Note: Descriptions are shown in the official language in which they were submitted.
~ ; ? ~
~lo 95116702 2 1 7 8 9 2 7 p ~ 7. ~1~45
--1--
iUlSS~:KIit' 1. lVN
PEPTIDE lN~ ORS OF CXC T~T~p~'RTl~ ~C~T.P!.q;
13ACKGRO~ND OF ~IE lNV~l~LlVN
The U. S. government owns rights in the present
invention pursuant to grant number ROI-HL 403650 from
10 NHLB I .
1. Field of the Invention
The present invention relates generally to the f ield
15 of cytokine actions and more particularly concerns
methods and compositions for inhibiting and modulating
the actions of CXC intercrine molecules. Disclosed are
peptide compositions which inhibit interleukin 8 ( IL- 8 )
and, particularly, which preferentially inhibit IL-8-
20 induced ralease of degradative enzymes by neutrophils. -
These compositions may be employed to treat various
infli tory diseases and disorders including the Adu~t
Respiratory Distress Syndrome (ARDS) and cystic fibrosis.
25 2. De~3criPtion of the Related Art
IL-8 is a member of the CXC intercrine family of
cytokines, so named due to elements of their N-terminal
sequences. This family also includes, amongst others,
30 peptide molecules known as growth related oncogene (GRO,
or GRO/MGSA) and macrophage infl: tory protein 2~
(MIP2,(~). IL-8 is a peptide of approximately 8 kD, and is
about 72 amino acids in length, with this length varying
according to the post-translational processing in
35 different cell types (Yoshimura et al., 1989; Hebert et
al., 1990; Strieter et al., 1989) . The IL-8 gene was
first identified by analyzing the genes transcribed by
2 1 7~927
WO 95116702 ~ ; ~; P~ Yall~L45
--2--
human blood i ~lear cells stimulated with
Staphylococcal enterotoxin A (Schmid & Weissman, 1987).
IL-8 production is known to be induced by tumor necrosis
factor and interleukin 1 (Strieter et al., 1990).
IL- 8 interacts with at least two distinct receptors
on neutrophils (Holmes et al., 1991; Murphy & Tiffany,
1991). The receptors are coupled to GTP-binding
proteins, allowing transmission of the .IL-8 signal into
10 the cell (Wu et al., 1993). While most of the members of
the intercrine family, such as GRO and MIP2~, bind to one
of the receptors, IL- 8 binds to both of the IL- 8
receptors (LaRosa et al., 1992; Cerretti et al., 1993).
The three dimensional structure of IL-8 has been
15 elucidated by NMR (Clore et al., 1990) and by X-ray
crystallography (Clore ~ Gronenborn, 1992; Baldwin et
al_, 1991). A freely movable amino terminal end is
followed:by three beta pleated sheets and an alpha helix
is located at the carboxyl-terminal end (Dppenheim et
20 al., 1991). Several lines of evidence suggest that both
the amino- and carboxyl-terminal ends are involved in
binding to its receptors (Clore et al., 1990; Clark-Lewis
et al ., 1991; Moser et al ., 1993 ) .
Certain functions of the CXC intercrines have been
elucidated by several laboratories (Yoshimura et al.,
1989; Schroder et al., 1988; Peveri et al., 1988). For
example, the major functions of the IL-8 peptide appear
to be related to its ability to stimulate neutrophil
chemotaxis and activation (Larsen et al., 1989; Schroder
et al., 1988; Peveri et al., 1988; Yoshimura et al.,
1987) and to promote angioge~nesis (Koch et al., 1992).
If neutrophils are `primed', e.g., by agents such as
surface adherence or 1~. coli endotoxin (also known as
lipopolysaccharide or LPS), IL-8 also stimulates the
release of neutrophil enzymes such as elastase and
myeloperoxidase .
WO95/16702 21 78q27 P ~ i~4~
--3--
Although the neutrophil inflammatory response is
essential for the destruction of bacteria which are
invading the body, inappropriate neutrophil activation
causes several problems. For example, if the neutrophils
are properly primed when attracted to the lungs, they
release destructive enzymes into the lung tissue. This
can lead to the development of adult respiratory distress
2Iylldr~ - (ARDS) (Weiland et al., 1986; Idell et al.,
1985). ARDS attacks between 150,000 and 200,000
Americans per year, with a mortality rate of 50-809~ in
the best clinical facilities (Balk & Bone, 1983). ARDS
is initiated by bacterial infections, sudden severe
dropping Qf the blood pressure (shock), and many other
insults to the body. Recent studies have demonstrated
that IL-8 is the major neutrophil activator in the lungs
of patients with ARDS (Miller et al., 1992), and primate
models of endotoxin shock also implicate IL-8 as a
causative agent (Van Zee et al., 1991).
Xigh ~ nr~ntrations of IL-8 have also been found in
inflammatory exudates in other disorders and pathological
conditions in which IL- 8 is thought to play an important
pathogenic role (Brennan et al., 1990; Miller & Idell,
1993; Miller et al., 1992). For example, IL-8 has also
been implicated as a possible mediator of inflammation in
rheumatoid arthritis (Brennan et al., 1990; Seitz et al.,
1991) and pseudogout (Miller & Brelsford, 1993); and to
have a role in cystic fibrosis (McElvaney et al., 1992;
Nakamura et al., 1992; Bedard et al., 1993). Therefore,
modulation of I~-8 function appears to be good strategy
to control a variety of pathological conditions.
Some progress has recently been made in identifying
compounds capable of reducing IL-8 synthesis. Such
cornrol1nrl~ include IL-4, oxygen radical scavengers,
secretory leukoprotease inhibitor and interferon gamma
(Standiford e~ al., 1990; DeForge et al., 1992; McElvaney
21 78927
WO Y5/16702 ~- . t ~ Y~ 4
--4 --
et al., 1992; Cassatella et al., 1993a; 1993b), however,
such studies do not concern~ IL-8 inhibitors . Other
diverse compositions, including protein kinase C
inhibitors, IL-4, and anti-IL-8 antibodies, have also
5 been reported to modulate IL-8 actions (Lam et al., 1990;
Standiford et al., 1992; Mulligan et al., 1993).
Unfortunately, these compou~ds are far from ideal a6
candidates for use a~ IL-8 inhibitors in a clinical
setting .
Certain progress has also been made in identifying
peptide IL-8 inhibitors, however, most of such work has
focused on portions of the IL-8 molecule itself (Miller
et al., 1990; Gayle et al., 1993) . For example, the
15 present inventors have shown that synthetic peptides, and
particularly, IL-8 amino t~rn;n;ll peptides, inhibit IL-8
binding to neutrophils and ~eutrophil chemotaxis (Miller
et al., 1990; Miller et al., 1993). An N-terminal
pentapeptide IL-8 inhibitor has also been reported
20 (Goodman et al_, 1991). ~:rnfortunately, to date, the .
inhibitory function of IL-8 derived peptides has proven
incomplete and insuf f icient .
As particularly effective peptide inhibitors of CXC
25 intercrines such as IL-8 have yet to be identified, it
seems to be clear that compositions other than the IL- 8
molecule itself now need to be investigated. The
identification of peptide inhibitors capable of
preferentially inhibiti3lg neutrophil enzyme release in
30 comparison to chemotaxis would be a particularly
advantageous discovery as this would enable neutrophils
to enter the lungs and defend against bacterial invasion
and yet not cause tissue damage.
~095116702 ~ 21 7~27 r~".J.,7~
--5--
S~RY OF l~IE lNVI:.l`l I lU~Y
The present invention seeks to overcome the
drawbacks inherent in the prior art by providing new
5 methods and compositions for modulating and inhibiting
the actions of CXC intercrine molecules such as IL- 8, GRO
(GRO/MGSA) and MIP2~. The peptides and pharmacological
compositions disclosed reduce IL-8, GRO and MIP2~B binding
to neutrophils and inhibit IL-8-induced neutrophil
10 activation. These peptide formulations are particularly
advantageous as they are capable of inhibiting IL-8-
induced enzyme release at significantly lower
r~nf-~ntratione than is required to inhibit neutrophil
chemotaxis. Also provided are methods for treating
15 various diseases and disorders, particularly inf lammatory
diseases, in which the unrestrained actions of CXC
intercrines play a role.
The invention is generally based upon the inventors
20 surprising discovery that relatively small peptides
including the amino acid se~uence Arg Arg Trp Trp Cys
Xaal (RR~WCX; SBQ ID NO:23), wherein Xaal is any amino
acid residue, are potent inhibitors of CXC intercrine
molecules such as IL-8. As used herein, the terms "CXC
25 intercrine family molecules" and "CXC intercrines'~ are
used collectively to ref er to the group of peptide
intercrines which include the CXC sequence motif in their
N-terminal regions. CXC intercrines are known to include
IL-8, GRO, MIP2~, MIP2,(3 and ENA78, all of which
3 0 molecules, and any other intercrine polypeptides that
include the CXC motif, will be understood to fall within
this term as used in the present application.
The inhibitory peptides of the present invention may
35 be termed "antileukinates". Certain hexamer peptides of
the sequence RRWWCX (SEQ ID NO:23) have been previously
shown to have anti-bacterial activity against
2 1 78927
WO 95116702 . ~ /I~-15
--6--
Staphylococcal aureus ~Houghten e~ al., l99l) . However,
there was no previously rlnc~ nted information to suggest
that any such peptides would have the advantageous anti-
cytokine/intercrine, anti-neutrophil and anti-
5 inf lammatory activities disclosed herein.
In certain aspects, the present invention theref orernn~-PrnR methods for inhibiting CXC intercrines, such as
GRO and MIP2~ or MIP2~, and most particularly, methods
lO for inhibiting IL-8. As used herein, the term
~inhibiting CXC intercrines" refers to the processes by
which the biological actions of the CXC intercrines are
reduced This may be particularly assessed by inhibiting
their binding to one of the I3~- 8 receptors on their
15 target cells, such as neutrophil~, although any mode of
determining CXC intercrine inhibition may be employed.
The term IL- 8 is used to ref er to the cytokine
compositions previously known as neutrophil-activating
20 factor, monocyte-derived neutrophil-acti~ating peptide,
monocyte-derived neutrophil-chemotactic factor and
neutrophil-activating peptide-l As used herein, the
term "inhibiting IL-8" generally refers to the processes
by which the biological actionR of IL- 8 are reduced or
25 lessened. This includes the inhibition of any or all of
the known actions of IL- 8 . These actions include
modulating sub-~ r effects, 3uch as receptor binding
or altering cytosolic calcium levels; modulating cellular
effects such as granulocyte recruitment and activation;
30 and also affecting phybiological effects, such as
inf lammation and angiogenesis .
In preferred embodiments, the inhibition of IL-8
function referred to in this application is the
35 inhibition of IL-8 action on granulocytes such as
neutrophils (polymorphonuclear neutrophils, PMN) This
may be determined in many cellular and physiological
Wo 95116702 ` ~ ` 2 ~ 7 ~ ~ 2 7 ~ _~/U~9~1~5
ways, as disclosed herein. For example, by measuring
inhibition of I~- 8 binding to purif ied receptor
compositions or neutrophils; by determining the
inhibition of IL-8-induced neutrophil chemotaxis or
5 diapedesis; by measuring the inhibition of IL- 8 -
stimulated neutrophil enzyme release (e.g.,
myeloperoxidase, ~-glucuronidase or elastase release) or
superoxide production; or by assaying for anti-
;nfli tory effects in vivo, e.g., using a rabbit model
lO of dermal inflammation.
The preferred manner of detrrmin;n~ IL-8 inhibition,
or indeed GRO or MIP2,~ inhibition, is to assay for a
reduction in the intercrine binding to neutrophils, which
15 is the most simple and straightforward method. In
addition, binding of the particular intercrine to its
receptor (s) must precede any other action that it has on
neutrophils or other cell types . " Inhibition" of
intercrines, as exemplified by the inhibition of I~-8,
20 G~O or MIP2cY or MIP2~ binding to neutrophils, refers to
the capacity of a given peptide or composition to inhibit
intercrine binding to any detectable degree, i . e . to
reduce binding below the levels observed in the absence
of the peptide or composition.
The inhibition of CXC intercrine binding to
neutrophils may be expressed as a 9~ Binding Inhibition
value, with the higher figures representing the more
effective inhibitors. The preferred peptides will
30 generally have the higher ~ binding inhibition figures.
Naturally, the 96 binding inhibition calculated will
depend upon the precise assay conditions, such as the
rrnr~ntration of CXC intercrine and the concentration of
the given peptide or composition. Conditions such as
35 those used to generate the data of Tables lA, lB, 5A and
5B, may be employed to determine whether a given peptide
has any inhibitory activity. Howev-r, one may choose to
WO95116702 ; ~ ~ 2 1 78q27 .~ Y~/1 ~5
. ,.,, , --
--8--
employ more discriminatory conditions, such as those
using lower peptide concentrations, e.g., on- the order of
about 20 ~lM (aæ used to generate the data of Figures 1
and 9), where one desires to obtain particularly accurate
5 quantitative or comparative data. In any event, the
determination of whether a peptide or analogue is capable
of inhibiting a CXC intercrine, such as IL-8, is a
straightforward matter readily achieved using assays such
as those disclosed herein.
Alt~ough an understanding of the --~h~n; r~ of action
of the CXC intercrine ;nh;~;tnrs iB not relevant in terms
of their practical utility, it is, however, important to
note that the peptide inhibitors of this invention ~are
15 capable of preferentially inhibiting IL-8-induced
neutrophil enzyme release at lower r~nne~ntrations than
IL-8-induced chemotaxis. In this sense, the term
"inhibiting", when used in connection with this
inventio~, also means "modulating" in that certain
20 neutrophil functions are more significantly inhibited
than others .
The ability of the peptides to inhibit IL-8-induced
neutrophil degradative enzyme release at about a 25 times
25 lower concentration than is re~uired to inhibit IL-8-
induced neutrophil chemotaxis is an important discovery
that could not have been predicted from prior studies.
This means that neutrophils may still be recruited to a
site of injury, but that the detrime~tal effects of the
30 enzymes that they would normally release will be
aignif icantly reduced. This property, coupled with their
small size, renders these type of peptides ideal for use
in various treatment protoc~ls and especially in the
treatment of lung inj ury .
To achieve CXC intercrine inhibition, such as IL-8,
GRO or MIP2 inhibition, or~ to preferentially reduce
~0 9511~702 2 ~ 7 ~ 9 ,~ 7 p~ y~l~4~
neutrophil enzyme release in comparison to neutrophil
chemotaxis, in accordance with this invention one would
generally contact the CXC intercrine family molecule or
or intercrine target cells, such as granulocytes or
5 neutrophils, with a biologically effective amount of a
composition comprising a peptide of the family disclosed
herein. The "contact" process is the process by which
the active peptide or peptides from within the
composition contact either the CXC intercrine peptide or
lO one of their receptors present on a target cell, or both,
and reduce or inhibit their functional interaction.
Although of scientific interest, the mech;lni ! by which
the CXC intercrine signals transmitted to a given cell
are reduced are not relevant to the practice of the
15 invention.
To contacl: a CXC intercrine or intercrine target
cell ~ith a peptide~ nt~;ning composition one may simply
add the peptide or composition to target cells, such as
20 neutrophils, and intercrines in vitrc. Alternatively,
one may administer a biologically effective amount of a
pharmacologically acceptable form of the peptide or
composition to an animal , where it will contact , e . g .,
neutrophils or macrophages and intercrines in a
25 = biological fluid in vivo. In this context, "contact" is
achieved simply by administering the composition to the
animal. Virtually any pharmaceutical peptide formulation
may be used, including, but not limited to, formulations
for parenteral administration, such as for intravenous,
30 intramuscular and subcutaneous administration; inh;~ ntc,
aerosols and spray formulations; formulations of peptides
for topical use, such as in creams, oin tc and gels;
and other formulations such as peptides with lipid tails,
peptides encapsulated in micelles or liposomes and drug
35 release capsules including the active peptides
incorporated within a bio~ tihle coating designed for
slow-release .
2 1 7~927
Wo 95116702 I ~ Ui~Y.~ 45
-10-
Increased levels of IL-8 are known to be present in
lung edema fluids in patients with ARDS (Miller et al.,
1992) and in the sputum of patients with cystic fibrosis
(Richman-Eisenstat et al . , 1993 ); in pleural spaces of
5 patients with pleural effusions (Miller h Idell, 1993);
in joint fluids from patients with several kinds of joint
disease (Brennan et al., l990; Miller & Brelsford, 1993),
in psoriatic pla~ues and in synovial fluid from arthritic
patients (Lam et al, 1990). Inappropriate neutrophil
lO activation is connected with all such disorders and with
ischemic and reperfusion injuries (DeForge et al., 1992) .
As the inhibition of IL- 8 neutrophil recruitment has been
shown to reduce lung 1 nfl iqmrniltion ln vivo (Mulligan et
al., 1993), and as the type of in vitro studies employed
15 herein are accepted as being predictive of i~ vivo
activity (see U.S. Patent, 5,079,228, incorporated herein
by reference), the highly successful inhibition of IL-8-
induced neutrophil activation disclosed in the
application supports the broad clinical utility of these
2 o p~ptides . . ~ . .
The present i~vention theref ore also provides
methods f or treating a wide variety of diseases and
disorders in which CXC intercrines, particularly IL- 8,
25 play a role, especially those which have an ;nfl; tory
component . This includes treating subj ects with lung
injuries and disorders, including bronchial ;nfli tion,
such as chronic bronchitis, cystic fibrosis, pleural
effusions, asthma, and ARDS; skin disorders such as
30 psoriasis and dermatitis; diseases of the joints,
including rheumatoid arthritis; and generally reducing
inflammation in other clinical settings, such as in the
treatment of pseudogout, inflammatory bowel disease or
reperfusion cardiac damage after myocardial infarction.
35 These peptides could even be used as anti-proliferative
agents to downregulate lymphocyte proliferation, for
example, in the treatment of cancer and other diseases
~V095116702 ; r~ ,3 ~b~ ~ P~.l/tJ~Y~I~JS
~ ~7~927
and disorders associated with increa3ed t-t?l 1l1 Ar
proliferation .
To treat any one o~ the above conditions, or any
5 other disorder influenced by neutrophil activity and
characterized by inflammation, one would identify a
patient having the particular inf~: tt~ry or I~-8-linked
disease and then administer to the patient, preferably
parenterally, a biologically effective amount of a
10 pharmaceutical composition which includes one or more
peptides of the family disclosed herein.
Naturally, one would generally tailor the particular
pharmaceutical formulation according to the disease or
15 disorder being treated. For example, in methods to treat
skin disorders, a topical cream or gel formulation would
be used, whereas in methods to treat pulmonary disorders,
injectable formulations, or even a spray, aerosol or
;nh tl ;-nt, may be employed. In methods to re~uce
20 ;nfl tion in other areas of the body, one may use
peptides formulated for parenteral administration or
peptides incorporated in a bioct _ ~t;hle coating designed
for slow-release. T ;r~ Rt~mt~-encapsulation may be
employed, which is known to increase the efficacy and
25 significantly prolong t~e half-life of ad~inistered
compounds, particularly those of lower m~'ecular weight
such as the peptides disclosed herein. ~3rious
compositions and technir;ues for preparinr~ all such
pharmaceutical formulations will generally be known to
3 0 those of skill in the art in light of the present
disclosure. For a detailed listing of suitable
- pharmacological compositions and associated
administrative techniques one may wish to refer to
Re~7tngton'~: Pharmaceutical Sciences, 16th ed., 1980, Mack
35 Publishing Co., incorporated herein by reference.
WO 95116702 2 ~ 7 8 9 2 7 . ~ iY~/i~45
--12--
I~-8 or CXC intercrine inhibition is achieved by
using a biologically effective amount of the inhibitory
peptide or peptides AY used herein, a "biologically
effective amount" of a peptide or composition refers to
5 an amount effective to inhibit the actions of I~-8 or the
particular intercrine. For~example, in regard to Il,-8
inhibtion, an appropriate amount would be that effective
to reduce neutrophil enzyme release, particularly in
comparison to chemotaxis As disclosed herein, a variety
10 of different peptide concentrations are very effective in
~ltro, such as those between about lQ0 ,uM and about
20 /lM. Clinical doses which result in similar a local
concentration of peptides are therefore contemplated to
be particularly useful.
Naturally, in a clinical context, the quantity and
volume ~f the peptide composition administered will
depend on the host animal and condition to be treated and
the route of administration. The precise amounts of
20 ac'cive peptide required to be administered will depend on
the judgment of the practitioner and may be peculiar to
each individual. ~lowever, in light of the data presented
herein, the determination of a suitable dosage range for
use in humans will be straightforward. For example, in
25 treating ARDS or cystic fibrosis, doses in the order of
about 0 83 mg/kg body weight/hour (mg~kgJhr) to about
16.56 mg/kg/hr, preferably about 0.83 mg/kg/hr to about
4.14 mg/kg/hr, and more preferably about 1 66 mg/kg/hr of
active ingredient peptide per individual are
30 contemplated.
The compositions for use in inhibiting CXC
intercrines, such as I~-8, GR0 and MIP2~ or MIP~, in
accordance with the present invention will be
35 compositions that contain a relatively small peptide,
generally of from 6 to about 14 residues in length/ which
includes within its sequence the amino acid sequence
Wo9S/16702 ~ 2 1 ~ Y3Ji~
RRWWCX ~SEQ ID N0:23) . The term "a peptide~ in this
sense means at least one peptide, and may refer to one or
more such peptides which include a sequence in compliance
with the general formula RRWWCX (SEQ ID N0:23).
The relatively small peptides Pn~0~3ed by the
present invention may be any length between six residues
and about 14 or 15 or so residue6 in length, with the
precise length not being an important feature of the
10 invention. There are many advantages to using smaller
peptides, for example, the cost and relative ease of
large scale synthesis, and their improved pharmacological
properties, such as the ease with which they can
penetrate tissues and their low immunogenicity.
In addition to including an amino sequence in
accordance with the sequence RRWWCX (SEQ ID N0:23), the
peptides may include other short peptidyl sequences of
various amino acids. For example, in certain
20 embodiments, the peptides may include a repeat of the
sequence RRWWCX (SEQ ID N0:23) or RRWWCXX (SEQ ID N0:57) .
They may also contain additional sequences including,
e.g., short targeting sequences, tags, labelled residues,
amino acids contemplated to increase the half life or
25 stability of the peptide, or indeed, any additional
residue desired for any purpose, 80 long as they still
function to inhibit intercrines such as IL-8 - which can
be readily determined by a simple assay such as those
described herein.
Amino acids which may incorporated into the peptides
include all o :the commonly occurring amino acids. Two
designations~ for amino acids are used interchangeably
throughout this application, as is common practice in the
35 art: Alanine = Ala (A); Arginine = Arg (R); Aspartic
Acid = Asp (D); Asparagine - Asn (N); Cysteine = Cys (C);
Glutamic Acid = Glu (E); Glutamine = Gln (Q); Glycine =
W095/16702 . j - 2 1 78927 1~ Y.~ 45
--14--
Gly (G); Xistïdine = His (H); Isoleucine = Ile (I);
Leucine = Leu (L); Lysine - Lys (K); Methionine = Met
(M); Phenyl ~1 ~n; nP = Phe (F); Proline = Pro (P); Serine =
Ser (S); Threonine= Thr (T); Tryptophan = Trp (W);
5 Tyrosine = Tyr (Y); Valine= Val (V).
Any of the so-called rare or modified amino acids
may also be incorporated into a peptide oi the invention,
including the following: 2-Pm;n~ ;ric acid,
10 3 -PnM nr~ ; ric acid, beta-Alanine (beta-Aminopropionic
acid), 2-Aminobutyric acid, 4-Aminobutyric acid
(piperidinic acid), 6-Aminocaproic acid, 2-7'n~;n~ Prtanoic
acid, 2-Aminoisobutyric acid, 3-Aminoisobutyric acid,
2-Aminopimelic acid, 2,4-Diaminobutyric acid, Desmosine,
15 2, 2 ' -Diaminopimelic acid, 2, 3-Diaminopropionic acid,
N-Ethylglycine, N-Ethylasparagine, Hydroxylysine, allo-
Xydroxylysine, 3-Hydru~y~Luline, 4-Hydroxyproline
Isodesmosine, allo=Isoleucine, N-Methylglycine
(sarcosine), N-Methylisoleucine, N-Methylvaline,
20 Norvaline, Norleucine and Qrnithine.
The inhibitory compositions of the invention may
include a peptide modified ~to render it biologically
protected. Biologically protected peptides; have certain
25 advantages over unprotected peptides when administered to
human subjects and, as disclosed in U.S. Patent 5,028,592
(incorporated herein by reference), protected peptides
often exhibit increased pharmacological activity, as was
found to be true in the present case.
The present invention theref ore encompasses
compositions comprising an acylated peptide or peptides,
and preferably, a peptide acylated at the N-terminus.
Although virtually any acyl group may be employed in this
35 context, the inventors have ~ound that the addition of an
acetyl group to the N-termInus of a given peptide also
renders the resultant peptide surprisingly effective at
wo 95116702 ' 2 ~ 7 8 9 2 7 . ~l,U~YJil~
.
--15--
inhibiting intercrines such as IL- 8 . The inhibitory
peptide compositions may also include a peptide (s) which
iB amidated at the C-terminus, i.e., to which an NH2
group has been added. In particularly preferred
5 embodiments, peptides which have both an acylated N-
terminal and an amidated C-terminal residue are preferred
as they are believed to most closely mimic natural
protein and peptide structure.
Compositions for use in the present invention may
also comprise peptides which include all L-amino acids,
all D-amino acids or a mixture thereof. The finding that
peptides composed entirely of D-amino acids have potent
inhibitory activity is particularly important as such
peptides are known to be resistant to proteases naturally
found within the human body and are less immunogenic and
can therefore be expected to have longer biological half
lives .
The anti-intercrine and anti-IL-8 compositions of.
the present invention will generally comprise one or more
peptides which include an amino acid sequence in
accordance with those set forth in SEQ ID NO:l or SEQ ID
NOS: 24 through 42 . In certain embodiments, short hexamer
peptides may be preferred. In such cases, the inhibitory
compositions will generally comprise one or more peptides
which have an amino acid se~uence in accordance with
those set iorth in SEQ ID NO: l or SEQ ID NOS: 24 through
42, presented below:
Arg Arg Trp Trp Cys Arg ~ SEQ ID NO: l )
- Arg Arg Trp Trp Cys Ala (SEQ ID NO:24)
Arg Arg Trp Trp Cys Cys (SEQ ID NO:25)
- Arg Arg Trp Trp Cys Asp (SEQ ID NO:26)
Arg Arg Trp Trp Cys Glu (SEQ ID NO:27)
Arg Arg Trp Trp Cys Phe ( SEQ ID NO: 2 8 )
Arg Arg Trp Trp Cys Gly ~SEQ ID NO:29)
WO9~116702 . .; 2~789~7 ~ ,Y3~ 4~ ~
-16-
Arg Arg Trp Trp Cy9 His ( SEQ ID NO: 3 0 )
Arg Arg Trp Trp Cys Ile (SEQ ID NO:31)
Arg Arg Trp Trp Cys Lys (SEQ ID NO:32)
Arg Arg Trp Trp Cys Leu (SEQ ID NO:33)
Arg Arg Trp Trp Cys Met (SEQ ID NO:34)
Arg Arg Trp Trp Cys Asn (SEQ ID NO:35)
Arg Arg Trp Trp Cys Pro (SEQ ID NO:36)
Arg Arg Trp Trp Cys Gln (SEQ ID NO:37)
Arg Arg Trp Trp Cys Ser (SEQ ID NO:38)
Arg Arg Trp Trp Cys Thr (SEQ ID NO:39)
Arg Arg Trp Trp Cys Val (SEQ ID NO:40)
Arg Arg Trp Trp Cys Trp (SEQ ID NO:41)
Arg Arg Trp Trp Cys Tyr (SEQ ID NO:42)
In other: ',o~;~onts, the inhibitory compositions of
the invention may include one or more peptides which
include a sequence in accordance with the amino acid
sequence Arg Arg Trp Trp Cys Arg Xaa2 (SEQ ID NO:2). In
20 these cases one of the variable positions has been
defined as arginine and the: L~ in;n~ Xaa2 may be any
amino acid residue. Such sequellces are exemplified by
those set forth in SEQ ID NOS:3 through 22. Where~short
heptamer peptides are preferred, the compositions will
generally comprise one or more peptides which have an
amino acid sequence in accordan~ with those set f orth
below:
Arg Arg Trp Trp Cys Arg Ala ~SEQ ID NO: 3 )
3 0 Arg Arg Trp Trp Cys Arg Cys (SEQ ID NO: 4 )
Arg Arg Trp Trp Cys Arg Asp (SEQ ID NO:5)
Arg Arg Trp Trp Cys Arg Glu (SEQ ID NO:6)
Arg Arg Trp Trp Cys Arg Phe (SEQ ID NO:7)
Arg Arg Trp Trp Cys Arg Gly (SEQ ID NO: 8 )
Arg Arg Trp Trp Cys Arg His (SEQ ID NO: 9~
Arg Arg Trp Trp Cys Arg Ile (SEQ ID NO:10)
Arg Arg Trp Trp Cys Ar~ Lys (SEQ ID NO :11)
~j095116702 ` ' ~ , ~l 7~927 .~ Y~ ~S
--17--
Arg Arg Trp Trp Cyb Arg Leu (SEQ ID N0 :12 )
Arg Arg Trp Trp Cys Arg Met (SEQ ID N0 :13 )
Arg Arg Trp Trp Cyb Arg Asn (SEQ ID N0 :14 )
Arg Arg Trp Trp Cyb Arg Pro (SEQ ID N0:15)
Arg Arg Trp Trp Cyb Arg Gln ~SEQ ID N0:16)
Arg Arg Trp Trp Cyb Arg Arg (SEQ ID N0:17)
Arg Arg Trp Trp Cyb Arg Ser (SEQ ID N0:18)
Arg Arg Trp Trp Cyb Arg Thr (SEQ ID N0:19)
Arg Arg Trp Trp Cyb Arg Val ( SEQ ID N0: 2 0 )
Arg Arg Trp Trp Cyb Arg Trp (S~Q ID N0:21)
Arg Arg Trp Trp Cyb Arg Tyr (SEQ ID N0:22)
The invention also contemplates the ube of peptideb
15 having the amino acid sequence Gln Ile Pro Arg Arg Ser
Trp Cyb Arg Phe Leu Phe (SEQ ID N0:52), either alone, or
more preferably, in combination with one or more of the
other peptides debcribed above. The successful ube of
thib dodecamer illubtrateb both the fact that longer
20 peptideb are buccessful and that certain biologicaily
functional equivalent peptides are active. All such
active equivalents therefore fall under the scope of the
present invention.
The compositions for use in the inhibitory methods
debcribed herein may contain only a single active
peptidyl species. Alternatively, they may contain more
than one peptide, up to and including about 40 or 45 or
so distinct peptideb. Any and all of the varioub
3 0 combinationb are contemplated, such as compositions
comprising 2, 3, 5, 10, 15, 20, 30 or 45 or so distinct
peptides .
Compobitionb compribing peptideb having the amino
acid sequence Arg Arg Trp Trp Cys Arg (SEQ ID N0:1)
and/or the amino acid sequence Arg Arg Trp Trp Cys Arg
Cyb (SEQ ID N0:4) are contemplated to be particularly
W095116N2 ` . ``' 2 1 7 ~ 9 2 7 ~ Y~ 245
useful, although the invention is not limited to these
peptides in any way. In this regard, it is important to
note that considerations other than in vitro activity,
such as plasma half life and stability, may be considered
5 in ultimately choosing peptides which are preferred for
clinical ~mho~l;r-ntR. The effects of different amino
acid substitutions on these parameters may be readily
determined and the results used to design the optimum
peptide or combination of peptides for use in vivo.
The RRWWCX (SEQ ID N0:23) sequence element is an
important feature of the peptides of this invention.
However, this does not exclude certain biological
functional equivalents from falling within the scope of
15 the invention. For example, the inventors have
discovered that the first tryptophan in RRWWCX (SEQ ID
N0:23) can be exchanged, e.g., by replacing with serine,
with only modest loss of activation. Therefore, one
example of equivalents ,onl- ~RRed by the inventio~ are
20 peptides of the sequence RRXWCX (SEQ ID NO:58) .
"Equivalent amino acids" may be defined as amino acids
whose hydrophilic or hydropathic index are within * 2;
more preferably, within ~ 1, and most preferably, within
: 0.5 of each other. Of course, to be a ~functional
25 equivalent", a peptide must still retain its intercrine
or IL-8 inhibitory activity, as may be easily determined
using assays such as those disclosed herein.
In addition to the peptidyl compounds described
30 herein, the inventors also contemplate that other =
sterically similar compounds, called peptidomimetics, may
be formulated to mimic the key portions of the peptide
structure. Such compounds may be used in the same manner
as the peptides of the invention and hence are also
35 functional equivalents. The generation of a structural
functional equivalent may be achieved by the techniques
of modelling and chemical design known to those of skill
Wo 95/16702 ~ 7 8 9 2 7 ~ IS
--19--
in the art. It will be understood that all such
sterically similar constructs fall within the scope of
the present invention.
The peptides and compositions for use in the
invention may be prepared by any one of a variety of
different methods. One preferred method for preparing
peptides in accordance with the present invention is
contemplated to be via automated peptide synthesis. A
synthetic peptide may be straightforwardly prepared using
an automated peptide synthesizer, the operation of which
will be generally known to those of skill in the art.
This method is one of those generally preferred for
preparing large quantities of a given peptide , e . g ., once
a particular peptide has been chosen for therapeutic use.
Another preferred method for preparing inhibitory
peptides, and the biological functional equivalents
thereof, is to use a combinatorial peptide library
method, as described by Houghten et al. (l991) and
disclosed in International Patent Application PCT
WO 92/09300, the entire disclosure of which is
specifically incorporated herein by reference. These
methods are particularly useful for preparing and
analyzing a plurality of peptides having a substantially
predetermined sequence, such as RRWWC, to which is
appended a variety of different amino acids at one or
more positions. These methods may be used to synthesize
a peptide mixture for direct use in the fQrmulation of a
composition in accordance with the invention or to
identify a particularly active peptide for subsequent
indivi dua l synthe s i s .
- If desired, peptides may also by prepared by
35 molecular biological means and the "recombinant" peptide
obtained from reco"~h; nilnt host cells which express the
peptide. o achieve this, one would prepare a specific
W09511670Z ~1 7~27
--20--
oligonucleotide, based upon the sequence of the desired
peptide, as is known to those of skill in the art, and
then insert the oligonucleotide into an expression
vector, such as any one of the many expression vectors
5 currently available commercially. One would then
transform a prokaryotic or eukaryotic host cell with the
vector, where it will direct the expression of the 80- .
called recombinant version of the peptide, which may then
be purified from the recombinant host cell This
lO methodology is standard practice in the art (see e_g.,
S ambrook e t al ., 1 9 8 9 ) .
BRIEF DES~.:K~ ~,. OF THE Dl?A~T~
The following drawings~ form part of the present
specification and are inrl~ 1 to further:demonstrate
certain aspects of the present invention. The invention
may be better understood by reference to one or more of
20 these drawings in combination with the detailed
description of specif ic l~mhorl i t c presented herein .
Flgure l. Binding Inhibition by Ac-RRWWCX ~SEQ ID NO:23)
Series. Twenty peptides which have structure of Ac-RRWWC
25 (SEQ ID NO:56) plus one of the 20 standard protein amino
acids in the sixth position were tested. The notation on
the x axis indicates the residue at the carboxy-terminal
position (SEQ ID I~O l and 24 through 42) . In this study,
neutrophils were incubated with l pM l25I-labeled IL-8
3 0 and 2 0 ~M of each peptides . ~
Figure 2. IL-8 Binding Inhibition by Ac-RRWWCR-NX2 (SEQ
ID NO:l) . Neutrophils (lx106) in 0.2ml of PBS c~nt~;ninr~
0.1% BSA were incubated with lnM l25I-labeled IL-8 and
35 increasing cnnrPntrations of Ac-RRWWCR-NH2 (SEQ ID NO:l)
for 90 min at 4C. These data are representative of four
studies .
~ O 95/16702 ~ 7 ~ 9 2 7 ~ Y~/1~5
-21 -
Figure 3A. Saturation Studies in the Presence of Ac-
RRWWCR-NH2 (SEQ ID NO:1), Binding Isotherms with Best Fit
Curve Calculated Using Lundon I. Binding assays were
performed in the absence of the peptide (O) and in the
5 presence o~ lO~LM (--), 20~LM (v), or 40/1M (-) peptide with
increasing nnnF~ntrations of 125I-labeled IL-8. Each
data point represent specific binding which was computed
by subtracting nonæpecif ic binding in the presence of
excess unlabeled IL-8 from total binding.
Figure 3B. Saturation Studies in the Presence of Ac-
RRWWCR-NH2 (SEQ ID NO:1), Scatchard Plots. Binding
assays were perf ormed in the absence of the peptide (O)
and in the presence of lO~M (--), 2011M (v), or 4011M (-)
15 peptide with increasing concentrations of 125I-labeled
IL-8 .
Figure 4. Binding Inhibition Studies in the Presence of
Ac-RRWWCR-NH2 (SEQ ID NO:1). Neutrophils were incubated
20 with lnM of 125I-labeled IL-8 and increasing
rAn~-~ntration of unlabeled IJ.-8 in the presence (--) or
the absence (O) of lOIlM Ac-RRWWCR-NH2 (SEQ ID NO:1) .
Figure 5. Effect of Ac-RRWWCR-NH2 (SEQ ID NO:1) on
25 Binding of IL-8, C5a, and Leukotriene B4 to Neutrophils.
The bindings assays were performed with lnM 125I-labeled
IL8 (--), 0.25nM 125I-labeled C5a (v), or 0.4nM 3H-labeled
leukotriene B4 ( ) and increased concentration of Ac-
RRWWCR-NH2 (SEQ ID NO:1) . Analysis of variance was used
30 for multiple comparison. When there was significant
difference, the differences between binding without the
- peptide and those with peptide were tested using Sheffés
test; *~, p~0.001.
35 Figure 6. Cytotoxicity Test. Chromium-labeled
neutrophils were ;n-llh~te-l with increasing ~nn~ntrations
of Ac-RRWWCR-NH2 (SEQ ID NO:1) in PBS containing 0.1~ BSA
WO 95/16702 ~ ~ ~ ; t ~ 2 1 7 8 9 2 7 P~~ Y~i~5
--22-
for 90 min at 4C ¦--) or in RPMI-1640 media cnnt~;nin~ 196
BSA for 30 min at 37C (O). Analysis of variance was
used for multiple comparison. When there was significant
difference, the differences between ~ lysis without the
5 peptide~and those with peptide were tested using Sheffés
test; ***, p~0.001~
Figure 7. The Effect of Ac-RRWWCR-NH2 (SEQ ID NO:1) on
Neutrophil Chemotaxis. Chemotaxis was performed in the
10 presence of increasing concentration of Ac-RRWWCR-NH2
(SEQ ID NO:1) in both upper and lower chamber. Stimulants
added to lower chamber were lOnM IL-8 (--), lOnM fMLP (--),
or media alone (O) as control. Analysis of variance was
used for multiple comparison. When there was significant
15 difference, the differences between distance migrated
without the peptide and those with peptide were tested
using Sheffés test; *, O.Ol~pc0.05, ***, p~0.001.
Figure 8. The Effect of Ac-RRWWCR-NH2 (SEQ ID NO:1) on
2 ,~-glucuronidase Release. Neutrophils pretreated with
cytochalasin B were incubated with lOOnM IL-8 (--), lOOnM
fMLP (--), lOOnM C5a (v) or lO~nM leukotriene B4 (-) or
without any st; m~ nt (O) in the presence of increasing
concentration of Ac-RRWWCR=NH2 (SEQ ID NO:1) for 30 min
25 at 37C. ~-glucuronidase activity of supernatants were
measured using phenolphthalein-glucuronic acid as
substrate. Analysis of variance was used for multiple
comparison. When there was significant difference, the
differences between distance migrated without the peptide
30 and those with peptide were tested using Sheffés test;
*, p~o.o5, **, p~0.01, ***, pcO.001.
Figure 9. Binding Inhibition by All D-amino Acid Ac-
rrwwcrx-NH2 (SEQ ID NO:2) Series. Ac-RRW~CR (SEQ ID
35 NO:1) was synthesi~ed using D-amino acids and added each
of the 20 standard protein D-amino acids were added at
the seventh position (SEQ ID NO:3 through 22) . The
WO95/16702 ~ ` 21 7~ 927 r~ Y~ 4s
.
--23--
notation on the x axis indicate i:he residue at the
carboxyterminal position. The binding 6tudy was
performed using 10 IlM of each peptide. Ac-RRWWCR-NH2
(SEQ ID NO:1) made with L-amino acids was used as a
5 control.
Figure 10. Ac-RRWWCX (SEQ ID NO:23) Inhibits Binding of
GRO and MIP2~ to human neutrophils. Radioiodinated MIP2,(~
and GRO/MGSA were mixed with various concentration of
10 Ac-RRWWCR-NH2 and incubated at room temperature for
15 minutes. Neutrophil suspension (1 X 106 cells in
160~1 PBS cr nt~;n;ng 0.1~ BSA) was added to 40~1 of the
mixture and incubated for 90 minutes on ice. The
radioactivity bound to the cells was separated from free
15 radioactivity by centrifugation through an oil layer.
The ~ bLnding inh;h;t;on was calculated as follows:
9~ binding inhibition = ~ 1 _ B NSP ¦ x 100
where B is bound radioactivity in the presence of the
25 peptiae, T is bound radioactivity in the absence of the
peptide, and NSP is bound radioactivity in the presence
of excess nonlabelled ligand.
3 0 D3T~TT Rn DES~:K~ N OF THE ~ ENBODINEN-TS
CXC Intercrine~, IL-8 Action~ and Inhibitory Peptide~
IL-8 has been identified as a neutrophil activating
molecule (Schroder et al., 1988; Peveri et al., 1988;
Yoshimura et al., 1987). It is produced mainly by
monocyte-macrophage and endothelial cell by the stimuli
such as bacterial lipopolysaccharide (LPS), tumor
necrosis factor or interleukin 1, and shares common
neutrophil activating properties with chemotactic
Wo 95/167~2 ~ i ; 2 ~ ~ 9~ Y~
--24--
agonists, such as fM~P, C5a or leukotriene B4 ~Baggiolini
et al., 1992) . I~-8 can stimulate chemotaxis of
neutrophils as well as enzyme relea3e and respiratory
burst. I~-8 is one member Df the family of peptide
5 molecules termed CXC intercrines, which all have the CXC
se~uence motif in their N-terminal region. The CXC
intercrines also include GR0, MIP2~ or MIP2~ and, more
recently, ENA78.
The functions of I~-8 ~are~mediated ~y IL-8 re~ceptors
on the neutrophil surf ace membrane . Recent studies
showed that I~-8 birlds to at least two distinct
receptors, whereas most of ~the other members of the
intercrine family, e.g., GR0 and MIP2~, bind to one of
15 the receptors with high aff~inity (Holmes et al., 1991;
Murphy & Tiffany, 1991; ~aRosa et a7., 1992; Cerretti et
al., 1993). These receptors are different from the
receptors for other chemotactic agonists (Dohlman et al.,
1987) .
II,-8 has been found in high concentrations in joint
fluids from patients with several kinds of joint disease
(Brennan et al. , 1990~, in ~pleural spaces of some
patients with pleural effusions (Miller & Idell, 1993),
25 and lung edema fluids from patients with the adult
respiratory distress ~ylldl~ (ARDS~ (Miller et al.,
1992) . Increased IL-8 levels have also been clearly
documented in various recent studies of patients with
cystic fibrosis (Richman-Eisenstat et al., 1993;
McElvaney et al., 1992; Nakamura et al., 1992; Bedard et
aL., 1993 ) .
I~-8 activates ~eutrophils and, although they are
powerful antimicrobial cells, neutrophils can also cause
35 considerable tissue damage through the release of certain
enzymes. IL-8 is therefore believed to be important in
pathogenesis of these and other infl; tory disorders.
WO 95116702 ~ 9 2 7 ~ Y.)~ 45
--25--
The inventors therefore hypothesized that modulation of
IL-8 function would be a good strategy to control various
diseases and pathological conditions, particularly ARDS
and cystic f ibrosis .
Recently, results from various studies attempting to
modify IL-8 function have been reported. These include
studies showing some success in inhibiting II.-8 synthesis
(Standiford et al., 1992; Lam et al., l990) . Also, anti-
10 human IL-8 has been reported to ameliorate lung
inflammation in rats suffering from an immunologic injury
(Mulligan et al., 1993) . IL-8 secretion in airways of
patients with cystic fibrosis was reportedly reduced with
the secretory leukoprotease inhibitor of neutrophil
elastase (McElvaney et al., 1992). In addition, IL-8
production has been shown to be suppressed in LPS-
stimulated whole human blood by oxygen radical scavengers
(DeForge et al., 1992) . Finally, two cytokines,
interferon gamma (Cassatella et al., 1993a; 1993b) and
20 interleukin 4 (Standiford et al., l990) inhibited the
synthesis of IL-8. However, such work has yet to yield a
particularly effective IL-8 inhibitor, or an inhibitor
capable of preferentially inhibiting certain neutrophil
responses over others.
Studies have also been conducted on the interaction
of IL- 8 with its receptors . This has led to the
identification of certain peptide inhibitors with
structures corresponding to portions of the IL-8
30 molecule. For example, Gayle and colleagues found that
the 44 amino acids at the amino-t~rm;n~l end of the
- rabbit IL- 8 receptor was a moderately good inhibitor of
IL-8 binding and function (Gayle et al., 1993) . The
- present inventors have shown that synthetic peptides, and
35 particularly, IL-8 amino t~ n~l peptides, inhibit IL-8
binding to neutrophils and neutrophil chemotaxis (Miller
et al., 1990; Miller et al.. 1993). An N-ter~Linal
2 1 78927
WO 9iS116702 ~ U~Y~ W5
--26--
pentapeptide I~-8 inhikitor has also been reported
(Goodman et al., l99l) .
Nonetheless, effective peptide inhibitors have not
5 yet been developed from a knowledge of the structure of
the IL-8 molecule. Therefore, in pursuing this aim, the
present inventors assayed various other peptide
compositions for I~-8 inhikitory activity by screening a
library of 400 groups of hexapeptides. In these
lO screening a3says, l2~I-labeled interleukin-8 (lO-l2 M)
was mixed with lO0 ~lM peptide, then added to neutrophils
and incukated at 4OC for 90 min. The bound radioactivity
was separated from unbound by centrifugation through a
dense cushion of a mixture of paraffin and silicon oils
15 and the l25I bound to neutrophils was counted in a gamma
radiation spectrometer, allowing the results to be
expressed as the percent of II~- 8 binding which was
inhibited .
In these studies, hexamerG of the sequence RRWWCX
(SEQ ID NO:23), where the t~rm;niql position may be any
amino acid, were found to be effective I~-8 inhibitors.
Although certain RRWWCX-type peptides had previously been
found to exhibit ar~ti-Staphylococcal properties (Houghten
et al., l99l), no other functional properties have been
reported which would suggest these peptides to have
either anti-cytokine or anti-;nfli tory activities.
The present inventors also showed that RRWWCR ef fectively
inhibits other CXC intercrines, such as GRO and MIP2, as
evidenced by reducing GRO and MIP2~ binding to human
neutrophils .
Although all RRWWCX (SEQ ID ~o: 23 ) series peptides
have anti-I~-8 activity (Tables lA and lB)V the peptide
RRWWCR (SEQ ID NO:l) was found to be particularly
effective. A form of this peptide with an amino-terminus
acetyl group and a carboxy-terminus amino group
Wo95116702 ~ ` 21 7892~ J~l~u~Yall~15
--27--
(AC-RRWWCR-NH2; SEQ ID NO:1), thus modified in order to
resemble peptides present among longer sequence of
protein, became one of the focal points of these studies.
AC-RRWWCR-NH2 (SEQ ID NO:1) was found to inhibit the
specific binding of 125I-labeled IL-8 to neutrophils with
an apparent KI of approximately 10 IlM, and to be almost
twice as effective as the non-acetylated form of the same
peptide. A preci3e KI value could not be obtained due to
the presence of positive cooperativity. The binding
isotherm of IL-8 in the absence of the peptide fitted
one-site model best, when it was analyzed using the
computer program Lundon I. One possible explanation of
the lower IL-8 concentration data in the Scatchard plots
is that cooperativity masked the high affinity bindin~ at
low IL-8 concentrations. Recent studies, however, have
shown that IL- 8 bound to two distinct classes of IL- 8
receptors with almost similar affinity (Lee et al., 1982;
Srh~ rhf~r et al., 1992) . Therefore, it is more likely
that the estimated Bmax for this binding site as one-site
model represents the total Bmax of two class of receptors
and that the estimated Kd is common for these receptors.
The bindin~ isotherms in the presence of the peptide
fit two-site model best. The analysis of binding
isotherms in the presence of Ac-RRWWCR-NH2 showed that
thiE~ peptide suppressed the binding of IL-8 to two
classes of receptors dif f erently . The estimated values
of binding parameters showed that af f inity of one class
of receptors was suppressed by lO~M peptide, which
suggested competitive inhibition. Higher r~mr~on~ration
of peptide is needed to inhibit the other class of
receptor non-competitively.
-
The activity of the present inhibitory peptides is
specif ic f or IL- 8 . Ac-RRWWCR-NH2 does not inhibit the
binding of C5a or leukotriene B4 to neutrophils,
WO 95/16702 i .~ 2 ~ 7 ~ 9 2 7 ~ Ya/1~4S
-28 -
chemotaxis induced by formyl-L-Met-L-Leu-L-Phe (fMhP), or
~-glucuronidase release induced by fMLP, C5a or
leukotriene B4. It also has no intrinsic ability to
cause neutrophil chemotaxis or enzyme release. These
5 observations suggest that peptides such as Ac-RRWWCR-NH2
can strongly inhibit human neutrophil activations induced
by IL- 8 as a result of modulation of its receptor
binding .
Fur~h, ~, hexamer and heptamer peptides based
upon the Ac-RRWWCR-NX2 structure are contemplated to
preferentially inhibit enzyme release over chemotaxis.
Indeed, Ac-RRWWCR-NH2 is herein shown to significantly
suppress neutrophil chemotaxis induced by lOnM IL-8 at a
15 concentration of 50 ~lM and ~-glucuronidase release at
2 llM, even though 10 times more IL- 8 is re~uired tQ cause
enzyme release. This preferential inhibition of enzyme
release at lower conc~nt~;nnq than chemotaxis is a
particularly important and surprising discovery that
20 makes these types of peptides ideal for the treatment of
lung injury in patients with the adult respiratory
distress syndrome.
The above f indings also suggest that distinct
25 neutrophil functions may reS~uire II,-8 binding to
different classes of receptors. Although not important
to the utility of the invention per se, this also makes
the present peptides suitable for use as investigational
tools to further elucidate IL-8 receptor and neutrophil
30 functions.
The inventors next examined the inhibitory activity
of a second set of peptides which contained Ac-rrwwcrx-
NH2 (SEQ ID NO:2), with all D-amino acids. Again, all
35 RRWWCRX (SEQ ID NO:2) series peptides were found to have
anti-IL-8 activity (Tables 5A and 53) . However, the
peptide Ac-rrwwcrc-N~I2 (SEQ ID NO:4) was found to be the
WOg5/16702 ~ ` 2 1 78 927 1 ~IJ~,~Y~i~4S
--29 -
best inhibitor, being almost four times as potent an
inhibitor as Ac-rrwwcr-NH2 (SEQ ID NO:1). This
observation is potentially of great signif icance as
l; ~n proteages cannot degrade D-amino acid peptides
5 and proteins (Togo et al., 1989) . Therefore, D-amino
acid peptide inhibitors are expected to have a longer
half life in vivo.
Various other synthetic peptides were also tested
lO for their ability to inhibit IL-8 binding to neutrophils
in the standard assay. These peptides were either
homologues of the amino - terminal end of IL- 8, or were
segments of proteins found in the protein data bases (PIR
or Swiss-Prot) which had five of the six residues in
15 RRWWCR (SEQ ID NO:2). The latter peptides were
identif ied by searching the PIR and Swiss-Prot databases
for RRWWCR (SEQ ID NO:1), using the IGSUITE program to
search the databases present on the CR~Y computer at the
Center for High Performance Computing in Austin Texas.
20 None of the proteins in the data bases ,-~ntA;npd all si~
of the amino acids.
Previous studies have shown that the residues Glu,
Leu and Arg at the 4, 5, and 6 positions of the 72 amino
25 acid of IL-8 are critical for the binding to neutrophils
(Clore et al. , 1992; Clark-Lewis et al. , 1991), and that
the amino terminal peptide of IL-8 inhibits IL-8 binding
to neutrophils and chemotaxi6 ~liller et al., 1993). The
inventors therefore ~Y~m; n.~d the IL-8 homologues
30 Ac-RELRCQ-NH2 (SEQ ID NO:54) and ELRCQCIRTY (SEQ ID
NO:49, including the C-X-C motif characteristic of
intercrine peptides), along with its two non-Cys-
cr nt~;n;n~ analogues, ELRSQSKTY (SEQ ID NO:50) and
- ELRMQM};TY (SEQ ID NO:51). None of these peptides had the
35 ability to inhibit IL-8 binding to neutrophils.
WO95/16702 ~ , 2 1 7~ 92~ ,V~Y~I~4~ ~
--30 -
The inventors next searched the protein databases to
determine if RRWWCR (SEQ ID NO:l~ might occur in other
peptides which might have relevant physiologic functions
in relation to IL- 8 . Two peptides were identif ied and
5 chosen for investigation because they ~nntA;n~ five of
the six residues in RRWWCR (SEQ ID NO:1) and were ~
cnntA;n~-l in known proteins. They were ~iw~Rww~ AVLY (SEQ
ID NO:53) and QIP~S~FLF ~SEQ ID NO:52) . The former
peptide is contained in "cell surface glycoprotein :CDllc
10 precursor - human leukocyte adhesion receptor pl50, 95
alpha chain" (Corbi et al., 1990; A~c~As;nn number,
A36534\A35543\S00864) and the latter is 3' ,5' -cyclic GMP
phosphodiesterase beta chain - bovine (Ovnh;nn;knv et
al , 1987; Accession Number, S00251~.
Although one of these homologues, namely
QIPRRSWCRFLF (SEQ ID NO:52), inhibited IL-8 binding to
neutrophils by about 609~, this activity is less than
Ac-RRWWCR-NH2 (SEQ ID NO:1) . It is not thought likely
20 that the protei~ from which this sequence was extracted,
bovine retinal 3'5'-cyclic GMP phosphodiesterase, has a
physiological role in IL-8 function, but this cannot be
totally ruled out. The most important data from this
study suggest that the first tryptophan in RRWWCR (SEQ ID
25 NO:l) can be modified with only modest loss of
activation. The lack of activity of the other peptide,
~ww~:~AVLY (SEQ ID NO:53), suggests that ch_nging the
last Arg in RRWWCR (SEQ ID NO:1) to Asp significantly
reduces its ability to inhibit IL-8 binding to
3 0 neutrophils, as supported by the observation that
Ac-rrwwcd (SEQ ID NO:26) is only m;n;r-l ly active.
Thus, the examples set forth herei~ detail the
identiiication of a new series of peptide inhibitors
35 capable o~ inhibiting IL-8 binding to neutrophils and
neutrophil activation. The peptide inhibitors of the
RRWWCX (SEQ ID NO:23) and RRWWCRX (SEQ ID NO:2) types
_ _ _ _ , , . .. . .. . .. -- -- --
Wo95116702 ` 2 1 78 927 1~,1/LI~Y3/1 ~45
have the distinct advantage over previously described
inhibitors that they are only six or seven residues long
and that the D-amino acid analogues are also active. It
is contemplated that this class of inhibitors will be
5 useful in the therapy of di3eases caused by the
unre~3trained action Ih- 8, such as the adult respiratory
distress syndrome. These inhibitors have the distinct
advantage that they will likely permit neutrophils to
enter the lungs, via chemotaxis which is not readily
lO inhibited, but that they will then preferentially prevent
the detrimental enzyme release (McGuire et al., lg82) .
In addition to their many and varied therapeutic
uses, the peptides and compositions of the present
15 invention have utility in other embodiments. The3e
include, for example, their use in vari~us bioassays,
e.g., as positive controls in assays of II-8 inhibitors
or neutrophil inhibitors; uses in further ~1P1 ;nP~ting
I~-8 receptor interactions and functionsi generating
20 antibodies, and the like.
R;olo~ics~l Functional Equivalentfi
Certain biological functional equivalents of the
25 RRWWCXX-type peptides are contemplated within the scope
of this invention. The concept of biologically
functional equivalent amino acids is well known to those
of skill in the art, and iR embodied in the knowledge
that modifications and changes may be made in the
30 structure of a protein or peptide and still obtain a
molecule having like or otherwise desirable
- characteristics.
- However, it is also well understood by skilled
35 artisans that, inherent in the definition of a
biologically functional equivalent protein or peptide, is
the concept that there is a limit to the number of
.
Wo 9~116702 ~ ~ 2 ~ 7 8 9 2 7 A ~ Y~/1~45
-32--
changes that may be made within a def ined portion of the
molecule and still result in a molecule with an
acceptable level of equivalent biological activity and
that key active site or structurally vital residues
5 cannot be exchanged. Biologically functional equivalent
peptides are therefore defined herein as those peptides
in which certain, not most or all, of the amino acids may
be substituted. In particular, where hexamer or heptamer
peptides are concerned, it is contemplated that only
lO about two, or more preferably, a single amino acid change
would be made within a given peptide. Of course, a
plurality of distinct peptides with different
substitutions may easily be made and used in accordance
with the invention.
In regard to changing a limited number of residues
within a peptide, it is known that certain amino aclds
may be substituted for other amino acids without
appreciable loss of~ function, as may be measured by the
2 0 interactive binding capacity f or structures such as
receptors and cells, or the ability to compete with other
molecules for binding to these sites. Since it is the
interactive and competitive capacity of a protein or
peptide that defines its biological functional activity,
25 certain amino acid substitutions can be made in a I?eptide
sequence (or, of course, its underlying DNA coding
sequence) and nevertheless obtain a peptide with like, or
even improved properties.
Amino acid substitutions are generally based on the
relative similarity of the amino acid side-chain
substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. An analysis
of the size, shape and type of the amino acid side-chain
substituents reveals that arginine, lysine and histidine
are all positively charged residues; that alanine,
glycine and serine are all a similar size; and that
wo95/16702 ` '~ S 2 ~ 78927 r~ Y3/l~4s
phenyl ;31 ~n;ns, tryptophan and tyrosine all have a
generally similar shape. Therefore, based upon these
conEiderations, arginine, lysine and histidine; alanine,
glycine and serine; and phenylalanine, tryptophan and
5 tyrosine; are defined herein as biologically functional
equivalents .
To effect more quantitative changes, the hydropathic
index of amino acids may be considered. Each amino acid
10 has been assigned a hydropathic index on the basis of
their hydrophobicity and charge characteristics, these
are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenyli3lAn;n~ (+2.8); cysteine/cystine (+2.5); methionine
( + 1 . 9 ); al anine ( + 1 . 8 ); glycine ( - 0 . 4 ); threonine ( - 0 . 7 );
serine (-0.8); tryptophan ~-0.9); tyrosine (-1.3);
proline ( -1 . 6); histidine (-3 . 2); glutamate (glutamic
acid) (-3.5); glutamine (-3.5); aspartate (aspartic acid)
( -3 . 5); asparagine (-3 . 5); lysine ( -3 . 9); and arginine
(-4 .5) .
The importance of the hydropathic amino acid index
in conferring interactive biological function on a
protein is generally understood in the art (Kyte &
Doolittle, 1982, incorporated herein by reference). It
25 is known that certain amino acids may be substituted for
other amino acids having a similar hydropathic index or
score and still retain a similar biological activity. In
making changes based upon the hydropathic index, the
substitution of amino acids whose hydropathic indices are
30 within +2 is preferred, those which are within $1 are
particularly preferred, and those within $0 . 5 are even
- more particularly preferred.
- Substitution of like amino acids can also be made on
3~ the basis of hydrophilicity, as disclosed in U.S. Patent
4,554,101, incorporated herein by reference. In U.S
Patent 4,554,101, the following hydrophilicity values are
WO 95/16702 '~ 2 1 7 8 9 2 7 , ~ Y~ ~S
--34--
assigned to amino acid residues: arginine (+3 . 0); lysine
(+3 . 0); aspartate (aspartic acid) (+3 . 0 _ 1); glutamate
(glutamic acid) (+3.0 _ 1); serine (+0.3); asparagine
(+0.2); glutamine (+0.2); glycine (0); proline (-0.5
+ 1); threonine (-0.4); alanine (-0.5); histidine (-0.5);
eysteine (-1.0); methinnin~ (-1.3); valine 1-1.5);
leucine (-1.8); isoleueine (-1.8) j tyrosine (-2.3);
phenyl a l an i ne ( - 2 . 5 ); tryp tophan ( - 3 . 4 ) .
It is well understood ~that an amino acid can be
substituted for another having a similar hydrophilicity
value and ætill obtain a biologically functional
equivalent protein or peptide. In making changeæ baæed
upon similar hydrophilicity values, the substitution of
amino acids whose hydrophilieity values are within _2 is
preferred, those whieh are=within _l are particularly
preferred, and those within _0.5 are even more
particularly preferred
20 Pl~rr~ eutiCal Formulation~
.
The peptides and eompositions of the invention may
be used for treating a yariety of diseases and disorders
in whieh CXC intererines, sueh as IL-8, or neutrophils
25 are involved or in whieh there i5 an inappropriate or
inereased inf lammatory response . The invention is
partieularly suitable for the treatment of lung
inflammation sueh as that eonneeted with asthma,
bronehitis, eystic f ibrosis and ARDS . To treat disorders
30 such as ARDS and cystic fibrosis, parenteral
administration, such as intravenous, intramuscular or
subcutaneous inj ection i5 contemplated to be the most
preferred route, although one may also use aerosols or
inl~ ntf~ Sprays, aerosols and inhalants, are only
35 effeetive if the droplets are sufficiently fine and
uniform in size so that the mist reaches the bronchioles.
Particle size iæ of major importance in the
WO 95/16702 ~ 2 1 7 8 9 2 7 r~ Y~i~5
-35 ~
administration of therapeutic agents via aerosols or
;n~ ntq. The optimum particle size for penetration
into the pulmonary cavity is of the order of 0 . 5 to 7 ILm.
As fine mists are produced by pressurized aerosols, their
5 use is considered advantageous. Such treatment
strategies and therapeutic formulations are described in
detail hereinbelow in Example VIII.
As the invention may be employed to treat
10 inflammation in various clinical settings, many types of
pharmaceutical peptide formulations are contemplated.
Therapeutic or pharmacological compositions of the
present invention, whether for pulmonary or other
treatments, will generally comprise~ an effective amount
15 of a relatively small intercrine- or IL-8-inhibiting
peptide or peptides, dissolved or dispersed in a
pharmaceutically acceptable medium. The phrase
~pharmaceutically acceptable~ refers to molecular
entities and compositions that do not produce an
20 allergic, tcxic, or otherwise adverse reaction when
administered to a human. Pharmaceutically acceptable
media or carriers include any and all solvents,
dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the
25 like. The use of such media and agents for
pharmaceutical active substances is well known in the
art . Except insof ar as any conventional media or agent
is incompatible with the active ingredient, its use in
the therapeutic compositions is contemplated.
Supplementary active ingredients can also be
- incorporated i~to the therapeutic compositions of the
present invention. ~or example, the intercrine-, IL-8-
- and neutrophil-inhibiting peptides may also be combined
35 with other agents such as IL-8-derived N-terminal
peptides, IFN-~, oxygen radical scavengers and the like,
to create peptide cocktails for treatment.
Wo 95/16702 ; ~ 2 l 7 ~ 9 2 7 ~~ Y3ll~45
-36-
The preparation of ~pharmaceutical or pharmacological
compositions cnnt~;n;n~ an intercrine-, I~-8- and
neutrophil-inhibiting peptide or peptides, including
dextrorotatory peptides, as an active ingredients will be
5 known to those of skill in the art in light of the
present disclosure. If desired, such compositions may be
prepared as injectables, either as liquid solutions or
suspensions; solid forms suitable for solution in, or
suspension in, liquid prior~ to injection; as tablets or
10 other solids for oral administration; as time release
capsules; or in any other form currently used, including
cremes, lotions and mouthwashes, and the inhalents and
aerosols of Example VIII
Solutions of the active peptides and compounds as
free base or pharmacologically acceptable salts can be
prepared in water suitably mixed with a surfactant, such
as hydroxypropylcellulose. ~ Dispersions can also be
prepared in glycerol, liquid polyethylene glycols, and
20 mixtures thereof and in oils. IJnder ordinary conditions
of storage and use, these preparations contain a
pre6ervative to prevent the growth of microorganisms.
Sterile solutions suitable for injection are
25 contemplated to be useful ~ in treating various diseases
and may be administered into the blood stream or into the
precise site of the inf lammation . The pharmaceutical
forms suitable for injectable use include sterile aqueous
solutions or dispersions and sterile powders for the
30 extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be
sterile and must be fluid to the extent that easy
syringability exists. It must be stable under the
conditions of manufacture and storage and must be
35 preserved against the rnnt~mi n~ting action of
microorganisms, such as bacteria and fungi.
0 95/16702 2 1 7 8 ~ 2 7 A ~ y
~W
--37--
A peptide or peptides can be formulated into a
composition in a neutral or salt form. Pharmaceutically
acceptable salts, include the acid addition salts (formed
with the free amino groups of the peptide) and which are
formed with inorganic acids such as, for example,
hydrochloric or ~hn~phnric acids, or such organic acids
as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups can also be
derived from inorganic bases such as, for example,
10 sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine, and the like.
The carrier can also be a solvent or disper~ion
15 medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid
polyethylene glycol , and the like), suitable mixtures
thereof, and vegetable oils. The proper fluidity can be
maintained, for example, by the use of a coating, such a~
20 lecithin, by the m-~i nt.on~n~ of the required particle
size in the case of dispersion and by the use of
surfactants. The prevention of the action of
microorganisms can be brought about by various
antibacterial ad antifungal agents, for example,
25 parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to
include isotonic agents, for example, sugars or sodium
chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the
30 compositions of agents delaying absorption, for example,
aluminum monostearate and gelatin.
Sterile inj ectable solutions are prepared by
incorporating the active compounds in the required amount
35 in the ~LU~r iate solvent with various of the other
ingredients enumerated above, as required, followed by
filtered sterilization. Generally, dispersions are
. ;,, ~ 2 1 78~27
Wo s5/l6702 ~ 9~ 45
. ., --
--38-
prepared by incorporating the various sterilized active
ingredients into a sterile vehicle which contains the
basic dispersion medium and the required other
ingredients from those enumerated above. In the case of
5 sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are-
vacuum-drying and freeze-drying techni~ues which yield a
powder of the active ingredient plus any additional
desired ingredient from a previously sterile-filtered
10 601ution thereof.
The preparation of more, or highly, concentrated
solutions for intramuscular injection is also
contemplated. In this regard, the use of DMS0 as solvent
15 is preferred as this will result in extremely rapid
penetration, delivering high concentrations of the active
peptide, peptides or agents to a small area.
The formulation of peptides for topical use, such as
20 in creams, Qintments and gels iS also contemplated. The
preparation of oleaginous or water-soluble ointment bases
is also well known to those~in the art. For example,
these compositions may include vegetable oils, animal
fats, and more preferably, semisolid hydrocarbons
25 obtained from petroleum. Particular components used may
include white ointment, yellow ointment, cetyl esters
wax, oleic acid, olive oil, paraffin, petrolatum, white
petrolatum, spermaceti, starch glycerite, white wax,
yellow wax, lanolin, anhydrous lanolin and glyceryl
30 monostearate. ~7arious water-soluble ointment bases may
also be used, including glycol ethers and derivative~,
polyethylene glycols, polyoxyl 40 stearate and
polysorbate~. Even delivery through the skin may be
employed if desired, e.g., by using transdermal patches,
35 iontophoresis or electrotransport.
~ ogs/l6702 2 ~ ~927 1~1~V~Y~/I~5
Buffered orllt~lm;c solutionb aiso fall within the
scope of the invention. These may be used in connection
with patients suffering from disorders connected with
increased retinal angiogenesis. The buffering is
5 nPcf~g~y due to pH changes the peptide may cause.
Orl~tl~l mi c preparations may be created in accordance with
conventional pharmaceutical practice, see for example
"Remington' 8 Pharmaceutical Sciences" 15th Edition, pages
1488 to 1501 (Mack Publishing Co., Easton, PA) .
Suitable orht~l m; c preparations will generally
contain a novel dipeptide, peptide or agent as disclosed
herein in a concentration from 'about 0 . 01 to about 19~ by
weight, and preferably from about 0.05 to about 0.5%, in
15 a pharmaceutically acceptable solutio~, suspension or
ointment. The ophthalmic preparation will preferably be
in the form of a sterile buffered solution containing, if
desired, additional ingredients, for example
preservatives, buffers, tonicity agents, antioxidants and
20 stabilizers, nonanoic wetting or clarifying agents,
viscosity-increasing agents and the like
Suitable preservatives for use in such a solution
include b~on7~lk~n;um chloride, benzethonium chloride,
25 chlorobutanol, thimerosal and the like. Suitable buffers
include boric acid, sodium and potassium bicarbonate,
sodium and potassium borates, sodium and potassium
carbonate, sodium acetate, sodium biphosphate and the
like, in amounts sufficient to n~;nt~;n the pH at between
30 about pH 6 and pH 8, and preferably, between about pH 7
and pH 7 . 5 . Suitable tonicity agents are dextran 40,
- dextran 70, dextrose, glycerin, potassium chloride,
propylene glycol, sodium chloride, and the like, such
- that the sodium chloride equivalent of the ophthalmic
35 solution is in the range 0.9 plus or minus 0.2%.
Suitable antioxidants and stabilizers include sodium
bisulfite, sodium metablsulfite, sodium thiosulfate,
~ 1 78927
W095/16702 ~ r~ Y~ 15
--40-
thiourea and the like. Suitable wetting and clarifying
agents include polysorbate 80, polysorbate 20, poloxamer
282 and tyloxapol. Suitable viscosity-increasing agents
include dextran 40, dextran 70, gelatin, glycerin,
5 llydL~y~thylcellulose~ hydroxmethylpropylcellulose,
lanolin, methylcellulose, petrolatum, polyethylene
glycol, polyvinyl alcohol, polyvinylpyrrolidone,
carboxymethylcellulose and the like.
Upon formulation, therapeutics will be administered
in a manner compatible with the dosage f~ t;on, and
in such amount as is pharmacologically effective. The
formulations are easily administered in a variety of
dosage forms, such as the type of in~ectable solutions
15 described above, but drug release capsules and the like
can also be employed.
A minimal volume of a composition required to
disperse the peptide is typically utilized. Suitable
20 regimes for administration are also variable, but would
be typified by initially administering the compound and
monitoring the results and then giving further controlled
doses at further intervals. For example, for parenteral
administration, a suitably buffered, and if necessary,
25 isotonic aqueous solution would be prepared and used for
intravenous, intramuscular, subcutaneous or even
intraperitoneal administration. One dosage could be
dissolved in 1 mL of isotonic NaCl solution and either
added to lOOOmL of hypodermoclysis fluid or injected at
30 the proposed site of infusion, (see for example,
"Remington' 8 Pharmaceutical Sclences" 15th Edition, pages
1035-1038 and 1570-1580).
In certain embodiments, active compounds may be
35 administered orally. This is contemplated for agents
which are generally resistant, or have been rendered
resistant, to proteolysis by digestive enzymes. Such
.. . . . . .. . . . .. . .. _ .. _ _ . .... _ _ _ ....
f,
~o 9sll6702 2 t 7 8 9 2 7 PCT/17S93/~224S
--41--
compounds are contemplated to include dextrorotatory
peptides; chemically designed or modified agents; and
peptide and lipo60mal formulations in time release
capsules to avoid peptidase and lipase degradation.
oral formulations may include compounds in
eombination with an inert diluent or an assimilable
edible carrier; those enclosed in hard or soft shell
gelatin capsules; those compressed into tablets; or those
10 incorporated directly with the food of the diet. For
oral therapeutic administration, the active compounds may
be incorporated with excipients and used in the form of
ingestible tablets, buccal tables, troches, capsules,
elixirs, suspensions, syrups, wafers, and the like. Such
15 compositions and preparations should generally contain at
least 0.19~ of active compound. The percentage of the
compositions and preparations may, of course, be varied
and may conveniently be between about 2 to about 60~ of
the weight of the unit. The amount of active compounds
20 in such therapeutically useful compositions is such that
a suitable dosage will be obtained.
Ta}alets, troches, pills, capsules and the like may
also contain the following: a binder, as gum tragacanth,
25 acacia, cornstarch, or gelatin; excipients, such as
dicalcium phosphate; a disintegrating agent, such as corn
starch, potato starch, alginic acid and the like; a
lubricant, such as magnesium stearate; and a sweetening
agent, such as sucrose, lactose or saccharin may be added
3 o or a f lavoring agent, such as peppermint, oil of
wintergreen, or cherry flavoring. When the dosage unit
form is a capsule, it may contain, in addition to
materials of- the above type, a li~uid carrier. Various
- other materials may be present as coatings or to
35 otherwise modify the physical form of the dosage unit.
For instance, tablets, pills, or capsules may be coatèd
with shellac, sugar or both. A syrup of elixir may
2 1 ~927
Wo 95116702 ~ Y~ 45
--42--
contain the active ~ sucrose as a SwoP~nirl~
agent methyl and propylparab~ens as preservatives, a dye
and flavoring, such as cherry or orange flavor. Of
course, any material used in preparing any dosage unit
5 form should be pharmaceutically pure and substantially
non-toxic in the amounts emE~loyed. In addition, the
active compounds may be incorporated into sustained-
release preparation and formulations.
The following examples=are included to demonstrate
preferred embodiments of the invention. It should be
appreciated by those of skill in the art that the
techniques disclosed in the examples which follow
15 represent techniques discovered by the inventor to ~
function well in the practice of the invention, and thus
can be considered to constitute preferred modes for its
practice. However, those of skill in the art should, in
light of the present disclosure, appreciate that many
20 changes can be made in the specific embodiments which are
disclosed and still obtain a like or similar result
without departing from the spirit and scope of the
invention.
EXANPLE I
HEXAMER PEPTIDE LN~L~l~J~S OF TT-8
A series of studies was f irst carried out to
30 determine whether hexamer peptides of the seS~uence RRWWCX
(SEQ ID NO:23), where X may be any amino acid, would act
as inhibitors of II,-8. The=assays of the initial screen
are based upon determining the ability of a given peptide
to inhibit the binding of IL-8 to human neutrophils.
wossJ~6702 ' 2 1 78 927 ~ Y~ 4$
A. PreParation o~ ~uman Neutrophils
The use of human subjects for acquisition of
neutrophils was approved by the Institutional Review
5 Board f or human experimentation . For the preparation of
neutrophilæ, human blood was anticoagulated with heparin
for enzyme release studies and with 0 . 3396 30dium citrate
for other studies. For chemotactic and enzyme releaEe
studies neutrophils were 6eparated by dextran
10 sedimentation and erythrocyte lysis by the method of
Boyum (Kohler & Milstein, 1975). For binding studies and
cytotoxic studies, the neutrophils were further purified
in gradients of Percoll (Pharmacia Fine Chemicals,
Piscataway, NJ) to a purity of 90-9396 (Kohler & Milstein,
1975).
B . IL- 8 Bindincr AssaYs
Recombinant human IL-8 (72 amino acids; Pepro Tech
20 Inc., Rocky Hill, NJ) was radioactively labeled with 125I
` ' by the chloramine T method of Xunter and Greenwood
(Hunter & Greenwood, 1962) . Binding studies were
performed according to Besemer et al. (1989), as follows:
Neutrophils in phosphate buffered saline pH 7.4 (PBS)
25 c~)nt~;n;ng 0.1% bovine serum albumin (BSA) were incubated
with labeled ligand in the presence or absence of the
peptide being tested for 90 min at 4C to reach
equilibrium and then centrifuged at 12, 000 x g for 3 mln
in Beckman B microfuge (Beckman Instruments, Fullerton,
3 0 CA), through a cushion consisting of a mixture of
paraffin oil (Fisher Scientific, Fair ~awn, NJ) and
- silicon oil (Serva Co., N.Y., NY). The pellet and
supernatant were then counted in a gamma radiation
- spectrometer. The non-specific binding was estimated to
35 measure the binding in the presence of 100-fold excess of
non-labeled ligand. The binding constants were
WO 95/1 6702 ~ 8 9 ~ 7 ~ Y~ 45
--44--
calculated using the computer programs I,undon I and
Lundon I I .
C. Pe~tide~a ~
The RRWWCR-type peptides were synthesized by
Houghten Pharmaceutical Company in San Diego using tBOC
for protection of the ~-amino group (Stewart & Young,
1969). All synthetic peptides were purified on high
lO performance liquid chromatography (HP~C) using a
preparative C18 reverse phase column (Waters Co., New
Bedford, MA) . Peptides were eluted using a gradient from
0.1~6 trifluoroacetic acid (TFA) to 80g6 acetonitrile in
O .19~ TFA. The composition of the peptides was conf irmed
15 by amino acid analysis and sequencing.
D. Stati~tic~
In this and all of the following examples, the data
20 are expressed as the mean and the variation as the
standard deviation (S.D.). Significance was determined
by the Shef f és test when the variances were e~ual and the
pOplll A~ n~ were normaily distributed and only 2 groups
were compared. Multiple comparisons were made using the
25 analysis of variance and Sheffe' 8 test.
E. Re~ult~
In these studies, a screening for inhibition was
30 carried out at 100 IlM concentration of the peptide being
tested, with a 10 12 M concentration of I~-8. In the
first series of studies, twenty peptides were synthesized
with the carboxyl-terminal residue of RRWWCX (SEQ ID
NO: 23 ) being changed to each of the standard protein
35 amino acids in turn. In the ~irst set of studies,
several of the peptides totally inhibited IL-8 binding,
as shown in Table lA a~d Table 1~. The information
WO95116702 2 1 7 8 ~ 2 7 P~ Y.~ IS
--45--
presented in Table lA and Table lB is the same data, with
Table lA being li6ted in order~of 96 inhibition and
Table lB being listed in order of SEQ ID N0, to enable
straightforward comparisons. It can be clearly seen that
5 all hexamer peptides other than RRWWCD (SEQ ID N0:26);
RRWWCE (SEQ ID N0:27); RRWWCN (SEQ ID N0:35) and RRWWCQ
(SEQ ID N0:37), have very significant inhibitory
activity. It should also be noted that even though the
preliminary data in Table 1 shows RRWWCN (SEQ ID NO:35)
lO not to have inhibitory activity, subsequent studies
showed this peptide did indeed exhibit certain inhibitory
properties (Pigure l).
2 t 789~7
WO 95/16702 ~ Ya/l~45
--46--
TA3I.E lA
PEPTIDE SEQ ID N~: % IN~IIB.
RRWWCR 1 112 . 2
RRWWCK 32 110 . 3
RRWWCT 3 9 8 8 . 4
RRWWCP 3 6 8 8 . 3
RRWWCH 3 0 87 . 9
RRWWC~ 3 3 8 6 . 9
RRWWCG 29 86 . 8
RRWWCV 4 0 81.1
RRWWCF 2 8 8 0 . 2
RRWWCS 3 8 7 9 . 3
RRWWCY 42 77 . 9
RRWWCC 25 75.1
RRWWCM 34 69 . 0
RRWWCW 41 6 6 . 4
RRWWCA 24 51. 9
RRWWCI 31 45.8
RRWWCQ 3 7 19 . 2
RRWWCE 2 7 17 . 1
RRWWCD 26 11. 8
RRWWC~7 3 5 -12 . O
WO95116702 2 1 7 8 9 ? 7 r~ y~l~S
--47--
TAB~E lB
PEPTIDE SEQ ID N0: 96 INE~IB.
RRWWCR 1 112 . 2
RRWWCA 24 51 . 9
RRWWCC 2 5 7 5 .1
RRWWCD 26 11. 8
RRWWCE 27 17 . 1
RRWWCF 2 8 8 0 . 2
RRWWCG 2 9 8 6 . 8
RRWWCH 3 0 8 7 . 9
RRWWCI 31 45 . 8
RRWWCK 3 2 110 . 3
RRWWCI. 3 3 8 6 . 9
RRWWCM 34 69 . (3
RRWWCN 3 5 -12 . O
RRWWCP 3 6 8 8 . 3
RRWWCQ 3 7 19 . 2
RRWWCS 3 8 7 9 . 3
RRWWCT 3 9 8 8 . 4
RRWWCV 40 81 . 1
RRWWCW 41 6 6 . 4
RRWWCY 42 77 . 9
2 1 7~927
WO 95116702 ' ~ Y3/i~ 5
--48 -
As shown in Tables lA and lB, several of the
peptides totally inhibited IL-8 binaing in the first
studies. To assess relative effectiveness, the group was
therefore re-evaluated with lower concentrations of the
5 peptides. In these studies, Ac-RRWWCR-NH2 (SEQ ID NO:1)
was f ound to be the most potent inhibitor of binding of
IL-8 to neutrophils (Figure 1) . The data presented in
Figure 1 shows RRWWCR (SEQ ID NO:1) to be significantly
better than the other peptides under these conditions.
In another initial study, the acetylated derivative
of RRWWCR (SEQ ID NO:1; Ac-RRWWCR-NH2) emerged to be
almost twice as effective as the non-acetylated peptide
in inhibiting IL-8 binding to neutrophils.
EI~AMPLE II
RINETICS OF RRWWCR IL-8 BINDING LN~l~ILL~N
In the studies described in this Example, the same
methodology as that detailed in Example I was employed.
An average EC50 was detprnlin~l from multiple replicates
of the bi~ding inhibition curve to be 13 . 7 i 0 . 6 ~M (a
representative curve from these assays is shown in
Figure 2 ) . If the mechanism of inhibition were purely
competitive with a single site model, the estimated KI
was approximately 10 ~M. However, the mechanism was more
complicated. A steeper binding inhibition curve than
usual single site model as well as high Hill coefficient
value (1.5 - ~.7) suggested the presence of positive
cooperativity . The curve did not f it either one-, two -
or three-site model well w~en it was analyzed using the
Lundon II computer program, although IL-8 is known to
react with at least two distinct receptors on
neutrophils. Therefore, it was difficult to estimate
individual KI values for each class of receptor.
WO95JI6702 ~ 7 8 9 2 7 P~./V~Y~
The kinetics of IL - 8 binding i~hibition by RRWWCR
(S~Q ID N0:1) were determined. IL-8 binding isotherms in
the absence of Ac-RRWWCR-NE~2 and in the presence of three
concentrations of the peptide are shown in Figure 3a.
5 The Scatchard plot in the absence of the peptide failed
to show the IL- 8 binding to two distinct receptors
(Figure 3b). The plots in the presence of Ac-RRWWCR-NEI2
(SEQ ID N0:1) were non-linear and were compatible with a
two site model when they were analyzed using the Lundon I
10 computer program. The estimated changes in Kd and Bmax
are shown in Table 2. The data indicate that one group
of IL-8 receptors decreased its affinity at 10 ILM of the
peptide and the other group decreased Bmax with
increasing concentrations of the peptide.
For further characterization of binding kinetics, a
binding inhibition assay using non-labeled IL-8 in the
presence of lO~M Ac-RRWWCR (SEQ ID N0:1) was performed
(Figure 4). Both the binding inhibition curves in the
20 presence and absence of Ac-RRWWCR (SEQ ID N0:1) were
analyzed using Lundon II and showr~ to best fit a two-site
model . There was no evidence f or appearance of a low
affinity state of the receptors. Estimated changes in Kd
and Bmax indlcate that only the high affinity sites
25 decreased both their affinity and Bmax in the presence of
lO~LM peptide (Table 3) .
WO 95116702 ~ , "" , :~ 2 1 7 8 q 2 7 ~ YJl12~15
--~0 -
D O r
O ,~ V
N iD
O O N ~1
O N ~ ~
., .
I` N ~i 0
N d~~ +~ +l
o N O N
~1 +
q~
N 1` r N a.)
r, o o
+ +1 +1 +1 +~ 0
O ~ a~
~ V
,~ . C U~
, .
O ' ~5
N t'l V
O
'~ V r~i
q~ 0
O ~
o o o V
m
H E ~ E ~ ~ N
O V V .
1 3 ~ x o x ~ ~ x r v
o ul o
In ;
WO 9S/16702 2 1 7 ~ 9 2 7 r~l,u~y~l~5
-51-
O
O o O
+l +l +l +l
N "~
~ ~ N L~
HH ~ O
~D o ~ O
W
~d +l +l +l +
~U 0 ~ ~ ~
:~, H r~1 o ~O o
.4; - ' .
O ' . W W
~1 _ L1
,, o E o E
a~
E
q~
~I N
X X
' m ' m
-
Wo 95/16702 ~ 2 1 7 9 2 7 PCrNS93/1224
E~AMPLE III
SPECIFICITY OF R~WWCR TO IL - 8 BIN3ING ~HIBITION
To examine the Epecificity of Ac-RRWWCR-NH2 (SEQ ID
5 NO:1) on ligand-receptor binding, the inventors tested
whether the peptide reduced the binding of human 125I-C5a
or 3H-leukotriene B4 to human neutrophils.
Recombinant human ~ifth component of complement
10 (C5a) (Sigma Chemical Co., St. Louis, MO) was
radioiodinated enzymatically using Enzymobead (Bio Rad,
Richmond, CA). Tritiated leukotriene B4 was purchased
from Du Pont Co., Wilmington, DE. Binding assays for C5a
and leukotriene B4 were performed as described for IL-8
15 binding in Example I, except that the buffers used were
PBS containing lmM CaC12, O . 5mM MgC12, O . 596 BSA, and XBSS
,~n~ilin;ng 0.1~ ovulabumin=and 10mM HEPES (pH7.3),
respectively, as previously described (Braunwalder et
a,l., 1992; Sherman et al., 1988). The radioactivity was
20 measured with liquid scintillation counter for the
leukotriene B4 assay . The ~binding of both ligands were
saturable and inhibited by the non-labeled agonists in
dose dependent manners.
In these studies it was found that at 100~LM
Ac-RRWWCR-NH2 (SEQ ID NO:1) did not affect the binding of
C5a nor leukotriene B4, but that it suppressed the IL- 8
binding significantly at a concentration of 100nM
(Figure 5) .
El~aMPLE IV
~,~L~JrL`v~lwl~ OF RRWWCR TO N~:UL~U~llJS
The inventors next ~ m; n~l the cytotoxic capacity
of Ac-RRWWCR-NH2 (SEQ ID NO:1). This was achievea by
Wo95/16702 ` ~ 2 1 7~927 P~ Y~i~5
--53--
measuring the amount of 51Cr released from neutrophils,
as follows: The neutrophil preparation (2x107 /ml) in
RPMI-1640 media containing 1096 donor' s plasma wa6
incubated with 50011Ci of Na251CrO4 (Du Pont Co. ) for 60
min at 37C. The cells were washed 3 times, resuspended
in the media at the concentration of lxlO7/ml, and then
incubated for 30 min at 37C to allow spontaneous lysis
of marginally viable cells. After washing twice, a 100
~1 aliquot of the 51Cr-labeled neutrophils (5xl06/ml) was
mixed with 100 fLl of Ac-RRWWCR-NE~2 (SEQ ID NO:l) in a
siliconized microcentrifuge tube. The buffer and the
conditions of incubation simulate~ ~ither the binding
assay or the chemotactic assay. After the incubation,
the tubes were centrifuged at 300xg for 7 min at 4C and
the radioactivity in the supernatant was then counted in
a gamma radiation spectrometer. Triplicate tubes
c~-ntA;n;n~ buffer alone or 29~i SDS were used to determine
spontaneous and maximum release, respectively. The
percentage lysis was calculated by using the following
2 0 f ormula:
LYSIS = (EXPERIMENTAI CPM - SPONTAIIEOUS CPM) X100
(MAXIMUM CPM - SPONTANEOUS CPM)
When chromium-labeled cells were incubated in PBS
,-,.n~;,;n;ng 0.196 BSA for 90 min at 4C, it was found that
the percentage of cells lysed remained near control level
up to 500~uM of the peptide (~igure 6). Under conditions
used in chemotaxis, 10011M of the peptide had no effect,
however, 500~M peptide damaged almost 2596 of the cells.
-
Wo95/16702 ~ t 2 1 7 ~ 2 7 PCrlUS93/12245
--54 -
3XAMPLE V
EFFECT OF RRWWCR ON N~:u-.:cOEHlJ J!U_._J1~,_.;j
The effect of RRWWCR on neutrophil chemotaxi6 and
enzyme release was next ~-~Arrli n~d.
A. Chemotaxis
Chemotaxis was performed using the leading front
method as described by Zigmond and Hirsh (Zigmond &
Hirsch, 1973). IL-8 or controls were placed in the lower
well of a Boyden chamber. ~ five micron pore size,
100 llm thick cellulose nitrate filter (Sartorius Filter,
Inc., San Francisco, CA) was placed on the surface~and
the chamber was then assembled. A 200 ~Ll aliquot of the
neutrophil preparation (lxl06 cells/ml) in RPMI-1640
media containing lg6 BSA was added to the top of the
filter and incubated at 37C for 30 min. The filter was
then fixed, stained and mounted on a glass microscope
slide. The leading front was determined by the position
of the leading two cells. The distance that the leading
two cells had moved through the ~ilter was measured for
six fields on each filter. The measurements were made
with four filters for each set of conditions.
B. ~ ~,,hil E~zYme Relea~e
Neutrophil enzyme release was studied by a
modif ication of the method of Goldstein and colleagues
(Goldstein et al., 1973) . Cy~o~~h;31Aq;n B (Sigma Chemical
Co. ) was stored in dimethyl sulfoxide at a concentration
of 5 mg/ml and was diluted to a concentration of 50 ~g/ml
in Hank' s RA1 An~'fi Salt Solution (HBSS) immediately
before use. Cyto~h~lAR;n B, 200 Ill, was added to l ml of
suspension of neutrophils, 6.25xlO6 cells/ml in HBSS, to
achieve a final cytochalasin B concentration of l0 ~Lg/ml.
Wo 95116702 ~ 1 8 9 2 7 ~ Y3/1~5
The solution was then incubated in 96 well plates at room
temperature for 10 min. The st; l~nt, 100 ~l, was
added, and this cell suspension was incubated for 30 min
at 37C. The plates were centrifuged and 100 /11 of
5 8Uplorn~t~nt wag removed. Alir~uots of ~0 ~11 of
supernatant were mixed with 101l1 of 0 . OlM
phenolphthalein-glucuronic acid (Sigma Chemical Co. ) and
40~1 of 0 . lM sodium phosphate pH 4 . 6 in 96-well plates
for ~-glucuronidase measurement. After 16 hours
;ncllh~tion at 37C, 200~1 of 0.2M Glycine in 0.2M NaCl,
pH10 . 4 was added to each well and OD540 was measured as
the enzyme activity.
C. Result~
It was found that the Ac-RRWWCR-NH2 (SEQ ID NO:l)
had no effect on neutrophil chemotaxis or enzyme release.
A checkerboard analysis of cell v indicated that
it was not chemotactic for neutrophils (Table 4). In
20 control studies shown in Figures 7 and 8, the peptide had
no effect on rh k;n~is of neutrophils and ~id not
stimulate ,~-glucuronidase release from the cells.
To conf irm that the inhibition of IL- 8 binding by
25 Ac-RRWWCR-NH2 is related to suppression of neutrophil
activation by IL-8, the inventors ~oY~m;nPri the effects of
the peptide on chemotaxis and ~-glucuronidase release.
Ac-RRWWCR-NH2 (SEQ ID NO:1) significantly inhibited
chemotaxis of neutrophils stim~lated with 10 nM of IL-8
30 at a r~nrPntration of Ac-RRWWCR-NH2 (SEQ ID NO:1) of
- 50~M, whereas it had no effect on chemotaxis induced by
formyl-L-Methionyl-L-Leucyl-L-Phenylalanine (fMLP)
(Figure 7) . Ac-RRWWCR-NH2 (SEQ ID NO:1) inhibited
~-glucuronidase release sti l i~tPfi by lOOnM of IL-8 at
35 2~LM, a lower concentration than required for inhibition
of chemotaxis, however, it did not affect the enzyme
W095/16702 ~ ` 7 1 78 927 P~ Y.S/ ~45
relea~e ~timulated by fMLP, C5a or leukotriene B4 (Figure
8) .
. r ~
WO95/16702 ' 2 1 78~2'7 ~ V~Y~/1~45
--57--
~1 ~ Ul
N N N
O rr~ I` r~ r~
N
.. +l +l +l +
rn r~ 1-
N N N N
W N t` ~1
N N N ~1 -
ul O ~ rn r~ ~
r ~1 ~ Ln ~ ~
+l +l +l + ~ ~
~ ~D N rn rn - V
-- N N N N ~1 ~1
h Kl ~
a) o . o
V r-~ V
~P p ~ rn rD 1` V ~ ~ ~
-- ~ o t` N r rn K -- C
E~ +l +l +l +l ~ '
~~ rD rn t~
~ N N N N ~ ~r
- . O O
O ~ ~ ~ .,
N N N ~ r
O o ~ r~
C +l +l +l +l ~ '
r~ ~D O rn
I` O
N N N ~ ~: V
L~ O O
. J N V
O+l - ~ J
O ~
N -- J
h IR .
-~ O ~ ^ o In O ~ ~ .
o ~ m r~ m h h
.. . . . . .. _ .. .
WO 95/16702 ~ ' ^ ^ ~ ~ '` 2 1 7 PJ 9 2 7 ~ Y~1~45
EXaMPLE VI
FIJRT~ER PEPTIDE lN~ ~S OF IL-8
Further series of studies were carried out to
5 determine whether other peptides related to AC-RRWWCR-NH2
(SEQ ID NO:l) would act as inhibitors of Il:,-8, and to
determine their relative effectivenees. In the studies
described in this Example, the same methodology as that
detailed in Example I was employed.
A. ~ePta~ner PePtides
In this series of studies, the inventors ~r~m;nF~l
D-amino acid analogues of RRWWCR (SEQ ID NO:l) with an
15 added seventh amino acid. In the heptamer studies,
twenty peptides were synthesized with the carboxyl-
terminal rexidue of RRWWCRX (SEQ ID NO:2) being changed
to each of the standard protein amino acids in turn. In
the first set of heptamer studies, several of the~
20 peptides exhibited very strong inhibition of I~-8
binding, as shown in Table 5A and Tabie 5B. The
information presented in Table 5A and Table 5B is the
same data, with Table 5A being listed in order of %
inhibition and Table 5B being listed in order of SEQ ID
25 NO, to enable straightforward comparisons. It can be
clearly seen that all heptamer peptides (SEQ ID NOS:3
through 22) have significant inhibitory activity under
these con~1~Lon~ ~T~b1e s~1.
WO 95116702 ~ 217 8 9 2 7 r~ Y~
--59 -
TAB~E 5A
PEPTIDE SEQ ID ~6 INHIB.
N0:
RRWWCR 1 112 . 2
rrwwcrk 11 113 .1
5rrwwcrn 14 110 . 8
rrwwcrg 8 10 8 . 0
rrwwcrc 4 107 . 7
rrwwcrd 5 107 . o
rrwwcrh 9 107 . 0
rrwwcrw 21 106 . 3
rrwwcrf 7 105 . 6
rrwwcrr 17 102 . 8
rrwwcre 6 99.1
rrwwcrl 12 99.1
rrwwcrm 13 96 . 2
rrwwcra 3 95 . 3
rrwwcri 10 93 . 9
rrwwcrq 16 89.2
rrwwcr~ 2 0 8 9 . 2
rrwwcrp 15 87.3
rrwwcry 22 85.4
rrwwcr~ 18 70 . 4
rrwwcrt 19 56 . 3
.
WO9~/16702 ~ 2 1 78 927 P~ Y~/I~45
. . ~1
--60-
TAB~E 5B
PEPTIDE SEQ ID 96 I~HIB.
N0:
RRWWCR 1 112 . 2
rrwwcra 3 95 . 3
rrwwcrc 4 107 . 7
rrwwerd 5 107 . 0
rrwwcre 6 9 9 .1
rrwwcrf 7 105 . 6
rrwwcrg 8 10 8 . O
rrwwcrh 9 107. 0
rrwwcri 10 93 . 9
rrwwcrk 11 113
rrwwerl 12 99.1
rrwwcrm 13 96 . 2
rrwwern 14 110 . 8
rrwwerp 15 87 . 3
rrwwerq 16 89.2
rrwwerr 17 102 . 8
rr~wcr~ 18 70 . 4
rrwwcrt 19 56 . 3
rrwwcrv 20 89.2
rrwwerw 21 10 6 3
rrwwery 2 2 8 5 . 4
WO95/16702 ~ 61- J~~ Y3/i~4~
In order to determine the relative effectiveness of
the heptamers, their inhibitory effects were determined
at lower ~ n~ ntrations. The peptide with D-cysteine
present at the carboxyl-terminal end (RRWWCRC; SEQ ID
5 NO:4) was found to be almost 56% more effective than the
next best peptide in this group and to be more effective
than Ac-RRWWCR-Ni~2 (SEQ ID NO:1; Figure 9) .
Ac-rrwwcrc-NH2 (SEQ ID NO:4) prevented 80% of the binding
of IL-8 to neutrophils as compared to 20% inhibition by
10 the L=amino acid peptide Ac-RRWWCR-NH2 (SEQ ID NO:1) at
101lM .
B. Other Pe~tide~
The inventors also tested several additional
peptides which were either related to the amino terminal
portion of IL-8 or were found in other proteins and had
five of the six residues in RRWWCR (SEQ ID NO:1) . The
peptides ELRCQCIKTY, ELRSQSIKTY, EI.RMQMIKTY,
20 QIPRRSWCRFLF, and ~w~wwwAVLY (SEQ ID NOS:49 through 53,
respectively) were synthesized at The University of Texas
Health Center at Tyler llt;l;~;n~ an 431 Peptide
Synthesizer (Applied Biosystems, Foster City, CA), using
the 9-fluorenylmethoxycarbonyl (fMOC) group to protect
25 the o~-amino group as described by Meienhofer and
coworkers (Meienhofer et al., 1979) and Arshady et al.
(1979). All synthetic peptides were purified on high
performance liquid cllrl t-~raphy (HP~C) using a
preparative C18 reverse phase column (Waters Co., New
30 Bedford, MA). Peptides were eluted using a gradient from
0.1~6 trifluoroacetic acid (TFA) to 80% acetonitrile in
0.1% TFA. The composition of the peptides was confirmed
by amino acid analysis and sequencing by the Protein Core
- facility at UTHC.
Wo 95116702 ~ 2 1 7 8 ~ 2 7 ~ Y.~112,L.~5
--62-
In this Rerie~ of studieR, only Ac-RRWWCR-NH2 (SEQ
ID NO:1) and QIPRRSWCRFLF (SEQ ID NO:52) inhibited
binding of IL-8 to neutrophil~ (Table 6).
TABLE 6
Inhibition of IL-8 Binding to 11__' ~llils
by SYnthetic Pel~tides
Peptide Tested SEQ ID NO: % Binding
Inhibition
10 Ac-KELRCQ 54 . -0.4 i 11.8
QIPRRSWCRFLF 52 61. 5 i 1. O
~iw~ww~AVLY 53 -12 . 9 i 2 . 9
EBRCQCIKTY 49 7 . 6 i 2 .1
ELRSQSIKTY 50 5 . 8 i 3 - 9
15 ELRMQMIKTY 51 -6 . 9 i 3 . O
Ac-RRWWCR 1 98 . 6 i O . 9
RXXXXX 4 3 11 . 5
XXXXXR 44 5 . 8
XRXXXX 45 9 . 9
2 o XXWXXX 4 6 1 . 4
XXXWXX 4 7 1 8 . 5
XXXXCX 4 8 7 . 1
EXAMP~E VII
OF GRO and MIP21g NEurrROPHIL BINDING
AC-RRWWCX-NH2 (SEQ ID~0:23) was also ,=~ o.l for
30 the ability to inhibit other CXC intercrineF~. The
pre~ent example demon~trate~ that, in addition to IL- 8
inhibition, Ac-RRWWCX-NH2 ~SEQ ID NO:23) effectively
inhibit~ GRO and MIP2~B binding to human neutrophils.
WO gSil6101 ~ ? ~ 2 1 7 8 9 2 7 PcT/rJs93~l~5
.
--63--
MIP2~ and GRO/MGSA were radioiodinated using Bolten
Hunter reagent. The radioiodinated components were mixed
with various concentration of the Ac-RRWWCX-NH2 (SEQ ID
NO:23) peptide and incubated at room temperature for
15 minutes. Neutrophil suspension (1 X 106 cells in
160~L1 PBS cnnt:~;n;n~ 0.196 BSA) was added to 40~1 of the
mixture and incubated for 90 minutes on ice. The
radioactivity bound to the cells was separated from free
radioactivity by centrifugation through an oil layer.
The bound radioactivity is an indication of bound CXC
intercrine peptide. The ~ binding inhibition in the
presence of Ac-RRWWCX-NH2 (SEQ ID NO:23) was calculated
as ollows:
96 binding inhibition = ¦ 1 _ B NSP ¦ x 100
T-NSP
where B is bound radioactivity in the presence of
the peptide, T i8 bound radioactivity in the absence of
the peptide; and NSP i8 bound radioactivity in the
presence of excess nonlabelled ligand.
In these studies, it was confirmed that Ac-RRWWCX-
NH2 (SEQ ID NO:23) inhibited binding of 1 nM IL-3 to
neutrophils in a dose dependent manner, with an EC50 of
almost 25 llM (Figure 10). Ac-RRWWCX-NH2 (SEQ ID NO:23)
was also found to effectively suppress the binding of
1 nM GRO and 1 nM MIP2~ to neutrophils in a simlar
manner, as shown in Figure 10.
-
3~ EXAMPLE VIII
l~ER~PE~ITIC F0R~U~AT~ONS AND T~ ~ PROTOCOLS
This example is directed to the techniques
c~intP~ ted by the inventors for use in further
W095/16702 ` ' ~ ~ 21i~9~7 ~ 45
--64--
characterizing the in vivo actions of the II.- 8 inhibitors
and their use in animal or ~human treatment protocols .
A. ISffects o_ IL-8 Inhibitor~ OD Tnfl tio rn Vivo
Prior to animal model ~studies, in vitra stability
examinations may be performed on the peptides including,
for example, pre-incubation in human serum and plasma;
treatment with various proteases; and also temperature-
lO and pH-stability analyses.: It is already known that
D-amino acid peptides are active and that these would
likely have enhanced stabiIity in vivo.
The inventors propose ~to examine the in vivo ~
15 properties and ef f ects of the I~- 8 peptide inhibitors in
animal models prior to moving onto clinical trialfi. The
most suitable form, dose and any possible toxicity of the
peptides will be determined in animal studies, as is
routinely employed in the art. For example, the bio-
20 availability and half lives of the peptides administeredin various ways may be determined using radioactively
labeled peptides and ~ min;n~ their longevity and tissue
distribution. If further stability ,onll~n~ t was
desired, the peptides could also be administered in the
25 form of lipid-tailed peptides, surfactant-like micelles,
peptide multimers or in semi-permeable drug release .
capsules .
The biological effects of the peptides may be
30 determined in various models of human disease. For
example, II.-8 has been shown to cause the accumulation of
neutrophils and edema in rabbit skin (Rampart et al.,
1989). Therefor~, a rabbit dermal inflammation model
will be employed to determine what dose of the peptides
35 can effectively inhibit the neutrophil accumulation and
edema. This model is useful because of the ease o~
assessment of i f lammation . The most suitable route of
WO Y5/16N2 , ,`.` 2 1 7 8 9 2 7 I ~ Y~I~S
--65--
peptide administration may be easily determined by
comparative in vivo tests,
In one particular example, New Zealand albino
5 rabbits may be injected with 125I-labeled human serum
albumin through the lateral ear vein. Certain sites may
then be inj ected intradermally with the test compounds,
i e., an agonist to attract neutrophils and an IL-8
inhibitor peptide; the agonist and a control peptide; and
10 the agonist alone. About two hours later, full thickness
skin samples 1 cm in diameter may be punched out, f ixed
and stained with Wright-Giemsa or for myeloperoxidase and
the histology ~x;1~1i n,~,1 for neutrophil accumulation and
edema or tissue damage. Other skin biopsies may be
15 counted in a gamma counter to assess the amount of
albumin f lux into the inj ected skin . Skin inf lammation
after administration of the inhibitor can then be
compared to the time-matched controls, ideally be
performed in the same animal. At least 4 replicates for
2 o each experimental arm are recl n~
B . IL- 8 Inhibitor Trea t o~ A~n~
The best human model of Adult Respiratory Distress
25 Syndrome (ARDS) is probably the introduction of gram
negative bacteria into the circulation of minipigs. This
model will be employed to determine if II--8 is the major
neutrophil activator in this model of ARDS, as it is in
human ARDS, using studies similar to those carried out in
30 man. The effects of Ih-8 administration through
intravenous and intr~ ry routes in minipigs will be
assessed .
- In the fist step, the minipigs will be treated with
35 I~-8 through the most appropriate route to cause
neutrophil influx and enzyme release into the lungs as
assessed above. The peptides of interest will be
.
WO95/16702 2 ' 78927 1~1/.J~Y~11~5
administered to determine appropriate doses for use in
impeding these neutrophil functions, especially the dose
with which the peptide suppresses enzyme release, but not
the neutrophil influx.
In the next step, the acute lung damage model of
minipigs caused by gram negative bacteria in the
circulation will be employed. The peptides of interest
will be administered ~:o th~ animals and the effect: of the
lO peptides on the prevention of lung damage will be
assessed The number of neutrophils in bronchoalveolar
fluids, the amount of enzyme released into lung
parenchyma, and the degree of protein leakage f rom
circulation to lung will be used as indicators in this
15 study.
If IL-8 causes neutrophil influx, enzyme release
into the lungs, and/or ARDS - like tissue damage to the
lungs, as expected, the peptides of interest will be
20 administered to determine the appropriate doses for use
in impeding these neutrophil functions. In these
studies, various intravenous doses of radioactively
labelled peptides will be administered initially. Plasma
c~noPntrations and forms of the radioactivity will then
25 be determined. From these data, plasma clearance, half
life and steady state volume of distribution will be
measured and used to determine the most effective do3e
ranges .
30 C. Treatment Protocols
Due to precautions which are nP~-PAsA~ily attendant
to every new pharmaceutical, the I~- 8 peptide inhibitore
and compositions of the present invention have not yet
35 been tested in a clinical setting in human subjects.
Xowever, their clear ill vi tro activity in accepted models
is believed to demonstrate the utility of the present
~WO 95/167D2 - 6 7 ~ Y~ tS
invention as an anti-inflammatory agent. Clinical trials
will be conducted in due course and, naturally, will be
in accordance with the FDA procedures. The following
embodiments represent the best modes currently
5 contemplated by the present inventors for carrying out
the practice of the invention in various clinical
settings .
It is believed that pharmaceutical compositions
lO which include peptide inhibitors of I~-8 will,prove to be
useful in the treatment of various conditions, ;nr~ l;n~
pulmonary disorders such as bronchial inflammation,
cystic fibrosis, pleural effusions, asthma, bronchitis
and ARDS; skin disorders such as psoriasis and
15 dermatitis; diseases of the joints, including rheumatoid
arthritis; and in the treatment of pseudogout,
;n~l. tory bowel disease, reperfusion cardiac damage or
even in the treatment of cancer and other diseases and
disorders associated with increased cellular
2 0 prol if erat ion .
As these peptides are thought to be particularly
suitable for the inhibition of pulmonary inflammation,
such as occurs in ARDS, chronic bronchitis and cystic
25 fibrosis, suitable treatment methods for these disorders
will be described. For the treatment of ARDS or cystic
fibrosis, one would preferably employ parenteral
administration, such as by using intravenous,
intramuscular or subcutaneous injections. However, one
30 may also use aerosols or inhalants. The preparation of
peptide foL li~t;~n~ for parenteral administration,
particularly those for~lated as injectables, is
described in detail in the Preferred Embodiments section
of the present application. The following describes
35 certain inhalant formulations, should one desire to use
such methods in connection with the present invention.
WO 95116702 ~ t C 2 1 7 8 9 2 7 PCrlUS93/12245
--68--
Inhalations and inh~l~ntc: are pharmaceutical ~
preparations designed for delivering a drug or compound
into the respiratory tree of a patient. A vapor or mist
is administered and reaches the affected area to give
5 relief from symptoms of bronchial and nasal congestion.
Inhalations may be administered by the nasal or oral
respiratory routes. The administration of inhalation
solutions is only effective if the droplets are
sufficiently fine and uniform in size so that the mist
lO reaches the bronchioles.
Another group of products, also known as
inhalations, and sometimes called insufflations, consists
of f inely powdered or liquid drugs that are carried into
15 the respiratory passages by the use of special delivery
systems, such as pharmaceutical aerosols, that hold a
solution or suspension of the drug in a liguefied gas
propellant. When released through a suitable valve and
oral adapter, a metered dose of the inhalation is
20 prbpelled into the respiratory tract of the patient.
Particle size is of major importance in the
administration of this type of preparation. It has been
reported that the optimum particle size for penetration
25 into the pulmonary cavity is of the order of 0 . 5 to 7 ILm .
Fine mists are produced by pressurized aerosols and hence
their use in considered advantageous.
The intravenous administration of one, or a
30 combination, of the anti-I1,-8 peptides described i~ this
application is contemplated to be capable of attenuating
inflammation in A~DS and cystic fibrosis. The range of
doses to be administered is estimated to be in the range
of about 500 to about lOOOmg/day, or between about 0 . 83
35 mg/kg body weight/hour (mg/kg/hr) to about
16 . 56 mg/kg/hr.
wo95/16702 - ~ 2 1 78~27 P~ Y.JI~
-69-
of course, one must not lose sight of the fact that
various other pharmaceutical formulations of the IL-8
inhibitors may be prepared and used to treat many other
disorders connected with neutrophil activation and
5 ;n~l. tion.
* * *
All of the compositions and methods disclosed and
claimed herein can be made and executed without undue
experimentation in light of the present disclosure.
While the compositions and methods of this invention have
15 been described in terms of preferred c.mhnrl; ~c, it will
be apparent to those of skill in the art that variations
may be a~plied to the composition, methods and in the
steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and
20 scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically
and physiologically related may be substituted for the
agents described herein while the same or similar results
would be achieved. A11 such similar substitutes and
25 modifications apparent to those skilled in the art are
deemed to be within the spirit, scope and concept of the
invention as defined b~ the appended claims.
'
.
WO 95/16702 2 ~ 7 8 9 2 7 I~ J~ /l2~.15
-70-
R~ :S
The following references, to the extent that they
provide exemplary procedural or other details
supplementary to those set forth herein, are specif ically
incorporated herein by reference.
Arshady, R., Atherton, E., Gait, M.J., I,ee, K., and
Sheppard, R.C. (1979) .J. Chem. Soc. Chem. Commun.,
23, 423-425.
Baggiolini, M., Imboden, P., and Detmers, P. (1992) in
Cytokine~: Tnterleukin= 8 (NAP-l) and Related
Chemotactic Cytokine~ (Gabbiolini, M. and Sorg, C.,
eds) pp. 1-17, Karger, Basel.
Baldwin, E.T., Weber, I.T., St. Charles, R., Xuan, J.-C.,
AE~ella, E., Yamada, M., Matsushima, K., Edwards,
B . F . P ., Clore , G . M ., Groenenborn , A . M ., and
. 20 Wlodawer, A. (l991) Proc. Natl. Acad. Sci. USA, 88,
502 -506 .
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--72--
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--74-
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WO 95/16702 '- ~ 1'! r~ ~' r ~ 2 1 7 8 ~ 2 7 P~ ~S
-78--
UUL~ i I,ISTING
( 1 ) GENERAL INFORMATION: ~
(i) APPLICANTS:
(A) NAME: BOl~RD OF REGENTS,
THE ~NIVERSITY OF TEXAS
(B) STREET: 201 West 7th Street
(C) CITY: Austin
( D ) STATE: TEXAS
(E) COUNTRY: UNITED STATES OF AMERICA
(F) POSTAL CODE: 78701
and
(ii) APPLICANT: CO~EN, Allen B.
MI~LER, Ed~und r.
2 0 KURDOWSKA, Anna
~YASHI, Shinichiro
TUTTLE, Ronald R.
(iii) TIT~E OF INVENTION: PEPTIDE INHIBITORS OF CXC
2 5 INTERCRINE MO~ECULES
(iv) NUMBER OF ~ ?U~N~:S: 58
(v) CORRESPONDENCE ADDRESS:
(A) ~nn~C~: Arnold, White & Durkee
(B) STREET: P. O. Box 4433
(C) CITY: Houston
(D) STATE: Texas
3 5 ( E ) COU~TRY: USA
(F) ZIP: 77210
WO 95/16702 ; ~ C 2 1 7 ~ ~ 2 7 P~_l/v~Y~ 15
--79-
(vi ) COMPUTER Rr~'An~Rr.r~ FORM:
~A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS/ASCII
(D) SOFTWARE: WordPerfect 5.1
(vii) CURRENT APPLICATION DATA:
(A) APPLICATION Nt~MBER: Unknown
(B) FILING DATE: Concurrently herewith
(C) CLASSIFICATION: Unknown
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: KITCHELL, Bar~ara S.
. (B) REGISTRATION NUMBER: 33,928
(C) REFERENCE/DOCKET NUMBER: rJTFNol6pcT
2 0 ( ix ) TELECOMMUNI CATION INFORMAT ION:
(A) TELEPHONE: (512) 320-7200
(B) TELEFAX: (713) 789-2679
(C) TELEX: 79-0924
( 2 ) INFORMATION FOR SEQ ID NO :1:
(i) SEQrJENCE (~T~R~RT~TIcs:
(A) LENGTH: 6 amino acid~
(B) TYPE: amino acid
(C) STRANDEDNESS: ~ingle
- (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
2 ~ 78927
WO 95/16702 . . . ~~ Y.~112245
-80 -
(xi) SEQUENCE D13SCRIPTIO~: SEQ ID NO:l:
Arg Arg Trp Trp Cys Arg
(2) INFORMATION FOR SEQ ID NO:2:
IU~;N~; CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) sTRhND~nN~c~c: single
l S ( D ) TOPOLOGY: l inear
(ii) MOLECULE TYPE: peptide
(Xi) ~ U~;N~ DESCRIPTIO~: SEQ ID NO:2:
. Arg Arg.Trp Trp Cys Arg Xaa~
25 (2) INFORM~TION FOR SEQ ID NO:3:
;12U~;N~:~; CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) sTR~ND~nN~s: single
(D) TOPO~OGY: linear
(ii) MOLECULE TYPE: peptide
~ ~ t~ - ~ r
~ O 9511670Z ~ 1 7 8 9 2 7 PCI~/US93112Z45
(Xi) ~ U~;N~:~; DESCRIPTION: SEQ ID NO:3:
Arg Arg Trp Trp Cys Arg Ala
( 2 ) INFORMATION FOR SEQ ID NO: 4:
( i ) ~;~;5~U~:N~:~ CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) sTR~l~n~nN~s single
(D) TOPOLOGY: lillear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Arg Arg Trp Trp Cys Arg Cys
( 2 ) INFORMATION FOR SEQ ID NO: 5:
( i ) ~;~5,)U~:N~,'~ CH~RACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) ~OLECULE TYPE: peptide
WO 95/16702 ~ '~ 2 1 7 8 ~ 2 7 PCT/US93/12245
--82--
(xi) ~ U~:N(:~: DESCRIPTION: SEQ ID NO:5:
Arg Arg Trp Trp Cys Arg Asp
( 2 ) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 7 amino acids
(B) TYPE: amino acid
(C) STR~Nn~nNF~ C: single
(D) TOPOLOGY: linear
( ii ) MOI.ECULE TYPE: peptide
(Xi) ~;~UU~:N~ DESCRIBTION: SEQ ID NO 6:
Arg Arg Trp Trp Cys Arg Glu
(2) INFORMATION FOR SEQ ID NO:7:
(i) ~;~;UU~;N~ RA~'T~RT~TIcs:
(A) ~ENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STR~Nn~l)N~ : single
( D ) TOPO~OGY: l inear
(ii) MO~ECU~E TYPE: peptide
W0 9~ 6N2 ; ~ 2 1 7 8 9 2 7 P., llu~73~ 45
-83 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Arg Arg Trp Trp Cys Arg Phe
( 2 ) INFORMATION FOR SEQ ID NO: 8:
( i ) ~i ~;(,2 U~;N ~ R ~ ~'T~ R T .q T I CS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STR~NnFi~nN~q~q: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(Xi) ~ U~;N~ DESCRIPTION: SEQ ID NO:8:
2 0 ~rg Arg Trp Trp Cys Arg Gly
( 2 ) IN~ORMATION FOR SEQ ID NO: 9:
( i ) SEQUENCE ('T~Z~R ~ t'T~R T .qT I CS:
(A) LENGTE~: 7 amino acids
(B) TYPE: amino acid
(C) sTR~Nn~.nN~c.q: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE- peptide
wo 95rl6702 ; ~ ~ - 2 1 7 8 ~ 2 7 ~ YJ/12245
-84--
(Xi) ~ ;1.2U~;I\I~ DESCRIPTION: SEQ ID NO:9:
Arg Arg Trp Trp Cys Arg His
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CH~RACTERISTICS:
(A) ~ENGTH: 7 amino aclds
(B) TYPE: amino acid
(C) STRANDEDNESS: slng~e
(D) TOPOLOGY: linear
( ii ) MO~.ECU~E TYPE: peptide
(xi)~ Y~:UU~N~; DESCRIPTION: SEQ ID NO:10:
20 Arg Arg Trp Trp Cys Arg Ile
(2) INFORMATION FOR SEQ ID NO:ll:
U~;N~; CH~RACTERISTICS:
(A) ~ENGTH: 7 amino acids
(.3) TYPE: amino acid
(C) STRZ~NnRTlNF~ : 8ingle
(D) TOPOLOGY: linear
(ii) MO~ECULE TYPE: peptide
W095ll6~02 ~ 2 ~ 7~92~ ,IIU~Y3/I~ 45
--85--
~xi) SEQUEN-OE DBSCRIPTION: SEQ ID NO:11:
Arg Arg Trp Trp Cys Arg Lys
(2) INFORMATION FOR SEQ ID NO:12:
(i) Y~;~U~;N~; (7TARAt~T~RT~qTIcs:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) sTR~Nn~nN~q~q single
(D) TOPOLOGY: linea~
(ii) MOLECULE TYPE: peptide
(xi) Y~Uu~No:~; DESCRIPTION: SEQ ID NO:12:
2 0 Arg Arg Trp Trp Cy8 Arg Leu
1 5
(2) INFORMATION FOR SEQ ID NO:13: -~
(i) Y~;~U~N~:~; CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANn~nNl~qq: single
(D) TOPOLOGY: linear
( ii ) MOLECULE TYPE: peptide
WO95/16702 , ~ 2~78927 p ~ Y~ 4s
--86--
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Arg Arg Trp Trp CYB Arg Met
(2) INFORMATION FOR SEQ ID ~0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STR~Nn~.nNR.~.~: single
(D) TOPOl.OGY: linear
(ii) MOLECULE TYPE: peptlde
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
2 0 Arg Arg Trp Trp CYB Arg Asn
.
(2) INFORMATION FOR SEQ ID NO:15-
( i ) B ~;U U ~:N ~; CHARACTERI ST I CS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
.
~095116702 ~ ? ~ 2 1 7~ 9 27 r~ Y~ 45
--87--
(Xi) ~ U~;N~.:~ DESCRIPTION: SEQ ID NO:15:
Arg Arg Trp Trp Cys Arg Pro
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENOE C~RACTERISTICS:
(A) I ENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STR~NnRnNRss ~ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENOE DESCRIPTION: SEQ ID NO:16:
2 0 Arg Arg Trp Trp Cy~ Arg Gln
(2) INFORMATION FOR SEQ ID NO:17:
( i ) SEQUENCE ~R z~(~TRR T .STI CS:
(A) I-ENGTH: 7 amino acidq
(B) TYPE: amino acid
(C) STRANDEDNESS: ~ingle
( D ) TOPO~OGY: 1 inear
~ii) MOI,ECULE TYPE: peptide
WO 95/16702 '' ` ~ 2 ~ 7 8 9 2 7 PCr/US93112245
-88-
(xi) SEQUENCE DESCRIPTION: SEQ rD NO:17:
Arg Arg Trp Trp Cys Arg Arg
(2) INFORMATION FOR SEQ ID NO:18:
JU~N~ CHARACTERISTICS:
(A) ~ENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: linear
(ii) MO~ECUIE TYPE: peptide
(Xi) ~ U~:N-:~ DESCRIPTION: SEQ ID NO:18:
2 0 Arg Arg Trp Trp Cys Arg Ser
.
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE ~ ARA~TRRTqTICS:
(A) ~ENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRAl~n~:nN~.q.q: single
( D ) TOPO~OGY: l inear
(ii) MOl.ECU~E TYPE: peptide
~O 9511670~ ' ' 2 1 7 8 9 2 7 r~ 45
--89--
(xi~ U~N-:~ DESCRIPTION: SEQ ID NO:19:
- Arg Arg Trp Trp Cys Arg Thr
(2) INFORMATION FOR SEQ ID NO:20:
U~;N~:~; CHARACTERISTICS:
(A) ~ENGTH: 7 amino acidæ
(B) TYPE: amino acid
(C) STRANl)RnNR.C~.~: single
( D ) TOPO~OGY: 1 inear
(ii) MO~ECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION-- SEQ ID NO:20:
2 0 . Arg Arg Trp Trp Cys Arg Val
(2) INFORMATION FOR SEQ ID NO:21:
U~;N0:~: CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOI-ECULE TYPE: peptide
WO 95/16702 ~ 7 ~ ~ 2 7 P~ Y~ 45 ~
-so-
(Xi) ~ ;s2U~;N(~; DESCRIPTION: SEQ ID NO:21:
Arg Arg Trp Trp Cys Arg Trp
5 .
(2) INFORMATION FOR SEQ ID NO:22:
2U~;N~ CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TY~E: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: pep~ide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
2 0 Arg Arg Trp Trp Cy8 Arg Tyr
(2) INFORMATION FOR SEQ ID NO:23:
( i ) SEQUENCE CE~RACTERISTICS:
(A) LENGT~: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
WO 95/16702 ~ r~~ 2 1 7 ~3 9 2 7 ~ ~45
-91-
(xi) ~ 5.?U~N~:~; DESCRIPTION: SEQ ID NO:23:
Arg Arg Trp Trp Cy3 Xaa
(2) INFORMATION FOR SEQ ID NO:24:
(i) Y~:~UI~;N~I:; CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STR~Nn~nN~cc 3ingle
(D) TOPOI.OGY: linear
(ii) MOLECULE TYPE: peptide
(xi) S~ U~;N~ DESCRIPTION: SEQ ID NO:24:
2 0 Arg Arg Trp Trp Cys Al a
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENOE ~R~T~RT~sTIcs
(A) ~ENGTH: 6 amino acid3
(B) TYPE: amino acid
(C) STR~Nn~nN~clc 3ingle
( D ) TOPOLOGY: l inear
(ii) MOLECI~LE TYPE: peptide
WO 95/16702 "~ 7 8 9 2 7 PCr/US93/12245
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Arg Arg Trp Trp Cy8 Cy8
(2) INFORMATION FOR SEQ ID NO:26~:
( i ) ~;~;U U ~;N ~; CHARACTERI ST I CS:
(A) ~ENGTH: 6 amino~ acids
(B) TYPE: amino aci~
(C) STRANnF~nN~ single ~=
(D) TOPO:~OGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~ ;yu~:N~: DESCRIPTION: SEQ ID NO:26:
2 0 Arg Arg Trp Trp Cys Asp
(2) IN-FORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino~ acids
(B ) TYPE: amino acid
(C) sTR~Nn~nNE~c single
(D) TOPO:~OGY: linear
(ii) MO~ECUIE TYPE: peptide
WO 95/16702 ; ~ ` 2 1 7 8 9 2 7 ~ Y~1~5
--93--
(Xi) ~i~;UU~!;N~:~; DESCRIPTION: SEQ ID NO:27:
Arg Arg Trp Trp Cy8 Glu
(2) INFORMATION FOR SEQ ID NO:28:
U~:N~ CH~RACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STR~NnEnN~q~q single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(Xi) S~ U~;N~:~; DESCRIPTION: SEQ ID NO:28:
2 0 Arg Arg Trp Trp Cy9 Phe
(2) INFORMATION FOR SEQ ID NO:29:
U~:N~; CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STR~Nn~nNE.qq: single
(D) TOBOLOGY: linear
(ii) MOLECULE TYPE: peptide
W0 95/l6702 ; . ~ , 2 1 7 8 9 2 7 ~ Y~/12~45
.
--94--
(xi) SEQUENOE DESCRIPTION: SEQ ID NO:29:
Arg Arg Trp Trp Cys Gly
(2) INFORMATION FOR SEQ ID NO:30:
( i ) SEQUENCE rM~R ~ rT~R T .~T I CS:
(A) I.ENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STR~ND~nN~ : single
(D) TOPOLOGY: linear
(ii) MO~ECUIE TYPE: peptide
(Xi) ~;~uu~;N~; DESCRIPTION. SEQ ID NO:30:
2 0 . Arg Arg .Trp Trp Cy5 His
(2) INFORMATION FOR SEQ ID NO:31:
UJ:~N~:~; rM7~Rz~rTliRT~TIcs
(A) ~ENGTH: 6 amino acids
(B) TYPE: amino acid
(C) sTR~Nn~nN~ single
( D ) TOPOI-OGY: l inear
(ii) MOT.ECU~E TYPE: peptide
wo95116702 .. ~. ~ " ~ I. 2 1 78927 PCrlUS93112245
_95_
(Xi) ~ U~;N(~ DESCRIPTION: SEQ ID NO:31:
Arg Arg Trp Trp Cys Ile
(2) INFORMATION FOR SEQ ID NO:32:
U~;N~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRZ~NT)~nl~cs: single
( D ) TOPOLOGY: l inear
15 .
(ii) MOLECULE TYPE: peptide
(xi).SEQUENCE DESCRIPTION: SEQ ID ~0:32:
2 0 Arg Arg Trp Trp CYB Lys
(2) INFORMATION FOR SEQ ID NO:33:
;52U~;N~ RZ~l~T~RT.'~TICS
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
3 0 ( C) STR ~Nn~:nN~.~.S: S ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
WO 95/16702 ' ` '' r ! ~ 2 ~ 7 8 9 2 7 P~ ~ Y~I~s
-96-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Arg Arg Trp Trp Cy9 Leu
(2) INFORMATION FOR SEQ ID NO:34:
;UU~;NC:~: CHAR~CTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANn~.T)N~ : single
(D) TOPOLOGY: linear
(ii) MOI,ECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
' A~g Arg Trp Trp Cys Met
(2) INFORMATION FOR SEQ ID NO:35:
;UU~;N(.I~ R1~'T~RT~cTIcs
(A) LENGTE~: 6 amlno acids
(B) TYPE: amlno acid
(C) STR~Nn~nN~c~s: single
(D) TOPOLOGY: linear
(ii) MOLECULE TY~E: peptïde
~0 95116702 ;~ t 7 8 q 2 7 . ~ Y~1~5
.
-97-
(Xi) Y~;l"U~;N~; DESCRIPTION: SEQ ID NO:35:
Arg Arg Trp Trp Cys Asn
( 2 ) INFORMATION FOR SEQ ID NO: 3 6:
(i) SEQUENOE (~T~RZ~('T~.RT.C~TICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRhNn~ : single
(D) TOPOLOGY: linear
(ii) MO~ECULE TYPE: peptide
(xi) Y~:~U~:N~; DESCRIPTION: SEQ ID NO:36:
2 O Arg Arg Trp Trp Cy5 Pro
(2) INFORMATION FOR SEQ ID NO:37
(i) Y~;~,2U~N-:~; CHARACTERISTICS:
(A) LENGTH: 6 amino acids =
(B) TYPE: amino acid
(C) STR~Nn~nNF~: single
( D ) TOPOI.OGY: 1 inear
(ii) MOLECULE TYPE: peptide
,
WO 95/16702 ' ". . 2 i 7 8 q 2 7 1 ~ Y.~/1~45
--98--
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Arg Arg Trp Trp Cys Gln
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acia
( C ) STR ;~NnRnNR.~: B lngl e
( D ) TOPOLOGY: l i near
(ii) MOLECULE TYPE: peptide
(xi). ~;5.?U~;N~'~; DESCRIPTION: SEQ ID NO:38:
,20 Arg Arg Trp Trp Cys Ser~
25 (2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STR;~NnF.nNF.. q.~: single
( D ) TOPOLOGY: l inear
( ii ) MOLECULE TYPE: peptide
t .~ 2 ~ 78927
WO ~5/16702 . ' ~ 7J~ 15
_99 _
(xi) ~:Uu~N~:~ DESCRIPTION: SEQ ID NO:39:
Arg Arg Trp Trp Cys Thr
(2) INFORMATION FOR SEQ ID NO:40:
(i) ~i~'UU~N(:~; CHARACTERISTICS:
(A) I-ENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STR~Nn~llN~CS single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(Xi ) ~;~;UU~iN~:~; DESCRIPTION: SEQ ID NO: 4 0:
Arg Arg Trp Trp Cys Val
' 1 5
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 arnino acids
(B) TYPE: amino acid
(C) STRANDEDN-ESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
WO 95/16702 ~ 2 ~ 7 8 ~ 2 7 r~ Y~112245
-100 -
(xi) SEQUENCE DESCRIPTIO~: SEQ ID NO:41.
Arg Arg Trp Trp Cys Trp
(2) INFORMATION FOR SEQ ID N~:42:
(i) SEQUENCE CHA~ACTERISTICS:
(A) LENGTH: 6 amino - acids
(B) TYPE: amino acid
(C) sT~NnFn~qq single
(D) TOPO~OGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~ U~N~:~ DESCRIPTIO~: SEQ ID NO:42:
2 0 Arg Arg Trp Trp Cys Tyr
(2) INFORMATION FOR SEQ ID NO:43:
U~:N~:~; CHARACTERISTICS:
(A) ~ENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
( D ) TOPOLOGY: l inear
(ii) MO~ECULE TYPE: peptide
O 95116702 ; ~ , 2 1 7 8 9 ~ 7 1 ~.,V~Y~1~4s
-101-
(xi) SEQUENOE DESCRIPTION: SEQ ID NO:43:
- Arg Xaa Xaa Xaa Xaa Xaa
( 2 ) INFORMATION FOR SEQ ID NO: 44:
;Uu~:N~:~; CXARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STR~NnRnNR.~.~: single
(D) TOPO~OGY: linear
(ii) MO~ECULE TYPE: pep~ide
(xi ) ~;Uu~;N~:~; DESCRIPTION-: SEQ ID N-O: 44:
2 0 Xaa Xaa Xaa Xaa Xaa Arg
(2) INFORMATION FOR SEQ ID NO:45:
( i ) SEQJENCE CE~RACTERISTICS:
(A) ~ENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STR~NnRnNR~S: single
(D) TOPOLOGY: linear
, (ii) MOl.ECl~E TYPE: pep:ide
WO 95116702 ` i ~ 2 1 7 8 9 2 7 ~ Y3/1~45
-102 -
(xi) ~i~;UU~N~ DESCRIPTION: SBQ ID NO:45:
Xaa Arg Xaa Xaa Xaa Xaa-
( 2 ) INFORMATION FOR SEQ ID NO: 4 6:
(i) ~i~;UU~;N~,~; CHPRACTERISTICS:
(A) LENGTH 6 amino- acids
(B) TYPE: amino~acid
(C) STRA~ n~ qq: single
(D) TOPOI-OGY: linear
( ii ) MO~ECU~E TYPE: peptide
(xi) ~;UU~N~:~; DESCRIPTION: SEQ ID NO:46:
2 0 Xaa Xaa Trp Xaa Xaa Xaa
l 5
( 2 ) INFORMATION FOR SEQ ID NO: 47:
UU~;N~:~ CHARACTERISTICS:
(A) :~ENGTE~: 6 amino acids
(B) TYPE: amino aci~
(C) STRZ~N~ ]N~:qq: slngle
(D) TOPO~-OGY: linear
(ii) MOl-ECULE TYPE: peptide
WO95/16702 . , ~ 2 1 78927 P. ~ s
- 103 -
(Xi) ~;~;5,)U~;N(.~; DESCRIPTION: SEQ ID NO:4~:
Xaa Xaa Xaa Trp Xaa Xaa
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) sTRAN[)~n~qs: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
2 0. Xaa Xaa Xaa Xaa Cys Xaa
(2) INFORMATION FOR SEQ ID NO:49:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STR~ m~ : single
( D ) TOPOLOGY: 1 inear
(ii) MO~ECULE TYPE: peptide
WO 9!;/i6702 1- . 2 17 ~ ~ 2 ~ r~ ~s
-104 -
(Xi) ~ ,2U~;N~:~ DESCRIPTION: SEQ ID NO:49:
Glu Leu Arg Cys Gln Cys Ile ~ys Thr Tyr
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino aci~
(C) STRANDEDNESS: single
( D ) TOPOLOGY: l inear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5Q:
Glu Leu Arg Ser Gln Ser Ile Lys Thr Tyr
lQ
25 (2) INFORMATION FOR SEQ ID NO:51:
( i ) ~i~;y U~;N ( :~; CH~RACTERIST I CS:
(A) LENGTH: 10 amino acids
(B ) TYPE: amino acid
(C) STR~NnF.nNF.. ~.c single
( D ) TOPOLOGY: l i near
, (ii) MOLECULE TYPE: peptlde
WO95116702 ; ~ ;. 21 78q27 .~1/",Y,~ 45
- 105 -
(xi) SEQUENCE DESCRIPTION: SEQ ID l`J0:51:
Glu Leu Arg Met Gln Met Ile Lys Thr Tyr
~2) INFORMATION FOR SEQ ID NO:52:
SyU~;NI_:~; CHA~ACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECIJLE TYPE: peptide
(xi) ~;~;yU~;N~; DESCRIPTION- SEQ ID NO:52:
- Gln Ile Pro Arg Arg Ser Trp Cys Arg Phe Leu Phe
(2) INFORMATION FOR SEQ ID NO:53:
;yU~;N( :~; CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
WO 95/16702 `~ J "~ 2 1 7 8 9 2 7 1 ~ y~ s
-106-
(Xi) ~ U~N(~ DESCRIPTIQN: SEQ ID NO:53:
Gly Trp Arg Arg Trp Trp Cy9 Asp Ala Val Leu Tyr
l Q
~2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) s~R~n~n~r~qq ~ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(Xi) ~;(,2U~;NO~; DESCRIPTION: SEQ ID NO:54:
20. I,y8 Glu Leu Arg CYE: Gln
25 (2) INFORMATION FOR SEQ ID NO:55:
(i) ~;~;(~U~;N~ R;~TERT~qTIcs:
(A) LENGTH: 13 amino acid~
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: line~:r
(ii) MOLECULE TYPE: peptide
W<)95116702 '~ 2178927 1~ Y.~ 45
.
-107-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
- Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys Pro Val
(2) INFORMATION FOR SEQ ID NO:56:
(i) iY~;UU~;N~; CH~RACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STR~NnFnNFcs gingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~;UU~;N~:~; DESCRIPTION: SEQ ID.NO:56:
2 0 ' Arg Arg Trp Trp Cys
(2) INFORMATION FOR SEQ ID ~0:57:
(i) SEQUENCE ('T~R~T~RT.~TICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STR~NnFnN~ : single
( D ) TOPOLOGY: l inear
(ii) MO~ECULE TYPE: peptide
WO 95116702 ; i ~ ~ - 2 1 7 8 9 2 7 ~ Y~ 4~
.
-108 -
(xi) Y~;UU~;N~:~; DESCRIPTION: SEQ ID NO:57:
Arg Arg Trp Trp Cy8 Xaa Xaa
(2) INFORMATION FOR SEQ ID NO:58:
;UU~;N(~ R~(~T~RT~TICS:
(A) LENGTH: 6 amino acids
(B) TYPE: anlino acid
(C) STRz~Nn~nN~ : ~ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
- Arg Arg. Xaa Trp Cy8 Xaa
