Note: Descriptions are shown in the official language in which they were submitted.
WO91/16919 PCT/US91/03026
2a~a~3 i ~
-- 1 --
SYNTHETIC PEPTIDES FOR ARTERIAL IMAGING
The subject matter of this application is a
continuation-in-part of USSN 189,130 and its continuations-
in-part USSN 518,215 and USSN 518,142; filed May 2, 1988;
May 3, 1990; and May 3, 1990; respectively.
BACKGROUND OF T~E INVENTION
The U.S. Government has rights in this invention
pursuant to NIH Grant No. HL3297S.
The invention relates to methods and means useful
for the early detection of vascular disease, such as
atherosclerosis, particularly, methods and means employing
labelled synthetic peptides to detect arterial injury.
Atherosclerosis is a disease which causes the
thickening and hardening of the arteries, particularly the -
larger artery walls. It is characterized by lesions or
raised ~ibrous plaques which form within the arterial lumen.
The plagues are most prevalent in the abdominal aorta, ;~
20 coronary arteries, or carotid arteries, and they increase `
progre5siVely with age. They commonly present dome-shaped,
opaque, glistening sur~aces which distort the lumen. A
lesion typically will consist of a central core of lipid and
necrotic cell debris, capped by a collagen fibromuscular
layer. Complicated lesions will also include calcified
deposits and exhibit various degrees of necrosis, -
thrombosis, and ulceration.
The injury at, or deformities of, the arterial lumen
presented by the plaque and associated deposits result in
30 occluded blood flow, and ultimately in angina, cerebral -
ischemia, renal hypertension, ischemic heart disease,
stroke, and diseases of other organs, if untreated. At
:'-'. .
W O 91/16919 PC~r/VS91/03026
2~89~3
- 2 -
present, coronary atherosclerosis is still the leading cause
of death in the United states, claiming the lives of over a
half million Americans annually, roughly twice as many as
are killed by cancer.
Unfortunately, there are no existing diagnostic
methods which can detect the early stages of atherosclerosis
and related vascular diseases which often are clinically
silent. Since lifestyle changes, drug therapy, and other
means exist for delaying or reducing vascular occlusion or
the stresses on various body organs which result from
atherosclerotic lesions, the early detection of atheromatous
plaques in the vascular system would be of considerable
value in permitting preventive intervention at a time when
it can be most effective.
Arteriography, the conventional approach to
diagnosing vascular disease, involves catheterization and
the injection of radiopaque substances into the bloodstream
in order to image obstructions in the arteries. This
procedure involves significant morbidity, in that infection,
ao perforation of the artery, arrhythmia, stroke, infarction,
and even death can occur. Because of the risks involved,
arteriograms typically are reserved for individual~ with
advanced or acute atherosclerotic disease.
A variety of less invasive techniques for the
diagnosis of vascular injury and disease have been proposed.
These techniques include plethysmography, thermography, and
ultrasonic scanning (Lees and Myers, Adv. Int. Med. 27:475,
1982).
Other non-invasive approaches to the diagnosis of
vascular injury which have been proposed by the presént
inventor involve the administration of labelled target-
seeking biologically active molecules or antibodies which
preferentially seek out arterial lesions (U.S. patent
. '
:
WO91/16919 PCT/US91/03026
2 ~ 3
- 3 -
Application ser. No. 929, 012, entitled "Detection of
Vascular Disease", filed Nov. 10, 1986) and the
administration of labelled low density lipoproteins (LDLs)
to the vascular system of a patient (U.S. Patent Nos.
4,647,445 and 4,660,563). LDLs circulating in the blood are
known to bind to atherosclerotic plaques (Lees et al., J.
Nucl . Ned . 24 :154, 1983). This binding most likely occurs
via apolipoprotein B-l00 (apo B-l00), the protein moiety of
the ~DL molecule, which is responsible for the removal of
LDL from the circulation by receptor-mediated uptake in a
variety of cell types. LDLs conjugated to a radioactive
label can be used to provide information on the location and
extent of plaque in the vascular system by imaging them with ~
a radiation detector. Alternatively, LDLs can be labelled -
with a non-radioactive, paramagnetic contrast agent capable
of detection in magnetic resonance imaging (MRI) systems.
One disadvantage to this method is that several days
are typically required to isolate LDLs from the patient's
blood and to label them. Often, such a delay in diagnosis
and subsequent treatment is detrimental for critically ill
patients. Further, àn additional risk of viral infection is
incurred lf donor blood is employed as an LDL source.
Consequently, there exists a need for better non-
invasive techniques and reagents capable of detecting and
mapping early, non-stenosing, non-flow-disturbing
atherosclerotic arterial lesions. :
Accordingly, it is an object of the present
invention to provide synthetic peptides which are useful for
detecting and imaging vascular disease or injury.
It is another object of the invention to provide
synthetic peptides useful for imaging which are inexpensive
and easy to prepare.
,
SUBSTITUTE SH~ET
.. .. .. . .. .. .. . . . . . . . . .
WO91/16919 PCT/US91/03026
2~80~93
It is yet another object of the invention to provide
an improved method of detecting and mapping vascular injury,
including vascular injury at its early stages.
Yet another object of the present invention is to
provide a method, which is non-invasive, of detecting and
mapping vascular injury.
Finally, it is an object of the present invention to
provide synthetic peptides for the prevention or treatment
of vascular damage.
SUMMARY OF THE INVENTION
In general, the invention features a peptide or
peptide analog having an affinity for, and propensity to
accumulate at, a site of vascular injury, wher2by the
peptide or peptide analog includes an amino acid sequence
selected from the group including:
Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asp-Ala-
Glu-Gly-Ala-Lys;
Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asn-Ala-
Glu-Gly-Ala-Lys;
Tyr-Arg-Ala-Leu-Val-Asp-Tyr-Leu-Lys-Phe-Val-Thr-Gln-Leu;
Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys;
Tyr-Ala-Lys-Phe-Arg-Glu-Thr-Leu-Glu-Asp-Thr-Arg-Asp-Arg- --
Met-Tyr;
Tyr-Ala-Ala-Leu-Asp-Leu-Asn-Ala-Val-Ala-Asn-Lys-Ile-Ala-
Asp-Phe-Glu-Leu;
. . ......... . . . .
: - ~- -- -- .... . . - - ~ . ....... . . ~.
:. -~ ::: . - - . . . . . .
WO91/16919 PCT/US91/03026 ~
2~$~3
- 5 -
Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Glu-Gln-
Ala-Lys-Gly-Ala; or ~ -
Tyr-Arg-Ala-Leu-Val-Asp-Thr-Glu-Phe-Lys-Val-Lys-Gln-Glu-
Ala-Gly-Ala-Lys. `;
,
By "peptide" is meant any chain of 30 amino acids or
less. By "peptide analog" is meant a peptide which dif~ers
in amino acid seguence from the native peptide only by
conservative amino acid substitutions, for example,
substitution of Leu for Val, or Arg for Lys, etc., or by one
or more non-conservative amino acid substitutions,
deletions, or insertions located at positions which do not
destroy the biological activity of the peptide (in this
case, the ability of the peptide to target vascular
lesions). A peptide analog, as used herein, may al90
include, as part or all of its sequence, one or more amino
acid analogues, molecules which mimic the structure of amino
acids, and/or natural amino acids found in molecules other
than peptide or peptide analogues.
In another aspect~ the invention features a peptide
or p~ptide analog having an affinity for, and a propenslty
to accumulate at, a 5ite of vascular injury; the peptide or -~
- peptide analog is derived from an amphiphilic domain,
preferably including an ~-helix, of apolipoprotein A-I
(apoA-I) and has a net charge of -2 or greater, such that
the peptide or peptide analog accumulates at the site of
injury.
By "net charge" is meant the total charge on a
peptide at neutral pH and is calculated by adding together
the charge (at neutral pH) on each of the amino acids of the
peptide. By "derived from" is meant having an amino acid
sequence identical or substantially identical to the
~'''` ~''
~'
WO91/16919 PCT/US91/03026
2~a~3
-- 6 --
sequence of, as used herein, apolipoprotein A-I. By
"substantially identical to" is meant having an amino acid
sequence which differs only by conservative amino acid
substitutions (as described above) or by non-conservative
amino acid substitutions, deletions, or insertions located
at positions which do not destroy the biological activity of
the peptide (also as described above).
In preferred embodiments, the peptide or peptide
analog has a net charge of -2 or greater and has an amino
acid sequence sufficiently duplicative of that of at least a
portion of an amphiphilic domain of apolipoprotein A-I such
that the peptide or peptide analog accumulates at sites of
vascular injury. A preferred peptide or peptide analog is:
Tyr-Val-Leu-Asp-Glu-Phe-Arg-GlU-Lys-Leu-Asn-Glu-Glu-Leu-
Glu-Ala-Leu-Lys-Gln-Lys.
In yet another aspect, the invention features a
peptide or peptide analog having an affinity for, and
propensity to accumulate at, a site of vascular injury; the
peptide or peptide analog includes a hydrophobic domain and
has a net charge of -~ or greater, such that t~e peptide or
peptide analog accumulates at the site of injury.
Preferably, the peptide or peptiade analog is derived from a
vascular-associated protein, for example, elastin.
By "derived from" is meant having an amino acid
25 sequence identical to or substantially identical to (as -
defined above) the sequence of, as used herein, a vascular-
associated protein. By a "vascular-associated protein" is
meant a protein that is naturally associated either with the -
vascular wall or with an extracellular component of the
vascular system, including the proteins elastin and
collagen, and carbohydrates such as proteoglycans.
.. . - . . :: - . :. . ~ . : : :: ~ , .
WO91/16919 PCT/US91/03026
'' ' ; ' "
2 ~ 3
- 7 - ::
In other preferred embodiments, the peptide or :-
peptide analog has an affinity for a vascular wall
component, for example, a collagen, a proteoglycan, or -
elastin; the peptide or peptide analog binds elastin with a
5 dissociation constant of lO 6 or less (i.e., or with greater .
affinity, as measured ln vitro by the method of Podet et
al., Arteriosclerosis a~d Thrombosis 1l:ll6, l99l); the
hydrophobic domain of the peptide or peptide analog includes :
a ~-strand, In yet other preferred embodiments, the
vascular-associated protein is a peptide or peptide analog
having a net charge of -2 or greater and an amino acid
sequence sufficiently duplicative of that of at least a :
portion of elastin such that the peptide or peptide analog .:.
accumulates at sites of vascular injury. A preferred
15 peptide or peptide analog may include the amino acid .
sequence: -
' ' . '
Ty~-(Val-Gly-Val-Ala-Pro-Gly)~,
wherein x is at least l and, preferably, 3; or the peptide
or peptide analog may include the amino acid sequence:
Tyr-~Val-Pro-Gly-Val-Gly)~,
wherein x is at least l and, preferably, 3 or, more -
preferably, 4.
In preferred embodiments of all aspects, the peptide
or peptide analog has an acetylated amino terminus and/or an
amidated carboxy terminus. Examples of such peptide or
peptide analogues include:
; '
".
~ . ''.
.. - . ~.... . . ... . , ,., . , .;, .. ... . . . .
WO 91tl6919 PCT/US91/03026
2~493
- 8 -
H2N-Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asp-
Ala-Glu-Gly-Ala-Lys-CONH2;
CH3CONH-Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-
Asp-Ala-Glu-Gly-Ala-Lys-CONH2;
H2N-Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asn-
Ala-Glu-Gly-Ala-Lys-CONH2;
cH3coNH-Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-A
Asn-Ala-Glu-Gly-Ala-Lys-CONH2
CH3CONH-Tyr-Arg-Ala-Leu-Val-Asp-Tyr-Leu-Lys-Phe-Val-Thr- .'
Gln-Leu-CONH2;
CH3CONH-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-CONH2;
CH3CONH-Tyr-Ala-Lys-Phe-Arg-Glu-Thr-Leu-Glu-Asp-Thr-Arg-
Asp-Arg-Met-Tyr-CONH2;
H2N-Tyr-Ala-Ala-Leu-Asp-Leu-Asn-Ala-Val-Ala-Asn-Lys-Ile-
~5 Ala-Asp-Phe-Glu-Leu-CONH2;
CH3CONH-Tyr-Arg-Ala-LeU-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-
Glu-Gln-Ala-Lys-Gly-Ala-CONH2;
CH3CONH-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Glu-Phe-Lys-Val-Lys- '' .
Gln-Glu-Ala-Gly-Ala-Lys-CONH2; `.
CH3CONH-Tyr-Val-Leu-Asp-Glu-Phe-Arg-Glu-Lys-Leu-Asn-Glu-
Glu-Leu-Glu-Ala-Leu-Lys-Gln-Lys-CONH2;
.- : ~ .. , . . . : . :
WO91/169t9 PCT/US91/03026
2 ~ 3
g ~''-
H2N-Tyr-Val-Pro-Gly-Val-Gly-Val-Pro-t;ly-Val-Gly-Val-Pro-
Gly-Val-Gly-Val-Pro-Gly-Val-Gly-CONH2; and
H2N-Tyr-Val-Gly-Val-Ala-Pro-Gly-Val-Gly-Val-Ala-Pro-Gly-
Val-Gly-Val-Ala-Pro-Gly-CoNH2.
: . ,
The synthetic peptide or peptide analogues are
useful for detecting and imaging injury in the vascular i
system of a subject. Other useful synthetic peptide or
peptide analogues may include: amino acid analogues,
molecules which mimic the structure of amino acids, and
natural amino acids found in molecules other than peptide or
peptide analogues.
In other preferred embodiments of all aspects, the
peptide or peptide analog is water soluble; or is soluble in
a physiological fluid, preferably, one which is at
physiological pH, for example, blood plasma; and the peptide
or peptide analog i9 linked to a detectable label to enable
its monitoring within the subject. Preferable labels
include a radioisotope, e.g., 131I, l25I l23~ In 99~TC
203Pb~ 198Hg, Ru97, or 201Tl; or a paramagnetic contrast
agent. Such labels may e~able the extracorporeal monitoring
- of synthetic peptide or peptide analogues within the
vascular system of the subject with, for example, a gamma
scintillation camera or an MRI system.
In another aspect, the invention features a method
for the detection of injury (for example, atherosclerosis)
in the vascular system of a subject involving introducing
into a subject a peptide or peptide analog of the forms set
forth above. The method may further involve administering a
second peptide or peptide analog of the forms set forth
.: , : - :. . . . :.: . - . ,. - .- . ., - , .
$ ~ . . - :- : ... . j .:
: ~ ' : - :-'` , . ' :- ~ .'. '' :
WO91/16919 PCT/US91/03026
20~ 3 - lO -
above. The peptide or peptide analog to be introduced may
be administrated by arterial or venous injection.
Alternatively, a non-hydrolyzable derivative may be
administered orally or nasally. The introduced synthetic
peptide or peptide analog is then allowed to circulate
within the vascular system of the subject, whereby at least
a portion of it accumulates at a site of injury. The
portion of the synthetic peptide or peptide analog which has
accumulated at a site of injury is then detected. The
detection step may further include quantitating the amount
of labelled peptide or peptide analog which has accumulated
at a site of vascular injury; or imaging the region of the
subject's vascular system at which the synthetic peptide or
peptide analog has accumulated, e.g., by extracorporeal
monitoring of a peptide or peptide analog having a
detectable label (e.g., a radioactive label or a
paramagnetic contrast agent) with a gamma scintillation
camera or a magnetic resonance imaging system.
In a final aspect, the invention includes a method
for inhibiting the binding of low density lipoprotein to the
vasoular wall~s) of a subject involving administering to the
sub~ect a therapeutically-effective amount o~ a peptide or
peptide a~alog of the forms set forth above.
Applicants have discovered that vascular diseases,
including asymptomatic atherosclerosis, can be diagnosed by
administering a synthetic peptide to a subject, and then
detecting the location, pattern, and concentration of the
peptide following its accumulation at sites of injury within
the subject's vascular system.- The technique affords a
30 number of advantages. It is non-invasive; it requires -
neither complex medical equipment, nor highly skilled
medical practitioners to be successfully accomplished; and
the peptides used to target vascular lesions may be produced
. '
:~ .
.
W O 91/16919 PC~r~US91/03026 :
- 11- 2~ 3
inexpensively, quickly, and in large quantity (e.g., by
recombinant DNA technology).
In additi~n, the peptides of the invention may be
used for the prevention or alleviation of vascular diseases
5 such as atherosclerosis. Administration of the peptides of :
the invention in therapeutically-effective doses can prevent
the accumulation of LDL by blocking LDL binding sites.
Other features and advantages of the invention wi}l
be apparent from the following description of the preferred
embodiments, and from the claims.
BRIEF DESCRIPTION OF T~E DRAWINGS
The foregoing and other objects of the invention,
the various features thereof, as well as the invention
itself, may be more fully understood from the following
description when read together with the accompanying
drawings in which:
FIG. 1 shows a schematic model of the apo B-100
configuration, when included in the LDL molecule, and
~urface-exposed regions;
FIG. 2 is a series of helical wheel diagrams
indicating the amphiphilic character of representative
synthetic peptides;
FIG. 3 shows a photograph ~A) and an onlay
autoradiograph ~B) of the abdominal aorta of a rabbit
treated with 125I-la~elled synthetic peptide, SP-17;
FIG. 4 shows a photograph (A) and an onlay
autoradiograph (B) of the abdominal aorta of a rabbit
treated with 125I-labelled synthetic peptide, SP-19a;
FIG. 5 shows a photograph (A) and an onlay
autoradiograph (B) of the abdominal aorta of a rabbit
treated with 125I-labelled synthetic peptide, SP-21a;
.:
. .
.. . . . . . .. . .
~: : . , . . ., .
: . - .; . . :
~- , ~ . . . ~ ,.
.. . . . . . . . . . .. . . . . .
WO 91/16919 P~/USgl/03026
2~ 3 - 12 - ~ .
FIG. 6 shows a photograph (A) and an onlay
autoradiograph (B) of the abdominal aorta of a rabbit
treated with l25I-labelled synthetic peptide, SP-28;
FIG. 7 shows a photograph (A) and an onlay
autoradiograph (B) of the abdominal aorta of a rabbit
treated with l25I-labelled synthetic peptide, SP-29; and
FIG. 8 shows a photograph (A) and an onlay
autoradiograph (B) of the abdominal aorta of a rabbit
treated with l25I-labelled synthetic peptide, SP-30.
DETAILED ~ESCRIPTION
This invention provides synthetic peptides which
have affinity for, and the propensity to accumulate at, a
site of vascular injury, and therefore are useful in
detecting, diagnosing, monitoring, and treating vascular
disease.
Specific examples of such synthetic peptides having
these characteristics may have an amino acid sequence that
is anal~gous to portions of known polypeptides which have an
a~finity for a site of vascular injury, i.e., have a
molecular conformat~on, charge, and/or size which is similar
to that part of the polypeptide (~.g., low density
lipoprotein or elastin) w~ich is responsible for its
a~finity for arterial lesions. Alternatively, the synthetic
peptLdes of the presQnt invention may be homologous with
portions of the apo B-100 moiety of LDL, the apo A-I moiety
of HDL, or elastin.
Design and Synthesis of Peptides
Peptides useful in the invention are those which
successfully target vascular lesions. Thus, it is
preferable to fashion such peptides after the sequence of a
protein which is "vascular-associated", i.e., naturally
associated with a vascular cell surface or with an
... . ... ~ .: . ~ .. , . .- - . .. . . . . . .. ..
W O 91/16919 PC~r/US91/03026
: ' .
2 ~
- 13 -
extracellular component of the vascular system (e.g.,
proteoglycans, collagen, or elastin). Proteins of this
class include: apolipoprotein B (i.e., the protein moiety of
low density lipoprotein) and elastin (a natural component of
the arterial wall). It is not necessary, and it is often
inconvenient, to use the entire protein molecule (see
above). Applicants have discovered that protein fragments
can also be used to effectively target vascular lesions.
Examples of useful fragments are described herein.
Applicants have shown that such fragments are of low net
charge (i.e., between -2 and ~2), allowing an interaction,
e.g., with the highly negatively-charged vascular wall.
Applicants have also shown that such peptides fall generally
into one of two classes: (1) peptides which include an
amphiphilic domain, preferably of ~-helical character; and
(2) peptides which include a hydrophobic domain (which
facilitates interaction with a vascular surface or
vascular-associated extracellular component) and a
hydrophilic domain of either positive charge or low negative
charge (i.e., -2 or greater; i.e., or more positive~ which
~acilitates solubility.
Preferred peptides of class I, i.e., those peptides
which include an amphiphilic domain (i.e., a domain which
has ~oth a hydropho~ic and a hydrophilic surface) are
identified, e.g., as described in Xaiser and Kezdy (Ann.
Rev. Biophys. Biophys. Chem. 16: 561, 1987; Science 223:249,
1984). Typically, the amphiphilic domain includes a region
of secondary structure, most commonly, an ~-helix or a ~-
strand. Because ~-helix-containing peptides are generally
more soluble than ~-strand-containing peptides, they are
preferred in the invention; increased solubility facilitates
in vitro peptide synthesis and peptide administration to a
patient.
, . .. .
.:' , ., . .: - ' .' ' ~ - . .. .
: ~ . : -: - . . . - -, . : , ~ ... .
:: : : , . . , . . . ,~ ... , ~ . :
~: - , ; . -; .,; . .: . . , . - . ., ., ;. . - .
WO91/16919 PCT/US91/03026
2~0~3
- 14 - ~ -
Preferred peptides of class II, i.e., those peptides
which include both (a) a hydrophobic domain which
facilitates interaction with a vascular cell surface or a
hydrophobic vascular-associated component (e.g., elastin)
and (b) a positively-charged or slightly negatively-charged
domain that facilitates solubility are identified using
e.g., the methods for predicting hydrophobicity and
hydrophilicity described below. Applicants have shown that
peptides of this class, even peptides including one or more
domains predicted to form ~-strands, may be administered to
a subject and used to efficiently target arterial lesions.
Peptides of this class likely interact with hydrophobic
vascular-associated extracellular components.
The net charge of a peptide is calculated by adding
together the charges on the amino acids of the peptide at
neutral pH. The local charged character (i.e., amphiphilic, -
hydrophilic, or hydrophobic nature, e.g., of a region of a
peptide) and secondary structure (i.e., the presence of an
~-hel~x or ~-strand) of a particular sequence of amino acids
may be predicted from its primary sequence using any of a
number of model-building approaches. For example, to
identify an amphiphilic ~-helix, one may construct an
"Edmundson wheel", and look for the presence o~ hydrophobic
and hydrophilic residues on opposite faces of the resultant
cylinder (Schiffer and Edmundson, Biophys. J. 7:121, 1967;
hereby incorporated by reference). Alternatively, to
identify an amphiphilic, hydrophobic, or hydrophilic domain ~--
or a region of secondary structure, one may use a semi-
empirical formula such as the Chou-Fasman method (Chou and
Fasman, Ann. Rev. Biochem. 47:251, 1978; hereby incorporated
by reference); or the program, PREDICT (based on the GOR -- -
method of secondary structure prediction) (Robson et al.,
Introduction to Proteins and Protein Engineering; Elsevier,
. . .
':
WO91/16919 PCT/US91/03026
1 5 2 ~ 3
New York, 1986; hereby incorporated by reference). Such a
program makes use of the equation:
':
~=j,8
Ij(X) = ~ I(Sj = X: X; RJ+m)
m=j-8
where I(S~=X:X;Rj+~) values are derived from a statistical
preference for a residue j to be in a conformation X. The
state of j is evaluated from a summation over m residues of
sequence on either side of j; parameter values are dependent
on the identity of the residue at each position and its
contribution to each of the four structural types. Values
are calculated for each of the states H, E, T, and C; the
highest value determines the predicted structure (either
H-~-helix, E=~-sheet, T=turn, or C=random coil). Finally,
amphiphilicity may be derived from a calculation of the
"hydrophobic moment", i.e., the measure of the
amphiphilicity perpendicular to the axis of a periodic
peptide structure; this approach is deæcribed in Eisenberg
(Ann . Rev. Biochem. ~: 595, 1984; hereby incorporated by
re~erence) . -'
lt has been shown that it is the charged character
(i.e., amphiphilic, hydrophilic, or hydrophobic) and/or
secondary structure of a protein, and not its part~cular
amino acid sequenca, which ~acilitates the protein's
~nteraction with other charged (or hydrophobic) surfaces
~see, e.g., Kaiser and Xezdy, Science 223:249, 1984).
Accordingly, it is possible to design any number of peptide
analogues, having different amino acid sequences, provided
that the local charge distribution (and overall net charge)
and secondary structuFe,~and hence the biological activity
,, -
~ ,.',
,:,.. ,.... : - : :: ~ : ~ , ~ .
W O 91/16919 PC~r/US91/030262~4~3
- 16 -
(in this case, the ability to target vascular lesions) is
maintained. Such peptide analogues will generally differ
from the native protein sequences by conservative amino acid
substitutions (e.g., substitution of Leu for Val, or Arg for
Lys, etc.) well known to those skilled in the art of
biochemistry. Moreover, peptides may be designed which
include a region~s) of amphiphilic, hydrophobic, hydrophilic
and/or secondary structure embedded within a longer amino
acid stretch. Generally, the charged character and
secondary structure of such a region is unaffected by the
surrounding amino acid residues. Again, only those peptides
which are capable of targeting vascular lesions are
considered to be useful in the invention.
Good candidates for peptides useful in the invention
are peptides based on surface-exposed protein domains (i.e.,
regions of the protein which are present on the external
surface of a protein molecule, preferably a vascular-
associated protein molecule) because such regions are most
likely to interact with the vascular wall or with a
vascular-associated extracellular component. The identity
of surface-exposed domains ~ay be determined by tryptic
digest analysis ~see below) and/or by calculation of a
region's degree of hydrophobicity/hydrophilicity (e.g., by
the Chou-Fasman method, Ann. Rev. ~iochem. 47:251, 1978); --
extracellular domains are generally hydrophilic or
amphiphilic in character; such domains are frequently
surrounded by hydrophobic stratches which correspond to ~-
transmembrane domains.
The peptides, once designed, can be synthesized by
any of a number of established proceduresj including, e.g.,
the expression of a recombinant DNA encoding that peptide in
an appropriate host cell. Alternatively, these peptides can -
be produced by the established procedure of solid phase ~ -
. .
- : ~ . - - . - . . ~. . .
.
. . - i :. :
:
WO91/16919 PCT/US91/03026
~ 17 ~
peptide synthesis. Briefly, this procedure entails the
sequential assembly of the appropriate amino acids into a
peptide of a desired sequence while the end of the growing
peptide is linked to an insoluble support. Usually, the
carboxyl terminus of the peptide is linked to a polymer from
which it can be liberated upon treatment with a cleavage
reagent. The peptides so synthesized are then labelled with
a reagent which enables the monitoring of the peptide after
its administration to a patient.
l0Peptides may be tested for their ability to
effectively target vascular lesions using an ln vivo animal
assay (e.g., that assay described herein). It is known that
LDL accumulates both in the balloon de-endothelialized
healing arterial wall of the rabbit and in human atheroma
15(Roberts et al., J. Lipid Res. 24 :1160, 1983; Lees et al.,
J. Nucle~r ~ed. 24:154, 1983). Accordingly, a rabbit whose
abdom~nal aorta has been balloon de-endothelialized
approximately four weeks prior may be used as a test model
for hu~an arterial disease. Other animals or experimental
systems can be used as well, such as Watanabe Heritable
Hyperlipemic rabbits which have inherited high blood
cholesterol secondary to a deficiency in LDL receptors.
This strain of rabbit develops spontaneous atherosclerosis
at about 2 months of age, and they often die of heart
attacks.
The rabbit model has been imaged both by onlay
autoradiography with l2sI-labelled LDL and by external
imaging with 99~Tc-labelled LDL using a gamma scintillation
camera. In each case, onlay autoradiography of the excised ~ -
rabbit aorta has been reliably predictive of the in vivo
results with extracorporeal imaging. In preparation for
vascular administration, each labelled synthetic peptide may
. . .
,. . .- . , .,, .. . . . . .. , ".. , .. ~, .~.. . , ~ ., . -
- - , , ~. ~ . - ;
- - -~ , ,. . . , ; . - :
: . , ~
WO91/16919 PCT/US91/03026
2Q~0~3
- 18 -
be injected in the free state or, alternatively, may be
bound to the surface of a lipid emulsion such as a
cholesterol ester phospholipid microemulsion. The emulsion
is then injected intravenously into the rabbit.
5 Approximately twenty-four hours later, the rabbit is -
subjected to extracorporeal monitoring appropriate for the
specific label on the peptide. Alternatively, the rabbit is
sacrificed, and its aorta removed and washed. The aorta is
either cut into sequential portions which are then monitored
in a liquid scintillation counter, or is dried, covered with
a layer of polyester wrap, and placed on a sheet of x-ray
film which is then developed to produce an onlay
autoradiogram after suitable storage time in the dark.
Use
The peptides of the invention may be used to -
diagnose vascular injury or, alternatively, to inhibit
binding oS LDL to vascular walls~ In either case, the i
peptide is first administered to a subject, e.g., a patient.
Administration may be accomplished by arterial or venous
in~ection. Alternatively a non-hydrolyzable derivative of
the peptide (e.g., a keto methylene derivative) may be
administered by mouth, or administration may be accomplished
nasally.
In preparation for vascular administration, labelled
synthetic peptide is suspended in a pharmaceutically-
acceptable carrier (e.g., a physiological saline solution)
or alternatively may be bound to the surface of a lipid
emulsion such as a cholesterol ester phospholipid
microemulsion (MV), and the emulsion is then injected
30 intravenously. -
For diagnostic use, the labelled peptide is
administered in an amount sufficient for later detection - -
(generally, 0.5-l mg intravenously or 5-lO0 mg orally). In
: .'
;' ~'",'."
.. .. . .
:
~: - - ~ - . : - . , - ,
WO91/16919 PCT/VS91/030t6
2Q~9~
-- 19 --
preferred emb~diments of the invention, the peptide is
labelled with, e.g., a radioisotope such as 123I, l25I or
99mTc, and peptide accumulation at a site of injury imaged
extracorporeally by radiation detection means such as a
gamma scintillation camera; alternatively, the synthetic
peptide is labelled with a non-radioactive, paramagnetic
contrast agent capable of being detected in MRI systems. In
such systems, a strong magnetic field is used to align the
nuclear spin vectors of the atoms in a patient's body. The
field is then disturbed and an image of the patient is read
as the nuclei return to their equilibrium alignments. In
the present invention, synthetic peptides can be linked to
paramagnetic contrast agents such as gadolinium, cobalt,
nickel, manganese or iron complexes, to form conjugate
diagnostic reagents that are imaged extracorporeally with an
MRI system.
For treatment of vascular disease (i.e., to inhibit
~D~ binding to vascular walls), the peptide is administered
~n a therapeutically-e~fective dose, generally 5-l00 mg
intravenously or intramuscularly. Treatment may be
repeated, as necessary, to prevent or alleviate vascular
damage.
DESCRI~TIO~ OF THE PREFERRE~ EMBODI~ENTS
There now follow9 a description of the design and
synthesis of sample peptides useful in the invention. There
also follows a description of an in vivo assay used to test
the ability of such peptides to target vascular injury.
These examples are provided to illustrate the invention and
should not be construed as limiting.
Apolipoprotein Pe~tides
Apoliprotein B (apoB) is the protein moiety of low
density lipoprotein. The primary structure of apo B-lOO has
.- - - . : . : . : . . :. . .. . .
; . . . - . . : . . , . .. -
WOgl/16919 PCT/US91/03026
2 Q ~ 3
- 20 -
become available by virtue of its cloning (see e.g., Knott
et al., Nature 323:734-742, 1986; Yang et al., Nature :
323:738, 1986; Carlsson et al., Nucl. Acids Res. 13:8813,
1985). Fur~her, enzymatic treatment of apo B-100 with
trypsin has enabled the identification of those surface
regions which are apparently involved in the binding of LDL
to various cells and tissues (Forgez et al., Biochem.
Biophys. Res. comim. 140:250, 1986; Knott et al., Nature
~ 734, 1986). The surface-exposed regions are shown
schematically in FIG. 1. The amino acid sequence analyses
of representative tryptic peptides are shown in TABLE 1.
- . -:' -
.
~, '~ ',
.
.
'
.,,; , ., . . . . . . . .. ,, . , ,, ., .,, .. ,. .. , . , . ... , .. . , , . , . .~ , . . . .. .
W O 91/16919 PC~r/US91/0302~ .
2 1
... . .
TABLE 1- 2 ~ ~ O ~- ~ 3 ~:
HPGC Correspondir~ to
Fr~ction Amino Acid Sequence Apo B Amino Acid
Ro ~esidue ~os
(Tp~
24 ~Lys)-Phe-Val-Thr-Gln-Al~- 1008-1016
Glu-Gly-Ala-Lys
1 0 123 (Lys)-Leu-Pro-Gln-Gln-Ala 201-2106
Asn-Asp-Tyr-Leu-Asn-Ser-Phe-
Asn-Asn-Clu-Arg
Leu-Pro-Gln-Gln-Alr-Asn-Asp- 201-2098
Tyr
1 5 49 ~Lys)-Phe-Arg Clu-Thr Leu- 2485 2493
Glu-Asp-Thr~Ar~
9 ~Ar9) ~le Ser Leu Pro Asp 267 2085
phe-Art
161 ~Arg)-Thr-Phe-Gln-lle-Pro- 3218 3236
2 0 Gly Tyr-Thr-Vrl-Pro-V~l-V~l-
Asn-Val-Glu-Val-Ser-Pro-Phc
134 Tyr-Thr-Val-Pro-Val-Val-Asn- 3224-3232
Val-Glu-Val-Ser-Pro-Phe-Thr-
lle-Glu-~et-Ser-Al--Phe-~Gly-
2 5 Tyr-V-l Phe-Pro Ly~)
18~ ~Arg)-V~l-Pro-S~r-Tyr Thr 3205-3275
Leu lle-Leu Pro ~Ser-Leu-Glu-
Leu-Pro-V l-Leu-H~c-Vel-Pro-
Aro~
3 0 59 ~Ly lle Ale ~-p Ph- O~u-L u- 3428 3841
Pro Th~ V~l Pro Glu
Oln Thr lle Glu lle-Pro-Ser
7 1l~
1C6 ~Ar9) A~n Leu Oln A~n A~n ~084 ~094
3 5 Al~ Glu Trp V~l Tyr Gln Oly
Al~ rO
e Reslduc number~ t~ken trrJn the complete jrimery sequence ot rpollpoprotein B
~ trom For9e~i et -l , ~k!g
.
Based on the data of Forgez et al. (ibid) the
published apo B sequence (described above), and information - --
~ known to those skilled in the art of biochemistry and
:`
. .
~ .
.
~' ' . . ' ' ' ' . ' ~ '.. ': ' ' . ' ' ,' " . ' ' . . . ':. ' ' '
W O 91/16919 PC~rlUS91/03026
2 Q ~ 3
- 22 -
peptide design (e.g., that described above), synthetic
peptides having an amino acid sequence analogous to the
amino acid sequences of surface regions of the apo B moiety
of LDL were designed. In some cases, the peptides were
amidated at their carboxy terminus and/or acetylated at
their amino termi~us (i.e., the "A" or "a" peptides). Eight
representative apo B peptides and their modified
counterparts are shown below. The numbers above the amino
acid residues refer to the primary sequence of apo B.
SP-6:
1000 ' , .
HN2-Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu
1010 , ...
Ala-Asp-Ala-Glu-Gly-Ala-Lys-CONH2;
SP-6A:
1000
CH3CONH-Tyr-Lys-Leu-Ala-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-
1010 .,
Leu-Ala-Asp-Ala-Glu-Gly-Ala-Lys-CONH2;
SP-8:
1000
H2N-Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu- ''-
1010 '' ' '
Ala-Asn-Ala-Glu-Gly-Ala-Lys-CONH2;
25 ~E~ '
1000 ',
CH3CONH-Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu ... '~
1010
Ala-Asn-Ala~Glu-Gly-Ala-Lys-CONH2;
30 SP-12A: -
1008
CH3CONH-Tyr-Arg-Ala-Leu-Val-Asp-Tyr-Leu-Lys-Phe-Val -
Thr-Gln-Leu-CONH2;
SP-14A:
CH3CONH-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-CONH2;
., '..
. . ; '' ,'' .' : ~
:
WO91/16919 PCT/US91/03026
2 ~ 3
- 23 -
SP-15a:
2485
CH3CONH-Tyr-Ala-Lys-Phe-Arg-Glu-Thr-Leu-Glu-Asp-Thr-Arg-
2495
Asp-Arg-Met-Tyr-CONH2;
SP-17:
3810
H2N-Tyr-Ala-Ala-Leu-Asp-Leu-Asn-Ala-val-Ala-Asn-Lys-Ile
3822
10 Ala-Asp-Phe-Glu-Leu-CONH2;
SP-19a:
1008
CH3CONH-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-
Glu-Gln-Ala-Lys-Gly-Ala-CONH2; and
SP-2la:
1002
CH3CONH-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Glu-Phe-Lys-Val-Lys-
Gln-Glu-Ala-Gly-Ala-Lys-CONH2. ~.
Amino acids 2-13 of the apoB-derived peptide, SP-4
~see, PCT/US89/01854), were conservatively substituted to
produce SP-6 and SP-8. SP-12 is a truncated form o~ SP-4 in
Which the last ~ive amino acid residueg wers replaced with a
sinqle leucine ~eu) residue. SP-14 is a truncated form of
SP-12 in which the last five amino acid residues have been
deleted and the tyrosine (Tyr) residue at position 7
replaced with a threonine (Thr) residue. SP-15a and SP-17
include amino acids 2483-2497 (i.e., including Tp 49) and
amino acids 3809-3825, (i.e., including part of Tp 59),
respectively. The sequences of SP-19a and SP-21a are
variations on the sequence of SP-4. Physical data obtained
for peptides SPl5a, SP17, SP19a, and SP21a are summarized in
Table 2. Helical wheel diagrams demonstrating the
amphiphilic and ~-helical nature of these peptides are shown
'
- - , : .. - :.. . . . ~ -, . .. . , . .. :-
WO91/16919 PCT/US91/03026
2~ 3
- 24 -
in FIG. 2; hydrophobic residues are encircled and charged
residues are indicated. Abbreviations are : A, alanine; D,
aspartate; E, glutamate; F, phenylalanine; G, glycine; K,
lysine; L, leucine; N, asparagine; Q, glutamine; R, -;
5 arginine; T, threonine; V, valine; Y, tyrosine; Ac, acetyl. ~-
Another amphiphilic peptide (i.e., SP34a) was
synthesized based on an apo A-I consensus peptide (termed
APOA-I CONSENSUS), i.e., an idealized ~-helix derived from a
number of regions of apolipoprotein A-I; the sequence of
this consensus peptide is published in Anantharamaiah (Meth.
Enzymol. 128:630, 1986). Unlike apolipoprotein A-I, the -
synthetic peptide is only weakly charged, and the sequence
is preceded by an animo-terminal tyrosine residue. This
peptide was amidated at its carboxy terminus and acetylated
at its amino terminus. This peptide has a weak net negative
charge (i.e., -2; see Table 2).
' '
SP-34a:
CH3CONH-Tyr-Val-Leu-Asp-Glu-Phe-Arg-Glu-Lys-Leu-
Asrl-Glu-Glu-Leu-Glu-Ala-Leu-Lys-Gln-Lys-CONH2. :.. -
Physical data obtained for SP-34a are summarized in
Table 2. A helical wheel diagram of SP-34a is shown in FIG.
2 tdescribed above).
. .: ' ,'
WO91/16919 PCT/US91/03026
2 ~ 3
- 25 -
TAsLE 2
P~rN)t Protein
Peptide Protein ~a H~D Charge
SP-15~ ~po B 2135.0~2135.5 -0.53 0
SP-17 ~po B 1950.0/1950.1 0.26 -1
SP 19~ 2051.1/2051.0 0 00 ~1
SP 21d 2po3 2094.1/2094.4 -0.18
SP 348 ~po A-l 2535.3t2535.4 -0.29 -2
SP-28 olllstin 1622.9/1622.9 0.62 -1
SP-30 el~stin 1818.0/1818.1 0.63 ~1
SP-29 e~astin 1408.8~1408.8 0.62 ~1
~oleculDr ~eight tcalculated/o~served); e~pressed as the parent ion ~H~H) ils determined by F~st Atcm
Barb~r~nent l~b8s Spectranetry
Dl~le-n N~drophob1city; c~lculated us~r~ th~ method ~nd scal- ot Eisaberg tJ. ~ol. 31Ol. 279:125 1984).
CCh~rge ~ expre~se~ ~s tho d1tferenc- betlleen positively ~rd r~atlvely charged gro4~o on the pept~do at
neutral p!l.
Elastin Pe~tides
Elastin is a major component of skin, arteries,
lung, and other tissues (Rosenbloom; Robert and Robert,
2S Fro~t~ers o~ Matrix B~ology, Vol. 8: Biology and Pathology
of Elastic Tissues, 1980; eddi, ~xtracellular ~atrlx:
Structure and Function, 14d5). Analysis of various elastin
sequences (see Rosenbloom, Meth. Enzymol. 144:172, 1987)
indicates that elastin proteins are generally composed of a
30 number of repeated units. Two such repeated units are the -:
pentapeptide, Val-Pro-Gly-Val-Gly (VPGVG), and the
hexapeptide, Val-Gly-Val-ala-Pro-Gly (VGVAPG).
Structurally, elastin repeats have been shown, by circular
- dichroism (Rahman et al., Coll . Czech . Chem. Comm . 52:1356,
35 1987) and by extensive nuclear magnetic resonance studies -
: '
-... - . - .. .. . . .. .
.... , - - . - . -.. - : , . ,.. -. -, .. . ...
, - . ~ - . . . . , ~ . . . . . . .
....
W091/l6919 PCT/US91/03~26
~ .
2~3~93 - 26 -
(Urry et al., Biopolymers 25:1939, 1986), to contain
repeating ~-turn structures (Biochem. Biophys. Res. Comm.
153:832, 1988). The hexapeptide has chemotactic properties
(Senior et al., J. Cell. Biol. 99:870, 1984).
Elastin-derived peptides useful for targeting
arterial lesions include a hydrophobic binding site; this
binding site facilitates interaction with a hydrophobic
extracellular vascular wall component (e.g., elastin) and/or
allows interaction of the peptide with the negatively- `
charged vascular wall. To facilitate solubility in
physiological fluids, the peptides preferably include a
hydrophilic domain or a net positive or weak negative
charge.
Three representative elastin peptides are shown
below. SP-28 includes three repeats of the elastin
hexapeptide VGVAPG. SP-30 and SP-29 include four and three
repeats, respectively, of the elastin pentapeptide VPGVG.
The SP-30 and SP-29 peptides were amidated at their carboxy
terminus.
SP-28:
H2N-Tyr-Val-Gly-Val-Ala-Pro-Gly-Val-Gly-Val-Ala-Pro-Gly-
Val-Gly-Val-Ala-Pro-Gly-OH;
:
SP-30:
H2N-Tyr-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro- ',.
25 Gly-Val-G~y-Val-Pro-Gly-Val-Gly-CONH2; and ;-
SP-29
H2N-Tyr-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro-
Gly--Val--Gly--CONH2 .
., ~ . .
- . : . . .
WO91/16919 PCT/US91!03026
3 : -
- 27 -
Physical dat~ obtained for elastin peptides i
summarized in Table 2. ~ -
Peptide Synthesis and Labelling
Peptides SP-6, SP-6A, SP-8, SP-8A, SP-12A, and SP-
14A were synthesized by solid phase peptide synthesis
according to the established method of Stewart and Young
(Solid Phase Peptide Synthesis, 2nd ed., pp. 53-123, 1984
The Pierce Chemical Co., Rockford, IL, hereby incorporated
by reference). These peptideg were synthesized using the
schedule listed in TABLE 2, but any one of the other
schedules listed in this reference may alternatively be used
to generate any desired peptides (e.g., any peptide
described herein).
. ' . . " `' '.' ', ' " ,. ' . ',, ' . . . , ~ ' ~' ' " " , ' ' ' ', ' ' '; '
" , ~ ' : " , " ' ' .. " ' ' . ' " . ' ' . .. . . ' ` ' . '
` ` ' ' ~'' '. '' "`' ,' . ' ' . ' ' . ' . ' , . ' ' ' ~ " ' ' '
' ' ' '" " `' , ' ' ' ~' . ' '. .
'' ' ' ' ' . ' ~ ~ " ' ' ' ' ' ' ' ' . ' ' ' ' '
.` ' ' ' '. ' " ' ' ~ . . ,
W O 91/16919 P ~ /US91/03026
2 ~ 9 3
~ 28 -- ~ .
TABLE 2 .,
SCHEDULE FOR SOLID PH~SE PEPTID~ SYIITHESIS
~Dioxane-HCI Deprotection: DCC Coupl ing~
5Step ~eagent No. RcpeatsVol~ml) Time~min.) :
dry CN2CI2, 4 25
2~ 50X T FA 1 25
2b 50X TF~ 1 25 20
1 0 3 dry CH2CI2 2 25
4 dry 2 propanal 2 25
CH2C12 3 25 1 . :
6 5X DIEA- 1 25 . 2
7 CN2CI2 2 25
8 5X DIEA 1 25 2
9 CN2CI2 5 25 1 ' . .
Introduc-
~yTm tric
r,h~r1d~ o~ ' '
2 0 ~oc Al-- 1 20 20
11 T~E~DIEA/
CH2Cl2(K~) 1 5 10
12 CH2Cl2 3 25
13 100X EtOH 3 25
2 5 _ _ -
..d~eycloha~ylcarbod~1mIdb
tr~tluoro cotIc aeld
dJ~opropylathylarin~
-tort butyloxycarbony~ ~mino ~cid
3 O ~2,2,2 tri~luoroethanol
Peptides SP-15a, SP-17, SP-19a, SP-21a, SP-34a, SP-
28, SP-30 and SP-29 were synthesized by manual solid-phase
methods using tert-butoxycarbonyl (t-Boc)-based chemistry.
(Barany and Merrifield, The Peptides: Analysis, synthesis,
35 Biology, Academic Press, New York, 1980; Stewart and Young, -
Solid-Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co.,
Rockford, IL, 1984). Sidechain protecting groups for amino
: ~ : : - : :.: .- . . ., : . ,, : . . -
W O 91/16919 P~r/US91/03026
2D~4~
- 29 -
acid derivatives included: benzyl esters for Asp and Glu,
benzyl ethers for Ser and Thr, chlorobenzyloxycarbonyl for
Lys, bromobenzyloxy for Tyr, and mesitylenesulfonyl for Arg.
The carboxyl terminal amino acid residue was attached to
s methylbenzhydrylamine resin with diisopropylcarbodiimide
(DIC~ by the method of stewart and Young (su~ra). The
peptide resin was washed twice with CH2Cl2 and once with 50%
TFA in CH2Cl2/1% dimethylsulfide, and the t-Boc group was
removed by treatment for 20 minutes with 50% TFA in
CH2C12/1% dimethylsulfide, or by treatment with 25% TFA in
CH2Cl2/1~ dimethylsulfide for 30 minutes. The peptide resin
was next washed five times with SX CH2Cl2; neutralized with
two wa~hes of 10% diisopropylethylamine (DIEA) in CH2Cl2;
and washed five times with CH2C12. The next amino acid was
coupled by treatment with either three equivalents of
symmetrical anhydride (see below) for 4S minutes or four
equivalents of active ester (see below) for 2 hours, in the
presence o~ 1.5 equivalents of DIEA. ~he peptide resin was
then washed four times with CH2Cl2; twice with 33% ethanol
in CH2Cl2; and twice with CH2Cl2.
Symmetrical anhydride-activated amino acids were
prepared by treating 6.1 equivalents of amino acid with
three aquivalents o~ DIC in CH2C12 for 20 minutes, on ice.
Active esters of hydroxybenzotriazole (HOBt) were prepared
from four equivalents each of amino acid, HOBt, and DIC in
dimethyl formamide (DMF) for 30 minutes on ice. Active
esters of ethylhydroxyiminocyanoacetate (EACNOx) were
prepared from four equivalents each of amino acid, EACNOx,
and DIC in CH2Cl2 for 30 minutes, on ice. Completion of
coupling at each step was verified by the Kaiser ninhydrin
test (Kaiser et al., Anal. Biochem. 34:595, 1970).
Incomplete couplings were repeated once or twice and, if
SU13STITUTE SH~-T
- .. , . . ,. . :............. , ` . . , - . .
. . , . . . : ,, .. . ... :
W O 91/16919 P~r/US91/03026
2 ~ 3
- 30 -
still incomplete, the peptide resin was acetylated with
acetic anhydride prior to continuation of synthesis.
Peptides were deprotected and cleaved from the resin either
by HF-treatment (performed as directed by Immunodynamics,
San Diego, CA) or by treatment with 1:10:1:0.5
trifluoromethanesulfonic acid:TFA:thioanisole:ethanedithiol
by the method of Yajima et al. (J. Chem. Soc., Chem. Comm.
p.107-108, 1974). Crude deprotected peptides were either
desalted on a column of Sephadex G-25 eluted with 5% acetic :
acid or were precipitated twice from the TFA solution with
10 to 100 volumes of ethyl ether. Peptides were then `
purified by reverse-phase HPLC using a Vydac C18 column and
a gradient of 0%-90% C~3CN/H2O containing 0.1% TFA.~`
Solutions of purified peptides were evaporated, redissolved
15 in water, and lyophilized to dryness. Identity of peptides -
was confirmed by Fast Atom Bombardment Mass Spectrometric
(FAB-MS) analysis.
The synthetic peptides SP-6, SP-6A, SP-8, SP-8A, SP-
12A, and SP-14A were radiolabelled by the chloramine T
20 method as described in Shih et al. (Proc. Natl. Acad. Sci. . .
USA 87:1436, l9gO, herein incorporated by reference).
Certain experiments required radiolabelled LDL. In these
cases LDL was labelled with 125-iodine by a previously
described modification of the NcFarlane iodine monochloride
technique described in Lees et al. (Proc. Natl. Acad. sci.
USA 80:5098, 1983, hereby incorporated by reference). The
radiolabelled lipoprotein or synthetic peptide was separated
from unbound radioisotope by passage through a gel
filtration "desalting" column of Sephadex G-25 or the
equivalent.
.. . . . . . . . . .. . . . . . .............. .. .
.-- , ; . . . : ::: , -- , :
WO 91/16919 PCr/US91/03026
2 ~ 3
-- 31 --
The synthetic peptides SP-15a, SP-17, SP-19a, SP-
21a, SP-34a, SP-28, SP29, and SP30 were radiolabelled with
5I using chloramine-T as follows.
The peptide (400 I g) was dissolved in 200 ul of 2.5
5 mM sodium phosphate/37.5 mM NaCl buffer, pH 7.4 and mixed
with 1 mCi (3~ 2sI. Chloramine-T (30 ~1, 8 mg/ml in H2O)
was added to the mixture and, after 35 seconds, the reaction
was quenched by the addition of sodium bisulfite (60 ~Ll, 8
mg/ml). Radiolabeled peptides were gel-filtered on a Bio-
10 Gel P-2 (Bio-Rad, Hercules, CA) column (1 cm X 30 cm) and
eluted with 0.1% BSA in 0.lM acetic acid. A lead fraction
of 5 ml was collected, followed by 0.45 ml fractions.
Iodinated peptide, which eluted at approximately fractions
9-12, was pooled and the pH adjusted to 5 with lN NaOH and
15 then to 7.5 with lM NaHCO3.
Iodination of SP17 was performed esser.tially as
described above except that the reaction mixture was
adjusted to a final concentration of 509c ethanol. Following
iodination, the radiolabeled peptide was precipitated by
20 addition o~ bovine serum albumin to a final concentration of
10%, and the precipitate was collected by centrifugation at
2000 rpm for 15 minutes. The pellet was then washed four
times with 1 ml. ~each) o~ water and, a~ter the final wash,
the precipitate was dissolved in 5 ml. of 10~6 BSA.
25 Alternatively, 20% BSA was added to the iodinated peptide
(in 50% ethanol) to a final concentration of 10% and a
volume not exceeding 5 ml. The solution was then passed
through a BioGel P-2 (10 cm X 1.5 cm) column and eluted with
0.1% BSA in 0.lM acetic acid as described above. Excess
30 buffer was removed using nitrogen pressure, the column was
washed with 5 ml of 0.1% BSA/ 0.lM acetic acid, and the most
highly radioactive fractions pooled for injection.
- . . ., . - , . .. .
~- .. - . . - . , , -. . - . - . . . . .
WO91/16919 PCT/US91/03026
2~ 3 - 32 -
In an alternative method, the synthetic peptides are
labelled either directly with technetium (Tc), or indirectly
through covalent attachment of a chelating group such as
diethylenetriamine pentaacetic acid (DTPA), which is ~nown
to chelate a variety of metals including radioisotopes such
as lll-indium. ;
Direct coupling to 99~Tc is carried out as follows.
50 mCi 99~Tc (in the form of 99~TcO~ ), in a 0.5 ml aqueous
solution, is added to 1-6 mg, but preferably to 2 mg,
synthetic peptide in 0.5 ml of a 0.2 M sodium bicarbonate
solution, pH 8.0, and mixed thoroughly for lO minutes at
room temperature. The pH is raised to 8.0 - 9.0 if
necessary with 0.25 M sodium hydroxide. To the mixture is
then added lO mg of reduced sodium dithionite (57.5 mmoles)
freshly dissolved in 0.5 ml distilled water. The mixture is
gently stirred for 30 minutes at room temperature.
The radiolabelled synthetic peptide fraction is
separated from uncoupled technetium and sodium dithionite by
molecular sieve chromatography. A l X 50 cm column of ~`
Sephadex G-25, equilibrated with a EDTA-bicarbonate buffer
(0.2 M sodium bicarbonate, pH 8.0, O.OOl M EDTA), is
suitable for separation. The column ls standardized with
blue dextran and potassium iodide to determ~ne the void
volume and the column volume, respectively. The reaction
mixture is applied to the column, and bicarbonate-EDTA
buffer is used to elute column fractions. The
macromolecular radioactive peak that elutes at a position
characteristic for the synthetic peptide is isolated and
ready for use.
Indirect coupling to 99'Tc is carried out as
follows. A chelating ligand, e.g., DTPA (as per Hnatowich
et al., Science 220:613, 1983) or bromoacetylparaaminobenzyl
.... . . - . - .; -: . -- : . : :
- . ~ , .
, ... ~ .. .. .
WOg1/16919 PCT/US~l/03026
2 ~ 3 ~ ~
- 33 - : -
EDTA (BABE; as per Meares et al., Analyt. Biochem. 142:142,
1984) is covalently bound to the N- or C-terminus of the
peptide. These references are hereby incorporated by
reference. Technetium is then chelated to the DTPA- or
BABE-synthetic peptide by the procedure described above for
direct labelling of synthetic peptide. Technetium, in the
form of 99~Tco~ is added to the DTPA-synthetic peptide, and
to the mixture is added a solution of reduced sodium
dit~ionite, pH 8.0-9Ø 99~Tc-labelled synthetic-DTPA
peptide is separated from uncomplexed 99~Tc and sodium
dithionite by column chromatography (as described above).
The preparations are then characterized by silica gel
chromatography essentially as described by Meares et al.
(ibid.) and by HPLC. The 99~Tc-labelled peptide is
administered either in a pharmaceutically-acceptable carrier
solution or bound to a lipid emulsion.
Structure
To determine whether the molecular conformation or
structure of the synthetic apoB peptides was analogous to
the conformation of the apoB moiety of LDL, a polyclonal
antiserum was raised to each peptide and its ability to bind
LDL tested. Antiserum raised to human LDL was used as the
control.
Specific anti-~DL antisera may be purchased from a
number of sources (e.g., Hoechst Pharmaceutical, Inc.,
Cincinnati, Ohio and Marburg-Lahn, West Germany; and Hyland
Laboratories, Inc., 4501 Colorado Boulevard, Los Angeles,
California). Alternatively, antisera may be prepared by any
number of protocols known to those skilled in the art. For
assays described herein, anti-LDL antiserum was produced as
follows. 5 - 20 mg of LDL, prepared according to the method -~
of Fischman et al. (Arteriosclerosis 7:361, 1987) in about 1 - ~ -
~: . - ; - , - -
.... - . -. , . . . : , : . . : ............... :
~ . . . : : . . .
WO91/16919 PCT/US91/03026
2~8~
- 34 - '
ml of saline or barbital buffer, was emulsified with an
equal volume of Freund's complete adjuvant (Difco -
Laboratories, Detroit, MI). This was most easily done by
placing the lipoprotein and the adjuvant in separate 5 ml
Luer-Lock syringes with 20~gauge needles and connecting the
two needles via a l-inch piece of 0.030 inch inner diameter
polyethylene tubing. The contents of the syringes are then
force~ully expelled from one syringe into the other several
dozen times through the two needles and the connecting
tubing. A stable creamy emulsion was produced which was
finally passed entirely into one of the syringes, and the
connecting tubing is removed. The emulsified antigen was
injected subcutaneously into the bacX of a laboratory
rabbit. If several rabbits were to be injected with the
same antigen, larger syringes and larger quantities of
materials were used and each rabbit injected with 2 ml of
the emulsion representing l ml of the original antigen
solution. An alternative method for preparing emulsion in
quantity is to place equal volumes of antigen solution and
adjuvant into one tube of a Mickle disintegrator (Mickle
Company, Hampton, Middlesex, England; Brinkmann Instruments,
Inc., We9tbury, NY) which is stoppered and placed on one of
the steel reeds of this magnetic vibrator. A second sample
or a water balance is placed on the other reed, the machine
turned on, and the reeds tuned to maximal excursion for
about ten minutes. The resulting emulsion is drawn into
syringes through a blunt 18-gauge needle and injected
subcutaneously through 20-gauge needles.
For the preparation of antisera of high antibody
titer the animal may be "boosted" every 3-5 weeks exactly as
for the first injection. Good antiserum is usually obtained
after two injections. Animals treated in this way may be
maintained for long periods in the i~mune state and will
, ~ . . .. .- . ,
,
-: - . : . . . : . . ,, : .
, ~: . - , , , - - . . . .
. , . ~ ... .. .. . .
WOg1/16919 PCTIUS91/~3026
3 -:
- 35 -
yield very large amounts of antiserum. If quantities of
antiserum in the range of 1 liter or more are needed, sheep
may be used in the same manner, except that two to three
times the amount of immunizing antigen is required. The
animals are bled 6-10 days after each booster injection. A
small test bleeding may be made to check the antibody level
and purity if desired.
The blood i5 allowed to clot at room temperature for
~evQral hours and i9 then placed overnight in the
refrigerator. The samples are centrifuged in the cold, the
clots removed with an applicator stick, recentrifuged to
sediment the remaining blood cells, and the serum is
decanted. One milligram per milliliter of sodium azide is
added as a preservative. Antisera in constant use may be
kept in the refrigerator, or stored at -15 to -20C.
To produce anti-synthetic peptide antisera, purified
synthetic peptide was dissolved in PBS, pH 7.4 at a
concentration of 1 mg/ml. The peptide solution was mixed
with an equal volume of complete Freund's ad~uvant and
vortexed thoroughly until a thick emulsion was produced.
New Zealand White rabbits (Millbrook Farms, Amherst, MA)
were injected with a total of 0.5 mg synthetic peptide
administered subcutaneously in the ~our dorsal quadrants.
The rabbits were given a boost (injected at the same sites)
with 0.5 mg peptide emulsified in incomplete Freund's
adjuvant 2-3 weeks later. Eight to ten days after the first
boost, the animals were given a second, identical boost and ;
were bled of 30 ml 8 to 10 days later.
To test for immunological cross-reactivity
microtiter plates (Immulon II Dynatech Labs, Chantilly, VA)
were coated with the purified synthetic peptide or LDL by an
overnight incubation at 4C with 100 ng peptide per well in
50 mM carbonate, pR 9.6, and blocked for nonspecific binding
... -: :
WO91/16~19 PCT/US91/03026 ~
2 ~
- 36 -
with an additional overnight incubation with phosphate-
buffered saline, p~ 7.4 (PBS), 1% bovine serum albumin
(8SA). Control wells were coated with 8SA alone. After
washing twice with PBS, the wells were filled with serial
dilutions (l:lO to l:lO0,000 made in PBS, 3~ BSA) of a
rabbit polyclonal antibody generated against the synthetic
peptide(s) and incubated fo~ 45 minutes at room temperature.
Following thorough washing (3X with P~S, 0.1% BSA), the
wells were filled with a l:2000 dilution of goat anti-rabbit
IgG-horseradish peroxidase conjugate (Atlantic Antibodies,
Scarborough, ME). After a final wash, the wells were filled
with 3,3'-5,5'- tetramethybenzidine (TMB) microwell
peroxidase substrate (Xirkegaard and Perry Labs,
Gaithersburg, MD) and read at 650 nm every two minutes on an
ELISA-5 automated plate reader (Physica, New York, NY).
Results were expressed as the initial velocity of substrate
conversion (change in OD6sO/hr), which was determined by a
linear regression of 15 data points per well. Each data
point represented the average of three measured data points
ao from the same plate, run at the same time.
ELISA plates were coated with LDL and treated with
SP-4 (see PCT/US89/01854) antiserum. Anti-SP-4 antiserum
was able to bind LD~ on the plates, providing immunological
confirmation that SP-4 and LDL have structural similarities.
In analogous experiments, anti-SP-4 antiserum was shown to
bind SP-4 and SP-4A as well as the conservatively
substituted peptides, SP-6, SP-6A, SP-8, SP-8A, SP-12A, and
SP-14A, demonstrating that these peptides have structural
similarities to SP-4 and therefore structural similarities
to LDL.
. , .; .. .. , - - ~, - .~ - :
WO91/16919 PCT/US91/03026
23~A~93
- 37 -
Animal Model:
The peptides described herein were assayed for their
ability to target sites of vascular injury as follows. Male
New Zealand white rabbits (2 to 3 kg each) were obtained
from ARI Breeding Labs, West Bridgewater, MA. To induce
vascular injury, their abdominal aortas were denuded of
endothelium by a modification of the Baumgartner technique
(Fischman et al., Arteriosclerosis 7:361, 1987). Briefly,
a~ter each animal was anesthetized with ketamine and ether
or, alternatively, with xylazine (20 mg/ml) and Xetalar ~50
mg/ml), the left fe~oral artery was isolated; a 4F Fogarty
embolectomy catheter (Model 12-040-4F, Edwards Laboratories
Incorporated, Santa Anna, CA) was introduced through an
arterotomy in the femoral artery and was advanced under
fluoroscopic visualization to the level of the diaphragm.
The catheter was inflated to a pressure of about 3 psi above
the balloon inflation pressure with radiographic contrast
medium (Conray, Mallinkrodt, St. Louis, M0). Three passes
were made through the abdominal aorta with the inflated
catheter to remove the aortic endothelium before removal of
the catheter, ligation of the femoral artery, and closure of
the wound. The animals were allowed to heal for a period of
4 to 5 weieks before injection of th~ labelled synthetic
peptides.
Watanabe Heritable Hyperlipemic (WHHL) rabbits were
also used as animal models. They were obtained from the
WHHL Rabbit Program of the National Heart Lung and Blood
Institute (Bethesda, MD) at about 3 months of age and
weighing about l.5 kg. The animals were raised until they
were 3-4 kg in weight. At this weight, they exhibited
marked aortic atherosclerosis. ~ -
'
.
-- ......... ; . .. .
,.............. . .. . ~
WO~1/16919 PCT/US91/03026
2 ~
- 38 -
Each labelled synthetic peptide preparation
(containing, for example, 150 to 400 or more ~Ci of l25I-
labelled peptide) in column elution buffer was injected into
the marginal ear vein of the ballooned and healing New
Zealand white rabbits or WHHL rabbits. Serial blood samples
were obtained from the opposite ear during the ensuing 0-24
hours and were analyzed for radioactivity. The labelled
peptide concentrations in the blood samples that were
withdrawn over the first 10 minutes after injection were
extrapolated to zero time to determine the time zero
radioactivity in the calculation of average plasma
radioactivity. Peptides SPlSa, SP17, SP19a, SP21a, SP28,
SP34a, and SP30 were cleared rapidly from the plasma with
half-lives of about one minute or less; after one hour, the
plasma levels were less than 10% of the injected dose and
fell by an additional 1~ over the next three hours.
Peptides SP15a, SP17, SP21a, SP28, SP34a, and SP30 leveled
off to a plasma level of 3-6%; peptide SP19a cleared more
quickly and leveled off to a concentration of 0.3% (at four
hours).
one to twenty-four hours after injection of the
labelled synthetic peptide preparations, each animal was
injeated intravenously with 4 ml of a 0.5% solution of Evans
blue dye ~Allied Chemical Company, National Aniline
Division, NY, NY) which stains areas of de-endothelialized
aorta blue. After 30 minutes, the animal was sacrificed by
a lethal injection of pentobarbital. After sacrifice, the
aorta was removed completely, washed in saline, and fixed in
10% trichloroacetic acid.
The washed and fixed aortas from the animals that
had been injected with radiolabelled synthetic peptide were
opened along the ventral surface. These segments were then
'--- - : ' ~ - . ' . .
~ - , . , ': '
WO91/16919 PCTtUS91/03026
~3~3
: ` '-: .
- 39 -
pinned out, fixed for 2 hours in l0~ trichloroacetic acid,
and photographed. The fixed, opened vessels were then
covered with a single layer of plastic (Saran) wrap, placed
on high speed x-ray film (Xodak Orthofilm OH-l), and stored
for 3 days to 4 weeks in a Kodak "X-Omatic cassette" (24 X
30 cm) at -70C before development in a 90 second X-OMAT. ;
Representative results are shown in FIGS. 8-13. De-
endothelialized arterial wall is stained blue (with Evans
blue dye, as described above) and appears as dark areas in
the photographs (FIGS. 3-8, A); accumulation of radiolabel
is indicated by dark areas in the autoradiographs (FIGS. 3-
8, B). All peptides accumulated focally at the leading
edges of regenerating endothelial tissue in a pattern
characteristic of LDL. In each case, the autoradiograph
(FIG 3-8, B) demonstrates clear-cut localization of the
synthetic peptide on the image at the healing (re-
endothelizing) edge of the aortic lesions produced by the
previous trauma. Since this lesion is known to resemble
human arteriosclerosis in many important respects, including
accumulation of lipoproteins and other pathological changes,
the ability of the 5ynthetic peptides to localize at the
trauma site, and to permit the imaging thereo~ demonstràtes
the utility of the present invention in imaging vascular
disease.
These results and others are summarized in TABLE 3
and TABLE 4. Control peptides used were SP-2 (part of the
heparin and LDL receptor binding site of apolipoprotein E)
and SP-llA which is a receptor and heparin binding domain of
apolipoprotein B. To compare the relative accumulation of
the l25I-labelled synthetic peptides in the aorta and
adrenal gland, it was necessary to correct for differences
in mean plasma concentration of the labelled compounds. The
.
. .
. .
:
- - . -
.: : . - . ~ - ~ .
WO91/16919 PCT/US91/03026
2Q~a~s3 ~"
- 40 -
mean concentration of synthetic peptide-associated ~25I
radioactivity was calculated by numerical integration of the
plasma decay curves and division by the time since injection
of the isotope.
$UBS~ITUTE SHEEr
~- ,, - ., . ... ; .................... . .
. ` . ., . - ` . . . - . . ` ` . ` ` .
.
,. ~ ~ . . ~ . . .
WO 91/16919 PCr/US91/03026
-- 41 --
TAsLE 32~ ; 93
Rabbit Compound DoseCir. T.~ Focal
ID Tested (yCi~ Isotope (hrs) ACC'1T.
~59 sp6A* + t~SV 312 l2sI 24 +
B127 sp6A~ 350 l25I 4 +
B122 sp6A* 348 l2~I 4 +
B139 sp6A~ 552 l25I 4 +
B146 sp6A* 550.4 ~25I 5 +
II.-4 sp6A~ 282 l25I 5 +
IL-l sp6A* 308 l25I 5 +
B55 sp6~ 529 ~25I24 + :
BllS sp6~ 918 l2cI 4 +
B115-1 ap6* 398.9 ~2CI 4 +
Blll sp8~ + MV 419 ~2~I 4 +
B103 sp8* 424 l25I 5 +
B124 sp8* 466.2 ~25I 4 +
B124-1 sp8S 445.8 ~2~I 4 +
B120 sp8A* 315.3 ~2~I 4 +
B120-1 8p8A* 364.5 ~2~I S
B137 8p8A* 402.6 ~2~I 4 + .
~135 Sp8A* 427.7 ~25I 5 +
C-14 spl2AS 132.S ~2~I 4 +
C-13 apl2AS 110 l2-I 4 + .
';' '~ ' ~
.
. .. ..
''.''','''.
-. :
'''''' '"'
..
- .
SU3S, , UTE SHEET
W~:> 91/16919 PCI`/US91/03026
- 42 -
2~493 TABLE 3 (Con't)
Rabbit Compoun~ Dose Cir. T. Focal
ID ~ es~ed ~Ci~ Isoto~_ (hrs)_~A~c'~.
';
BS90 spll 648.1 115I 4
B690 spll 363.0 l2~I 4
NZW-l spl2A* 260.0 l2~I 5
NZW-2 spl2A* 166.4 l2~I . 4
NZW-3 spl4A* 941.0 l2~I 4
WHHL-l sp2* 318.9 l2~
WHHL-6 spll~ 126.3 l26I 4
WHHL-7 spll* 117.1 l25I 4
.. . . .
MV cholesterol ester microvesicles
* . radioactive
~ cold
' ': '
' - .
.
~ . .
SUBSTITUTE SHEE~ -
W O 91/16919 PC~F/US9ltO30Z6
3 ; ;~
- 43 -
TA~LE 4
Conpoulda Ci r . T . Focsi
Tested Isotope (hours~ Acc m
.. . .
5 SP-3411 1ZS1 4 ~ :
SP-28 1251 4
SP-30 1251 4 ~ ~ .
~,., " ., .
All peptides hDve plllsm~ hslt lives ot approximl~tely oro mir~te. R~diol~olled peptides ~ere
10 inJcctod intrcvenously et r dose ot ~t~e~n 0.2 1.0 mCi.
at is claimed is~
., ,
,. . .~ ~ ., . -
' '
~, '"-.' '-'', .
~, .
