U.S. patent application number 12/502937 was filed with the patent office on 2010-12-30 for cyclic peptides as g-protein coupled receptor antagonists.
This patent application is currently assigned to PROMICS PTY LIMITED. Invention is credited to David Fairlie, Darren March, Ian Alexander Shiels, Stephen Maxwell Taylor, Michael Whitehouse.
Application Number | 20100331236 12/502937 |
Document ID | / |
Family ID | 3832159 |
Filed Date | 2010-12-30 |
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United States Patent
Application |
20100331236 |
Kind Code |
A1 |
Taylor; Stephen Maxwell ; et
al. |
December 30, 2010 |
CYCLIC PEPTIDES AS G-PROTEIN COUPLED RECEPTOR ANTAGONISTS
Abstract
The invention relates to novel cyclic compounds which have the
ability to modulate the activity of G protein-coupled receptors.
The compounds preferably act as antagonists. In preferred
embodiments, the invention provides cyclic peptidic and
peptidomimetic antagonists of C5a receptors, which are active
against C5a receptors on polymorphonuclear leukocytes and
macrophages. The compounds of the invention are both potent and
selective, and are useful in the treatment of a variety of
inflammatory conditions.
Inventors: |
Taylor; Stephen Maxwell;
(Bellbird Park, AU) ; Shiels; Ian Alexander;
(Muirlea, AU) ; Fairlie; David; (Springwood,
AU) ; March; Darren; (Banks, AU) ; Whitehouse;
Michael; (Holland Park W., AU) |
Correspondence
Address: |
HAYNES AND BOONE, LLP;IP Section
2323 Victory Avenue, Suite 700
Dallas
TX
75219
US
|
Assignee: |
PROMICS PTY LIMITED
Macquarie Park
AU
UNIQUEST PTY LIMITED
Brisbane
AU
|
Family ID: |
3832159 |
Appl. No.: |
12/502937 |
Filed: |
July 14, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10493117 |
Oct 24, 2005 |
7579432 |
|
|
PCT/AU02/01427 |
Oct 17, 2002 |
|
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12502937 |
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Current U.S.
Class: |
514/1.5 ;
514/1.7; 514/1.9; 514/16.4; 514/16.6; 514/17.7; 514/17.8; 514/17.9;
514/18.7; 514/21.1; 530/321 |
Current CPC
Class: |
A61P 17/06 20180101;
C07K 14/472 20130101; A61P 19/04 20180101; A61P 17/00 20180101;
A61P 29/00 20180101; A61P 11/06 20180101; A61P 9/10 20180101; A61P
11/00 20180101; A61K 38/12 20130101; A61P 25/28 20180101; A61P
37/00 20180101; A61P 37/02 20180101; A61P 19/02 20180101; A61P
19/00 20180101; A61P 31/04 20180101; A61P 25/00 20180101; A61P 7/00
20180101; A61P 37/06 20180101; A61P 1/02 20180101; A61P 43/00
20180101; C07K 7/56 20130101; A61P 17/04 20180101 |
Class at
Publication: |
514/1.5 ;
530/321; 514/21.1; 514/16.6; 514/16.4; 514/1.9; 514/17.9; 514/17.8;
514/1.7; 514/17.7; 514/18.7 |
International
Class: |
A61K 38/12 20060101
A61K038/12; C07K 7/64 20060101 C07K007/64; A61P 9/10 20060101
A61P009/10; A61P 25/28 20060101 A61P025/28; A61P 29/00 20060101
A61P029/00; A61P 19/02 20060101 A61P019/02; A61P 11/00 20060101
A61P011/00; A61P 11/06 20060101 A61P011/06; A61P 25/00 20060101
A61P025/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 17, 2001 |
AU |
PR 8334 |
Nov 7, 2002 |
AU |
PCT/AU02/01427 |
Claims
1. A compound which is an antagonist of a C5a G protein-coupled
receptor, which has no C5a agonist activity, and which is a cyclic
peptide or peptidomimetic of the general formula: ##STR00056##
wherein A is H, alkyl, aryl, NH-alkyl, N(alkyl).sub.2, NH-aryl,
NH-benzoyl, NHSO.sub.3, NHSO.sub.2-alkyl, NHSO.sub.2-aryl, OH,
O-alkyl, or O-aryl; B is an alkyl, aryl, benzyl, naphthyl or indole
group, or is the side chain of L-phenylalanine or L-phenylglycine;
C is a side chain of glycine, alanine, leucine, valine, proline,
hydroxyproline, or thioproline; D is a side chain of D-leucine,
D-homoleucine, D-cyclohexylalanine, D-homocyclohexylalanine,
D-valine, D-norleucine, D-homo-norleucine, D-phenylalanine,
D-tetrahydroisoquinoline, D-glutamine, D-glutamate, or D-tyrosine;
E is L-1-napthyl or L-3-benzothienyl alanine, or is a side chain of
an amino acid selected from the group consisting of
L-phenylalanine, L-tryptophan and L-homotryptophan; F is a side
chain of L-arginine, L-homoarginine, L-citrulline, or L-canavanine;
X is --(CH.sub.2).sub.nNH-- or (CH.sub.2).sub.n--S--, where n is an
integer of from 1 to 4; --(CH.sub.2).sub.2O--;
--(CH.sub.2).sub.3O--; --(CH.sub.2).sub.3--; --(CH.sub.2).sub.4--;
--CH.sub.2COCHRNH--; or --CH.sub.2--CHCOCHRNH--, and where R is a
side chain of any common or uncommon amino acid, with the proviso
that the compound is not AcF-[OPdChaWR] (compound 1).
2-18. (canceled)
19. The compound of claim 1, in which n is 2 or 3.
20. The compound of claim 1, in which A is an aminomethyl group, or
a substituted or unsubstituted sulphonamide group.
21. The compound of claim 20, in which A is a substituted
sulfonamide group and wherein the substituent on the substituted
sulfonamide is an alkyl chain of 1 to 6 carbon atoms, or a phenyl
or toluoyl group.
22. The compound of claim 21, in which A is a substituted
sulfonamide group, and wherein the substituent on the substituted
sulfonamide is an alkyl chain of 1 to 4 carbon atoms.
23. The compound of claim 1, in which the compound has antagonist
activity against a C5a receptor, a vasopressin receptor or a
neurokinin receptor.
24. The compound of claim 1, in which the compound has antagonist
activity at submicromolar concentrations.
25. The compound of claim 24, in which the compound has a receptor
affinity IC.sub.50<25 .mu.M, and an antagonist potency
IC.sub.50<1 .mu.M.
26. A compound selected from the group consisting of compounds 2,
10, 11 and 17. ##STR00057## ##STR00058##
27. The compound of claim 1 or claim 26, together with a
pharmaceutically-acceptable carrier or excipient.
28. The compound of claim 1, having the formula
hydrocinnamate-[Orn-Pro-dCha-Trp-Arg].
29. A composition comprising the compound of claim 1, and a
pharmaceutically-acceptable carrier or excipient.
30. A method of treatment of a pathological condition mediated by a
G protein-coupled receptor, comprising administering an effective
amount of a compound according to claim 1 to a mammal in need of
such treatment.
31. The method of claim 30, in which the condition mediated by a G
protein-coupled receptor is a condition mediated by a C5a
receptor.
32. The method of claim 31, in which the condition involves
overexpression or underregulation of C5a.
33. The method of claim 32, in which the condition comprises
rheumatoid arthritis, adult respiratory distress syndrome (ARDS),
systemic lupus erythematosus, tissue graft rejection, ischemic
heart disease, reperfusion injury, septic shock, gingivitis,
fibrosis, atherosclerosis, multiple sclerosis, Alzheimer's disease,
asthma, dementias, central nervous system disorders, lung injury,
extracorporeal post-dialysis syndrome, or dermal inflammatory
disorders.
34. The method of claim 33, in which the condition is reperfusion
injury.
35. A method of treating reperfusion injury, comprising
administering to a mammal in need of such treatment an effective
amount of a compound according to claim 1.
Description
[0001] The present application is a continuation of U.S.
application Ser. No. 10/493,117, filed Oct. 24, 2005 (now allowed),
which was the United States national stage of PCT International
Patent Application PCT/NZ2000/00064, filed Apr. 28, 2000 (now
expired), which claimed priority to New Zealand Provisional
Application No. 335553, filed Apr. 30, 1999 (now expired); the
contents of each of which is specifically incorporated herein in
its entirety by express reference thereto.
FIELD OF THE INVENTION
[0002] The invention relates to novel cyclic compounds which have
the ability to modulate the activity of G protein-coupled
receptors. The compounds preferably act as antagonists. In
preferred embodiments, the invention provides cyclic peptidic and
peptidomimetic antagonists of C5a receptors, which are active
against C5a receptors on polymorphonuclear leukocytes and
macrophages. The compounds of the invention are both potent and
selective, and are useful in the treatment of a variety of
inflammatory conditions. This invention relates to novel cyclic
compounds which have the ability to modulate the activity of G
protein-coupled receptors. The compounds preferably act as
antagonists. In preferred embodiments, the invention provides
cyclic peptidic and peptidomimetic antagonists of C5a receptors,
which are active against C5a receptors on polymorphonuclear
leukocytes and macrophages. The compounds of the invention are both
potent and selective, and are useful in the treatment of a variety
of inflammatory conditions.
BACKGROUND OF THE INVENTION
[0003] All references, including any patents or patent
applications, cited in this specification are hereby incorporated
by reference. No admission is made that any reference constitutes
prior art. The discussion of the references states what their
authors assert, and the applicants reserve the right to challenge
the accuracy and pertinency of the cited documents. It will be
clearly understood that, although a number of prior art
publications are referred to herein, this reference does not
constitute an admission that any of these documents forms part of
the common general knowledge in the art, in Australia or in any
other country.
[0004] G protein-coupled receptors are prevalent throughout the
human body, comprising approximately 60% of known cellular receptor
types, and mediate signal transduction across the cell membrane for
a very wide range of endogenous ligands. They participate in a
diverse array of physiological and pathophysiological processes,
including, but not limited to those associated with cardiovascular,
central and peripheral nervous system, reproductive, metabolic,
digestive, immunoinflammatory, and growth disorders, as well as
other cell-regulatory and proliferative disorders. Agents which
selectively modulate functions of G protein-coupled receptors have
important therapeutic applications. These receptors are becoming
increasingly recognised as important drug targets, due to their
crucial roles in signal transduction (G protein-coupled Receptors,
IBC Biomedical Library Series, 1996).
[0005] One of the most intensively studied G protein-coupled
receptors is the receptor for C5a. C5a is one of the most potent
chemotactic agents known, and recruits neutrophils and macrophages
to sites of injury, alters their morphology; induces degranulation;
increases calcium mobilisation, vascular permeability (oedema) and
neutrophil adhesiveness; contracts smooth muscle; stimulates
release of inflammatory mediators, including histamine,
TNF-.alpha., IL-1, IL-6, IL-8, prostaglandins, and leukotrienes,
and of lysosomal enzymes; promotes formation of oxygen radicals;
and enhances antibody production (Gerard and Gerard, 1994).
[0006] Overexpression or underregulation of C5a is implicated in
the pathogenesis of immune system-mediated inflammatory conditions,
such as rheumatoid arthritis, adult respiratory distress syndrome
(ARDS), systemic lupus erythematosus, tissue graft rejection,
ischaemic heart disease, reperfusion injury, septic shock,
psoriasis, gingivitis, atherosclerosis, Alzheimer's disease, lung
injury and extracorporeal post-dialysis syndrome, and in a variety
of other conditions (Whaley 1987; Sim 1933).
[0007] Agents which limit the pro-inflammatory actions of C5a have
potential for inhibiting chronic inflammation, and its accompanying
pain and tissue damage. For these reasons, molecules which prevent
C5a from binding to its receptors are useful for treating chronic
inflammatory disorders driven by complement activation. Such
compounds also provide valuable new insights into the mechanisms of
complement-mediated immunity.
[0008] In our previous application No. PCT/AU98/00490, the entire
disclosure of which is incorporated herein by this reference, we
described the three-dimensional structure of some analogues of the
C-terminus of human C5a, and used this information to design novel
compounds which bind to the human C5a receptor (C5aR), behaving as
either agonists or antagonists of C5a. It had previously been
thought that a putative antagonist might require both a C-terminal
arginine and a C-terminal carboxylate for receptor binding and
antagonist activity (Konteatis et al, 1994). In PCT/AU98/00490, but
we showed that in fact a terminal carboxylate group is not
generally required either for high affinity binding to C5aR or for
antagonist activity. Instead we found that a hitherto unrecognized
structural feature, a turn conformation, was the key recognition
feature for high affinity binding to the human C5a receptor on
neutrophils. We used these findings to design constrained
structural templates which enable hydrophobic groups to be
assembled into a hydrophobic array for interaction with a C5a
receptor.
[0009] By investigating the effect of varying the structure at each
amino acid residue in the most potent compound identified in our
previous application, we have now developed further examples of
cyclic antagonists of the C5a receptor on human neutrophils and
have identified potent C5aR antagonist activity for a range of
compounds. These compounds each comprise a cyclic scaffold which
satisfies the general three-dimensional structural requirements set
out in the earlier application No. PCT/AU98/00490, but we have now
found that certain substituents attached to the cycle surprisingly
lead to most unexpected results, producing both high and low
antagonist potencies which were not accurately predicted in the
previous application No. PCT/AU98/00490. These surprising new
findings allow us to refine and better define the required
pharmacophore for antagonism of C5a receptors. The unexpected
structure-activity relationships described herein help to define a
refined structural pharmacophore for active antagonism of C5a
receptors on human polymorphonuclear leukocytes (neutrophil
granulocytes). This pharmacophore is expected to be appropriate
also for C5a receptors on other human and mammalian cells.
SUMMARY OF THE INVENTION
[0010] According to a first aspect, the invention provides a
compound which is an antagonist of a G protein-coupled receptor,
which has substantially no agonist activity, and which is a cyclic
peptide or peptidomimetic of formula I:
##STR00001##
[0011] where A is H, alkyl, aryl, NH.sub.2, NH-alkyl,
N(alkyl).sub.2, NH-aryl, NH-acyl, NH-benzoyl, NHSO.sub.3,
NHSO.sub.2-alkyl, NHSO.sub.2-aryl, OH, O-alkyl, or O-aryl;
[0012] B is an alkyl, aryl, phenyl, benzyl, naphthyl or indole
group, or the side chain of a D- or L-amino acid such as
L-phenylalanine or L-phenylglycine, but is not the side chain of
glycine, D-phenylalanine, L-homophenylalanine, L-tryptophan,
L-homotryptophan, L-tyrosine, or L-homotyrosine;
[0013] C is a small substituent, such as the side chain of a D-, L-
or homo-amino acid such as glycine, alanine, leucine, valine,
proline, hydroxyproline, or thioproline, but is preferably not a
bulky substituent such as isoleucine, phenylalanine, or
cyclohexylalanine;
[0014] D is the side chain of a neutral D-amino acid such as
D-Leucine, D-homoleucine, D-cyclohexylalanine,
D-homocyclohexylalanine, D-valine, D-norleucine, D-homo-norleucine,
P-phenylalanine, D-tetrahydroisoquinoline, D-glutamine,
D-glutamate, or D-tyrosine, but is preferably not a small
substituent such as the side chain of glycine or D-alanine, a bulky
planar side chain such as D-tryptophan, or a bulky charged side
chain such as D-arginine or D-Lysine;
[0015] E is a bulky substituent, such as the side chain of an amino
acid selected from the group consisting of L-phenylalanine,
L-tryptophan and L-homotryptophan, or is L-1-napthyl or
L-3-benzothienyl alanine, but is not the side chain of
D-tryptophan, L-N-methyltryptophan, L-homophenylalanine,
L-2-naphthyl L-tetrahydroisoquinoline, L-cyclohexylalanine,
D-leucine, L-fluorenylalanine, or L-histidine;
[0016] F is the side chain of L-arginine, L-homoarginine,
L-citrulline, or L-canavanine, or a bioisostere thereof, i.e. a
side chain in which the terminal guanidine or urea group is
restrained, but the carbon backbone is replaced by a group which
has different structure, but is such that the side chain as a whole
reacts with the target protein in the same way as the parent
group;
[0017] X is --(CH.sub.2).sub.nNH-- or (CH.sub.2).sub.n--S--, where
n is an integer of from 1 to 4, preferably 2 or 3;
--(CH.sub.2).sub.2O--; --(CH.sub.2).sub.3O--; --(CH.sub.2).sub.3--;
--(CH.sub.2).sub.4--; --CH.sub.2COCHRNH--; or
--CH.sub.2--CHCOCHRNH--, and where R is the side chain of any
common or uncommon amino acid, with the proviso that the compound
is not compound 1 referred to below.
[0018] In C, both the cis and trans forms of hydroxyproline and
thioproline may be used.
[0019] Preferably A is an acetamide group, an aminomethyl group, or
a substituted or unsubstituted sulphonamide group.
[0020] Preferably where A is a substituted sulphonamide, the
substituent is an alkyl chain of 1 to 6, preferably 1 to 4 carbon
atoms, or a phenyl or toluoyl group.
[0021] Preferably the G protein-coupled receptor is a C5a receptor.
However, we have found that the leading compound of our earlier
application also has significant binding affinity at vasopressin
and neurokinin receptors, and therefore these receptors are also
within the scope of the invention.
[0022] In a particularly preferred embodiment, the compound has
antagonist activity against C5aR, and has no C5a agonist
activity.
[0023] The cyclic compounds of the invention are preferably
antagonists of C5a receptors on human, mammalian cells including,
but not limited to, human polymorphonuclear leukocytes and human
macrophages. The compounds of the invention preferably bind
potently and selectively to C5a receptors, and more preferably have
potent antagonist activity at sub-micromolar concentrations. Even
more preferably the compound has a receptor affinity
IC.sub.50<25 .mu.M, and an antagonist potency IC.sub.50<1
.mu.M.
[0024] Still more preferably the compound is selected from the
group consisting of compounds 2 to 6, 10 to 15, 17, 19, 20, 22, 25,
26, 28, 30, 31, 33 to 37, 39 to 45, 47 to 50, 52 to 58 and 60 to 70
described herein. Most preferably the compound is compound 33,
compound 60 or compound 45.
[0025] For the purposes of this specification, the term "alkyl" is
to be taken to mean a straight, branched, or cyclic, substituted or
unsubstituted alkyl chain of 1 to 6, preferably 1 to 4 carbons.
Most preferably the alkyl group is a methyl group. The term "acyl"
is to be taken to mean a substituted or unsubstituted acyl of 1 to
6, preferably 1 to 4 carbon atoms. Most preferably the acyl group
is acetyl. The term "aryl" is to be understood to mean a
substituted or unsubstituted homocyclic or heterocyclic aryl group,
in which the ring preferably has 5 or 6 members.
[0026] A "common" amino acid is a L-amino acid selected from the
group consisting of glycine, leucine, isoleucine, valine, alanine,
phenylalanine, tyrosine, tryptophan, aspartate, asparagine,
glutamate, glutamine, cysteine, methionine, arginine, lysine,
proline, serine, threonine and histidine.
[0027] An "uncommon" amino acid includes, but is not restricted to,
D-amino acids, homo-amino acids, N-alkyl amino acids, dehydroamino
acids, aromatic amino acids other than phenylalanine, tyrosine and
tryptophan, ortho-, meta- or para-aminobenzoic acid, ornithine,
citrulline, canavanine, norleucine, .delta.-glutamic acid,
aminobutyric acid, L-fluorenylalanine, L-3-benzothienylalanine, and
.alpha., .alpha.-disubstituted amino acids.
[0028] According to a second aspect, the invention provides a
composition comprising a compound according to the invention,
together with a pharmaceutically-acceptable carrier or
excipient.
[0029] The compositions of the invention may be formulated for use
in oral, parenteral, inhalational, intranasal, transdermal or other
topical applications, but oral or topical formulations are
preferred. For topical administration, vehicles such as
dimethylsulphonate or propylene glycol may be used. Other vehicles
may be preferred depending on the tissue surface to be treated.
[0030] It is expected that most if not all compounds of the
invention will be stable in the presence of metabolic enzymes such
as those of the gut, blood, lung or intracellular enzymes. Such
stability can readily be tested by routine methods known to those
skilled in the art.
[0031] Suitable formulations for administration by any desired
route may be prepared by standard methods, for example by reference
to well-known textbooks such as Remington: The Science and Practice
of Pharmacy, Vol. II, 2000 (20th edition), A. R. Gennaro (ed),
Williams & Wilkins, Pennsylvania.
[0032] In a third aspect, the invention provides a method of
treatment of a pathological condition mediated by a G
protein-coupled receptor, comprising the step of administering an
effective amount of a compound of the invention to a mammal or
vertebrate in need of such treatment.
[0033] Preferably the condition mediated by a G protein-coupled
receptor is a condition mediated by a C5a receptor, and more
preferably involves overexpression or underregulation
under-regulation of C5a. Such conditions include but are not
limited to rheumatoid arthritis, adult respiratory distress
syndrome (ARDS), systemic lupus erythematosus, tissue graft
rejection, ischaemic heart disease, reperfusion injury, septic
shock, gingivitis, fibrosis, atherosclerosis, multiple sclerosis,
Alzheimer's disease, asthma, dementias, central nervous system
disorders, lung injury, extracorporeal post-dialysis syndrome, and
dermal inflammatory disorders such as psoriasis, eczema and contact
dermatitis.
[0034] In one preferred embodiment the condition is rheumatoid
arthritis.
[0035] In a second preferred embodiment the condition is
reperfusion injury. In this second embodiment it will be clearly
understood that the proviso to formula I does not apply.
[0036] While the invention is not in any way restricted to the
treatment of any particular animal or species, it is particularly
contemplated that the compounds of the invention will be useful in
medical treatment of humans, and will also be useful in veterinary
treatment, particularly of companion animals such as cats and dogs,
livestock such as cattle, horses and sheep, and zoo animals,
including non-human primates, large bovids, felids, ungulates and
canids. Other species which may be amenable to treatment include
reptiles, fishes or amphibians.
[0037] The compounds may be administered at any suitable dose and
by any suitable route. Oral, topical, transdermal or intranasal
administration is preferred, because of the greater convenience and
acceptability of these routes. Topical applications could also
include the use of formulations such as pessaries or suppositories
for vaginal or rectal administration or the use of aqueous drops
for topical administration to ears or eyes. The effective dose will
depend on the nature of the condition to be treated, and the age,
weight, and underlying state of health of the individual treatment.
This be at the discretion of the attending physician or
veterinarian. Suitable dosage levels may readily be determined by
trial and error experimentation, using methods which are well known
in the art.
[0038] For the purposes of this specification it will be clearly
understood that the word "comprising" means "including but not
limited to", and that the word "comprises" has a corresponding
meaning.
BRIEF DESCRIPTION OF THE FIGURES
[0039] FIG. 1A, FIG. 1B, FIG. 1C, and FIG. 1D show the inhibition
of the vascular leakage associated with a dermal Arthus reaction by
intravenous (FIG. 1A), oral (FIG. 1B) and topical (FIG. 1C)
AcF-[OPdChaWR], and appropriate controls (FIG. 1D).
[0040] FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D show the inhibition
of the rise in circulating TNF-.alpha. associated with a dermal
Arthus reaction by intravenous (FIG. 2A), oral (FIG. 2B) and
topical (FIG. 2C) AcF-[OPdChaWR], and appropriate topical controls
(FIG. 2D).
[0041] FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D show the reduction of
the pathology index associated with a dermal Arthus reaction by
intravenous, oral and topical AcF-[OPdChaWR].
[0042] FIG. 4 shows the effect of a C5a antagonist on gut
ischemia-reperfusion induced intestinal edema.
[0043] FIG. 5 shows the effect of a C5a antagonist on gut
ischemia-reperfusion induced neutropenia.
[0044] FIG. 6 shows the effect of a C5a antagonist on gut
Ischemia-reperfusion induced serum TNF-.alpha. elevation.
[0045] FIG. 7 shows the effect of a C5a antagonist on gut
ischemia-reperfusion induced serum haptoglobin elevation.
[0046] FIG. 8 shows the effect of a C5a antagonist on gut
ischemia-reperfusion induced aspartate aminotransferase.
[0047] FIG. 9 shows the effect of a C5a antagonist on
histopathology of gut ischemia-reperfusion.
[0048] FIG. 10 shows the inhibition of arthritic right knee joint
swelling by AcF-[OPdChaWR] given orally on Days 2 to +14.
[0049] FIG. 11 shows the inhibition of right knee joint TNF-.alpha.
and IL-6 levels in joint lavage. "Untreated" refers to animals not
treated with AcF-[OPdChaWR] but with the right knee challenged with
antigen following sensitisation.
[0050] FIG. 12 shows that dermal application of 3D53 in
DMSO/distilled H.sub.2O or PG/H.sub.2O results in the appearance of
the C5a antagonist in the circulating plasma within 15 minutes, and
that significant levels persist for at least four hours. Points
represent the mean.+-.SEM in each group (n=6-8).
[0051] FIG. 13 shows the inhibition of C5a-induced neutropenia by
topical administration of C5a antagonists. The results are
expressed as percentage change from a zero time baseline.
[0052] FIG. 14A, FIG. 14B, and FIG. 14C that topical administration
of C5a antagonists inhibits systemic effects of intravenously
administered LPS in rats. The data show that administration of
various C5a antagonists, either i.v. (1 mg/kg), or topically by
dermal application (50 mg/kg total applied dose: solvent vehicle
50% DMSO/50% H.sub.2O), inhibits the neutropenia caused by i.v. LPS
(1 mg/kg). (FIG. 14A) 3D53 (compound 1); (FIG. 14B) compound 45;
(FIG. 14C) compound 17.
[0053] FIG. 15A and FIG. 15B show the effects of the C5a antagonist
AcF-[OPdChaWR] on increases in serum levels of (FIG. 15A) creatine
kinase (CK) and (FIG. 15B) lactate dehydrogenase (LDH) during
reperfusion in rats. Data represent the mean.+-.SEM (n=6-10).
*P<0.05 all drug-treated groups vs. ischemia/reperfusion
(I/R)-only; .dagger. P<0.05 all drug-treated groups vs.
sham-operated.
[0054] FIG. 16A and FIG. 16B show the effects of the C5a
antagonist, AcF-[OPdChaWR], on increases in serum levels of (FIG.
16A) alanine aminotransferase (ALT) and (FIG. 16B) aspartate
aminotransferase (AST) following 2, 3 and 4 hours reperfusion in
rats. Data represent the mean.+-.SEM (n=6-10). * P<0.05 all
drug-treated groups vs ischemia/reperfusion (I/R)-only; .dagger.
P<0.05 all drug-treated groups vs. sham-operated.
[0055] FIG. 17A, FIG. 17B, FIG. 17C, and FIG. 17D show the levels
of (FIG. 17A) circulating PMNs, (FIG. 17B) muscle myeloperoxidase
(MPO), (FIG. 17C) lung MPO and (FIG. 17D) liver MPO in rats. Levels
were measured in rats at the completion of the experiment. Data
represent the mean.+-.SEM (n=4-10). * P<0.05 vs.
ischemia/reperfusion (I/R)-only; .dagger. P<0.05 vs.
sham-operated.
[0056] FIG. 18 shows the levels of tumour necrosis factor-.alpha.
(TNF-.alpha.) in rat liver homogenate samples taken at the
completion of the experiment. Data represent the mean.+-.SEM
(n=4-10). * P<0.05 vs. ischemia/reperfusion (I/R)-only; .dagger.
P<0.05 vs. sham-operated.
[0057] FIG. 19 shows the amount of edema (wet-to-dry ratio) in the
hindlimb muscle of rats at the completion of the experiment. Data
represent the mean.+-.SEM (n=4-10). * P<0.05 vs.
ischemia/reperfusion (I/R)-only; .dagger. P<0.05 vs.
sham-operated.
DETAILED DESCRIPTION OF THE INVENTION
[0058] Generally, the terms "treating", "treatment" and the like
are used herein to mean affecting a subject, tissue or cell to
obtain a desired pharmacological and/or physiological effect. The
effect may be prophylactic in terms of completely or partially
preventing a disease or sign or symptom thereof, and/or may be
therapeutic in terms of a partial or complete cure of a disease.
"Treating" as used herein covers any treatment of, or prevention of
disease in a vertebrate, a mammal, particularly a human, and
includes: preventing the disease from occurring in a subject who
may be predisposed to the disease, but has not yet been diagnosed
as having it; inhibiting the disease, i.e., arresting its
development; or relieving or ameliorating the effects of the
disease, i.e., cause regression of the effects of the disease.
[0059] The invention includes various pharmaceutical compositions
useful for ameliorating disease. The pharmaceutical compositions
according to one embodiment of the invention are prepared by
bringing a compound of formula I, analogues, derivatives or salts
thereof and one or more pharmaceutically-active agents or
combinations of compound of formula I and one or more
pharmaceutically-active agents into a form suitable for
administration to a subject using carriers, excipients and
additives or auxiliaries.
[0060] Frequently used carriers or auxiliaries include magnesium
carbonate, titanium dioxide, lactose, mannitol and other sugars,
talc, milk protein, gelatin, starch, vitamins, cellulose and its
derivatives, animal and vegetable oils, polyethylene glycols and
solvents, such as sterile water, alcohols, glycerol and polyhydric
alcohols. Intravenous vehicles include fluid and nutrient
replenishers. Preservatives include antimicrobial, anti-oxidants,
chelating agents and inert gases. Other pharmaceutically acceptable
carriers include aqueous solutions, non-toxic excipients, including
salts, preservatives, buffers and the like, as described, for
instance, in Remington's Pharmaceutical Sciences, 20th ed. Williams
& Wilkins (2000) and The British National Formulary 43rd ed.
(British Medical Association and Royal Pharmaceutical Society of
Great Britain, 2002), the contents of which are hereby incorporated
by reference. The pH and exact concentration of the various
components of the pharmaceutical composition are adjusted according
to routine skills in the art. See Goodman and Gilman's The
Pharmacological Basis for Therapeutics (7th ed., 1985).
[0061] The pharmaceutical compositions are preferably prepared and
administered in dosage units. Solid dosage units include tablets,
capsules and suppositories. For treatment of a subject, depending
on activity of the compound, manner of administration, nature and
severity of the disorder, age and body weight of the subject,
different daily doses can be used. Under certain circumstances,
however, higher or lower daily doses may be appropriate. The
administration of the daily dose can be carried out both by single
administration in the form of an individual dose unit or by
administration of several smaller dose units, and also by multiple
administrations of subdivided doses at specific intervals.
[0062] The pharmaceutical compositions according to the invention
may be administered locally or systemically in a therapeutically
effective dose. Amounts effective for this use will, of course,
depend on the severity of the disease and the weight and general
state of the subject. Typically, dosages used in vitro may provide
useful guidance in the amounts useful for in situ administration or
the pharmaceutical composition, and animal models may be used to
determine effective dosages for treatment of the cytotoxic side
effects. Various considerations are described, e.g., Langer
(Science, 249:1527, 1990). Formulations for oral use may be in the
form of hard gelatin capsules, in which the active ingredient is
mixed with an inert solid diluent, for example, calcium carbonate,
calcium phosphate or kaolin. They may also be in the form of soft
gelatin capsules, in which the active ingredient is mixed with
water or an oil medium, such as peanut oil, liquid paraffin or
olive oil.
[0063] Aqueous suspensions normally contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients may be suspending agents such as
sodium carboxymethyl cellulose, methyl cellulose,
hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents, which may be (a) a naturally occurring phosphatide
such as lecithin; (b) a condensation product of an alkylen oxide
with a fatty acid, for example, polyoxyethylene stearate; (c) a
condensation product of ethylene oxide with a long chain aliphatic
alcohol, for example, heptadecaethylenoxycetanol; (d) a
condensation product of ethylene oxide with a partial ester derived
from a fatty acid and hexitol such as polyoxyethylene sorbitol
monooleate, or (e) a condensation product of ethylene oxide with a
partial ester derived from fatty acids and hexitol anhydrides, for
example polyoxyethylene sorbitan monooleate.
[0064] The pharmaceutical compositions may be in the form of a
sterile injectable aqueous or oleaginous suspension. This
suspension may be formulated according to known methods using
suitable dispersing or wetting agents and suspending agents such as
those mentioned above. The sterile injectable preparation may also
be a sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example, as a
solution in 1, 3-butanediol. Among the acceptable vehicles and
solvents which may be employed are water, Ringer's solution, and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed, including
synthetic mono- or triglycerides. In addition, fatty acids such as
oleic acid may be used in the preparation of injectables.
[0065] Compounds of formula I may also be administered in the form
of liposome delivery systems, such as small unilamellar vesicles,
large unilamellar vesicles, and multilamellar vesicles. Liposomes
can be formed from a variety of phospholipids, such as cholesterol,
stearylamine, or phosphatidylcholines.
[0066] Dosage levels of the compound of formula I of the present
invention will usually be of the order of about 0.5 mg to about 20
mg per kilogram body weight, with a preferred dosage range between
about 0.5 mg to about 10 mg per kilogram body weight per day (from
about 0.5 g to about 3 g per patient per day). The amount of active
ingredient which may be combined with the carrier materials to
produce a single dosage will vary, depending upon the host to be
treated and the particular mode of administration. For example, a
formulation intended for oral administration to humans may contain
about 5 mg to 1 g of an active compound with an appropriate and
convenient amount of carrier material, which may vary from about 5
to 95 percent of the total composition. Dosage unit forms will
generally contain between from about 5 mg to 500 mg of active
ingredient.
[0067] It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, rate of excretion, drug combination and
the severity of the particular disease undergoing therapy.
[0068] In addition, some of the compounds of the invention may form
solvates with water or common organic solvents. Such solvates are
encompassed within the scope of the invention.
[0069] The compounds of the invention may additionally be combined
with other compounds to provide an operative combination. It is
intended to include any chemically compatible combination of
pharmaceutically-active agents, of the compound of formula I of
this invention.
[0070] It will be clearly understood that the foregoing comments
regarding pharmaceutical formulations, routes of administration,
dosage levels and the like are equally applicable to compound
1.
[0071] Abbreviations used herein are as follows: [0072] BOP
benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorophosphate [0073] C5cR C5a receptor [0074] dH.sub.2O
distilled water [0075] D-Cha D-cyclohexylamine [0076] DIPEA
diisopropylethylamine [0077] DMF N,N-dimethylformamide [0078] DMSO
dimethylsulphoxide [0079] HBTU O-benzotriazole
N',N',N',N'-tetramethyluronium hexafluorophosphate [0080] HEPES
N-[2-hydroxyethyl]piperazine-N'-[2-ethane sulfonic acid] [0081]
HPLC high performance liquid chromatography [0082] RP-HPLC reverse
phase high performance liquid chromatography [0083] i.v.
intravenous [0084] LPS lipopolysaccharide [0085] PMN
polymorphonuclear granulocyte [0086] p.o. oral [0087] RMSD root
mean square deviation [0088] rp-HPLC reverse phase-high performance
liquid chromatography [0089] TFA trifluoroacetic acid;
[0090] Throughout the specification conventional single-letter and
three-letter codes are used to represent amino acids.
[0091] The terms "3D53" and "PMX53" are synonymous, and represent
the compound Ac-Phe-[Orn-Pro-dCha-Trp-Arg];
[0092] The terms "LP-10" and "PMX-201" are synonymous, and
represent the compound Ac-Phe-[Orn-Pro-dCHa-Trp-Cit]; and
[0093] The terms "LP-16" and "PMX-205" are synonymous, and
represent the compound EC--HC--[Orn-Pro-dCha-Trp-Arg], in which
"HC" indicates hydrocinnamate.
[0094] The invention will now be described by way of reference only
to the following general methods and experimental examples.
[0095] We have found that all of the compounds of formula I which
have so far been tested have broadly similar pharmacological
activities, which are similar to those of compound 1 (also referred
to herein as 3D53 or PMX53), although the physicochemical
properties, potency, and bioavailability of the individual
compounds varies somewhat depending on the specific substituents.
Thus we expect that results obtained in vitro or in vivo with
compound 1 will be reasonably predictive of activity of the
compounds of formula I in corresponding assays.
General Methods
[0096] Protected amino acids and resins were obtained from
Novabiochem. TFA, DIPEA and DMF (peptide synthesis grade) were
purchased from Auspep. All other materials were reagent grade
unless otherwise stated. Preparative scale reverse-phase HPLC
separations were performed on a Vydac C18 reverse-phase column
(2.2.times.25 cm), and analytical reverse-phase HPLC separations
were performed on a Waters Delta-Pak PrepPak C18 reverse-phase
column (0.8.times.10 cm), using gradient mixtures of solvent
A=water/0.1% TFA and solvent B=water 10%/acetonitrile 90%, 0.09%
TFA. The molecular weight of the peptides was determined by
electrospray mass spectrometry, recorded on a triple quadruple mass
spectrometer (PE SCIEX API III), as described elsewhere (Haviland
et al, 1995). .sup.1H-NMR spectra were recorded on either a Bruker
ARX 500 MHz or a Varian Unity 400 spectrometer. Proton assignments
were determined by 2D NMR experiments (DFCOSY, TOCSY, NOESY).
Compounds were analysed by mass spectrometry and by reversed phase
analytical HPLC.
Compound Synthesis
[0097] Linear peptide sequences were assembled by manual step-wise
standard solid-phase peptide synthesis (SPPS) techniques well known
to those skilled in the art. The amino acids or peptide termini
were activated with HBTU with DIEA in situ neutralisation.
Couplings were monitored by the standard quantitative ninhydrin
test. Boc chemistry was employed for temporary N.alpha.-protection
of amino acids with two 1 minute treatments with TFA for Boc group
removal. Peptides were synthesised on a Novabiochem
Boc-D-Arg(Tos)-PAM or Boc-L-Arg(Tos)-PAM resin with a substitution
value of approx. 0.2-0.5 mmol/g. The peptides were fully
deprotected and cleaved by treatment with liquid HF (10 mL),
p-cresol (1 mL) at -5.degree. C. for 1-2 hrs. Peptides were
purified by reversed phase HPLC (e.g., gradient: 0% B to 75% B over
60 min) and analysed by electrospray mass spectrometry.
[0098] Alternatively, the linear peptides can be synthesised by
Fmoc chemistry, using HBTU/DIEA activation on an Fmoc-D-Arg
(Mtr)-Wang resin. Fmoc group removal was effected using two 1 min
treatments with 50% piperidine/DMF. Cleavage and deprotection using
95% TFA/2.5% TIPS/2.5% H2O gives the Mtr-protected peptide, which
can be purified by RP-HPLC. A general procedure for cyclization of
linear peptides involves dissolving the peptide (1 equiv.) and BOP
(5 equiv.) in DMF (10 mM peptide concentration) and stirring
vigorously, followed by the addition of DIEA (15 equiv.). Solutions
are generally allowed to stir at room temperature overnight,
although in most cases the reaction was complete within 2 hrs. DMF
is removed under high vacuum at 30.degree. C. on a rotary
evaporator and then purified by RP-HPLC. For cyclic peptides
containing a free N-terminus, an Fmoc group was used as the
temporary N-terminal protecting group during the cyclization step.
DMF was removed under high vacuum at 30.degree. C. on a rotary
evaporator, and then the peptide was treated with 30%
piperidine/DMF for 1 hr at room temperature to remove the Fmoc
group. This was followed by solvent removal under high vacuum, and
purification by RP-HPLC. Representative examples of the synthesis
of the cycles are described below.
NMR Structure Determination
[0099] .sup.1H-NMR spectra were recorded for test compounds (3 mg
in 750 .mu.L, d.sub.6-DMSO, .delta. 2.50) referenced to solvent on
a Varian Unity 400 spectrometer at 24.degree. C. Two-dimensional
.sup.1H-NMR NOESY (relaxation delay 2.0 s, mix time 50-300 ms),
DFQ-COSY and TOCSY (mixing time 75 ms) experiments were acquired
and recorded in phase sensitive mode. Acquisition times=0.186 s,
spectral width=5500 Hz, number of complex points (t.sub.1
dimension)=1024 for all experiments. Data was zero-filled and
Fourier transformed to 1024 real points in both dimensions.
[0100] NMR data for compound 1 was processed using TRIAD software
(Tripos Assoc.) on a Silicon Graphics Indy work station. 2D NOE
cross peaks were integrated and characterised into strong (1.8-2.5
.ANG.), medium (2.3-3.5 .ANG.) and weak (3.3-5.0 .ANG.).
Preliminary three-dimensional structures were calculated from upper
and lower distance limit files using Diana 2.8 (69 distance
constraints, including 27 for adjacent residues and 6 further away)
with the redundant dihedral angle constraints (REDAC) strategy.
Upper and lower distance constraints were accurately calculated
using MARDIGRAS. At this stage the peptide was examined for
possible hydrogen bonds, and these were added as distance
constraints. The 50 lowest energy Diana structures were subjected
to restrained molecular dynamics (RMD) and energy minimisation
(REM). Initially, REM consisted of a 50 step steepest descent
followed by 100 step conjugate gradient minimisation. RMD was
performed by simulated heating of the structures to 300.degree. K
for 1 ps, followed by 500.degree. K for 1 ps. The temperature was
gradually lowered to 300.degree. K over 2 ps and finally for 2 ps
at 200.degree. K. REM was performed again with a 50 step steepest
descent, 200 step conjugate gradient followed by a 300 step Powell
minimisation. The final structures were examined to obtain a mean
pairwise rms difference over the backbone heavy atoms (N, C.alpha.
and C). Twenty of the 50 structures had a mean rmsd<.ANG. for
all backbone atoms (O, N, C).
Receptor-Binding Assay
[0101] Assays were performed with fresh human PMNs, isolated as
previously described (Sanderson et al, 1995), using a buffer of 50
mM HEPES, 1 mM CaCl.sub.2, 5 mM MgCl.sub.2, 0.5% bovine serum
albumin, 0.1% bacitracin and 100 .mu.M phenylmethylsulfonyl
fluoride (PMSF). In assays performed at 4.degree. C., buffer,
unlabelled human recombinant C5a (Sigma) or peptide, Hunter/Bolton
labelled .sup.125I-C5a (.about.20 pM) (New England Nuclear, MA) and
PMNs (0.2.times.10.sup.6) were added sequentially to a Millipore
Multiscreen assay plate (HV 0.45) having a final volume of 200
.mu.L/well. After incubation for 60 min at 4.degree. C., the
samples were filtered and the plate washed once with buffer.
Filters were dried, punched and counted in an LKB gamma counter.
Non-specific binding was assessed by the inclusion of 1 mM peptide
or 100 nM C5a, which typically resulted in 10-15% total
binding.
[0102] Data was analysed using non-linear regression and statistics
with Dunnett post-test.
Myeloperoxidase Release Assay for Antagonist Activity
[0103] Cells were isolated as previously described (Sanderson et
al, 1995) and incubated with cytochalasin B (5 .mu.g/mL, 15 min,
37.degree. C.). Hank's Balanced Salt solution containing 15%
gelatin and peptide was added on to a 96 well plate (total volume
100 .mu.L/well), followed by 25 .mu.L cells (4.times.106/mL). To
assess the capacity of each peptide to antagonise C5a, cells were
incubated for 5 min at 37.degree. C. with each peptide, followed by
addition of C5a (100 nM) and further incubation for 5 min. Then 50
.mu.L of sodium phosphate (0IM 0.1M, pH 6.8) was added to each
well, the plate was cooled to room temperature, and 25 .mu.L of a
fresh mixture of equal volumes of dimethoxybenzidine (5.7 mg/mL)
and H2O2 (0.51%) was added to each well. The reaction was stopped
at 10 min by addition of 2% sodium azide. Absorbances were measured
at 450 nm in a Bioscan 450 plate reader, corrected for control
values (no peptide), and analysed by non-linear regression.
In vivo Assays of Anti-Inflammatory Activity
[0104] The following well-known in vivo assay systems are used to
assess the anti-inflammatory activity of compounds of the
invention. All assay data are analysed using non-linear regression
analysis and Student's t-test, analysis of variance, with p<0.05
as the threshold level of significance.
(a) Carrageenan Paw Oedema
[0105] Anaesthetised (i.p. ketamine & xylazine) Wistar rats
(150-200 g) or mice are injected with sterilised air (20 mL day 1,
10 mL day 4) into the subcutaneous tissue of the back. The cavity
can be used after 6 days, whereupon carrageenan (2 mL, 1% w/w in
0.9% saline) is injected into the air pouch, and exudate is
collected after 10 hr. Test compounds are administered daily after
Day 6, and their anti-inflammatory effects assayed by differential
counting of cells in the air-pouch exudate. Animals are killed at
appropriate times after injection, and 2 mL 0.9% saline is used to
lavage the cavity; lavage fluids are transferred to heparinised
tube and cells are counted with a haemocytometer and Diff-Quik
stained cytocentrifuged preparation.
[0106] Alternatively, a routine carrageenan paw oedema developed in
Wistar rats by administering a pedal injection of carrageenan may
be used to elicit oedema which is visible in 2 h and maximised in 4
h. Test compounds are given 40 min before inflammagen and evaluated
by microcaliper measurements of paws after 2 & 4 hr. See
Fairlie, D. P. et al 1987). Also see Walker and Whitehouse
(1978).
(b) Adjuvant Arthritis
[0107] Adjuvant arthritis is induced in rats (3 strains) either
immunologically (injection of heat-killed Mycobacterium
tuberculosis) or chemically (with pyridine) by inoculation with the
arthritogenic adjuvant co-administered with oily vehicles, such as
Freund's adjuvants in the tail base (See Whitehouse, M. W.,
Handbook of Animal Models for the Rheumatic Diseases, Eds.
Greenwald, R. A.; Diamond, H. S.; Vol 1, pp. 3-16, CRC Press).
[0108] Within 13 days the adjuvant arthritis is manifested by local
inflammation and ulceration in the tail, gross swelling of all four
paws, inflammatory lesions in paws and ears, weight loss and fever.
These symptoms, which are similar to those of inflammatory disease
in humans (Winter and Nuss, 1966), can be alleviated by agents such
as indomethacin or cyclosporin, which also show beneficial effects
in man (e.g., Ward and Cloud, 1966). Without drug treatment at Day
14, arthritic rats had hypertrophy of the paws, reduced albumin but
raised acute phase reaction proteins in serum, and depressed
hepatic metabolism of xenobiotics as indicated by prolonged
barbiturate-induced sleeping times.
[0109] To assess activity, compounds are administered for 4 days
orally (<10 mg/kg/day) or intraperitoneally (i.p.) from Days
10-13 following inoculation with arthritogen (Day 0). If the
compound is active, the inflammation is either not visible, or is
very significantly reduced in rear or front paws, as assessed by
microcaliper measurements of paw thickness and tail volume, as well
as by gross inspection of inflammatory lesions. Animals are
sacrificed by cervical dislocation on Day 18 unless arthritis signs
are absent, whereupon duration of observations is continued with
special permission from the Ethics Committees. Experiments are
staggered to maximise throughput and allow early comparisons
between compounds. This routine assay is well-accepted as
identifying anti-inflammatory agents for use in humans.
Pharmacokinetics
[0110] Female and male Wistar rats (200-250 g) were anaesthetized
with 1 mL of zoletil (50 mg/kg) and xylazine (10 mg/kg; Lyppard,
Australia), which was injected intraperitoneally. An area of
5.times.10 cm was shaved and marked on the lower abdominal area of
the rat, on to which the dose of drug was applied. A stock solution
containing 10 mg/ml of C5a antagonist was dissolved in solvents
propylene glycol or dimethylsulfoxide at varying concentrations
with water and smeared evenly on the shaved abdominal area of the
rat with a spatula. A heating pad was used to maintain the body
temperature of the rats and blood samples were taken at 15 min
intervals for the first hour, and after that at 1 hr intervals for
a total period of 3 hours.
[0111] Blood samples were immediately added to tubes containing
heparin (500 Units/.mu.L) and centrifuged (11000.times.g). The
plasma layer of each sample was removed and stored at -20.degree.
C. A deuterated internal standard, .sup.2H3CO--F-[OPdChaWR] 50
.mu.L, 5 .mu.g/mL in 50% aeotonitrile/water), was added and
vortexed. The samples were further diluted 1:3 with high
performance liquid chromatography (HPLC) grade acetonitrile and
rapidly vortexed (20 sec), then centrifuged (11000.times.g). This
process resulted in precipitation of large plasma proteins in the
samples, and allowed the complete extraction of the drug from the
plasma. The fluid portions of the samples were placed in 1 mL
Eppendorf tubes and stored until analysed.
[0112] These samples were transferred to 96-well plates and
evaporated to dryness using a GeneVac centrifugal evaporator, then
reconstituted in the wells with mobile phase (20 .mu.L). Analysis
of samples was performed by liquid chromatography (LCMS) using an
Agilent 1100 series HPLC equipped with a well plate autosampler
coupled with a PE Sciex Qstar Pulsar ESI-TOF mass spectrometer.
Concentrations were determined from a standard curve of drug:
internal standard peak area ratios. Standards were prepared by
adding an appropriate amount of the drug and internal standard to
plasma from an untreated rat, and were extracted and prepared by
the same method as the experimental samples.
Example 1
Synthesis of Cyclic Compounds
[0113] Synthesis of cycle AcF-[OPdChaWR] (1). The linear peptide
Ac-Phe-Orn-Pro-dCha-Trp-Arg was synthesised by Boc chemistry on a
0.20 mmole scale using HBTU/DIEA activation and in situ
neutralization on a Boc-L-Arg(Tos)-PAM resin (338 mg, SV=0.591
mmol/g). Cleavage and deprotection of the resin (457 mg) was
achieved by treating the resin with HF (10 mL) and p-cresol (1 mL)
at -5 to 0.degree. C. for 1-2 hrs, to give crude peptide (160 mg,
90%). Cyclization involved stirring the crude peptide (41 mg, 45
.mu.mol), BOP (126 mg, 0.28 mmol) and DIEA (158 .mu.L, 0.9 mmol) in
DMF (57 mL) for 15 hrs. The solvent was removed in vacuo and the
cyclic peptide purified by rpHPLC (18.8 mg, 47%) Rt=10.8 min
(gradient: 70% A/30% B to 0% A/100% B over 30 min). MS:
[M+H]+(calc.)=896.5, [M+H]+(exper.)=896.5.
[0114] Synthesis of cycle AcF-[OPdPheWR] (33). The linear peptide
Ac-Phe-Orn-Pro-dPhe-Trp-Arg was synthesised by Boc chemistry usign
HBTU/DIEA activation and in situ neutralisation on a Boc-L-Arg
(Tos)-PAM resin. Cleavage and deprotection of the resin was
achieved by treating the resin with HF (10 mL) and p-cresol (1 mL)
at -5 to 0.degree. C. for 1-2 hrs, to give crude peptide.
Cyclization involved stirring the crude peptide (85 mg), BOP (200
mg) and DIEA (222 .mu.L) in DMF (10 mL) for 15 hrs. The solvent was
removed in vacuo and the cyclic peptide purified by rpHPLC (31 mg)
Rt=16. 7 min (gradient: 70% A/30% B to 0% A/100% B over 30 min).
MS: [M+H]+(calc.)=890.5, [M+H]+(exper.)=890.5.
[0115] Synthesis of cycle AcF-[OPdChaFR] (60). The linear peptide
Ac-Phe-Orn-Pro-dCha-Phe-Arg was synthesised by Boc chemistry using
HBTU/DIEA activation and in situ neutralization on a
Boc-L-Arg(Tos)-PAM resin. Cleavage and deprotection of the resin
was achieved by treating the resin with HF (10 mL) and p-cresol (1
mL) at -5 to 0.degree. C. for 1-2 hrs, to give crude peptide.
Cyclization involved stirring the crude peptide (104 mg), BOP (57
mg) and DIEA (103 mL) in DMF (1 mL) for 15 hrs. The solvent was
removed in vacuo and the cyclic peptide purified by rpHPLC (52 mg).
Rt=11.37 min (gradient: 70% A/30% B to 0% A/100% B over 15 min).
MS: [M+H]+(calc.)=857.5, [M+H]+(exper.)=857.4.
[0116] Synthesis of cycle AcF-[OpdCha(N-Me-Phe)R] (64). The linear
peptide Ac-Phe-Om-Pro-dCha-(N-Me-Phe)-Arg-OH was synthesised by
Fmoc chemistry using HBTU/DIEA activation and in situ
neutralisation on a Fmoc-L-Arg (pbf)-Wang resin (0.35 mmol/g) from
Novabiochem using methyl-L-Phe (281 mg, 2 equiv), Fmoc-dCha (275
mg, 2 equiv), Fmoc-Pro (472 mg, 4 equiv), Fmoc-Orn (Boc) (477 mg, 3
equiv), Fmoc-Phe (542 mg, 4 equiv) and Ac2O (4 equiv). Cleavage and
deprotection of the resin was achieved by treating the resin with
95% TFA (15 mL) for 1 h to give crude peptide (150 mg) after
precipitation with diethyl ether. Cyclization involved stirring the
rpHPLC purified peptide (100 mg), BOP (200 mg) and DIEA (222 .mu.L)
in DMF (2 mL) for 4 hrs. The solvent was removed in vacuo and the
cyclic peptide purified by rpHPLC (50 mg) Rt=33 min (gradient: 70%
A/30% B to 0% A/100% B over 30 min). MS: [M+H]+(calc.)=871.5, [M+H]
(exper.)=871.5.
[0117] Synthesis of cycle AcF-[{Orn-(.delta.N-Me)}PdChaWR] (66).
Boc-(.delta.N-Me-Orn)-OH was synthesized as reported (Pol. J. Chem.
1988, 62, 257-261). The linear peptide
Ac-Phe-[Orn-(.delta.N-MeCbz)]-Pro-dCha-Trp-arg-OH was synthesised
by Boc chemistry using HBTU/DIEA activation and in situ
neutralisation on Boc-L-Arg(tosyl)PAM resin (0.41 mmol/g) from
Novabiochem. Cleavage and deprotection of the resin was achieved by
treating the resin with HF/pCresol for 2 h to give crude peptide
after precipitation with diethyl ether. Cyclization involved
stirring the RP-HPLC purified peptide (100 mg), BOP (200 mg) and
DIEA (222 .mu.L) in DMF (2 mL) for 4 hrs. The solvent was removed
in vacuo and the cyclic peptide purified by rpHPLC. Rt=11.5 min
(35% B). MS: [M+H]+(calc.)=910.5, [M+H]+(exper.)=910.5.
Purification and Characterization.
[0118] Crude peptides were purified using preparative rp-HPLC using
a Vydac C18 reverse-phase column (2.2.times.25 cm). Gradients of 1
mL/min of solvent A to solvent B were employed and monitored at 214
nm. Fractions were collected and tested by ion spray mass
spectrometry (ISMS) for the correct molecular weight, and purity
was checked by analytical rp-HPLC on a Waters Delta-Pak
PrepPak.RTM. C18 reverse-phase column (0.8.times.10 cm) (varying
gradients such as: 0 to 75% over 60 min). The acetonitrile was HPLC
grade (BDH Laboratories) and TFA was synthesis grade (Auspep).
[0119] Table 1 shows examples of reactions used to prepare cyclic
compounds 1-70, and their characterisation by electrospray mass
specrometry (Mass Spec Found) and reversed phase HPLC (rp-HPLC)
retention times (Rt mins) under specified elution conditions.
[0120] Table 2 depicts the structures of the respective compounds,
and lists their respective receptor binding affinities and
antagonist potencies for the C5a receptor on human
polymorphonuclear leukocytes (neutrophils), as measured by
myeloperoxidase assay.
TABLE-US-00001 TABLE 1 Summary of Synthesis and Characterisation of
Cyclic Compounds listed in Table 2 Amount Mass Mass Linear Amount
Amount Amount Yield Spec Spec rpHPLC Compound Peptide BOP DIPEA DMF
Cycle Calcd Found rpHPLC conditions Rt (min) 1 41 mg 126 mg 158
.mu.L 57 mL 19 mg 895.5 896.5 30.fwdarw.100% B 30 m 10.8 2 957.5
958.5 30.fwdarw.100% B 30 m 14.8 3 78 mg 36 mg 71 .mu.L 1 mL 29 mg
937.6 938.5 30.fwdarw.100% B 30 m 13.1 4 65 mg 29 mg 57 .mu.L 1 mL
966.6 967.5 30.fwdarw.100% B 60 m 18.5-20.5 5 81 mg 36 mg 71 .mu.L
1 mL 967.5 968.5 30.fwdarw.100% B 90 m 23.2 6 25 mg -- 0.25 mL 2M
0.5 mL Quantitative As above 19.3 NaOH MeOH 7 92 mg 200 mg 222
.mu.L 10 mL 29 mg 910.5 910.3 30.fwdarw.100% B 30 m 23.0-23.8 8 87
mg 200 mg 222 .mu.L 10 mL 18 mg 896.5 898.4 30.fwdarw.100% B 30 m
13.1-14.2 9 155 mg 200 mg 222 .mu.L 10 mL 5 mg 912.5 912.5
30.fwdarw.100% B 30 m 3.2-4.2 10 105 mg 200 mg 222 .mu.L 10 mL 51
mg 932.5 932.7 30.fwdarw.100% B 30 m 6.0-8.2 11 88 mg 200 mg 222
.mu.L 10 mL 27 mg 1008.5 1008.7 30.fwdarw.100% B 30 m 10.4-11.7 12
72 mg 200 mg 222 .mu.L 10 mL 15 mg 882.5 882.5 30.fwdarw.100% B 30
m 7.0-8.0 13 53 mg 28 mg 56 .mu.L 1 mL 21 mg 805.5 806.5
30.fwdarw.100% B 60 m 7.6 14 51 mg 200 mg 222 .mu.L 10 mL 12 mg
914.5 914.5 30.fwdarw.100% B 30 m 8.6-9.4 15 90 mg 200 mg 222 .mu.L
10 mL 33 mg 914.5 914.5 30.fwdarw.100% B 30 m 7.3-8.5 16 81 mg 200
mg 222 .mu.L 10 mL 22 mg 935.5 935.5 30.fwdarw.100% B 30 m
21.8-22.2 17 90 mg 46 mg 91 .mu.L 1 mL 46 mg 838.5 839.4
30.fwdarw.100% B 60 m 13.4-17 18 60 mg 31 mg 61 .mu.L 1 mL 25 mg
836.5 837.4 30.fwdarw.100% B 60 m 15.1-18 19 53 mg 33 mg 65 .mu.L 1
mL 691.4 692.4 30.fwdarw.100% B 60 m 9-10.5 20 66 mg 200 mg 222
.mu.L 10 mL 15 mg 898.5 898.5 30.fwdarw.100% B 30 m 14.2-14.7 21 81
mg 200 mg 222 .mu.L 10 mL 22 mg 912.5 912.5 30.fwdarw.100% B 30 m
17.2-18.0 22 59 mg 200 mg 222 .mu.L 10 mL 20 mg 945.5 946.7
30.fwdarw.100% B 30 m 24 1110 mg 200 mg 222 .mu.L 10 mL 37 mg 952.6
952.4 30.fwdarw.100% B 30 m 16.7-17.2 25 82 mg 200 mg 222 .mu.L 10
mL 20 mg 912.5 912.5 30.fwdarw.100% B 30 m 7.2-8.3 26 71 mg 200 mg
222 .mu.L 10 mL 16 mg 928.5 928.5 30.fwdarw.100% B 30 m 6.7-7.6 27
75 mg 200 mg 222 .mu.L 10 mL 21 mg 896.5 896.7 30.fwdarw.100% B 30
m 25.9-26.3 28 130 mg 62 mg 122 .mu.L 2 mL 90 mg 909.5 910.4
30.fwdarw.100% B 60 m 14.6-17.6 29 143 mg 73 mg 144 .mu.L 2 mL 60
mg 841.4 842.5 30.fwdarw.100% B 60 m 7.4-8.9 30 135 mg 72 mg 142
.mu.L 2 mL 813.4 814.4 30.fwdarw.100% B 60 m 16.8-18.6 31 102 mg 52
mg 101 .mu.L 2 mL 855.5 856.6 30.fwdarw.100% B 60 m 8.2-9.7 32 49
mg 200 mg 222 .mu.L 1 mL 8 mg 928.5 929.6 30.fwdarw.100% B 30 m
12.7-13.7 33 85 mg 200 mg 222 .mu.L 10 mL 31 mg 889.5 890.5
30.fwdarw.100% B 30 m 16.5-16.9 34 122 mg 57 mg 113 .mu.L 10 mL
901.5 902.2 30.fwdarw.100% B 15 m 6.0 35 106 mg 49 mg 95 .mu.L 2 mL
901.5 902.2 30.fwdarw.100% B 15 m 10.7 36 120 mg 61 mg 122 .mu.L 2
mL 855.5 856.4 30.fwdarw.100% B 60 m 9.2-10.7 39 70 mg 200 mg 222
.mu.L 2 mL 28 mg 906.5 906.7 30.fwdarw.100% B 60 m 40 62 mg 34 mg
66 .mu.L 10 mL 799.4 800.6 30.fwdarw.100% B 60 m 9-9.7 44 100 mg 48
mg 93 .mu.L 1 mL 909.5 910.6 30.fwdarw.100% B 60 m 13.7-16.3 45 94
mg 45 mg 89 .mu.L 1 mL 896.5 897.8 30.fwdarw.100% B 60 m 14.0-15.2
49 55 mg 29 mg 58 .mu.L 1 mL 796.4 797.4 30.fwdarw.100% B 60 m
17.2-18.6 50 75 mg 30 mg 0.06 mL 1 mL 35 mg 862.5 863.7
30.fwdarw.100% B 90 m 21-23 51 64 mg 27 mg 0.05 mL 1 mL 25 mg 822.5
823.7 30.fwdarw.100% B 90 m 14.5-17 52 169 mg 94 mg 184 .mu.L 1 mL
67 mg 906.5 907.5 30.fwdarw.100% B 60 m 17-18.4 53 177 mg 84 mg 166
.mu.L 2 mL 906.5 907.6 30.fwdarw.100% B 60 m 16.1-20.4 54 79 mg 200
mg 222 .mu.L 2 mL 22 mg 944.5 945_5 30.fwdarw.100% B 30 m 56 161 mg
79 mg 156 .mu.L 10 mL 868.5 869.2 30.fwdarw.100% B 60 m 57 70 mg 39
mg 70 .mu.L 2 mL 846.5 847.4 30.fwdarw.100% B 60 m 15.3-17.6 58 160
mg 76 mg 150 .mu.L 1 mL 912.5 913.3 30.fwdarw.100% B 60 m 15.4-19.6
59 150 mg 73 mg 143 .mu.L 2 mL 895.5 896.6 30.fwdarw.100% B 15 m
10.4 60 856.5 857.4 30.fwdarw.100% B 15 m 11.4 61 160 mg 80 mg 156
.mu.L 2 mL 870.5 871.4 30.fwdarw.100% B 60 m 13.9-17.4 62 180 mg 91
mg 180 .mu.L 2 mL 84 mg 850.4 851.4 30.fwdarw.100% B 60 m 8.9-11.3
63 174 mg 83 mg 164 .mu.L 2 mL 75 mg 900.5 901.4 30.fwdarw.100% B
60 m 13.4-15.3 64 100 mg 200 mg 222 .mu.L 2 mL 50 mg 870.5 871.5
30.fwdarw.100% B 60 m 33.0 65 100 mg 200 mg 222 .mu.L 2 mL 903.5
904.5 35% B 8.2 66 100 mg 200 mg 222 .mu.L 2 mL 909.5 910.5 35% B
11.5 67 50 mg 50 mg 100 .mu.L 5 mL 22 mg 861.6 862.6 30.fwdarw.100%
B 30 m 20.5 68 50 mg 50 mg 100 .mu.L 5 mL 18 mg 881.5 882.4
30.fwdarw.100% B 30 m 29.5 69 50 mg 50 mg 100 .mu.L 5 mL 19 mg
863.0 864.0 30.fwdarw.100% B 30 m 23.2 70 50 mg 50 mg 100 .mu.L 5
mL 4 mg* 884.0 885.0 30.fwdarw.100% B 30 m 29.5 *Tyr-O-Benzyl was
the amino acid used, but the product involved a rearrangement to a
meta-C-substituted tyrosine with a benzyl substituent (see
structure of compound 70).
Example 2
Antagonist Activity of Cyclic Compounds
[0121] Table 2 shows the structures of the compounds synthesised in
Example 1, as well as their respective receptor binding affinities
and antagonist potencies for the C5a receptor on human
polymorphonuclear leukocytes (neutrophils), as measured by the
myeloperoxidase assay.
[0122] Compounds 1-9, 16-18, 20, 21, 23, 24, 27-32, 36, 38, 44, 51,
and 59 are within the broad scope of the general structure set out
in our earlier patent application, Intl. Pat. Appl. No.
PCT/AU98/00490. However, Table 2 demonstrates that in fact, of
these compounds only 1-6, 17, 20, 28, 30, 31, 36 and 44 have
appreciable antagonist potency (IC.sub.50<1 .mu.M) against the
C5a receptor on human neutrophils. The other compounds, 7-9, 16,
18, 21, 23, 24, 27, 29, 32, 38, 51, and 59, do not show appreciable
antagonist potency and/or receptor affinity, with IC.sub.50>1
.mu.M in all cases.
[0123] On the other hand, compounds 10-15, 19, 22, 25, 26, 33-35,
37, 39-43, 45, 47-50, 52-58, and 60-70 are not included within the
scope of Intl. Pat. Appl. No. PCT/AU98/00490, although they do
involve the same or similar cyclic scaffolds to those disclosed
therein. Table 2 shows that of these new compounds, 10-12, 14, 15,
25, 33, 35, 40, 45, 48, 52, 58, 60, 66 and 68-70 have appreciable
antagonist potency (IC.sub.50<1 .mu.M). However, the other
compounds (13, 19, 22, 26, 34, 37, 39, 41-43, 47, 49, 50, 53-57,
61-65, and 67) do not show appreciable antagonist potency and/or
receptor affinity, with >1 .mu.M in all these cases.
[0124] The results shown in Table 2 enable us to define further and
to refine the limitations on the active pharmacophore for C5a
receptor antagonist activity, in order to obtain or predict
sub-micromolar antagonist potency.
TABLE-US-00002 TABLE 2 Str.mu.ct.mu.res and Activities of 70
Examples of Cyclic Antagonists of C5a Receptors on H.mu.man
Polymorphon.mu.clear Le.mu.kocytes ##STR00002## 1 C5aR Binding
IC.sub.50: 0.45 .mu.M C5aR Antagonist IC.sub.50: 28 nM ##STR00003##
2 C5aR Binding IC.sub.50: 1.1 .mu.M C5aR Antagonist IC.sub.50: 110
nM ##STR00004## 3 C5aR Binding IC.sub.50: 0.84 .mu.M C5aR
Antagonist IC.sub.50: 0:30 nM ##STR00005## 4 C5aR Binding
IC.sub.50: 0.25 .mu.M C5aR Antagonist IC.sub.50: 62 nM ##STR00006##
5 C5aR Binding IC.sub.50: 0.84 .mu.M C5aR Antagonist IC.sub.50: 38
nM ##STR00007## 6 C5aR Binding IC.sub.50: 0.45 .mu.M C5aR
Antagonist IC.sub.50: 23 nM ##STR00008## 7 C5aR Binding IC.sub.50:
1000 .mu.M C5aR Antagonist IC.sub.50: ND ##STR00009## 8 C5aR
Binding IC.sub.50: 28.7 .mu.M C5aR Antagonist IC.sub.50: ND
##STR00010## 9 C5aR Binding IC.sub.50: 0.** .mu.M C5aR Antagonist
IC.sub.50: ND ##STR00011## 10 C5aR Binding IC.sub.50: 0.47 .mu.M
C5aR Antagonist IC.sub.50: 34 nM ##STR00012## 11 C5aR Binding
IC.sub.50: 0.96 .mu.M C5aR Antagonist IC.sub.50: 291 nM
##STR00013## 12 C5aR Binding IC.sub.50: 0.76 .mu.M C5aR Antagonist
IC.sub.50: 151 nM ##STR00014## 13 C5aR Binding IC.sub.50: 37 .mu.M
C5aR Antagonist IC.sub.50: ND ##STR00015## 14 C5aR Binding
IC.sub.50: 0.52 .mu.M C5aR Antagonist IC.sub.50: 38 nM ##STR00016##
15 C5aR Binding IC.sub.50: 0.39 .mu.M C5aR Antagonist IC.sub.50: ND
##STR00017## 16 C5aR Binding IC.sub.50: 19.2 .mu.M C5aR Antagonist
IC.sub.50: ND ##STR00018## 17 C5aR Binding IC.sub.50: 0.22 .mu.M
C5aR Antagonist IC.sub.50: 31 nM ##STR00019## 18 C5aR Binding
IC.sub.50: 9.9 .mu.M C5aR Antagonist IC.sub.50: ND ##STR00020## 19
C5aR Binding IC.sub.50: 16.1 .mu.M C5aR Antagonist IC.sub.50: ND
##STR00021## 20 C5aR Binding IC.sub.50: 0.68 .mu.M C5aR Antagonist
IC.sub.50: ND ##STR00022## 21 C5aR Binding IC.sub.50: 2.9 .mu.M
C5aR Antagonist IC.sub.50: ND ##STR00023## 22 C5aR Binding
IC.sub.50: 2.4 .mu.M C5aR Antagonist IC.sub.50: ND ##STR00024## 23
C5aR Binding IC.sub.50: 2.4 .mu.M C5aR Antagonist IC.sub.50: ND
##STR00025## 24 C5aR Binding IC.sub.50: >1000 .mu.M C5aR
Antagonist IC.sub.50: ND ##STR00026## 25 C5aR Binding IC.sub.50:
0.27 .mu.M C5aR Antagonist IC.sub.50: ND ##STR00027## 26 C5aR
Binding IC.sub.50: 75.5 .mu.M C5aR Antagonist IC.sub.50: ND
##STR00028## 27 C5aR Binding IC.sub.50: 144 .mu.M C5aR Antagonist
IC.sub.50: ND ##STR00029## 28 C5aR Binding IC.sub.50: 0.39 .mu.M
C5aR Antagonist IC.sub.50: 40 nM ##STR00030## 29 C5aR Binding
IC.sub.50: 13 .mu.M C5aR Antagonist IC.sub.50: ND ##STR00031## 30
C5aR Binding IC.sub.50: 145 .mu.M C5aR Antagonist IC.sub.50: 37 nM
##STR00032## 31 C5aR Binding IC.sub.50: 1.1 .mu.M C5aR Antagonist
IC.sub.50: 35 nM ##STR00033## 32 C5aR Binding IC.sub.50: 30.1 .mu.M
C5aR Antagonist IC.sub.50: ND ##STR00034## 33 C5aR Binding
IC.sub.50: 11.26 .mu.M C5aR Antagonist IC.sub.50: 22 nM
##STR00035## 34 C5aR Binding IC.sub.50: 32.1 .mu.M C5aR Antagonist
IC.sub.50: ND ##STR00036## 35 C5aR Binding IC.sub.50: 9.2 .mu.M
C5aR Antagonist IC.sub.50: 25 nM ##STR00037## 36 C5aR Binding
IC.sub.50: 0.53 .mu.M C5aR Antagonist IC.sub.50: 30 nM ##STR00038##
37 C5aR Binding IC.sub.50: 77 .mu.M C5aR Antagonist IC.sub.50: 77
nM ##STR00039## 38 C5aR Binding IC.sub.50: 77 .mu.M C5aR Antagonist
IC.sub.50: 77 nM ##STR00040## 39 C5aR Binding IC.sub.50: >1000
.mu.M C5aR Antagonist IC.sub.50: ND ##STR00041## 40 C5aR Binding
IC.sub.50: 2.16 .mu.M C5aR Antagonist IC.sub.50: 30 nM ##STR00042##
41 C5aR Binding IC.sub.50: >100 .mu.M C5aR Antagonist IC.sub.50:
ND ##STR00043## 42 C5aR Binding IC.sub.50: 1082 .mu.M C5aR
Antagonist IC.sub.50: ND ##STR00044## 43 C5aR Binding IC.sub.50: 77
.mu.M C5aR Antagonist IC.sub.50: 77 nM ##STR00045## 44 C5aR Binding
10.sub.50: 1.36 .mu.M C5aR Antagonist IC.sub.50: 160 nM
##STR00046## 45 C5aR Binding IC.sub.50: 6.0 .mu.M C5aR Antagonist
IC.sub.50: 690 nM ##STR00047## 56 C5aR Binding IC.sub.50: 4.0 .mu.M
C5aR Antagonist IC.sub.50: 28 nM ##STR00048## 57 C5aR Binding
IC.sub.50: 0. .mu.M C5aR Antagonist IC.sub.50: 28 nM ##STR00049##
58 C5aR Binding IC.sub.50: 0. .mu.M C5aR Antagonist IC.sub.50: 28
nM ##STR00050## 59 C5aR Binding IC.sub.50: 0. .mu.M C5aR Antagonist
IC.sub.50: 28 nM ##STR00051## 60 C5aR Binding IC.sub.50: 0. .mu.M
C5aR Antagonist IC.sub.50: 28 nM ##STR00052## 61 C5aR Binding
IC.sub.50: 0. .mu.M C5aR Antagonist IC.sub.50: 28 nM ##STR00053##
62 C5aR Binding IC.sub.50: 0. .mu.M C5aR Antagonist IC.sub.50: 28
nM ##STR00054## 63 C5aR Binding IC.sub.50: 0. .mu.M C5aR Antagonist
IC.sub.50: 28 nM ##STR00055## 64 C5aR Binding IC.sub.50: 0. .mu.M
C5aR Antagonist IC.sub.50: 28 nM
[0125] "C5a Binding IC.sub.50" refers to the concentration of
compound required to achieve 50% maximum binding to human PMNs.
"C5a Antagonist IC.sub.50" refers to the concentration of compound
required to achieve 50% antagonism of myeloperoxidase release from
C5a-stimulated human PMNs. Boxed regions indicate the location of
relative changes between structures. Compound 1 is the lead
compound from our previous application PCT/AU98/00490, and is
included for purposes of comparison.
Example 3
Cyclic Antagonists of C5a
[0126] Some examples of these cyclic antagonists and their apparent
receptor-binding affinities and antagonist potencies are given in
Table 3, in which the single letter code for amino acids is used.
"d" indicates the dexto (D) form of an amino acid. "ND" indicates
not determined.
TABLE-US-00003 TABLE 3 NEW COMPOUNDS AS C5a ANTAGONISTS AcPhe
Replacements Compound n Binding Antagonist MsF [OP-dCha-WR] 10 3
0.47 34 TsF [OP-dCha-WR] 11 3 0.96 291 AcPhg [Opd-Cha-WR] 12 3 0.76
151 AcG [OP-dCha-WR] 13 3 37.2 ND Ac(o-fluoro)F[OP-dCha-WR] 14 3
0.52 38 Ac(m-fluoro)F[OP-dCha-WR] 15 1 0.39 ND HC [OP-dCha-WR] 17 3
0.22 31 Hydrogen [OP-dCha-WR] 19 3 >1000 ND Ms = Mesyl, Ts =
Tosyl, MeSuc = Methylsuccinate, Suc = Succinate, Ahx =
6-Aminohexanoate, HPhe = Homophenylalanine, Phg = Phenylglycine, HC
= Hydrocinnamate ND = not done Compound Binding Antagonist Pro
Replacements Number N (.mu.M) (nM) AcF[O-Hyp-dCha-WR] 25 3 0.27 ND
AcF[O-Thp-dCha-WR] 26 1 75 5 ND AcF[O-Phe-dCha-WR] 22 3 2.43 ND Hyp
= trans-Hydroxyproline, Thp = cis-Thioproline Antagonist D-Cha
Replacements Lab Code N Binding (.mu.M) (nM) AcF[OP-dCha-WR] 3D53,
1 13 0.45 28 AcF[O-dLeu-WR] 31 3 1.13 35 AcF[OPGWR] 42 3 1082 ND
AcF[OP-dVal-WR] 29 3 13.0 ND AcF[OP-dNle-WR] 36 3 0.53 30
AcF[OP-dTic-WR] 35 3 9.18 15,000 AcF[OP-aic-WR] 34 3 22.71 ND
AcF[OP-DTyr-WR] 40 3 2.16 300 AcF[OP-dArg-WR] 41 3 >100 ND
AcF[OP-dPhe-WR] 33 3 0.26 22 AcF[OP-dhCha-WR] 28 3 0.39 40.5
Aic--aminoindanecarboxylio acid Tic--tetrahydroisoquino1ine
dhCha--D-homocyclohexylalanine Antagonist Trp Replacements Lab Code
N Binding (.mu.M) (nM) AcF[OP-dCha-HR] 57 3 23.5 ND AcF[OP-dCha-FR]
60 3 0.25 32 AcF[OP-dCha-LR] 51 3 18.9 3,000 AcF[OP-dCha-Cha-R] 50
3 11.9 4,500 AcF[OP-dCha-hPhe-R] 61 3 11.5 ND AcF[OP-dCha-2Nal-R]
53 3 15.8 ND AcF[OP-dCha-Bta-R] 58 3 0.28 172 AcF[OP-dCha-Flu-R] 54
3 28.9 ND AcF[OP-dCha-1Nal-R] 52 3 0.71 46.6 AcF[OP-dCha-Tic-R] 56
3 3.73 10,900 AcF[OP-dCha-G-R] 55 3 >1000 ND AcF[OPdCha-dTrp-R]
59 3 30.4 ND hPhe = Homophenylalanine, 2Nal = 2-Naphthylalanine,
1Nal = 1-Naphthylalanine, Bta = Benzothienylalanine, Flu =
Fluorenylalanine, Tyr-O-alkyl = O-alkylated analogue of tyrosine.
Tic = tetrahydroisoquinoline Binding Antagonist Arg Replacements
Lab Code N (.mu.M) (nM) AcF[OPdChaW-Cit] 45 3 6.00 690
AcF[OpdChaW-K] 47 3 24.15 ND AcF[OpdChaW-hArg] 44 3 1.36 ND Can =
L-canavanine, Cit = Citrulline, hArg = homoarginine Multiple
Binding Antagonist Replacements Lab Code N (.mu.M) (nM)
AcF[OP-dPhe-dleu-Nal-R] 105 3 3.1 ND AcF[OP-dPhe-FR] 62 3 5.2 5,210
AcF[DapOPdChaWRC] 151 3 1.84 100 AcF[OP-dPhe-lNal-R] 63 3 3.1 ND
AcF[OP-dPhe-Y-R] 150 3 69.2 ND 1Nal = 1-Naphthylalanine, Dap =
2'3-diaminopropionic acid, dPhe = D-phenylalanine
Example 4
Pharmacophore Refinement
[0127] On the basis of the results in Table 2, we can develop a
refined pharmacophore for active antagonism of the C5a receptor on
human polymorphonuclear leukocytes, as follows:
[0128] Position "A" can tolerate a very large number of groups,
including H (e.g., compound 17, 18), alkyl, aryl, NH.sub.2,
NHalkyl, N(alkyl).sub.2, NHaryl, NHacyl (e.g., compounds 1, 3, 4,
5, 6), NHbenzoyl (e.g., compound 2), OH, Oalkyl, Oaryl,
NHSO.sub.2alkyl (e.g., compound 10), NHSO.sub.2aryl (e.g., compound
11), without an adverse effect on activity.
[0129] The wide tolerance to substitution at position "A" indicates
that there is considerable space in the receptor for appendages to
the cyclic peptide scaffold. This position can therefore be used
for adding substituents in order to vary the water and lipid
solubility of the antagonist, thereby enhancing oral or transdermal
absorption of the antagonist. This position also allows attachment
of labels such as fluorescent tags, agonists or polypeptide
sequences which confer high affinity for target cells, such as
sequences similar to amino acids 1-69 of C5a.
[0130] Position "B" can be alkyl, aryl, phenyl, benzyl, naphthyl or
indole, or the side chain of a D- or L-amino acid such as
L-phenylalanine (compound 1), or L-phenylglycine (compound 12). It
should not be the side chain of D-phenylalanine (compound 8),
L-homophenylalanine (compound 7), L-tyrosine (compound 9),
L-homotyrosine, glycine (compound 13), L-tryptophan (compound 16),
or L-homotryptophan.
[0131] Position "B" does not tolerate a wide range of
substitutents. It appears that the benzyl group of L-phenylalanine
cannot tolerate much substitution, and cannot be made much bulkier.
This position seems to be required for receptor binding, rather
than being important for antagonism per se, since the greatest
effects on modification were on receptor affinity, as measured by
IC.sub.50.
[0132] Position "C" should be a small substitutent, such as the
side chain of a D- or L-amino acid such as proline (compound 1),
L-valine (compound 20), alanine, trans-hydroxyproline (compound
25), or cis-thioproline (compound 26). It should not be a bulky
substituent such as the side chains of L-isoleucine (compound 21),
D- or L-phenylalanine (compounds 22, 23), L-cyclohexylalanine
(compound 24).
[0133] Position "D" should be a bulky substituent, such as the side
chain of a D-amino acid like D-Leucine (compound 31),
D-homoleucine, D-cyclohexylalanine (compound 1),
D-homocyclohexylalanine (compound 28), valine (compound 29),
D-norleucine (compound 36), D-homonorleucine, D-phenylalanine
(compound 33), D-tetrahydroisoquinoline (compound 35), D-glutamine
(compound 37), D-glutamate (compound 38), or D-tyrosine (compound
40). It should not be a smaller substituent, such as the side chain
of glycine, D-alanine (compound 30), a bulky planar side chain like
D-tryptophan (compound 32), a bulky charged side chain like
D-arginine (compound 39) or D-Lysine (compound 43), or an L-amino
acid like L-cyclohexylalanine (compound 27). Small D-amino acids
and small or large L-amino acids at position "D" on the scaffold
lead to greatly reduced affinity for the C5a receptor.
[0134] Position "E" is chosen from among the bulky side chains of
L-Tryptophan (compound 1) and L-homotryptophan, but not
D-tryptophan (compound 59) or L-N-methyltryptophan (compound 47);
L-phenylalanine (compound 60) but not L-homophenylalanine (compound
61); L-naphthyl (compound 52) but not L-2-naphthyl (compound 53);
L-3-benzothienylalanine (compound 58). It should not be the side
chain of L-cyclohexylalanine (compound 50), D-leucine (compound
51), L-fluorenylalanine (compound 54), glycine (compound 55),
L-tetrahydroisoquinoline (compound 56), or L-histidine (compound
57).
[0135] Substituents at position "E" on the cyclic peptide scaffold
are crucial for antagonism of the C5a receptor. Substituents at
this position may limit the conformational changes usually
associated with agonist responses. This may be a "blocking"
residue, which fixes the antagonist in the receptor and prevents
the conformational reorganization in the receptor which is
necessary for agonism.
[0136] Position "F" may be the side chain of L-arginine (compound
1), L-homoarginine (compound 44), L-citrulline (compound 45); or
L-canavinine (compound 48). It should not be D- or L-lysine
(compound 47), D- or L-homolysine, or glycine (compound 49). The
size of the substitutent at this position is important for
conferring high receptor affinity. The citrulline compound has no
charged side chain, yet still possesses appreciable antagonist
potency compared to arginin at this position.
[0137] Position "X" may be --(CH.sub.2).sub.nNH-- or
--(CH.sub.2).sub.n--S--, where n is an integer from 1 to 4,
preferably 2 or 3, --(CH.sub.2).sub.2O--, --(CH.sub.2).sub.3O--,
--(CH.sub.2).sub.3--, --(CH.sub.2).sub.4--, --CH.sub.2COCHRNH--, or
--CH.sub.2--NHCOCHRNH-- where R is the side chain of any common or
uncommon L- or D-amino acid.
[0138] This group provides the cyclization link, for example
between the Arg and Phe residues of compound 1, and thus influences
the structure of the cyclic backbone. In addition, substituents
such as R on this linker could potentially interact with receptor
residues to increase affinity of the antagonists.
[0139] N-methylation of the amino acid components of the cycles
tends to reduce the receptor binding affinity and antagonist
potency of the compounds (e.g., 64, 65), although N-methylation of
the delta nitrogen of ornithine has virtually no effect on
antagonist potency.
[0140] Multiple changes on the scaffold can be detrimental to
obtaining increased antagonist potency relative to 1. Thus although
L-Phe was a suitable replacement (e.g., 60) for L-Trp in 1, with
little change in antagonist potency, a combination of changes to 1,
such as L-Phe for Trp and either L-HomoPhe for Arg (e.g., 67) or
p-chloro-phenylalanine for Arg (e.g., 68), led to reduced affinity
for the receptor and reduced antagonist potency. Similarly when
L-Trp in 1 was replaced by L-Phe and D-Cha was also replaced by
D-Phe, the compound lost substantial potency (e.g., 62). While a
change from D-Cha in 1 to D-Phe led to retention of the antagonist
potency (e.g., 33), this change is detrimental when coupled with
replacement of L-Trp by L-Phe (62).
[0141] Clearly there is cooperativity in the binding of these
residues to the receptor, since either of the single changes (e.g.,
33, 60) results in substantially higher potency than when the
changes are made together (e.g., 62). When the Arg was replaced by
an aromatic group still containing a charged amine (69), there was
a significant loss in activity, as was observed when Phe of 60 was
replaced by a substituted tyrosine (70).
[0142] All these changes are indicative of what can and cannot be
tolerated on the cyclic scaffold used to engender affinity for the
human PMN C5a receptor and antagonist potency. It is recognised
that C5a receptors on other types of cells may have different
tolerances for side chains, but the cyclic scaffold will still form
the basis of active compounds.
Example 5
Reverse Passive Arthus Reaction in the Rat
[0143] A reverse passive peritoneal Arthus reaction was induced as
previously described (Strachan et al., 2000), and a group of rats
were pretreated prior to peritoneal deposition of antibody with
AcF-[OPdChaWR] (1) by oral gavage (10 mg kg.sup.-1 dissolved in 10%
ethanol/90% saline solution to a final volume of 200 .mu.L) or an
appropriate oral vehicle control 30 min prior to deposition of
antibody. Female Wistar rats (150-250 g) were anaesthetised with
ketamine (80 mg kg.sup.-1 i.p.) and xylazine (12 mg kg.sup.-1
i.p.).
[0144] The lateral surfaces of the rat were carefully shaved and 5
distinct sites on each lateral surface clearly delineated. A
reverse passive Arthus reaction was induced in each dermal site by
injecting Evans blue (15 mg kg.sup.-1 i.v.), chicken ovalbumin (20
mg kg.sup.-1 i.v.) into the femoral vein 10 min prior to the
injection of antibody. Rabbit anti-chicken ovalbumin (saline only,
100, 200, 300 or 400 .mu.g antibody in a final injection volume of
30 .mu.L) was injected in duplicate at two separate dermal sites on
each lateral surface of the rat, giving a total of 10 injection
sites per rat. Rats were placed on a heating pad, and anaesthetic
was maintained over a 4 h-treatment period with periodic collection
of blood samples. Blood was allowed to spontaneously clot on ice,
and serum samples were collected and stored at -20.degree. C. Four
hours after induction of the dermal Arthus reaction, the
anaesthetised rat was euthanased and a 10 mm.sup.2 area of skin was
collected from the site of each Arthus reaction. Skin samples were
stored in 10% buffered formalin for at least 10 days before
histological analysis using haematoxylin and eosin stain.
Additionally, a second set of skin samples were placed in 1 mL of
formamide overnight, and the absorbance of Evans blue extraction
measured at 650 nm, as an indicator of serum leakage into the
dermis. FIG. 1A, FIG. 1B, FIG. 1C, and FIG. 1D show the optical
density of dermal punch extracts following intradermal injection of
rabbit anti-chicken ovalbumin at 0-400 .mu.g site.sup.-1 following
pretreatment with AcF-[OPdChaWR] intravenously, orally or
topically. Data are shown as absorbance at 650 nm as a percentage
of the plasma absorbance, as mean values.+-.SEM (n=3-6). *indicates
a P value .ltoreq.0.05 when compared to Arthus control values.
[0145] Rats were pretreated with the C5aR antagonist,
AcF-[OPdChaWR] (1) as the TFA salt, either intravenously (0.3-1 mg
kg.sup.-1 in 20 .mu.L saline containing 10% ethanol, 10 min prior
to initiation of dermal Arthus), orally (0.3-10 mg kg.sup.-1 in 200
.mu.L saline containing 10% ethanol by oral gavage, 30 min prior to
initiation of dermal Arthus in rats denied food access for the
preceding 18 hours) or topically (200-400 .mu.g site.sup.-1 10 min
prior to initiation of dermal Arthus reaction), or with the
appropriate vehicle control. Topical application of the antagonist
involved application of 20 .mu.L of a 10-20 mg mL.sup.-1 solution
in 10% dimethyl sulphoxide (DMSO), which was then smeared directly
onto the skin at each site, 10 min prior to induction of the Arthus
reaction.
[0146] The saline-only injection site from rats treated with Evans
blue only served as antigen controls, the saline-only injection
site from rats treated with Evans blue plus topical DMSO only
served as a vehicle control, the saline-only injection site from
rats treated with Evans blue plus either intravenous, oral or
topical antagonist only served as antagonist controls, and Evans
blue plus dermal rabbit anti-chicken ovalbumin served as antibody
controls. Topical application of the peptide AcF-[OPGWR] which has
similar chemical composition and solubility to that of
AcF-[OPdChaWR] (1), but with an IC.sub.50 binding affinity of >1
mM in isolated human PMNs, served as an inactive peptide control.
AcF-[OPGWR] was also dissolved in 10% DMSO and applied topically at
400 .mu.g site.sup.-1 10 min prior to initiation of the Arthus
reaction.
TNF-.alpha. Measurement
[0147] Serum TNF-.alpha. concentrations were measured using an
enzyme-linked immunosorbent assay (ELISA) kit. Antibody pairs used
were a rabbit anti-rat TNF-.alpha. antibody coupled with a
biotinylated murine anti-rat TNF-.alpha. antibody. FIG. 2A, FIG.
2B, FIG. 2C, and FIG. 2D show the serum TNF-.alpha. concentrations
at regular intervals after initiation of a dermal Arthus reaction,
with group of rats pretreated with AcF-[OPdChaWR] intravenously,
orally or topically. Data are shown as mean values.+-.SEM (n=3-6).
*indicates a P value of .ltoreq.0.05 when compared to Arthus
control values.
Interleukin-6 Measurement
[0148] An ELISA method as described previously was used to measure
serum and peritoneal lavage fluid interleukin-6 (IL-6)
concentrations (Strachan et al., 2000).
Pathology Assessment
[0149] Rat skin samples were fixed in 10% buffered formalin for at
least 10 days, and stained with haematoxylin and eosin using
standard histological techniques. Dermal samples were analysed in a
blind fashion for evidence of pathology, and the degree of rat PMN
infiltration was scored on a scale of 0-4. Initiation of a dermal
Arthus reaction resulted in an increase in interstitial
neutrophils, which as quantified in the following manner. Sections
were given a score of 0 if no abnormalities were detected. A score
of 1 indicated the appearance of increased PMNs in blood vessels,
but no migration of inflammatory cells out of the lumen. A score of
2 and 3 indicated the appearance of increasing numbers of PMNs in
the interstitial tissue and more prominent accumulations of
inflammatory cells around blood, vessels. A maximal score of 4
indicated severe pathological abnormalities were present in dermal
sections, with excessive infiltration of PMNs into the tissues and
migration of these cells away from blood vessels. FIG. 3A, FIG. 3B,
FIG. 3C, and FIG. 3D show the intradermal injection of increasing
amounts of antibody leads to a dose-responsive increase in the
pathology index scored by dermal samples (FIG. 3A). Data are shown
for dermal samples intradermally injected with saline or 400 .mu.g
site.sup.-1 antibody (n=5) in rats pretreated with AcF-[OPdChaWR]
intravenously (FIG. 3B) (n=3), orally (FIG. 3C) (n=3) and topically
(FIG. 3D) (n=3). Data are shown as mean values.+-.SEM. *
P.ltoreq.0.05 when compared to Arthus values using a non-parametric
t-test.
Example 6
Effect of C5a Antagonist on Model of Intestinal
Ischemia-Reperfusion Injury in Rats
[0150] Female Wistar rats (250-300 g, n=132) were fasted and given
only water for 12 h prior to all experiments. Animals were
anaesthetised by the intraperitoneal injection of 80 mg kg.sup.-1
ketamine and 10 mg kg.sup.-1 xylazine and throughout the procedure
rats were placed on a heating pad to maintain normal body
temperature. The abdomen was opened through a midline incision to
expose the superior mesenteric artery (SMA). A non-traumatic
occlusive device for the artery was fashioned from a silk suture
looped though a length of polyethylene tubing. The SMA was occluded
by applying traction to the ends of the loop. A polyethylene
catheter was inserted in the femoral vein to allow the infusion of
either 1 mg kg.sup.-1 AcF-[OPdChaWR] (1) in 5% ethanol or sterile,
pyrogen-free saline in 0.2 mL volume. Infusions were made over a
2-min period. Oral dosing of AcF-[OPdChaWR] (1) (0.3, 1, 10 mg
kg.sup.-1) was achieved by gavage (0.2 mL saline in 25% ethanol) 60
min prior to SMA occlusion. Intestinal ischemia was induced by
clamping the SMA for 30 min, after which the occlusive suture was
removed, then reperfusion was monitored for another 120 min.
Sham-operated rats were treated in an identical fashion, with the
omission of vascular occlusion, and were infused with 0.2 mL of
sterile, pyrogen-free saline or gavaged with 0.2 mL of saline in
25% ethanol. Blood samples (50 .mu.L) were collected into
heparinised Eppendorf tubes at regular intervals over the 180-min
duration of the experiments for the estimation of PMN numbers. In a
different series of identical experiments, whole blood was
collected at regular intervals over the 180 min and allowed to clot
on ice, and serum or plasma samples collected and stored at
-20.degree. C. for later measurement of tumour necrosis
factor-.alpha. (TNF-.alpha.), haptoglobin (Hp) and aspartate
aminotransferase (AST) concentrations. At the end of the 120 min
reperfusion period, the animals were euthanased with an overdose of
pentobarbital. A section of the occluded ileum was removed, the
lumen rinsed with saline, and the intestine was blotted dry, then
weighed. Specimens were dried in an oven for 24 hours at 80.degree.
C. to obtain the tissue dry weight. Intestinal oedema was
determined by assessing the wet and dry tissue weight ratio.
Additionally, segments of both ischaemic and normal intestine were
harvested and rinsed with saline and immediately fixed in 10%
buffered formaldehyde-saline for histological studies.
[0151] FIG. 4 shows that the wet-to-dry weight ratio of the small
intestine is significantly elevated after ischemia-reperfusion
compared to sham-operated animals. Treatment with the C5a receptor
antagonist AcF-[OPdChaWR] 1 mg/kg i.v. and 10 mg/kg p.o.
significantly reduced tissue edema compared to untreated
ischemia-reperfusion (I/R) animals. Data are shown as means.+-.SEM
(n=4-6 in each group). *, P<0.05 vs. sham-operated animals. +,
P<0.05 vs. I/R animals.
Neutropenia Assay
[0152] Blood (50 .mu.L) for PMW was placed into heparinised tubes
and then layered over an equal volume of Histopaque 1083 (Sigma,
U.S.A.), PMNs were isolated and cell number counted on a
haemocytometer. Concentrations of PMNs values are presented as mean
percentage.+-.SEM of the values obtained immediately prior to SMA
occlusion.
[0153] FIG. 5A and FIG. 5B show that the gut ischemia-reperfusion
caused significant reduction in circulating PMN concentrations
compared with sham-operated animals (FIG. 5A and FIG. 5B).
Pretreatment of rats with C5a receptor antagonist AcF-[OPdChaWR] 1
mg/kg i.v. (FIG. 5A) and 10 mg/kg p.o. (FIG. 5B) significantly
inhibited ischemia-reperfusion induced neutropenia. 1 mg/kg p.o,
(FIG. 5B) treated animals showed no inhibition of neutropenia. Data
are shown as means.+-.SEM (n=6 in each group). *equals P<0.05
vs. Control animals. Bar shows 30-min period of ischemia.
Tumor Necrosis Factor-.alpha. Measurement
[0154] Serum TNF-.alpha. concentrations were measured using an
enzyme-linked immunosorbent assay, (Pharmingen, USA), according to
the manufacturers instructions. Concentrations of TNF-.alpha. in
serum samples were determined by linear regression analysis from
the standard curve.
[0155] FIG. 6 shows that the gut ischemia-reperfusion resulted in
significant elevation in serum TNF-.alpha. compared with
sham-operated animals. Pre-treatment of rats with the C5a receptor
antagonist AcF-[OPdChaWR] 1 mg/kg i.v. and 1-10 mg/kg p.o.
completely inhibited the change in serum TNF-.alpha. levels. Data
are shown as means.+-.SEM (n=6 in each group). *=P<0.05 vs.
sham-operated animals. The bar shows 30-min period of ischemia.
Haptoglobin Assay
[0156] Serum Hp was measured by using an Hp assay kit (Tridelta.
Development, Ltd., U.K.), according to the manufacturers'
instructions. Concentrations of Hp in the serum samples were
determined by linear regression analysis from the standard
curve.
[0157] FIG. 7 shows that gut ischemia-reperfusion resulted in a
significant increase in serum haptoglobin compared to sham-operated
animals. Pretreatment of rats with C5a receptor antagonist
AcF-[OpdChaWR] 1 mg/kg i.v. and 1, 10 mg/kg p.o. significantly
inhibited the increase in serum haptoglobin levels. Data are shown
as means.+-.SEM (n=4-6 in each group). *, P<0.05 vs.
sham-operated animals. +, P<0.05 vs. I/R animals.
Aspartate Aminotransferase Assay
[0158] Plasma AST (AST/GOT; Sigma, USA) concentrations were
measured according to manufacturer's instructions within 48 h of
collecting plasma. Plasma AST concentrations were derived from
calibration curve. Results are expressed in Sigma-Franke (SF)
units/ml.
[0159] FIG. 8 shows that the gut ischemia-reperfusion resulted in a
significant increase in plasma aspartate aminotransferase compared
to sham-operated animals. Treatment with the C5a receptor
antagonist AcF-[OPdChaWR] 1 mg/kg i.v. and 1, 10 mg/kg
significantly reduced gut ischemia-reperfusion induced aspartate
aminotransferase compared to untreated I/R animals. Data are shown
as means.+-.(n=6 in each group). *, P<0.05 vs. sham-operated
animals. +, P<0.05 vs. I/R animals.
Histopathology
[0160] Specimens were fixed in 10% formaldehyde-saline, embedded in
paraffin wax, sectioned serially, and stained with haematoxylin and
eosin. Tissues were read and scored by a trained observer in a
blinded fashion. The degree of intestinal tissue injury was
determined using a previously described graded scale ranging from
0-8 (Chiu et al., 1970).
[0161] FIG. 9 shows that gut ischemia-reperfusion resulted in
significant damage to the intestine compared to sham-operated
animals. Treatment with C5a receptor antagonist AcF-[OPdChaWR] 1
mg/kg i.v. and 1, 10 mg/kg significantly reduced gut
ischemia-reperfusion induced tissue damage compared to untreated
I/R animals. Data are shown as mean.+-.SEM (n=6 in each group). *,
P<0.05 vs. I/R animals.
Example 7
Rat Monoarticular Antigen-Induced Arthritis
[0162] Female Wistar rats (150-250 g) were obtained from the
Central Animal Breeding House, University of Queensland. Methylated
bovine serum albumin (mBSA) (0.5 mg) was dissolved in Freund's
complete adjuvant (0.5 mg) and sonicated to produce a homogenous
suspension. Each rat received a subcutaneous injection of this
suspension (0.5 mL) on days 1 and 7. On day 12-28, rats were
separated into separate cages, and body weight and food and water
intake monitored daily. Rats received either ordinary tap water or
drinking water containing AcF-[OPdChaWR] (1). Body weight and water
intake were monitored daily, and rats received a daily dose of 1
mg/kg/day of the C5aR antagonist AcF-[OPdChaWR] (1) for days 12-28
of the trial. On day 14, rats were anaesthetised and their hind
limbs shaved. Each rat received an intra-articular (100 .mu.L)
injection of mBSA (0.5 mg) in the left knee, and saline in the
right knee. The saline-only knee from rats receiving normal
drinking water served as a saline control, the saline only knee
from rats receiving AcF-[OPdChaWR] (1) in the drinking water served
as an antagonist control.
[0163] Rats were euthanased on day 28, and whole blood collected
into an Eppendorf tube and allowed to clot on ice. Blood samples
were centrifuges (11,000 rpm.times.3 min) and serum collected and
stored at -20.degree. C. until analysis of serum cytokines using an
ELISA. Each knee capsule was lavaged with 100 .mu.L saline, and the
total cell count determined using a haemocytometer. In addition, an
aliquot of the knee joint lavage fluid was dropped onto a glass
slide, and allowed to air dry. Once dry, cells were stained with a
differential stain (Diff Quick) and a differential cell count was
performed using a 40.times. dry lens microscope. The remaining
lavage fluids from each joint were stored at -20.degree. C. until
later analysis of intra-articular cytokine levels using an ELISA.
Each knee joint was removed and the skin was split with a scalpel
blade to allow fixation. Knee samples were stored in 10% buffered
formalin for .gtoreq.10 d. Knees were then rinsed with distilled
water and placed in a saturated solution of EDTA solution for 21 d
for decalcification before being embedded in paraffin wax.
[0164] Knee tissue samples were prepared using standard
histological techniques as described above in Example 6 and stained
using an heamotoxylin and eosin stain. Histological slides were
analysed in a blinded fashion. Tissue sections were scored from
0-4, with a score of 0 indicating no abnormalities, and increasing
scores with the appearance of synovial cell proliferation,
inflammatory cell infiltration, cartilage destruction and
haemorrhage. In no samples was there evidence of significant bone
erosion. Samples of serum and synovial fluid were thawed on the day
of ELISA analysis for TNF-.alpha. and IL-6 levels. Concentrations
were determined from a standard curve, using an ELISA as described
previously in Example 6.
[0165] FIG. 10 shows the inhibition of arthritic right knee joint
swelling by AcF-[OPdChaWR] given orally on Days-2 to +14, while
FIG. 11 shows the inhibition of right knee joint TNF-.alpha.; and
IL-6 levels in joint lavage. Untreated refers to animal not treated
with AcF-[OPdChaWR] but with right knee challenged with antigen
following sensitisation.
Example 8
Topical Dermal Administration of C5a Antagonists
[0166] The invention teaches that topical administration of C5a
antagonists may be used for the treatment of topical inflammatory
involving activation of the complement system. In this example we
demonstrate that topical application of C5a antagonists can also
result in systemic pharmacological actions and the appearance of
pharmacologically relevant concentrations of C5a antagonists in the
circulation.
[0167] The in vivo pharmacological properties of cyclic antagonists
were examined following topical dermal administration. A model of
endotoxaemia was used, in which 1 mg/kg Escherichia coli
liposaccharide (LPS; serotype 55: B5, Sigma, USA, stored at 100 mg
mL.sup.-1 in dH.sub.2O, 4.degree. C.), was injected i.v. into a
rat, resulting in acute changes in circulating PMN levels and blood
pressure. These parameters were measured in the presence and
absence of C5aR antagonists (1 mg/kg i.v. or 50 mg/kg/rat
topically). This study shows that topical administration of C5a
receptor antagonists is an effective method of delivery of these
compounds, with systemic pharmacological activity being
observed.
[0168] Female Wistar rats weighing between 200-250 g were used for
in vivo testing of all C5a receptor antagonists. Rats were
anaesthetized using the procedure described above, and transferred
on to a heating pad to ensure that body temperature was maintained
throughout the experiment. A catheter was inserted in the femoral
vein, secured with a suture and flushed with 100 .mu.L of
heparinised saline. Rats were dosed with either the antagonist or
vehicle, intravenously (1 mg kg.sup.-1 in 200 .mu.L saline
containing 10% ethanol, 10 min prior to LPS challenge) or topically
(10 mg site-1 in 50% dimethyl sulphoxide/H.sub.2O 60 min prior to
complement challenge). Rats were infused i.v. via the femoral
catheter with either LPS (1 mg kg.sup.-1 i.v. in 100 .mu.L saline),
recombinant human C5a (2 .mu.g kg.sup.-1 in 100 .mu.L), or vehicle
control 10 minutes after the iv administration or 60 min after
topical administration, of antagonist or vehicle control. All
agents infused i.v. were injected over a 2 min period, and were
followed with a subsequent injection of 100 .mu.L of saline to
ensure complete delivery of the drug.
[0169] Whole blood samples (0.1 mL) were collected from the tail
vein at the time of drug and LPS or C5a administration. Samples
were collected at -15, 0, 5, 10, 15, 30, 60, 90, 120 and 150 min
relative to the injection of LPS (zero time), and placed in tubes
containing heparin (500 Units/mL). To isolate PMNs, 100 .mu.L of
blood was layered on to 200 .mu.L of Histopaque 1077 (Sigma U.S.A.)
solution and centrifuged at 400.times.g for 30 min min at room
temperature (25.degree. C.). The supernatant layer of platelet-rich
plasma, the monocyte and lymphocyte interface and the separation
layer of Histopaque were removed and discarded, leaving the PMN and
red blood-cell rich layer. 9 mL of cold distilled water (4.degree.
C.) was added to the remaining pellet and shaken for 40 sec to lyse
the red blood cells. Dulbecco's Phosphate-buffered saline
(10.times. concentration), was added to restore isotonicity. The
cells were then centrifuged at 400.times.g for 15 min at 10.degree.
C. The resulting supernatant was discarded, leaving a pellet of
PMNs. The PMNs were further washed in 9 mL of saline, and
centrifuged again at 400.times.g for 10 min at 10.degree. C. Again
the resulting supernatant was discarded, and the remaining pellet
was resuspended in 100 .mu.L or saline and mixed well. The number
of PMNS was counted on a haemocytometer. The number of cells at
each time point was calculated as a percentage of the total number
of cells at time zero, prior to complement challenge or LPS.
[0170] The female Wistar rats chosen for this study were divided
into the following treatment groups:
[0171] (a) LPS alone, in which 100 .mu.L 5% ethanol (-15 min)+1
mg/kg LPS (0 min) were infused;
[0172] (b) ethanol control, in which 100 .mu.L 5% ethanol was
infused at -15 min to examine the influence of ethanol on the
parameters measured in the rat;
[0173] (c) antagonist control, in which 1 mg/kg of cyclic
antagonist was infused i.v. at -15 min;
[0174] (d) i.v. antagonist+LPS, in which 1 mg/kg cyclic antagonists
(-15 min) and 1 mg/kg LPS (0 min) were infused; and
[0175] (e) topically-applied antagonist+LPS, in which 10 mg/rat of
antagonist was smeared on the abdominal area of the rat, followed
by i.v. infusion of LPS at 0 min.
[0176] Administration of C5a antagonists such as 3D53 (PMX53) to
the dermis of rats in an applied dose of 50 mg/kg results in the
detection of pharmacologically significant levels of the drug in
the circulating plasma. The application of the drug in a variety of
solvents, such as dimethyl sulphoxide (DMSO), propylene glycol (PG)
and water, in various combinations leads to the appearance of
pharmacologically-relevant concentrations of the drug in the
circulation, as illustrated in FIG. 12. This shows that dermal
application of 3D53 in DMSO/distilled H.sub.2O or propylene glycol
(PG)/H.sub.2O results in the appearance of the C5a antagonist in
the circulating plasma within 15 min, and that significant levels
persist for at least 4 hr.
Example 9
Systemic Effects of C5a Antagonists Following Dermal
Application
[0177] As described in Example 8, topical application of C5a
antagonist to the dermis of an animal results in
pharmacologically-relevant levels of the drug in the circulation.
To show that these levels have systemic activity, the ability of
these circulating levels of C5a antagonist to inhibit the
neutropenic effects of C5a administered i.v. was determined.
[0178] The C5a receptor antagonist 3D53 was administered (10 mg/per
rat) in 50% propylene glycol and 50% distilled H.sub.2O. The
composition was smeared evenly over the abdominal skin (4.times.8
cm.sup.2) for 30 minutes, then C5a was administered i.v. in a dose
of 2 .mu.g/kg in 200 .mu.L saline solution. Blood samples were
taken at time points: -30 (before drug administration), 0 (before
injection of C5a) and 5, 15, 30, 60, and 120 minutes for
determination of circulating PMNs. As shown in FIG. 13, topical
administration of compound 3D53 did indeed prevent the neutropaenic
effects of C5a.
Example 10
Topical Administration of C5a Antagonists Inhibits the Systemic
Effects of LPS
[0179] The C5a antagonists, 3D53 (compound 1), as well as compound
17 and compound 45 were applied topically at a dose of 10 mg/rat in
50% DMSO/50% H.sub.2O, as described above. LPS was injected 1 mg/kg
i.v. 60 min after dermal application of the antagonists.
Circulating PMN levels were monitored for 150 min following LPS
injection, and the percentage change in PMN levels from zero time,
when LPS was injected, was calculated. The results, illustrated in
FIG. 14A, FIG. 14B, and FIG. 14C, show that each C5a antagonist
applied topically inhibited the neutropenia response to i.v. LPS,
and that the inhibition was comparable to that observed following
i.v. administration of the drugs.
Example 11
Effect of C5a Antagonist on Ischemia-Reperfusion Injury
[0180] Lower limb ischemia-reperfusion (I/R) injury is a serious
problem following the surgical repair of abdominal aortic aneurysm,
as well as following traumatic crush injuries (Kerrigan and
Stotland, 1993). Ischemia and the subsequent reperfusion of the
skeletal muscle tissue stimulates an inflammatory response in the
affected muscle, as well as inducing injury in other tissues (Gute
et al, 1998). In severe cases of limb ischemia, the resulting
reperfusion is associated with high mortality, resulting from
multiple system organ failure (Defraigne and Pincemail, 1997). In
order to investigate the capacity of a potent C5a receptor
antagonist to inhibit various parameters of local and remote organ
injury following lower limb ischemia-reperfusion (I/R) in rats, rat
hindlimbs were subjected to 2 hours ischemia and 4 hours
reperfusion. This tourniquet shock model has been widely used as a
model of lower limb I/R injury.
[0181] Rats were subjected to 2 hours bilateral hindlimb ischemia
and 4 hours reperfusion. Drug-treated rats received AcF-[OPdChaWR]
(1 mg/kg) i.v. either 10 min before ischemia or 10 min prior to
reperfusion, or orally (10 mg/kg) 30 min prior to ischemia. Levels
of circulating creatine kinase (CK), lactate dehydrogenase (LDH),
alanine and aspartate aminotransferase (ALT/AST), creatinine, blood
urea nitrogen (BUN), polymorphonuclear leukocytes (PMNs) and
calcium (Ca.sup.++) and potassium (K.sup.+) ions were determined.
These biochemical indices are known to reflect tissue or organ
injury following I/R events. Other parameters measured included
urinary protein levels, muscle edema and myeloperoxidase (MPO)
concentrations in the lung, liver and muscle along with liver
homogenate TNF-.alpha. concentrations. No significant changes were
observed in any of these markers compared to sham-operated animals,
indicating that the drug alone had no adverse effects as defined by
changes in these markers. Limb I/R injury was characterized by
significant elevations of CK, LDH, ALT, AST, creatinine, BUN,
proteinuria, PMNs, serum K.sup.+, muscle edema, organ MPO and liver
homogenate TNF-.alpha. concentrations, but a significant reduction
in serum Ca.sup.++ concentrations. When rats were treated with
AcF-[OpdChaWR], there were significant improvements in all these
parameters.
[0182] The study was performed in accordance with guidelines from
the National Health & Medical Research Council of Australia,
and the experimental protocol approved by the University of
Queensland Animal Ethics Committee. Female Wistar rats weighing
250-300 g were fasted overnight before being anaesthetized with the
i.p. injection of 6 mg/kg xylazine and 120 mg/kg ketamine.
Anesthesia was maintained throughout the study by additional
injections of ketamine. Rats were placed on a heating pad to
maintain normal body temperature, and a polyethylene catheter was
inserted into the right jugular vein for the infusion of the C5a
antagonist or 7% ethanol/saline. Bilateral hindlimb ischemia was
then induced through the application of latex o-rings (marking
rings; Hayes Veterinary Supplies, Brisbane, Australia) above the
greater trochanter of each hind limb. Following 2 hours of
ischemia, the latex rings were cut and removed and limbs allowed to
reperfuse for 4 hours. Six experimental groups were used:
[0183] (a) sham-operated,
[0184] (b) ischemia-only,
[0185] (c) I/R-only,
[0186] (d) I/R+C5a antagonist (1 mg/kg, i.v.) administered 10 min
prior to ischemia,
[0187] (e) I/R+C5a antagonist (10 mg/kg, p.o.) administered 30 min
prior to ischemia, and
[0188] (I/R+C5a antagonist (1 mg/kg, i.v.) administered 10 min
prior to reperfusion.
[0189] Sham-operated animals did not undergo any ischemia or
reperfusion, and ischemia-only animals had tourniquets applied for
2 hours without subsequent reperfusion. All other groups underwent
2 hours of ischemia and 4 hours of reperfusion. Sham-operated,
ischemia-only and I/R groups were infused with 7% ethanol/saline 10
min prior to ischemia, instead of drug. Blood was collected
throughout the study from the tail vein, and serum or plasma was
stored at either 4.degree. C. or -20.degree. C. for later
biochemical assays. Urine was collected over the last hour of the
study for the determination of urinary protein levels. At the
completion of the experiment, rats were euthanased, and sections of
the lungs, liver and gastrocnemius muscle removed and weighed for
edema, neutrophil accumulation and liver TNF-.alpha. studies.
[0190] All experimental results are expressed as means standard
error of the mean (SEM). Data analysis was performed using GraphPad
Prism 3.0 software (GraphPad Software, Inc. USA). Statistical
comparisons were made to sham-operated and I/R-only groups, using a
one-way analysis of variance followed by a Dunnett comparison
post-test analysis. Statistical significance was assessed at
P<0.05.
(a) Inhibition of Creatine Kinase and Lactate Dehydrogenase
[0191] Circulating levels of creatine kinase (CK) were measured in
serum samples taken immediately after ischemia, following 1, 2 and
3 hours reperfusion, and at the completion of the study using a CK
kit (Sigma, St. Louis, USA) according to the manufacturer's
instructions. Serum was also taken 10 min after tourniquet release
for CK measurement in I/R-only animals. A 1:5 dilution was used for
samples taken after 2 hours reperfusion.
[0192] Circulating levels of lactacte dehydrogenase (LDH) were
measured in serum samples taken immediately after ischemia,
following 1, 2 and 3 hours reperfusion, and at the completion of
the study. Serum was also taken 10 min after tourniquet release for
LDH measurement in I/R-only animals. Concentrations of LDH were
determined with a LDH kit (Sigma), according to the manufacturer's
instructions, with a 1:4 dilution of samples taken after 2 hours
reperfusion. For both enzymes, all samples were stored at 4.degree.
C. and analyzed within 24 hours of collection. Results were
expressed as Sigma-Franke (SF) units/mL.
[0193] As illustrated in FIG. 15A and FIG. 15B, bilateral hindlimb
I/R resulted in elevation of circulating levels of both CK and LDH
after 1, 2, 3 and 4 hours of reperfusion, with peaks of both
enzymes reached after 4 hours. Ischemia-only rats showed no
significant elevation of either CK or LDH levels (CK, 58.3.+-.23.5
units/mL; LDH, 269.5.+-.72.8 units/mL; P>0.05; n=4) compared to
sham-operated rats. In I/R-only rats there was no significant
increase in the levels of these enzymes 10 min after tourniquet
release (CK, 73.9.+-.28.1 units/mL; LDH, 395.3.+-.123.2 units/mL;
P>0.05; n=4), compared to sham-operated rats, indicating a
reperfusion-dependent elevation over the 4 hour time period.
Reperfusion significantly elevated the plasma levels of both CK and
LDH (P<0.05). Rats treated prior to ischemia with the C5a
antagonist, either i.v. (1 mg/kg) or orally (10 mg/kg), had similar
significantly decreased levels of both CK and LDH compared to
I/R-only rats (P<0.05). In addition, rats treated i.v. with the
C5a antagonist (1 mg/kg) just prior to reperfusion also displayed
significant inhibition of CK and LDH levels, of similar magnitude
to pre-ischemia treated rats (P<0.05). Levels of these enzymes
during reperfusion in all the drug-treated rats were significantly
higher than in sham-operated rats, indicating partial inhibition by
the C5a antagonist (P<0.05).
(b) Inhibition of Alanine Transaminase and Aspartate
Aminotransferase
[0194] Circulating levels of alanine aminotransferase (ALT) and
aspartate aminotransferase (AST) were measured in plasma samples
taken at the completion of the study and following 2 and 3 hours
reperfusion. Serum was also taken 10 min after tourniquet release
for measurement of and AST in I/R-only animals. Concentrations of
ALT and AST were determined with an ALT/AST kit (GPT/GOT; Sigma),
according to the manufacturer's instructions, within 24 hours of
collecting plasma, which was stored at 4.degree. C. Results were
expressed as SF units/mL.
[0195] As shown in FIG. 16A and FIG. 16B, limb I/R resulted
elevation of ALT and AST in the plasma after 2, 3 or 4 hours of
reperfusion, with peaks of both enzymes reached after 4 hours. Rats
receiving 2 hours of ischemia alone showed no significant elevation
of either ALT or AST levels (ALT, 12.9.+-.4.6 unit/mL; AST,
91.4.+-.10.4 units/mL; P>0.05; n=4) compared to sham-operated
rats. In I/R-only rats, 10 min after tourniquet release, there was
no significant increase in levels of these enzymes (ALT,
18.1.+-.4.4 units/mL; AST, 105.9.+-.21.3 units/mL; P>0.05; n=4),
compared to sham-operated rats, again indicating a
reperfusion-dependent elevation. C5a antagonist-treated rats in all
3 groups were found to have similar significant decreases in ALT
and AST levels compared to I/R-only rats (P<0.05). After 4 hours
of reperfusion, but not at 2 or 3 hours, drug-treated rats had
significantly increased levels of ALT compared to sham-operated
rats (P<0.05; FIG. 16A). In contrast, these drug-treated rats
had increased AST levels compared to sham-operated rats at all time
points measured, indicating the differential inhibition of the C5a
antagonist for ALT and AST (P<0.05; FIG. 16B).
(c) Inhibition of Changes in Serum Levels of Potassium and Calcium
Ions
[0196] Serum levels of potassium ion (K.sup.+) were measured with a
flame photometer (Corning 435; Corning, U.S.A.) after the
completion of each experiment, and 10 min after tourniquet release
for I/R-only animals. Serum calcium ion (Ca.sup.++) concentrations
were also measured at the completion of the study and 10 min after
tourniquet release for I/R-only animals, using a calcium kit
(Sigma). Samples were stored at -20.degree. C., and analyzed for
K.sup.+ and Ca.sup.++ levels within 2 weeks of collection. Results
were expressed as mmol/L, and are summarized in Table 4.
TABLE-US-00004 TABLE 4 Alterations in serum cation levels following
ischemia and 4 hours reperfusion in rats Serum K.sup.+ Serum
Ca.sup.++ Experimental Group n.sup.a (mmol/L) (mmol/L)
Sham-operated 8 4.84 .+-. 0.29* 2.66 .+-. 0.06* Ischemia-only 4
4.70 .+-. 0.17* 2.52 .+-. 0.10* I/R.sup.b-only 10 7.53 .+-.
0.34.sup..dagger. 2.23 .+-. 0.09 I/R + 1 mg/kg 8 6.13 .+-. 0.18*
2.46 .+-. 0.04* i.v. pre-ischemia I/R + 10 mg/kg 6 5.57 .+-. 0.58*
2.49 .+-. 0.04* p.o. pre-ischemia I/R + 1 mg/kg 6 5.02 .+-. 0.32*
2.45 .+-. 0.10* i.v. pre-reperfusion Data represent the mean .+-.
SEM. .sup.aNumber of rats .sup.bIschemia/reperfusion *P < 0.05
vs. I/R-only .sup..dagger.P < 0.05 vs. sham-operated
Inhibition of Blood Urea Nitrogen, Creatinine and Urinary
Protein
[0197] Circulating levels of blood urea nitrogen (BUN) were
measured in serum samples taken at the completion of the study
using a urea nitrogen kit (Sigma) according to the manufacturer's
instructions. Samples were stored at -20.degree. C. and were
analysed within 2 weeks of collection. Circulating levels of
creatinine were measured in serum samples taken at the completion
of the study using a creatinine kit (Sigma) according to the
manufacturer's instructions. Protein concentrations in urine
samples collected over a 1 hour period prior to the completion of
the study were determined with a protein kit (Sigma) according to
the manufacturer's instructions. Samples were stored at 4.degree.
C. and analyzed within 24 hours of collection. Results for all
three parameters, expressed as mg/dL, are shown in Table 5.
TABLE-US-00005 TABLE 5 Alterations in kidney injury markers
following ischemia and 4 hours reperfusion in rats Plasma Serum
BUN.sup.b Creatinine Proteinuria Experimental Group n.sup.a (mg/dL)
(mg/dL) (mg/dL) Sham-operated 8 21.9 .+-. 1.2* 0.79 .+-. 0.12* 10.7
.+-. 2.2* Ischemia-only 4 22.8 .+-. 2.5* 0.62 .+-. 0.26* 13.3 .+-.
7.0* I/R.sup.c-only 10 41.9 .+-. 3.1.sup..dagger. 1.66 .+-.
0.09.sup..dagger. 120.3 .+-. 18.9.sup..dagger. I/R + 1 mg/kg i.v. 8
23.6 .+-. 1.6* 1.07 .+-. 0.07* 36.4 .+-. 11.4* pre-ischemia I/R +
10 mg/kg p.o. 6 21.8 .+-. 2.1* 1.14 .+-. 0.14* 45.8 .+-. 9.8*
pre-ischemia I/R + 1 mg/kg i.v. 6 22.0 .+-. 2.2* 1.22 .+-. 0.05*
41.2 .+-. 11.4* pre-reperfusion Data represent the mean .+-. SEM.
.sup.aNumber of rats .sup.bBlood urea nitrogen
.sup.cIschemia/reperfusion .sup.dNot detectable *P < 0.05 vs.
I/R-only .sup..dagger.P < 0.05 vs. sham-operated
[0198] Hyperkalaemia was observed in I/R-only rats compared to
sham-operated rats after 4 hours of reperfusion (P<0.05). Rats
in all 3 drug-treatment groups had significantly lower K.sup.+
levels than I/R-only rats, with rats treated just prior to
reperfusion showing near-normal levels (P<0.05). Following
ischemia and 4 hours of reperfusion, I/R-only rats had
significantly decreased serum concentrations of Ca.sup.++ compared
to sham-operated animals (P<0.05). Rats in all 3 drug-treatment
groups showed a similar inhibition of the FR-induced decrease in
Ca.sup.++ levels (P<0.05). Levels of both K.sup.+ and Ca.sup.++
in drug-treated I/R rats, as well as ischemia-only rats, were not
significantly different from those in sham-operated rats
(P>0.05). Levels of these ions 10 min after the release of the
tourniquet in I/R-only rats (K.sup.+, 5.27.+-.0.49 mmol/L;
Ca.sup.++, 2.50.+-.0.17 mmol/L; P<0.05; n=4) were also not
significantly different from those in sham-operated rats.
[0199] Following ischemia and 4 hours reperfusion, I/R-only rats
had significantly elevated serum BUN and creatinine levels, as well
as increased urinary protein concentrations, compared to
sham-operated rats (P<0.05). Two hours of ischemia alone caused
no increase in any of these parameters compared to sham-operated
rats (P>0.05). Rats treated with the C5a antagonist in all 3
groups had significantly lower levels of these parameters compared
to I/R-only rats (P<0.05), and these levels were not
significantly different from those in sham-operated rats
(P<0.05).
Inhibition of Polymorphonuclear Leukocyte Numbers and Neutrophil
Accumulation
[0200] The numbers of circulating PMNs were measured in heparinised
blood samples taken just prior to ischemia and at the completion of
the study, as described by Strachan et al., (2000). Numbers of PMNs
in the final samples were expressed as a mean percentage SEM of
pre-ischemia numbers.
[0201] As shown in FIG. 17A, FIG. 17B, FIG. 17C, and FIG. 17D, the
number of circulating PMNs was found to be significantly elevated
in I/R-only rats following 4 hours of reperfusion, compared to
sham-operated rats (P<0.05). Ischemia-only rats had no
significant elevation of PMNs compared to sham-operated rats
(P>0.05). PMN numbers in rats from all 3 drug-treated groups
were not significantly different from those in sham-operated rats
(P>0.05), and were significantly decreased compared to those in
I/R-only rats (P<0.05), with rats treated i.v. (1 mg/kg)
pre-ischemia displaying the greatest inhibitory effect.
[0202] The infiltration of neutrophils into the liver, lung and
muscles of rats was determined by measuring the level of
myeloperoxidase (MPO) activity. Sections of lung (.about.0-5 g),
liver (.about.1 g) and the left lower limb muscle (.about.1 g)
obtained at the completion of the study were weighed and then
homogenized with 1 mL phosphate-buffered saline (PBS). Samples were
then sonicated for 20 seconds for liver and lung samples or 60
seconds for muscle samples. Following centrifugation
(14,000.times.g, 10 min, 22.degree. C.), the resulting supernatants
were tested immediately for MPO levels. The assay mixture consisted
of o-dianasidine (2.85 mg/mL; Sigma), hydrogen peroxidase (0.85%)
and a 1:40 dilution of samples in PBS. Absorbances were read at 450
nM, 5 min after substrate addition, and results expressed as
absorbance units/g tissue.
[0203] The level of MPO activity in the hindlimb muscles, lungs and
liver of rats were taken as a measurement of neutrophil
sequestration into the tissue (Kyriakides et al., 2000). As shown
in FIG. 17B, FIG. 17C and FIG. 17D, there were significant
elevations in MPO activity in the hindlimb muscles, lungs and liver
of I/R-only rats compared to those in sham-operated rats
(P>0.05), whereas sham-operated rats, ischemia-only rats had no
significant increase in MPO activity in any of these tissues
(P>0.05). Drug-treated rats in all 3 groups had significant
decreases in MPO activity in all tissues compared to I/R-only rats
(P<0.05), and the levels were not significantly different from
those in sham-operated rats (P>0.05).
Inhibition of Liver Homogenate TNF-.alpha.Levels of TNF-.alpha.;
were measured in liver homogenate supernatant samples, using an
enzyme-linked immunosorbent assay kit (OptEIA, Pharmingen, USA) as
previously described (Strachan et al., 2000). A 1:10 dilution of
supernatant from liver homogenate samples was used in the assay.
Supernatant was stored at -20.degree. C., and samples were analyzed
within 2 weeks of collection. Results were expressed as ng/g
tissus. As shown in FIG. 18, liver homogenate samples from I/R-only
rats had significantly increased TNF-.alpha.concentrations compared
to those from sham-operated rats (P<0.05). Levels of TNF-.alpha.
in ischemia-only rats were not significantly different from those
from sham-operated rats (P>0.05). In drug-treated rats, all 3
groups had a similar decrease in TNF-.alpha. concentrations
compared to I/R-only rats (P<0.05), which were not significantly
different from those from sham-operated animals (P>0.05).
Inhibition of Muscle Edema
[0204] Sections of the right lower limb muscle (.about.1 g)
obtained at the completion of the study were weighed and placed in
an oven for 24 hours at 80.degree. C. before weighing again. The
wet-to-dry weight ratio was determined and taken as a measurement
of muscle edema. As illustrated in FIG. 19, wet-to-dry weight
ratios of the hindlimb muscle in I/R-only rats were significantly
increased compared to those in sham-operated animals (P<0.05).
Ratios in ischemia-only animals were not significantly different
from sham-operated animals (P>0.05). In all 3 groups of
drug-treated rats, there was a similar decrease in wet-to-dry
ratios compared to those in I/R-only rats (P<0.05), and these
values were not significantly different from those in sham-operated
animals (P>0.05).
[0205] The results show that rats subjected to 2 hours of
tourniquet-induced bilateral hindlimb ischemia and 4 hours
reperfusion suffered both local injury and injury to the lungs,
liver and kidney, as measured by various indices of tissue stress.
Rats subjected to ischemia alone had no significant alterations in
disease markers compared to sham-operated animals. Blood taken from
I/R-only animals after only 10 min of reperfusion also had no
significant changes in the plasma or serum levels of CK, LDH, AST,
ALT, K+ or Ca++ compared to sham-operated animals. The severity of
local skeletal muscle injury was assessed by measuring increases in
muscle edema and neutrophil accumulation following 4 hours of
reperfusion, as well as serum CK and LDH throughout the reperfusion
period. The cytosolic enzyme CK is found predominantly in muscle,
and is-a reliable marker of muscle tissue damage (Tay et al.,
2000). Lactate dehydrogenase is also a cytosolic enzyme found in
the muscle, but is present in many other tissues as well (Carter et
al., 1998). Consequently, LDH was a less specific measure of muscle
injury, but still provided a measure of general tissue injury.
[0206] Indices of remote organ injury were detected in the lungs,
liver and kidneys of animals subjected to ischemia and reperfusion
episodes. The potential for lung injury was assessed by measuring
increases in neutrophil accumulation in lung parenchyma. Hepatic
injury was also quantified by measuring the increase in hepatic
TNF-.alpha. and neutrophil accumulation, and by measuring increases
in plasma levels of ALT and AST. Although increases in plasma
levels of ALT and AST have typically been used as markers of liver
pathology, both of these enzymes are also found within the muscle,
and thus any increases may in part be attributed to muscle, rather
than liver damage (Tay et al., 2000). Kidney dysfunction following
skeletal muscle I/R is common (Tanaka et al., 1995). We found
increases in serum BUN and plasma creatinine in I/R rats. However,
creatinine, and in particular BUN, are also derived from the
muscle, and the observed increases may also be attributed to muscle
injury (Carter et al., 1998). We found a sizeable increase in
proteinuria in I/R rats, indicating some degree of kidney
injury.
[0207] The C5a antagonist AcF-[OPdChaWR] was found to inhibit a
multitude of disease markers of local tissue and remote organ
injury in this model. These results indicate a key role for C5a in
the pathophysiology of skeletal muscle I/R injury. Given the high
incidence of complications following lower limb ischemia and
reperfusion in humans, the C5a receptor antagonists of the
invention represent a possible future treatment of these
complications, especially when I/R injury is anticipated, such as
in surgical procedures. The ability of C5a antagonists to block
both proinflammatory cytokine production and neutrophil trafficking
may be key factors in their disease-modifying properties. The oral
activity demonstrated here is a useful drug property for its
widespread use in clinical situations.
DISCUSSION
[0208] This invention describes a series of
conformationally-constrained turn-containing cyclic molecules which
are pre-organized for binding to cells which also bind human C5a.
The principal feature of the compounds of the invention is the
pre-organized turn conformation presented by the cyclic scaffold,
which assembles at least three hydrophobic groups into neighbouring
space, creating a hydrophobic surface `patch`.
[0209] This turn conformation of the antagonist may permit the
cyclic peptide to bind in the transmembrane region of the C5a
receptor at, or close to, the location which is also bound by the
C-terminal end of human C5a.
[0210] The results described herein enable the design and
development of even more potent conformationally constrained small
molecule antagonists of C5a. In principle the features of these
cyclic antagonists are also useful for designing unrelated
non-peptidic templates which similarly project substituents,
corresponding to or similar to those attached to the cyclic peptide
scaffolds described herein, into similar three-dimensional space as
that occupied by these C5a receptor antagonists when bound to the
receptor.
[0211] Cyclic peptides have several important advantages over
acyclic peptides as drug candidates (Fairlie et al., 1995, Fairlie
et al., 1998, Tyndall and Fairlie, 2001). The cyclic compounds
described in this specification are stable to proteolytic
degradation for at least several hours at 37.degree. C. in human
blood or plasma, in human or rat gastric juices, or in presence of
digestive enzymes such as pepsin, trypsin and chymotrypsin. In
contrast, short linear peptides composed of L-amino acids are
rapidly degraded to their component amino acids within a few
minutes under these conditions. A second advantage lies in the
constrained single conformations adopted by the cyclic and
non-peptidic molecules, in contrast to acyclic or linear peptides,
which are flexible enough to adopt multiple structures in solution
other than the one required for receptor-binding. Thirdly, cyclic
compounds such as those described in this invention are usually
more lipid-soluble and more pharmacologically bioavailable as drugs
than acyclic peptides, which can rarely be administered orally.
Fourthly, the plasma half-lives of cyclic molecules are usually
longer than those of peptides.
[0212] It will be apparent to the person skilled in the art that
while the invention has been described in some detail for the
purposes of clarity and understanding, various modifications and
alterations to the embodiments and methods described herein may be
made without departing from the scope of the inventive concept
disclosed in this specification.
[0213] References cited herein are listed on the following pages,
and are incorporated herein by this reference.
REFERENCES
[0214] Carter, W. O., Bull, C., Bortolon, E., et al., "A murine
skeletal muscle ischemia-reperfusion injury model: differential
pathology in BALB/c and DBA/2N mice," J. Appl. Physiol.,
85:1676-1683, 1998. [0215] Defraigne, J. O., and Pincemail, J.,
"Local and systemic consequences of severe ischemia and reperfusion
of the skeletal muscle," Physiopathol. Prevent. Acta. Chir. Belg.,
97:176-186, 1997. [0216] DeMartino, J. A., Konteatis, Z. D.,
Siciliano, S. J., Van Riper, G., Underwood, D. J., Fischer, P. A.,
Springer, M. S. J., Biol. Chem., 270:15966-15969, 1995. [0217]
Demartino, J. A., Van Riper, G., Siciliano, S. J., Moineaux, C. J.,
Konteatis, Z. D., Rosen, H. Springer, M. S., J. Biol. Chem.,
269:14446-14450, 1994. [0218] Ember, J. A., Sanderson, S. D.,
Taylor, S. M., Kawahara, M. and Hugli, T. E., J. Immunol.,
148:3165-3173, 1992. [0219] Fairlie, D. P., Wong, A. K.; West, M.
W., Curr. Med. Chem., 5:29-62, 1998. [0220] Fairlie, D. P.,
Abbenante, G. and March, D., Curr. Med. Chem., 2:672-705, 1995.
[0221] Finch, A. M., Vogen, S. M., Sherman, S. A., Kirnarsky, L.,
Taylor, S. M., and Sanderson, S. D., J. Med. Chem., 40:877, 1997.
[0222] Gerard, C. and Gerard, N. P., Ann. Rev. Immunol.,
12:775-808, 1994. [0223] Gerard, N. and Gerard, C., Nature,
349:614-617, 1991. [0224] Gute, D. C., Ishida, T, Yarimizu, K,
Korthuis, R. J., "Inflammatory responses to ischemia and
reperfusion in skeletal muscle," Mol. Cell. Biochem., 179:169-187,
1998. [0225] Haviland, D. L., McCoy, R. L., Whitehead, W. T.,
Akama, H., Molmenti, E. P., Brown, A., Haviland, J. C., Parks, W.
C., Perlmutter, D. H. and Wetsel, R. A., J. Immunol, 154:1861-1869,
1995. [0226] Kawai, M., Quincy, D. A., Lane, B., Mollison, K. W.,
Luly, J. R., Carter, G-W., J. Med. Chem., 34:2068-71, 1991. [0227]
Kawai, M., Quincy, D. A., Lane, B., Mollison, K. W., Or, Y.-S.,
Luly, J. R., and Carter, G-W., J. Med. Chem., 35:220-223, 1992.
[0228] Kerrigan, C. L., and Stotland, M. A., "Ischemia reperfusion
injury: a review," Microsurgery, 14:165-175, 1993. [0229] Kohl, J.,
Lubbers, B., Klos, A. et al., Eur. J. Immunol., 23:646-652, 1993.
[0230] Konteatis, Z. D., Sicilian, S. J., Van Riper, G., Molineaux,
C. J., Pandya, S., Fischer, P., Rosen, H., Mumford, R. A., and
Springer, M. S., J. Immunol., 153:4200-4204. 1994. [0231]
Kyriakides, C, Austen, W G, Jr., Wang, Y, et al., "Neutrophil
mediated remote organ. injury after lower torso Ischemia and
reperfusion is selectin and complement dependent," J. Trauma,
48:32-38, 2000. [0232] Morgan, E. L., Sandersorl, S. D., Schloz,
W., Noonal, D. J., W Weigle, W. O. and Hugli, T. E., J. Immunol.,
48:3937-3942, 1992. [0233] Sanderson, S. D., Ember, J. A.,
Kirnarsky, L., Sherman, S. A., Finch, A. M., Taylor, S. M., J. Med.
Chem., 37:3171-3180, 1994. [0234] Sanderson, S. D., Kirnarsky, L.,
Sherman, S. A., Vogen, S. M., Prakesh, O., Ember, J. A., Finch, A.
M. and Taylor, S. M., J. Med. Chem., 38:3669-3675, 1995. [0235]
Siciliano, S. J., Rollins, T. E., DeMartino, J., Konteatis, Z.,
Malkowitz, L., VanRiper, G., Bondy, S., Rosen, H. and Springer, M.
S., Proc. Nat. Acad. Sci. USA, 91:1214-1218. 1994. [0236] Sim, E.
The Natural Immune System. Humoral Factors, IRL Press, Oxford
University Press, Oxford, 1993. [0237] Strachan, A. J., Haaima, G,
Fairlie, D. P. and S. M. Taylor, "Inhibition of the reverse passive
Arthus reaction and endotoxic shock in rats by a small molecule C5a
receptor antagonist," J. Immunol., 164:6560-6565, 2000. [0238]
Strachan, A. J., Woodruff, T. M., Haaima, G, et al., "A new small
molecule C5a receptor antagonist inhibits the reverse-passive
Arthus reaction and endotoxic shock in rats," J. Immunol.,
164:6560-6565, 2000. [0239] Tanaka T, Kita T, Liu R, Tanaka N.,
"Protective effect of peptide leukotriene antagonist on renal
failure induced by a tourniquet in rabbits," Forensic Sci. Int.,
71:57-64, 1995. [0240] Tay, S. K., Ong, H. T., Low, P. S.
"Transaminitis in Duchenne's muscular dystrophy," Ann. Acad. Med.
Singapore, 29:719-722, 2000. [0241] Tempero, R. M., Hollingsworth,
M. A., Burdick, M. D., Finch, A. M., Taylor S. M., Vogen, S. M.,
Morgan, E. L., and Sanderson, S. D., J. Immunol., 158:1377-1382,
1997. [0242] Tyndall, J. D. A.; Fairlie, D. P., Curr. Med. Chem.,
8:893-907, 2001. [0243] Whaley, K. Complement in Health and
Disease, Immunology and Medicine Series, Ed. Reeves, W. G., MTP
Press Ltd, Lancaster, 1987.
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