Note: Descriptions are shown in the official language in which they were submitted.
CA 02321164 2000-08-11
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METHOD OF PROMOTING NEURONAL CELL PROLIFERATION AND
DIFFERENTIATION
Cross Reference
This application is a continuation in part of U.S. Application Serial No.
60/075,232
filed February 19, 1998.
1 o Field of the Invention
This present invention relates to methods and kits for accelerating the
proliferation
and/or differentiation of neuronal cells.
Background of the Invention
Stem cells in the central nervous system (hereinafter referred to as "CNS")
have the
potential to differentiate into neurons, astrocytes, and oligodendrocytes and
to self renew.
(McICay, Science 276:66-71, 1997; hereby incorporated by reference in its
entirety). CNS
progenitor cells have a more restricted potential than a stem cell, while CNS
precursor cells
comprise any non-fully differentiated CNS cell type (McKay, 1997).
2o Mammalian fetal precursor cells that give rise to neurons and glia have
been isolated
(Frederiksen et al., Neuron 1:439 (1988); Reynolds and Weiss, Science 255:1707
(1992);
Davis and Temple, Nature 372:263 (1994)). The adult CNS also contains
multipotential
precursor cells for neurons, astrocytes and oligodendrocytes (McKay, 1997).
Cultured cells
from both the fetal and adult CNS that have proliferated in vitro can
differentiate to show
morphological and electrophysiological features characteristic of neurons
(Gritti et al., J.
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Neurosci. 16:1091 (1996); Vicario-Abejon et al., Neuron 15:105 (1995)). These
data show
the multipotential nature of cells derived from the CNS.
Multipotential cells from the fetal brain have been demonstrated to be both
homogenous and stable. (Johe et al., Genes Dev. 10:3129 (1996)). In vitro,
these cells divide
daily and efficiently generate neurons and glia for at least the first month
of culture. These
cells can be considered to be stem cells because they fulfill the criteria of
multipotency and
self renewal.
Cells from the adult brain proliferate and differentiate into neurons and glia
in tissue
culture with the same efficiency for neuronal differentiation as found in
fetal stem cells and
to the same response to extracellular ligands (McKay, 1997). Thus, similar
general mechanisms
control the differentiation of stem cells from fetal or adult brain. The
proliferation of
precursor cells in the adult forebrain can be stimulated by the direct
application of mitogenic
growth factors in vivo, and in animals treated in this way, proliferating
cells in the
subventricular zone differentiate into neurons and glia (Craig et al., J.
Neurosci. 16:2649
(1996)). However, in vivo less than 3% of the proliferating cells labeled with
bromodeoxyuridine differentiate into neurons (McKay, 1997). The discrepancy
between the
efficient neuronal differentiation of adult stem cells in vitro and their
inefficient
differentiation in vivo is a critical but unresolved question in the field
(Id.) The lack of
differentiating neurons may not be a consequence of the lack of cells with the
appropriate
2o potential but rather a function of the signaling environment in the adult
brain (Id.)
The long term delivery of proteins in the brain is a major goal in gene
therapy.
Transplantation of cells engineered to produce growth factors shows the
potential of grafted
cells as vectors for protein delivery (Beck et al., Nature 373:339 (1995);
Tomac et al., Nature
373:335 (1995); Moore et al., Nature 382:76 (1996)). It is possible to
generate many different
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immortal cell lines from the developing CNS. It is also possible to graft
primary cells
expanded in vitro. Experiments have suggested that primary adult cells derived
from the
hippocampus and cultured for long periods in vitro can still differentiate
into neumns when
re-implanted into the migratory pathway used to replenish neurons in the adult
olfactory bulb
(Suhonen et al., Nature 383:624 (1996)). Experimental grafts in animal models
suggest that
the integration of grafted neurons into the circuitry of the host may be
possible, as other
studies illustrate the use of in vitro manipulated donor cells that
differentiate in vivo into
oligodendrocytes (Tontsch et al., Proc. Natl. Acad. Sci. 91:11616 ( 1994);
Groves et al.,
Nature 362:453 (1993)). Furthermore, clinical trials show that neuron
replacement therapies
for neurodegenerative diseases, such as Parkinson's and Huntington's disease,
are feasible
(Kordower et al., New Engl. J. Med 332:1118 (1995); Lindvall et al., Ann.
Neurol. 35:172
( 1994)). Thus, for clinical applications, cell culture offers an important
opportunity to use
sophisticated genetics in cell-based therapies for neural disease (McKay,
1997).
CNS stem cells have been expanded in vitro by using epidermal growth factor
(EGF)
and basic fibroblast growth factor (bFGF) (McKay, 1997). In vitro, EGF has
also been shown
to be a differentiation factor for astrocytes.
While these studies demonstrate the potential for stem cell and neuron
replacement
therapy, a careful analysis of adult neuronal stem cells has only just begun.
Further
characterization of the mechanisms that control the multipotentiality, self
renewal and fate
restriction of neuronal stem cells is clearly important to develop new
therapies for cell
regeneration and replacement in the adult nervous system. (Johe et al., 1996.)
Methods that increase the in vitro and ex vivo proliferation and
differentiation of
neuronal stem and progenitor cells will greatly increase the utility of neuron
replacement
therapy in various neurodegenerative conditions such as Parkinson's and
Alzheimer's
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Diseases, and amyotrophic lateral sclerosis. Similarly, methods that increase
in vivo
proliferation and differentiation of neuronal stem and progenitor cells will
enhance the utility
of neuron replacement therapy by rapidly increasing local concentrations of
neuronal stem
and progenitor cell at the site of therapy.
Summary of the Invention
The present invention provides methods that increase the proliferation or
differentiation of neuronal stem and progenitor cells that are useful in
rapidly providing a
large population of such cells for use in neuron replacement therapy and for
making a large
1o population of transfected cells for use in neuron replacement therapy.
In one aspect, the present invention provides methods that promote neuronal
cell
proliferation or differentiation by contacting the cells with angiotensinogen,
angiotensin I
(AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AII),
All analogues,
All fragments or analogues thereof or All AT2 type 2 receptor agonists, either
alone or in
~ 5 combination with other growth factors and cytokines.
In another aspect of the present invention, an improved cell culture medium is
provided for the proliferation or differentiation of neuronal cells, wherein
the improvement
comprises addition to the cell culture medium of an effective amount of
angiotensinogen, AI,
AI analogues, AI fragments and analogues thereof, AII, All analogues, All
fragments or
2o analogues thereof or All ATZ type 2 receptor agonists.
In a further aspect, the present invention provides kits for the propagation
or
differentiation of neuronal cells, wherein the kits comprise an effective
amount of
angiotensinogen, AI, AI analogues, and/or AI fragments and analogues thereof,
AII, All
analogues, All fragments or analogues thereof, and/or All ATz type 2 receptor
agonists, and
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instructions for culturing the cells. Preferred embodiments of the kit fiuther
comprise cell
culture growth medium, a sterile container, and an antibiotic supplement.
Brief Description of the Figures
Figure 1. Effect of All on human neuronal progenitor neurite outgrowth.
Figure 2. Effect of AII, AII(1-7), and Ala4-AIII on the proliferation of
normal human
neural progenitors.
Detailed Description of the Preferred Embodiments
As defined herein, the term "neuronal cells" include either primary cells or
established
cell lines with the potential to differentiate into neurons, astrocytes, and
oligodendrocytes and
to self renew, and also to differentiated cells derived therefrom, including
fully differentiated
CNS and peripheral nervous system ("PNS") cell types. Examples of neuronal
stem and
progenitor cells include, but are not limited to, those described in Gritti et
al., J. of
Neuroscience 16:1091-1100 (1996); Frederiksen et al., (1988); Reynolds and
Weiss, (1992);
Davis and Temple, (1994); McKay, (1997);. Vicario-Abejon et al., (1995); Craig
et al.,
(1996); Tontsch et al. (1994); Graves et al., (1993) and Johe et al., Genes
and Develop.
10:3129-3142 ( 1996), all references hereby incorporated in their entirety. As
defined herein,
"proliferation" encompasses both cellular self renewal and cellular
proliferation with
2o accompanying differentiation.
Unless otherwise indicated, the term "active agents" as used herein refers to
the group
of compounds comprising angiotensinogen, angiotensin 1 (AI), AI analogues, AI
fragments
and analogues thereof, angiotensin II (AII), All analogues, All fragments or
analogues thereof
and All AT2 type 2 receptor agonists.
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Within this application, unless otherwise stated, the techniques utilized may
be found
in any of several well-known references such as: Molecular Cloning: A
Laboratory Manual
(Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene
Expression~Technology
(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991, Academic Press,
San
Diego, CA), "Guide to Protein Purification" in Methods in Enrymology (M.P.
Deutshcer, ed.,
(1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and
Applications {Innis, et
al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of
Basic
Technigue, 2"d Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY), Gene
Transfer and
Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc.,
Clifton, N.J.),
1 o and the Ambion 1998 Catalog (Ambion, Austin, TX).
U.S. Patent 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 appiication of All to wound tissue
significantly increases the
rate of wound healing, leading to a more rapid re-epithelialization and tissue
repair. The term
All 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. The
substance so
formed is a decapeptide called angiotensin I (AI) which is converted to All by
the converting
2o enzyme angiotensinase which removes the C-terminal His-Leu residues from AI
(Asp-Arg_
Va1-Tyr-Ile-His-Pro-Phe-His-Leu [SEQ ID N0:37]). All is a known pressor agent
and is
commercially available. The use of All analogues and fragments, AT2 agonists,
as well as
AIII and AIII analogues and fragments in wound healing has also been
described. (U.S.
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Patent No. 5,629,292; U.S. Patent No. 5,716,935; WO 96/39164; all references
herein
incorporated by reference in their entirety.)
Studies have shown that All 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. Patent 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); Naftiian, et al., ,I. 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
1o Madri, Am. J. Pathol. 137:7-12 (1990). In addition, All 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 ).
Therefore, All may
accelerate wound repair through increased neovascularization, growth factor
release,
reepithelialization and/or production of extracellular matrix.
All has also been implicated in both cell growth and differentiation (Meffert
et al.,
Mol. and Cellul. Endocrin. 122:59 (1996)). Two main classes of All receptors,
ATE and ATZ
have been identified (Meffert, 1996). The growth-promoting effects of All have
been
attributed to mediation by the AT1 receptor, while some evidence suggests that
the AT2
receptor may be involved in mediation of the cell differentiation effects of
All (Bedecs et al.,
2o Biochem. J. 325:449 (1997)).
The effects of All receptor and All receptor antagonists have been examined in
two
experimental models of vascular injury and repair which suggest that both All
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 ); Kauffman, et
al., Life Sci.
7
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49:223-228 (1991); Viswanathan, et al., Peptides 13:783-786 (1992); Kimura, et
al., BBRC
187:1083-1090 (1992).
Many studies have focused upon AII(1-7) (AII residues 1-7) or other fragments
of All
to evaluate their activity. All{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
( I 993 ); 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. I 11:652-
654 (1994).
Studies have shown that All inhibits proliferation of both an immortalized
neuronal
to cell line (the pheochromocytoma derived PC12W; Meffert, 1996), and of
dissociated primary
cultures of retrochiamatic hypothalamus from 18-day old rat embryos
(Jirikowski et al.,
Develop. Brain Res. 14:179-183 (1984)). Other studies have shown that the
proportion of
AT, and ATZ receptors in rat brain changes during development, with fetal
tissue expressing
far more ATZ receptor subtype, while adult animals express far more AT,
subtype (Meffert,
t5 1996). However, little is known about All effects on most CNS or peripheral
nervous system
("PNS") cell types. Furthermore, it is not known what All receptor subtypes
are expressed by
neuronal stem and progenitor cells, nor what effect All has on their
proliferative capacity.
A peptide agonist selective for the AT2 receptor (AII has 100 times higher
affinity for
AT2 than AT1) is p-aminophenylalanine6-All ["(p-NHZ-Phe)6-AII)"], Asp-Arg-Val-
Tyr-Ile-
2o Xaa-Pro-Phe [SEQ ID N0.36] wherein Xaa is p-NH2-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).
8
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The active AI, AI analogues, AI fragments and analogues thereof, All
analogues,
fragments of All 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~-R8 in the sequence of general
formula I
Ri-R2-R3-Ra-Rs-R6-R~-Ra
in which R' and R2 together form a group of formula
X-R~-RB-,
wherein X is H or a one to three peptide group,
to RA is suitably selected from Asp, Glu, Asn, Acpc (1-aminocyclopentane
carboxylic acid), Ala, Me2Gly, Pro, Bet, Glu(NH2), Gly, Asp(1VH2) and Suc,
RB is suitably selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-
Lys;
R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly,
Pro,
~ 5 Aib, Acpc, Lys, and Tyr;
R4 is selected from the group consisting of Tyr, Tyr(P03)Z, Thr, Ser, homoSer,
Ala, and azaTyr;
RS is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and
Gly;
R6 is His, Arg or 6-NHZ-Phe;
20 R' is Pro or Ala; and
R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr,
excluding sequences including R4 as a terminal Tyr group.
Compounds falling within the category of AT2 agonists useful in the practice
of the
invention include the All analogues set forth above subject to the restriction
that R6 is p-NHZ-
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Phe. 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.
Particularly preferred combinations for RA and RB 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 N0:2]; AII(3-8), also
known as desl-
AIII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID N0:3]; All{1-7), Asp-Arg-Val-Tyr-
Ile-His-
Pro {SEQ ID N0:4]; AII(2-7). Arg-Val-Tyr-Ile-His-Pro [SEQ ID NO:S]; AII(3-7),
Val-Tyr-
Ile-His-Pro [SEQ ID N0:6]; AII(5-8), Ile-His-Pro-Phe [SEQ ID N0:7]; AII(1-6),
Asp-Arg-
to Val-Tyr-Ile-His [SEQ ID N0:8]; AII(1-S), Asp-Arg-Val-Tyr-Ile [SEQ ID N0:9];
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 )D N0:12]
and Arg-
Val-Tyr-norLeu-His-Pro-Phe [SEQ ID N0:13]. Still another preferred embodiment
encompassed within the scope of the invention is a peptide having the sequence
Asp-Arg-Pro-
i5 Tyr-Ile-His-Pro-Phe [SEQ ID N0:31]. AII(6-8), His-Pro-Phe [SEQ ID N0:14]
and AII(4-8),
Tyr-Ile-His-Pro-Phe [SEQ ID NO:15] were also tested and found not to be
effective.
In another preferred embodiment, the present invention provides a method for
promoting neuronal cell proliferation or differentiation comprising contacting
neuronal cells
with an amount effective to promote proliferation or differentiation of at
least one active agent
zo comprising a sequence consisting of the general formula:
Ri-ARG-VAL-TYR-R2-HIS-PRO-R3
wherein R1 is selected from the group consisting of H or Asp;
R2 is selected from the group consisting of Ile, Val, Leu, norLeu and Ala;
R3 is either Phe or H.
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In a most preferred embodiment, the active agent is selected from the group
consisting
of SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:13, SEQ ID N0:18, SEQ ID
N0:19, SEQ ID N0:26, and SEQ ID N0:34.
Another class of compounds of particular interest in accordance with the
present
invention are those of the general formula II
R2_R3_Ra_Rs_R6-R~_Rs
in which R2 is selected from the group consisting of H, Arg, Lys, Ala, Orn,
Ser(Ac), Sar, D-Arg and D-Lys;
R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly,
Pro,
t o Aib, Acpc and Tyr;
R4 is selected fiom the group consisting of Tyr, Tyr(P03)2, Thr, Ser, homoSer
and azaTyr;
Rs is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and
Gly;
R6 is His, Arg or 6-NH2-Phe;
R' is Pro or AIa; and
Rg is selected from the group consisting of Phe, Phe(Br), Ile and Tyr.
A particularly preferred subclass of the compounds of general formula II has
the
formula
R2-R3-Tyr-Rs-His-Pro-Phe [SEQ ID N0:16]
2o wherein R2, R3 and Rs are as previously defined. Particularly preferred is
angiotensin
III of the formula Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID N0:2J. Other preferred
compounds
include peptides having the structures Arg-Val-Tyr-Gly-His-Pro-Phe [SEQ ID
N0:17J and
I1
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Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID N0:18J. The fragment AII(4-8) was
ineffective in
repeated tests; this is believed to be due to the exposed tyrosine on the N-
terminus.
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:
TABLE 1
Abbreviation for Amino Acids
MezGlv N,N-dimeth 1 1 c 1
Bet 1-carboxy-N,N,N-trimethylmethanaminium hydroxide
inner salt '
betaine)
Suc Succin I
Phe Br) -bromo-L- henvlalan 1
azaTyr aza-a'-homo-L-tyros 1
Ac c 1-aminoc clo entane carbox lic acid
Aib 2-aminoisobutvric acid
Sar N-meth 1 1 c I (sarcosine)
It has been suggested that All and its analogues adopt either a gamma or a
beta turn
to (Regoli, et al., Pharmacological Reviews 26:69 (1974). In general, it is
believed that neutral
side chains in position R3, RS and R' may be involved in maintaining the
appropriate distance
between active groups in positions R4, R6 and R8 primarily responsible for
binding to
receptors and/or intrinsic activity. Hydrophobic side chains in positions R3,
RS and Rg may
also play an important role in the whole conformation of the peptide and/or
contribute to the
formation of a hypothetical hydrophobic pocket.
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Appropriate side chains on the amino acid in position R2 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.
For purposes of the present invention, it is believed that R3 may be involved
in the
formation of linear or nonlinear hydrogen bonds with RS (in the gamma turn
model) or R6 (in
the beta turn model). R3 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
1o conformation. Accordingly, R3 may suitably be selected from Val, Ala, Leu,
norLeu, Ile, Gly,
Pro, Aib, Acpc and Tyr. In another preferred embodiment, R3 is Lys.
With respect to R", conformational analyses have suggested that the side chain
in this
position (as well as in R3 and RS) contribute to a hydrophobic cluster
believed to be essential
for occupation and stimulation of receptors. Thus, R'~ is preferably selected
from Tyr, Thr,
t5 Tyr (P03)2, homoSer, Ser and azaTyr. 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). In a further preferred
embodiment, R4 is Ala.
In position R5, an amino acid with a (i aliphatic or alicyclic chain is
particularly
desirable. Therefore, while Gly is suitable in position R5, it is preferred
that the amino acid in
2o this position be selected from Ile, Ala, Leu, norLeu, Gly and Val.
In the AI, AI analogues, AI fragments and analogues thereof, AII, All
analogues,
fragments and analogues of fragments of particular interest in accordance with
the present
invention, R6 is His, Arg or 6-NHZ-Phe. The unique properties of the imidazole
ring of
histidine (e.g., ionization at physiological pH, ability to act as proton
donor or acceptor,
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aromatic character) are believed to contribute to its particular utility as
R6. 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'. Similarly, it is presently considered that R' should be Pro in order to
provide the most
desirable orientation of R8. In position R8, 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.
Analogues of particular interest include the following:
TABLE 2
1 o Angiotensin II Analogues
All AnalogueAmino Acid Sequence Sequence
Name Identifier
Analogue Asp-Arg-Val-Tyr-Val-His-Pro-Phe SEQ ID NO:
1 19
Analogue Asn-Arg-Val-Tyr-Val-His-Pro-Phe SEQ ID NO:
2 20
Analogue Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-PheSEQ ID NO:
3 21
Analogue Glu-Arg-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO:
4 22
Analogue Asp-Lys-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO:
5 23
Analogue Asp-Arg-Ala-Tyr-Ile-His-Pro-Phe SEQ ID NO:
6 24
Analogue Asp-Arg-Val-Thr-Ile-His-Pro-Phe SEQ ID NO:
7 25
Analogue Asp-Arg-Val-Tyr-Leu-His-Pro-Phe SEQ ID NO:
8 26
Analogue Asp-Arg-Val-Tyr-Ile-Arg-Pro-Phe SEQ ID NO:
9 27
Analogue Asp-Arg-Val-Tyr-Ile-His-Ala-Phe SEQ ID NO:
28
Analogue Asp-Arg-Val-Tyr-Ile-His-Pro-Tyr SEQ ID NO:
11 29
Analogue Pro-Arg-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO:
12 30
Analogue Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe SEQ ID NO:
13 31
Analogue Asp-Arg-Val-Tyr(P03)2-Ile-His-Pro-PheSEQ ID NO:
14 32
Analogue Asp-Arg-norLeu-Tyr-Ile-His-Pro-Phe SEQ ID NO:
33
Analogue Asp-Arg-Val-Tyr-norLeu-His-Pro-Phe SEQ ID NO:
16 34
Analogue Asp-Arg-Val-homoSer-Tyr-Ile-His-Pro-PheSEQ ID NO:
17 35
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
14
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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.
In general, these methods involve the sequential addition of protected amino
acids to a
growing peptide chain (U.S. Patent 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.
1o 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.
In one aspect of the present invention, a method of increasing in vivo, in
vitro and ex
vivo neuronal stem and progenitor cell proliferation by exposure to
angiotensinogen, AI, AI
analogues, AI fragments and analogues thereof, All analogues, All fragments or
analogues
thereof or All AT2 type 2 receptor agonists ("active agents") is disclosed.
Experimental
conditions for the isolation, purification, in vitrolex vivo growth and in
vivo mobilization of
neuronal stem and progenitor cells have been reported (Frederiksen et al.,
1988; Reynolds and
Weiss, 1992; Gritti et al., 1996; Vicario-Abejon et al., 1995; Johe et al.,
1996; Craig et al.,
1996; Suhonen et al., Nature 383:624-627, 1996; and Tontsch et al., 1994).
Proliferation 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), 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
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a preferred embodiment, proliferation of neuronal stem and progenitor cells is
assessed by
reactivity to an antibody directed against 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, California).
In one embodiment, neuronal cells are isolated from primary cell masses
according to
standard methods (Jirikowski et al., 1984; Reynolds and Weiss, 1992; Johe et
al., 1996; Gritti
et al., 1996; Vicario-Abejon et al., 1995; Kordower et al., 1995; Nauert and
Freeman, Cell
Transplant 3:147-151, 1994; Freeman and Kordower, In: Lindvall et al., eds.
Intracerebral
1 o Transplantation in Movement Disorders. New York: Elsevier Science, 163-
170, 1991 ),
suspended in culture medium and incubated in the presence of, preferably,
between about 0.1
ng/ml and about 10 mg/ml of the active agents of the invention. The cells are
expanded for a
period of between 8 and 21 days and cellular proliferation is assessed as
described above.
In a preferred embodiment, neuronal stem and progenitor cells are isolated
from
primary cells which are isolated from the adult rat mammalian forebrain or rat
embryonic
hippocampus (Johe et al., 1996). The cell mass is dissociated by either
mechanical trituration
or by incubating minced tissue in Hank's Buffered Saline Solution (HBSS). The
cells are
collected by centrifugation and resuspended in a serum-free medium containing
Dulbecco's
modified Eagle medium (DMEM)/F12, glucose, glutamine, sodium bicarbonate, 25
pg/ml of
2o insulin, 100 pg/ml of human apotransferrin, 20 nm progesterone, 100 pm
putrescene, 30 nm
sodium selenite (pH 7.2), plus 10 ng/ml of recombinant basic fibroblast growth
factor (bFGF;
R&D, Inc.) (Johe et al., 1996). The cells are plated into tissue culture
plates precoated with
cell attachment factors, as is well known in the art. BFGF is added daily, and
the medium is
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changed every two days. Cells are passaged at SO% confluence by briefly
incubating them in
HBSS and scraping with a cell scraper.
Alternatively, neuronal stem and progenitor cells are isolated from the
dissociated cell
mass by antibody-mediated cell capture ("panning"; Barres et al., Cell 70:31-
46, 1992).
s Antibodies that can be used to isolate neuronal stem and precursor cells
include, but are not
limited to, nestin antibody (Vicario-Abejon et al., 1995). The cells are then
treated as above.
The neuronal stem and progenitor cells exposed to the active agents as
described
above can be used for neuron replacement therapy, to treat disorders
including, but not limited
to, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral
sclerosis. The cells are
l0 cultured in vitro or ex vivo as described above. The cells are rinsed to
remove all traces of
culture fluid, resuspended in an appropriate medium and then pelleted and
rinsed several
times. After the final rinse, the cells are resuspended at between 0.7 x 106
and SO x 106 cells
per ml in an appropriate medium and used for transplantation according to
previously
described methods. (Kordower et al., 1995; Freed et al., N. Engl. J. Med.
327:1549-1555,
15 1992; Ann. Neurol. 31:155-165, 1992; Peschanski et al., Brain 117:487-499,
1994; Spencer et
al., N. Engl. J. Med 327:1541-1548, 1992; Henderson et al., Arch. Neurol.
48:822-827, 1992;
Hitchcock et al., Exp. Neurol. 129:3, 1994; Lindvall et al., Science 247:574-
577, 1990;
Widner et al., N. Engl. J. Med. 327:1556-1563, 1992; Bankiewicz et al., J.
Neurosurg.
72:231-244, 1990; Kordower et al., Ann. Neurol. 29:405-412, 1991)
20 In a preferred embodiment, the neuronal stem and progenitor cells used for
transplantation are transfected with an expression vector so as to express a
therapeutic protein,
including but not limited to glial-cell-line-derived neurotrophic factor
(GDNF; Beck et al.
Nature 373:339-341, 1995; Tomac et al., Nature 373:335-339, 1995) after
transplantation.
I7
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In a further aspect of the present invention, the effect of the active agents
on neuronal
stem and progenitor cell differentiation is assessed by examination of changes
in gene
expression, phenotype, morphology, or any other method that distinguishes stem
and/or
progenitor cells from fully differentiated cells. Examples of such
differentiation markers
against which antibodies are available include, but are not limited to, neuron-
specific
microtubule-associated protein 2 (MAP2; Vicario-Abejon et al., 1995; antibody
available
from Boehringer Manheim, Germany), astroglial-specific glial flbrillary acidic
protein
(GFAP; Vicario-Abejon et al., 1995; antibody available from Incstar,); neuron-
specific tau
protein (Johe et al., 1996; antibody available from Sigma, St. Louis, MO);
neurofilaments L
1o and M (Joke et al., 1996; antibody available from Boehringer Manheim); and
oligodendrocyte-specific 04 and galactocerebroside (GaIC; Johe et al., 1996).
The DNA
sequences for all of these differentiation-specific markers are known, and
thus PCR
amplification and/or hybridization studies to evaluate gene expression of the
differentiation
markers can be performed according to standard methods in the art. (Johe et
al., 1996)
In a preferred embodiment, neuronal stem and progenitor cells are isolated and
cultured as described above. Differentiation is initiated by contacting the
cells with the
active agents as described above, in serum-free medium in the absence of bFGF.
Differentiation is assessed at various times by immunodetection of
differentiation-specific
markers, using the antibodies described above (Johe et al., 1996).
Alternatively,
2o differentiation is assessed morphologically by measurement of neurite
outgrowth.
In another aspect of the present invention the active agents are used to
increase in vivo
neuronal stem and progenitor cell proliferation. For use in increasing
proliferation of
neuronal stem and progenitor cells, the active agents may be administered by
any suitable
route, including orally, parentally, by inhalation spray, rectally, or
topically in dosage unit
18
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formulations containing conventional pharmaceutically acceptable carriers,
adjuvants, and
vehicles. The term parenteral as used herein includes, subcutaneous,
intravenous, intra-
arterial, infra-ventricular, intramuscular, intrasternal, intratendinous,
intraspinal, intracranial,
intrathoracic, infusion techniques or intraperitoneally.
The active agents may be made up in a solid form (including granules, powders
or
suppositories) or in a liquid form (e.g., solutions, suspensions, or
emulsions) and may be
subjected to conventional pharmaceutical operations such as sterilization
and/or may contain
conventional adjuvants, such as preservatives, stabilizers, wetting agents,
emulsifiers, buffers
etc.
to While the active agents can be administered as the sole active agent, they
can also be
used in combination with one or more other compounds. When administered as a
combination, the active agents and other compounds can be formulated as
separate
compositions that are given at the same time or different times, or the active
agents and other
compounds can be given as a single composition.
1 s For administration, active agents are ordinarily combined with one or more
adjuvants
appropriate for the indicated route of administration. The compounds may be
admixed with
lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic
acid, talc,
magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric
and sulphuric
acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or
polyvinyl alcohol, and
2o tableted or encapsulated for conventional administration. Alternatively,
the compounds of
this invention may be dissolved in saline, water, polyethylene glycol,
propylene glycol,
carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil,
cottonseed oil,
sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes
of
administration are well known in the pharmaceutical art. The carrier or
diiuent may include
19
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time delay material, such as glyceryl monastearate or glyceryl distearate
alone or with a wax,
or other materials well known in the art.
Formulations suitable for topical administration include liquid or semi-liquid
preparations suitable for penetration through the skin (e.g., liniments,
lotions, ointments,
creams, or pastes) and drops suitable for administration to the eye, ear, or
nose.
The dosage regimen for increasing in vivo proliferation or differentiation of
neuronal
stem and progenitor cell with the active agents of the invention is based on a
variety of
factors, including the type of injury or deficiency, the age, weight, sex,
medical condition of
the individual, the severity of the condition, the route of administration,
and the particular
t0 compound employed. Thus, the dosage regimen may vary widely, but can be
determined
routinely by a physician using standard methods. Dosage levels of the order of
between 0.1
ng/kg and 10 mg/kg angiotensinogen, AI, AI analogues, AI fragments and
analogues thereof,
AII, All analogues, All fragments and analogues thereof and/or All ATZ type 2
receptor
agonists per body weight are useful for all methods of use disclosed herein.
In a preferred embodiment of the present invention, the active agents are
administered
by unilateral infusion directly into the mammalian brain lateral ventricle
using an osmotic
pump (Alza Palo Alto, CA) attached to a 30 gauge cannulae implanted at the
injection
coordinate, as described in Craig et al., J. of Neuroscience 16:2649-2658
(1996). A suitable
injected dose of active ingredient of the active agents is preferably between
about 0.1 ng/kg
2o and about 10 mg/kg administered twice daily. The active ingredient may
comprise from
0.001 % to 10% w/w, e.g., from 1 % to 2% by weight of the formulation,
although it may
comprise as much as 10% w/w, but preferably not more than S% w/w, and more
preferably
from 0.1 % to 1 % of the formulation.
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In another aspect of the present invention, an improved cell culture medium is
provided for the proliferation and differentiation of neuronal cells, wherein
the improvement
comprises addition to the cell culture medium of an effective amount of the
active agents, as
described above. Any cell culture media that can support the growth of
neuronal stem and
progenitor cells 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~ Reduced-Serum Medium, RPMI Medium, and Macrophage-SFM
Medium or combinations thereof.
1o The improved cell culture medium can be supplied in either a concentrated
(ie: 10X)
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)
~5 In a further aspect, the present invention provides kits for the
propagation of neuronal
stem and progenitor cells, wherein the kits comprise an effective amount of
the active agents,
as described above.
In a preferred embodiment, the kit further comprises cell culture growth
medium. Any
cell culture media that can support the growth of neuronal stem and progenitor
cells can be
Zo used with the present invention. Examples of such cell culture media are
described above.
The improved cell culture medium can be supplied in either a concentrated (ie:
10X)
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.
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In another preferred embodiment, the kit further comprises a sterile
container. The
sterile container can comprise either a sealed container, such as a cell
culture flask, a roller
bottle, or a centrifuge tube, or a non-sealed container, such as a cell
culture plate or microtiter
plate (Nunc; Naperville, IL).
In a further preferred embodiment, the kit further comprises an antibiotic
supplement
for inclusion in the reconstituted cell growth medium. Examples of appropriate
antibiotic
supplements include, but are not limited to actimonycin D, Fungizone~,
kanamycin,
neomycin, nystatin, penicillin, streptomycin, or combinations thereof (GIBCO).
The present invention may be better understood with reference to the
accompanying
to examples that are intended for purposes of illustration only and should not
be construed to
limit the scope of the invention, as defined by the claims appended hereto.
Example 1. All Effect on the Proliferation and Differentiation of Normal Human
Neuronal
Progenitor Cells
Normal human progenitor cells were purchased from Clonetics (San Diego, CA)
and
cultured in Neural Progenitor Cell Maintenance Medium (NPMM) (Neural
Progenitor Basal
Medium containing human recombinant fibroblast growth factor beta, human
recombinant
epidermal growth factor, neural survival factors, gentamycin and amphotericin
B). The cells
2o were thawed, diluted into NPMM and cultured for 24 hours in a 75 cm2 flask.
The cells were
cultured in dedifferentiaited spheroids until studies were conducted to assess
differentiation.
When the cells were cultured in suspension culture in the presence of 1, 10,
or i00 pg/ml All
for 4-7 days prior to placement of the cells on a culture substrate that
allowed adherence and
differentiation (as described further below), an increase in the number of
cells able to undergo
differentiation (ie: proliferation) was observed (Table I).
22
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Table I. Effect of All on the Proliferation of Human Neuronal Progenitor Cells
All Concentration _ Cells ner Well Cells er Well on
on Da 4 Da 7
0 ~/ml 66 63
I ml 93 gs
ml 89 86
100 ml 91 86
Assessment of Differentiation
In order to assess the differentiation of neuronal cells, the cells were
seeded upon
5 wells coated with 0.05% polyethyleneimine (PEI) substrate in borate buffer
solution. The
wells of a 96 well plate were coated with 0.05 ml of this solution overnight
at room
temperature. After the incubation, the substrate was removed by aspiration,
rinsed with sterile
water and allowed to dry before seeding of the cells.
After culture of cells for 4-7 days in the presence of All (to assess
proliferation), the
i o cells were washed and placed in PEI-coated wells to assess
differentiation. Four days after
plating, the number of cells undergoing differentiation, as assessed by
neurite outgrowth, was
counted (see Table 1 ).
In an additional study, the effect of All on the differentiation of neuronal
progenitor
cells was assessed. After adherence to PEI substrate, the cells cease
proliferation and undergo
differentiation and neurite outgrowth. One thousand cells were placed in each
well ir. the
presence and absence of various concentrations of AII. Four and seven days
after initiation of
culture, the size of the neurites (by measurement with an ocular micrometer)
on the cells
undergoing differentiation and the number of cells undergoing differentiation
was assessed.
Culture of the cells in the presence of All increased the rate of neurite
outgrowth (see Figure
2o I) and the number of cells undergoing differentiation by approximately 50%
(data not
shown).
23
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These studies demonstrate that exposure to All promotes the proliferation and
differentiation of normal human neuronal progenitor cells.
Example 2. Ef~'ect of All, All(1-7), and ALa4 Alll on the proliferation of
normal human
neural progenitors
Normal human progenitor cells were purchased from Clonetics (San Diego, CA)
and
cultured in Neuronal Progenitor Cell Maintenance Medium (NPMM) (Neural
Progenitor
Basal Medium containing human recombinant fibroblast growth factor beta, human
to recombinant epidermal growth factor, neural survival factors, gentamycin,
and amphotericin
B). The cells were thawed, diluted into NPMM and cultured for 24 hours in a 75
cm2 flask.
Until studies to assess differentiation, the cells were cultured in
dedifferentiated spheroids. If
the cells were cultured in suspension culture in the presence of 10 pg/ml All
(SEQ ID NO:I ),
AII(1-7) (SEQ ID N0:4), or Ala4-AIII (SEQ ID N0:18) for 7 days prior to
placement of the
cells on collagen-coated plates to allow adherence, an increase in the number
of cells able to
undergo proliferation was observed (Figure 2). The increase in the number of
human neural
progenitors in the wells was 300% in the presence of AII, 175% in the presence
of Ala4-AIII,
and 100% in the presence of AII(1-7), while the increase was only 33% in the
control wells.
These studies show that each of these peptides promoted the proliferation of
normal human
2o neuronal progenitor cells.
The present invention, by providing a method for enhanced proliferation of
neuronal
cells, will greatly increase the clinical benefits of neuronal stem and
progenitor
transplantation. This is true both for increased "self renewal" of neuronal
stem cells, which
will provide a larger supply of stem cells capable of differentiation into
various neuronal cell
types, and for proliferation with differentiation, which will provide a larger
supply of neuronal
progenitor and differentiated cells at the appropriate site. Similarly,
methods that increase in
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vivo proliferation of neuronal stem, progenitor, and differentiated cells are
beneficial in
treating many neurological disorders, including Parkinson's disease,
Alzheimer's disease, and
amyotrophic lateral sclerosis.
The method of the present invention also increases the potential utility of
neuronal
stem and progenitor cells as vehicles for gene therapy in central and
peripheral nervous
system disorders by more efficiently providing a large number of such cells
for transfection,
and also by providing a more efficient means to rapidly expand transfected
neuronal stem and
progenitor cells.
The present invention is not limited by the aforementioned particular
preferred
to 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.
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SEQUENCE LISTING
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII
<400> 1
Asp Arg Val Tyr Ile His Pro Phe
1 5
<210> 2
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (2-8)
<400> 2
Arg Val Tyr Ile His Pro Phe
1
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1 5
<210> 3
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (3-8)
<400> 3
Val Tyr Ile His Pro Phe
1 5
<210> 4
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (1-7)
2
SUBSTITUTE SHEET (RULE 26)
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<400> 4
Asp Arg Val Tyr Ile His Pro
1 5
<210> 5
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (2-7)
<400> 5
Arg Val Tyr Ile His Pro
1 5
<210> 6
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
3
SUBSTITUTE SHEET (RULE 26)
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<223> Description of Artificial Sequence:AII (3-7)
<400> 6
Val Tyr Ile His Pro
1 S
<210> 7
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (5-8)
<400> 7
Ile His Pro Phe
1
<210> 8
<211> 6
<212> PRT
<213> Artificial Sequence
4
SUBSTITUTE SHEET (RULE 26)
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<220>
<223> Description of Artificial Sequence:AII (1-6)
<400> 8
Asp Arg Val Tyr Ile His
1 5
<210> 9
<211> S
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (1-5)
<400> 9
Asp Arg Val Tyr Ile
1 5
<210> 10
<211> 4
SUBSTITUTE SHEET (RULE 26)
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<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (1-4)
<400> 10
Asp Arg Val Tyr
1
<210> 11
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (1-3)
<400> 11
Asp Arg Val
1
6
SUBSTITUTE SHEET (RULE 26)
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<210> 12
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
<220>
<221> MOD RES
<222> (2)
<223> Nle
<400> 12
Arg Xaa Tyr Ile His Pro Phe
1 5
<210> 13
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
7
SUBSTITUTE SHEET (RULE 26)
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<223> Description of Artificial Sequence:AII analogue
<220>
<221> MOD RES
<222> (4)
<223> Nie
<400> 13
Arg Val Tyr Xaa His Pro Phe
1 S
<210> 14
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (6-8)
<400> 14
His Pro Phe
1
8
SUBSTITUTE SHEET (RULE 26)
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<210> 15
<211> 5
<212> PRT
<2I3> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (4-8)
<400> 15
Tyr Ile His Pro Phe
1 5
<210> 16
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
class
<220>
9
SUBSTITUTE SHEET (RULE 26)
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<221> UNSURE
<222> (1)
<223> Xaa at poistion 1 can be Arg, Lys, Ala, Orn, Ser,
MeGly, D-Arg, or D-Lys
<220>
<221> UNSURE
<222> (2)
<223> Xaa at position 2 can be Val, Ala, Leu, Nle, Ile,
Gly, Pro, Aib, Acp, or Tyr
<220>
<221> UNSURE
<222> (4 )
<223> Xaa at position 4 can be Ile, Ala, Leu, Nle, Val,
or Gly
<400> 16
Xaa Xaa Tyr Xaa His Pro Phe
1 5
<210> 17
<211> 7
SUBSTITUTE SHEET (RULE 26)
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<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
<400> 17
Arg Val Tyr Gly His Pro Phe
1 5
<210> 18
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
<400> 18
Arg Val Tyr Ala His Pro Phe
1 5
11
SUBSTITUTE SHEET (RULE 26)
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<210> 19
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 1
<400> 19
Asp Arg Val Tyr Val His Pro Phe
1 5
<210> 20
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 2
<400> 20
Asn Arg Val Tyr Val His Pro Phe
1 5
12
SU9STITUTE SHEET (RULE 2fi)
CA 02321164 2000-08-11
WO 99/42123 PCT/US99/03772
<210> 21
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 3
<400> 21
Ala Pro Gly Asp Arg Ile Tyr Val His Pro Phe
1 5 10
<210> 22
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 4
<400> 22
13
SUBSTITUTE SHEET (RULE 26)
CA 02321164 2000-08-11
WO 99/42123 PCT/US99/03772
Glu Arg Val Tyr Ile His Pro Phe
1 S
<210> 23
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 5
<400> 23
Asp Lys Val Tyr Ile His Pro Phe
1 5
<210> 24
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 6
14
SUBSTITUTE SHEET (RULE 26)
CA 02321164 2000-08-11
WO 99/42123 PCT/US99/03772
<400> 24
Asp Arg Ala Tyr Ile His Pro Phe
1 5
<210> 25
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 7
<400> 25
Asp Arg Val Thr Ile His Pro Phe
1 5
<210> 26
<211> 8
<212> PRT
<213> Artificial Sequence
SUBSTITUTE SHEET (RULE 25)
CA 02321164 2000-08-11
WO 99/42123 PCT/US99/03772
<220>
<223> Description of Artificial Sequence:AII analogue 8
<400> 26
Asp Arg Val Tyr Leu His Pro Phe
1 5
<210> 27
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 9
<400> 27
Asp Arg Val Tyr Ile Arg Pro Phe
1 5
<210> 28
<211> 8
<212> PRT
16
SU6STITUTE SHEET (RULE 26)
CA 02321164 2000-08-11
WO 99/42123 PCT/US99/03772
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 10
<400> 28
Asp Arg Val Tyr Ile His Ala Phe
1 5
<210> 29
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 11
<400> 29
Asp Arg Val Tyr Ile His Pro Tyr
1 5
<210> 30
17
sues sHESr tRU~ Zs)
CA 02321164 2000-08-11
WO 99/42123 PCT/ITS99/03772
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 12
<400> 30
Pro Arg Val Tyr Ile His Pro Phe
1 5
<210> 31
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 13
<400> 31
Asp Arg Pro Tyr Ile His Pro Phe
1 5
18.
suesmu~ sHeEr tRU~ 2s~
CA 02321164 2000-08-11
WO 99/42123 PCT/US99/03772
RUI
<210> 32
<211> $
<212 > PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 14
<220>
<221> MOD RES
<222> (4)
<223> PHOSPHORYLATION
<400> 32
Asp Arg Val Tyr Ile His Pro Phe
1 5
<210> 33
<211> $
<212> PRT
<213> Artificial Sequence
19
SUBSTITUTE SHEET (RULE 26)
CA 02321164 2000-08-11
WO 99/42123 PCT/US99/03772
<220>
<223> Description of Artificial Sequence:AII analogue 15
<220>
<221> MOD RES
<222> (3)
<223> Nle
<400> 33
Asp Arg Xaa Tyr Ile His Pro Phe
1 5
<210> 34
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue Z6
<220>
<221> MOD RES
<222> (S)
SUBSTrTUTE SHEET (RULE 26)
CA 02321164 2000-08-11
WO 99/42123 PCT/US99/03772
<223> Nle
<400> 34
Asp Arg Val Tyr Xaa His Pro Phe
1 5
<210> 35
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 17
<220>
<221> MOD RES
<222> (4)
<223> homo Ser
<400> 35
Asp Arg Val Ser Tyr Ile His Pro Phe
1 5
21
SU8ST1TUTE SHEET (RU~.E 28)
CA 02321164 2000-08-11
WO 99/42123 PCT/US99/03772
<210> 36
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial
Sequence:p-aminophenylalanine 6 All
<220>
<221> MOD RES
<222> (6)
<223> p-aminophenylalanine
<400> 36
Asp Arg Val Tyr Ile Xaa Pro Phe
1 5
<210> 37
<211> 10
<212> PRT
<213> Artificial Sequence
22
SUBSTITUTE SHEET (RULE 2B)
CA 02321164 2000-08-11
WO 99/42123 PCT/US99/03772
<220>
<223> Description of Artificial Sequence:angiotensin I
<400> 37
Asp Arg Val Tyr Ile His Pro Phe His Leu
1 5 10
23
SU65TITUTE SHEET (RULE 2B)
