic amino acids include Ala, Leu, Val and Ile.
Examples of non-
encoded aliphatic amino acids include Nle.
"Hydrophilic Amino Acid" refers to an amino acid having a side chain that is
attracted by aqueous solution. Examples of genetically encoded hydrophilic
amino acids
include Ser and Lys. Examples of non-encoded hydrophilic amino acids include
Cit and
hCys.
"Acidic Amino Acid " refers to a hydrophilic amino acid having a side chain pK
value of less than 7. Acidic amino acids typically have negatively charged
side chains at
physiological pH due to loss of a hydrogen ion. Examples of genetically
encoded acidic
amino acids include aspartic acid (aspartate) and glutamic acid (glutamate).
"Basic Amino Acid" refers to a hydrophilic amino acid having a side chain pK
value of greater than 7. Basic amino acids typically have positively charged
side chains at
physiological pH due to association with hydronium ion. Examples of
genetically encoded
basic amino acids include arginine, lysine and histidine. Examples of non-
genetically
encoded basic amino acids include the non-cyclic amino acids ornithine, 2, 3-
diaminopropionic acid, 2.4-diaminobutyric acid and homoarginine.
"Polar Amino Acid" refers to a hydrophilic amino acid having a side chain that
is
uncharged at physiological pH, but which has a bond in which the pair of
electrons shared
in common by two atoms is held more closely by one of the atoms. Examples of
genetically encoded polar amino acids include asparagine and glutamine.
Examples of
non-genetically encoded polar amino acids include citrulline, N-acetyl lysine
and
methionine sulfoxide.
"Cysteine-Like Amino Acid" refers to an amino acid having a side chain capable
of
forming a covalent linkage with a side chain of another amino acid residue,
such as a
disulfide linkage. Typically, cysteine-like amino acids generally have a side
chain
containing at least one thiol (SH) group. Examples of genetically encoded
cysteine-like
amino acids include cysteine. Examples of non-genetically encoded cysteine-
like amino
acids include homocysteine and penicillamine.
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As will be appreciated by those having skill in the art, the above
classifications are
not absolute -- several amino acids exhibit more than one characteristic
property, and can
therefore be included in more than one category. For example, tyrosine has
both an
aromatic ring and a polar hydroxyl group. Thus, tyrosine has dual properties
and can be
included in both the aromatic and polar categories. Similarly, in addition to
being able to
form disulfide linkages, cysteine also has apolar character. Thus, while not
strictly
classified as a hydrophobic or apolar amino acid, in many instances cysteine
can be used to
confer hydrophobicity to a peptide.
Certain commonly encountered amino acids which are not genetically encoded of
which the peptides and peptide analogues of the invention may be composed
include, but
are not limited to, (3-alanine (B-Ala) and other omega-amino acids such as 3-
aminopropionic acid (Dap), 2.3-diaminopropionic acid (Dpr), 4-aminobutyric
acid and so
forth; a-aminoisobutyric acid (Aib); E-aminohyxanoic acid (Aha);8-aminovaleric
acid
(Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline
(Cit); t-
butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (MeIle);
phenylglycine
(phg); cyclohexylalanine (Cha); norleucine (Nle); 2-naphythylalanine (2-Nal);
4-
chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)); 3-
flurophenylalanine
(Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); 1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid (Tic); (3-2-thienylalanine (Thi);
methionine
sulfoxide (MOS); homoarginine (hArg); N-acetyl lysine (AcLys); 2,3-
diaminobutyric acid
(Dab); 2,3-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe(pNHz)); N-
methyl
valine (MeVal); homocysteine (hCys) and homoserine (hSer). These amino acids
also fall
conveniently into the categories defined above.
The classifications of the above-described genetically encoded and non-encoded
amino acids are summarized in Table l, below. It is to be understood that
Table 1 is for
illustrative purposes only and does not purport to be an exhaustive list of
amino acid
residues which may comprise the peptides and peptide analogues described
herein. Other
amino acid residues which are useful for making the peptides and peptide
analogues
described herein can be found, e.g., in Fasman, 1989, CRC Practical Handbook
of
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Biochemistry and Molecular Biology, CRC Press, Inc., and the references cited
therein.
Amino acids not specifically mentioned herein can be conveniently classified
into the
above-described categories on the basis of known behavior and/or their
characteristic chemical and/or physical properties as compared with amino
acids
specifically identified.
Table 1
Classification Genetically Encoded Genetically Non-Encoded
Hydrophobic
Aromatic F, Y, W Phg, Nal, Thi, Tic, Phe(4-Cl),
Phe(2-F), Phe(3-F), Phe(4-F);
Pyridyl Ala, Benzothienyl Ala
Apolar M, G, P T-BuA, T-BuG, MeIle, Nle,
Aliphatic A, V, L, I MeVal, Cha, bAla, MeGly,
Aib
Hydrophilic
Acidic D, E Dpr, Orn, hArg, Phe(p-NHZ),
Basic H, K, R DBU, AZBU
Polar Q, N, S, T, Y Cit, AcLys, MSO, hSer
Cysteine-Like C Pen, hCys, (3-methyl Cys
The designation Z" in each case represents an amino acid or other moiety
capable
of forming covalent linkages with other Z" so as to allow cyclization of the
peptide.
Examples of amino acid residues which are capable of forming covalent linkages
with one
another include cysteine-like amino acids such as Cys, hCys, (3-methyl Cys and
Pen, which
are capable of forming disulfide bridges with one another. Preferred cysteine-
like amino
acid residues include Cys and Pen.
Amino acids used to cyclize a peptide need not be cysteine-like amino acids.
Pairs
of amino acids that have side chain functional groups capable of forming
covalent linkages
with one another can also be used. Such pairs of functional groups are known
to those of
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skill in the art and include, inter alia, -COOH and -OH, -COOH and -NHZ, and -
COOH
and -SH. Thus, pairs of amino acids that can be used to cyclize a peptide
include, inter
alia, Asp and Lys; Glu and Lys; Asp and Arg; Glu and Arg; Asp and Ser; Glu and
Ser;
Asp and Thr; Glu and Thr; Asp and Cus; and Glu8 and Cys. Other pairs of amino
acids
which can be used to cyclize the peptide will be apparent to those skilled in
the art.
It will also be recognized that Zn groups used to cyclize a peptide need not
be
amino acids. Thus, Zn may be any molecule having three functional groups --
one
functional group capable of forming a covalent linkage with a terminus of the
peptide, a
second functional group capable of forming a covalent linkage with the second
functional
group of another Z~, and a third functional group capable of forming a
covalent linkage
with hydrophobic moieties Bn. Molecules having a suitable functional groups
will be
apparent to those skilled in the art. Examples of functional groups capable of
forming a
covalent linkage with the amino terminus of a peptide include carboxylic acids
and esters.
Examples of functional groups capable of forming a covalent linkage with the
carboxyl
terminus of a peptide include -OH, -SH, -NHZ and -NHR where R is (C, -C~)
alkyl, (C, -
C6) alkenyl and (C, -C~) alkynyl.
A variety of interlinkages useful to cyclize a peptide can be generated by
reaction
between two Z~ Z~ with functional groups suitable for forming such
interlinkages, as well
as reaction conditions suitable for forming such interlinkages, will be
apparent to those of
skill in the art. Preferably, the reaction conditions used to cyclize the
peptides are
sufficiently mild so as not to degrade or otherwise damage the peptide.
Suitable groups for
protecting the various functionalities as necessary are well know in the art
(see, e.g., Green
& Wuts, 1991, 2"d ed., John Wiley & Sons NY), as are various reaction schemes
for
preparing such protected molecules.
The destination Bn in each case represents a hydrophobic moiety. While not
intending to be bound by any particular theory, it is believed that when
placed in aqueous
solution, these hydrophobic moieties interact so as to confer the peptide with
structural
stability. A significant hydrophobic interaction for conferring structural
stability is
thought to be stacking of aromatic rings. Thus, in a preferred embodiment,
such B"
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designates a peptide of 1-6 amino acids, at least one of which is an aromatic
amino acid or
an aromatic or heteroaromatic moiety. Bp may be illustrated as X32 -X33 -X3a -
Xss -X36 -
X3~= wherein X~ is an amino acid at least one of which is an aromatic amino
acid. More
preferably, X32 -X33 -X34 -Xss -X36 are absent and X3~ is an aromatic amino
acid. Suitable
aromatic amino acids include Tyr, Phe and Trp, with Tyr and Phe being
preferred.
Suitable aromatic or heteroaromatic moieties include phenyl, naphthyl, purine,
pyrimidine,
and the like.
In the peptides of formulae (II) - (IV), the symbol "-" between amino acid
residues
X" generally designates a backbone interlinkage. Thus, the symbol "-" usually
designates
an amide linkage (-C(O)-NH). It is to be understood, however, that in all of
the peptides
described in the specific embodiments herein, one or more amide linkages may
optionally
be replaced with a linkage other than amide, preferably a substituted amide or
an isostere
of an amide linkage. Thus, while the various Xn have generally been described
in terms of
amino acids, one having skill in the art will recognize that in embodiments
having non-
amide linkages, the term "amino acid" refers to other bifunctional moieties
having side-
chain groups similar to the side chains of the amino acids. For example, in
embodiments
having non-amide linkages, the phrase "acidic amino acid" refers to a
bifunctional
molecule capable of forming the desired backbone interlinkages and which has a
side
chain group similar to the side chain of an acidic amino acid. Substituted
amides generally
include groups of the formula -C(O)-NR, where R is (C, -C~) alkyl, (C, -C~)
alkenyl, (C, -
C~) alkynyl, substituted (C, -C~) alkyl, substituted (C, -C~) alkenyl or
substituted (C, -C~)
alkynyl. Isosteres of amide generally include, but are not limited to, -CHZNH-
, -CHZS-, -
CHZCH2, -CH=CH- (cis and traps), -C(O)CHZ-and -CHZS)-.
Compounds having such linkages and methods for preparing such compounds are
well-known in the art (see, e.g., Spatola" Vega Data 1 (3); 1983, for a
general review);
Spatola, "Peptide Backbone Modifications" In: Chemistry and Biochemistry of
Amino
Acids Peptides and Proteins (Weinstein, ed.), Marcel Dekker, New York, p. 267
(general
review) 1983; Morley, Trends Pharm. Sci. 1:463468, 1980; Hudson et al." Int.
J. Prot.
Res. 14:177-185 (-CHZNH-, -CHzCH2-)1979; Spatola et al., Life Sci. 38:1243-
1249 (-
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CHZ -S), 1986; Hann, J. Chem. Soc. Perkin Trans. I. 1:307-314 (-CH=CH-, cis
and trans) ,
1982; Jennings-White et al., Tetrahedron. Lett. 23:1392-1398 (-COCHz-);
European Patent
Application EP 45665 (1982) CA:97:39405 (-CH(OH)CHZ-); Holladay et al.,
Tetrahedron
Lett. 24:4401-4404, 1983, (-C (OH)CHZ-); and Hruby, Life Sci. 31:189-199, 1982
(-CHZ-
S-).
As will be discussed in more detail below, the interlinkage designated by "_"
between residues Bn and/or Zn and/or Xn in the compounds of formulae (II) -
(IV) may also
be a linker. Typically, a linker is a bifunctional molecule that spaces one
moiety from
another. Such linkers, which may be flexible, semi-rigid or rigid, are well-
known in the
art and include polypeptides such as poly-Gly and poly-Pro, bifunctional
hydrocarbons
such as aminocaproic acid, 8- aminovaleric acid and (3-alanine, carbohydrates,
nucleic
acids, and the like.
In one specific illustrative embodiment, the compounds of formula (II) are
defined
as follows:
B~-Z =X3-X4
XS
B ~ o- Z9= Xg-- X7
(II)
wherein:
B, and Boo are each independently a peptide of 1-6 amino acids, at least one
of
which is a hydrophobic amino acid, an aromatic moiety or a heteroaromatic
moiety;
ZZ is a moiety that is capable of forming a covalent linkage with B,, X3 and
Z9;
Z~ is a moiety that is capable of forming a covalent linkage with B,o, X8 and
Z2;
X3 is absent or a hydrophilic amino acid;
X4 is a hydrophobic amino acid;
XS is a hydrophilic amino acid;
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X6 is a hydrophilic amino acid;
X~ is a hydrophobic or hydrophilic amino acid;
Xg is a hydrophobic or hydrophilic amino acid;
"-" is an amide, substituted amide or an isostere of amide thereof;
"--" is a covalent linkage; and
"_" is a covalent linkage.
In a preferred embodiment of the invention, the compounds are those of formula
(II) wherein:
B, and B,o are each independently a peptide of 1-3 amino acids, at least one
of
which is an aromatic amino acid;
Zz and Z~ are each independently a Cys-like amino acid;
X3 is absent or an acidic amino acid;
X4 is an aromatic or apolar amino acid; '
XS is a polar amino acid;
X~ is a polar amino acid;
X, is an aromatic or polar amino acid;
Xg is an aromatic, apolar or polar amino acid;
"-" is an amide linkage;
"--" is a disulfide linkage; and
"_" is an amide linkage.
In particularly preferred embodiment, the compounds used in the invention are
those of formula (II) wherein:
B, and Blo are each independently Tyr or Phe;
ZZ and Z~ are each Cys;
X3 is absent or Glu;
X4 is Trp or Leu;
XS is Ser;
X~ is Gln;
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X~ is Tyr or Asn;
X8 is Tyr or Leu;
"-" is an amide linkage;
"--" is a disulfide linkage; and
"_" is an amide linkage.
Particularly preferred peptides used in the invention include the following:
YCELSQYLCY (SEQ ID N0:12)
YC WSQNLCY (SEQ ID N0:13)
YC WSQNYCY (SEQ ID N0:14)
YC WSQYLCY (SEQ ID NO:15)
In a second illustrative embodiment, the compounds of formula (III) are
defined as
follows:
B11Z12=X13-X14
XI~
(III) Xl ~
18
B22= z21= X20'- X19
wherein:
15 BI, and Bzz are each independently a peptide of 1-6 amino acids, at least
one of
which is a hydrophobic amino acid, an aromatic moiety or a heteroaromatic
moiety;
Z,z is a moiety that is capable of forming a covalent linkage with B", X,3 and
Zz,;
Zz, is a moiety that is capable of forming a covalent linkage with Bzz, Xzo
and Z,z;
X,3 is absent or hydrophobic amino acid;
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X14 is absent or a hydrophilic amino acid;
X15 is a hydrophilic or hydrophobic amino acid;
X16 is hydrophilic amino acid;
X~~ is absent or a hydrophobic amino acid;
X18 is a hydrophilic amino acid;
X19 is a hydrophilic amino acid;
XZO is a hydrophilic amino acid;
"-" is an amide, a substituted amide or an isostere of amide thereof;
In a preferred embodiment, the compounds are those of formula (III) wherein:
B11 and Bz2 are each independently a peptide of 1-3 amino acids, at least one
of
which is an aromatic amino acid;
Zlz and Zzl are each independently a Cys-like amino acid;
X13 is absent or an aromatic amino acid;
X14 is absent or a polar amino acid;
X15 is a basic, polar or apolar amino acid;
X,~ is a polar amino acid;
X1~ is absent or an apolar amino acid;
X1g is an acidic amino acid;
X,~ is a polar amino acid;
Xzo is a basic amino acid;
"-" is an amide linkage;
"--" is a disulfide linkage; and
"_" is an amide linkage.
In particularly preferred embodiment, the compounds are those of formula
(III),
wherein:
B1, and B22 are each independently Tyr or Phe;
Z,~ and Zzl are each Cys;
X13 is absent or Phe;
X,4 is absent or Thr;
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X15 is Ala, Asn or Arg;
X16 is Ser;
X1~ is absent or Val;
X1g is Glu;
X,~ is Asn;
XZO is Arg or His;
"-" is an amide linkage;
"--" is a disulfide linkage; and
"_" is an amide linkage.
Particularly preferred
peptides used in
the invention include
the following:
YC FTASENH CY (SEQ. ID N0:16)
YC FTNSENH CY (SEQ. ID N0:17)
YC FTRSENH CY (SEQ. ID N0:18)
FC ASENH CY (SEQ. ID N0:19)
YC ASENH CY (SEQ. ID N0:20)
FC NSENH CY (SEQ. ID N0:21 )
FC NSENR CY (SEQ. ID N0:22)
FC NSVENR CY (SEQ. ID N0:23)
In a third illustra tive embodiment, the compounds of formula
(IV) are defined as
follows:
B23= z24= X25- X2G
X27
X28
(I'1) j 29
B33 Z32=X31 X30
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wherein:
Bz3 an B33 are each independently a peptide of 1-6 amino acids, at least one
of
which is a hydrophobic amino acid, an aromatic moiety or a heteroaromatic
moiety;
Zz4 is a moiety that is capable of forming a covalent linkage with Bz3, X25
and Z3z;
Z3z is a moiety that is capable of forming a covalent linkage with B33, X3,
and Zz4;
Xz5 is absent or a hydrophilic amino acid;
Xzb is a hydrophilic amino acid;
Xz, is a hydrophilic amino acid;
Xzg is a hydrophobic amino acid;
Xz~ is a hydrophobic amino acid;
X3o is absent or a hydrophilic amino acid;
X3, is absent or a hydrophobic amino acid;
"-" is an amide, a substituted amide or an isostere of amide;
"-" is a covalent linkage; and
"_" is a covalent linkage.
In a preferred embodiment, the compounds are those of formula (IV) wherein:
Bz3 an B33 are each independently a peptide of 1-3 amino acids, at least one
of
which is a an aromatic acid;
Zz4 and Z3z are each independently a Cys-like amino acid;
Xzs is absent or a basic amino acid;
Xzb is a basic amino acid;
Xz, is an acidic amino acid;
Xz8 is an apolar amino acid;
Xz9 is an apolar amino acid;
X3o is absent or a polar amino acid;
X3, is absent or a apolar amino acid;
"-" is an amide linkage;
"--" is a disulfide linkage; and
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"_" is an amide linkage.
In a particularly preferred embodiment, the compounds used in the invention or
analogues thereof are those of formula (IV), wherein:
Bz3 an B33 are each independently Tyr or Phe;
Zz4 and Z3z are each Cys;
Xz5 is absent or Arg;
Xz~ is Lys;
Xz~ is Glu;
Xzg is Leu, Pro or Met;
Xz~ is Gly;
X3o is absent or Gln;
X3~ is absent or Val;
"-" is an amide linkage;
"--" is a disulfide linkage; and
"_" is an amide linkage.
Particularly preferred peptides used in the invention include the following:
YC RKELGOV CY (SEQ. ID N0:24)
YC KEPGQ CY (SEQ. ID N0:25)
YC RKEMG CY (SEQ. ID N0:26)
FC RKEMG CY (SEQ. ID N0:27)
In all of the aforementioned embodiments of the invention, it is to be
understood
that the phrase "amino acid" also refers to bifunctional moieties having amino
acid-like
side chains, as previously described.
Generally, active peptides or peptide analogues used in the invention are
those that
exhibit at least about 15% inhibition of TNF-R:TNF interactions as measured in
vitro
assays such as those described, infra. Preferably, active peptides used in the
invention or
analogues thereof will exhibit at least about 20% to 50% or even 80% or more
inhibition
of TNF-R:TNF-a binding interactions.
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Preparation Of Peptides And Peptide Analogues
Chemical Synthesis
The peptides used in the invention or analogues thereof, may be prepared using
virtually any art-known technique for the preparation of peptides and peptide
analogues.
For example, the peptides may be prepared in linear or non-cyclized form using
conventional solution or solid phase peptide syntheses and cyclized using
standard
chemistries. Preferably, the chemistry used to cyclize the peptide will be
sufficiently mild
so as to avoid substantially degrading the peptide. Suitable procedures for
synthesizing the
peptides described herein as well as suitable chemistries for cyclizing the
peptides are well
known in the art.
Formation of disulfide linkages, if desired, is generally conducted in the
presence
of mild oxidizing agents. Chemical, enzymatic or photolytic oxidation agents
may be
used. Various methods are known in the art, including those described, for
example, by
Tam, J.P. et al., Synthesis 955-957, 1979; Stewart et al., Solid Phase Peptide
Synthesis. 2d
Ed., Pierce Chemical Company Rockford, IL , 1984; Ahmed et al., J. Biol. Chem.
250:8477-8482, 1975; and Pennington et al. Peptides 1990 164-166, Giralt and
Andrew
Eds., ESCOM, 1991; Leiden, The Netherlands. An additional alternative is
described by
Kamber et al., Helv Chim Acta, 63:899-915, 1980. A method conducted on solid
supports
is described by Albericio, Int.J. Peptide Protein Res., 26:92-97, 1985. Any of
these
methods may be used to form disulfide linkages in the peptides of the
invention. Preferred
methods for effecting disulfide-bridge formation for the peptides described
herein are
provided in the examples.
Recombinant Synthesis
If the peptide is composed entirely of gene-encoded amino acids, or a portion
of it
is so composed, the peptide or the relevant portion may also be synthesized
using
conventional recombinant genetic engineering techniques. The isolated
peptides, or
segments thereof, are then condensed, and oxidized, as previously described,
to yield a
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cyclic peptide.
For recombinant production, a polynucleotide sequence encoding a linear form
of
the peptide is inserted into an appropriate expression vehicle, i.e., a vector
which contains
the necessary elements for the transcription and translation of the inserted
coding
sequence, or in the case of an RNA viral vector, the necessary elements for
replication and
translation. The expression vehicle is then transfected into a suitable target
cell which will
express the linear form of the cyclic peptide. Depending on the expression
system used,
the expressed peptide is then isolated by procedures well-established in the
art. Methods
for recombinant protein and peptide production are well known in the art (see,
e.g.,
Maniatis et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor
Laboratory,
N.Y., 1989; and Ausubel et al., Current Protocols in Molecular Biology, Greene
Publishing Associates and Wiley Interscience, N.Y., 1989).
A variety of host-expression vector systems may be utilized to express the
peptides
described herein. These include, but are not limited to, microorganisms such
as bacteria
transformed with recombinant bacteriophage DNA or plasmid DNA expression
vectors
containing an appropriate coding sequence; yeast or filamentous fungi
transformed with
recombinant yeast or fungi expression vectors containing an appropriate coding
sequence;
insect cell systems infected with recombinant virus expression vectors (e.g.,
baculovirus)
containing an appropriate coding sequence; plant cell systems infected with
recombinant
virus expression vectors (e.g., cauliflower mosaic virus or Tobacco mosaic
virus) or
transformed with recombinant plasmid expression vectors (e.g., Ti plasmid)
containing an
appropriate coding sequence; or animal cell systems.
The expression elements of the expression systems vary in their strength and
specificities. Depending on the host/vector system utilized, any of a number
of suitable
transcription and translation elements, including constitutive and inducible
promoters, may
be used in the expression vector. For example, when cloning in bacterial
systems,
inducible promoters such as pL of baceriophage ~,, plac, ptrp, ptac (ptrp-lac
hybrid
promoter) and the like may be used; when cloning in insect cell systems,
promoters such
as the baculovirus polyhedron promoter may be used; when cloning in plant cell
systems,
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CA 02380009 2002-O1-25
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promoters derived from the genome of plant cells (e.g., heat shock promoters;
the
promoter for the small subunit of RUBISCO; the promoter for the chlorophyll
a/b binding
protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat
protein
promoter of TMV) may be used; when cloning in mammalian cell systems,
promoters
derived from the genomes of mammalian cells (e.g., metallothionein promoter)
or from
mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5
K promoter)
may be used; when generating cell lines that contain multiple copies of
expression product,
SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable
marker.
In cases where plant expression vectors are used, the expression of sequences
encoding the peptides of the invention may be driven by any of a number of
promoters.
For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV
(Brisson et al., Nature 310: 511-514, 1984), or the coat protein promoter of
TMV
(Takamatsu et al., EMBO J., 6:307-311, 1987) may be used; alternatively, plant
promoters
such as the small subunit of RUBISCO (Coruzzi et al., EMBO J. 3:1671-1680,
1984;
Brogue et al., Science 224:838-843, 1984) or heat shock promoters, e.g.,
soybean hsp17.5-
E or hsp17.3-B (Gurley et al., Mol. Cell. Biol. 6:599-565, 1986) may be used.
These
constructs can be introduced into plant cells using Ti plasmids, Ri plasmids,
plant virus
vectors, direct DNA transformation, microinjection, electroporation, etc. For
reviews of
such techniques see, e.g., Weissbach & Weissbach, Methods for Plant Molecular
Biology,
Academic Press, NY, Section VIII, pp. 421-463, 1988; and Grierson & Corey,
Plant
Molecular Biology, 2°d Ed., Blackie, London, Ch. 7-9, 1988.
In one insect expression system that may be used to produce the peptides of
the
invention, Autographa californica nuclear polyhidrosis virus (AcNPV) is used
as a vector
to express the foreign genes. The virus grows in Spodoptera frugiperda cells.
A coding
sequence may be cloned into non-essential regions (for example the polyhedron
gene) of
the virus and placed under control of an AcNPV promoter (for example, the
polyhedron
promoter). Successful insertion of a coding sequence will result in
inactivation of the
polyhedron gene and production of non-occluded recombinant virus (i. e., virus
lacking the
proteinaceous coat coded for by the polyhedron gene). These recombinant
viruses are then
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CA 02380009 2002-O1-25
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used to infect Spodoptera frugiperda cells in which the inserted gene is
expressed. (e.g.,
see Smith et al, J. Virol., 46:584, 1983; Smith, U.S. Patent No. 4,215,051).
Further
examples of this expression system may be found in Current Protocols in
Molecular
Biology, Vol. 2, Ausubel et al., eds., Greene Publish. Assoc. & Wiley
Interscience.
In mammalian host cells, a number of viral based expression systems may be
utilized. In cases where an adenovirus is used as an expression vector, a
coding sequence
may be ligated to an adenovirus transcription/translation control complex,
e.g., the late
promoter and tripartite leader sequence. This chimeric gene may then be
inserted in the
adenovirus genome by in vitro or in vivo recombination. Insertion in a non-
essential
region of the viral genome (e.g. Region E1 or E3) will result in a recombinant
virus that is
viable and capable of expressing peptide in infected hosts. (e.g., See Logan &
Shenk, Proc.
Natl. Acad. Sci. USA 81:3655-3659, 1984). Alternatively, the vaccinia 7.5 K
promoter
may be used, (see, e.g., Mackett et al., Proc. Natl. Acad. Sci. USA, 79:7415-
7419, 1982;
Mackett et al., J. Virol., 49:857-864, 1984; Panicali et al., Proc. Natl.
Acad. Sci. 79:4927-
4931, 1982).
Other expression systems for producing linear or non-cyclized forms of the
cyclic
peptides used in the invention will be apparent to those having skill in the
art.
Purification of the P~tides and Peptide Analogues
The peptides and peptide analogues used in the invention can be purified by
art-
known techniques such as high performance liquid chromatography, ion exchange
chromatography, gel electrophoresis, affinity chromatography and the like. The
actual
conditions used to purify a particular peptide or analogue will depend, in
part, on factors
such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent
to those
having skill in the art.
For affinity chromatography purification, any antibody which specifically
binds the
peptides or peptide analogues may be used. For the production of antibodies,
various host
animals, including but not limited to rabbits, mice, rats, etc., may be
immunized by
injection with a linear or cyclic peptide. The peptide may be attached to a
suitable carrier,
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such as BSA, by means of a side chain functional group or linkers attached to
a side chain
functional group. Various adjuvants may be used to increase the immunological
response,
depending on the host species, including but not limited to Freund's (complete
and
incomplete), mineral gels such as aluminum hydroxide, sufrace active
substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet
hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG
(bacilli
Calmette-Duerin) and Corynebacterium parvum.
Monoclonal antibodies to a peptide may be prepared using any technique which
provides for the production of antibody molecules by continuous cell lines in
culture.
These include but are not limited to the hybridoma technique originally
described by
Koehler and Milstein, Nature, 256:495-497, 1975; the human B-cell hybridoma
technique,
Kosbor et al., Immunology Today, 4:72, 1983; Cote et al., Proc. Natl. Acad.
Sci., USA,
80:2026-2030, 1983; and the EBV-hybridoma technique (Cole et al., Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). In
addition,
techniques developed for the production of "chimeric antibodies" (Morrison et
al., Proc.
Natl. Acad. Sci., USA, 81: 6851-6855, 1984; Neuberger et al., Nature, 312:604-
608, 1984;
Takada et al., Nature, 314:452-454, 1985) by splicing the genes from a mouse
antibody
molecule of appropriate antigen specificity together with genes from a human
antibody
molecule of appropriate biological activity can be used. Alternatively,
techniques
described for the production of single chain antibodies (U.5. Patent No.
4,946,778) can be
adapted to produce cyclic peptide-specific single chain antibodies.
Antibody fragments which contain deletions of specific binding sites may be
generated by known techniques. For example, such fragments include but are not
limited
to F(ab')Z fragments, which can be produced by pepsin digestion of the
antibody molecule
and Fab fragments, which can be generated by reducing the disulfide bridges of
the F(ab')Z
fragments. Alternatively, Fab expression libraries may be constructed (Huse et
al.,
Science 246: 1275-1281, 1989) to allow rapid and easy identification of
monoclonal Fab
fragments with the desired specificity for the cyclic peptide of interest.
The antibody or antibody fragment specific for the desired cyclic peptide can
be
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attached, for example, to agarose, and the antibody-agarose complex is used in
immunochromatography to purify cyclic peptides of the invention. See, Scopes,
Protein
Purification: Principles and Practice, Spriger-Verlag New York, Inc., NY,
1984;
Livingstone, Methods Enzymologw Immunoaffinity Chromato~raphy of Proteins
34:723-
731, 1974.
Formulation and Route of Administration
The compounds of the invention, may be administered to a subject per se or in
the
form of a pharmaceutical composition. Pharmaceutical compositions comprising
the
compounds of the invention may be manufactured by means of conventional
mixing,
dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping
or lyophilizing processes. Pharmaceutical compositions may be formulated in
conventional manner using one or more physiological acceptable Garners,
diluents,
excipients or auxiliaries which facilitate processing of the active peptides
or peptide
analogues into preparations which can be used pharmaceutically. Proper
formulation is
dependent upon the route of administration chosen.
For topical administration the compounds of the invention may be formulated as
solutions, gels, ointments, creams, suspensions, etc. as are well-known in the
art.
Systemic formulations include those designed for administration by injection,
e.g.
subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal
injection, as well
as those designed for transdermal, transmucosal, oral or pulmonary
administration.
For injection, the compounds of the invention may be formulated in aqueous
solutions, preferably in physiologically compatible buffers such as Hanks's
solution,
Ringer's solution, or physiological saline buffer. The solution may contain
formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the compounds may be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
For transmucosal administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art.
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For oral administration, the compounds can be readily formulated by combining
the active peptides or peptide analogues with pharmaceutically acceptable
carriers well
known in the art. Such carriers enable the compounds of the invention to be
formulated as
tablets, pills, dragees, capsules, liquids gels, syrups, slurnes, suspensions
and the like, for
oral ingestion by a patient to be treated. For oral solid formulations such
as, for example,
powders, capsules and tablets, suitable excipients include fillers such as
sugars, such as
lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize
starch, wheat
starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium, carboxymethylcellulose, and/or
polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If
desired,
disintegrating agents may be added, such as the cross-linked
polyvinylpyrrolidone, atgar,
or alginic acid or a salt thereof such as sodium alginate.
If desired, solid dosage forms may be sugar-coated or enteric-coated using
standard
techniques.
For oral preparations such as, for example, suspensions, elixirs and
solutions,
suitable carriers, excipients or diluents include water, glycols, oils,
alcohols, etc.
Additionally, flavoring agents, preservatives, coloring agents and the like
may be added.
For buccal administration, the compounds may take the form of tablets,
lozenges,
etc. formulated in conventional manner.
For administration by inhalation, the compounds for use according to the
present
invention are conveniently delivered in the form of an aerosol spray from
pressurized
packs or a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other
suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined by
providing a valve
to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use
in an inhaler
or insufflator may be formulated containing a powder mix of the compound and a
suitable
powder base such as lactose or starch.
The compounds may also be formulated in rectal or vaginal compositions such as
suppositories or retention enemas, e.g., containing conventional suppository
bases such as
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cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also
be
formulated as a depot preparation. Such long acting formulations may be
administered by
implantation (for example subcutaneously or intramuscularly) or by
intramuscular
injection. Thus, for example, the compounds may be formulated with suitable
polymeric
or hydrophobic materials (for example as an emulsion in an acceptable oil) or
ion
exchange resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble
salt.
Alternatively, other pharmaceutical delivery systems may be employed.
Liposomes and emulsions are well known examples of delivery vehicles that may
be used
to deliver peptides and peptide analogues of the invention. Certain organic
solvents such
as dimethylsulfoxide also may be employed, although usually at the cost of
greater
toxicity. Additionally, the compounds may be delivered using a sustained-
release system,
such as semipermeable matrices of solid polymers containing the therapeutic
agent.
Various of sustained-release materials have been established and are well
known by those
skilled in the art. Sustained-release capsules may, depending on their
chemical nature,
release the compounds for a few weeks up to over 100 days. Depending on the
chemical
nature and the biological stability of the therapeutic reagent, additional
strategies for
protein stabilization may be employed.
As the compounds of the invention may contain charged side chains or termini,
they may be included in any of the above-described formulations as the free
acids or bases
or as pharmaceutically acceptable salts. Pharmaceutically acceptable salts are
those salts
which substantially retain the antimicrobial activity of the free bases and
which are
prepared by reaction with inorganic acids. Pharmaceutical salts tend to be
more soluble in
aqueous and other protic solvents than are the corresponding free base forms.
Effective Dosages
The compounds of the invention will generally be used in an amount effective
to
achieve the intended purpose. For use to treat or prevent osteoclastogenesis
or osteoclast
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activity, the compounds of the invention, or pharmaceutical compositions
thereof, are
administered or applied in a therapeutically effective amount. By
therapeutically effective
amount is meant an amount which is effective to ameliorate, or prevent the
symptoms of
the disease or disorder, or prolong the survival of the patient being treated.
Determination
of a therapeutically effective amount is well within the capabilities of those
skilled in the
art, especially in light of the detailed disclosure provided herein.
For systemic administration, a therapeutically effective dose can be estimated
initially from in vitro assays. For example, a dose can be formulated in
animal models to
achieve a circulating concentration range that includes the ICso as determined
in cell
culture (i.e., the concentration of test compound that inhibits 50% of TNF-R:
TNF-binding
interactions). Such information can be used to more accurately determine
useful doses in
humans.
Initial dosages can also be estimated from in vivo data, e.g., animal models,
using
techniques that are well known in the art. One having ordinary skill in the
art could
readily optimize administration to humans based on animal data.
Dosage amount and interval may be adjusted individually to provide plasma
levels
of the compounds which are sufficient to maintain therapeutic effect. Usual
patient
dosages for administration by injection range from about 0.1 to 5 mg/kg/day,
preferably
from about 0.5 to 1 mg/kg/day. Therapeutically effective serum levels may be
achieved by
administering multiple doses each day.
In cases of local administration or selective uptake, the effective local
concentration of the compounds may not be related to plasma concentration. One
having
skill in the art will be able to optimize therapeutically effective local
dosages without
undue experimentation.
The amount of compound administered will, of course, be dependent on the
subject
being treated, on the subject's weight, the severity of the affliction, the
manner of
administration and the judgment of the prescribing physician.
Toxicity
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Preferably, a therapeutically effective dose of the compounds described herein
will
provide therapeutic benefit without causing substantial toxicity.
Toxicity of the compounds described herein can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals, e.g., by
determining
the LDSO (the dose lethal to 50% of the population) or the LD,o° (the
dose lethal to 100% of
the population). The dose ratio between toxic and therapeutic effect is the
therapeutic
index. Compounds which exhibit high therapeutic indices are preferred. The
data
obtained from these cell culture assays and animal studies can be used in
formulation a
dosage range that is not toxic for use in human. The dosage of the compounds
described
herein lies preferably within a range of circulating concentrations that
include the effective
dose with little or not toxicity. The dosage may vary within this range
depending upon the
dosage form employed and the route of administration utilized. The exact
formulation,
route of administration and dosage can be chosen by the individual physician
in view of
the patient's condition. (See, e.g., Fingl et al., In: The Pharmacological
Basis of
Therapeutics, Ch.l, p.1, 1975).
The invention having been described, the following examples are offered by way
of
illustration and not limitation.
EXAMPLES
Example 1
Recently, therapeutic peptidomimetics that interfere with the TNF/TNF receptor
interaction have been developed based on atomic structures deduced from the
crystal
structures of TNF-a and the TNF(3/TNF receptor complex (Takasaki et al. Nature
Biotechnology, 15:1266-1270, 1997). The most critical TNF-a recognition site
was
localized to the first loop of the third domain of TNF receptor (residues 107-
114). A
peptidomimetic (WP9QY) engineered to mimic this recognition site efficiently
antagonized the effects of TNF-binding to the TNF-a receptor in L929
lymphocytes.
WP9QY peptide was tested at concentrations ranging from about 5 pM to about
500 ~M for its effect on osteoclast formation using the co-culture system
induced by
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1,250H2D3 (lalpha,25-dihydroxyvitamin D3) and PGE2. Osteoclastogenesis was
dose-
and time-dependently inhibited by the peptide WP9QY (ICSO = 250 gM), but this
ICSO was
50-fold higher than what was required for the TNF/TNF receptor interaction
(SpM). This
difference suggests that the WP9QY peptide inhibits osteoclastogenesis by
interfering not
with the TNF/TNF receptor interaction but with another related ligand-receptor
pair such
as TRANCE/RANK. This was confirmed by demonstrating that WP9QY inhibits
TRANCE-induced marrow cultures. There was a reciprocal dose-dependence of
WP9QY
and TRANCE. Thus, WP9QY is capable of interfering not only with the TNF/TNF
receptor interaction but also with the RANK Ligand/RANK interaction, thereby
decreasing
the osteoclastogenic potential of this cytokine.
Example 2
Materials and Methods
Human recombinant TNF-a and'ZSI-labeled TNF-a were obtained from Amersham
Life Science, Inc. (Arlington Heights, IL). TNF-R(I) or p55 extracellular
domain-IgG
heavy chain chimeric protein was prepared by expression of a cDNA construct
(Peppel et
al., J. Exp. Med. 174:1483, 1991; Williams et al., Immunol. 84:433, 1995).
Anti-TNF-a
monoclonal antibody was prepared according to Doring et al. (Molecular
Immunol.
31:1059, 1994) and anti-TNF-R(I) monoclonal antibody (htr-9) was obtained from
BMA
Biomedicals AG (Augst, Switzerland).
Molecular Modeling
Computer modeling was performed using Quanta 4.0 (Molecular Simulation Inc.,
MA). The model peptides were constructed from their sequences and folded using
CHARMM. The side chains of amino acid residues were first positioned to
permitted
conformation using Ponders rotamer (Ponder et al., J. Mol. Biol. 193:775-791,
1987)
database provided in QUANTA. Then, the folded peptides were minimized to
convergence with the dielectric constant set to 80.
The crystal structure of the TNF-(3/TNF-R(I) complex (Banner et al., Cell
73:431,
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1993) was utilized to determine the binding sites of TNF-R for TNF-a. The
first (residues
56-73) and second (residues 76-83) loops of domain 2 and the first loop
(residues 107-114)
of domain 3 of the TNF-R were explored for use in designing peptides. The
essential
amino acid sequences of TNF-R for binding interactions with TNF-a were
identified as
structural templates by superimposing TNF-a to TNF-(3 complexed with its
cognate
receptor. Then, 5-8 amino acid-long peptides derived from TNF-R as shown in
Table 2
were used as templates for the design of exocyclic peptides. Additional
peptides were
derived from CDR sequences of a light chain of an anti-TNF-a neutralizing
antibody,
CDR1L of Di62 (boring et al., Mol. Immunol. 31:1059, 1994). Exocyclic
modifications
such as peptide cyclization and addition of aromatic amino acids such as Phe
and Tyr to
the ends of each peptide were performed as described (Zhang et al., Nature
Biotech.
14:472, 1996; Zhang et al., Nature Biotech 15:150, 1997).
Peptide Synthesis, Cyclization and Purification
Linear peptides were synthesized by solid-phase methods, deprotected, and
released from the resin utilizing standard methodology well known to those
skilled in the
art. Peptides were precipitated and purified by high performance liquid
chromatography
(HPLC) utilizing a C18 column and then lyophilized. The purity of such
peptides was
greater than 95% as measured by HPLC analysis.
The peptides containing internal Cys residues were oxidized by dissolving them
at
100 ~g/ml in distilled water adjusted or buffered to pH 8.0 - 8.5, for
example, by
(NH4)ZC03 with stirnng and exposure to air at 4°C for <10 days until
95% formation of
intramolecular disulfide bonds had ben confirmed by DTNB (Sigma, St. Louis,
MO)
which determined free sulfhydryls in peptides (Habeeb, Anal. Bioch. 56:60,
1973;
Angeletti et al., In Techniques in Protein Chemistry VII, Ed. Marsak, Academic
Press, San
Diego, CA., pp. 81-91, 1996). Briefly, peptides (100 ~g/ml, 50 ~,l) and DTNB
(lOmM,
50 p1) were added to 0.1 M sodium phosphate buffer (pH 8.0, 1 ml), incubated
in the dark
for 30 minutes, and the absorbance at 420 nm was determined and compared with
the
linear unoxidized peptides.
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The cyclized peptides were lyophilized, purified by HPLC utilizing a C18
preparative column and a size exclusion column Protein-Pak 60 (Waters,
Milford, MA).
The purity of the peptides was shown to be greater than 95% by HPLC analysis.
The
concentration of each cyclized peptide was calculated based on LTV intensity
versus the
corresponding linear peptide by HPLC analysis.
Amino acid sequences corresponding to three TNF-a-binding loops of TNF-R were
used as templates for the synthesis of a number of peptides. Cys residues were
included in
the linear peptides to enable their cyclization. Their identity was verified
by mass
spectrometry. Various exocyclic peptides are listed in Table 2.
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Table 2
Amino Acid Sequences of TNF-a Binding Sites in TNF-R and Exocyclic Peptides
Derived from These Sites.
TNF-a Binding Sites in the Receptor Name Exocyclic Peptides* S.LD. #
Binding Site - 5
~_a WP5 YC FTASENH CY 16
53E 82R 85VSY87 125Q 127E WPSN YC FTNSENH CY 17
WPSR YC FTRSENH CY 18
Receptor (loop 1 of domainWPSJ FC ASENH CY 19
2)
60 FTASENH 66 WPSJY YC ASENH CY 20
WPSJN FC NSENH CY 21
WPSJR FC NSENR CY 22
WPSVR FC NSVENR CY 23
WP1** YC SQSVSND CF 28
WP1R** FC VSNDR CY 29
Binding site - 8
TNF-a
65K 67Q 113P 115Y 143L
145A
WP8L YC RKELGQV CY 24
Receptor (loop 2 of domain WP8JP YC KEPGQ CY 25
2)
76 CRKEMGOV 83 WP8J YC RKEMG CY 26
WP8JF FC RKEMG CY 27
Binding site - 9
TNF-a
72THVL75 77T 97I 137 N
WP9Q YC WSQNL CY 13
Receptor (loop 1 of domainWP9ELY YCELSQYL CY 12
3)
107 WSENL 111 WP9Y YC WSQNY CY 14
WP9QY YC WSQYL CY 15
* Peptides were cyclized with cysteine disulfide bridges
** WP1 and WP1R were derived from an anti-TNF-a antibody (Di62, CDRIL) and the
template sequence is QSVSNDV.
Example 3
Identification of Osteoclasts Formed in Vitro
TRAP refers to tartrate resistant acid phosphatase which identifies osteoclast-
like
cells. Osteoprotegerin (OPG) is a naturally occurring secreted protein with
homology to
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members of the TNF receptor family. Administration of OPG in vivo inhibits
osteoclastogenesis and associated bone resorption and blocks the pathological
increase in
osteoclast numbers and activity seen in animal models that mimic osteopenic
disorders in
humans. OPG can be used as a positive control in the TRAP assay.
Cytochemical staining for TRAP is widely used for identifying osteoclasts in
vivo
and in vitro. Naphthol AS-MX phosphate 5 mg. Sigma, St. Louis, MO) is resolved
in 0.5
ml of N, N-dimethylformamide (Wako). Thirty milligrams of fast red violet LB
salt
(Sigma) and 50 ml of 0.1 M sodium acetate buffer (pH 5.0) containing 50 mM
sodium
tartratet are added to the mixture (the TRAP-staining solution). Cells are
fixed with 3.7%
(v/v) formaldehyde in Caz-- and MgZ+-free phosphate-buffered saline [PBS(-)]
for 10 min.
fixed again with ethanol-acetone (50:50, v:v) for 1 min. and incubated with
the TRAP-
staining solution for 10 min. at room temperature. TRAP-positive osteoclasts
appear as
red cells. The incubation period longer than 10 min. should be avoided since
cells other
than osteoclasts become weakly positive with time. After staining, cells are
washed with
distilled water, and TRAP-positive multinucleated cells having three or more
nuclei are
counted as osteoclasts under a microscope. (G.C. Nicholson, J.M. Mosely, P.M.
Sexton,
F.A.O. Mendelssohn, and T.J. Martin, J. Clin. Invest. 78, 355, 1986, which is
incorporated
herein by reference).
The present invention is not to be limited in scope by the exemplified
embodiments
ZO which are intended as illustrations of single aspects of the invention and
any sequences
which are functionally equivalent are within the scope of the invention.
Indeed, various
modifications of the invention in addition to those shown and described herein
will
become apparent to those skilled in the art from the foregoing description and
accompanying drawings. Such modifications are intended to fall within the
scope of the
appended claims.
All publications cited herein in incorporated by reference in their entirety.
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SEQUENCE LISTING
<110> Aoki, Kazuhiro
Horne, William Carle
Baron, Roland
Greene, Mark I.
Murali, Ramachandran
The Trustees of the University of Pennsylvania
<120> Methods of Inhibiting Osteoclastogenesis
<130> UPN3833
<140>
<141>
<150> 60/146,090
<151> 1999-07-28
<160> 29
<170> PatentIn Ver. 2.1
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Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His
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Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln
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Val Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys
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Arg Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys
50 55 60
Phe Asn Cys Ser Leu Cys Leu Asn Gly Thr
65 70

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<210> 2
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Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala
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Glu Glu Ile Ala Pro Cys Thr Ser Lys Arg Lys Thr Gln Cys Arg Cys
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Gln Pro Gly Met Phe Cys Ala Ala Trp Ala Leu Glu Cys Thr His Cys
50 55 60
Glu Leu Leu Ser Asp Cys Pro Pro Gly Thr Glu Ala Glu
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Cys Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val Val Ser Ala
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Ser Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg Cys Ala Tyr
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Cys Arg Lys Gln Cys Glu Pro Asp Tyr Tyr Leu Asp Glu Ala Asp Arg
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Cys Thr Ala Cys Val Thr Cys Ser Arg Asp Asp Leu Val Glu Lys Thr
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Pro Cys Ala Trp Asn Ser Ser Arg Val Cys Glu Cys Arg Pro Gly Met
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Phe Cys Ser Thr Ser Ala Val Asn Ser Cys Ala Arg Cys Phe Phe His
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Ser Val Cys Pro Ala Gly Met Ile Val Lys
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Cys Arg Lys Gln Cys Glu Pro Asp Tyr Tyr Leu Asp Glu Ala Gly Arg
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Pro Cys Ala Trp Asn Ser Ser Arg Thr Cys Glu Cys Arg Pro Gly Met
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Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu Asp Thr Trp Asn Arg
1 5 10 15
Glu T hr His Cys His Gln His Lys Tyr Cys Asp Pro Asn Leu Gly Leu
20 25 30
Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp Thr Ile Cys Thr Cys
35 40 45
Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys Glu Ser Cys Val Leu
50 55 60
His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys
65 70 75
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Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His
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Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly
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Leu Glu
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Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys
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Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg
20 25 30
Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Asp Thr Val Cys Arg Cys

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35 40 45
Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr
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Gly Gln Glu Leu Thr
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<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 15
Tyr Cys Trp Ser Gln Tyr Leu Cys Tyr
1 5
<210> 16
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 16
Tyr Cys Phe Thr Ala Ser Glu Asn His Cys Tyr
1 5 10
7

CA 02380009 2002-O1-25
WO 01/08699 PCT/US00/20510
<210> 17
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 17
Tyr Cys Phe Thr Asn Ser Glu Asn His Cys Tyr
1 5 10
<210> 18
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 18
Tyr Cys Phe Thr Arg Ser Glu Asn His Cys Tyr
1 5 10
<210> 19
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 19
Phe Cys Ala Ser Glu Asn His Cys Tyr
1 5
<210> 20
<211> 9
<212> PRT
<213> Artificial Sequence
8

CA 02380009 2002-O1-25
WO 01/08699 PCT/US00/20510
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 20
Tyr Cys Ala Ser Glu Asn His Cys Tyr
1 S
<210> 21
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 21
Phe Cys Asn Ser Glu Asn His Cys Tyr
1 5
<210> 22
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 22
Phe Cys Asn Ser Glu Asn Arg Cys Tyr
1 S
<210> 23
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 23
Phe Cys Asn Ser Val Glu Asn Arg Cys Tyr
1 5 10
9

CA 02380009 2002-O1-25
WO 01/08699 PCT/iJS00/20510
<210> 24
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 24
Tyr Cys Arg Lys Glu Leu Gly Gln Val Cys Tyr
1 5 10
<210> 25
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 25
Tyr Cys Lys Glu Pro Gly Gln Cys Tyr
1 5
<210> 26
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 26
Tyr Cys Arg Lys Glu Met Gly Cys Tyr
1 5
<210> 27
<211> 9
<212> PRT
<213> Artificial Sequence

CA 02380009 2002-O1-25
WO 01/08699 PCT/US00/20510
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 27
Phe Cys Arg Lys Glu Met Gly Cys Tyr
1 5
<210> 28
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 28
Tyr Cys Ser Gln Ser Val Ser Asn Asp Cys Phe
1 5 10
<210> 29
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Novel Sequence
<400> 29
Phe Cys Val Ser Asn Asp Arg Cys Tyr
1 5