Note: Descriptions are shown in the official language in which they were submitted.
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ANALOGS OF PARATHYROID HORNONE
Backqround of the Invention
n Parathyroid hormone ("PTH") is a polypeptide
5 produced by the parathyroid glands. The mature
circulating form of the hormone is comprised of 84 amino
acid residues. The biological action of PTH can be
reproduced by a peptide fragment of its N-terminus (e.g.
amino acid residues 1 through 34). Parathyroid hormone-
10 related protein ("PTHrP") is a 139 to 173 aminoacid-protein with N-terminal homology to PTH. PTHrP
shares many of the biological effects of PTH including
binding to a common PTH/PTHrP receptor. Tregear, et al.,
Endocrinol., 93:1349 (1983). PTH peptides from many
15 different sources, e.g., human, bovine, rat, chicken,
have been characterized. Nissenson, et al., Receptor,
3:193 (1993).
PTH has been shown to both improve bone mass and
quality. Dempster, et al., Endocrine Rev., 14:690
(1993); and Riggs, Amer. J. Med., 91 (Suppl. 5B):37S
(1991). The anabolic effect of intermittently
a~m;n;~tered PTH has been observed in osteoporotic men
and women either with or without concurrent
antiresorptive therapy. Slovik, et al., J. Bone Miner.
25 Res., 1:377 (1986); Reeve, et al., Br. Med. J., 301:314
(1990); and Hesch, R-D., et al., Calcif. Tissue Int'l,
44:176 (1989).
Summary of the Invention
In one aspect, the invention relates to peptide
30 variants of PTH(1-34) of the following generic formula:
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R
1~
V 1 A Glu-A5-Gln-A7-A8-His-Asn-Al1 A12 Ly 15
5 R2
A16 A17 Al8 - Al9 - Arg - A2l - Glu - A23 - A24 - Arg - Lys - A27 - A
A30--A31--A32--A33--A34--R3
wherein
A1 is Ser, Ala, or Dap;
o A3 is Ser, Thr, or Aib;
A5 is Leu, Nle, Ile, Cha, ~-Nal, Trp, Pal, Phe or
p-X-Phe, in which X is OH, a halogen, or CH3;
A7 is Leu, Nle, Ile, Cha, ~-Nal, Trp, Pal, Phe, or
p-X-Phe in which X is OH, a halogen, or CH3;
A8 is Met, Nva, Leu, Val, Ile, Cha, or Nle;
All is Leu, Nle, Ile, Cha, ~-Nal, Trp, Pal, Phe or
p-X-Phe in which X is OH, a halogen, or CH3;
A12 is Gly or Aib;
A15 is Leu, Nle, Ile, Cha, ~-Nal, Trp, Pal, Phe,
20 or p-X-Phe in which X is OH, a halogen, or CH3;
A16 is Ser, Asn, Ala, or Aib;
A17 is Ser, Thr, or Aib;
A18 is Met, Nva, Leu, Val, Ile, Nle, Cha, or Aib;
Alg is Glu or Aib;
25 A21 is Val, Cha, or Met;
A23 is Trp or Cha;
A24 is Leu or Cha;
A27 is Lys, Aib, Leu, hArg, Gln, or Cha;
A28 is Leu or Cha;
30 A30 is Asp or Lys;
A31 is Val, Nle, Cha, or deleted;
A32 is His or deleted;
A33 is Asn or deleted;
A34 is Phe, Tyr, Amp, Aib, or deleted;
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each of R1 and R2 is, independently, H, Cl_l2
alkyl, C2_12 alkenyl, C7_20 phenylalkyl, Cll_20
napthylalkyl, Cl_l2 hydroxyalkyl, C2_12 hydroxyalkenyl, C7_
20 hydroxyphenylalkyl, or Cll_20 hydroxynapthylalkyl; or
one and only one of Rl and R2 is COE1 in which El is Cl_l2
alkyl, C2_12 alkenyl, C7_20 phenylalkyl, Cll_20
napthylalkyl, C1_12 hydroxyalkyl, C2_12 hydroxyalkenyl, C7_
20 hydroxy-phenylalkyl, or Cll_20 hydroxynapthylalkyl; and
R3 is OH, NH2, Cl_l2 alkoxy, or NH-Y-CH2-Z in which
o Y is a C1_12 hydrocarbon moiety and Z is H, OH, C02H, or
CoNH2;
provided that (i) at least one of A5, A7, A8, A11,
Al5~ A18, A21, A23, A24, A27, A28, and A31 is Cha, or at
least one of A3, A12, Al6, Al7, A18, Alg, and A34 is Aib;
lS or that (ii) at least Al is Dap, A7 is ~-Nal, Trp, Pal,
Phe, or p-X-Phe, A15 is ~-Nal, Trp, Pal, Phe, or p-X-Phe,
A27 is hArg, or A31 is Nle; or a pharmaceutically
acceptable salt thereof.
A subset of the compounds covered by the above
20 formula are those in which at least one of A5, A7, All,
Als~ Al8, A2l, A23, A24, A27, A28, and A31 is Cha. For
example, A3 is Ser; A5 is Ile; A7 is Leu or Cha; A8 is
Met, Nva, Leu, Val, Ile, or Nle; A11 is Leu or Cha; A12 is
Gly; A15 is Leu or Cha; A16 is Asn or Aib; A17 is Ser; A18
2s is Met or Nle; A21 is Val; A27 is Lys, hArg, or Cha; A32
is His; A31 is Val, Nle, or Cha; A33 is Asn; A34 is Phe,
Tyr, Amp, or Aib; Rl is H; R2 is H; and R3 is NH2;
provided that at least one of A5, A7, A8, All, Al5, A18,
A2l, A23, A24, A27, A28, and A31 is Cha, or at least one of
30 A3~ A12, A16, A17, Al8, Alg, and A34 is Aib. If desired,
at least one of A7 and A11 can be Cha; or at least one of
A1s~ A23, A24, A27, A28, and A31 is Cha.
In another subset, at least one of A3, A12, A16,
A17, A18, Alg, and A34 is Aib. For example, A3 is Ser or
3s Aib; A5 is Ile; A7 is Leu or Cha; A8 is Met, Nva, Leu,
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Val, Ile, or Nle; All is Leu or Cha; A15 is Leu or Cha,
A16 is Asn or Aib; A18 is Met, Aib, or Nle; A21 is Val; A27
is Lys, Aib, Leu, hArg, or Cha; A31 is Val, Nle, or Cha;
A32 is His; A33 is Asn; A34 is Phe, Tyr, Amp, or Aib; R1
5 is H; R2 is H; and R3 is NH2; provided that at least one
A5~ A7~ A8, A11~ A15, A18, A21, A23~ A24~ A27, A25, and
A31 is Cha, or at least one of A3, A12, A16~ A17, A18, A19,
and A34 is Aib. If desired, at least one of A7 and A11
can be Cha; or at least one of A1s, A23, A24~ A27~ A28~ and
o A31 is Cha.
In a still further subset, at least one of A5, A7,
A8, Al1, A15, A18~ A21~ A23, A24, A27, A28, and A31 is Cha,
or at least one of A3, A12, A16, A17, A18~ A1s~ and A34 is
Aib. For example, A3 is Ser or Aib; A5 is Ile; A7 is Leu
15 or Cha; A8 is Met, Nva, Leu, Val, Ile, or Nle; All is Leu
or Cha; A15 is Leu or Cha; A16 is Asn or Aib; A18 is Met,
Aib, or Nle; A21 is Val; A27 is Lys, Aib, Leu, hArg, or
Cha; A31 is Val, Nle, or Cha; A32 is His; A33 is Asn; A34
is Phe, Tye, Amp, or Aib; R1 is H; R2 is H; and R3 is NH2.
20 If desired, at least one of A7 and All is Cha and at least
one of A16, Alg, and A34 is Aib; or at least one of A24,
A28, and A31 is Cha and at least one of A16 and A17 is Aib.
In yet another subset, at least one of A1 is Dap,
A7 is ~-Nal, Trp, Pal, Phe or p-X-Phe, A13 is ~-Nal, Trp,
25 Pal, Phe, or p-X-Phe. For example, Al is Ser, Gly, or
Dap; A3 is Ser or Aib; A8 is Met, Nva, Leu, Val, Ile, or
Nle; A16 is Asn or Aib; A18 is Met, Aib, or Nle; A21 is
Val; A27 is Lys, Aib, Leu, hArg, or Cha; A31 is Val, Nle,
or Cha; A32 is His; A33 is Asn; A34 is Phe, Tyr, Amp, or
30 Aib; Rl is H; R2 is H; and R3 is NH2
The following are examples of the peptide of this
invention as covered by the above formula: [Cha7]hPTH(1-
34)NH2; [Chall]hPTH(1-34)NH2; [Chal5]hPTH(1-34)NH2; [cha7
( 34)NH2i [Cha7~ 11, Nle8, 18 Tyr34]h~5 [Cha23]hPTH(1-34)NH2; [Cha24]hPTH(1-34)NH2; [Nle3~ 18,
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Cha27]hPTH(1--34)NH2; [Cha28]hPTH(1--34)NH2; [Cha31]hPTH(l--
34)NH2; [Cha27]hPTH(1--34)NH2; [Cha27~ 29]hPTH(1--34)NH2;
[Cha28]bPTH(1-34)NH2; [Cha28]rPTH(1-34)NH2; [Cha24~ 28~
31]hPTH(1--34)NH2; [Aibl6]hPTH(1--34)NH2; [Aibl9]hPTH(l--
34)NH2; [Aib34]hPTH(1--34)NH2; [Aib16~ 19]hPTH(1--34)NH2;
[Aibl6' 19, 34]bPTH(1--34)NH2; [Aibl6' 34]hPTH(1--34)NH2;
[Aibl9~ 34]hPTH(l-34)NH2; [Cha7~ 11, Nle8~ 13 Aib16, 19
Tyr34]hpTH(l-34)NH2; tCha7~ 11, Nle8, 18, 31 Aib16, 19
Tyr34]hPTH(1-34)NH2; [Cha7, Aibl6]hPTH(1-34)NH2; [Chal1,
o Aibl6]hPTH(1-34)2; [Cha7, Aib34]hPTH(1-34)NH2; [Chall,
Aib34]hPTH(1-34)NH2; [Cha27, Aibl6]hPTH(1-34)NH2; [Cha27,
Aib34]hPTH(1-34)NH2; [Cha23, Aibl6]hPTH(1-34)NH2; [Cha28,
Aib34]hPTH(1-34)NH2; [Nle31]hPTH(1-34)NH2; [hArg27]hPTH(l-
34)NH ; [Dap1 Nle8~ 18, Tyr34]hPTH(1~34)NH2;
[Nle31]bPTH(1-34)NH2; [Nle3l]rPTH(1-34)NH2; [hArg27]bPTH(l-
34)NH2; [hArg27]rPTH(1-34)NH2; [Cha7~ 11, Aib19,
Lys30]hPTH(1-34)NH2; [Aibl2]hPTH(1-34)NH2; [Cha24~ 28~ 31,
Lys30]hPTH(1-34)NH2; [Cha28~ 31]hPTH(1-34)NH2; [Cha7~ 11,
Nle8~ 18, Aib34]hPTH(1-34)NH2; [Aib3]hPTH(1-34)NH2;
[Cha8]hPTH(1-34)NH2; [Chal5]hPTH(1-34)NH2; [Cha7~ 11,
Aibl9]hPTH(1-34)NH2; [Cha7~ 1l, Aibl6 ]hPTH(1-34)NH2;
[Aib17]hPTH(1-34)NH2; [Cha5]hPTH(1-34)NH2;[Cha7
15]hPTH(1-34)NH2; [Cha7~ 11, Nle8~ 18, Aibl9,
Tyr ]hPTH(1-34)NH2; [Cha7~ l1, Nle8~ 13 Aibl9 L 30
25 Tyr34]hPTH(1-34)NH2; [Cha7~ 15]hPTH(1-34)NH2;
[Aibl7]hPTH(1-34)NH2; [Cha7~ 11, Leu27]hPTH(1-34) NH2;
[Cha7~ 1l, 15, Leu27]hPTH(1-34)NH2; [Cha7~ ll, 27] hPTH(1-
Ch 7, 11, 15, 27]hPTH (1-34)NH2; [Trp ]
34)NH2; [Nall5]hPTH(1-34) NH2; [Trpl5, Cha23]hPTH(1-34)NH2;
[Chal5~ 23]hPTH(1-34)NH2; [Phe7~ 11]hPTH(1-34)NH2; [Nal7
]hPTH(1-34)NH2; [Trp7~ ll]hPTH (1-34)NH2; [Phe7
5]hPTH(1-34)NH2; [Nal7~ l5]hPTH(1-34)NH2; [Trp7
]hPTH(1-34)NH2; and [Tyr7~ l5]hpTH(l-34)NH
In another aspect, this invention relates to
35 peptides covered by the following formula:
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Al - val - A3 - Glu - A5 - Gln - A7 - A8 - His - Alo - All - Al2 - Lys A14 A15
5 R2
A16--A17-A18-Alg~Arg~Arg~A22~A23~A24~A25~A26~A27~A28
A29--A30--A31--A32--A33--A34--R3
wherein
A1 is Ala, Ser, or Dap;
A3 is Ser or Aib;
A5 is His, Ile, or Cha;
A7 is Leu, Cha, Nle, ~-Nal, Trp, Pal,
Phe, or p-X-Phe in which X is OH, a halogen, or CH3;
A8 is Leu, Met, or Cha;
A1o is Asp or Asn;
Al1 is Lys, Leu, Cha, Phe, or ~-Nal;
A12 is Gly or Aib;
A14 is Ser or His;
A15 is Ile, or Cha;
A16 is Gln or Aib; .
A17 is Asp or Aib;
A18 is Leu, Aib, or Cha;
Alg is Arg or Aib;
A22 is Phe, Glu, Aib, or Cha;
A23 is Phe, Leu, Lys, or Cha;
A24 is Leu, Lys, or Cha;
A25 is His, Aib, or Glu;
A26 is His, Aib, or Lys;
A27 is Leu, Lys, or Cha;
A28 is Ile, Leu, Lys, or Cha;
A29 is Ala, Glu, or Aib;
A30 is Glu, Cha, Aib, or Lys;
A31 is Ile, Leu, Cha, Lys, or deleted;
A32 is His or deleted;
A33 is Thr or deletedj
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A34 is Ala or deleted;
each of R1 and R2 is, independently, H,
Cl_l2 alkanyl, C7_20 phenylalkyl, C11_20 napthyalkyl, Cl_l2,
hydroxyalkyl, C2_12 hydroxyalkenyl, C7_20
hydroxyphenylalkyl, or Cll_20 hydroxynapthylalkyl; or one
and only one of Rl and R2 is COEl in which El is Cl_l2
alkyl, C2 12 alkyl, C2_12 alkenyl~ C7_20 phenYlalkYl' 11-20
napthylalkyl, Cl_l2 hydroxyalkyl, C2_12 hydroxyalkenyl, C7_
20 hydroxyphenylalkyl, or Cll_20 hydroxynapthylalkyl; and
lo R3 is OH, NH2, Cl_l2 alkoxy, or NH-Y-CH2-Z in which
Y is a C1_12 hydrocarbon moiety and Z is H, OH, C02H or
CONH2;
provided that (i) at least one of A5, A7, A8, All,
Als~ A18~ A22~ A23~ A24, A27, A28, A30, or A31 is Cha, or at
15 least one of A3, A12, A16, A17, A18, Als~ A22~ A2s~ A26~
A29, A30, or A34 is Aib; or that (ii) at least one of A23,
A24, A27, A28, or A31 is Lys; or a pharmaceutically
acceptable salt thereof. In one embodiment, at least one
of A7 and All is Cha. In another embodiment, at least one
20 of A16 or Alg is Aib. Specific examples of peptides of
the just-recited formula include, but are not limited to,
[Cha7]hPTHrP(1-34)NH2; [Cha11]hPTHrP(1 -34)NH2; [Cha7
]hPTHrP(1-34)NH2; [Aib16, Tyr34hPTHrP(1-34)NH2;
[Aibl9]hPTHrP(1-34)NH2; [Aibl6~ 19]hPTHrP(1-34)NH2; [Cha7~
25 11, Aibl6hPTHrP(1-34)NH2; [Cha7~ 1l, Aibl9]hPTHrP(1-34)NH2;
[cha22 LeU23, 28, 31, Glu25, 29, Lys26~ 3o]hpTHrp(l-34)NH
[Glu ~ , 29, LeU23, 28, 31, LyS26, 27, 30]hpTH
[Cha 2~ 23, GlU25~ 29, Leu28, 31 LyS26~ 30]hPTHrP(1 34)NH
[GlU22, 25 LeU23, 28, 31, Aib29, Ly526~ 3o]hpTHrp(l-34)NH
[GlU22, 25, 29 Lys23, 26, 30, Leu28~ 31] hPTHrP(1-34)NH2;
[GlU22, 25, 29 Leu23, 28, 31, LyS26, Cha30]hPTHrP(l-34)NH2;
22, 25, 29 Leu23, 28, 31, LyS26, Aib3o]hpTHrp(l-34)NH2;
[GlU22, 25, 29 Leu23, 31, LyS26, 28~ 30]hPTHrP(1-34) NH2;
[cha22, 23, 24, 27, 28, 31, Glu25, 29, Lys26~ 30]hPTHrP(1-
35 34)NH2; [Glu22~ 25~ 29, Cha23, 24, 28, 31 LyS26, 27,
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30]hPTHrP(1-34)NH2; tGlU22~ 25, 29, Cha23, 24, 27, 31 LyS26,
28~ 30]hpTHrp(l-34)NH2; [Glu22~ 25, 29 Lys23, 26, 30 Cha24,
27~ 28~ 31]hPTHrP(1-34)NH2; tCha22, Leu23~ 28~ 31, Glu25~ 29,
Lys26~ 27~ 30] hPTHrP(1-34)NH2; [Cha22, Leu23- 31, Glu25~ 29,
5 Lys26~ 28~ 30]hPTHrP(1-34)NH2; [Cha22, Lys23~ 26~ 30, Glu25
29, Leu28~ 31]hPTHrP(1-34)NH2; [Cha22 Leu23~ 28~ 31 Glu25
Lys26~ 30, Aib29] hPTHrP(1-34)NH2; [Cha22, Leu23~ 28~ 31,
Glu25~ 29 Lys26 Aib30]hPTHrP(1-34)NH2; [Glu22~ 25, Leu23
28~ 31, Lys26~ 27~ 30, Aib29]hPTHrP(1-34)NH2; [Glu22~ 25,
23 26, 30 Leu 28, 31 Aib29] hPTHrP(1-34)NH2; [Glu
Leu23, 31 Lys 26, 28, 30 Aib29]hpTHrp(l-34)NH2; [Cha7' 1
GlU22, 25, 29 Leu23, 28, 31, LyS26~ 30] hPTHrP(1-34)NH2;
[Cha ' , 22, LeU23, 28, 31, Glu25' 29 Lys26' 30]h
34)NH2; [Cha7~ 11, Glu22, 25, 29, Leu23~ 28, 31 LyS26, 27, 30]
15 hPTHrP(1-34)NH2; [Cha7~ 11, 22, 23 GlU25, 29 LeU28, 31
Lys26~ 3~]hPTHrP(1-34)NH2; [Cha7~ 11, GlU22, 25, 29 LyS23,
26~ 30, Leu28~ 31]hPTHrP(1-34)NH2; [Cha7~ 11, Glu22~ 25~ 29,
LeU23, 31 LyS26, 28, 30] hPTHrP(1-34)NH2; [Cha7~ 11, Glu22'
25 Leu23' 28, 31 Aib29, Lys26~ 3~]hPTHrP(1-34)NH2; [Cha7'
, Glu , 25, 29, Leu23~ 28, 31 Lys26 Aib30]hPTH P(
34)NH2; [Chal5, G1U22~ 25, 29, Leu23~ 28, 31 LyS26,
3~]hPTHrP(1-34) NH2; [Chal5~ 22, Leu23, 28, 31 GlU25, 29
Lys26~ 3~]hPTHrP(1-34)NH2; [chal5, Glu22, 25, 29 LeU23, 28,
31, Lys26~ 27~ 30]hPTHrP(1-34)NH2; [Chal5~ 22~ 23, Glu25~ 29,
25 LeU28~ 31 Lys26~ 3~]hPTHrP(1-34) NH2; [Chal5~ Glu22~ 25
LeU23, 28, 31 Aib29, Ly526~ 3~]hpTHrP(l-34)NH2; [Chal5,
Glu22, 25, 29 Lys23~ 26, 30, Leu25' 31]hPTHrP(1-34) NH2;
Ch 15 Glu22~ 25~ 29 Leu23~ 28, 31, LyS26, Aib30] hPTHrp
34)NH2; [Chal5, Glu22, 28, 29, Leu23, 31 LyS26, 28,
30 3~]hPTHrP(1-34)NH2; [chal5~ 30, GlU22, 25, 29 Leu23, 28, 31
Lys26]hPTHrP(1-34)NH2; [Cha7~ 8~ 22, Leu23~ 25, 31 Glu25~ 29
Lys26~ 30]hPTHrP(1-34)NH2; [Cha7~ 8, Glu22~ 25~ 29 Leu23~ 25
31, Lys26~ 27~ 30]hPTHrP(1-34)NH2; [Cha7~ 8~ 22~ 23, Glu25~
29 LeU28, 31 LyS26~30] hPTHrP (1-34)NH2; [cha7~ 8~ Glu22'
35 25, 29 LeU23, 28, 31 Lys26,30] hpTHrp(l-34)NH2; [cha7~ 8,
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GlU22, 25 LeU23, 28, 31, Aib29 , LyS26, 30]hPTHrP(1-34) NH2;
tCha7~ 8 GlU22, 25, 29 LyS23, 26~ 30, LeU2s, 31]hPTHrP(1-
34)NH2; [Cha7~ 8, Glu22~ 25~ 29, LeU23, 28, 31 LyS26 Aib30]
hPTHrP(1-34)NH2; [Cha7~ 8, Glu22~ 25, 29, Leu23~ 31 LyS26,
28~ 3o]hpTHrp(l-34)NH2; [Cha7~ 8, 30 GlU22, 25, 29 LeU23, 28,
31, Lys26]hPTHrP(1-34)NH2; [Serl, Ile5, Cha7~ 11, 22, Met8,
Asn , Hisl4, Leu23, 28, 31, Glu25~ 29 LyS26, 30]hpT
NH2; [Ser , Ile5, Cha7~ 11, Met8 Asn10 Hi514 Gl 22 25
29 LeU23, 28, 31 Ly526~ 27~ 3~]hpTHrp(1-34)NH2; [Ser1,
lo Ile5, Cha7~ 11, Met8, Asn10, Hi514, GlU22, 25, 29 LeU23, 31
Lys26~ 28~ 3~]hPTHrP(1-34)NH2; Serl, Ile5, Cha7~ 11, Met8,
Asn , His14, G1U22~ 25, 29 LYS23, 26, 30 L 28
( 34)NH2; [Ser1, Ile5, Cha7~ 11 Metg A 1o
Hisl4 GlU22, 25 LeU23, 28, 31, Aib29, Lys26~ 3~]hPTHrP(1-34)
15 NH2; [Ser , Ile5, Cha7~ 11, Met8 Asn1o His14 Gl 22 25
29 LeU23, 28, 31 Ly526~ Aib30] PTHrP(1-34)NH2; [Ser1,
Iles Cha7~ 11, 22, 23 Met8, A5nlo~ Hisl4, Glu25~ 29, Leu23
31, Lys26~ 30]hPTHrP(1-34) NH2; [Serl, Ile5, Cha7~ 11, 15,
Met3, Asn10, Hisl4]hPTHrP(1-34)NH2; [Serl, Ile5, Met3,
20 Asn10, Leull, Hisl4, Aibl6]hPTHrP (1-i4)NH2; [Serl~ Ile5,
M t8 A5n1~ LeU11, 28, 31, His14, Cha22~ 23, Glu ~ ,
LyS26, 30]hPTHrP (1-34)NH2; [Ser1, Ile5, Cha7~ 11 Met8
Asnl~, Hisl4, GlU22~ 25~ 29, Leu23, 28, 31 LyS26, 30]hpTH p
(1-34)NH2; [Serl, Ile5, Met8, Asnl~ Hisl4 Chal5 Gl 22
25 25, 29 LeU23, 28, 31, LyS26, 3o]hpTHrp (1-34)NH2; [Ser
Ile5, Cha7~ 8, Asn10, Hi5l4, Glu22, 25, 29 LeU23, 28, 31
Lys26~ 30]hPTHrP (l-34)NH2;[Glu22~ 25, 29 LeU23, 28, 31
Lys24~ 26~ 30]hPTHrP(1-34)NH2; [Aib22, Leu23~ 28~ 31, Glu25
29, Lys26~ 30]hPTHrP(1-34)NH2; [Glu22~ 29, Leu23~ 28~ 31,
30 Aib25, Lys26~ 30]hPTHrP(1-34)NH2; [Glu22~ 25~ 29, LeU23~ 28~
31, Aib26, Lys30]hPTHrP (1-34)NH2; [Glu22, 25, 29 LeU23, 28
- Lys 26~ 30, 31] hPTHrP(1-34)NH2; [Ser1, Ile5, Met8, Asn10,
Leul1, 23, 28, 31, Hisl4, Cha22, Glu25' 29, Lys26~ 30]
~ hPTHrP(1-34)NH2; [Ser1, Ile5, Met8, Asn10, Leul1~ 28~ 31,
35 HiS14 GlU22, 25, 29, Lys23, 26, 30]pTHrp(l-34)NH2; [Ser1~
CA 02226l77 l997-l2-30
W 097r02834 PCT~US96/11292
-- 10 --
Il 5 Met3 Asn10 Leull~ 23~ 28~ 31, Hisl4, Glu2 ~ ~ ,
Lys26~ 27~ 30] hPTHrP(1-34)NH2; [Serl, Ile5, Met5, Asnl0,
Leu1l~ 23~ 31, Hi514, Glu22, 25, 29 LyS26, 28, 30] hPTH P(1
34)NH2; tSer1, Ile5, Met5, A5nlo~ Leull, 23, 28, 31 HiS14
5 GlU22, 25 Aib29, LyS26~ 30] hpTHrp(l-34)NH2; [Serl, Ile5,
Met8 Asnl~ LeUll, 23, 28, 31, His14, Glu22~ 25~ 29, Lys
Aib30] hPTHrP(1-34)NH2; or [Ser1, Ile5, Met3]hPTHrP(1-
34)NH2.
With the exception of the N-terminal amino acid,
lo all abbreviations (e.g. Ala or A1) of amino acids in this
disclosure stand for the structure of -NH-CH(R)-C0-,
wherein R is a side chain of an amino acid (e.g., CH3 for
Ala). For the N-terminal amino acid, the abbreviation
stands for the structure of =N-CH(R)-C0-, wherein R is a
15 side chain of an amino acid. ~-Nal, Nle, Dap, Cha, Nva,
Amp, Pal, and Aib are the abbreviations of the following
~-amino acids: ~-(2-naphthyl)alanine, norleucine, ~,~-
diaminopropionic acid, cyclohexylalanine, norvaline, 4-
amino-phenylalanine, 3-pyridinylalanine, and ~-
20 aminoisobutyric acid, respectively. In the aboveformula, hydroxyalkyl, hydroxyphenyl-alkyl, and
hydroxynaphthylalkyl may contain 1-4 hydroxy
substituents. Also, COE1 stands for -C=O E1. Examples of
-C=O E1 include, but are not limited to, acetyl and
25 phenylpropionyl.
A peptide of this invention is also denoted herein
by another format, e.g., [Cha7~ ll]hPTH(1-34)NH2, with the
substituted amino acids from the natural sequence placed
between the second set of brackets (e.g., Cha7 for Leu7,
30 and Chal1 for Leu11 in hPTH). The abbreviation hPTH
stands for human PTH, hPTHrP for human PTHrP, rPTH for
rat PTH, and bPTH for bovine PTH. The numbers between
the parentheses refer to the number of amino acids
present in the peptide (e.g., hPTH(1-34) is amino acids 1
35 through 34 of the peptide sequence for human PTH). The
CA 02226l77 l997-l2-30
W O 97/02834 PCT~US96/11292
sequences for hPTH(1-34), hPTHrP(1-34), bPTH(1-34), and
rPTH(1-34) are listed in Nissenson, et al., Receptor,
3:193 (1993). The designation "NH2" in PTH(1-34)NH2
indicates that the C-terminus of the peptide is amidated.
~ 5 PTH(1-34), on the other hand, has a free acid C-terminus.
Each of the peptides of the invention is capable
of stimulating the growth of bone in a subject (i.e., a
mammal such as a human patient). Thus, it is useful in
the treatment of osteoporosis and bone fractures when
lo administered alone or concurrently with antiresorptive
therapy, e.g., bisphosphonates and calcitonin.
The peptides of this invention can be provided in
the form of pharmaceutically acceptable salts. Examples
of such salts include, but are not limited to, those
formed with organic acids (e.g., acetic, lactic, maleic,
citric, malic, ascorbic, succinic, benzoic,
methanesulfonic, toluenesulfonic, or pamoic acid),
inorganic acids (e.g., hydrochloric acid, sulfuric acid,
or phosphoric acid), and polymeric acids (e.g., tannic
20 acid, carboxymethyl cellulose, polylactic, polyglycolic,
or copolymers of polylactic-glycolic acids).
A therapeutically effective amount of a peptide of
this invention and a pharmaceutically acceptable carrier
substance (e.g., magnesium carbonate, lactose, or a
25 phospholipid with which the therapeutic compound can form
a micelle) together form a therapeutic composition (e.g.,
a pill, tablet, capsule, or liquid) for administration
(e.g., orally, intravenously, transdermally, pulmonarily,
vaginally, subcutaneously, nasally, iontophoretically, or
30 by intratracheally) to a subject. The pill, tablet, or
capsule that is to be administered orally can be coated
~ with a substance for protecting the active composition
from the gastric acid or intestinal enzymes in the
~ stomach for a period of time sufficient to allow it to
pass undigested into the small intestine. The
CA 02226177 1997-12-30
W 097/02834 PCT~US96/11292
therapeutic composition can also be in the form of a
biodegradable or nonbiodegradable sustained release
formulation for subcutaneous or intramuscular
a~r;n;~tration. See, e.g., U.S. Patents 3,773,919 and
5 4,767,628 and PCT Application No. WO 94/15587.
Continuous a~m; n; ~tration can also be achieved using an
implantable or external pump (e.g., INFUSAID~ pump). The
administration can also be conducted intermittently,
e.g., single daily injection, or continuously at a low
lo dose, e.g., sustained release formulation.
The dose of a peptide of the present invention for
treating the above-mentioned diseases or disorders varies
depending upon the manner of administration, the age and
the body weight of the subject, and the condition of the
subject to be treated, and ultimately will be decided by
the attending physician or veterinarian.
Also contemplated within the scope of this
invention is a peptide covered by the above generic
formula for use in treating diseases or disorders
20 associated with deficiency in bone growth or the like,
e.g., osteoporosis or fractures.
Other features and advantages of the present
invention will be apparent from the detailed description
and from the claims.
Detailed Description of the Invention
Based on the description herein, the present
invention can be utilized to its fullest extent. The
following specific examples are to be construed as merely
illustrative, and not limitative of the remainder of the
30 disclosure in any way whatsoever. Further, all
publications cited herein are incorporated by reference.
Structure
PTH(1-34) has been reported to have two
amphophilic alpha helical dom~;n~ See, e.g., Barden, et
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W O 97/02834 PCTrUS96/11292
al., Biochem., 32:7126 (1992). The first ~-helix is
formed between amino acid residues 4 through 13, while
the second ~-helix is formed between amino acid residues
21 through 29. Some peptides of this invention contain
- 5 the substitution of Cha for one or more residues within
or near these two regions of PTH(1-34), e.g., Cha7 and
Cha11 within the first ~-helix or Cha27 and Cha23 within
the second ~-helix.
Also covered by this invention are variants of
lo PTH(1-34) with the substitution of Aib for a residue
adjacent to the ~-helixes, e.g., Aib16, Aib19, and Aib34;
hArg27 and Nle31, or the substitution of Dpa for the N-
terminal residue.
Svnthesis
The peptides of the invention can be prepared by
standard solid phase synthesis. See, e.g., Stewart,
J.M., et al., Solid Phase Synthesis (Pierce Chemical Co.,
2d ed. 1984). The following is a description of how
[Aib34]hPTH(1-34)NH2 was prepared. Other peptides of the
20 invention can be prepared in an analogous manner by a
person of ordinary skill in the art.
The peptide was synthesized on an Applied
Biosystems (Foster City, CA) model 430A peptide
synthesizer which was modified to do accelerated Boc-
25 chemistry solid phase peptide synthesis. See Schnoize,et al., Int. J. Peptide Protein Res., 90:180 (1992). 4-
Methylbenz-hydrylamine (MBHA) resin (Peninsula, Belmont,
CA) with the substitution of 0.93 mmol/g was used. The
Boc amino acids (Bachem, CA, Torrance, CA; Nova Biochem.,
30 LaJolla, CA) were used with the following side chain
protection: Boc-Arg(Tos)-OH, Boc-Asp(OcHxl)-OH, Boc-
Asn(Xan)-OH, Boc-Glu(OcHxl)-OH, Boc-His(DNP)-OH, Boc-Asn-
GH, Boc-Val-OH, Boc-Leu-OH, Boc-Ser-OH, Boc-Gly-OH, Boc-
Met-OH, Boc-Gln-OH, Boc-Ile-OH, Boc-Lys(ZClZ)-OH, Boc-
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Ser(Bzl)-OH, and Boc-Trp(Fm)-OH. The synthesis was
carried out on a 0.14 mmol scale. The Boc groups were
removed by treatment with 100% TFA for 2 x 1 min. Boc
amino acids (2.5 mmol) were pre-activated with HBTU (2.0
mmol) and DI~A (l.o mL) in 4 mL of DMF and were coupled
without prior neutralization o~ the peptide--resin TFA
salt. Coupling times were 5 min except for the Boc-Aib-
OH and the following residue, Boc-Asn(Xan)-OH, wherein
the coupling times were 20 min.
At the end of the assembly of the peptide chain,
the resin was treated with a solution of 20%
mercaptoethanol/10% DIEA in DMF for 2 x 30 min. to remove
the DNP group on the His side chain. The N-terminal Boc
group was then removed by treatment with 100% TFA for 2 x
15 Z min. After neutralization of the peptide-resin with
10% DIEA in DMF (1 x 1 min.), the formyl group on the
side chain of Trp was removed by treatment with a
solution of 15% ethanolamine/15% water/70% DMF for 2 x 30
min. The partially-deprotected peptide-resin was washed
20 with DMF and DCM and dried under reduced pressure. The
final cleavage was done by stirring the peptide-resin in
10 mL of HF containing 1 mL of anisole at 0~C for 75 min.
HF was removed by a flow of nitrogen. The residue was
washed with ether (6 x 10 mL) and extracted with 4N HOAc
(6 x 10 mL).
The peptide mixture in the aqueous extract was
purified on a reversed-phase preparative high pressure
liquid chromatography (HPLC) using a reversed phase
Vydac~ C18 column (Nest Group, Southborough, MA). The
30 column was eluted with a linear gradient (10% to 45% of
solution B over 130 min.) at a flow rate of 10 mL/min
(Solution A = 0.1% agueous TFA; Solution B = acetonitile
contA;n;ng 0.1% of TFA). Fractions were collected and
checked on analytical HPLC. Those contA; n; ng pure
35 product were combined and lyophilized to dryness. 62.3
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W O 97/02834 PCTrUS96/11292
- 15 -
mg of a white solid was obtained. Purity was >99~ based
on analytical HPLC analysis. Electro-spray mass
spectrometer analysis gave the molecular weight at 4054.7
(in agreement with the calculated molecular weight of
5 4054-7)-
- The synthesis and purification of [Cha7~11]hPTH
(1-34)NH2 was carried out in the same manner as the above
synthesis of [Aib34]hPTH(1-34)NH2. The protected amino
acid Boc-Cha-OH was purchased from Bachem, CA. The
o purity of the final product was >98%, and the electron-
spray mass spectrometer gave the molecular weight at
4197.0 (calculated molecular weight is 4196.9).
The full names for the abbreviations used above
are as follows: Boc for t-butyloxycarbonyl, HF for
hydrogen fluoride, Fm for formyl, Xan for xanthyl, Bzl
for benzyl, Tos for tosyl, DNP for 2,4-dinitrophenyl, DMF
for dimethylformamide, DCM for dichloromethane, HBTU for
2-(lH-Benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate, DIEA for diisopropylethylamine, HOAc
for acetic acid, TFA for trifluoroacetic acid, 2ClZ for
2-chlorobenzyloxycarbonyl and OcHxl for O-cyclohexyl.
The substituents R1 and R2 ~f the above generic
formula may be attached to the free amine of the N-
terminal amino acid by standard methods known in the art.
For example, alkyl groups, e.g., C1_12 alkyl, may be
attached using reductive alkylation. Hydroxyalkyl
groups, e.g.,
C1_12 hydroxyalkyl, may also be attached using reductive
alkylation wherein the free hydroxy group is protected
30 with a t-butyl ester. Acyl groups, e.g., COEl, may be
attached by coupling the free acid, e.g., E1COOH, to the
free amine of the N-terminal amino acid by m i~;ng the
- completed resin with 3 molar equivalents of both the free
acid and diisopropylcarbodiimide in methylene chloride
r 35 for one hour and cycling the resulting resin through
steps (a) to (f) in the above wash program. If the free
acid contains a free hydroxy group, e.g., p-
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W O 97/02834 PCT/US96/11292
- 16 -
hydroxyphenylpropionic acid, then the coupling should be
performed with an additional 3 molar equivalents of HOBT.
Other peptides of this invention can be prepared
in an analogous manner by a person of ordinary skill in
5 the art.
Functional Assays
A. Binding to PTH Receptor
The peptides of the invention were tested for
their ability to bind to the PTH receptor present on
lo SaOS-2 (human osteosarcoma cells). SaOS-2 cells
(American Type Culture Collection, Rockville, MD; ATCC
#HTB 85) were maintained in RPMI 1640 medium (Sigma, St.
Louis, MO) supplemented with 10~ fetal bovine serum (FBS)
and 2 mM glutamine at 37~C in a humidified atmosphere of
15 5% CO2 in air. The medium was changed every three or
four days, and the cells were subcultured every week by
trypsinization.
SaOS-2 cells were maintained for four days until
they had reached confluence. The medium was replaced
20 with 5~ FBS in RPMI 1640 medium and incubated for 2 hrs
at room temperature with 10 x 104 cpm mono-l25I-[Nle8~l8,
Tyr34(3-125I)] bPTH(1-34)NH2 in the presence of a
competing peptides of the invention at various
concentrations between 10-1lM to 10-4 M. The cells were
25 washed four times with ice-cold PBS and lysed with 0.1 M
NaOH, and the radioactivity associated with the cells was
counted in a scintillation counter. Synthesis of mono-
3 18 T 34(3-l25I)] bPTH(1-34)NH2 was c
as described in Goldman, M.E., et al., Endocrinol.,
30 123:1468 (1988).
The binding assay was conducted with various
peptides of the invention, and the IC50 value, (half
maximal inhibition of binding of mono-l25I-[Nle
Tyr34(3-l25I)]bPTH(1-34)NH2, for each peptide was
35 calculated.
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- 17 -
As shown in Table I, all of the tested peptides
had a high binding affinity for the PTH receptor on the
SaOS-2 cell.
B. Stimulation of Adenylate Cyclase Activity
s The ability of the peptides of the invention to
- induce a biological response in SaOS-2 cells were
measured. More specifically, any stimulation of the
adenylate cyclase was determined by measuring the level
of synthesis of cAMP (adenosine 3',5'-monophosphate) as
10 described previously in Rodan, et al., J. Clin. Invest.
72: 1511 (1983) and Goldman, et al., Endocrinol.,
123:1468 (1988). Confluent SAOS-2 cells in 24 wells
plates were incubated with 0.5 ~Ci [3H]adenine (26.9
Ci/mmol, New England Nuclear, Boston, MA) in fresh medium
15 at 37~C for 2 hrs, and washed twice with Hank's balanced
salt solution (Gibco, Gaithersburg, MD). The cells were
treated with 1 mM IBMX [isobutylmethyl-xanthine, Sigma,
St. Louis, MO] in fresh medium for 15 min, and the
peptides of the invention were added to the medium to
20 incubate for 5 min. The reaction was stopped by the
addition of 1.2 M trichloroacetic acid (TCA) (Sigma, St.
Louis, MO) followed by sample neutralization with 4 N
KOH. cAMP was isolated by the two-column chromatographic
method (Salmon, et al., 1974, Anal. Biochem. 58, 541).
25 The radioactivity was counted in a scintillation counter
(Liquid Scintillation Counter 2200CA, PACKARD, Downers
Grove, IL).
The respective EC50 values (half maximal
stimulation of adenylate cyclase) for the tested peptides
30 were calculated and shown in Table I. All tested
peptides were found to be potent stimulators of adenylate
cyclase activity, which is a biochemical pathway
~ indicative as a proximal signal for osteoblast
proliferation (e.g., bone growth).
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- 18 -
TABLE I
PEPTIDE Kd (~LM) ECSo (nM)
Cha7~ 11]hPTH(1-34)NH2 0.01 0.6
lCha231hPTH(1-34)NH2 0.2 20
ICha24]hPTH(1-34)NH2 Ø1 10
Nlc8~ 18, Cha27]hPTH(1-34)NH2; 0.05 2
lCha28]hPTH(1-34)NH2 0 05 25
¦Cha31]hPTH(1-34)NH2 Q 03 4
IAibl61hPTH(1-34)NH2; O.OW 0.7
10 lAibl9]hPTH(1-34)NH2; O.OOS 0.6
[Aib34]hPTH(1-34)NH2; 0.007 3
[Nle31]hPTH(1-34)NH2; O.OW 0.7
[hArg27]hPTH(1-34)NH2 0 007
[Dap, Nle8~ 18. Tyr34]hPTH(1-34)NH2 0.1S0 10
15 [cha24, 28, 31, Lys30]hPTH(1-34)NH2; 05 7
[Cha ~ , Nle ~ , Tyr ]hPTH(1-34)NH2 0.006 0.6
[Cha7~ 11 Nle8~ 18 Ajbl6~ 19, Tyr34]hPTH (1-34)NH2 ~.~~S 15
[Cha7~ 11 Nle8, 18, 31 Ajbl6, 19, Tyr34]hPTH(1-34)NH2 O.W 4
lChall]hPTH(1-34)NH2 0.005 2
20 [Cha28~ 31]hPTH(1-34)NH2 0.06 7
[Cha7~ 11,Nle8~ 18, Aib34]hPTH(1-34)NH2 0.03 15
[ChalS]hPT11(1-34)NH2 Q oo5 1.3
[Cha7~11, Aibl9]hPTH(1-34)NH2 0.007 05
[Cha7~11, Aibl6]hPTH(1-34)NH2 O.OW 1.1
2 5 [Aib1fi~ 19]hPTH(1-34)NH2 0.004 0.6
[Aib12]hPTH(1-34)NH2 O.OOS 2
[Aib3]hPTH(1-34)NH2 O.OW 1.1
[Cha7~11, Aibl9, Lys30]hPTH(1-34)NH2 0.004 2
[Cha7]hPTH(1-34)NH2 0.02 2.3
30 [Cha24~28~ 31]hPTH(1-34)NH2 1.0 30
[Aibl7]hPTH(1-34) 0.05 3
[Cha7~ 15]hPTH(1-34) 0.01 lA
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W O 97/02834 PCTrUS96111292
-- 19 --
Other Embodiments
It is to be understood that while the invention
has been described in conjunction with the detailed
description thereof, that the foregoing description is
intended to illustrate and not limit the scope of the
invention, which is defined by the scope of the appended
claims. Other aspects, advantages, and modifications are
within the claims.
