Note: Descriptions are shown in the official language in which they were submitted.
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Regulation Of Cell Proliferation And Differentiation
Using Topically Applied Peptides
Background of the Invention
Field of tlae Invehtioh
This invention relates to the regulation of cell differentiation and
proliferation, e.g., for treating hyperproliferative skin disorder, such as
psoriasis, for enhancing wound healing, for stimulating hair growth, and
inhibiting hair growth by topical administration of parathyroid hormone
(PTH), parathyroid related peptide (PTHrP), or fragment, analog or derivative
thereof, and salts thereof, encapsulated by particular liposomes.
Related Art
U.S. Pat. Nos. 5,527,772, 5,840,690 and 6,066,618 describe methods
of inhibiting proliferation and enhancing differentiation of mammalian cells,
inducing proliferation of mammalian cells, enhancing wound healing, and
stimulating hair growth using a peptide which has a 10% or greater homology
to a region of human PTH or human PTHrP. Certain fragments and analogs
(e.g. PTH (1-34), PTH (3-34) and PTHrP (1-34)) were found to act as agonists
of PTH and PTHrP and inhibit proliferation and enhance differentiation of
mammalian cells. Qther fragments and analogs (e.g. PTH (7-34) and PTHrP
(7-34) are antagonists of PTH and PTHrP and enhance the proliferation of
mammalian cells. The agonists are useful for treatment of hyperproliferative
skin diseases such a psoriasis and the antagonists are useful for wound
healing,
particularly wounds of the skin, enhancing or maintaining hair growth,
particularly following chemotherapeutic treatment of a mammal, and
stimulating epidermal regrowth. Methods of administration include oral,
nasal, intravenous, subcutaneous, parenteral and intraperitoneal
administration.
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The peptides may be administered by subcutaneous pumps, patches, tapes, or
by liposomal carriers.
A variety of PTH and PTHrP analogs and derivatives thereof have
been made. See U.S. Pat. Nos. 4,086,196, 4,423,037, 4,771,124, 4,833,125,
S 4,968,669, 5,001,223, 5,087,562, 5,093,233, 5,116,952, 5,149,779, 5,171,670,
5,229,489, 5,317,010, 5,382,658, 5,393,869, 5,434,246, 5,527,772, 5,589,452,
5,807,823, 5,821,255, 5,840,690, 5,977,070, 6,025,467, 6,051,868, and
6,066,618; W094/02510, WO00/23594, and WO00/31137; and EP 477,885.
Methods for determining whether a particular analog is an agonist or
antagonist of PTH and PTHrP are described in U.S. Pat. Nos. 5,527,772,
5,840,690 and 6,066,618.
Active vitamin D compounds are useful for treating hyperproliferative
skin diseases and other conditions. A large number of such active vitamin D
compounds axe known. See U.S. Patent Nos. 5,457,217, 5,414,098, 5,384,313,
1S 5,373,004, 5,371,249, 5,430,196, 5,260,290, 5,393,749, 5,395,830,
5,250,523,
5,247,104, 5,397,775, 5,194,431, 5,281,731, 5,2S4,S38, 5,232,836, S,18S,1S0,
5,321,018, 5,086,191, 5,036,061, 5,030,772, 5,246,925, 4,973,584, 5,354,744,
4,927,815, 4,857,518, 4,851,401, 4,851,400, 4,847,012, 4,755,329, 4,940,700,
4,619,920, 4,594,192, 4,588,716, 4,564,474, 4,552,698, 4,588,528, 4,719,204,
4,719,205, 4,689,180, 4,505,906, 4,769,181, 4,502,991, 4,481,198, 4,448,726,
4,448,721, 4,428,946, 4,411,833, 4,367,177, 4,336,193, 4,360,472, 4,360,471,
4,307,231, 4,307,025, 4,358,406, 4,305,880, 4,279,826, and 4,248,791.
Summary of the Invention
The invention provides two important therapeutic methods one
involving inhibition of cell proliferation and enhancement of skin cell
differentiation (the agonist activity), which is useful in the treatment of
psoriasis, ichthyosis, actinic keratoses, skin cancer, inhibiting hair growth
or
preventing hair regrowth. A second method involves enhancement of cell
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proliferation (the antagonist activity), which is useful in wound healing,
stimulating epidermal regrowth and hair growth. In addition, the invention
provides methods for enhancing wound healing and hair growth based on in
vivo wound healing activity or in vitro or in vivo hair growth activity rather
than strict agonist or antagonist activity in vitro.
The first method of the invention generally involves inhibiting
proliferation and enhancing differentiation of mammalian skin cells by
contacting the cell with a liposomal preparation comprising a peptide
(preferably at least 3, and more preferably at least 8, amino acids long)
which
has 10% or greater (more preferably, 50% or greater, and most preferably 75%
or greater) sequence identity with a region (preferably within the amino-
terminal 34 amino acid region) of human PTH or human PTHrP, and which is
capable of inhibiting proliferation or enhancing the differentiation in vitro
of
cultured human keratinocytes; or ivc vivo in mouse skin by inhibiting skin
cell
proliferation or hair cycle progression or hair growth. In preferred
embodiments of this method, the peptide is hPTH (1-84), hPTH (1-34), hPTH
(3-34), hPTHrP (I-34), hPTHrP (1-141), hPTHrP (1-139) or hPTHrP (1-173).
This method has particular application in the treatment of hyperproliferative
skin disorders such as psoriasis. The method may also be useful in the
treatment of certain skin cancers, by the inhibition of cancer cell
proliferation
and by the induction of differentiation and inhibition of hair growth.
The second method of the invention generally involves enhancing
proliferation of mammalian skin cells by contacting the skin cells with a
liposomal preparation comprising a peptide (preferably at least 3, and more
preferably at least 8, amino acids long) which has 10% or greater (more
preferably, 50% or greater, and most preferably 75% or greater) sequence
identity with a region (preferably within the amino-terminal 34 amino acid
region) of hPTH or hPTHrP, and which is capable of blocking the
differentiation or the inhibition of proliferation in vitro of cultured human
keratinocytes by PTH (1-34) or 1,25(OH)2D3 or PTHrP (1-34); or in vivo in
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mouse skin by stimulating skin cell proliferation or accelerating hair cycle
progression or stimulating hair growth. In a preferred embodiment of this
method, the peptide is PTH (7-34), hPTH (5-34) or hPTHrP (5-34). In a
related method of the invention, proliferation of mammalian skin cells, e.g.,
during wound healing, is enhanced by contacting the cell or wound with a
liposomal preparation comprising a peptide (preferably at least 3, and more
preferably at least 8, amino acids long) which has 10% or greater (more
preferably, 50% or greater, and most preferably, 75% or greater) sequence
identity with a region (preferably, within the amino-terminal 34 amino acid
region) of hPTH or hPTHrP, and which is capable of enhancing wound
healing in an in vivo skin punch assay. In preferred embodiments of this
method, the peptide is hPTH (1-84), hPTH (1-34), hPTH (7-34), hPTH (5-34),
hPTH (5-36), hPTHrP (1-34), or hPTHrP (7-34). These related methods have
particular application in the enhancement of wound healing and also have
applications in the promotion of skin growth in patients with burns or skin
ulcerations as well as in the stimulation of epidermal regrowth in people who
have decreased epidermal cell proliferation due to aging.
Hair growth is stimulated by administering to a mammal a liposomal
preparation comprising a peptide (preferably at least 3, and more preferably
at
least 8, amino acids long) which has 10% or greater (more preferably, 50% or
greater, and most preferably, 75% or greater) sequence identity with a region
(preferably, within the amino-terminal 34 amino acid region) of hPTH or
hPTHrP, and which is capable of stimulating hair growth in vitro or i~c vivo.
In
preferred embodiments of this method, the peptide is hPTH (7-34), hPTH (5-
34) or hPTH (5-36). This method has applications in the promotion of new
hair growth or stimulation of the rate of hair growth, e.g., following
chemotherapeutic treatment or for treating a form of alopecia, e.g., male or
female pattern baldness.
In particular, the invention relates to a method of inhibiting
proliferation or enhancing differentiation of a mammalian skin or hair cell,
the
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method comprising topically administering to the mammalian skin or hair cell
in need of inhibited proliferation or enhanced differentiation with a
proliferation-inhibiting or differentiation-enhancing amount of a peptide or a
salt or derivative thereof encapsulated within a liposome, wherein the peptide
is at least 3 amino acids long, has at least 10% sequence identity with the 34
amino acid N-terminal region of hPTH or hPTHrP, and is capable of inhibiting
proliferation or enhancing differentiation in vitro of cultured human
keratinocytes; or in vivo in mouse skin by inhibiting skin cell proliferation
or
hair cycle progression or hair growth; wherein the liposome comprises at least
two distinct lipids, a primary lipid and a secondary lipid, the primary lipid
constituting the greatest proportion, by weight, of any single lipid material
forming the bilayers of said vesicle, the primary lipid being selected from
the
group consisting of Clz -Cls fatty alcohols, Clz -Clg glycol monoesters, Clz -
Cl~
gyceryl mono-and diesters, and mixtures thereof, and the primary lipid further
having the property that it will to form a lipid vesicle in the absence of the
secondary lipid, and the secondary lipid being present in an amount sufficient
to allow formation of the lipid vesicles, the secondary lipid being selected
from the group consisting of quaternary dimethyldiacyl amines,
polyoxyethylene acyl alcohols, polyglycerols, sorbitan fatty acid esters,
fatty
acids and their salts, and mixtures thereof.
The invention also relates to a method of inhibiting proliferation or
enhancing differentiation of a skin or hair cell of a mammal, comprising
administering to the mammal in need thereof a proliferation-inhibiting or
differentiation-enhancing amount of a peptide or a salt or derivative thereof
and an active vitamin D compound, wherein the peptide is at least 3 amino
acids long, has at least 10% sequence identity with the 34 amino acid N-
terminal region of hPTH or hPTHrP, and is capable of inhibiting proliferation
or enhancing differentiation in vitro of cultured human keratinocytes, or ih
vivo in mouse skin by inhibiting skin cell proliferation or hair cycle
progression or hair cell growth.
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The invention also relates to a method of inducing proliferation of a
mammalian skin or hair cell, the method comprising topically administering to
the mammalian skin or hair cell in need of proliferation with a proliferation-
inducing amount of a peptide or a salt or derivative thereof encapsulated
within a liposome, wherein the peptide is at least 3 amino acids long, has at
least 10% sequence identity with the 34 amino acid N-terminal region of
hPTH or hPTHrP, and is capable of blocking the inhibition of proliferation or
stimulation of differentiation in vitro of cultured human keratinocytes by PTH
(1-34), 1,25(OH)2D3 or PTHrP (1-34), or ih vivo in mouse skin by stimulating
skin cell proliferation or accelerating hair cycle progression or stimulating
hair
growth; wherein said liposome comprises at least two distinct lipids, a
primary
lipid and a secondary lipid, the primary lipid constituting the greatest
proportion, by weight, of any single lipid material forming the bilayers of
said
vesicle, the primary lipid being selected from the group consisting of C,Z -
C,8
fatty alcohols, C,2 -Cl8 glycol monoesters, Cla -Cl8 gyceryl mono-and
diesters,
and mixtures thereof, and the primary lipid further having the property that
it
will to form a lipid vesicle in the absence of the secondary lipid, and the
secondary lipid being present in an amount sufficient to allow formation of
the
lipid vesicles, the secondary lipid being selected from the group consisting
of
quaternary dimethyldiacyl amines, polyoxyethylene acyl alcohols,
polyglycerols, sorbitan fatty acid esters, fatty acids and their salts, and
mixtures thereof.
The invention also relates to a composition comprising a proliferation-
inhibiting or differentiation-enhancing amount of a peptide ~or a salt or
derivative thereof encapsulated within a liposome, wherein the peptide is at
least 3 amino acids long, has at least 10% sequence identity with the 34 amino
acid N-terminal region of hPTH or hPTHrP, and is capable of inhibiting
proliferation or enhancing differentiation in vitro of cultured human
keratinocytes, or in vivo in mouse skin by inhibiting skin cell proliferation
or
hair cycle progression or hair growth; wherein said liposome comprises at
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least two distinct lipids, a primary lipid and a secondary lipid, the primary
lipid constituting the greatest proportion, by weight, of any single lipid
material forming the bilayers of said vesicle, the primary lipid being
selected
from the group consisting of Clz -Cls fatty alcohols, Clz -CIS glycol
monoesters, C,z -C,s gyceryl mono-and diesters, and mixtures thereof, and the
primary lipid further having the property that it will to form a lipid vesicle
in
the absence of the secondary lipid, and the secondary lipid being present in
an
amount sufficient to allow formation of the lipid vesicles, the secondary
Iipid
being selected from the group consisting of quaternary dimethyldiacyl amines,
polyoxyethylene aryl alcohols, polyglycerols, sorbitan fatty acid esters,
fatty
acids and their salts, and mixtures thereof.
The invention also relates to a composition comprising a proliferation-
inducing amount of a peptide or a salt or derivative thereof encapsulated
within a liposome, wherein the peptide is at least 3 amino acids Iong, has at
least 10% sequence identity with the 34 amino acid N-terminal region of
hPTH or hPTHrP, and is capable of blocking the inhibition of proliferation or
stimulation of differentiation in vitro of cultured human keratinocytes by PTH
(1-34), 1,25(OH)2D3 or PTHrP (1-34), or ih vivo in mouse skin by stimulating
skin cell proliferation or accelerating hair cycle progression or stimulating
hair
growth; wherein said liposome comprises at least two distinct lipids, a
primary
lipid and a secondary lipid, the primary lipid constituting the greatest
proportion, by weight, of any single lipid material forming the bilayers of
said
vesicle, the primary lipid being selected from the group consisting of CIZ -
Cis
fatty alcohols, Clz -Cls glycol monoesters, C,z -Cls gyceryl mono-and
diesters,
and mixtures thereof, and the primary lipid further having the property that
it
will to form a lipid vesicle in the absence of the secondary lipid, and the
secondary lipid being present in an amount suff cient to allow formation of
the
lipid vesicles, the secondary lipid being selected from the group consisting
of
quaternary dimethyldiacyl amines, polyoxyethylene acyl alcohols,
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polyglycerols, sorbitan fatty acid esters, fatty acids and their salts, and
mixtures thereof.
The invention also relates to a composition comprising a proliferation-
inhibiting or differentiation-enhancing amount of a peptide or a salt or
derivative thereof and an active vitamin D compound, optionally encapsulated
within a liposome, wherein the peptide is at least 3 amino acids long, has at
least 10% sequence identity with the 34 amino acid N-terminal region of
hPTH or hPTHrP, and is capable of inhibiting proliferation or enhancing
differentiation in vitro of cultured human keratinocytes, or ih vivo in mouse
skin by inhibiting skin cell proliferation or hair cycle progression or hair
growth.
Other features and advantages of the invention will be apparent from
the following description of the preferred embodiments thereof, and from the
claims.
Brief Description of the Figures
Fig. 1 depicts a bar graph showing the macroscopic effects of topical
PTH (7-34) in Novasome on C57BL/6 mice (7 days).
Fig. 2 depicts a bar graph showing the effects of PTH (7-34) in
Novasome on BRDU stained hair follicle cells (7 days).
Fig. 3 depicts a bar graph showing the topical effects of PTH (1-34) in
Novasome on BRDU stained hair follicle cells.
Fig. 4 depicts a bar graph showing the effect of 60 days of topical PTH
(I-34) on tritiated thymidine incorporation into epidermal DNA in SKH-I
hairless mice.
Fig. 5 depicts a bar graph showing the effect of 60 days of topical PTH
(7-34) in Novasome on tritiated thymidine incorporation into epidermal DNA
in SKH-1 hairless mice.
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Description of the Preferred Embodiments
Synthesis and Selection of Peptides
The peptides used in the methods of the invention are all easily
synthesized, using recombinant DNA or solid phase peptide synthesis
techniques, and some are available commercially as well, or can be derived
from commercially available peptides. For example, there is reproduced below
a section of the Bach Chem catalog, listing a number of available human, rat,
and bovine analogs and fragments. (The Peninsula Laboratory catalog also
lists available fragments.)
PTHrP - (1-40)
HaN-Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-IIe-Gln-
Asp-Leu-Arg-Arg-Arg-Phe-Phe-Leu-His-His-Leu-Ile-Ala-Glu-Ile-His-Thr-
Ala-Glu-Ile-Arg-Ala-Thr-Ser-OH (SEQ ID NO:1)
PTH, Bovine (bPTH) (84 amino acids)
HZN-Ala-VaI-Ser-Glu-Ile-GIn-Phe-Met-His-Asn-Leu-GIy-Lys-His-Leu-Ser-
Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-GIn-Asp-Val-His-Asn-
Phe-Val-Ala-Leu-Gly-Ala-Ser-Ile-Ala-Tyr-Arg-Asp-Gly-Ser-Ser-Gln-Arg-
Pro-Arg-Lys-Lys-GIu-Asp-Asn-Val-Leu-Val-Glu-Ser-His-Gln-Lys-Ser-Leu-
Gly-Glu-Ala-Asp-Lys-Ala-Asp-Val-Asp-Val-Leu-Ile-Lys-Ala-Lys-Pro-Gln-
OH (SEQ ID N0:2)
[Tyr63]-hPTH (63-84)
HZN-Tyr-Glu-Lys-Ser-Leu-Gly-Glu-Ala-Asp-Lys-Ala-Asp-
Val-Asn-Val-Leu-Thr-Lys-Ala-Lys-Ser-Gln-OH (SEQ ID NO:3)
hPTH (64-84)
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H2N-GIu-Lys-Ser-Leu-Gly-GIu-Ala-Asp-Lys-Ala-Asp-Val-Asn-Val-Leu-Thr-
Lys-Ala-Lys-Ser-GIn-OH (SEQ ID N0:4)
[Tyr69]-hPTH (69-84)
HZN-Tyr-Ala-Asp-Lys-Ala-Asp-Val-Asn-Val-Leu-Thr-Lys-Ala-Lys-Ser-Gln-
OH (SEQ ID NO:S)
hPTH (70-84)
HZN-Ala-Asp-Lys-Ala-Asp-Val-Asn-Val-Leu-Thr-Lys-Ala-Lys-Ser-Gln-OH
(SEQ ID N0:6)
Human Bone Gla Protein (37-49) (BGP 37-49)
HZN-Gly-Phe-Gln-Glu-Ala-Tyr-Arg-Arg-Phe-Tyr-Gly-Pro-Val-OH (SEQ ID
NO:7) (Poser, J. W. et al, (1980) PNAS 255:8685)
[Tyr36 , Phe4a,a6]-H~~ Bone Gla Protein (38-49)
H2N-Tyr-Gln-Glu-Ala-Phe-Arg-Arg-Phe-Phe-Gly-Pro-Val-OH (SEQ ID
N0:8)
Human Bone Gla Protein (45-49)
HZN-Phe-Tyr-Gly-Pro-Val-OH (SEQ ID N0:9)
hPTH (84 amino acids)
HZN-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-
Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-VaI-His-Asn-
Phe-Val-Ala-Leu-GIy-Ala-Pro-Leu-Ala-Pro-Arg-Asp-Ala-GIy-Ser-Gln-Arg-
Pro-Arg-Lys-Lys-Glu-Asp-Asn-Val-Leu-Val-Glu-Ser-His-GIu-Lys-Ser-Leu-
Gly-Glu-Ala-Asp-Lys-Ala-Asp-Val-Asn-Val-Leu-Thr-Lys-Ala-Lys-Ser-Gln-
OH (SEQ ID NO:10) (Kimura, T. et al, (1983) BBRC 114493; Fairwell, T. et
al, (1983) Biochemistry 222691)
Rat PTH (rPTH) (84 amino acids)
HZN-Ala-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Ala-
Ser-Val-Glu-Arg-Met-Gln-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-
Phe-Val-Ser-Leu-GIy-VaI-GIn-Met-Ala-Ala-Arg-Glu-Gly-Ser-Tyr-Gln-Arg-
Pro-Thr-Lys-Lys-Glu-Asp-Asn-Val-Leu-Val-Asp-Gly-Asn-Ser-Lys-Ser-Leu-
Gly-Glu-Gly-Asp-Lys-Ala-Asp-Val-Asp-Val-Leu-Val-Lys-Ala-Lys-Ser-Gln-
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OH (SEQ ID NO:l 1 (Heinrich, G. et al, (1984) J. Biol. Chem. 2593320)
bPTH (1-34)
HZN-Ala-Val-Ser-Glu-Ile-Gln-Phe-Met-His-Asn-Leu-Gly-Lys-His-Leu-Ser-
Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-
Phe-OH (SEQ ID N0:12) (Tregear, G. W. et al, (1977) Biochemistry 162817)
hPTH (1-34)
HZN-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-
Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-
Phe-OH (SEQ ID N0:13) (Takel, T. et al, (1979) Peptide Chemistry)
[NleB°'8, Tyr34]-bPTH (1-34), Amide
HZN-Ala-Val-Ser-Glu-Ile-Gln-Phe-Nle-His-Asn-Leu-Gly-Lys-His-Leu-Ser-
Ser-Nle-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-
Tyr-NHa (SEQ ID N0:14) (Colrora, M. D. et al, (1981) J. Biol. Chem.
256:10.555; Rosenblatt, M. et al, (1977) Endocr. Res. Comm. 4:115;
Rosenblatt, M. et al, (1976) J. Biol. Chem. 251:159)
[NleB°'$, Tyr34] hPTH (1-34)
HZN-Ser-Val-Ser-Glu-Ile-Gln-Leu-Nle-His-Asn-Leu-Gly-Lys-His-Leu-Asn-
Ser-Nle-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-
Tyr-OH (SEQ ID NO:15)
[NleB°z', Tyr34]-rPTH (1-34), Amide
HZN-Ala-Val-Ser-Glu-Ile-Gln-Leu-Nle-His-Asn-Leu-Gly-Lys-His-Leu-Ala-
Ser-Val-Glu-Arg-Nle-Gln-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-
Tyr-NHZ (SEQ ID N0:16)
[Tyr']-hPTH (1-34)
HZN-Tyr-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-
Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-
Phe-OH (SEQ ID N0:17)
hPTH (1-38)
HZN-Sex-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-
Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-
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Phe-Val-Ala-Leu-Gly-OH (SEQ ID NO:18) (Heech, R. D. et al, (1984) Horm.
Metab. Res. 16:556)
hPTH (1-44)
HZN-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-
Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-GIn-Asp-Val-His-Asn-
Phe-Val-Ala-Leu-Gly-Ala-Pro-Leu-Ala-Pro-Arg-OH (SEQ ID N0:19)
(Kimura T. et al, (1981) Biopolymers 20:1823)
bPTH (3-34)
HZN-Ser-GIu-Ile-Gln-Phe-Met-His-Asn-Leu-Gly-Lys-His-Leu-Ser-Ser-Met
Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH
(SEQ ID N0:20) (Lowrik, C. et al, (1985) Cell Calcium 6:311)
[NleB°lg, Tyr34]-bPTH (3-34), Amide
HZN-Ser-Glu-Ile-Gln-Phe-Nle-His-Asn-Leu-Gly-Lys-His-Leu-Ser-Ser-Nle-
Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Tyr-NHZ
(SEQ ID N0:21) (Rosenblatt, M. et al, (1977) J. Biol. Chem. 252:5647)
[NleB°'$, Tyr34]-bPTH (7-34), Amide
HZN-Phe-Nle-His-Asn-Leu-Gly-Lys-His-Leu-Ser-Ser-Nle-Glu-Arg-Val-Glu-
Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Tyr-NHz (SEQ ID N0:22)
[Tyr34]-bPTH (7-34), Amide
HZN-Phe-Met-His-Asn-Leu-Gly-Lys-His-Leu-Ser-Ser-Met-Glu-Arg-Val-Glu-
Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Tyr-NHZ (SEQ ID N0:23)
hPTH (13-34)
HzN-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-
Gln-Asp-Val-His-Asn-Phe-OH (SEQ ID N0:24)
[Tyrz']-hPTH (27-48)
H2N-Tyr-Leu-Gln-Asp-Val-His-Asn-Phe-Val-Ala-Leu-Gly-Ala-Pro-Leu-Ala-
Pro-Arg-Asp-Ala-Gly-Ser-OH (SEQ ID N0:25)
hPTH (28-48)
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HZN-Leu-Gln-Asp-Val-His-Asn-Phe-Val-Ala-Leu-Gly-Ala-Pro-Leu-Ala-Pro-
Arg-Asp-Ala-Gly-Ser-OH (SEQ ID N0:26) Rosenblatt, M. et al, (1977)
Biochemistry 16:2811)
hPTH (53-84)
HZN-Lys-Lys-Glu-Asp-Asn-Val-Leu-Val-Glu-Ser-His-Glu-Lys-Ser-Leu-Gly-
Glu-Ala-Asp-Lys-Ala-Asp-Val-Asn-Val-Leu-Thr-Lys-Ala-Lys-Ser-Gln-OH
(SEQ ID N0:27) (Rosenblatt, M. et al, (I978) Endocrinology 103:976)
In addition, the peptides and peptide derivatives disclosed in the
following documents can also be used: U.S. Pat. Nos. 4,086,196, 4,423,037,
4,771,124, 4,833,125, 4,968,669, 5,001,223, 5,087,562, 5,093,233, 5,116,952,
5,149,779, 5,171,670, 5,229,489, 5,317,010, 5,382,658, 5,393,869, 5,434,246,
5,527,772, 5,589,452, 5,807,823, 5,821,255, 5,840,690, 5,977,070, 6,025,467,
IS 6,051,868, and 6,066,618; W094/02510, WO00/23594, and WO00/31137;
and EP 477,885.
When selecting a candidate peptide for a method of this invention, a
preferred first step is to choose a peptide which includes a fragment which
has
at least 10%, and more preferably 50% or greater, sequence identity with an 8
or greater amino acid long fragment within the amino terminal 34 amino acid
region of hPTH or hPTHrP. The term "sequence identity" refers to a measure
of the identity of nucleotide sequences or amino acid sequences. In general,
the sequences are aligned so that the highest order match is obtained.
"Identity" per se has an art-recognized meaning and can be calculated using
published techniques. (See, e.g.: Computational Molecular Biology, Lesk,
A.M., ed., Oxford University Press, New York, 1988; Biocomputing:
Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New
York, 1993; Computes Analysis of Sequence Data, Part I, Griffin, A.M., and
Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in
Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence
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Analysis Prime, Gribskov, M. and Devereux, J., eds., M Stockton Press, New
York, 1991). While there exist a number of methods to measure identity
between two polynucleotide or polypeptide sequences, the term "identity" is
well known to skilled artisans (Carillo, H. & Lipton, D., SIAMJApplied Math
48:1073 (1988)). Methods commonly employed to determine identity or
similarity between two sequences include, but are not limited to, those
disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press,
San Diego, 1994, and Carillo, H. & Lipton, D., SIAMJApplied Math 48:1073
(1988). Methods to determine identity and similarity are codified in computer
programs. Preferred computer program methods to determine identity and
similarity between two sequences include, but are not limited to, GCG
program package (Devereux, J., et al., Nucleic Acids Research 12(i):387
(1984)), BLASTP, BLASTN, FASTA (Atschul, S.F., et al., J Molec Biol
215:403 (1990)).
Therefore, as used herein, the term "identity" represents a comparison
between a test and reference polypeptide. More specifically, reference test
polypeptide is defined as any polypeptide that is 10% or more identical to a
reference polypeptide. As used herein, the term at least 10% identical to
refers
to percent identities from 10 to 99.99 relative to the reference polypeptides.
Identity at a level of 10% or more is indicative of the fact that, assuming
for
exemplification purposes a test and reference polynucleotide length of 100
amino acids, that no more than 90% (i.e., 90 out of 100) amino acids in the
test
polypeptides differ from that of the reference polypeptides. Such differences
may be represented as point mutations randomly distributed over the entire
length of the amino acid sequence of the invention or they may be clustered in
one or more locations of varying length up to the maximum allowable amino
acid difference. Differences are defined as amino acid substitutions, or
deletions.
Because of the high degree of homology among human PTH and PTH
of other species, non-human as well as human fragments or analogs can be
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used. Further, the fragment can be modified in any of a variety of standard
chemical ways, e.g., the carboxy-terminal amino acid residue can be made into
a terminal amide group; the amino-terminal residue can be modified with
groups to, e.g., enhance lipophilicity; the peptide can be chemically
glycosylated to increase solubility or in vivo half life; and D-amino acids
can
be substituted for L-isomers in the peptide.
Candidate peptides may be tested for suitability as inhibitors of cell
proliferation and enhancers of differentiation using cultured human
keratinocytes, as described in U.S. Pat. Nos. 5,527,772, 5,840,690 and
6,066,618. Briefly, those peptides which inhibit proliferation and induce
differentiation in cultured keratinocytes are those potentially useful as
therapeutic agents in treating disorders, e.g., psoriasis and cancer, where
suppression of cell proliferation is desired. Candidate peptides may be tested
for suitability as enhancers of cell proliferation using cultured human
keratinocytes or i~ vivo mouse model. Those peptides which block the effect
of agonist peptides or 1,25(OH)2D3 on cultured keratinocyte proliferation are
those potentially useful as therapeutic agents in treating disorders, e.g.,
wounds, burns, or skin ulcerations, where maintenance or stimulating of cell
proliferation is desired.
Candidate peptides may be tested for their ability to enhance wound
healing by carrying out a skin punch biopsy test, as described in U.S. Pat.
Nos.
5,527,772, 5,840,690 and 6,066,618.
Candidate peptides may be tested for suitability as stimulators of hair
growth using an in vitro hair growth assay, as described in U.S. Pat. Nos.
5,527,772, 5,840,690 and 6,066,618. Those peptides which stimulate hair
growth in vitro are those potentially useful for the stimulation of hair
growth
in vivo, e.g., for the stimulation or maintenance of hair growth during or
following chemotherapy or to treat a form of alopecia, e.g., male pattern
baldness.
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Alternatively, in vivo assays may be carried out as described herein and
in Schilli, M.B. et al., J. Invest. Dermatol. 108:928-932 (1997); Holick,
M.F.,
et al., Proc. Natl. Acad. Sci. 91:8014-8016 (1994); Paus, R. and Cotsarelis,
G., N. Engl. J. Med. 341: 491-497 (1999); and Paus, R., et al. Laboratory
Invest. 60: 365-369 (1989).
Peptides which block antiproliferative compounds can also be useful in
conjunction with chemotherapeutic agents in the treatment of skin cancer;
many chemotherapeutic agents are effective only against dividing cells, and
the blocking peptides can have the effect of inducing division of otherwise
dormant cells, rendering them vulnerable to the chemotherapy. Blocking
peptides can also be useful in promoting growth of new cells, e.g., skin
cells,
in topical skin creams. Differentiation-inducing peptides can be used as
immunostimulants, by inducing maturation of monocytes and lymphocytes
bearing PTH receptors, while blocking peptides can be used to inhibit
lymphocyte maturation, and thus can be used to treat conditions, e.g.,
autoimmune diseases such as juvenile diabetes, rheumatoid arthritis, and
allograft rejection, where mature lymphocytes are a causative agent.
The peptides are administered in therapeutically effective amounts to
mammals in need of them. The peptides may be administered as part of
liposomal preparations described in U.S. Pat. 5,260,065. Such liposomes
comprise at least two distinct lipids, a primary lipid and a secondary lipid,
the
primary lipid constituting the greatest proportion, by weight, of any single
lipid material forming the bilayers of said vesicle, the primary lipid being
selected from the group consisting of Clz -Cl8 fatty alcohols, Clz -Cl8 glycol
monoesters, Clz -Cls gYceryl mono-and diesters, and mixtures thereof, and the
primary lipid further having the property that it will to form a lipid vesicle
in
the absence of the secondary lipid, and the secondary lipid being present in
an
amount sufficient to allow formation of the lipid vesicles, the secondary
lipid
being selected from the group consisting of quaternary dimethyldiacyl amines,
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polyoxyethylene acyl alcohols, polyglycerols, sorbitan fatty acid esters,
fatty
acids and their salts, and mixtures thereof.
The preferred primary lipids are Clz -C1$ fatty alcohols, glyceryl mono
and distearate, glyceryl dilaurate, and glycol stearate. While any of the
secondary lipids could be used with any of the primary lipids, preferred
combinations include polyoxyethylene 10-20 acyl alcohols or quaternary
dimethyldiacyl amines as the secondary lipids to be used in conjunction with
the fatty alcohols. Matching chain lengths in terms of carbon content and
unsaturations is an important factor to consider for selection of the
secondary
lipid. These same acyl alcohols and dimethyldiacyl (specifically distearyl)
amines are also useful with the glycol stearate, glyceryl monostearate,
glyceryl
distearate and the glyceryl dilaurate. However, the glyceryl distearate and
glyceryl dilaurate may also use sodium laurate sarcosinates, as well as other
matching sarcosinate salts (all being water soluble), or lauryl sarcosinates
as
secondary lipids.
In certain instances, primarily the stearate derivatives, a sterol such as
cholesterol is a particularly useful additive. The addition of cholesterol
appears
to make the vesicles population more uniform in terms of size and shape.
Even cholesterol is not sufficient, in itself, to allow vesicle formation.
This is
contrast to the materials described in U.S. Pat. No.4,917,951 which only
require cholesterol to make vesicles. In certain circumstances, cholesterol
will
allow these materials which will not otherwise form a lamellar phase to form a
lamellar phase but they cannot be formed into vesicles without the addition of
the secondary lipid. In fact, some of the most prefeiTed secondary lipids,
e.g.,
dimethyldistearyl amine, water soluble polyoxyethylene aryl alcohols, and
acyl sarcosinate salts, will not form vesicles or lamellar phases either.
According to Example 1 of U.S. Pat. 5,260,065, a variety of materials
may be blended in order to make vesicles. Table 1 shows the composition,
water uptake level, and oil uptake under hot and cold loading techniques of
five different compositions. According to U.S. Pat. 5,260,065, none of the
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primary lipids used, e.g., glyceryl dilaurate (GDL), glyceryldistearate (GDS),
cetyl alcohol (CA), stearyl alcohol (SA), or glycol stearate (GS) will form
vesicles or lamellar phase on their own.
Table 1
Composition Water UptakeOil Uptake
(ml/ml)
(ml/ml)
Hot Cold
GDL/C 16Q/Chol 13 .5 z 7.2 z 2.7
( 1.0/0.05/0.05)
GDS/POElOSA/Chol 12.5 z6.9 z6.5
(1.0/0.5/0.25)
CA/POElOCA/Chol 9.5 z4.2 z4.2
(1.0/0.2/0.1)
SA/C 18 Q/Chol 13 .5 z 6.5 z 6. 5
(1.0/0.2/0.1)
GS/POElOSA/Chol 13.5 z6.5 z6.5
(1.0/0.2/0.1)
The first compound shown in Table 1 is a blend of glyceryl dilaurate,
dimethyldicetyl quaternary amine (C16Q), and cholesterol (Chol) in a
1.0:0.05:0.05 molar ratio. According to U.S. Pat. 5,260,065, the water uptake
is 13.5 ml/ml of lipid and the hot load and cold loading values were >_7.2 and
>_2.7 ml of oil/ml of lipid, respectively. According to U.S. Pat. 5,260,065,
the
vesicles were made by blending the two lipids and the cholesterol at
70°-75°
C. with the aqueous phase at 65° C. According to U.S. Pat. 5,260,065,
the Iipid
phase was placed in one syringe, the aqueous phase was placed in another
syringe, and the two syringes were connected by a stopcock. According to
U.S. Pat. 5,260,065, the material was shear mixed by blending from one
syringe to another through the stopcock forming vesicles in less than two
minutes. According to U.S. Pat. 5,260,065, for the cold loading technique, the
preformed vesicles were mixed with 20% and 50% V/V mineral oil (Drakeol
19) using the same syringe technique to load the oil. According to U.S. Pat.
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5,260,065, for the hot loading technique, the oil was heated to 70°-
75° C.,
blended with the lipophilic phase prior to hydration by the aqueous phase, and
then the combined lipophilic/water immiscible oily phase was hydrated by the
aqueous phase. According to U.S. Pat. 5,260,065, either hot loading or cold
loading techniques may be used for a mineral oil but with a highly volatile
oil
which would not survive the 70°-75° C. heating, the cold loading
technique,
which can be carried out a ambient temperature, is preferred.
According to U.S. Pat. 5,260,065, the second compounded tested was a blend
of glyceryl distearate, Polyoxyethylene 10 stearyl alcohol (POElOSA), and
cholesterol in a 1.0:0.5:0.25 molar ratio. This blended material had a water
uptake of 12.5 mllml lipid and the oil uptake for either hot and cold loading
was >6.5 ml/ml using the same techniques previously described.
According to U.S. Pat. 5,260,065, the third material tested was a blend of
cetyl
alcohol, polyoxyethylene 10 cetyl alcohol (POElOCA), and, cholesterol in a
1:0.2:0.1 molar ratio. Water uptake was 9.5 ml/ml and both hot and cold oil
uptake was >4.2 ml/ml lipid.
According to U.S. Pat. 5,260,065, the fourth combination tested was a
blend of stearyl alcohol, dimethyldistearyl quaternary amine (C18Q), and
cholesterol on a 1:0.2:0.1 ratio. Water uptake was 13.5 ml/ml and oil uptake
on both a hot and cold basis was >6.5 ml/ml lipid.
According to U.S. Pat. 5,260,065, the f fth compound tested was a
blend of glycol stearate, polyoxyethylene 10 stearyl alcohol, and cholesterol
in
a 1:0.2:0.1 ratio. Again, the water uptake was approximately 13.5 ml/ml and
the oil uptake was >6.5 ml/ml under both hot and cold loading techniques.
According to Example 2 of U.S. Pat. 5,260,065, retinoic acid, a water
insoluble material in a water immiscible carrier, was used in lieu of the
mineral oil of Example 1 in the amorphous central cavity of the paucilamellar
lipid vesicles. Retinoic acid has a substantial number of dermatological uses
including, potentially, the reduction of facial wrinkles.
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Table 2
A B
Cetyl Alcohol 4.7 g
Glycol Stearate 11.5 g
POE10 Cetyl Alcohol 2.35 g.
POE10 Stearyl Alcohol 2.3 g
Cholesterol 1.2 g 1.15 g
Petrolatum 10.9 g
Paraffin Wax 11.6 g
Soybean Oil 21.8 g
Retinoic Acid 0.25 g 0.25 g
Deionized Water 69 g 63 g
According to U.S. Pat. 5,260,065, Table 2 shows the formulas for two
different retinoic acid formulations, one using a cetyl
alcohol/polyoxyethylene
cetyl alcohol blend and the other using a glycol stearate/polyoxyethylene
5 10 stearyl alcohol blend as the vesicles formers. According to U.S. Pat.
5,260,065, both formulas include cholesterol while one uses a mixture
petrolatum and paraffin wax as a carrier for the retinoic acid while the other
uses a soybean oil carrier. According to U.S. Pat. 5,260,065, in both cases,
the
retinoic acid was dissolved in the carrier at 65°-75° C.
According to U.S. Pat.
10 5,260,065, the lipids and the cholesterol were then heated and blended to
homogeneity and the retinoic acid mixture was added and blended therein.
According to U.S. Pat. 5,260,065, an aqueous phase consisting of the
deionized water was then heated to approximately 65° C. and the
resulting
phases were shear mixed to form the vesicles. According to U.S. Pat.
5,260,065, while the syringe method described in Example 1 could be used, a
NovaMix~ vesicle forming machine manufactured by Micro Vesicular
Systems, Inc., Nashua, N.H. was used. This machine, which is described in
more detail in U.S. Pat. No. 4,895,452, has a substantially cylindrical
central
chamber with an axial outflow tube and tangentially located inflow tubes.
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According to U.S. Pat. 5,260,065, the phases are injected into the central
chamber, under pressure sufficient to form turbulent flow and shear mixing,
rapid vesicle formation occurs, and the vesicles are removed through the
outflow tube.
Alternatively, the apparatus described in U.S. Pat. 5,013,497 may be
used to prepare the liposomes.
According to Example 3 of U.S. Pat. No. 5,260,065, two different
formulations for encapsulating anthralin, an antipsoriatic, were tested. Table
3
lists the ingredients used in these formulations. According to the present
invention, a peptide agonist or antagonist may be substituted for anthralin.
Table 3
C D
Glyceryl Distearate 9.4 g
Cetyl Alcohol 6.85 g
Dimethyl Distearyl Ammonium Chloride0.3 g
POE10 Cetyl Alcohol 1.35 g
Sodium Lauryl Sarcosinate 1.4 g
Cholesterol 1.0 g 0.7 g
Petrolatum 15.7 g 17.3 g
Paraffin Wax 16.8 g 18.5 g
Anthralin 0.5 g 0.5 g
Deionized Water 54.9 g 54.8 g
According to U.S. Pat. 5,260,065, in formulation C, the petrolatum and
paraffin are melted together and the anthralin is dissolved into the carrier
mixture. According to U.S. Pat. 5,260,065, this also the case of formulation
D.
According to U.S. Pat. 5,260,065, this petrolatum/paraffin wax mixture
appears to be particularly advantageous in that micro-crystals form rather
than
the macroscopic crystals which normally appear when anthralin cools.
According to U.S. Pat. 5,260,065, in formulation C, however, the glyceryl
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distearate, cholesterol and dimethyldistearyl ammonium chloride are blended
together at approximately 75° C. until clear and the anthralin solution
(forming
a water immiscible phase) is then mixed therein. According to U.S. Pat.
5,260,065, the aqueous phase is fornied by heating the deionized water to
approximately 65° C. and dissolving the secondary lipid, the sodium
lauryl
sarcosinate, therein. According to U.S. Pat. 5,260,065, the aqueous phase and
the lipid phase are then shear mixed, using a NovaMix~ machine as described
in Example 2, to form vesicles. According to U.S. Pat. 5,260,065, in contrast,
in formulation D, the cetyl alcohol, polyoxyethylene 10 cetyl alcohol and the
cholesterol are blended together at an elevated temperature, the anthralin
solution is mixed in, and the aqueous which consists merely of the deionized
water is shear mixed using the NovaMix~ machine to form the vesicles.
According to U.S. Pat. 5,260,065, the difference in the procedure is that the
non-ionic lipids of formulation D cannot be carried in the aqueous solution as
is the ionic sodium lauryl sarcosinate of formulation C. According to U.S.
Pat.
5,260,065, either formulation forms acceptable anthralin carrying vesicles.
According to Example 4 of U.S. Pat. 5,260,065, three different
materials, Vitamin E acetate, levamisole base, and a butter flavor oil were
carried in the central cavity of vesicles of the invention. Table 4 shows the
formulas for these vesicles. According to the present invention, a peptide
agonist or antagonist may be substituted for vitamin E acetate.
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Table 4
E F G
Glyceryl Distearate 11.2 g 4.35
g
Glycol Stearate 7,5
g
POE10 Stearyl Alcohol 5.6 g 1.5 2.2 g
g
Cholesterol 2.8 g 0.75 1.1 g
g
Soybean Oil 8.5
g
Vitamin E ~ 2.2 g
Levamisole Base 4.63
g
Butter Flavor Oil 20.0
g
Deionized Water 78.2 g 74.12 72.35
g g
According to U.S. Pat. 5,260,065, formulation E uses glyceryl
distearate, polyoxyethylene 10 stearyl alcohol, and cholesterol as the
lipophilic
phase which are blended at 70° C. to obtain a clear, homogeneous
solution.
According to U.S. Pat. 5,260,065, the Vitamin E acetate was dissolved therein
and the mixture was hydrated with 65° C. water using the NovaMix~
machine
as described in Example 2.
According to U.S. Pat. 5,260,065, formulation F used a levamisole
base (a sheep dip) in soybean oil at 75° C. to form the water
immiscible phase.
According to U.S. Pat. 5,260,065, the glycol stearate, polyoxyethylene stearyl
alcohol and cholesterol were heated together at 75° C. to obtain a
clear,
homogeneous solution and the levamisole/soybean oil mixture was blended
therewith. According to U.S. Pat. 5,260,065, the deionized water was heated to
approximately 65° C. and used as a hydrating solution for the lipids,
again
using the previously described NovaMix''~'~ machine.
According to U.S. Pat. 5,260,065, in formulation G, the lipids and
cholesterol were melted together at 75° C. and the butter oil dissolved
therein.
According to U.S. Pat. 5,260,065, again, the deionized water was heated to
approximately 65° C. and used as a hydrating solution in a NovaMix~
machine.
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According to Example 5 of U.S. Pat. 5,260,065, three different
formulations for vesicles using retinoic acid, with both cationic and anionic
vesicles may be used. Table 5 lists the formulations for each vesicle.
According to the present invention, a peptide agonist or antagonist may be
substituted for retinoic acid.
Table 5
H I J
Glyceryl Distearate 9.4 g
Glycol Stearate 13.2 13.2
g g
Dimethyl Distearyl Ammonium 0.3 g
Chloride
Dimethyl Dicetyl Ammonium Chloride 0.6 g
Sodium Oleate 1.0
g
Petrolatum 15.7
g
Paraffin Wax 16.8
g
Soybean Oil 22.0 22.0
g g
Retinoic Acid 0.25 0.25 0.25
g g g
Deionized Water 56.55 62.75 63.35
g g g
According to U.S. Pat. 5,260,065, formulation H uses the paraffin
wax/petrolatum carrier for the retinoic acid, with the retinoic acid being
dissolved in the carrier at approximately 65°-75° C. According
to U.S. Pat.
5,260,065, the lipophilic phase is formed of glyceryl distearate, cholesterol,
and the dimethyl distearyl ammonium chloride. According to U.S. Pat.
5,260,065, the carrier containing the retinoic acid is blended into the
lipophilic
phase and is hydrated with the deionized water using the NovaMix~ machine
as described in Example 2.
According to U.S. Pat. 5,260,065, formulations I and J use the soybean
oil carrier and the same materials except for the secondary lipid. According
to
U.S. Pat. 5,260,065, in formulation I, the secondary lipid, which forms part
of
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the initial lipophilic phase, is dimethyl dicetyl ammonium chloride while in
formulation J, the secondary lipid, which is incorporated into the aqueous
phase, is sodium oleate. According to U.S. Pat. 5,260,065, in either case, the
retinoic acid is dissolved in the soybean oil at elevated temperatures, the
soybean oil is blended into the lipophilic phase, and the combined phase is
then hydrated using the aqueous phase. According to U.S. Pat. 5,260,065,
formulation J forms anionic vesicles while formulation I forms cationic
vesicles. However, according to U.S. Pat. 5,260,065, both are effective in
encapsulating the retinoic acid.
The liposome encapsulted peptides can be admixed with a
pharmacologically inert topical carrier such as one comprising a gel, an
ointment or a cream, including such carriers as water, glycerol, alcohol,
propylene glycol, fatty alcohol, triglycerides, fatty acid ester or mineral
oils.
~ther possible carriers are liquid petrolatum, isopropylpalmitate,
polyethylene
glycol ethanol 95%, polyoxyethylene monolaurate 5% in water, sodium lauryl
sulfate 5% in water, and the like. Materials such as antioxidants, humectants,
viscosity stabilizers and the like may be added, if necessary.
The peptides can be provided in the form of pharmaceutically
acceptable salts. Examples of preferred salts are those of therapeutically
acceptable organic acids, ~e.g., acetic, lactic, malefic, citric, malic,
ascorbic,
succinic, benzoic, salicylic, methanesulfonic, toluenesulfonic, or pamoic
acid,
as well as polymeric acids such as tannic acid or carboxymethyl cellulose, and
salts with inorganic acids such as hydrohalic acids, e.g, hydrochloric acid,
sulfuxic acid, or phsophoric acid.
Dosage will be dependent upon the age, health, and weight of the
recipient; kind of concurrent treatment, if any; frequency of treatment; and
the
nature of the effect desired. Generally, daily dosage will be from about
0.0001
micrograms/kg to 100 micrograms/kg, preferably 0.001 to 10.0
micrograms/kg. The topical dosage will be from about 0.01 micrograms/cm2 to
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100 micrograms/cm2, preferably 0.1 to 10 micrograms/cm2. The liposomal
formulations may be applied by one or more applications per day.
The invention also relates to compositions comprising a proliferation
inhibiting or differentiation-enhancing amount of a peptide or a salt or
derivative thereof, an active vitamin D compound and a pharmaceutical
caxrier, wherein the peptide is at least 3 amino acids long, has at least 10%
sequence identity with the 34 amino acid N-terminal region of hPTH or
hPTHrP, and is capable of inhibiting proliferation or enhancing
differentiation
in vitro of cultured human keratinocytes, or ih vivo in mouse skin by
inhibiting
skin cell proliferation or hair cycle progression or hair growth. A large
number of active vitamin D compounds are known which can be used in the
practice of the present invention. See U.S. Patent Nos. 5,457,217, 5,414,098,
5,384,313, 5,373,004, 5,371,249, 5,430,196, 5,260,290, 5,393,749, 5,395,830,
5,250,523, 5,247,104, 5,397,775, 5,194,431, 5,281,731, 5,254,538, 5,232,836,
5,185,150, 5,321,018, 5,086,191, 5,036,061, 5,030,772, 5,246,925, 4,973,584,
5,354,744, 4,927,815, 4,857,518, 4,851,401, 4,851,400, 4,847,012, 4,755,329,
4,940,700, 4,619,920, 4,594,192, 4,588,716, 4,564,474, 4,552,698, 4,588,528,
4,719,204, 4,719,205, 4,689,180, 4,505,906, 4,769,181, 4,502,991, 4,481,198,
4,448,726, 4,448,721, 4,428,946, 4,411,833, 4,367,177, 4,336,193, 4,360,472,
4,360,471, 4,307,231, 4,307,025, 4,358,406, 4,305,880, 4,279,826, and
4,248,791. A preferred active vitamin D compound is calcipotriene. In this
embodiment, any conventional liposome may be used including the liposomes
described in U.S. Pat. 4,235,871, 4,241,046, 4,247,411, 4,356,167, 4,377,567,
4,544,545, 4,551,288, 4,610,868, 4,731,210, 4,744,989, 4,772,471, 4,897,308,
4,917,951, 5,021,200, 5,032,457, and 5,260,065.
The invention relates as well to a method of inhibiting proliferation or
enhancing differentiation of a skin or hair cell of a mammal, comprising
administering to the mammal in need thereof a proliferation-inhibiting or
differentiation-enhancing amount of a peptide or a salt or derivative thereof
and an active vitamin D compound, wherein the peptide is at least 3 amino
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acids long, has at least 10% sequence identity with the 34 amino acid N-
terminal region of hPTH or hPTHrP, and is capable of inhibiting proliferation
or enhancing differentiation in vitro of cultured human keratinocytes, or in
vivo in mouse skin by inhibiting skin cell proliferation or hair cycle
progression or hair cell growth. Surprisingly, it has been discovered by
recalcitrant psoriasis responds to the administration of the peptide and
active
vitamin D compound. In this embodiment, the peptide and the active vitamin
D compound may be administered as part of single or separate pharmaceutical
compositions. Either one or both of the peptide and active vitamin D
compound may be administered topically or parenterally. In a preferred
embodiment, the peptide is administered first followed by the active vitamin D
compound.
The following examples are illustrative, but not limiting, of the method
and compositions of the present invention. Other suitable modifications and
adaptations of the variety of conditions and parameters normally encountered
in clinical therapy and which are obvious to those skilled in the art are
within
the spirit and scope of the invention.
Example 1
Process for the Production of Liposomes
PTH (1-34) and PTH (7-34) was formulated in Novasome A (obtained
from IGI, Inc., Buena, N~. The formulation included dissolving the PTH
analog in doubly distilled water at a concentration of 1 mg/100 ~,1. This
solution was mixed with 3 ml of Novasome A.
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Example 2
Effect of Liposome Encapsulated Peptides oh Skih and Hair Growtla iu Mice
Evaluation of the effect of topically administered PTH (1-34) and PTH (7
34) formulated in Novasome A oh skin and hair growth
C57 BL/6 were purchased from Jackson Labs and were in their telogen
state of hair development. The animals were depilated as previously described
(1). Groups of six animals received daily a topical application of either 10
~,g
of PTH (1-34) in 30 ~1 of Novasome or their depilatated backs, 10 ~,g of PTH
(7-34)/30 ~,1 of Novasome, or 30 ~.1 of Novasome. The animals were dosed
daily for seven days. Macroscopic evaluation and tritiated thymidine and
bromouridine analysis was made on day seven as previously described
(1,2,3,4).
Results
Macroscopic evaluation of the effect of PTH (7-34) on C57 BL/6 mice
demonstrated an increase in the progression of the hair follicles into their
proliferative anagen state (Fig. 1). There was a 155% increase in the anagen
area in the group of animals receiving 10 ~,g of PTH (7-34) formulated in
Novasome A. An evaluation of BRDU staining of the hair follicles revealed
that there was an increase in BRDU staining of the hair follicle which was an
indication of increased proliferation of the hair follicle (Fig. 2). Animals
that
received the PTH (1-34) topically showed a decrease in hair growth
progression (Fig. 1). An evaluation of the BRDU staining in hair follicles
from the mice that received topically 10 ~,g of PTH (1-34) demonstrated a
decrease (Fig. 3).
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Conclusion
These results demonstrate that the topical application of PTH (7-34) is
able to accelerate the hair cycle and stimulate the hair follicles to
proliferate.
The animals that received topically PTH (1-34) revealed a decrease in hair
growth progression and a decrease in the proliferation of the hair follicles.
Next, the effect of topically applying PTH (1-34) or PTH (7-34)
formulated in Novasome A on SKH-1 hairless mice for 60 days was
determined.
SKH-1 hairless mice received topically daily for 60 days either 10 ~,g
of PTH (1-34) formulated in Novasome A, PTH (7-34) formulated in
Novasome A, or Novasome A without any PTH analog to serve as the control
group. The animals received 3H-tritiated thymidine for the evaluation of
epidermal proliferation as previously described.
Results
As can be seen in Fig. 4, the animals that received topically PTH (1-
34) in Novasome A showed a decrease of 25% in DNA synthesis
(proliferation) compared to the control group.
Evaluation of 3H-thymidine in the epidermis of the mice that received
topically PTH (7-34) showed a more than two fold enhancement in DNA
synthesis (proliferation) compared to the control group 10 ~.g of PTH (7-34)
formulated in Novasome, respectively.
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Example 3
Effect of Liposome Encapsulated Peptides oh Skiu and Hair Growth ih
Humans
Nine patients with chronic psoriasis were enrolled in a double blind
placebo controlled trial. Two comparable lesions received either PTH (1-34)
formulated in Novasome at a concentration of 20 ~.g/0.1 ml and the
contralateral lesion received 100 ~.l of Novasome A placebo for a period of
two months. The percentage of clinical improvement of scaling, erythema,
and induration for the lesion treated with placebo was 17.2, 15, and 30%,
respectively. For the lesion treated with PTH (1-34), there was a marked
improvement in scaling of 30.9%, erythema 34%, and induration 52%,
respectively. An evaluation of the skin biopsies analyzed by hemotoxilin and
eosin staining and by immunohistochemistry for proliferating cell nuclear
antigen and transglutaminase demonstrated that the lesions treated with PTH
(1-34) compared to the placebo treated lesions showed a marked decrease in
the proliferation of the epidermis, restoration of the PCNA staining in the
basal layer, and restoration of transglutaminase staining in the upper
granular
layer only of the lesions treated with PTH (1-34) compared to the lesion
treated with placebo control Novasome. These data indicate that the topical
application of PTH (1-34) in Novasome A restored psoriatic proliferation and
differentiation to normal.
One patient with chronic psoriasis received topically PTH (1-34) for
two months. He had a minimal response. The patient returned two weeks
later and, on the lesion treated with PTH (1-34) and the placebo lesion,
received topically calcipotriene cream. After two months of treatment, there
was complete resolution of the psoriatic lesion that had previously received a
topical application of PTH (1-34) for two months. There was no significant
improvement in the lesion that received calcipotriene therapy and had
received, previously, topical Novasome placebo. Therefore, the combination
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therapy of PTH (1-34) followed by calcipotriene or other activated vitamin D
compounds can be an effective therapeutic approach for treating psoriasis.
Referetzces:
(1) Schilli, M.B. Ray, S., Paus, R., Obi-Tabot, E. and Holick, M.F. Control of
hair growth with parathyroid hormone (7-34). J. Invest. Dermatol.
108:928-932, 1997.
(2) Holick, M.F., Ray, S., Chen, T., Tian, X., and Persons, K. Novel functions
of a parathyroid hormone antagonist: stimulation of epidermal
proliferation and hair growth in mice. Proc. Natl. Acad. Sci. 91:8014-
8016, 1994.
(3) Paus, R. and Cotsarelis, G. The biology of hair follicles. N. Engl. J.
Med.
341: 491-497, 1999.
(4) Paus, R., Stenn, K.S., and Link, R.E. The induction of anagen hair growth
in telogen mouse skin by cyclosporine A administration. Laboratory Invest.
60: 365-369, 1989.
Having now fully described this invention, it will be understood by
those of ordinary skill in the art that the same can be performed within a
wide
and equivalent range of conditions, formulations and other parameters without
affecting the scope of the invention or any embodiment thereof. All patents,
patent applications and publications cited herein are fully incorporated by
reference herein in their entirety.
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SEQUENCE LISTING
<110> Holick, Michael F.
<120> Regulation Of Cell Proliferation And Differentiation Using Topically
Applied Peptides
<130> 1539.031PC01
<150> US 60/213,247
<l51> 2000-06-22
<160> 27
<l70> PatentIn version 3.0
<210> 1
<211> 40
<212> PRT
<213> Artificial Sequence
<220>
<221> mist feature
<223> PTHrP - (1-40)
<400> 1
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln
1 5 10 15
Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His
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20 25 30
Thr Ala Glu Ile Arg Ala Thr Ser
35 40
<210>2
<211>84
<212>PRT
<213>Bos
sp.
<220>
<221> misc feature
<223> bPTH
<400> 2
Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His Leu Ser
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Phe Val Ala Leu Gly Ala Ser Ile Ala Tyr Arg Asp Gly Ser Ser
35 40 45
Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Gln
50 55 60
Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asp Val Leu Ile Lys
65 70 75 80
Ala Lys Pro Gln
<210> 3
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
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-3-
<221> mist feature
<223> [Tyr63] -hPTH (63-84)
<400> 3
Tyr Glu Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu
1 5 10 15
Thr Lys Ala Lys Ser Gln
<210> 4
<211> 21
<2l2> PRT
<213> Homo Sapiens
<220>
<221> mist feature
<223> hPTH (64-84)
<400> 4
Glu Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr
1 5 10 15
Lys Ala Lys Ser Gln
<210> 5
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<221> mist feature
<223> [Tyr69] -hPTH (69-84)
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<400> 5
Tyr Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys Ala Lys Ser Gln
1 5 10 15
<210> 6
<211> 15
<212> PRT
<213> Homo Sapiens
<220>
<221> mist feature
<223> hPTH (70-84)
<400> 6
Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys Ala Lys Ser Gln
1 5 10 15
<210> 7
<211> 13
<212> PRT
<213> Homo Sapiens
<220>
<22l> mist feature
<223> Human Bone Gla Protein (37-49) (BGP 37-49)
<400> 7
Gly Phe Gln Glu Ala Tyr Arg Arg Phe Tyr Gly Pro Val
1 5 10
<210> 8
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<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<22l> mist feature
<223> [Tyr36 , phe42,4s ] -Human Bone Gla Protein (38-49)
<400> 8
Tyr Gln Glu Ala Phe Arg Arg Phe Phe Gly Pro Val
1 5 10
<210>9
<211>5
<212>PRT
<213>Homo Sapiens
<220>
<221> mist feature
<223> Human Bone Gla Protein (45-49)
<400> 9
Phe Tyr Gly Pro Val
1 5
<210> 10
<211> 84
<212> PRT
<2135 Homo Sapiens
<220>
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<221> mist feature
<223> hPTH
<400> 10
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Phe Val Ala Leu Gly A1a Pro Leu Ala Pro Arg Asp Ala Gly Ser
35 40 45
Gln Arg Pro Arg Lys Lys G1u Asp Asn Val Leu Val Glu Ser His Glu
50 55 60
Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys
65 70 75 80
Ala Lys Ser Gln
<210> 11
<211> 84
<212> PRT
<213> Rattus sp.
<220>
<221> mist feature
<223> Rat PTH (rPTH)
<400> 11
Ala Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Ala
1 5 10 15
Ser Val Glu Arg Met Gln Trp Leu Arg Lys Lys Leu Gln Asp Va1 His
20 25 30
Asn Phe Val Ser Leu Gly Val Gln Met Ala Ala Arg Glu Gly Ser Tyr
35 40 45
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_'7_
Gln Arg Pro Thr Lys Lys Glu Asp Asn Val Leu Val Asp Gly Asn Ser
50 55 60
Lys Ser Leu Gly Glu Gly Asp Lys Ala Asp Va1 Asp Val Leu Val Lys
65 70 75 ~ 80
Ala Lys Ser Gln
<210> 12
<211> 34
<212> PRT
<213> Bos Sp.
<220>
<22l> misC feature
<223> bPTH (1-34)
<400> 12
Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His Leu Ser
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Phe
<210>13
<211>34
<212>PRT
<213>Homo Sapiens
<220>
<221>mist feature
<223>hPTH (1-34)
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_g_
<400> 13
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Phe
<210> 14
<211> 34
<2l2> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (8)..(8)
<223> Nle
<220>
<221> MOD RES
<222> (18)..(18)
<223> Nle
<220>
<221> mist feature
<223> [N1e8~18~ Tyr34~ -bpTH (1-34)
<400> 14
Ala Val Ser Glu Ile Gln Phe Xaa His Asn Leu Gly Lys His Leu Ser
l 5 10 15
Ser Xaa Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
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20 25 30
Asn Tyr
<210> l5
<21l> 34
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (8)..(8)
<223> Nle
<220>
<221> MOD RES
<222> (18)..(18)
<223> Nle
<220>
<221> mist feature
<223> [NleB~18, Tyr34] hPTH (1-34)
<400> 15
Ser Val Ser Glu Ile Gln Leu Xaa His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Xaa Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Tyr
<210> 16
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<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (8) . . (8)
<223> Nle
<220>
<221> MOD RES
<222> (21) . . (21)
<223> Nle
<220>
<221> mist feature
<223> [Nlee-zl Tyr34] _rpTH (1-34)
<400> 16
Ala Val Ser Glu Ile Gln Leu Xaa His Asn Leu Gly Lys His Leu Ala
1 5 10 15
Ser Val Glu Arg Xaa Gln Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Tyr
<210> 17
<211> 34
<212> PRT
<213> Artificial Sequence
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<220>
<22l> misC feature
<223> [Tyre] -hPTH (1-34)
<400> 17
Tyr Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Phe
<210>18
<211>38
<212>PRT
<213>Homo Sapiens
<220>
<221>mist feature
<223>hPTH (l-38)
<400> 18
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Phe Val Ala Leu Gly
<210> 19
<211> 44
<212> PRT
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<213> Homo Sapiens
<220>
<221> misc feature
<223> hPTH (1-44)
<400> 19
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg
35 40
<210> 20
<211> 32
<212> PRT
<213> Bos Sp.
<220>
<221> mist feature
<223> bPTH (3-34)
<400> 20
Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His Leu Ser Ser Met
1 5 10 15
Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Phe
20 25 30
<210> 21
<211> 32
<212> PRT
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<213> Artificial Sequence
<220>
<221> MOD RES
<222> (6) . . (6)
<223> Nle
<220>
<221> MOD RES
<222> (16)..(16)
<223> Nle
<220>
<221> mist feature
<223> [Nlee~m~ Tyr34] -bPTH (3-34)
<400> 21
Ser Glu Ile Gln Phe Xaa His Asn Leu Gly Lys His Leu Ser Ser Xaa
1 5 10 15
Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Tyr
20 25 30
<210> 22
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (2) .. (2)
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-14-
<223> Nle
<220>
<221> MOD RES
<222> (12)..(12)
<223> Nle
<220>
<221> misc feature
<223> [Nlee~le Tyr34] _bpTH (7-34)
<400> 22
Phe Xaa His Asn Leu Gly Lys His Leu Ser Ser Xaa Glu Arg Val Glu
1 5 10 15
Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Tyr
20 25
<210> 23
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<221> mist feature
<223> [Tyr34] -bPTH (7-34)
<400> 23
Phe Met His Asn Leu Gly Lys His Leu Ser Ser Met Glu Arg Val Glu
1 5 10 15
Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Tyr
20 25
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<210>24
<211>22
<212>PRT
<213>Homo Sapiens
<220>
<221>mist feature
<223>hPTH (13-34)
<400> 24
Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu
1 5 10 15
Gln Asp Val His Asn Phe
<210> 25
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<221> mist feature
<223> [Tyrz'] -hPTH (27-48)
<400> 25
Tyr Leu Gln Asp Val His Asn Phe Val Ala Leu Gly Ala Pro Leu Ala
1 5 10 15
Pro Arg Asp Ala Gly Ser
<210> 26
<211> 21
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<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> hPTH (28-48)
<400> 26
Leu Gln Asp Va1 His Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro
1 5 10 15
Arg Asp Ala Gly Ser
<210> 27
<211> 32
<212> PRT
<213> Homo Sapiens
<220>
<221> mist feature
<223> hPTH (53-84)
<400> 27
Lys Lys Glu Asp Asn Val Leu Va1 Glu Ser His Glu Lys Ser Leu Gly
1 5 10 15
Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys Ala Lys Ser Gln
20 . 25 30
