U.S. patent application number 10/174443 was filed with the patent office on 2004-02-19 for method of promoting production of living tissue equivalents.
This patent application is currently assigned to The University of Southern California. Invention is credited to DiZerega, Gere, Rodgers, Kathleen E..
Application Number | 20040033956 10/174443 |
Document ID | / |
Family ID | 26759341 |
Filed Date | 2004-02-19 |
United States Patent
Application |
20040033956 |
Kind Code |
A1 |
Rodgers, Kathleen E. ; et
al. |
February 19, 2004 |
Method of promoting production of living tissue equivalents
Abstract
The present invention provides methods, improved cell culture
medium and kits for accelerating the generation of tissue
equivalents, and for improving the quality of tissue equivalents,
by growth in the presence of angiotensinogen, AI, AI analogues, AI
fragments and analogues thereof, AII, AII analogues, AII fragments
and analogues thereof and/or AII AT.sub.2 type 2 receptor
agonists.
Inventors: |
Rodgers, Kathleen E.; (Long
Beach, CA) ; DiZerega, Gere; (Pasadena, CA) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Assignee: |
The University of Southern
California
Los Angeles
CA
|
Family ID: |
26759341 |
Appl. No.: |
10/174443 |
Filed: |
June 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10174443 |
Jun 18, 2002 |
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09266293 |
Mar 11, 1999 |
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6498138 |
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60077499 |
Mar 11, 1998 |
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60089064 |
Jun 12, 1998 |
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Current U.S.
Class: |
514/16.5 ;
514/15.4; 514/16.4; 514/16.7; 514/17.1; 514/17.2; 514/21.1 |
Current CPC
Class: |
C12N 5/0629 20130101;
C12N 2533/54 20130101; C12N 2533/70 20130101; C12N 2501/32
20130101 |
Class at
Publication: |
514/16 |
International
Class: |
A61K 038/08 |
Claims
We claim:
1. An improved method for producing a tissue equivalent, the
improvement comprising contacting the tissue equivalent with an
amount effective to accelerate generation of tissue equivalents of
at least one active agent comprising a sequence consisting of at
least three contiguous amino acids of groups R.sup.1-R.sup.8 in the
sequence of general formula I
R.sup.1-R.sup.2-R.sup.3-R.sup.4-R.sup.5-R.sup.6-R.sup.7-R.sup.8 in
which R.sup.1 and R.sup.2 together form a group of formula
X-R.sup.A-R.sup.B-, wherein X is H or a one to three peptide group
R.sup.A is selected from Asp, Glu, Asn, Acpc, Ala, Me.sup.2Gly,
Pro, Bet, Glu(NH.sub.2), Gly, Asp(NH.sub.2) and Sue; R.sup.B is
selected from Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys;
R.sup.3 is selected from the group consisting of Val, Ala, Leu,
norLeu, Ile, Gly, Pro, Aib, Acpc, Lys and Tyr; R.sup.4 is selected
from the group consisting of Tyr, Tyr(PO.sub.3).sub.2, Thr, Ser,
Ala, homoSer and azaTyr; R.sup.5 is selected from the group
consisting of Ile, Ala, Leu, norLeu, Val and Gly; R.sup.6 is His,
Arg or 6-NH.sub.2-Phe; R.sup.7 is Pro or Ala; and R.sup.8 is
selected from the group consisting of Phe, Phe(Br), Ile and Tyr,
excluding sequences including R.sup.4 as a terminal Tyr group.
2. The method of claim 1 wherein the active agent is selected from
the group consisting of angiotensinogen, SEQ ID NO:1, SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID
NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ
ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,
SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID
NO:33, SEQ ID NO: 34; SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ
ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID
NO:42.
3. The method of claim 1 wherein the active agent is SEQ ID NO:1,
SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:31 SEQ ID NO:38, SEQ ID NO:39,
SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42.
4. The method of claim 1 wherein the concentration of active agent
is between about 0.1 ng/kg and about 1.0 mg/kg.
5. The method of claim 1 where the tissue equivalent is selected
from the group consisting of a skin, dermis, bone, bone marrow,
pancreas, heart valve, vascular graft, cartilage, ligament, liver,
and kidney tissue equivalent.
6. An improved chemically defined medium for the culture of tissue
equivalents, wherein the improvement comprises contacting the
tissue equivalent with an amount effective to accelerate generation
of tissue equivalents of at least one active agent comprising a
sequence consisting of at least three contiguous amino acids of
groups R.sup.1-R.sup.8 in the sequence of general formula I
R.sup.1-R.sup.2-R.sup.3-R.sup.4-R.sup.5-R.s- up.6-R.sup.7-R.sup.8
in which R.sup.1 and R.sup.2 together form a group of formula
X-R.sup.A-R.sup.B-, wherein X is H or a one to three peptide group
R.sup.A is selected from Asp, Glu, Asn, Acpc, Ala, Me.sup.2Gly,
Pro, Bet, Glu(NH.sub.2), Gly, Asp(NH2) and Suc; R.sup.B is selected
from Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys; R.sup.3 is
selected from the group consisting of Val, Ala, Leu, norLeu, Ile,
Gly, Pro, Aib, Acpc, Lys and Tyr; R.sup.4 is selected from the
group consisting of Tyr, Tyr(PO.sub.3).sub.2, Thr, Ser, Ala,
homoSer and azaTyr; R.sup.5 is selected from the group consisting
of Ile, Ala, Leu, norLeu, Val and Gly; R.sup.6 is His, Arg or
6-NH.sub.2-Phe; R.sup.7 is Pro or Ala; and R.sup.8 is selected from
the group consisting of Phe, Phe(Br), Ile and Tyr, excluding
sequences including R.sup.4 as a terminal Tyr group.
7. The improved chemically defined medium of claim 6 wherein the
active agent is selected from the group consisting of
angiotensinogen, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,
SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ
ID NO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34;
SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID
NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42.
8. The improved chemically defined medium of claim 6 wherein the
active agent is SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:18, SEQ ID
NO:31,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or
SEQ ID NO:42.
9. The improved chemically defined medium of claim 6 wherein the
concentration of active agent is between about 0.1 ng/ml and about
1.0 mg/ml.
10. The improved chemically defined medium of claim 6 where the
tissue equivalent is selected from the group consisting of a skin,
dermis, bone, bone marrow, pancreas, heart valve, vascular graft,
cartilage, ligament, liver, and kidney tissue equivalent.
11. An improved kit for the culture of tissue equivalents, wherein
the improvement comprises providing a). an amount effective to
accelerate generation of tissue equivalents of at least one active
agent comprising a sequence consisting of at least three contiguous
amino acids of groups R.sup.1-R.sup.8 in the sequence of general
formula I
R.sup.1-R.sup.2-R.sup.3-R.sup.4-R.sup.5-R.sup.6-R.sup.7-R.sup.8 in
which R.sup.1 and R.sup.2 together form a group of formula
X-R.sup.A-R.sup.B-, wherein X is H or a one to three peptide group
R.sup.A is selected from Asp, Glu, Asn, Acpc, Ala, Me.sup.2Gly,
Pro, Bet, Glu(NH.sub.2), Gly, Asp(NH.sub.2) and Suc; R.sup.B is
selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;
R.sup.3 is selected from the group consisting of Val, Ala, Leu,
norLeu, Ile, Gly, Pro, Aib, Acpc, Lys and Tyr; R.sup.4 is selected
from the group consisting of Tyr, Tyr(PO.sub.3).sub.2, Thr, Ser,
Ala, homoSer and azaTyr; R.sup.5 is selected from the group
consisting of Ile, Ala, Leu, norLeu, Val and Gly; R.sup.6 is His,
Arg or 6-NH.sub.2-Phe; R.sup.7 is Pro or Ala; and R.sup.8 is
selected from the group consisting of Phe, Phe(Br), Ile and Tyr,
excluding sequences including R.sup.4 as a terminal Tyr group; and
b) and instructions for use of the active agent to accelerate
generation of tissue equivalents.
12. The kit of claim 11 further comprising tissue culture
medium.
13. The kit claim 11 wherein the active agent is selected from the
group consisting of angiotensinogen, SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ
ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,
SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ
ID NO: 34; SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,
SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42.
14. The kit of claim 11 wherein the active agent is SEQ ID NO:1,
SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:31, SEQ ID NO:38, SEQ ID
NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42.
15. The kit of claim 11 wherein the concentration of active agent
is between about 0.1 ng/ml and about 1.0 mg/ml.
16. An improved method for producing a tissue equivalent, the
improvement comprising contacting the tissue equivalent with an
amount effective to accelerate generation of tissue equivalents of
at least one active agent comprising a sequence of the following
general formula: R1-Arg-R2-R3-R4-His-Pro-R5 wherein R1 is selected
from the group consisting of H, Gly and Asp; R2 is selected from
the group consisting of Val, Pro, and Acpc; R3 is selected from the
group consisting of Tyr and Tyr(PO.sub.3).sub.2; R4 is selected
from the group consisting of Ala, Val, Ile, Leu, and norLeu; and R5
is Phe, Ile, or is absent.
17. The method of claim 16 wherein the active agent is selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4,
SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:31, SEQ ID
NO:32, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ
ID NO:41, or SEQ ID NO:42.
18. The method of claim 16 wherein the concentration of active
agent is between about 0.1 ng/kg and about 1.0 mg/kg.
19. The method of claim 16 where the tissue equivalent is selected
from the group consisting of a skin, dermis, bone, bone marrow,
pancreas, heart valve, vascular graft, cartilage, ligament,
collagen lattice, liver, and kidney tissue equivalent.
20. The method of claim 16 where the tissue equivalent is selected
from the group consisting of a collagen lattice and dermis tissue
equivalent.
21. An improved chemically defined medium for the culture of tissue
equivalents, wherein the improvement comprises contacting the
tissue equivalent with an amount effective to accelerate generation
of tissue equivalents of at least one active agent comprising a
sequence of the following general formula:
R1-Arg-R2-R3-R4-His-Pro-R5 wherein R is selected from the group
consisting of H, Gly and A sp; R2 is selected from the group
consisting of Val, Pro, and Acpc; R3 is selected from the group
consisting of Tyr and Tyr(PO.sub.3).sub.2; R4 is selected from the
group consisting of Ala, Val, Ile, Leu, and norLeu; and R5 is Phe,
Ile, or is absent.
22. The chemically defined medium of claim 21 wherein the active
agent is selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:26, SEQ ID
NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:39, SEQ
ID NO:40, SEQ ID NO:41, or SEQ ID NO:42.
23. The method of claim 21 wherein the concentration of active
agent is between about 0.1 ng/kg and about 1.0 mg/kg.
24. The method of claim 21 where the tissue equivalent is selected
from the group consisting of a skin, dermis, bone, bone marrow,
pancreas, heart valve, vascular graft, cartilage, ligament,
collagen lattice, liver, and kidney tissue equivalent.
25. The method of claim 21 where the tissue equivalent is selected
from the group consisting of a collagen lattice and dermis tissue
equivalent.
26. An improved kit for the culture of tissue equivalents, wherein
the improvement comprises providing a). an amount effective to
accelerate generation of tissue equivalents of at least one active
agent comprising a sequence of the following general formula:
R1-Arg-R2-R3-R4-His-Pro-R5 wherein R1 is selected from the group
consisting of H, Gly and Asp; R2 is selected from the group
consisting of Val, Pro, and Acpc; R3 is selected from the group
consisting of Tyr and Tyr(PO.sub.3).sub.2; R4 is selected from the
group consisting of Ala, Val, Ile, Leu, and norLeu; and R5 is Phe,
Ile, or is absent; and b) instructions for use of the active agent
to accelerate generation of tissue equivalents.
27. The kit of claim 26 wherein the active agent is selected from
the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32,
SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, or SEQ ID NO:42.
28. The kit of claim 26 wherein the concentration of active agent
is between about 0.1 ng/kg and about 1.0 mg/kg.
29. The kit of claim 26 where the tissue equivalent is selected
from the group consisting of a skin, dermis, bone, bone marrow,
pancreas, heart valve, vascular graft, cartilage, ligament,
collagen lattice, liver, and kidney tissue equivalent.
30. The kit of claim 16 where the tissue equivalent is selected
from the group consisting of a collagen lattice and dermis tissue
equivalent.
Description
CROSS REFERENCE
[0001] This application is a continuation in part of U.S.
Application Serial No. 60/077,499 filed Mar. 11, 1998 and
60/089,064 filed Jun. 12, 1998.
FIELD OF THE INVENTION
[0002] This present invention relates to methods, tissue culture
medium, and kits for accelerating the production living tissue
equivalents.
BACKGROUND OF THE INVENTION
[0003] Recently, various systems for the in vitro production of
tissue equivalents have been described. As used herein, the term
"tissue" comprises any group or layer of cells which together
perform one or more certain functions. Such tissue equivalents
include, but are not limited to, equivalents of epithelial tissue,
connective tissue, cartilage, bone, organs, vascular grafts, glands
and blood vessels and comprise living cells and extracellular
matrix molecules, principally collagen, and may optionally be
provided with components not typically found in normal tissue.
[0004] Three-dimensional cell culture systems have been described
which can be used to culture a variety of different cells and
tissues in vitro for prolonged periods of time (U.S. Pat. Nos.
5,624,840; 5,541,107; 5,521,087; 5,516,681, 5,516,680; 5,512,475,
herein incorporated by reference in their entirety). Cells derived
from a desired tissue are inoculated and grown on a pre-established
stromal support matrix. The stromal support matrix comprises
stromal cells, such as fibroblasts, actively growing on a
three-dimensional matrix. Stromal cells may also include other
cells found in loose connective tissue such as endothelial cells,
macrophages/monocytes, adipocytes, pericytes, reticular cells found
in bone marrow stroma, etc. The stromal matrix provides the
support, growth factors, and regulatory factors necessary to
sustain long-term active proliferation of cells in culture. When
grown in this three-dimensional system, the proliferating cells
mature and segregate properly to form components of adult tissues
analogous to counterparts found in vivo.
[0005] These inventions are based, in part, on the discovery that
growth of stromal cells in three dimensions will sustain active
proliferation of cells in culture for longer periods of time than
will monolayer systems (U.S. Pat. No. 5,510,254). This may be due,
in part, to the increased surface area of the three-dimensional
matrix which results in a prolonged period of active proliferation
of stromal cells. These proliferating stromal cells elaborate
proteins, growth factors and regulatory factors necessary to
support the long term proliferation of both stromal and
tissue-specific cells inoculated onto the stromal matrix. In
addition, the three-dimensionality of the matrix allows for a
spatial distribution which more closely approximates conditions in
vivo, thus allowing for the formation of microenvironments
conducive to cellular maturation and migration. The growth of cells
in the presence of this support may be further enhanced by adding
proteins, glycoproteins, glycosaminoglycans, a cellular matrix, and
other materials to the support itself or by coating the support
with these materials.
[0006] Similarly, tissue equivalents comprising a hydrated collagen
lattice contracted by a contractile agent, such as fibroblast cells
or blood platelets, in combination with a variety of cell types to
form the tissue equivalent are disclosed in U.S. Pat. Nos.
4,485,096; 4,485,097; 4,539,716; 4,546,500; 4,604,346; and
4,835,102; 4,837,379; 5,256,418; 5,536,656; and RE 35,399, all of
which are incorporated herein by reference.
[0007] In specific embodiments of the invention, bone marrow, bone,
skin, dermis, liver, kidney, cartilage, ligament, tendon, pancreas,
and heart valve tissues may be grown in the three dimensional
culture system. The resulting cultures have a variety of
applications ranging from transplantation or implantation, in vivo,
of cells grown in the cultures, cytotoxicity testing and screening
compounds in vitro, and the design of "bioreactors" for the
production of biological materials in vitro. For example, skin
tissue equivalents can be used as grafts to treat bum victims or
ulcer patients, while kidney and liver tissue equivalents can be
used for transplanting where disease has caused organ damage or
failure. For diffuse tissues such as bone marrow, the proliferating
cells could be isolated from the culture system for
transplantation. Tendon, ligament, and cartilage tissue equivalents
can be used for transplantation or prosthetics for seriously
damaged tissue.
[0008] While the methods described above have met with some
success, improved methods that accelerate the generation or improve
the quality of tissue equivalents would be useful for more rapidly
providing usable tissue equivalents. In particular, it would be
useful to provide improved methods that promote more rapid
acceleration of the cell type of interest and that accelerates the
production of extracellular matrix by stromal cells in the tissue
equivalent, and also improves the quality of the extracellular
matrix produced.
SUMMARY OF THE INVENTION
[0009] The present invention provides methods that increase the
production of tissue equivalents that are useful in transplantation
therapy, drug testing, cytotoxicity testing of compounds,
production of cellular compounds in quantity, and laboratory
testing of tissue systems. In one aspect, the present invention
provides improved methods for accelerating the production of tissue
equivalents by contacting the tissue equivalent with
angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and
analogues thereof, angiotensin II (AII), AII analogues, AII
fragments or analogues thereof or AII AT.sub.2 type 2 receptor
agonists.
[0010] In another aspect of the present invention, an improved cell
culture medium is provided for the production of tissue
equivalents, wherein the improvement comprises addition to the
culture medium of an effective amount of angiotensinogen, AI, AI
analogues, AI fragments and analogues thereof, AII, AII analogues,
AII fragments or analogues thereof or AII AT.sub.2 type 2 receptor
agonists.
[0011] In a further aspect, the present invention provides kits for
the production of tissue equivalents, wherein the kits comprise an
effective amount of angiotensinogen, AI, Al analogues, and/or AI
fragments and analogues thereof, AII, AII analogues, AII fragments
or analogues thereof, and/or AII AT.sub.2 type 2 receptor agonists,
and instructions for culturing the tissue equivalents.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 is a graph showing the concentration-dependent effect
of AII on epidermal thickness.
[0013] FIG. 2 is a graph showing the effect of exposure to AII
during the outgrowth period on the area of an artificial dermis
that is covered with keratinocytes.
[0014] FIG. 3 is a graph showing the effect of AII on collagen
lattice formation by fibroblasts (750,000 cells per well).
[0015] FIG. 4 is a graph showing the effect of AII on collagen
lattice formation by fibroblasts (250,000 cells per well).
[0016] FIG. 5 is a graph showing the effect of on collagen lattice
formation by fibroblasts (75,000 cells per well).
[0017] FIG. 6 is a graph showing the effect of AII and AII
analogues and fragments on keratinocyte number on integra
susbstrate.
[0018] FIG. 7 is a graph showing the effect of AII and AII
analogues and fragments on keratinocyte number on Integra
susbstrate.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0019] As defined herein, the term "tissue equivalents" refers to
three-dimensional cell and tissue culture systems used for the long
term proliferation of cells and tissues in vitro in an environment
that more closely approximates that found in vivo. Such tissue
equivalents include, but are not limited to, equivalents of
epithelial tissue, connective tissue, cartilage, bone, organs,
vascular grafts, bone marrow, skin, dermis, liver, kidney,
cartilage, ligament, tendon, pancreas, heart valve tissues, glands
and blood vessels and comprise living cells and extracellular
matrix molecules, principally collagen, and may optionally be
provided with components not typically found in normal tissue.
[0020] Unless otherwise indicated, the term "active agents" as used
herein refers to the group of compounds comprising angiotensinogen,
angiotensin I (AI), AI analogues, AI fragments and analogues
thereof, angiotensin II analogues, AII fragments or analogues
thereof and AII AT.sub.2 type 2 receptor agonists.
[0021] U.S. Pat. No. 5,015,629 to DiZerega (the entire disclosure
of which is hereby incorporated by reference) describes a method
for increasing the rate of healing of wound tissue, comprising the
application to such tissue of angiotensin II (AII) in an amount
which is sufficient for said increase. The application of AII to
wound tissue significantly increases the rate of wound healing,
leading to a more rapid re-epithelialization and tissue repair. The
term AII refers to an octapeptide present in humans and other
species having the sequence Asp-Arg-Val-Tyr-Ile-His-Pro-- Phe [SEQ
ID NO:1]. The biological formation of angiotensin is initiated by
the action of renin on the plasma substrate angiotensinogen
(Circulation Research 60:786-790 (1987); Clouston et al., Genomics
2:240-248 (1988); Kageyama et al., Biochemistry 23:3603-3609;
Ohkubo et al., Proc. Natl. Acad. Sci. 80:2196-2200 (1983); all
references hereby incorporated in their entirety). The substance so
formed is a decapeptide called angiotensin I (AI) which is
converted to AII by the converting enzyme angiotensinase which
removes the C-terminal His-Leu residues from AI [SEQ ID NO:37]. AII
is a known pressor agent and is commercially available.
[0022] Studies have shown that AII increases mitogenesis and
chemotaxis in cultured cells that are involved in wound repair, and
also increases their release of growth factors and extracellular
matrices (dizerega, U.S. Pat. No. 5,015,629; Dzau et. al., J. Mol.
Cell. Cardiol. 21:S7 (Supp III) 1989; Berk et. al., Hypertension
13:305-14 (1989); Kawahara, et al., BBRC 150:52-9 (1988); Naftilan,
et al., J. Clin. Invest. 83:1419-23 (1989); Taubman et al., J.
Biol. Chem. 264:526-530 (1989); Nakahara, et al. BBRC 184:811-8
(1992); Stouffer and Owens, Circ. Res. 70:820 (1992); Wolf, et al.,
Am. J. Pathol. 140:95-107 (1992); Bell and Madri, Am. J. Pathol.
137:7-12 (1990).
[0023] In addition, AII was shown to be angiogenic in rabbit
corneal eye and chick chorioallantoic membrane models (Fernandez,
et al., J. Lab. Clin. Med. 105:141 (1985); LeNoble, et al., Eur. J.
Pharmacol. 195:305-6 (1991). Additionally, AII and angiotensin III
analogs and fragments thereof have been shown to be effective in
tissue repair. (U.S. Pat. No. 5,629,292; International Application
No. WO 95/08565; International Application WO 95/08337;
International Application No. WO 96/39164; all references hereby
incorporated in their entirety.)
[0024] Although AII has been shown to increase the proliferation of
a number of cell types in vitro, it does not necessarily increase
the proliferation of all cell types. AII has been shown to increase
cellular proliferation in hair follicles in the area of a thermal
injury. (Rodgers et al., J. Burn Care Rehabil. 18:381-388 (1997).
The effect of AII on a given cell type has been hypothesized to be
dependent, in part, upon the AII receptor subtypes the cell
expresses (Shanugam et al., Am. J. Physiol. 268:F922-F930 (1995);
Helin et al., Annals of Medicine 29:23-29 (1997); Bedecs et al.,
Biochem J. 325:449-454 (1997)). These studies have shown that AII
receptor subtype expression is a dynamic process that changes
during development, at least in some cell types (Id.) While the
preceding studies suggest that AII and other AII receptor agonists
may accelerate wound repair through increased neovascularization,
growth factor release, reepithelialization and/or production of
extracellular matrix, the effect of angiotensinogen, AI, AI
analogues, AI fragments and analogues thereof, AI, AII analogues,
AII fragments or analogues thereof or AII AT.sub.2 type 2 receptor
agonists on accelerating the generation of tissue equivalents is
unknown.
[0025] A peptide agonist selective for the AT2 receptor (AII has
100 times higher affinity for AT2 than AT1) has been identified.
This peptide is p-aminophenylalanine6-AII ["(p-NH2-Phe)6-AII)"],
Asp-Arg-Val-Tyr-Ile-Xaa-- Pro-Phe [SEQ ID NO.36] wherein Xaa is
p-NH.sub.2-Phe (Speth and Kim, BBRC 169:997-1006 (1990). This
peptide gave binding characteristics comparable to AT2 antagonists
in the experimental models tested (Catalioto, et al., Eur. j
Pharmacol. 256:93-97 (1994); Bryson, et al., Eur. J. Pharmacol.
225:119-127 (1992).
[0026] The effects of AII receptor and AII receptor antagonists
have been examined in two experimental models of vascular injury
and repair which suggest that both AII receptor subtypes (AT1 and
AT2) play a role in wound healing (Janiak et al., Hypertension
20:737-45 (1992); Prescott, et al., Am. J Pathol. 139:1291-1296
(1991); Kauffinan, et al., Life Sci. 49:223-228 (1991);
Viswanathan, et al., Peptides 13:783-786 (1992); Kimura, et al.,
BBRC 187:1083-1090 (1992).
[0027] Many studies have focused upon AII(1-7) (AII residues 1-7)
or other fragments of AII to evaluate their activity. AII(1-7)
elicits some, but not the full range of effects elicited by AII.
Pfeilschifter, et al., Eur. J Pharmacol. 225:57-62 (1992); Jaiswal,
et al., Hypertension 19(Supp. II):II-49-II-55 (1992); Edwards and
Stack, J. Pharmacol. Exper. Ther. 266:506-510 (1993); Jaiswal, et
al., J. Pharmacol. Exper. Ther. 265:664-673 (1991); Jaiswal, et
al., Hypertension 17:1115-1120 (1991); Portsi, et a., Br. J.
Pharmacol. 111:652-654 (1994).
[0028] As hereinafter defined, a preferred class of AT2 agonists
for use in accordance with the present invention comprises AI, AI
analogues, AI fragments and analogues thereof, AII, AII analogues
or active fragments thereof having p-NH-Phe in a position
corresponding to a position 6 of AII. In addition to peptide
agents, various nonpeptidic agents (e.g., peptidomimetics) having
the requisite AT2 agonist activity are further contemplated for use
in accordance with the present invention.
[0029] The active AI, AI analogues, AI fragments and analogues
thereof, AII analogues, fragments of AII and analogues thereof of
particular interest in accordance with the present invention are
characterized as comprising a sequence consisting of at least three
contiguous amino acids of groups R.sup.1-R.sup.8 in the sequence of
general formula I
R.sup.1-R.sup.2-R.sup.3-R.sup.4-R.sup.5-R.sup.6-R.sup.7-R.sup.8
[0030] in which R.sup.1 and R.sup.2 together form a group of
formula
X-R.sup.A-R.sup.B-,
[0031] wherein X is H or a one to three peptide group,
[0032] R.sup.A is suitably selected from Asp, Glu, Asn, Acpc
(1-aminocyclopentane carboxylic acid), Ala, Me.sup.2Gly, Pro, Bet,
Glu(NH.sub.2), Gly, Asp(NH.sub.2) and Suc,
[0033] R.sup.B is suitably selected from Arg, Lys, Ala, Orn,
Ser(Ac), Sar, D-Arg and D-Lys;
[0034] R.sup.3 is selected from the group consisting of Val, Ala,
Leu, Lys, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;
[0035] R.sup.4 is selected from the group consisting of Tyr,
Tyr(PO.sub.3).sub.2, Thr, Ser, Ala, homoSer and azaTyr;
[0036] R.sup.5 is selected from the group consisting of Ile, Ala,
Leu, norLeu, Val and Gly;
[0037] R.sup.6 is His, Arg or 6-NH.sub.2-Phe;
[0038] R.sup.7 is Pro or Ala; and
[0039] R.sup.8 is selected from the group consisting of Phe,
Phe(Br), Ile and Tyr, excluding sequences including R.sup.4 as a
terminal Tyr group.
[0040] Compounds falling within the category of AT2 agonists useful
in the practice of the invention include the AII analogues set
forth above subject to the restriction that R.sup.6 is
p-NH.sub.2-Phe.
[0041] Particularly preferred combinations for R.sup.A and R.sup.B
are Asp-Arg, Asp-Lys, Glu-Arg and Glu-Lys. Particularly preferred
embodiments of this class include the following: AII, AIII or
AII(2-8), Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]; AII(3-8), also
known as des1-AIII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:3];
AII(1-7), Asp-Arg-Val-Tyr-Ile-His-Pro {SEQ ID NO:4]; AII(2-7).
Arg-Val-Tyr-Ile-His-Pro [SEQ ID NO:5]; AII(3-7),
Val-Tyr-Ile-His-Pro [SEQ ID NO:6]; AII(5-8), Ile-His-Pro-Phe [SEQ
ID NO:7]; AII(1-6), Asp-Arg-Val-Tyr-Ile-His [SEQ ID NO:8];
AII(1-5), Asp-Arg-Val-Tyr-Ile [SEQ ID NO:9]; AII(1-4),
Asp-Arg-Val-Tyr [SEQ ID NO:10]; and AII(1-3), Asp-Arg-Val [SEQ ID
NO:11]. Other preferred embodiments include:
Arg-norLeu-Tyr-Ile-His-Pro-Phe [SEQ ID NO:12] and
Arg-Val-Tyr-norLeu-His-- Pro-Phe [SEQ ID NO: 13]. Still another
preferred embodiment encompassed within the scope of the invention
is a peptide having the sequence Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe
[SEQ ID NO:31]. AII(6-8), His-Pro-Phe [SEQ ID NO:14] and AII(4-8),
Tyr-Ile-His-Pro-Phe [SEQ ID NO:15] were also tested and found not
to be effective.
[0042] In a particularly preferred embodiment, the active compounds
of the present invention are selected from those comprising the
following general formula:
R1-Arg-R2-R3-R4-His-Pro-R5,
[0043] wherein R1 is selected from the group consisting of H, Gly
and Asp;
[0044] R2 is selected from the group consisting of Val, Pro, and
Acpc;
[0045] R3 is selected from the group consisting of Tyr and
Tyr(PO.sub.3).sub.2;
[0046] R4 is selected from the group consisting of Ala, Val, Ile,
Leu, and norLeu; and
[0047] R5 is Phe, Ile, or is absent.
[0048] Another class of compounds of particular interest in
accordance with the present invention are those of the general
formula II
R.sup.2-R.sup.3-R.sup.4-R.sup.5-R.sup.6-R.sup.7-R.sup.8
[0049] in which R.sup.2 is selected from the group consisting of H,
Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys;
[0050] R.sup.3 is selected from the group consisting of Val, Ala,
Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;
[0051] R.sup.4 is selected from the group consisting of Tyr,
Tyr(PO.sub.3).sub.2, Thr, Ser, homoSer and azaTyr;
[0052] R.sup.5 is selected from the group consisting of Ile, Ala,
Leu, norLeu, Val and Gly;
[0053] R6 is His, Arg or 6-NH.sub.2-Phe;
[0054] R.sup.7 is Pro or Ala; and
[0055] R.sup.8 is selected from the group consisting of Phe,
Phe(Br), Ile and Tyr.
[0056] A particularly preferred subclass of the compounds of
general formula II has the formula
R.sup.2-R.sup.3-Tyr-R.sup.5-His-Pro-Phe [SEQ ID NO: 16]
[0057] wherein R.sup.2, R.sup.3 and R.sup.5 are as previously
defined. Particularly preferred is angiotensin III of the formula
Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]. Other preferred
compounds include peptides having the structures
Arg-Val-Tyr-Gly-His-Pro-Phe [SEQ ID NO: 17] and
Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO:18]. The fragment AII(4-8)
was ineffective in repeated tests; this is believed to be due to
the exposed tyrosine on the N-terminus.
[0058] In the above formulas, the standard three-letter
abbreviations for amino acid residues are employed. In the absence
of an indication to the contrary, the L-form of the amino acid is
intended. Other residues are abbreviated as follows:
1TABLE 1 Abbreviation for Amino Acids Me.sup.2Gly
N,N-dimethylglycyl Bet 1-carboxy-N,N,N-trimethylmethanaminium
hydroxide inner salt (betaine) Suc Succinyl Phe(Br)
p-bromo-L-phenylalanyl azaTyr aza-.alpha.'-homo-L-tyrosyl Acpc
1-aminocyclopentane carboxylic acid Aib 2-aminoisobutyric acid Sar
N-methylglycyl (sarcosine)
[0059] It has been suggested that AII and its analogues adopt
either a gamma or a beta turn (Regoli, et al., Pharmacological
Reviews 26:69 (1974). In general, it is believed that neutral side
chains in position R.sup.3, R.sup.5 and R.sup.7 may be involved in
maintaining the appropriate distance between active groups in
positions R.sup.4, R.sup.6 and R.sup.8 primarily responsible for
binding to receptors and/or intrinsic activity: Hydrophobic side
chains in positions R.sup.3, R.sup.5 and R.sup.8 may also play an
important role in the whole conformation of the peptide and/or
contribute to the formation of a hypothetical hydrophobic
pocket.
[0060] Appropriate side chains on the amino acid in position
R.sup.2 may contribute to affinity of the compounds for target
receptors and/or play an important role in the conformation of the
peptide. For this reason, Arg and Lys are particularly preferred as
R2.
[0061] For purposes of the present invention, it is believed that
R.sup.3 may be involved in the formation of linear or nonlinear
hydrogen bonds with R.sup.5 (in the gamma turn model) or R.sup.6
(in the beta turn model). R.sup.3 would also participate in the
first turn in a beta antiparallel structure (which has also been
proposed as a possible structure). In contrast to other positions
in general formula I, it appears that beta and gamma branching are
equally effective in this position. Moreover, a single hydrogen
bond may be sufficient to maintain a relatively stable
conformation. Accordingly, R.sup.3 may suitably be selected from
Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr.
Furthermore, Lys has surprisingly been found to be suitable at
R.sup.3 (see Examples).
[0062] With respect to R.sup.4, conformational analyses have
suggested that the side chain in this position (as well as in
R.sup.3 and R.sup.5) contribute to a hydrophobic cluster believed
to be essential for occupation and stimulation of receptors. Thus,
R.sup.4 is preferably selected from Tyr, Thr, Tyr (PO.sub.3).sub.2,
homoSer, Ser and azaTyr. Furthermore, Ala has surprisingly been
found to be suitable at the R.sup.4 position (See Examples). In
this position, Tyr is particularly preferred as it may form a
hydrogen bond with the receptor site capable of accepting a
hydrogen from the phenolic hydroxyl (Regoli, et al. (1974),
supra).
[0063] In position R.sup.5, an amino acid with a .beta. aliphatic
or alicyclic chain is particularly desirable. Therefore, while Gly
is suitable in position R.sup.5, it is preferred that the amino
acid in this position be selected from Ile, Ala, Leu, norLeu, Gly
and Val.
[0064] In the AI, AI analogues, AI fragments and analogues thereof,
AII analogues, fragments and analogues of fragments of particular
interest in accordance with the present invention, R.sup.6 is His,
Arg or 6-NH.sub.2-Phe. The unique properties of the imidazole ring
of histidine (e.g., ionization at physiological pH, ability to act
as proton donor or acceptor, aromatic character) are believed to
contribute to its particular utility as R.sup.6. For example,
conformational models suggest that His may participate in hydrogen
bond formation (in the beta model) or in the second turn of the
antiparallel structure by influencing the orientation of R.sup.7.
Similarly, it is presently considered that R.sup.7 should be Pro in
order to provide the most desirable orientation of R.sup.8. In
position R.sup.8, both a hydrophobic ring and an anionic carboxyl
terminal appear to be particularly useful in binding of the
analogues of interest to receptors; therefore, Tyr and especially
Phe are preferred for purposes of the present invention.
[0065] Analogues of particular interest include the following:
2TABLE 2 Angiotensin II Analogues AII Analogue Sequence Name Amino
Acid Sequence Identifier Analogue 1 Asp-Arg-Val-Tyr-Val-His-Pro-Phe
SEQ ID NO:19 Analogue 2 Asn-Arg-Val-Tyr-Val-His-Pro-Phe SEQ ID
NO:20 Analogue 3 Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe SEQ ID
NO:21 Analogue 4 Glu-Arg-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO:22
Analogue 5 Asp-Lys-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO:23 Analogue 6
Asp-Arg-Ala-Tyr-Ile-His-Pro-Phe SEQ ID NO:24 Analogue 7
Asp-Arg-Val-Thr-Ile-His-Pro-Phe SEQ ID NO:25 Analogue 8
Asp-Arg-Val-Tyr-Leu-His-Pro-Phe SEQ ID NO:26 Analogue 9
Asp-Arg-Val-Tyr-Ile-Arg-Pro-Phe SEQ ID NO:27 Analogue 10
Asp-Arg-Val-Tyr-Ile-His-Ala-Phe SEQ ID NO:28 Analogue 11
Asp-Arg-Val-Tyr-Ile-His-Pro-Tyr SEQ ID NO:29 Analogue 12
Pro-Arg-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO:30 Analogue 13
Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe SEQ ID NO:31 Analogue 14
Asp-Arg-Val-Tyr(PO.sub.3).sub.2-Ile-His-Pro-Phe SEQ ID NO:32
Analogue 15 Asp-Arg-norLeu-Tyr-Ile-His-Pro-Phe SEQ ID NO:33
Analogue 16 Asp-Arg-Val-Tyr-norLeu-His-Pro-Phe SEQ ID NO:34
Analogue 17 Asp-Arg-Val-homoSer-Tyr-Ile-His-Pro-- Phe SEQ ID
NO:35
[0066] The polypeptides of the instant invention may be synthesized
by methods such as those set forth in J. M. Stewart and J. D.
Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co.,
Rockford, Ill. (1984) and J. Meienhofer, Hormonal Proteins and
Peptides, Vol. 2, Academic Press, New York, (1973) for solid phase
synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1,
Academic Press, New York, (1965) for solution synthesis. The
disclosures of the foregoing treatises are incorporated by
reference herein.
[0067] In general, these methods involve the sequential addition of
protected amino acids to a growing peptide chain (U.S. Pat. No.
5,693,616, herein incorporated by reference in its entirety).
Normally, either the amino or carboxyl group of the first amino
acid and any reactive side chain group are protected. This
protected amino acid is then either attached to an inert solid
support, or utilized in solution, and the next amino acid in the
sequence, also suitably protected, is added under conditions
amenable to formation of the amide linkage. After all the desired
amino acids have been linked in the proper sequence, protecting
groups and any solid support are removed to afford the crude
polypeptide. The polypeptide is desalted and purified, preferably
chromatographically, to yield the final product.
[0068] In one aspect of the present invention, a method of
accelerating the production of tissue equivalents by exposure to
angiotensinogen, AI, AI analogues, AI fragments and analogues
thereof, AII analogues, AII fragments or analogues thereof or AII
AT.sub.2 type 2 receptor agonists (the "active agents") is
disclosed. Experimental conditions for the production of various
tissue equivalents have been reported as follows: liver tissue
equivalents (U.S. Pat. No. 5,624,840), bone marrow tissue
equivalents (U.S. Pat. No. 5,541,107), ligament tissue equivalents
(U.S. Pat. No. 5,521,087), kidney tissue equivalents (U.S. Pat. No.
5,516,680), blood vessel tissue equivalents (U.S. Pat. No.
5,256,418), vascular graft equivalents (U.S. Pat. No. 5,628,781 and
5,387,236) and skin tissue equivalents (5,512,475, 4,835,102, and
RE 35,399), all references herein incorporated by reference in
their entirety.
[0069] In one embodiment, tissue equivalents are prepared according
to standard methods (U.S. Pat. Nos. 5,624,840, 5,541,107,
5,521,087, 5,516,680, 5,256,418, 5,512,475, 4,835,102, and RE
35,399) and incubated in the presence of, preferably, between about
0.1 ng/ml and about 1 mg/ml of the active agents of the
invention.
[0070] In another embodiment, a dermal equivalent is formed by the
inoculation of fibroblasts onto a three-dimensional matrix and
their growth to subconfluence (U.S. Pat. No. 5,578,485,
incorporated by reference herein in its entirety). The
three-dimensional support may be of any material and/or shape that:
(a) allows cells to attach to it (or can be modified to allow cells
to attach to it); and (b) allows cells to grow in more than one
layer. A number of different materials may be used to form the
matrix, including but not limited to: nylon (polyamides), dacron
(polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl
compounds (e.g., polyvinylchloride), polycarbonate (PVC),
polytetrafluorethylene (PTFE; teflon), thermanox (TPX),
nitrocellulose, cotton, polyglycolic acid (PGA), cat gut sutures,
cellulose, gelatin, dextran, etc. Any of these materials may be
woven into a mesh, for example, to form the three-dimensional
matrix. Certain materials, such as nylon, polystyrene, etc., are
poor substrates for cellular attachment. When these materials are
used as the three-dimensional support matrix, it is advisable to
pre-treat the matrix prior to inoculation of stromal cells in order
to enhance the attachment of stromal cells to the matrix. For
example, prior to inoculation with stromal cells, nylon matrices
could be treated with 0.1M acetic acid, and incubated in
polylysine, FBS, and/or collagen to coat the nylon. Polystyrene
could be similarly treated using sulfuric acid.
[0071] Where the three-dimensional culture is itself to be
implanted in vivo, it may be preferable to use biodegradable
matrices such as poly glycolic acid, catgut suture material, or
gelatin, for example (U.S. Pat. No. 5,578,485). Where the cultures
are to be maintained for long periods of time or cryopreserved,
non-degradable materials such as nylon, dacron, polystyrene,
polyacrylates, polyvinyls, teflons, cotton, etc. may be preferred.
A convenient nylon mesh which could be used in accordance with the
invention is Nitex, a nylon filtration mesh having an average pore
size of 210 mu m and an average nylon fiber diameter of 90 mu m
(#3-210/36, Tetko, Inc., N.Y.).
[0072] In a preferred embodiment, the fibroblasts are allowed to
proliferate until the entire growth substrate is covered, although
only approximately 60% confluency of the fibroblasts on the
three-dimensional matrix is required to support the growth of
epidermal cells later inoculated. The fibroblasts will continue to
divide even after they have reached confluency because the
three-dimensional culture permits the exit of cells, thereby
preventing contact inhibition. Although any fibroblasts may be
utilized in the inoculum, it is advantageous to use skin
fibroblasts, as these will deposit the appropriate types of
collagen and elaborate other dermal components. Fibroblasts may be
allogeneic or autologous. Skin fibroblasts may be readily obtained
from cellular suspensions prepared by mechanical and/or enzymatic
disaggregation of dermal tissue. When the cellular suspension
obtained is plated, the fibroblasts will adhere more quickly than
other cells, and thus, can be grown to confluence, lifted by mild
enzymatic treatment and inoculated onto the three-dimensional
matrix.
[0073] While the use of fibroblasts alone is sufficient to form a
three-dimensional stromal matrix that functions as a dermal
equivalent, additional types of stromal cells may be used to
inoculate the three-dimensional matrix. These include, but are not
limited to endothelial cells, pericytes, macrophages, monocytes,
lymphocytes, plasma cells, adipocytes, etc.
[0074] In a further preferred embodiment, epidermal cells are
inoculated onto the dermal equivalent to provide full thickness
skin equivalents (U.S. Pat. No. 5,578,485, incorporated by
reference herein in its entirety). To this end, melanocytes and
keratinocytes may be inoculated simultaneously, or preferably, in
sequence. For example, keratinocytes can be inoculated onto
subconfluent melanocytes which were previously inoculated onto the
stromal matrix.
[0075] Melanocytes and keratinocytes may be allogeneic or
autologous in their relationship to fibroblast stromal cells, can
be isolated from skin using known procedures which involve
incubating skin in a digestive enzyme, such as trypsin, in order to
separate dermal and epidermal layers.
[0076] In one embodiment, keratinocytes and melanocytes may be
isolated as follows. A tissue sample, e.g. foreskin, may be trimmed
so that the entire surface may be easily exposed to antibiotics.
Tissue may be first washed in a concentrated antibiotic solution
for twenty minutes, followed by two subsequent washes of ten
minutes each. The outer portion of the tissue may then be cut into
small pieces, and then placed in a 0.15% trypsin solution (in PBS
without calcium or magnesium), quickly removed, placed in a fresh
container of the same trypsin solution (such that all the tissue is
covered by solution), and refrigerated overnight at between about
2.degree. C. and 8.degree. C. The next day, the tissue pieces may
be removed from the trypsin solution, and the epidermis separated
from the dermis using curved forceps. The epidermis may be placed
in a conical tube, and about 0.15% trypsin in PBS (without calcium
or magnesium) may be used to digest the tissue into a single cell
suspension; to facilitate this process, the sample may be
repeatedly aspirated into and out of a Pasteur pipette. When the
sample appears to be a single cell suspension, it may be
centrifuged at 1400 g for about 7 minutes and then resuspended in
either growth media or in growth media containing 0.01 mg/ml PMA,
which selects for melanocytes. Accordingly, cultures of
keratinocytes or melanocytes may be produced. The epidermal cells
can be suspended and used to inoculate the dermal equivalent.
Alternatively, the epidermal cell suspension can be plated and
melanocytes and keratinocytes separated based upon their
differential attachment qualities. Isolated melanocytes may first
be inoculated onto the dermal equivalent and allowed to grow for a
few days prior to inoculation of keratinocytes. This "tissue" grows
rapidly and can be maintained in nutrient media without exogenous
growth factors.
[0077] In a preferred embodiment, a dermal equivalent produced as
set forth supra may be inoculated with keratinocytes as follows
(U.S. Pat. No. 5,478,739, incorporated by reference herein in its
entirety). Fresh dermal equivalent cultures, or dermal equivalent
cultures removed from the freezer and rinsed with PBS in order to
remove dimethyl sulfoxide (DMSO), may be allowed to equilibrate in
stratification medium (DMEM with 5 percent fetal bovine serum; 100
mu g/ml ascorbate (Sigma) and 0.5 mu g/ml hydrocortisone (Sigma))
for about 24-48 hours. Keratinocytes may then be seeded onto the
dermal equivalent at a density of about 5.times.10.sup.5
keratinocytes per cm of dermal equivalent. The keratinocyte/dermal
equivalent co-cultures may then be incubated submerged in
stratification medium for 5-7 days, then raised such that
keratinocytes may differentiate at the air/liquid interface. After
about 12-14 days in culture, a cholesterol-rich lipid supplement
(Sigma) (0.5%) may be added to the stratification medium and the
cultures may be grown for an additional 12-21 days until a
multi-layered stratum corneum is formed.
[0078] In another embodiment, bone marrow cells are grown on a
three-dimensional support in co-cultures with stromal cells
comprising fibroblasts (of either fetal or bone marrow origin) or a
mixture of cell types which comprise the stromal components of
normal marrow, including fibroblasts, macrophages, reticular cells,
and adipocytes (U.S. Pat. No. 5,541,107). Factors derived from
media of splenic and/or hepatic (liver) macrophage cultures or from
subsets of stromal cells may optionally be added to the culture.
The three-dimensional culture system of the present invention
appears to maximize the proliferation of multipotential
hematopoietic stem cells which have the capability of repopulating
bone marrow when the bone marrow has been destroyed by
intrinsically or environmentally-mediated disease or by the
treatment of such disease with chemotherapy and/or radiation.
[0079] Alternatively, a liver tissue equivalent is produced by
inoculation and culturing of liver parenchymal cells on a
pre-established three-dimensional stromal tissue (U.S. Pat. No.
5,624,840). The stromal tissue comprises stromal cells grown on a
three-dimensional matrix or framework. The stromal cells comprise
fibroblasts with or without additional cells and/or elements. The
fibroblasts and other cells and/or elements that comprise the
stroma may be fetal or adult in origin, and may be derived from
convenient sources such as skin, liver, pancreas, etc. Such tissues
and/or organs can be obtained by appropriate biopsy or upon
autopsy. In fact, cadaver organs may be used to provide a generous
supply of stromal cells and elements.
[0080] In another embodiment, bone, ligament, cartilage and tendon
equivalents, are produced by forming a collagen gel having living
connective tissue cells dispersed therein (U.S. Pat. No.
5,521,087). The cells are capable of contracting the gel. The gel
is maintained under conditions suitable for contraction by the
connective tissue cells, while simultaneous contraction of the gel
is restrained to define an axis of predetermined length for cell
alignment. The connective tissue cells align along the defined axis
to produce an oriented tissue-equivalent having increased
mechanical strength in the direction of the axis.
[0081] In another embodiment, kidney tissue equivalents are
prepared by culturing kidney parenchymal cells cultured on a living
stromal tissue prepared in vitro, said living stromal tissue
comprising stromal cells and connective tissue proteins naturally
secreted by the stromal cells attached to and substantially
enveloping a framework composed of a biocompatible, non-living
material formed into a three-dimensional structure having
interstitial spaces bridged by the stromal cells (U.S. Pat. No.
5,516,680).
[0082] In another embodiment, pancreatic tissue equivalents are
formed by mincing pancreatic tissue and washing in calcium-free,
magnesium-free buffer (U.S. Pat. No. 5,578,485, incorporated by
reference herein in its entirety.) The minced tissue fragments are
incubated in a solution of trypsin and collagenase. Dissociated
cells may be filtered using a 20 mu m nylon mesh, resuspended in a
suitable buffer such as Hanks balanced salt solution, and pelleted
by centrifugation. The resulting pellet of cells can be resuspended
in minimal amounts of appropriate media and inoculated onto the
three-dimensional stroma.
[0083] In another embodiment, living vascular graft equivalents,
comprising a polymeric backbone and endothelial cells are prepared
as described in U.S. Pat. Nos. 5,628,781 and 5,387,236, both
references hereby incorporated in their entirety. In a preferred
embodiment, a polymeric substrate for the vascular prosthesis is
dispersed in a solution of biological tissue fragments such as
vascular tissues, connective tissues, fat tissues and muscular
tissues and/or cells such as vascular endothelial cells, smooth
muscle cells and fibroblast cells. (U.S. Pat. No. 5,387,236) The
cells and/or tissue fragments are deposited and captured within the
wall and on the inner surface of the vascular prosthesis substrate
wall from the outside and/or the inside of the vascular prosthesis
substrate by providing a pressure differential between the outside
and the inside of the substrate. The interior space of the vascular
prosthesis can be vacuumized or pressurized, and the cells and/or
tissue fragments are deposited and captured in and on the walls
either outside or inside of the prosthesis substrate. In a
preferred embodiment, the living vascular graft further comprises
vascular smooth muscle cells and fibroblasts.
[0084] Methods for producing heart valve equivalents are described
in U.S. Pat. Nos. 5,713,950; 5,480,424; 5,192,312; and 5,052,934,
all references herein incorporated by reference in their
entirety.
[0085] In order to accelerate the generation of the all of the
above tissue equivalents, they are exposed to preferably, between
about 0.1 ng/ml and about 1 mg/ml of the active agents of the
invention as described above. Acceleration of tissue equivalent
generation by exposure to the lo active agents occurs via increased
proliferation of the cells that comprise the tissue equivalent,
increase in growth factor production by cells of the tissue
equivalent, and via increasing production of extracellular matrix
components by stromal cells within the tissue equivalents.
[0086] Proliferation of cells in the tissue equivalent can be
quantitated using any one of a variety of techniques well known in
the art, including, but not limited to, bromodeoxyuridine
incorporation (Vicario-Abejon et al., 1995), .sup.3H-thymidine
incorporation (Fredericksen et al., 1988), or antibody labeling of
a protein present in higher concentration in proliferating cells
than in non-proliferating cells. In a preferred embodiment,
accelerated production of tissue equivalents is assessed by
antibody detection of a protein known to be present in higher
concentrations in proliferating cells than in non-proliferating
cells, including but not limited to proliferating cell nuclear
antigen (PCNA, or cyclin; Zymed Laboratories, South San Francisco,
Calif.).
[0087] Increased production of growth factors, including but not
limited to transforming growth factor beta, fibroblast growth
factor, and epidermal growth factor, by cells of the tissue
equivalent can be quantitated by standard immunohistochemical
techniques using antibodies to the growth factors (DAKO,
Carpernterica, Calif.; Genzyme, Cambridge, Mass.; Sigma Chemical
Co., St. Louis, Mo.).
[0088] Increasing production of extracellular matrix components by
the stromal component of the tissue equivalent can be quantitated
by antibody labeling of extracellular matrix components, including
but not limited to fibronectin, elastin, glycosaminoglycans, and
laminin (DAKO, Carpernterica, Calif.; Pharmingen, San Diego,
Calif.).
[0089] In another aspect of the present invention, an improved cell
culture medium is provided for accelerated production of tissue
equivalents, wherein the improvement comprises addition to the cell
culture medium of an effective amount of active agents, as
described above. For any given active agent, the optimum
concentration for a given formulation may be readily determined
empirically. In general, a concentration of active agent suitable
for use in accordance with the present invention preferably ranges
between about 0.1 ng/ml and about 1 mg/ml active agents.
[0090] Any cell culture media that can support the growth of tissue
equivalents can be used with the present invention. Such cell
culture media include, but are not limited to Basal Media Eagle,
Dulbecco's Modified Eagle Medium, Iscove's Modified Dulbecco's
Medium, McCoy's Medium, Minimum Essential Medium, F-10 Nutrient
Mixtures, Opti-MEM.RTM. Reduced-Serum Medium, RPMI Medium, and
Macrophage-SFM Medium or combinations thereof. In a preferred
embodiment, the defined cell culture medium described in U.S. Pat.
No. 5,712,163 is used.
[0091] The improved cell culture medium can be supplied in either a
concentrated (ie: 10.times.) or non-concentrated form, and may be
supplied as either a liquid, a powder, or a lyophilizate. The cell
culture may be either chemically defined, or may contain a serum
supplement. Culture media is commercially available from many
sources, such as GIBCO BRL (Gaithersburg, Md.) and Sigma (St.
Louis, Mo.).
[0092] In a further aspect, the present invention provides kits for
the propagation of tissue equivalents, wherein the kits comprise an
effective amount of the active agents of the invention, and
instructions for their use in accelerating the production of tissue
equivalents.
[0093] In a preferred embodiment, the kit further comprises cell
culture growth medium. Any cell culture media that can support the
growth of tissue equivalents can be used with the present
invention. Examples of such cell culture media are described
above.
[0094] The improved cell culture medium can be supplied in either a
concentrated (ie: 10.times.) or non-concentrated form, and may be
supplied as a liquid, a powder, or a lyophilizate. The cell culture
may be either chemically defined, or may contain a serum
supplement. In a further embodiment, the kit further comprises a
three-dimensional support material.
EXAMPLE 1
Effect of AII on Epidermal Layer Thickness
[0095] The purpose of this study was to determine, by quantitative
histology, the effect of presoaking a living skin equivalent (LSE)
in a lactated Ringer's solution (LRS)-Dextrose containing AII, on
the thickness of the epidermis on day 23 post-grafting.
[0096] Twenty three male Swiss nude mice (22-24 grams) were
purchased from Taconic Laboratories and quarantined at least two
days prior to surgery, and divided into 4 groups of mice. The mice
were anesthetized with an intramuscular injection of Ketamine
(ketaset; Phoenix Pharmaceuticals, Inc.) and Rompun (xylazine;
Phoenix Pharmaceuticals, Inc.) and a 1 cm.times.1 cm full thickness
skin excision was made on their dorsal surface. Each group received
an Apligraph LSE (Organogenesis; Canton, Mass.) that had been
soaked for 10 minutes in concentrations of AII ranging from 0 to
1.0 mg/ml. The LSE was placed in the defect and trimmed with
microscissors so that no gap was observed between the edges of the
mouse skin and the LSE. After the graft was placed, the dorsal
surface of the mouse was covered by petroleum embedded gauze
(Dermacea) followed by two adhesive bandages (Baxter). After
recovery from anesthesia, the mice were returned to their
individual cages and observed daily until euthenasia. The mice
received intramuscular analgesia for the first three days after
surgery. No mouse lost their bandage prior to bandage removal on
day 7 and day 23. At necropsy, the degree of graft taken and the
appearance of the grafted tissue was noted prior to placement of
the biopsy in 10% buffered formalin in preparation for paraffin
embedding and sectioning for hematoxylin and eosin staining.
[0097] AII grafts appeared healthy (except one control which had
lost its graft) and inosculation was noted for 80-100% of the graft
edges. On day 7, one of the AII-treated grafts had numerous vessels
attached to its underside (against the fascia of the nude mice
after full thickness excision). This was not noted on any of the
other mice.
[0098] Measurement of the thickness of the epidermis at day 23 was
accomplished with an ocular micrometer in a 10.times. ocular at the
10.times. magnification on the objective (100.times. total
magnification). The thickest part of each graft was measured and
the thickness of the graft at one-half of a 10.times. field fo each
side of the thickest portion was also measured. As can be seen in
FIG. 1, the epidermal thickness increased in a
concentration-dependent manner after AII administration.
Example 2
Effect of AII on the Outgrowth of Keratinocytes from Explants of
Human Skin
[0099] Human skin explants were obtained from surgery of split
thickness skin grafts and cut by scalpel in to 2 mm.times.2 mm
squares. Ten explants per condition were placed dermal side down on
frozen and thawed Dermagraft (Advanced Tissue Sciences, San Diego,
Calif.) and cultured at the air-liquid interface using
Keratinocyte-SFM medium (Gibco BRL, Grand Island, N.Y.)
supplemented with epidermal growth factor and bovine pituitary
extract according to the manufacturer's instructions. The culture
medium contained penicillin (50 U/ml) and streptomycin (50
.mu.g/ml) and extra calcium chloride to a final concentration of 1
mM. Angiotensin II was added to the culture medium at a final
concentration of 1 or 10 .mu.g/ml. The cultures were maintained at
37.degree. C. with 5% CO.sub.2 and medium with and without AII and
the medium was changed twice weekly. After 7 days, the cultures
were fixed in formalin for one hour, stained with hematoxylin and
washed in tap water. The dermal replacements appeared dark purple
except for the area where the keratinocytes exclude the stain.
Cultures were photographed using a dissection microscope at a fixed
magnification and the area of outgrowth was quantified by
gravimetric planimetry. As shown in FIG. 2, exposure to AII during
the outgrowth period increased the area of the artificial dermis
covered with keratinocytes.
EXAMPLE 3
Contraction of Collagen Lattices by Human Fibroblasts
[0100] Contraction of collagen lattices is a first step in the
preparation of an artificial dermis, where keratinocytes are
subsequently grown in the collagen matrix. Collagen lattices can
also be implanted sub-cutaneously and used as a bulking-up agent
for plastic surgery applications.
[0101] Normal human fibroblasts were purchased from Clonetics (San
Diego, Calif.) and were thawed and cultured in Fibroblast Growth
Medium (Clonetics) according to the manufacturer's instructions.
Once the cells reached confluence in the flask, they were harvested
by trypsinization and utilized for the studies described below.
[0102] Rat tails were harvested from 200 gram female rats and
frozen until use. The frozen rat tails were thawed in 70% (vol/vol)
ethanol for 20 minutes. The tendon bundles were excised in 70%
ethanol in a vertical laminar flow hood. The individual tendons
were pulled out of the tendon sheath, minced, and placed in dilute
acetic acid (1:1000) using 250 ml per tail. The mixture was
incubated at 4.degree. C. for 48 hours, at which point the minced
tendons had swelled to the volume of the dilute acetic acid. The
viscous mixture was centrifuged at 23,000 rpm in a Beckman L
ultracentrifuge for 1 hour. The supernatant was harvested and
further refined. This crude collagen solution was mixed with 0.1M
NaOH in a 6:1 ratio to neutralize the acetic acid and precipitate
the collagen. The mixture was then centrifuged at 1500 rpm for 5
minutes. The supernatant was discarded and an equal volume of fresh
acetic acid (1:1000) was introduced to resolubilize the collagen
over 48 hours. This solution was stored at 4.degree. C. as refined
collagen. The protein concentration was determined by BCA
assay.
[0103] The collagen lattices for the assessment of contraction by
fibroblasts and angiotensin II were formed in 60 mm Falcon
bacteriological dishes. Each dish contained 1 ml 4.times. DMEM High
glucose medium, 1 ml fetal calf serum, 0.25 ml NaOH, 1.5 ml of 500
.mu.g/ml refined collagen and 1 ml of fibroblasts in Fibroblast
Growth Medium (7.5.times.10.sup.4 cells/ml to 7.5.times.10.sup.5
cells/ml). In these cultures, various concentrations of AII (1 to
10 .mu.g/ml) were added to assess the effect of AII on the
formation of an artificial dermis. The cultures were placed in an
incubator at 37.degree. C. in an atmosphere of 5% CO.sub.2 in air.
At various times after culture initiation, the diameter of the
formed lattice was measured. As there are slight differences in
diameter at various points (ie: the lattices were not always
perfectly round), the average of the largest and the smallest
diameters were taken. As shown in FIGS. 3-5, AII accelerated the
contraction of collagen lattices.
[0104] Similar experiments were conducted using AII analogues and
fragments, except that ten .mu.g/ml of each of the peptides shown
in Table 3 were added to 1.times.10.sup.5 of fibroblasts/well. At
various times after culture initiation, the diameter of the formed
lattice was measured. The results of these experiments are shown in
Table 4, and demonstrate that each of the AII analogues and
fragments accelerated the contraction of collagen lattices.
3TABLE 3 Designation for Analogues/Fragments Abbreviation Sequence
SEQ ID NO: Gly.sup.1-AII GRVYIHPF SEQ ID NO:39 NorLeu.sup.4-AIII
--RVYnLHPF SEQ ID NO:40 Acpc.sup.3-AII DR(Acpc)YIHPF SEQ ID NO:4l
Ile AII DRVYIHPI SEQ ID NO:38 Ala.sup.4-AIII --RVYAHPF SEQ ID NO:18
AII(1-7) DRVYIHP-- SEQ ID NO:4 AII DRVYIHPF SEQ ID NO.1
[0105]
4TABLE 4 Results of Collagen Lattice Contraction with AII Analogues
and Fragments Day of Culture Diameter of Lattice (cm) Peptide Day 1
Day 2 Day 3 Day 4 None 3.5 2.8 2.3 1.9 Ala4-AIII 3.4 2.7 1.9 1.6
Gly1 AII 3.4 2.2 1.6 1.5 NorLeu4 AIII 3.4 2.0 1.6 1.5 Acpc3 AII 3.5
2.0 1.6 1.4 AII(1-7) 3.4 2.4 2.0 1.7 AII 3.3 2.4 1.6 1.4 Ile8 AII
3.4 2.4 1.7 1.4
EXAMPLE 4
Effect of AII, AII Analogues and Fragments, and AII Fragment
Analogues on Keratinocyte Number on an Artificial Dermis
[0106] Integra, a commercially available artificial dermis of
chondrotin sulfate and collagen, was obtained (Integra Life
Sciences) and used in culture as a matrix for keratinocyte growth.
The Integras was washed free of preservative by a sterile saline
for injection and cut to size to fit snugly into the bottom of 24
well plates. After trimming, the pieces of Integra were placed
silicone-side down into the wells.
[0107] Human keratinocytes were purchased from Clonetics and thawed
and cultured as described in Example 3, except that they were grown
in Keratinocyte Growth Medium, as per the manufacturer's
instructions. Once the cells reached confluence in the flask, they
were detached from the tissue culture flasks by trypsinization. The
cells were resuspended at 1.times.10.sup.5 cells/ml in Keratinocyte
Growth Medium or Keratinocyte Basal Medium with or without 10
.mu.g/ml of various AII-related peptides (AII, AII(1-7), Pro3
AII(1-7), Ala4-AIII, and Pro3-AII; see Table 5 below). One ml of
these cell preparations was added to wells of the 24 well plates
containing Integra membrane. The cultures were placed in an
incubator at 37.degree. C. in an atmosphere of 5% CO.sub.2 in air.
At various times after initiation of the cultures, the number of
keratinocytes on the surface of the Integra membrane in 1
500.times. fields was assessed under phase contrast microscopy. The
data are summarized in FIGS. 6-7 and demonstrate that each of the
peptides tested increased the proliferation of keratinocytes on
this artificial membrane.
5TABLE 5 Designation for Analogues/Fragments Name Abbreviation
Sequence SEQ ID NO: GSD 24B Pro.sup.3-AII DRPYIHPF SEQ ID NO:31 2GD
Pro.sup.3-AII(1-7) DRPYIHP SEQ ID NO:42 GSD 22A Ala.sup.4-AIII
RVYAHIPF SEQ ID NO:18 AII(1-7) DRVYIHP-- SEQ ID NO:4 AII DRVYIHPF
SEQ ID NO.1
[0108] The present invention, by providing a method for enhanced
production of tissue equivalents, will greatly increase the
clinical benefits of tissue equivalent transplantation, as well as
increasing the utility of drug and cytotoxicity testing on tissue
equivalents, production of cellular compounds in quantity, and
laboratory testing of tissue equivalent systems.
[0109] The present invention is not limited by the aforementioned
particular preferred embodiments. It will occur to those ordinarily
skilled in the art that various modifications may be made to the
disclosed preferred embodiments without diverting from the concept
of the invention. All such modifications are intended to be within
the scope of the present invention.
Sequence CWU 1
1
42 1 8 PRT Artificial Sequence Description of Artificial
SequenceAII 1 Asp Arg Val Tyr Ile His Pro Phe 1 5 2 7 PRT
Artificial Sequence Description of Artificial SequenceAII (2-8) 2
Arg Val Tyr Ile His Pro Phe 1 5 3 6 PRT Artificial Sequence
Description of Artificial SequenceAII (3-8) 3 Val Tyr Ile His Pro
Phe 1 5 4 7 PRT Artificial Sequence Description of Artificial
SequenceAII (1-7) 4 Asp Arg Val Tyr Ile His Pro 1 5 5 6 PRT
Artificial Sequence Description of Artificial SequenceAII (2-7) 5
Arg Val Tyr Ile His Pro 1 5 6 5 PRT Artificial Sequence Description
of Artificial SequenceAII (3-7) 6 Val Tyr Ile His Pro 1 5 7 4 PRT
Artificial Sequence Description of Artificial SequenceAII (5-8) 7
Ile His Pro Phe 1 8 6 PRT Artificial Sequence Description of
Artificial SequenceAII (1-6) 8 Asp Arg Val Tyr Ile His 1 5 9 5 PRT
Artificial Sequence Description of Artificial SequenceAII (1-5) 9
Asp Arg Val Tyr Ile 1 5 10 4 PRT Artificial Sequence Description of
Artificial SequenceAII (1-4) 10 Asp Arg Val Tyr 1 11 3 PRT
Artificial Sequence Description of Artificial SequenceAII (1-3) 11
Asp Arg Val 1 12 7 PRT Artificial Sequence Description of
Artificial SequenceAII analogue 12 Arg Xaa Tyr Ile His Pro Phe 1 5
13 7 PRT Artificial Sequence Description of Artificial SequenceAII
analogue 13 Arg Val Tyr Xaa His Pro Phe 1 5 14 3 PRT Artificial
Sequence Description of Artificial SequenceAII (6-8) 14 His Pro Phe
1 15 5 PRT Artificial Sequence Description of Artificial
SequenceAII (4-8) 15 Tyr Ile His Pro Phe 1 5 16 7 PRT Artificial
Sequence Description of Artificial SequenceAII analogue class 16
Xaa Xaa Tyr Xaa His Pro Phe 1 5 17 7 PRT Artificial Sequence
Description of Artificial SequenceAII analogue 17 Arg Val Tyr Gly
His Pro Phe 1 5 18 7 PRT Artificial Sequence Description of
Artificial SequenceAII analogue 18 Arg Val Tyr Ala His Pro Phe 1 5
19 8 PRT Artificial Sequence Description of Artificial SequenceAII
analogue 1 19 Asp Arg Val Tyr Val His Pro Phe 1 5 20 8 PRT
Artificial Sequence Description of Artificial SequenceAII analogue
2 20 Asn Arg Val Tyr Val His Pro Phe 1 5 21 11 PRT Artificial
Sequence Description of Artificial SequenceAII analogue 3 21 Ala
Pro Gly Asp Arg Ile Tyr Val His Pro Phe 1 5 10 22 8 PRT Artificial
Sequence Description of Artificial SequenceAII analogue 4 22 Glu
Arg Val Tyr Ile His Pro Phe 1 5 23 8 PRT Artificial Sequence
Description of Artificial SequenceAII analogue 5 23 Asp Lys Val Tyr
Ile His Pro Phe 1 5 24 8 PRT Artificial Sequence Description of
Artificial SequenceAII analogue 6 24 Asp Arg Ala Tyr Ile His Pro
Phe 1 5 25 8 PRT Artificial Sequence Description of Artificial
SequenceAII analogue 7 25 Asp Arg Val Thr Ile His Pro Phe 1 5 26 8
PRT Artificial Sequence Description of Artificial SequenceAII
analogue 8 26 Asp Arg Val Tyr Leu His Pro Phe 1 5 27 8 PRT
Artificial Sequence Description of Artificial SequenceAII analogue
9 27 Asp Arg Val Tyr Ile Arg Pro Phe 1 5 28 8 PRT Artificial
Sequence Description of Artificial SequenceAII analogue 10 28 Asp
Arg Val Tyr Ile His Ala Phe 1 5 29 8 PRT Artificial Sequence
Description of Artificial SequenceAII analogue 11 29 Asp Arg Val
Tyr Ile His Pro Tyr 1 5 30 8 PRT Artificial Sequence Description of
Artificial SequenceAII analogue 12 30 Pro Arg Val Tyr Ile His Pro
Phe 1 5 31 8 PRT Artificial Sequence Description of Artificial
SequenceAII analogue 13 31 Asp Arg Pro Tyr Ile His Pro Phe 1 5 32 8
PRT Artificial Sequence Description of Artificial SequenceAII
analogue 14 32 Asp Arg Val Tyr Ile His Pro Phe 1 5 33 8 PRT
Artificial Sequence Description of Artificial SequenceAII analogue
15 33 Asp Arg Xaa Tyr Ile His Pro Phe 1 5 34 8 PRT Artificial
Sequence Description of Artificial SequenceAII analogue 16 34 Asp
Arg Val Tyr Xaa His Pro Phe 1 5 35 9 PRT Artificial Sequence
Description of Artificial SequenceAII analogue 17 35 Asp Arg Val
Ser Tyr Ile His Pro Phe 1 5 36 8 PRT Artificial Sequence
Description of Artificial Sequencep-aminophenylalanine 6 AII 36 Asp
Arg Val Tyr Ile Xaa Pro Phe 1 5 37 10 PRT Artificial Sequence
Description of Artificial Sequenceangiotensin I 37 Asp Arg Val Tyr
Ile His Pro Phe His Leu 1 5 10 38 8 PRT Artificial Sequence
Description of Artificial Sequence1GDIle8-AII 38 Asp Arg Val Tyr
Ile His Pro Ile 1 5 39 8 PRT Artificial Sequence Description of
Artificial SequenceGly1-AII 39 Gly Arg Val Tyr Ile His Pro Phe 1 5
40 7 PRT Artificial Sequence Description of Artificial
SequencenorLeu4-AIII 40 Arg Val Tyr Xaa His Pro Phe 1 5 41 8 PRT
Artificial Sequence MOD_RES (3) Xaa at position 3 is
1-aminocyclopentane carboxylic acid 41 Asp Arg Xaa Tyr Ile His Pro
Phe 1 5 42 7 PRT Artificial Sequence Description of Artificial
Sequence2GD Pro3-AII(1-7) 42 Asp Arg Pro Tyr Ile His Pro 1 5
* * * * *