CA2080493A1 - Synthetic peptides for arterial imaging - Google Patents

Abstract

Vascular disease including asymptomatic atherosclerosis can be diagnosed by administering a synthetic peptide or peptide analog having an affinity for, and propensity to accumulate at, a site of vascular injury to a patient, and then detecting the location of the peptide or peptide analog within the patient's vascular system. The synthetic peptide or peptide analog may include an amino acid sequence sufficiently duplicative of the amino acid sequence of a region of either the apolipoprotein B, apolipoprotein A-I, or elastin proteins such that the peptide or peptide analog accumulates at a site of vascular injury.

Description

2a~a~3 i ~

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 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 . ' :

2 ~ 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.

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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;

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WO91/16919 PCT/US91/03026 ~
2~$~3 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 ~'''` ~'' ~' 2~a~3 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.

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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:

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WO 91tl6919 PCT/US91/03026 2~493 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 .: , : - :. . . . :.: . - . ,. - .- . ., - , .
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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;

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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 : ' .
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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.

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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, . . .

':

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 (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 ~ -. .

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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 . . .

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2Q~0~3 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 : .' ;' ~'",'."

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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 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~ .

... . .
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 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:

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:

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 ... '~

Ala-Asn-Ala~Glu-Gly-Ala-Lys-CONH2;
30 SP-12A: -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;
., '..

. . ; '' ,'' .' : ~

:

2 ~ 3 SP-15a:

CH3CONH-Tyr-Ala-Lys-Phe-Arg-Glu-Thr-Leu-Glu-Asp-Thr-Arg-Asp-Arg-Met-Tyr-CONH2;
SP-17:

H2N-Tyr-Ala-Ala-Leu-Asp-Leu-Asn-Ala-val-Ala-Asn-Lys-Ile 10 Ala-Asp-Phe-Glu-Leu-CONH2;
SP-19a:

CH3CONH-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Glu-Gln-Ala-Lys-Gly-Ala-CONH2; and SP-2la:

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 ' - - , : .. - :.. . . . ~ -, . .. . , . .. :-2~ 3 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).
. .: ' ,' 2 ~ 3 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 ;-H2N-Tyr-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly--Val--Gly--CONH2 .

., ~ . .
- . : . . .

WO91/16919 PCT/US91!03026 3 : -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 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~

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 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 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.

- . . ., . - , . .. .
~- .. - . . - . , , -. . - . - . . . . .

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 - ~ -~: . - ; - , - -.... - . -. , . . . : , : . . : ............... :

~ . . . : : . . .

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 -:

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 ~

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.

. , .; .. .. , - - ~, - .~ - :

23~A~93 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. ~ -' .

-- ......... ; . .. .

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WO~1/16919 PCT/US91/03026 2 ~

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 : ` '-: .

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 .
. .
. .
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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.

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WO 91/16919 PCr/US91/03026 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 + .

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W~:> 91/16919 PCI`/US91/03026 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 ' ': ' ' - .
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SUBSTITUTE SHEE~ -W O 91/16919 PC~F/US9ltO30Z6 3 ; ;~

TA~LE 4 Conpoulda Ci r . T . Focsi Tested Isotope (hours~ Acc m .. . .
5 SP-3411 1ZS1 4 ~ :

SP-30 1251 4 ~ ~ .
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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~

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Claims (63)

Claims

1. A peptide or peptide analog having an affinity for, and propensity to accumulate at a site of vascular injury, said peptide or peptide analog comprising an amino acid sequence selected from the group comprising ;
;
;
;
;
;
; and .

2. The peptide or peptide analog of claim 1, wherein said amino acid sequence comprises .

3. The peptide or peptide analog of claim 1, wherein said amino acid sequence comprises .

4. The peptide or peptide analog of claim 1, wherein said amino acid sequence comprises .

5. The peptide or peptide analog of claim 1, wherein said amino acid sequence comprises .

6. The peptide or peptide analog of claim 1, wherein said amino acid sequence comprises .

7. The peptide or peptide analog of claim 1, wherein said amino acid sequence comprises .

8. The peptide or peptide analog of claim 1, wherein said amino acid sequence comprises .

9. The peptide or peptide analog of claim 1, wherein said amino acid sequence comprises .

10. A peptide or peptide analog having an affinity for, and propensity to accumulate at a site of vascular injury, said peptide or peptide analog comprising an amphiphilic domain of apolipoprotein A-I and having a net charge of -2 or greater, whereby said peptide or peptide analog accumulates at said site.

11. The peptide or peptide analog of claim 10, wherein said amphiphilic domain further comprises an .alpha.-helix.

12. The peptide or peptide analog of claim 10, having a net charge of -2 or greater and an amino acid sequence sufficiently duplicative of that of at least a portion of said amphiphilic domain of apoA-I, whereby said peptide or peptide analog accumulates at said site of vascular injury.

13. The peptide or peptide analog of claim 10, said peptide or peptide analog having an amino acid sequence comprising .

14. A peptide or peptide analog having an affinity for, and propensity to accumulate at a site of vascular injury, said peptide or peptide analog comprising a hydrophobic domain and having a net charge of -2 or greater, whereby said peptide or peptide analog accumulates at said site of vascular injury.

15. The peptide or peptide analog of claim 14, wherein said peptide or peptide analog is derived from a vascular-associated protein.

16. The peptide or peptide analog of claim 15, wherein said vascular-associated protein is elastin.

17. The peptide or peptide analog of claim 14, wherein said peptide or peptide analog has an affinity for a vascular wall component.

18. The peptide or peptide analog of claim 17, wherein said vascular wall component is a collagen.

19. The peptide or peptide analog of claim 17, wherein said vascular wall component is a proteoglycan.

20. The peptide or peptide analog of claim 17, wherein said vascular wall component is elastin.

21. The peptide or peptide analog of claim 20, wherein said peptide or peptide analog binds elastin with a dissociation constant of 10-6 or less.

22. The peptide or peptide analog of claim 14, wherein said hydrophobic domain comprises a .beta.-strand.

23. The peptide or peptide analog of claim 14, having a net charge of -2 or greater and an amino acid sequence sufficiently duplicative of that of at least a portion of elastin, whereby said peptide or peptide analog accumulates at said site.

24. The peptide or peptide analog of claim 16, said peptide analog having an amino acid sequence comprising , wherein X is at least 1.

25. The peptide or peptide analog of claim 24, wherein said x is 3.

26. The peptide or peptide analog of claim 16, wherein said amino acid sequence comprises , wherein x is at least 1.

27. The peptide or peptide analog of claim 26, wherein said x is 4.

28. The peptide or peptide analog of claim 26, wherein said x is 3.

29. The peptide or peptide analog of claim 1, 10, or 14, wherein the amino terminus of said peptide or peptide analog is acetylated.

30. The peptide or peptide analog of claim 1, 10, or 14, wherein the carboxy terminus of said peptide or peptide analog is amidated.

31. The peptide or peptide analog of claim 1, 10, or 14, wherein said peptide or peptide analog is water soluble.

32. The peptide or peptide analog of claim 1, 10, or 14, wherein said peptide or peptide analog is soluble in a physiological fluid.

33. The peptide or peptide analog of claim 32, wherein said physiological fluid is at physiological pH.

34. The peptide or peptide analog of claim 33, wherein said physiological fluid is blood plasma.

35. The peptide or peptide analog of claim 1, 10, or 14, further comprising a detectable label linked thereto.

36. The peptide or peptide analog of claim 35, wherein said label is radioactive.

37. The peptide or peptide analog of claim 36, wherein said radioactive label is selected from the group comprising 131I, 125I 123I, 111In, 99mTc, 203Pb, 198Hg, 97Ru, and 201T1.

38. The peptide or peptide analog of claim 37, wherein said radioactive label is 99mTc.

39. The peptide or peptide analog of claim 35, wherein said label comprises a paramagnetic contrast agent.

40. A method for the detection of injury in the vascular system of a subject, said method comprising the steps of:

(a) introducing into said subject a peptide or peptide analog of claims 1, 10, or 14, (b) allowing said introduced peptide or peptide analog to circulate within said vascular system, whereby at least a portion of said peptide or peptide analog accumulates at said site; and (c) detecting the location in said vascular system of said peptide or peptide analog which has accumulated at said site.

41. The method of claim 40, wherein said vascular injury in the vascular system comprises atherosclerosis.

42. The method of claim 40, wherein said peptide or peptide analog comprises an amphiphilic domain of apolipoprotein A-I and has a net charge of -2 or greater, whereby said peptide or peptide analog accumulates at said site of vascular injury.

43. The method of claim 42, wherein said amphiphilic domain further comprises an .alpha.-helix.

44. The method of claim 40, wherein said peptide or peptide analog comprises a hydrophobic domain and has a net charge of -2 or greater, whereby said peptide or peptide analog accumulates at said site of vascular injury.

45. The method of claim 44, wherein said peptide or peptide analog is derived from a vascular-associated protein.

46. The method of claim 45, wherein said vascular-associated protein is elastin.

47. The method of claim 40, wherein said introducing step (a) comprises introducing into said subject a peptide or peptide analog having an acetylated amino terminus.

48. The method of claim 40, wherein said introducing step (a) comprises introducing into said subject a peptide or peptide analog have an animated carboxy terminus,

49. The method of claim 40, wherein said introducing step (a) comprises administering a peptide or peptide analog having a detectable label linked thereto.

50. The method of claim 49, wherein said detectable label is a paramagnetic contrast agent.

51. The method of claim 49, wherein said detectable label is a radioactive label.

52. The method of claim 51, wherein said radioactive label is selected from the group consisting of 131I, 125I 123I, 111In, 99mTc, 203Pb, 198Hg, 97Ru, and 201T1.

53. The method of claim 40, wherein said introducing step (a) comprises the administration of said peptide or peptide analog by arterial injection.

54. The method of claim 40, wherein said introducing step (a) comprises the administration of said peptide or peptide analog by venous injection.

55. The method of claim 40, wherein said introducing step (a) comprises the oral administration of said peptide or peptide analog.

56. The method of claim 40, wherein said introducing step (a) comprises the nasal administration of said peptide or peptide analog.

57. The method of claim 40, wherein said detecting step (c) further comprises imaging a region of said vascular system at which said peptide or peptide analog has accumulated.

58. The method of claim 40, further comprising the step of quantitating a detected amount of said labelled peptide or peptide analog.

59. The method of claim 40, wherein said introducing step (a) comprises administering a second peptide or peptide analog having an affinity for, and propensity to accumulate at a site of arterial injury.

60. The method of claim 40, wherein said detecting step (c) further includes the extracorporeal monitoring of said labelled peptide or peptide analog.

61. The method of claim 51, wherein said detecting step (c) comprises the extracorporeal monitoring of said radioactive label with a gamma scintillation camera.

62. The method of claim 50, wherein said detecting step (c) comprises the extracorporeal monitoring of said paramagnetic contrast agent with a magnetic resonance imaging system.

63. A method for inhibiting the binding of low density lipoprotein to a vascular wall of a subject comprising administering to said subject a therapeutically-effective amount of the peptide or peptide analog of claim 1, 10, or 14.