Note: Descriptions are shown in the official language in which they were submitted.
WO95/10532 PCTIGB94/02230
~ - 217385~
COMPOUNDS USEFUL IN ANTI-ALLERGY TREATMENT
Backqround of the invention
l. Field of the invention
The invention relates to compounds for use in anti-allergy
treatment and, particularly, to a class of peptides useful in
treating IgE-mediated allergies.
2. Description of the related art
Allergic symptoms are brought about through the release of
vasoactive amines (mediators), notably histamine, from cells
into the surrounding tissue and vascular structures. Histamine
is normally stored in special cells known as mast cells and
basophil leucocytes. The mast cells are dispersed throughout
animal tissue whilst the basophils circulate within the vascular
system. Th~se cells manufacture and store histamine within the
cell unless a specialised sequences of events occurs to trigger
its release.
The role of immunoglobulin E (IgE) antibodies in mediating
allergic reactions is well known. IgE is a complex arrangement
of polypeptide chA;n~ which, as in other immunoglobulins
consists of two light and two heavy chains linked together by
disulphide bonds in a "Y" shaped configuration. Each light
chain has two domains, one variable (VL) ~0~ i n linked to a
domain with a relatively invariant amino acid sequence termed
a constant domain (CL) . Heavy chains, by contrast, have one
variable domain (VH) and in case of IgE, four constant domains
(CHl, CH2, CH3, CH4, also known as CE1~ C~2, C~3, C~4). ~he two
"arms" of the antibody are responsible for antigen binding,
having regions where the polypeptide structure varies, and are
termed Fab' fragments (fragment - antigen - binding) or F(ab')2
which represents two Fab' arms linked together by disulphide
bonds. The 'Itail'' or central axis o~ the antibody contains a
fixed or constant sequence of peptides and is termed the Fc
fragment (fragment - crystalline). The Fc fragment contains the
.
WO95/10~32 PCT/GB9~/02230
2 ~ 7 ~ 8 ~ ~
antibody's biologically active sites which enable the antibody
to communicate with other immune system molecules or cells by
binding to their Fc receptors. Fc receptors are molecules which
bind with high affinity and specificity to molecular active
sites within immunoglobulin Fc regions. Fc receptors may exist
as integral membrane proteins within a cell's outer plasma
membrane or may exist as free "soluble" molecules which freely
circulate in blood plasma or other body fluids. Figure l of the
drawings shows the structure of an antibody molecule and the
location of the antigen binding sites (Fab' arms), the Fc
fragment, and the active sites which includes the cell binding
site.
Active sites, depending on their function, may already be
exposed and therefore able to bind to cellular receptors.
Alternatively, they may be hidden until the antibody binds to
the antigen, whereupon the antibody may change in structure and
subsequently expose other active sites which can then trigger
a specific~,immune activity.
The allergic (immunologic) release of histamine within the
organism from the mast cells and basophils can only occur under
the following circumstances. An IgE molecule must lock onto or
attach itself at its Fc end to the cellular Fc receptor site,
thus securing the IgE molecule to the mast cell or basophil
(Figure 2a~. The Fab' portions of the cell-bound IgE molecules
must be cross-linked by a particular compatible antigen (the
allergen). Should such an interaction occur (Figure 2b), the
mast cell or basophil is automatically triggered to release
histamine to the local environment, manifesting familiar
allergic symptoms.
Conventional approaches to allergy treatment have involved
systemic therapy withanti-histamines orattempts to desensitise
patients, approaches with have not addressed themselves to the
basic IgE-mast cell/basophil interaction.
Other prior art has concerned itself with the production of
polypeptide ch~; n.~ capable of blocking the binding of the IgE
WO95/10532 PCT/GB94102230
1 7 3 8 5 6
antibody to the Fc receptors on the cell surfaces and displacing
IgE from binding sites upon which IgE is already bound (Figure
3).
In PCT Patent Application WO 90/15878 (Stanworth, et al) it has
been suggested that a certain part of one of the constant
~om~in~ of human IgE, namely amino acid residues 497-506 of the
C~4 chain, acts as an effector of histamine release, but only
when the IgE has undergone a postulated change in conformation
as a result of interaction with the allergen.
Assuming the validity of the "second receptor" hypothesis, it
is not clear how to prevent the interaction between the
postulated "effector site" on the anaphylactic (IgE) antibody
molecule and this llsecond receptor" on the cell surface.
Presumably the cross linking of mast cell bound IgE antlbody
molecules by a specific antigen (allergen) induces a
conformational change within their Fc regions bringing the
"effector ~site" into close juxtaposition with the "second
receptor". In this event, any attempt to block this interaction
could encounter problems of steric hindrance in the region of
the supposed effector site.
The patent application proposes an anti-allergy treatment based
on in vivo stimulation of production of antibodies to a peptide
of sequence which is identical with or similar to tha~ of the
"497-506" effector portion of the IgE. This effector sequence
(SEQ ID NO: l) is as follows:
Lys Thr Lys Gly Ser Gly Phe Phe Val Phe
l 5 lO
The effector peptide, which has SEQ ID NO: l, is administered
in some suitably "non-self" form, e.g. as a conjugate to a
protein. Antibodies and polyclonal antiserum to this peptide
have also been shown to inhibit IgE-mediated allergic reactions.
With the exception of Stanworth, generally the prior art has
concerned itself with the development of "blocking peptides" for
WO95/1053~ PCT/GB9~/02230
; 2~ 73856 ~
-
the prevention of the binding o~ IgE to mast cells and
basophils. The development o~ ~anti-binding site antibodies'~,
has elucidated the FC binding site and subse~uently, blocking
anti-peptides were developed [Burg, E. et al, Molecular
Immunology, 24, pp. 379-389 (1987)].
Hahn (WO 86/01211: Merck Patent GmbH) sugges~ed that
immunotherapeutic agents might be ~ound amongst a class o~
compounds which block immune complex binding to immunoglobulin
Fc receptors and/or which block immunoglobulin binding to
lymphocyte FC receptors. Hahn's reason for looking at putative
FC blocking peptides is stated as ~ollows:
"It is only after antibodies bind to antigen or are
otherwise caused to aggregate that active sites within the
Fc region are able to bind to and activate Fc receptors. FC
receptors are, therefore, the critical link between
antibodies and the r~m~in~er of the immune system. FC
receptor,binding to antibody FC region active sites may thus
be characterized as the "final common pathway" by which
antibody functions are mediated. If an antigen-bound
antibody does not bind to an Fc receptor, the antibody is
unable to activate the other portions of the immune system
and is therefore rendered functionally inactive.
Any peptide with the ability to bind to immunoglobulin Fc
receptors has therapeutic usefulness as an immunoregulator
by virtue of the peptide's ability to regulate binding to
the receptor. Such an Fc receptor "blocker" occupies the
immunoglobulin-binding site o~ the Fc receptor and thus
"short circuits" the immunoglobulin's activating ability".
Hahn lists many thousands of compounds in the patent
application, all of which are alleged to be active site
compounds which block immune complex binding to Fc receptors.
However, there is no teaching of any theory or principle of
selection of e~ective Fc binding "blocker" compounds.
It is known that the IgE antibody is often firmly bound to the
WO95/10532 PCT/GB94/02230
~ ~ r~ 2 1 7 3 8 5~
~ .
mast cell or basophil [Stanworth, D.R., Nature 233, pp. 310-316
(1971)], even without the presence of an allergen, and it is
only when the allergen is present that the supposed "second
receptor" is triggered and the histamine is released. Thus,
following the teaching of ~ahn, merely blocking the site at
which IgE binds to the mast cells would inhibit IgE function of
only those IgE molecules which ~reely circulate and are not yet
attached to the mast cells or basophils. This approach would
be unsuitable where cell-bound IgE is already present, unless
such a blocking peptide is also able to displace already bound
IgE. This approach was taken by Hamburger who reported that a
pentapeptide from the ~H2 domain of human IgE was capable of
competing with IgE specific binding sites on mast cells in human
skin [Hamburger, R.N., Science 189, pp. 389-390 (1975)]. This
result could not however be confirmed by other investigators
[Bennich, H.H., et al, Int. Arch. Allergy Appl. Immunol., 53,
pp. 459-468 (1977)]. It is to be noted that a patient who has
already been exposed to allergen will have allergen-specific
cell-bound,IgE.
The leading anti-allergy drugs are disodium cromoglycate (DSCG)
and nedocromil sodium, marketed by Fisons PLC as "Intal" and
"Tilade". It is desirable to find alternatives to these drugs
for the benefit of patients who do not tolerate them well and
because their insolubility limits the methods by which they can
be ~mi n; stered, e.g. by inhaler or aerosol respectively.
Summary of the invention
It has now surprisingly been found that certain short peptides
show anti-allergic activity when administered as they are, i.e.
without any attempt to raise antibodies to them. These peptides
comprise two negatively charged amino acid residues, especially
of glutamic acid (Glu), separated by an uncharged amino acid
such as proline (Pro) or glycine (Gly). Three-~;m~nqional models
suggest strongly that the mode of action of these peptides is
to bind to a part of the effector peptide, SEQ ID NO: 1, namely
to the Lys Thr Lys region. The evidence of modelling suggests
strongly that negative charges on the peptides of the invention
WO95/10532 PCT/GB9~/02230
~ J ~ ~s 2173856
bind strongly to the doubly charged Lys Thr Lys "head" of the
effector site, thus neutralising its effector activity and
therefore the release of histamine triggered by the action of
the IgE on mast cells.
~,
A further constraint is a requirement ~or the residue or
residues spacing the negatively charged groups of the compound
according to the invention to react via non-covalent forces with
the Thr residue separating the key Lys residues of the peptide
SEQ ID NO: l (the effector or "trigger" peptide).
From these considerations, it follows that other small
compounds, which are not peptides, but which have negatively
charged groups or atoms spaced apart by about the same distance
as those in the preferred tripeptide Glu Pro Glu, will act in
the same way to combat IgE-mediated allergies. These
considerations are made even more credible by the fact that the
marketed drugs DSCG and nedocromil sodium have negacive charges
spaced by 1~5 and lO Angstrom units (l.5 and l.0 nm) respectively
and that these separations are similar to those ln the said
tripeptide.
In a first aspect, therefore, the present invention provides
small compounds for use in the treatment of IgE-mediated
allergies comprising a first negatively charged atom or group
and a second negatively charged atom or group, separated by a
spacing group effective conformationally to position said
negatively charged atoms or groups so that they will neutralize
the lysine residues of the amino acid sequence Lys Thr Lys at
positions 497-499 of the C~4 constant domain of cell-bound human
IgE, but excluding any compounds already known for this purpose
such as DSCG and nedocromil sodium and toxic compounds. For the
purposes of this application the term "small" is defined as a
molecular weight (MW) less than 1200.
In a second aspect, the present invention provides short
peptides of the following formula I (in which the left-hand side
represents the N-terminus and the right-hand side the C-
terminus):
WO95/10532 -` ~ PCT/GB94/02230
~ 2 ~ 73856
R1m Xaa1 Sp Xaa2 R2n (Formula I)
wherein:
represents a residue of an amino acid or of a sequence of
two or three amino acids, which may be the same or different;
R2 represents a residue of an amino acid or of a sequence of two
or three amino acids, which may be the same or different;
(but preferably any amino acid residue of R1 or R2 adjacent to
an Xaa residue is neither positively nor negatively charged);
Xaa1 represents a residue o~ a negatively charged amino acid,
preferably Glu;
Sp represents a spacing residue, preferably of a noncharged
amino acid, preferably Pro, or of a non-charged dipeptide, which
provides the spacing required for the negatively charged groups
of the Xaa residues to be sufficiently proximal to the lysine
residues of the amino acid sequence Lys Thr Lys at positions
497-499 of~he C~4 constant domain of cell-bound human IgE to
neutralise them;
Xaa2 represents a residue of a negatively charged amino acid,
preferably Glu; and
m and n denote the number of amino acids in R1 and R2
respectively and each of m and n independently is 0, l, 2 or 3,
and the sum o~ m plus n is ~rom 0 to 3;
and their terminal functional derivatives. For the purposes of
this application the term "short" is defined as 6 or less amino
acids (aa).
The invention includes such peptides both ~er se and for use in
therapy, especially for treating IgE-mediated allergies. Where
legally permissible, the invention includes a method of treating
a patient having symptoms of an IgE-mediated allergy, which
comprises administering to said patient a therapeutically
effective amount of a com~ound as defined above, especially a
peptide as defined above.
Further aspects of the present invention are defined in the
claims.
WO95/10532 2 ~ 7 3 8 5 6 PCT/GB9~/02230
i- r ~
... ,. _
The term '~terminal functional derivative" as used herein
includes any derivative formed by reaction of the terminal amino
or carboxy group and which does not alter adversely the
character imparted to the peptide by the other amino acid
residues. Thus, any conventional derivative, especially N-
acetyl and/or C-amide, can be used in this invention.
Description of the preferred embodiments
The peptides used in this invention have a length of from 3 to
6 amino acid residues (or spacing e~uivalent when Sp is not an
amino acid or dipeptide residue).
The essential feature of the peptides used in this invention is
that they should contain two negatively charged amino acids
spaced apart by an appropriate spacer group. The spacer group
should perform the function of spacing the negative charges on
the negativ~ely charged amino acids, so that they lie adjacent
to the positive charges on the lysines of the IgE chain referred
to above. The positive charge in lysine lies on the amino group
nitrogen atom of a 4-aminobutyl side chain. Computer modelling
by the inventors predicts that these nitrogens lie about 1.4 nm
apart and that the negative charges in DSCG and nedocromil
sodium are about 1.5 and 1.0 nm apart respectively, suggesting
that these distances are about the maximum and m;nimllm spacings
for neutralisation of the lysines. In the preferred tripeptide
Glu Pro Glu, the spacing between the negatively charged oxygens
in the 2-carboxyethyl side-~hAin~ of glutamate is about 1.3 nm.
Here, the spacing group is the single amino acid residue
proline. If another amino acid were included in the spacing
group, i.e. if it were a dipeptide, the spacing between the
negative charges on the glutamates would increase by about the
length of two C-N bonds, roughly 0.3 nm, to 1.6 nm. which is a
little too great for the best neutralising effect.
However, if the negatively charged amino acids were aspartates,
which have shorter, carboxymethyl, side ~h~in~, the spacing
group would have to be made correspondingly longer, i.e. usually
WO95/10532 ~ f i,~; ' 2 1 7 3 8 5 6 PCT/GB9~/02230
a dipeptide residue. It would be possible for the peptides to
have different negatlvely charged residues, such as one
glutamate and one aspartate, but this particular combination may
give rise to some spacing problems as the single amino acid
spacer is a little short, while the dipeptide spacer is a little
long, although it is likely that the effects of the invention
would be obtained in some degree with either. Using these
guidelines, the person of average skill in the art will be able
to obtain the benefits of this invention for a variety of
different peptides.
The amino acids herein can have the (D) or (L) configuration,
but are preferably selected from the 20 naturally occurring (L-)
amino acids. Thus, the preferred negatively charged amino acids
are aspartic (Asp) or glutamic (Glu). The spacing group Sp
preferably comprises (consists of when it is a single amino acid
residue or includes when it is a dipeptide residue) proline
(Pro) or a ring-substituted derivative thereof.
~'
The peptides are thought to bind to the patient's IgE, after the
IgE has supposedly undergone a conformational change as a
consequence of interaction with the allergen, thereby blocking
its subsequent action of triggering mast cells. Such triggering
initiates release of hist~mine~
Therefore, it is considered essential to the invention that the
two negatively chargedamino acid residues, appropriatelyspaced
apart, should be presented to the IgE. Accordingly, the peptide
should not contain any amino acids which are likely to interfere
with such presentation or cause the peptide to bind to other
proteins. Thus, the spacing group preferably does not comprise
cysteine, which might cause binding to other proteins.
The peptide need not be any longer than a tripeptide, as in
preferred peptides Glu Pro Glu and Glu Gly Glu, but it can have
additional, flanking amino acid(s) at either or both sides of
the essential motif Xaal Sp Xaa2. The nature of any flanking
amino acid or peptide chain represented by Rl or R2 is not
critical to the invention, although an amino acid externally
adjacent to either of the negatively charged amino acids, i.e.
WO95/10532 , ~ 2 ~ 73 8 5 6 PcT/GBg~/0223n
to the N-terminus of Xaal or to the C-terminus of Xaa2 is
preferably not negatively charged, especially not Glu or Asp,
or positively charged, especially not Lys, Arg or His. From the
viewpoint of ease or synthesis, it is preferred that any
flanking amino acids be the same within each flank or both
flanks, e.g. as in another peptide of the invention, having the
sequence (SEQ, ID NO: 2):
Ala Ala Ala Glu Pro Glu
l 5
The peptides of the invention may be prepared by standard
methods, e.g. the well-known Fmoc. method.
The peptides used in the invention are preferably administered
as such, i.e. without any carrier such as a conjugated protein
or a branched peptide type of carrier, as it is not desirable
that the patient should raise antibodies against them. It may
well be desirable, however, that they be administered in a slow
release or depot form, for example in liposomes or nanoparticles
(isobutyl and isodecylcyanoacrylate carriers used for anti-
cancer drugs).
The peptides used in the invention may be ~m;n; stered by any
of the conventional routes of giving drugs, for example orally,
by intravenous or intramuscular injection or as eyedrops or as
a nasal spray.
Pharmaceutical compositions of the invention comprise a peptide
of the invention and a carrier or diluent. Oral preparations
include solid forms such as tablets, pills, lozenges and
capsules, and liquid forms such as solutions in containers and
sprays, e.g. nasal and throat sprays, all of which can be
formulated in any conventional way.
Injectable preparations include sterile solutions, especially
in physiological saline. As the peptides are readily soluble,
they can be ~m;n; stered very easily in a liquid form.
WO95/10532 ~ ~ 2 1 7 3 8 5 6 PCT/GB94102230
It will usually be desirable to give the peptide in several
doses over an extended period, each dose typicaily containing
~ from 0.01 to 1 mg. of the peptide. The peptide is best given
prophylactically, so as to be available to block the IgE
effector site immediately the allergen binds to the IgE, but the
invention also includes therapeutic treatment.
Turning now to the non-peptide compounds for use in treating
allergies, the compounds should aim to mimic the tripeptide Glu
Pro Glu structurally and therefore one class of preferred
compounds has the general formula (II):
MOOC-CH2-CH2-Y-CH2-CH2-COOM (II)
wherein M represents a hydrogen atom or a non-toxic cation such
as sodium or potassium and Y represents a group which spaces the
two 2-carboxyethyl groups apart so that the negative charges
(when in anionic form) are separated by a distance of from about
1.0 to less than 1.6 nm. Roughly stated, the length of Y is
about equal to six saturated C-C or C-M bond lengths, about 0.15
x 6 = 0.9 nm. The Y residue could simply be an aliphatic chaln,
as in a 1,5pentylenediamine N,N-di-(3-propionate), but
preferably has some rigidity to mimic the planar carbonyl groups
in peptide linkages and possibly additional rigidity as
con~erred by the pyrrolidine ring in the preferred proline
spacer amino acid. This may be provided by at least one
carbocyclic or heterocyclic ring within Y, as in 1,2-
cyclopentylene and 1,2-pyrrolidinylene compounds or in bicyclic
ring systems.
The compounds must, of course, be non-toxic and stable to rapi~
metabolic degradation. They may be organic or organometallic
in nature.
The formulation and administration of the non-peptide compounds
are in principle no different from that of the peptides, as
described above, but variations and limitations may be necessary
according to the type of compound chosen.
WO95/10532 ~ 2 ~ 73 8 5 6 PCT/GB9~/02230
In particular, the dosage may need to be higher, e.g. dosages
of 1 to 100 mM may be required.
The following Examples illustrate the invention.
EXAMPLE 1
This Example relates to the testing of the peptide Glu Pro Glu
for anti-allergy activity.
Part A: In vitro rat mast cell test
Rats were experimentally sensitised by injection with allergen
(ovalbumin) and Bordetella Pertussis, employing a regimen
previously described by D.R.Stanworth et al., The Lancet. 336,
1279-1281 (1990). Their peritoneal mast cells were isolated by
washing out the peritoneum twice with cold (4 deg. C.) calcium
ion-free HEPES-buffered Tyrode salt solution (HBT), pH 7.2. (HBT
contains 13,.7 mm sodium chloride; 2.7 mM potassium chloride;
0.64 mM sodium dihydrogen phosphate dihydrate; 0.05 mM magnesium
chloride hexahydrate; 0.1 mM calcium chloride dihydrate; 1 mM
HEPES; 0.56 mM D-glucose and 1 mg./ml. gelatin). The resulting
cell suspension, at 4 deg. C., was centrifuged for 5 minutes at
2000 r.p.m., the supernatant was discarded and the solid residue
re-washed with the cold buffer. This process was repeated three
times. If the supernatant was not clear at the end of the final
wash, the cells were discarded. The cells were finally re-
suspended in HBT buffer, this time containing 0.1 mM calcium
ions, provided as the chloride. The cells were then
recentrifuged and resuspended in the calcium ion-containing HBT
buffer at a concentration o~ about 20,000 cells per 100
microlitres in 1.5 ml. tubes.
The mast cells were incubated with different concentrations of
the peptide under test for 15 minutes at 37 deg. C. An allergen
to which rats had been experimentally sensitised, namely
ovalbumin, was then added, as a 0.5 mg./ml. solution in calcium
ion-containing HBT buffer and the cells were then incubated for
a further 15 minutes at the same temperature.
W095/10532 ~ r ~ - PCT/GB91tO2230
~ 1~ 2 ~ 7385~
At the end of the period of incubation, l ml. of cold calcium
ion-free HBT buffer was added to each sample to quench the
reaction and the tubes were then centrifuged at low speed in an
MSE Microfuge centrifuge for l minute. The supernatants were
removed by fine-tipped pipettes and added to tubes containing
l ml. of 0.8M perchloric acid. The tubes were then centrifuged
at 2,000 r.p.m. for 5 minutes and stored at 4 deg. C. overnight
or at -20 deg. C. over longer periods. l ml. of 0.4M perchloric
acid was added to the pellet. The tube was then whirled for 20
seconds to mix th~ contents and sonicated for 5 minutes to
thoroughly lyse the cells. The cells were then centrifuged in
the MSE Microfuge at high speed for l minute. The cell pellets
thus obtained were stored as described above for the
supernatants.
The percentage of histamine recovered from the mast cell
supernatants and lysed pellets by solvent extraction was then
reacted with ortho-phthalaldehyde to produce a fluorophore and
measured using a spectrafluorimetr-.-= assay, as first described
by D.P.Evans et al., Life Sciences ~~2, Part II, 327-336 (1973).
The amounts of histamine in the supernatants, i.e. released by
the samples incubated with the peptide, were expressed as a
percentage of the total histamine available (from supernatants
plus cell pellets) and a percentage inhibition was determined
by reference to the amount of histamine released by a control
sample to which buffer was added without any peptide. The
results are shown in Table l below. All results are the mean
of duplicates.
WO95/10532 PCTIGB9~/02230
5 . 2 ~ 73856
Table 1: Inhibition of histamine release ~rom actively
sensitised rat peritoneal mast cells
Conc. of test Histamine released Inhibition
pe~tide (mM) (as ~ total available) (as ~ of
control)
3.20 12.0 45.9
1.60 11.3 49.1
0.80 12.6 43.2
0.40 11.2 49.8
0.20 12.8 42.3
0.10 13.4 39.6
0.05 13.1 41.0
Control 22.2 0.0
(buffer only)
Part B: Repeat of in vitro rat mast cell test, with the addition
of a comparison with nedocromil sodium
The test of Part A was repeated, but using different
concentrations of the peptide and also performing runs in which
the cells were incubated with various concentrations of the
known drug nedocromil sodium. The results, which are the mean
of duplicates, are shown in Table 2 below.
WO95/10532 ~ . . ; PCTtGB94/02230
2~ 738~
Table 2: Inhibition of histamine release from actively
sensitised rat peritoneal mast cells
Conc. of test Histamine released Inhibition
comDound (mM) (as ~ total available) (as ~ of
control)
Glu Pro Glu
1.300 27.2 22.1
0.540 23.6 32.4
0.270 25.6 26.7
0.135 22.4 35.8
0.027 21.0 39.8
0.014 30.1 13.7
0.003 33.6 3.7
Nedocromil
sodium
2.50 18.4 47.3
1.25 21.6 38.0
0.25 23.5 32.7
Control 34.9 o.o
(buffer only)
W095/lOS32 2 ~ 7 3 8 5 6 PCTIGB9~/02230
. -; ,. ~
EXAMPLE 2
This Example relates to the testing of the peptide Glu Pro Glu
with various other substances for comparison, by the method of
Example 1. The results shown in Table 3 below are the mean of
duplicates.
Table 3: Inhibition of hist~ e release in actively sensitised
rat peritoneal mast cells
Peptide ~ inhibition of histamine release
under at peptide concentrations, mM
test 3.0 1.0 0.33 0.17
Glu Pro Glu 8.2 28.7 30.3 23.8
Comparative ~ inhibition of
aqents histamine release
Murine monoclonal
antibody to peptide
SEQ ID NO: 1 30.3
(diluted l:lOO)
Rat antiserum to
peptide SEQ ID NO: 1
(undiluted) 23.8
Nedocromil sodium
(0.25 mg./ml. = 0.6mM) 23.8
It will be seen that the preferred Glu Pro Glu peptide of the
invention gave at least as good a result as nedocromil sodium
and the antisera to the peptide of SEQ ID NO:1 mentioned above.
=~
WO95/10~32 ~ PCT/GB94/02230
-~ 2~ 7385~
EXAMPLE 3
This Example relates to the testing of several peptides of the
invention by a different method, which depends on the inhibition
of allergen-induced histamine release from passively sensitised
human lung mast cells.
A piece of normal human lung tissue (about 7 g.) was stored at
4 deg. C. in calcium ion-free HEPES-buffered Tyrode (CFHBT)
solution until use. The piece of lung was washed three times
with CFHBT to remove any blood cells and blood clots. It was
then cut into 1 cm. x 2 cm. pieces, which were then chopped in
a McIlwain tissue chopper, making five passages of the blade and
employing 2 micrometre cuts on high-speed and medium blade
force.
The resulting chopped tiss-=~e was pooled and aliquots placed into
a 50 ml. tube containinc cold (4 deg. C.) CFHBT. The pooled
chopped ti~Ssue, which prcvided the lung mast cells, was then
washed with the cold CFHBT and the cells thoroughly dispersed
with a plastic Pasteur pipette. The dispersion was centrifuged
at 2000 r.p.m. for 5 minutes at 4 deg. C. The supernatants were
discarded and fresh cold CFHBT added. This washing was repeated
thrice. If the supernatant was not clear by the end of the
final wash, the cells were discarded. Allergic serum from a
rabbitsensitive individual ("IVL") was added to washed chopped
human lung (l ml. serum/g. wet weight of cellsi 1 g. cells/5 ml.
CFHBT). The cells were then mixed for 18 hours at room
te~perature in a Denley Spiramix (which keeps them continuously
suspended as a result of its gentle rolling/rotating motion).
The cells were finally made up in HBT at 4 deg. C., using 100
microlitres of cells (about 50 mg. per 1.5 ml. tube).
It will be seen from Table 4 that in this test the peptides of
the invention gave the same or better results than the marketed
drug nedocromil sodium. While N-acetylation or Camidation
indi~idually did not seem to affect the inhibition of histamine
markedly, there was a reduction in the inhibition when both
termini were blocked in these ways.
WO95/10532 2 1 7 3 8 5 6 PCT/GB9~/02230
e
Table 4: Inhibition of histamine release from passively
sensitised human lung-mast cells
Peptides ~ inhibition of histamine release
under at peptide conce~trations, m~
test 2.0 0.2 0.02 0.002 0.0002 0,00002
Glu Pro Glu 53,7 40.6 51.4 20.0 13.1 15.4
Glu Gly Glu 8.0 10.3
Ac-Glu Pro Glu 45.1 45.1 17.7
Glu Pro Glu-NH2 39.4 22.9
Ac-Glu Pro Glu-NH2 22.9 6.9 17.1
Comparative,a~ent
Nedocromil
sodium 4.0 29,7 11.4
EXAMPLE 4
This example relates to the testing of 24 peptides according to
the invention in a comparison against the known compound
Nedocromil Sodium, Table 5 lists the tested compounds and shows
the charge spacing in Angstrom units A. St~n~Ard single letter
codes for amino acids are used throughout the Table, The test
uses an ELISA screen to determine the concentration (mM) of
compound required to achieve a set level (25~ or 50~) of
inhibition of the peptide SEQ ID NO:1 (the "effector'i peptide
for histamine release).
It can be seen from the results ~hat the peptides of the
invention as shown have effectiveness comparable to that of the
known compound nedocromil sodium, and in some cases the peptides
are more effective (i.e. lower dosage required for .he same
WO95/10532 ~,' - - 2 1 7 3 8 5 6 PCTIGB94/02230
19
level o~ inhibition).
- Table 5:
- Com~ound Charge Inhib. Conc. (mM)
Spacing A 25~ 50
Nedocromil 12.1 0.25 1.25
Sodium
F229 AcEPE 12 0.16 2.7
F237 EGFGE 10.6 0.5 >2
F239 EGFE 14.1 0.26 0.15
F241 EGFK 12.3 0.5
F242 AcEGKE 12 0.4 1.6
F250 EKE 10.2 0.15 0.3
F252 AcEKE 10.2 0.3 >2
F241 AcEGFK 12.3 0.2 0.5
F288 DAAPD 12.8 0.6 0.1
F303 EPPE,~ 14.6 0.4 0.8
F249 EFE 10.2 0.3 1.2
F285 DAPD 11.8 0.15 1.2
F291 DAAAPD 12 0.6 0.2
F254 AEPEA 12.2 0.6 0.5
F236 EEKGE 12.8 1 1.3
F255 EPEA 13.2 0.4 1.1
F259 AEPE 12.1 0.3 1.1
F248 AcEKGE 12.8 0.3 0.8
F251 AcEFE 10.2 0.3 1.2
F228 EGE 13 0.4 0.7
F230 AcEGE 13 0.1 1.5
F235 EGFGE 13.5 0.2 0.5
F78 EPE 12 0.3 1.3
F237 AcEGFGE 13.5 0.5 0.7
WO95/10532 ~ ` 2 1 7 3 8 5 6 PCT/GB94/02230
The following claims define some important features of the
invention but should not be construed as detracting from the
generality of the inventive concepts hereinbefore indicated.
WO95110532 ~ 2 1 7 3 8 5 ~ PCT/GB94/02230
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: PEPTIDE THERAPEUTICS LIMITED
(B) STREET: 321 CAMBRIDGE SCIENCE PARK
(C) CITY: CAMBRIDGE
(D) STATE: CAMBRIDGE
(E) COUNTRY: ENGLAND
(F) POSTAL CODE (ZIP): CB4 4WG
(G) TELEPHONE: 01223 423333
(H) TELEFAX: 01223 423111
(ii) TITLE OF INVENTION: COMPOUNDS USEFUL IN ANTI-ALLERGY
TREATMENT
(iii) NUMBER OF SEQUENCES: 2
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
WO95/10532 ~ 2 1 7 3 8 5 6 PCT/GB94/02230
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
Lys Thr Lys Gly Ser Gly Phe Phe Val Phe
l 5 l0
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
~B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ala Ala Ala Glu Pro Glu
l 5
