Note: Descriptions are shown in the official language in which they were submitted.
CA 02324988 2000-10-06
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METHODS FOR TREATMENT AND PREVENTION OF INFECTIONS
Cross-Reference
This application is a continuation-in-part of U.S. Provisional Application
Serial Nos. 60/081,262 filed April 9, 1998 and 60/089024 filed June 12, 1998,
both
references herein incorporated by reference in their entirety.
Background of the Invention
1o Humans are susceptible to infection by a variety of pathogens, including
bacteria, viruses, and other parasites. Such infections pose a significant
health risk
for the human population in general. One example of bacterial infection is
sepsis,
which can occur in hospitalized patients having underlying diseases or
conditions
that render them susceptible to bloodstream invasion or in burn, trauma or
surgical
patients. (U.S. Patent No. 5,714,469, hereby incorporated by reference in its
entirety). In many cases of sepsis, the predominant pathogen is Escherichia
coli,
although other gram-negative bacteria such as the Klebsiella-Enterobacter-
Serratia
group and Pseudomona, and gram positive microbes such as Staphylococcus, can
be
the causative pathogen. The genitourinary tract, gastrointestinal tract and
respiratory
2o tract are the most frequent sources of sepsis. Other common foci are wound,
burn,
and pelvic infections and infected intravenous catheters.
A serious consequence of bacterial sepsis often is septic shock. Gram
negative sepsis is a disease syndrome resulting from the systemic invasion of
gram
negative rods and subsequent endotoxemia. (U.S. Patent No. 5,698,198,
1
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WO 99/52540 PCT/US99/07654
incorporated by reference herein in its entirety) The severity of the disease
ranges
from a transient, self limiting episode of bacteremia to a fulminant, life-
threatening
illness often complicated by organ failwe and shock. The disease is often the
result
of invasion from a localized infection site, or may result from trauma,
wounds,
ulcerations or gastrointestinal obstructions. The symptoms of gram negative
sepsis
include fever, chills, pulmonary failwe and septic shock (severe hypotension).
Gram negative infections are particularly common among patients receiving
anti-cancer chemotherapy and immunosuppressive treatment. (U.S. Patent No.
5,698,198) Infections in such immuno-compromised hosts characteristically
exhibit
resistance to many antibiotics, or develop resistance over the long course of
the
infection, making conventional treatment difficult. The ever increasing use of
cytotoxic and immunosuppressive therapy and the natural selection for drug
resistant
bacteria by the extensive use of antibiotics have contributed to gram negative
bacteria evolving into pathogens of major clinical significance.
Septic shock is a major cause of death in intensive care units. It is
estimated
that over 700,000 patients become susceptible to septic shock-causing
bacterial
infections each year in the United States alone. Of these, 160,000 actually
develop
septic shock, resulting in 50,000 deaths annually. (U.S. Patent No. 5,698,198)
Despite advances in respiratory support technology and antibiotic therapy, the
mortality rate for septic shock remains in excess of 40%. (L1.S. Patent No.
5,714,469) In fact, mortality for established septic shock has decreased very
little
over the past 50 years. (Arch. Intern. Med. 88:467-488 (1951)) Although
effective
antibiotics are available, and there is an increased awareness of the septic
shock
syndrome, the incidence of septic shock over the last several decades has
actually
2
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WO 99/52540 PCT/US99/07654
increased. With the appreciation that antimicrobial agents have failed to
completely
abrogate septic mortality, it is clear that other agents must be developed to
rectify
the deficiencies of current established therapy for septic shock, as well as
for other
types of infections, including but not limited to peritonitis, bacteremia,
endotoxemia,
and viral and parasitic infections.
Treatment of bacterial diseases with antibiotics is further complicated by the
ability of the organisms to develop antibiotic resistance. The magnitude of
the
problem is further amplified by the extreme difficulty of total eradication,
and the
frequent reappearance of the same strain even after apparently successful
1o elimination. The inability to eliminate the carrier state by any of the
currently known
methods and the prevalence of the new antibiotic resistant hospital strains
have
added a new dimension to the frustrating situation. The development of such
multiple antibiotic resistant strains of the organism further suggests the
desirability
of investigating additional means of combating bacterial infections.
Summary of the Invention
The present invention provides methods and kits for treating or preventing
infection comprising the administration of an amount effective for treating or
preventing an infection of angiotensinogen, angiotensin I (AI), AI analogues,
AI
2o fragments and analogues thereof, angiotensin II (AII), All analogues, All
fragments
or analogues thereof or All ATZ type 2 receptor agonists.
In another embodiment, the present invention provides improved methods
and pharmaceutical compositions for antibiotic therapy, wherein the
improvement
comprises the administration of an amount effective for treating a bacterial
infection
3
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WO 99/52540 PCTNS99/07654
of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII,
All
analogues, All fragments or analogues thereof or All AT2 type 2 receptor
agonists.
These aspects and other aspects of the invention become apparent in light of
the following detailed description.
s
Brief description of the drawings
Figure 1 is a graph showing the effect of All on host resistance to bacterial
peritonitis, based on the percentage of peritoneal sites without abscesses
associated
with infection.
1o Figure 2 is a graph showing the effect of All on host resistance to
bacterial
peritonitis, based on the mean overall abscess score.
Figure 3 is a graph showing the effect of All administration on abscess score
(mean
score).
Figure 4 is a graph showing the effect of All administration on abscess score
(rank
1 s order analysis).
Figure 5 is a graph showing the effect of All administration on abscess
incidence.
Figure 6 is a graph showing the effect of All with and without Ofloxacin on
abscess
formation (mean score).
Figure 7 is a graph showing the effect of All with and without Ofloxacin on
abscess
2o formation (rank order).
Figure 8 is a graph showing the effect of All with and without Ofloxacin on
abscess
free sites (mean score).
Figure 9 is a graph showing a comparison of AII, AII(1-7) and Neupogen in a
rat
peritonitis model (mean abscess score).
4
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WO 99/52540 PCT/US99/07654
Figure 10 is a graph showing a comparison of AII, AII(1-7) and Neupogen in a
rat
peritonitis model (rank order).
Figure 11 is a graph showing a comparison of AII, AII(1-7) and Neupogen in a
rat
peritonitis model (% abscess free).
Figure 12 is a graph showing a comparison of AII(1-7) analogues in the
infection
model (mean abscess score).
Figure 13 is a graph showing a comparison of AII(1-7) analogues in the
infection
model (rank order).
Figure 14 is a graph showing a comparison of AII(1-7) analogues in the
infection
to model (% abscess free).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
All references, patents and patent applications are hereby incorporated by
reference in their entirety.
1 s Infections, as used herein, are broadly defined to mean situations when
the
invasion of a host by an agent is associated with the clinical manifestations
of
infection including, but not limited to, at least one of the following:
abnormal
temperature, increased heart rate, abnormal respiratory rate, abnormal white
blood
cell count, fatigue, chills, muscle ache, pain, dizziness, dehydration,
vomiting,
2o diarrhea, and organ dysfunction. Such infections may be bacterial, viral,
or parasitic
m nature.
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
S
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WO 99/52540 PCT/US99/07654
(AII) in an amount which is sufficient for said increase. The application 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 (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); each reference hereby incorporated
in
its entirety). The substance so formed is a decapeptide called angiotensin I
(AI)
1o which is converted to All by the angiotensin converting enzyme (ACE) which
removes the C-terminal His-Leu residues from AI [SEQ ID NO: 37]. All is a
known pressor agent and is commercially available.
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. 21:57 (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 ai., 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). 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).
Additionally, All and angiotensin III analogs and fragments thereof have been
6
CA 02324988 2000-10-06
WO 99/52540 PCT/US99/07654
shown to be effective in tissue repair. (IJ.S. Patent 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.)
Studies have demonstrated that serum levels of ACE are decreased in
patients with Adult Respiratory Distress Syndrome CARDS), which is present in
patients with sepsis. (Rice et al., Circulatory Shock 11:59-63 (1983), and
that ACE
levels are increased during recovery of patients with bacterial pneumonia.
(Kerttula
and Weber, J. Clin. Pathol. 39:1250-1253 (1986)) Treatment of mice with an ACE
io inhibitor, which acts to prevent the formation of All from its precursor
angiotensin I,
after thermal injury resulted in greater survival and decreased bacterial
translocation
compared to controls. (Gennari et al., Shock 6:95-100 (1996))
All was used to identify changes in pulmonary microcirculation reactivity in
a rat model of sepsis. (Kirton et al., Intensive Care Med. 18:293-298 (1992))
The
effect of All on the underlying bacterial infection was not addressed. All has
been
used in the treatment of septic shock patients as a means to increase system
vascular
resistance (Thomas et al., Critical Care Medicine 19:1084-1086 (1991); Landow
J.
Cardiothor. Vasc. Anesth. 5:97-98 (1991)) with no indication of its effect on
the
underlying bacterial infection. All was also used as an adjunct to amrinone
2o treatment of refractory septic shock as a means to reverse the amrinone
vasodilatory
side effect; the successful treatment of the bacterial infection was
attributed entirely
to the effect of amrinone. (Ryding et al., Chest 107:201-203 (1995).
Based on all of the above, there is no indication in the art that
angiotensinogen, AI, AII, AI or All analogues or fragments or All AT2 type 2
7
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WO 99/52540 PCT/US99/07654
receptor agonists would be useful for the treatment and prevention of
bacterial,
viral, or parasitic infections, or that angiotensinogen, AI, AII, AI or All
analogues or
fragments or All AT2 type 2 receptor agonists would be useful as an
improvement
for antibiotic therapy.
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-
aminophenylalanine 6-All ["(p-NH2-Phe) 6-AII)"], Asp-Arg-Val-Tyr-Ile-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
1o 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).
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 ); Kauffrnan, et al., Life Sci. 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. AII( 1-7) elicits some, but not
the full
2o 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 17:111 S-1120
( 1991 ); Portsi, et a., Br. J. Pharmacol. 111:652-654 ( 1994).
8
CA 02324988 2000-10-06
WO 99/52540 PCT/US99/07654
As hereinafter defined, a preferred class of AT2 agonists for use in
accordance with the present invention comprises angiotensinogen, angiotensin I
(AI), AI analogues, AI fragments and analogues thereof, AII, All analogues,
All
fragments or analogues thereof or All AT2 type 2 receptor agonists 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.
The active All analogues, fragments of All and analogues thereof of
particular interest in accordance with the present invention comprise a
sequence
1o consisting of at least three contiguous amino acids of groups R'-Rg in the
sequence
of general
formula I
Ri _Rz_R3_R4_Rs_R6_R~Rs
in which R' and R2 together form a group of formula
X-RA-RB_
wherein X is H or a one to three peptide group,
RA is suitably selected from Asp, Glu, Asn, Acpc (1-
aminocyclopentane carboxylic acid), Ala, Me2Gly, Pro, Bet, Glu(NH2), Gly,
Asp{NH2) and Suc,
Rg 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, Lys,
norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;
9
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WO 99/52540 PCT/US99/07654
R4 is selected from the group consisting of Tyr, Tyr(P03)2, Thr, Ser,
Ala, 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 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-NH2-Phe.
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]; AII(1-7), Asp-Arg-Val-Tyr-Ile-His-Pro {SEQ ID N0:4]; AII(2-7). Arg-Val-
Tyr-Ile-His-Pro [SEQ ID N0:5]; 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-Val-Tyr-Ile-His
[SEQ
ID N0:8]; AII(1-5), 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:I 1]. Other preferred
embodiments include: Arg-norLeu-Tyr-Ile-His-Pro-Phe [SEQ ID 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-Tyr-Ile-His-Pro-Phe [SEQ ID N0:31 ]. AII(6-8), His-Pro-Phe [SEQ
CA 02324988 2000-10-06
WO 99/52540 PCT/US99/07654
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 a particularly preferred embodiment, the active compounds of the present
invention are selected from those comprising the following general formula:
Asp-Arg-R1-R2-Ile-His-Pro-R3, wherein
R1 is selected from the group consisting of Val, Pro, Lys, Norleu, and Leu;
R2 is selected from the group consisting of Ala, Tyr, and Tyr(P03)2; and
R3 is Phe or is absent.
Another class of compounds of particular interest in accordance with the
1 o 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, Aib, Acpc and Tyr;
R4 is selected from the group consisting of Tyr, Tyr(PO3)2, Thr, Ser,
homoSer and azaTyr;
Rs is selected from the group consisting of Ile, Ala, Leu, norLeu, Val
and Gly;
2o R6 is His, Arg or b-NH2-Phe;
R' is Pro or Ala; and
R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr.
11
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WO 99/52540 PCT/US99/07654
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]
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 N0:2].
Other
preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His-
Pro-Phe [SEQ ID N0:17] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID N0:18]. The
fragment AII(4-8) was ineffective in repeated tests; this is believed to be
due to the
exposed tyrosine on the N-terminus.
1o 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
Me2G1 N,N-dimethylglycyl
Bet 1-carboxy-N,N,N-trimethylmethanaminium
hydroxide
inner salt betaine
Suc Succin I
Phe Br -bromo-L- hen lalan 1
azaTyr aza-a'-homo-L- ros 1
Ac c 1-aminoc clo entane carbox lic acid
Aib 2-aminoisobu 'c acid
Sar N-meth 1 I c I sarcosine
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It has been suggested that All 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 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 R8 may also play an important
role
in the whole conformation of the peptide and/or contribute to the formation of
a
hypothetical hydrophobic pocket.
Appropriate side chains on the amino acid in position Rz may contribute to
1o 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 conformation. Accordingly,
R3 may
2o suitably be selected from Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc
and Tyr.
Lys has also been found to be effective at R3.
With respect to R4, 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, R4
is
13
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WO 99/52540 PCT/US99/07654
preferably selected from Tyr, Thr, 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). Ala has also been found to be effective at R4.
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 this position be selected from Ile, Ala, Leu, norLeu,
Gly and
Val.
In the angiotensinogen, AI, AI analogues, AI fragments and analogues
1o thereof, AII, All analogues, fragments and analogues of fragments of
particular
interest in accordance with the present invention, R~ is His, Arg or 6-NH2-
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 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 Rg. In position Rg, both a hydrophobic ring and
an
anionic carboxyl terminal appear to be particularly useful in binding of the
2o 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:
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WO 99/52540 PCT/US99/07654
TABLE 2
Angiotensin II Analogues
Analogue Amino Acid Sequence Sequence
Name Identifier
Analo As -Ar -Val-T -Val-His-Pro-Phe SE ID NO:
ue 1 19
Analo Asn-Arg-Val-Tyr-Val-His-Pro-Phe SE ID NO:
a 2 20
Analo Ala-Pro-Gl -As Ar -Ile-T -Val-His-Pro-PheSE ID NO:
ue 3 21
Analo Glu-Ar -Val-T -Ile-His-Pro-Phe SE ID NO:
ue 4 22
Analo As -L s-VaI-T -Ile-His-Pro-Phe SE ID NO:
ue 5 23
Analo As -Ar -AIa-T r-Ile-His-Pro-Phe SE ID NO:
a 6 24
Analo As -Ar -Val-Thr-Ile-His-Pro-Phe SE ID NO:
a 7 25
Analo As -Ar -Val-T -Leu-His-Pro-Phe SE ID NO:
a 8 26
Analo As -Ar -Val-T -IIe-Ar -Pro-Phe SE ID NO:
ue 9 27
Analo As -Ar -VaI-Tyr-Ile-His-Ala-Phe SE ID NO:
ue 10 28
Analo As -Ar -Val-Tyr-Ile-His-Pro-Tyr SE ID NO:
ue 11 29
Analo Pro-Ar -Val-T -IIe-His-Pro-Phe SE ID NO:
a 12 30
Analo As -Ar -Pro-T -Ile-His-Pro-Phe SE ID NO:
a 13 31
Analo As -Ar -Val-T r PO3 z-Ile-His-Pro-PheSE ID NO:
ue 14 32
Analo As -Ar -norLeu-Tyr-Ile-His-Pro-PheSE ID NO:
a 15 33
Analo As -Ar -Val-T -norLeu-His-Pro-Phe SE ID NO:
ue 16 34
Analo A -Ar -Val-homoSer-T -Ile-His-Pro-PheSE TD NO:
ue 17 35
The polypeptides of the instant invention may be produced by any standard
s method, including but not limited to recombinant DNA technology and
conventional
synthetic methods including, but not limited to, 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.
1o 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
15 reference in its entirety). Normally, either the amino or carboxyl group of
the first
CA 02324988 2000-10-06
WO 99/52540 PCTNS99/07654
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.
Preferably, peptides are synthesized according to standard solid-phase
methodologies, such as may be performed on an Applied Biosystems Model 430A
peptide synthesizer (Applied Biosystems, Foster City, Calif.), according to
manufacturer's instructions. Other methods of synthesizing peptides or
peptidomimetics, either by solid phase methodologies or in liquid phase, are
well
known to those skilled in the art.
In one aspect, the present invention provides methods and kits for treating
and preventing infections in a mammal comprising administering to the mammal
an
amount effective to treat or prevent an infection of 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
(the active agents).
2o The invention is appropriate for the treatment and prevention of all types
of
infection, including but not limited to septic shock, peritonitis, bacteremia,
endotoxemia, and viral and parasitic infections. The methods of the invention
are
applicable to infections resulting from any condition, including but not
limited to
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wounds, burns, infected intravenous catheters, trauma, ulcerations,
gastrointestinal
obstructions, or due to the immuno-compromised state of the host.
The active agents of the invention can be used alone or in a combination of
active agents, or may be used in combination with other anti-infective agents,
including but not limited to oflaxacin, granulocyte colony stimulating
factors,
gentamicin, augmentin or cephalosporins such as ceftazidime, amino-glycosides
(such as amikacin, tobramycin, netilmicin, and gentamicin), related beta-
lactam
agents such as maxalactam, carbopenems such as imipenem, monobactam agents
such as aztreonam; ampicillin and broad-spectrum penicillins, (e.g.,
penicillinase-
1o resistant penicillins, ureidopenicillins or antipseudomonal penicillin or
Augmentin)
that are active against P. aeruginosa, Enterobacter species, indole-positive
Proteus
species, and Serratia. Also included within the definition of anti-infective
agents are
antifungal agents, amphotericin and the like as well as anti-vital agents such
as
famvir and acyclovir.
In another embodiment, the present invention provides improved methods,
compositions, and kits for antibiotic therapy, wherein the improvement
comprises
the administration of an amount effective for treating a bacterial infection
of the
active agents.
The active agents may be administered by any suitable route, including
orally, parentally, . by inhalation spray, rectally, or topically in dosage
unit
formulations containing conventional pharmaceutically acceptable carriers,
adjuvants, and vehicles. The term parenteral as used herein includes,
subcutaneous,
intravenous, intraarterial, intramuscular, intrasternal, intratendinous,
intraspinal,
intracranial, intrathoracic, infusion techniques or intraperitoneally.
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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). The compounds of the invention may be applied in a variety of
solutions. Suitable solutions for use in accordance with the invention are
sterile,
dissolve sufficient amounts of the peptide, and are not harmful for the
proposed
application. In this regard, the compounds of the present invention are very
stable
but are hydrolyzed by strong acids and bases. The compounds of the present
invention are soluble in organic solvents and in aqueous solutions at pH 5-8.
The active agents may be subjected to conventional pharmaceutical
l0 operations such as sterilization andlor may contain conventional adjuvants,
such as
preservatives, stabilizers, wetting agents, emulsifiers, buffers etc. For
administration, the 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 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
diluent may
include time delay material, such as glyceryl monostearate or glyceryl
distearate
alone or with a wax, or other materials well known in the art.
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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 treating or preventing infections in a mammal with
the active agents is based on a variety of factors, including the type of
injury, the
age, weight, sex, medical condition of the individual, the severity of the
condition,
the route of administration, and the particular compound employed. Thus, the
dosage regimen may vary widely, but can be determined routinely by a physician
to using standard methods. Dosage levels of the order of between 0.1 ng/kg and
10
mg/kg body weight active agent are useful for all methods of use disclosed
herein.
The treatment regime will also vary depending on the infection being treated,
based on a variety of factors, including the type of infection, the age,
weight, sex,
medical condition of the individual, the severity of the condition, the route
of
administration, and the particular compound employed. For example, the active
agents are administered to a mammal suffering from bacteremia for two weeks.
The
therapy is administered for between two and five times per day at dosages as
described above.
In a preferred embodiment, the active agent is administered subcutaneously
or intraperitoneally. A suitable subcutaneous dose of active agent is
preferably
between about 0.1 ng/kg and about 10 mg/kg administered twice daily for a time
sufficient to treat or prevent infections in a mammal. In a more preferred
embodiment, the concentration of active agent is between about 100 ng/kg body
weight and about 10.0 mg/kg body weight. In a most preferred embodiment, the
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concentration of active agent is between about 10 p.g/kg body weight and about
10.0
mg/kg body weight. This dosage regimen maximizes the therapeutic benefits of
the
subject invention while minimizing the amount of antagonist needed. Such an
application minimizes costs as well as possible deleterious side effects.
For subcutaneous administration, the active ingredient may comprise from
0.0001 % 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 5% w/w, and
more preferably from 0.1 % to 1 % of the formulation.
In another preferred embodiment of the present invention, the active agent is
to administered topically. Suitable topical doses and active ingredient
concentration in
the formulation are as described for subcutaneous administration.
In a further aspect, the present invention provides kits for treating or
preventing infection in a mammal, wherein the kits comprise an effective
amount of
the active agents for treating or preventing infection in a mammal, and
instructions
for using the amount effective of active agent as a therapeutic. In a
preferred
embodiment, the kit further comprises a pharmaceutically acceptable Garner,
such as
those adjuvants described above. In another preferred embodiment, the kit
further
comprises a means for delivery of the active agent to a patient. Such devices
include, but are not limited to syringes, matrical or micellar solutions,
bandages,
wound dressings, aerosol sprays, lipid foams, transdermal patches, topical
administrative agents, polyethylene glycol polymers, carboxymethyl cellulose
preparations, crystalloid preparations (e.g., saline, Ringer's lactate
solution,
phosphate-buffered saline, etc.), viscoelastics, polyethylene glycols, and
polypropylene glycols. The means for delivery may either contain the effective
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amount of the active agent, or may be separate from the active agents, which
are
then applied to the means for delivery at the time of use.
The active agents can be administered alone, or may be combined with other
anti-infective agents in combinatorial therapy. In a preferred embodiment the
anti-
infective agent is selected from the group consisting of Ofloxacin and
granulocyte
colony stimulating factors.
In another aspect of the invention, the method comprises pharmaceutical
compositions for treating or preventing infections, comprising the active
agents of
the invention, an amount an amount effective to treat or prevent an infection
of an
anti-infective agent and a pharmaceutically acceptable carrier. In a preferred
embodiment, the anti-infective agent is select4ed from the group consisting of
Ofloxacin and granulocyte colony stimulating factors.
The methods and kits of the present invention provide significant benefits for
the treatment and prophylaxis of mammalian infections. The methods and kits of
the present invention may be particularly valuable in the hospital setting,
where
potentially serious bacterial infections are common, and may also enable
decreased
reliance on high doses of antibiotics, which can lead to the development of
antibiotic
resistant bacteria.
The present invention may be better understood with reference to the
2o accompanying example that is 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 in a rat model of bacterial peritonitis
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The following rat model of bacteremia was used in all subsequent examples.
Forty-five female Sprague Dawley rats, weighing between 175 and 225 grams
each,
were used in the study. Fifteen of the rats were used to produce fecal
material. The
rats were housed in the University of Southern California vivarium on a 12:12
hour
light/dark cycle and were quarantined at least two days prior to surgery. Food
and
water were available ad libitum except in the immediate postoperative period.
All was purchased from Bacem (Torrance, CA) and resuspended in saline on
the day of surgery. The peptide was placed in an Alzet miniosmotic pump (Model
1002, 0.5 ttl/hour for 14 days). The cecal contents and feces from rats fed
1o hamburger for two weeks were collected and mixed 1:1 with peptone yeast
glucose
broth (Scott Laboratories) and 10% barium sulfate. The amount of this fecal
preparation that caused mortality in 0 to 30% of the rats (LD2o) was
determined in
preliminary studies. The appropriate amount of material was added to a gelatin
capsule (Number 1, Eli Lilly Co., Indianapolis, IN). This capsule was then
placed in
a second larger capsule (Number 00, Eli Lilly Co.) This was referred to as a
double-
walled gelatin capsule.
The rats underwent a standardized procedure for laparotomy (intramuscular
anesthesia with ketamine/rompum, shaving with animal clippers, betadine scrub,
alcohol scrub). A 2 cm incision was then made on the midline. A double-walled
2o gelatin capsule was placed on the right side of the abdomen through the
incision. A
polyethylene tube (PE 60) was sutured to the left sidewall and attached to the
Alzot
miniosmotic pump containing either saline, 10 pg/kg/day AII, or 100 pg/kg/day
AII.
The pump was then placed in a subcutaneous pocket. The abdominal wall and skin
was then sutured closed using two layers of 4-0 Ethilon suture. Following
surgery,
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the rats received analgesic for three days and were observed twice daily for
signs of
morbidity and mortality.
Rats that died during the 11 day post-operative observation period were
necropsied to confirm the presence of an acute bacterial infection. The rats
that
survived the initial acute infection were terminated on day 12 after surgery.
Each rat
was examined for odor upon opening and splenomegaly. Additionally, four areas
of
the peritoneum were examined for abscess formation. These areas included the
liver, abdominal wall, bowel and omentum. The abscesses were scored at each
site
as follows:
l0 0 No abscess present at site
0.5 One very small abscess present at site
1 Several small abscesses present at site
2 Medium abscess present at site
3 Large or several medium abscesses present at site
4 One very large or several small abscesses present at site
The scoring was conducted in a blinded fashion by two separate observers
and the scores recorded. If there was a disagreement between the two observers
as
to the score at a particular site the more severe score was reported. The
results
(Figures 1-2 and Tables 3-5) demonstrate that although All administration did
not
affect mortality after exposure to the bacterial inoculum, it did reduce the
formation
of abscesses associated with the infection.
Table 3 Abscess Scores in Saline Treated Rats
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Table 4 Abscess Scores in Rats Treated with 10 ~g/kg/day All
Table 5 Abscess Scores in Rats Treated with 100 ~g/kg/day All
Example 2. All administration timing and route
The rat peritonitis model was generated as in Example 1. All (100
pg/kg/day) was given either: (1) subcutaneously (daily) three days before and
after
initiation of infection (SQ/SQ); (2) subcutaneously only after initiation of
infection
to (SQ Post); (3) subcutaneously (daily) three days before and
intraperitoneally after
initiation of infection (SQ/IP); (4) intraperitoneally only after initiation
of infection
(IP Post); or (5) intraperitoneally via Alzet pump starting at the initiation
of infection
throughout the post-infection interval (Pump).
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The results of these experiments are expressed in three different ways. The
first is the mean of the overall abscess scores in the abdomen of the animals
(FIG. 3:
Mean Score). These data are nonparametric and should therefore be analyzed by
a
nonparametric statistical test. We used an analysis of variance of the rank
order of
the overall abscess scores; thus, the second method of presenting data is as
the mean
and SEM of the rank (FIG. 4: Rank Order Analysis). Lastly, it is important to
determine whether the test compounds reduce the incidence of abscesses in
addition
to reducing abscess size. Therefore, the incidence of abscess free sites (FIG.
5:
Sites Abscess Free) is also graphically presented.
1 o The data for these experiments are presented in Figure 3-5. These data
indicate that a reduction in both the size and the occurrence of abscess
formation (ie:
reduced bacterial peritonitis) was observed after each treatment regimen.
Thus, the
data indicate that All is effective both for treating and preventing bacterial
peritonitis.
Example 3. Effect of All compared to and in combination with Ofloxacih
The rat peritonitis model was generated as in Example 1. All (100
wg/kg/day) was give alone or in combination {AII/Oflox) with Ofloxacin (Sigma
Chemical CO., St. Lotus, MO) (6.7 mg/kg/day), a broad spectrum antibiotic.
2o Pretreatment with All by subcutaneous injection was given to all animals
that
received post-infection All for three days. The routes of administration
tested
included subcutaneous (sq) and intraperitoneal via Alzet miniosmotic pump
(Pump).
Experimental results are presented as described for example 2. The results
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demonstrated that All treatment provided improved reduction in the size and
the
occurrence of abscess formation (ie: reduced occurrence and severity of
bacterial
peritonitis) relative to Ofloxacin. Furthermore, combined therapy with All and
Ofloxacin improved the efficacy of the Ofloxacin. (FIGURES 6-8)
Example 4. Comparison ofAll, All(1-7) and G-CSF
The rat peritonitis model was generated as in Example 1. All (1-100
pg/kg/day) and AII(1-7) (1-100 ~tg/kg/day) treatment were compared with G-CSF
treatment (Neupogen--Amgen, Thousand Oaks, CA) (0.1-10 wg/kg/day) for
~ o reduction in abscess size and occurrence. The experimental treatments were
given
by subcutaneous injection starting three days prior to initiation of infection
and
continued until the animals were euthanized.
Experimental results are presented as described for example 2. The results
demonstrated that at all concentrations tested, treatment with both All and
AII(1-7)
provided improved reduction in the size and the occurrence of abscess
formation (ie:
reduced occurrence and severity of bacterial peritonitis} relative to G-CSF
treatment.
(Figures 9-11)
Example S. All(1-7) analogue effect on abscess formation
2o The rat peritonitis model was generated as in Example 1. Subcutaneous
injections with the peptides listed in Table 6 (10-100 pg/kg/day) were
initiated three
days prior to the initiation of infection ~ and continued until the animals
were
euthanized.
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Experimental results are presented as described for example 2. The results
demonstrated that at all concentrations tested, treatment with each of the
AII(1-7)
analogues reduced. the size and occurrence of abscess formation (ie: reduced
occurrence and severity of bacterial peritonitis). (Figures 12-14)
Table 6 AII(1-7) Analogues used
Peptide Designation Sequence SEQ ID NO
1GD Ala4-AII(1-7) DRVAIHP SEQ ID N0:38
2GD Pro3-AII(1-7) DRPYIHP SEQ ID N0:39
1o SGD Lys3-AII(1-7) DRKYIHP SEQ ID N0:40
9GD NorLeu-AII(1-7)DR(nor)YIHP SEQ ID N0:41
All DRVYIHPF SEQ ID NO:1
It is to be understood that the invention is not to be limited to the exact
details of operation; or to the exact compounds, compositions, methods,
procedures
or embodiments shown and described, as obvious modifications and equivalents
will
be apparent to one skilled in the art, and the invention is therefore to be
limited only
by the full scope of the appended claims.
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SEQUENCE LISTING
<110> Kathleen Rodgers and Gere diZerega
<120> Method for Treatment and Prevention of Infections
<130> 98,017-M2
<140> To be assigned
<141> To be assigned
<160> 41
<170> PatentIn Ver. 2.0
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:angiotensin II
<400> 1
1
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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:AII2-8
<400> 2
Arg Val Tyr Ile His Pro Phe
1 5
2
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<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
3
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<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:ATI(1-7)
<400> 4
Asp Arg Val Tyr Ile His Pro
1 5
<210> 5
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
4
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<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>
<223> Description of Artificial Sequence:AII(3-7)
<400> 6
S
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Val Tyr Ile His Pro
1 5
<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
6
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<210> 8
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII(1-6)
<400> 8
Asp Arg Val Tyr Ile His
1 5
<210> 9
<211> 5
<212> PRT
7
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<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
<212> PRT
<213> Artificial Sequence
<220>
8
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<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
9
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1
<210> 12
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<221> Xaa at postion 2 is Nle
<222> 2
<223> Description of Artificial Sequence:AII analogue
<400> 12
Arg Xaa Tyr Ile His Pro Phe
1 5
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<210> 13
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<221> Xaa at position 4 is Nle
<222> 4
<223> Description of Artificial Sequence:AII analogue
<400> 13
Arg Val Tyr Xaa His Pro Phe
1 5
11
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<210> 14
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII(6-8)
<400> 14
His Pro Phe
1
<210> 15
<211> 5
<212> PRT
<213> Artificial Sequence
12
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<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>
<221> Xaa at position 1 can be Hydrogen, Arg, Lys, Ala, Or
n, Ser(Acetylated), MeGly, D-Arg, or D-Lys; Xaa at positi
on 2 can be Val, Ala, Leu, Nle, Ile, Gly, Pro, Aib, Acp, o
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r Tyr; Xaa at position 4 can be Ile, Ala, Leu, Nle, Val, o
r Gly
<222> 1-4
<223> Description of Artificial Sequence:AII analogue
class
<400> 16
Xaa Xaa Tyr Xaa His Pro Phe
1 5
<210> 17
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
14
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<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
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1 5
<210> 19
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue i
<400> 19
Asp Arg Val Tyr Val His Pro Phe
1 5
16
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<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
<210> 21
<211> 11
<212> PRT
<213> Artificial Sequence
17
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<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
18
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<400> 22
Glu Arg Val Tyr Ile His Pro Phe
1 5
<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
19
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1 5
<210> 24
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII Analogue 6
<400> 24
Asp Arg Ala Tyr Ile His Pro Phe
1 5
<210> 25
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<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII Analoge 7
<400> 25
Asp Arg Val Thr Ile His Pro Phe
1 5
<210> 26
<211> 8
<212> PRT
<213> Artificial Sequence
21
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<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
22
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<400> 27
Asp Arg Val Tyr Ile Arg Pro Phe
1 5
<210> 28
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII Analogue 10
<400> 2B
Asp Arg Val Tyr Ile His Ala Phe
1 5
23
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<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
24
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<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
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<220>
<223> Description of Artificial Sequence:AII Analogue 13
<400> 31
Asp Arg Pro Tyr Ile His Pro Phe
1 5
<210> 32
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<221> PHOSPHORYLATION
<222> 4
26
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<223> Description of Artificial Sequence:AII Analogue 14
<400> 32
Asp Arg Val Tyr Ile His Pro Phe
1 5
<210> 33
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<221> Xaa at position 3 is Nle
<222> 3
<223> Description of Artificial Sequence:AII Analogue l5
27
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<400> 33
Asp Arg Xaa Tyr Ile His Pro Phe
1 5
<210> 34
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<221> Xaa at position 5 is Nle
<222> S
<223> Description of Artificial Sequence:AII Analogue 16
<400> 34
Asp Arg Val Tyr Xaa His Pro Phe
28
CA 02324988 2000-10-06
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1 5
<210> 35
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> homo Ser
<222> 4
<223> Description of Artificial Sequence:AII Analogue 17
<400> 35
Asp Arg Val Ser Tyr Ile His Pro Phe
1 5
29
CA 02324988 2000-10-06
WO 99/52540 PCT/US99/07654
<210> 36
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<221> Xaa at position 6 is p-aminophenylalanine
<222> 6
<223> Description of Artificial
Sequence:p-aminophenylalanine 6 All
<400> 36
Asp Arg Val Tyr Ile Xaa Pro Phe
1 5
CA 02324988 2000-10-06
WO 99/52540 PCTNS99/07654
<210> 37
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:angiotensin I
<400> 37
Asp Arg Val Tyr Ile His Pro Phe His Leu
1 5 10
<210> 38
<211> 7
<212> PRT
<213> Artificial Sequence
31
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<220>
<223> Description of Artificial
Sequence:lGD: ALa4-AII(1-7)
<400> 38
Asp Arg Val Ala Ile His Pro
1 5
<210> 39
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial
Sequence:2GD: Pro3 AII(1-7)
32
CA 02324988 2000-10-06
WO 99/52540 PCTNS99/07654
<400> 39
Asp Arg Pro Tyr Ile His Pro
1 5
<210> 40
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial
Sequence:SGD: Lys3 AII(1-7)
<400> 40
Asp Arg Lys Tyr Ile His Pro
1 5
<210> 41
33
CA 02324988 2000-10-06
WO 99/52540 PCT/US99/07654
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<221> Xaa at position 3 is norLeu
<222> 3
<223> Description of Artificial
Sequence: 9GD: norLeu AII(1-7)
<400> 41
Asp Arg Xaa Tyr Ile His Pro
1 5
34
