JPH05507276A - Synthetic peptides for arteriography - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Synthetic peptides for arteriography The subject matter of this application was filed on May 2, 1988, May 3, 1990, and 1990, respectively. USSN 189.130 filed on May 3, 2013, and its continuation USS It is a 11m continuation of N518.215 and UssN)18.142.

Background of the invention The United States Government has granted rights to this invention pursuant to NIH Grant No. HL32975. has.

The present invention provides methods and methods useful for early detection of vascular diseases such as atherosclerosis. methods and methods, particularly the use of labeled synthetic peptides to detect arterial damage. and means.

Atherosclerosis is the thickening and hardening of the walls of arteries, especially larger arteries. It is a disease that causes It is a lesion or raised fibers that form within the arterial lumen. characterized by raised fibrous plaques . The plaques most commonly occur in the abdominal aorta, coronary arteries, or carotid arteries, and they vary with age. Both will gradually increase.

They generally exhibit a dome-shaped, opaque shimmer (surface) that distorts the lumen.

Lesions are typically keyed by a central core of lipids and a collagenous fibromuscular layer. Consists of capped necrotic cellular debris. Complex lesions are calcific The tumor may also include inflammation and may exhibit varying degrees of necrosis, thrombosis, and ulceration.

damage to or deformation of the arterial lumen caused by plaques and associated deposits; Therefore, blood flow is obstructed and, if untreated, can eventually lead to angina, cerebral ischemia, and renal It causes hypertension, ischemic heart disease, stroke, and other organ diseases. Currently, coronary Teloma atherosclerosis remains the leading cause of death in the United States. , more than 500,000 Americans die each year, nearly twice as many as die from cancer. take away.

Unfortunately, in the early stages of atherosclerosis, and often clinically nothing No diagnostic method exists that can detect related vascular diseases not mentioned.

Lifestyle changes, drug treatments, and other measures can reduce atherosclerotic lesions. Exists to delay or reduce vascular occlusion or stress on various body organs Therefore, early detection of plaques in the vasculature may be most effective. It would be of great value as a preventive intervention at the same time.

Arteriography, a conventional approach to the diagnosis of vascular disease, characterizes and obstructs arteries. involves injecting a radiopaque substance into the bloodstream to image the image. This method medical conditions, including infection, arterial perforation, arrhythmia, stroke, infarction; Furthermore, it can even lead to death. Because of the risks involved, arteriography is typically For individuals suffering from epigenetic or acute atherosclerosis An arteriogram is prepared.

Less invasive for the treatment of vascular injuries and diseases Various techniques have been proposed. These techniques include blethysmography, imaging and ultrasound scanning (Lees and Myers, Adv, Int. Med, 27:4. 75.1982).

Other non-invasive methods for diagnosis of vascular injury proposed in the present invention ive) approach seeks to label targets biologically active molecules or arterial lesions. Administration of antibodies that preferentially seek out (U,S, patent application no. 929.012, invention Name: “Detection of Vascular Disease” (Detection pof Vascular Di) sease), filed on November 10, 1986) and labeled low-density riboprotein (LD Administration of L) into the patient's vascular system (U, S, Patent No. 4.647.445 and No. 4.660.563). LDL circulating in the blood is atherosclerosis known to bind to inflammatory plaques (Lees et al., Journal Op. Bu Nuclia Medin (J, Nucl, Med, ), 24 +1 54.1983).

This binding probably occurs through receptor-mediated uptake in various cell types. Apoliboprotein B'-, the protein part of the LDL molecule that is responsible for removing LDL from 100 (apo B-100). Conduit to radioactive label by using regulated LDL and imaging them with a radiation detector. Information about the location and size of plaques in the vasculature can be obtained. alternative method As a non-radiating method, LDL is detected in a magnetic resonance imaging (MRI) system. It can be labeled with a radioactive paramagnetic contrast agent.

One drawback of this method is that it requires isolating LDL from the patient's blood and labeling them. This typically requires several days. Often such diagnoses and Delays in treatment and subsequent treatment are detrimental to the critically ill person. moreover, Additional risk of viral infection if donor blood is used as a source of LDL to cover.

As a result, non-stenotic, non-flow-impaired atherosclerotic arterial lesions can be detected early. Better non-erosive techniques and reagents that can be extracted and mapped are needed. Ru.

Therefore, it is an object of the present invention to provide An object of the present invention is to provide synthetic peptides.

Furthermore, an object of the present invention is to provide synthetic peptides that are inexpensive and easy to prepare and are useful for imaging. The goal is to provide the following.

Furthermore, it is an object of the present invention to detect vascular damage, including its initial vascular damage and It also provides an improved method for pinging.

Furthermore, it is an object of the present invention to detect vascular damage and pine pins that are non-erosive. It is also about providing a way to

Finally, it is an object of the present invention to provide synthetic peptides for the prevention or treatment of vascular damage. It is to be.

Summary of the invention In general, the present invention provides that the peptide or peptide analog is Tyr-Lys-Le u -A la -Leu -G lu -Ala -A la -Arg - Leu -Leu -A Ia -Asp-Ala-Glu-Gly-Ala- Lys';Tyr-Lys-Leu-Ala-Leu-Glu -Ala -Ala -Arg -Leu -Leu -Ala -Asn-A la-Glu-Gly-Ala-Lys; Tyr-Arg-Ala, -L eu -Val -Asp -Tyr -Leu -Lys -P he - 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 -A la -A, 1a -Leu -Asp -Leu -Asn -Ala -Val -Ala, -Asn -Lys -11e-Ala-A sp-Phe-Glu-Leu: Tyr-Arg-A, la-Leu -Val -Asp -Thr -Leu -Lys -Phe -Val -Thr -Glu-Gin-Ala-Lys-C; 1y-ALa + or T yr -Arg -, 411a -Leu -Val -Asp -Thr - G lu -P he -Lys -Val -L ys@- an atom selected from the group consisting of Gln-Glu-Ala-Gly-Ala-Lys; Contains a amino acid sequence that has an affinity for and accumulates at sites of vascular damage. Characterizes a peptide or peptide analog having

The term "peptide" means any chain of 30 or fewer amino acids. “Pe The term "petide analogue" refers to, for example, Val to Leu or Lys to -Ar. or by conservative amino acid substitutions such as without destroying the activity (in this case, the peptide's ability to target vascular lesions)! Nia Only by one or more non-conservative amino acid substitutions, deletions, or insertions peptides that differ in their amino acid sequences. As described herein Peptide analogues include one or more amino acid analogs, either as part of their sequence or in their entirety. molecules, molecules that closely resemble the structure of amino acids, and/or peptides or Contains naturally occurring amino acids in molecules other than peptide analogs.

Furthermore, the present invention has an affinity for the site of vascular injury and a property of accumulating at that site. is characterized by a peptide or peptide analog having: Alternatively, the peptide analogue is a preferred protein of apoliboprotein A-1 (apoA-1). Preferably, it is derived from an amphipathic domain containing an α-helix and has a net charge of −2 or more. As a result, the peptide or peptide analog accumulates at the site of injury.

The term "net charge" refers to the total charge of the peptide at neutral pH; Calculated by adding together the charge (at neutral pH) for each of the amino acids. be done. As used herein, the term "derived from" refers to apoliboprotein A- It means having an amino acid sequence that is the same or substantially the same as that of No. 1. " The term "substantially identical" means that, by conservative amino acid substitutions (as described above), or (as mentioned above) a non-conserved position that does not destroy the biological activity of the peptide. having an amino acid sequence that differs only by amino acid substitution, deletion, or insertion means.

In preferred embodiments, the peptide or peptide analog has ~2 or more net a charged amino acid of at least a portion of the amphipathic domain of apoliboprotein A-I; has an amino acid sequence that is sufficiently duplicated with the amino acid sequence , as a result, the peptide or peptide analog accumulates at the site of vascular injury. Like A new peptide or peptide analog is Tyr-Val　-Leu　-Asp　-Glu　-Phe-Arg　-Clu -Lys -Leu-Asn -G 1u-Glu-Leu-Glu-Ala -Leu-Lys-Gin-Lys.

Furthermore, the present invention provides an affinity for vascular injury sites and a tendency to accumulate at the sites. characterized by a peptide or peptide analog having the quality The analog contains a hydrophobic domain and has a net charge of −2 or more, so that the Peptides or peptide analogs accumulate at the site of injury. Preferably a peptide Alternatively, the peptide analog is derived from a vascular associated protein, such as elastin.

As used herein, the term "go" derived from (as shown) have the same or substantially the same amino acid sequence.

The term "vascular-associated protein" refers to proteins naturally occurring in either blood vessel walls or extracellular components of the vasculature. refers to proteins related to elastin and collagen, as well as proteoglycerides. Contains carbohydrates such as lycan.

In other preferred embodiments, the peptide or peptide analog is a blood vessel wall component, e.g. For example, having an affinity for collagen, proteoglycans or elastin; The peptide or peptide analog has a dissociation constant of 10-' or less (i.e., or Podet et al., Atherosclerosis and Thrombosis Ar. teriosclerosis and thrombosis), 11:1 16. Greater affinity when measured in vitro by the method of 1991 ) The hydrophobic domain of the peptide or peptide analog binds elastin Contains β-chain. Furthermore, in a preferred embodiment of Koike, the blood vessel-related protein comprises one or more positive Low taste charge and elastin (both have sufficient overlap with some amino acid sequences) A peptide or peptide analog having an amino acid sequence that Peptides or peptide analogs accumulate at sites of vascular injury. Preferred peptide or peptide analogs are amino acid sequences Tyr-(Val-Gly-Val-Ala-Pro-Gly).

[wherein X may contain at least 1, preferably 3: or the peptide Or the peptide analog has the amino acid sequence: Tyr-(Val-Pro-Gly- Val-Gly).

[wherein X is at least 1, preferably 3, or more preferably 4] Good too.

In a preferred embodiment of the invention as a whole, the peptide or peptide analog is It has a cetylated amino terminus and/or an aminated carboxy terminus. like this Also, examples of peptide analogs include HIN-Tyr-L ys -Leu -Ala -Leu -Glu -A la -Ala-A rg -Leu -Leu-Ala-, A1.5l)-, to 1a-Glu-Gl y-Ala-Lys-CONH2; CH, CON H-Tyr-Lys-Le u -, A'la -Leu -Glu -Ala-, Ala -Arg -L eu -Leu -Ala-Asp -Ala -Cr1u -Gly -A la-Lys-CONH! ;H, N -Tyr -Lys -Leu - Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu -, Ala-Asn-Ala-Glu-Gly-Ala-Lys-CONH2+ CH3C0NH-Tyr-Lys-Leu-, A, 1a-Leu-G lu　-Ala-Ala, -Arg　-Leu　-Leu　-Ala　-Asn -Ala -Glu -Gly -Ala -Lys -CONH2:CH 3CON H-Tyr-, Arg-Ala-Leu-Val-Asp-Tyr- Leu-Lys-Phe-VaL-Thr-Gin-Leu-C0NH2 ;CH3CONH-Tyr-Arg-Aha-Leu-Val-Asp -Thr -Leu -Lys -CONH2; CH3CONH-Tyr-Ala-Lys-Phe-Arg-Glu-Th r-Leu-Glu-Asp-Thr-Arg-, Asp-, Arg-M et-Tyr-C0NH2; H2N-Tyr-, A, la-Ala-Leu-A sp-Leu-, Asn-Ala-VaI-Ala-Asn-Lys-I1 e-Ala-Asp-Phe-Glu-Leu-CONH, ;CH3CONH- Tyr-Arg-Ala-Leu-Val-Asp-Thr-L eu-Lys-Phe-Val-Thr-Glu-Gin-Ala-Lys -Gly-Ala-CONH, ;CH3CONH-Tyr -Arg -Ala -Leu -Val-Asp -Thr -Glu -Phe -Lys - Val-Lys-Gin-Glu-Ala-G1. y-Ala-Lys -C0NH2; CH3CONH-Tyr -Val -Leu -Asp-G lu　-Phe　-Arg　-Glu　-Lys　-L　eu　-A　sn　- G1. u -Glu -Leu -Glu -A la -Leu -Lys -Gln-Lys-CONH,; H2N-Tyr-Val-Pro-Gly-Val-Gly-Val -Pro -Gly -Val -Gly-Val -Pro-Gly -V al　-Gly　-Val　-Pro　-Gly-Val　-Gly　-CON H2, and H4N -Tyr-Val-Gly-Val-, Ala-Pro-Gly-Va l-Guy-Val-AIa-Pro-Gly-Val-Gly-Val-Al a-Pro-Gly-CONH2 is mentioned.

The synthetic peptides or peptide analogs detect damage in a subject's vasculature. Useful for imaging and imaging. Other useful synthetic peptides or peptide analytes Logs include amino acid analogs, molecules that resemble amino acid structures, and peptides. or containing naturally occurring amino acids in molecules other than peptide analogs.

In other preferred embodiments of the invention as a whole, the peptide or peptide analog is aqueous. soluble or in physiological fluids, preferably at physiological pH, such as plasma. Soluble: The peptide or peptide analog can be conjugated to a detectable label that allows for conjugation. Preferred labels are radioisotopes, For example, +31 J, +! 51.1247,””Ins””Tcs "" P bs' "" Hg, Ru" or 201T7B or paramagnetic Examples include contrast agents. Such signs may include, for example, gamma scintillation cameras or or synthetic peptides or peptide analogs within the subject's vasculature in the MHI system. This may enable in vitro monitoring of species.

The present invention also provides for introducing a peptide or a peptide analog of the above form into a subject. Damage in the subject's vasculature, including atherosclerosis ) is characterized by a detection method. The method further comprises a second peptide of the above form or may include administering a peptide analog. the peptide to be introduced or Peptide analogs can also be administered by intraarterial or intravenous injection. alternative method Alternatively, non-hydrolyzable derivatives can also be administered orally or nasally.

The introduced synthetic peptide or peptide analog is then allowed to circulate within the subject's vasculature. ring, thereby causing at least a portion of it to accumulate at the site of injury. Next, loss A portion of the synthetic peptide or peptide analog that accumulates at the wound site is detected. Applicable The detection step further includes detecting labeled peptides or peptide analyzers that have accumulated at the site of vascular damage. Second, to quantify the amount of logs: or for example, with a gamma scintillation camera or or a label detectable by a magnetic resonance imaging system (e.g., a radioactive label or a paramagnetic by in vitro monitoring of peptides or peptide analogs with contrast agents). to create areas of the subject's vasculature where the synthetic peptide or peptide analog has accumulated. May include shadowing.

Finally, the present invention provides therapeutic properties of peptides or peptide analogues of the above-mentioned forms. Blood vessels of the subject, including administering an effective amount to the subject! binding of low-density lipoprotein to including methods for suppressing

We administered synthetic peptides to a subject and then Detecting the location, pattern and concentration of the peptide after its accumulation at the site of injury This allows us to diagnose vascular diseases, including asymptomatic atherosclerosis. I found it. The technology offers many advantages. It is non-erosive: it is Requires neither crude medical equipment nor highly skilled practitioners to perform successfully. No need; peptides used to target vascular lesions can be used cheaply and quickly , and can be produced in large quantities (eg, by recombinant DNA technology).

Furthermore, the peptides of the present invention can be used to prevent vascular diseases such as atherosclerosis. or may be used for relief. Administration of a therapeutically effective dose of a peptide of the invention can prevent LDL accumulation by blocking LDL binding sites. Ru.

Other features and advantages of the invention reside in the following description of preferred embodiments and in the claims. It should be clear from this.

Brief description of the drawing The foregoing and other objects of the invention, its various features, and the invention itself are described below. The following description will be better understood when read in conjunction with the accompanying drawings.

Figure 1 shows apo B-100 form when contained in LDL molecules and surface exposure. A schematic model of the area is shown.

Figure 2 shows a series of helical wheels demonstrating the amphiphilic characteristics of representative synthetic peptides. It is a diagram.

Figure 3 shows rabbit abdominal aorta treated with 1251-labeled synthetic peptide 5P-17. A photograph (A) and an onlay autoradiograph (B) are shown.

Figure 4 shows l! It　　[i1 Rabbit treated with synthetic peptides-19a A photograph (A) and an onlay autoradiograph (B) of the abdominal aorta are shown.

Figure 5 shows the abdominal movements of rabbits treated with 115■ labeled synthetic peptide 5P-21a. Pulse photographs (A) and onlay autoradiographs (B) are shown.

Figure 6 shows the abdominal movements of rabbits treated with I'lS■-labeled synthetic peptide 5P-28. Pulse photographs (A) and onlay autoradiographs (B) are shown.

Figure 7 shows rabbit abdominal aorta treated with +zs1-labeled synthetic peptide 5P-29. A photograph (A) and an autoradiograph (B) are shown.

Figure 8 shows rabbit abdominal aorta treated with +251-labeled synthetic peptide 5P-30. A photograph (A) and an onlay autoradiograph (B) are shown.

detailed description The present invention has an affinity for a vascular injury site and a property of accumulating at that site, Therefore, synthetic compounds that are useful in the detection, diagnosis, monitoring and treatment of vascular diseases Provides peptides.

Specific examples of such synthetic peptides with these characteristics are i.e., the point responsible for its affinity for arterial lesions. The amino acid configuration of a portion of a ribopeptide (e.g., low-density riboprotein or elastin) a known polypeptide with similar molecular structure, charge, and/or size to the sequence may have an amino acid sequence similar to that of the same class. Alternatively, the synthetic peptides of the invention Tides contain the apoB-100 part of LDL, the apoA-I part of HDL, or the apoA-I part of HDL. It may be homologous to a portion of lastin.

Design and synthesis of peptides Peptides useful in the present invention are those that successfully target vascular lesions. write are “vascular-related”, i.e., on the surface of vascular cells or on extracellular components of the vasculature; naturally associated with (e.g. proteoglycans, collagen, or elastin) It is preferable to create such peptides after the sequence of the protein. of this kind Proteins include apolipoprotein B (i.e., the protein part of low-density riboprotein) and and elastin (a natural component of artery walls). It is essential to use whole protein molecules. It is not necessary and is often a disadvantage (see above). The present inventors have demonstrated that protein fragments are We have also found that it can be used to effectively target ductal lesions. Examples of useful snippets Describe it here. We found that such fragments have a low net charge (i.e. - 2 to +2), such as very negatively charged blood vessels! to interact with and showed. The inventors also found that such peptides generally fall into two classes: ( 1) Peptides comprising an amphipathic domain, preferably of α-helical character; and ( 2) Hydrophobic domain (which promotes interaction with vascular surfaces or vascular-associated extracellular components) positive charge or low negative charge (i.e., −2 or more) to promote , or more positively) to one of the peptides containing a hydrophilic domain. It was also shown that

■ class, i.e., amphipathic domains (i.e., hydrophobic and hydrophilic surfaces) Preferred peptides of the peptides containing a domain with both domains include, for example, Kaiser and KezdyXAnn, Rev. B1ophys, B1ophys.

Chem, 16:561.1987; Science, 22 3:249.1984). Typically both A philic domain contains a region of secondary structure, most commonly an α-helix or a β-strand. nothing. α-helix-containing bebutides are generally more soluble than β-chain-containing bebutides. They are more preferred in the present invention because: the increased solubility - Facilitates peptide synthesis and peptide administration to patients.

(a) vascular cell surfaces or hydrophobic vascular-related components (e.g., (b) hydrophobic domain that promotes interaction with stin) and (b) promotes solubility. Preference is given to peptides containing both positively charged and slightly negatively charged domains. For example, suitable peptides can be found using the following methods for predicting hydrophobicity and hydrophilicity. and identify it. The present inventors predicted that this class of peptides would form β-chains. Even peptides that contain more than one domain may be administered to a subject and cause arterial disease. showed that it can be used to adequately target mutations. This classification of peptides is It appears to interact with hydrophobic vascular-associated extracellular components.

The net charge on a peptide is the charge on the amino acids of the peptide at neutral pH added together. Calculate. Localized charge characteristics (i.e. amphipathic properties of a region of a peptide, e.g. properties, hydrophilic, or hydrophobic properties) and the secondary structure of individual sequences of amino acids (i.e. i.e., the presence of α-helices or β-strands) is can be predicted from its primary sequence using For example, amphiphilic α-helices are In order to determine the hydrophobic residues and hydrophilic residues on opposite faces of the resulting cylinder. You can look for the presence of residues (Schiff-r-(Sehiffer) and Ed. E dmundson, Pkuiga Physical Journal (Bi ophys, J.), 7/121.1967; cited herein. ). Alternatively, amphipathic, hydrophobic or hydrophilic domains or regions of secondary structure In order to identify the area, the Chou-Fassman method (Chou-Fasman) was used. Chou and Fasman, Ann, Rev, Bi ochem, 47:251.1978; cited herein). Empirical formula or program PREDICT (based on GOR method of secondary structure prediction) (Robson et al., Introduction to Proteins and Protein Engineering) tion to Proteins and Protein Engine ring) ; Nilsveier (E1sevier), New York, 198 6: cited herein) may be used. Such a program is an equation [In the formula, I(S,=X:X:R,,) value is the residue that should be in structure X." derived from the statistical preference for use. The state of `` is determined from the sum of more than m residues in the sequence on either side of ``. The parameter values were evaluated based on the identity of the residue at each position and the four types of structures. It depends on the contribution to each. Values are calculated for each of states H, E, T and C. and the highest value is the predicted structure (H-α-helix, E=β-note, T-bicurve, or C= Determine the size of the silanum coil. Finally, amphiphilicity is defined as “hydrophobic calculation of the amphiphilicity perpendicular to the axis of the periodic peptide structure. can be derived from the This approach is based on Eisenberg (Ann, RevBiocheIQ, 53 595.1.984; herein (cited in ).

The charged properties of the protein (i.e. amphipathic, @aqueous or hydrophobic) and/or secondary Although the structure is known, it is unclear how the protein interacts with the charged (or hydrophobic) surface of the pond. The individual amino acid sequences that promote r) and Kezdy, Science, 223 : 249.1984).

Therefore, the local charge contribution (and total net charge) and secondary structure, thereby provided that biological activity (in this case the ability to target vascular lesions) is maintained. designed some of the many peptide analogs with different amino acid sequences to It is possible to do so. Such peptide analogs are readily available to those skilled in the art of biochemistry. Conservative amino acid substitutions well known to those skilled in the art (e.g., Val to Leu, or will generally differ from the native protein sequence by a Lys to Arg substitution) . Additionally, amphipathic, hydrophobic, and hydrophilic properties within longer amino acid stretches Peptides can be designed that include regions and/or secondary structures. In general, this The charged properties and secondary structure of the nucleated region are not affected by surrounding amino acid residues. . Moreover, only these peptides that can target vascular lesions are It is considered useful in the invention.

Preferred peptides useful in the present invention include surface-exposed protein domains. (i.e., a region of the protein on the outer surface of a protein molecule, preferably a blood vessel-associated protein molecule) based on peptides (peptides). This is because it is thought to interact with continuous extracellular components. Identity of surface exposed domains the trypton digestion analysis (see below) and/or the hydrophobicity/hydrophilicity of the region. (e.g., Koufusman method, Ann, Rev , Biochem, 47:251.1978): the extracellular domain is one Generally hydrophilic or amphiphilic in nature: such domains often have transmembrane Surrounded by hydrophobic stretches corresponding to the domain.

After designing the peptides, e.g. Synthesized by any of a number of established methods, including expression of recombinant DNA can be done. Alternatively, these peptides can be synthesized using established solid-phase peptide synthesis. It can be manufactured by the method described above. In other words, this method The appropriate amino acid is added to the peptide of the desired sequence while the end of the compound is linked to an insoluble support. Requires continuous incorporation into the compound. Usually, the carbomonyl end of the peptide The ends are connected to a polymer that can be released by treatment with cut straw. Then like this Monitoring of the synthesized peptides after administering them to a practitioner Label with a reagent that allows

Peptides can be used in in vivo animal assays (e.g., the assays described herein). ) can be tested for their ability to effectively target vascular lesions. LDL is Rabbit balloon de-endothelial ablation healing artery realized healing garterial wall) and human is known to accumulate in both atheromas (Roberts ) et al., Journal of Libido Research (J, L1pid Res, ), 24:1160.19831 Lees et al., Journal of , i+-”1J7-Medicine (J, Nuclear Med, ), 24 :154.1983). Therefore, about 4 weeks ago, the abdominal aorta was balloon-endothelialized. Separated rabbits can be used as a test model for human arterial disease. LDL receipt genetically inherited high blood cholesterol secondary to deletions in Watanabe Heritable Rabbit The same applies to pond animals such as hyperlipemic rabbits or experimental systems. It can be used in various ways. This strain of rabbits suddenly develops atherosclerosis at approximately 2 months of age. They develop arteriosclerosis and often die from heart attacks.

The rabbit model was subjected to onlay autoradiography using 125-labeled LDL. , and 99” Tc-labeled LDL using a gamma scintillation camera. Both images were imaged by extracorporeal imaging. In each case, excised rabbit aorta Onlay autoradiography ensures in vivo results with extracorporeal contrast I expected it. In preparations for vascular administration, each labeled synthetic peptide is injected in its free state. Alternatively, cholesterol ester phospholipid micro It may also be attached to the surface of a lipid emulsion, such as an emulsion. Then, the corresponding The emulsion is injected intravenously into the rabbit. After about 24 hours, the rabbit was treated with the A suitable in vitro monitor for the specific label on the titanium chloride is applied. Alternatively, Kill the heron, remove its aorta, and clean it. The aorta is divided into sequential parts (seq section) and then in a liquid scintillation column. Monitor under counter or dry and cover with a layer of polyester wrap. Invert and place on a sort of X-ray film, then store it after a suitable storage time in the dark. This is developed to obtain an onlay autoradiogram.

Tama-nuri The peptides of the invention can be used to diagnose vascular damage or, alternatively, to stimulate L into the vascular wall. It can be used to inhibit DL binding. In either case, the peptide is first , to a subject, eg, a patient. Administration is by arterial or intravenous injection. I can. Alternatively, non-hydrolyzable derivatives of the peptide (e.g. tyrene derivatives) may be administered via the mouth, or administration may be done intranasally.

In preparations for vascular administration, the labeled synthetic peptide is carried in a pharmaceutically acceptable carrier (e.g. (e.g., a saline solution) or alternatively, cholesterol on the surface of lipid emulsions such as tellurine lipid microemulsions (MV). The emulsion is then injected intravenously.

For diagnostic applications, labeled peptides are added in sufficient quantities (generally intravenously) for subsequent detection. Administer 05 to 10 days per day or orally. Preferred embodiments of the present invention In examples, the peptide is eg 1211111151 or 99"Tc. Labeled with a radioactive isotope, it inhibits peptide accumulation at the site of injury using gamma The image may be imaged externally using radiation detection methods such as a fusion camera, or alternatively. For example, synthetic peptides are labeled with a non-radioactive paramagnetic contrast agent that can be detected by an MRI system. Understand. Such systems use strong magnetic fields to stimulate the source in the patient's body. Align the child core spin vectors. The magnetic field is then disrupted and the patient's image is , measured as the return of the cores to their equilibrium alignment. In the present invention, synthetic peptides metals such as gadolinium, cobalt, nickel, magnesium or iron complexes. Conjugate diagnosis that is combined with a paramagnetic contrast agent and imaged externally using an MRI system Can form drug withdrawal.

For the treatment of vascular diseases (i.e., to suppress LDL binding to the vascular wall) ), the peptide is administered intravenously or intramuscularly in a therapeutically effective amount, generally 5 to 100 mg. Administer physically. If desired, the treatment can be repeated to prevent or reduce vascular damage. Ru.

Description of preferred specific examples The design and synthesis of sample peptides useful in the present invention are described below. . Instances used to test the ability of these peptides to target vascular damage In-vivo assays are also described. These examples are illustrative of the invention. This does not limit the present invention.

apolipoprotein peptides Apolipoprotein B (apoB) is the protein portion of low density lipoprotein. apo The primary structure of B-100 is available by cloning it (e.g. , Knott et al., Nature, 323: 73 4-742.1986: Yang et al., Nature, 323: 7 38.1986: Calsson et al. Nucleitsk Acids ・Research (Nucl, Ac1ds Res, ), 13:8813.198 (see 5). Furthermore, enzymatic treatment of apo B-100 with trypton can reduce LDL of these surface areas apparently involved in binding to various cells and tissues of (Forgez et al., Biochemical and ・Biochem, B1ophys, Res.

Come, ), 140:250.1986: Knott et al., Nature, 323 : 734.1986). The surface exposed area is shown schematically in FIG. Typical tripe Amino acid sequence analysis of the synpeptide is shown in Table 1.

Table 1 X “Residue numbers obtained from the complete primary sequence of apolipoprotein B.

From X Forges et al. (ibid.).

data from Forges et al. (ibid.), the published apo B sequence (above) and the Information known to those skilled in the fields of chemistry and peptide design (e.g., those listed above) ), the amino acid sequence of the surface region of the apoB portion of LDL is similar to that of the apoB portion of LDL. A synthetic peptide with a amino acid sequence was designed. In some cases, the peptides amidated at their carboxy termini and/or amines at their amino termini. cetylated (ie, "A" or raJ peptides). 8 types of representative ap.

B peptides and their modified counterparts are shown below. The top number of amino acid residues is Concerning the primary sequence of apoB.

Thr-Gin-Leu-CONH2; CH3CONB-Tyr-Arg-^1 a-LeuJal-Asp-Thr-Leu-Lys-CON[Iz　;Glu -Gin-Ala-Lys-Gly-^1a-CONt5: and apo B Derivative peptide, amino acids 2 to 5P-4 (see PCT/US89101854) Thirteen conservative substitutions were made to produce 5P-6 and 5p-s. 5P-12 is 5P-4 with one leucine (Leu) residue substituted for the last five amino acid residues It is a blunt-ended form of . 5P-14 lacks the last five amino acid residues, resulting in 7 5P-1 in which the tyrosine (Tyr) residue at position is replaced with a threonine (Thr) residue This is the blunt-ended form of 2. 5P-15a and 5P-17 each have amino acid 2 483-2497 (i.e., including Tp49) and amino acids 3809-38 25 (ie, including Tp59). 5P-19a and 5P-218 The sequence is a modification on the 5P-4 sequence. Peptide SPI; 3a, 5P17. 5P19a and 5P21. The physical data obtained for a are shown in Table 2. . A helical wheel diagram demonstrating the amphipathic and α-helical nature of these peptides. The helical wheel diagrams are shown in Figure 2. Residues are circled to indicate charged residues. The codes are as follows: A1 A Ranin; D1 aspartate: E1 glutamate, F1 phenylalanine: 01 Glycine: Ni1 Rin: L10 Isine: N1 Asparagine: Q1 Glutamine; R , arginine, T1 threonine, ■, valine; Y1 tyrosine; Ac, acetyl .

apoA-1 consensus peptide (referred to as APOA-I C0N5ENSUS) ), i.e., an idealized α-screw derived from many regions of apolipoprotein A-1. Another amphiphilic peptide (i.e., 5P34a) was synthesized based on this The consensus peptide sequence was determined by Anantharamaiah. iahXMeth.

Enzymol, 128:630.1986). apolipoprotein Unlike White A-1, the synthetic peptide is only slightly charged and the sequence is Preceded by a mino-terminal tyrosine residue. This peptide is The amino terminus was amidated and acetylated at the amino terminus. This peptide is weakly negative (i.e. -2, see Table 2).

5P-343: CH3C0NH-Tyr-Val-Leu-^sp-Glu-Phe-Arg- Glu-Lys-Leu-＾5n-Gl u-Glu-Leu-Gl u-A 1 Obtained for a-Leu-Lys-Gin-Lys-CONH2SP-34a The physical data obtained are shown in Table 2. 5P-34a spiral wheel diagram The system is shown in Figure 2 (described above).

Table 2 °Molecular weight (calculated/measured); parent ion measured by distant atomic bombardment mass spectrometry It was expressed as (M+H)'.

1 average hydrophobicity, Eisenberg (Journal of ・Molecular Biology (J, Mo1. B iol, ), 279 :125.1984) and the scale.

1 charge is expressed as the difference between the positively and negatively charged groups on the peptide at neutral pH. vinegar.

elastin peptide Elastin is a major component of skin, arteries, lungs, and other tissues (Rosenbru Rosenbloom; Robert & Robert and Robert), the field of matrix biology (Frontiers of Matrix Biology), Volume 8, Biology and Diseases of Elastic Tissues Biology and Pathology of Elastic Ti5sues), 1980: Reddi, Extracellular Matrix: Structure and Function (E xtracecellularMatrx: S tru culture and F function), 1985). Various elastin compounds Analysis of columns (Rosenbloom, Meth, Ezymol, 144:1 72.1987), elastin proteins are generally composed of many repeating units. Indicates that Two types of such repeating units are the pentapeptide, V al-Pro-Gly-Val-Gly (VPGVG) and hexapeptide, Val-Gly-Val-ala-Pro-Gly (■GVAPG).

Structurally, the nilastin repeats exhibit circular dichroism (Rahman Co., Ltd. of Czechoslovak Chemical Communications (Coil, Czech, Chem, Crown,) 1.52:135 6.1987) and extensive nuclear magnetic resonance studies (Urry et al., Pi. Opolymers (Biopolymers), 25 +1939.1986) Therefore, the repeating β-one rotation structure (biochemical and biophysical Search Communications (Biochem, B1ophys.

Res, Camm, ), 153: 832.1988). It was. Hexapeptides have chemotactic properties (Sennior et al., J.D. Null of Cell Biology (J, Ce1l. Biol, ), 99 : 870.1984).

Elastin-inducing peptides useful for targeting arterial lesions have hydrophobic binding sites. This binding site is compatible with hydrophobic extracellular vessel wall components (i.e., elastin). promote interaction and/or cause the peptide to interact with negatively charged blood vessel walls. let To promote solubility in physiological fluids, the peptide has a hydrophilic domain. Preferably, it contains a positive or weakly negative net charge.

Three representative elastin peptides are shown below. 5P-28 to elastin Contains three repeats of the xapeptide VG VAPG. 5P-30 and 5P-29 are four and three repeats of elastin beta peptide VPGVG, respectively. Including repeats. 5P-30 and 5P-29 peptides have their carboxy-terminus Amide 112 N-Tyr-Val-GlyJal-A 1a-Pro- Gly-Val -GlyJal-A 1a, -Pro-Gly-Val-G ly-Val-Ala-Pro-61y-OH; 5P-30: H2N-Tyr-Va l-Pro-Gl y-Va 1-Gl y-Va 1-Pro-GlyJa　1-Gl　y-Val　-Pro| Gly-Val-GlyJal-Pro-Gly-Val-Gly-CONH2 : and 5P-29: ■2N-TyrJal-Pro-Gly-Val-Gly-Val-Pro-G 1. y-Val-Gly-Val-Pro-Gly-Val-Gly-CONH 2 Physical data obtained for elastin peptides are shown in Table 2.

Peptide synthesis and labeling Established Steward and Young method (Solid Phase Peptide 5ynth esis), 2nd edition, pp. 53-123, 1984, The Pierce Chemical Co. Company (The Poerce Chemical, Co.), Rock Solid-phase peptide synthesis according to Ford, IL, cited herein) Peptides 5P-6, 5P-6A, 5P-8, 5P-8A, 5P-12 by A and 5P-14A were synthesized. These peps using the plan listed in Table 2. synthesized, but none of the other schemes listed in this literature could achieve any desired Also used to obtain peptides (e.g. any of the peptides described herein) can be done.

Table 2 ! Dichlorohequinolcarbonimide chitrifluoroacetic acid mdiisopropylethylamine ==tert-butyloxylponyl amino acid=Iα2.2.2-trifluoro using roethanol tert-butquine carbonyl (t-Boa)-based chemistry. manual solid-phase methods (Barany and Merrifield) iffield), peptide analysis, synthesis, biology (The Peptides) :Analysis, 5 synthesis, Biology), Academia Mitsuku Press, New York, 1980; Steward and Young, Solid Phase Peptide Ptide Synthesis, 2nd Edition, Pierce Chemical Company, Rockford, IL. Peptides 5P-15a, 5P-17, 5P-19a by Ishu, 1984) Combining SSP-21aSSP-34a, 5P-28, 5P-30 and 5P-29 accomplished. Side chain protecting groups for amino acid derivatives include bases for Asp and Glu. Benzyl esters, benzyl ethers for Set and Thr, Lys Chlorobenzyloxycarbonyl for, Promobenzyloxy for Tyr , as well as mesitylenesulfonyl for Arg. Steward Diisopropylcarbodiimide (DI In C), the carboxyl-terminal amino acid residue is attached to the methylbenzhydrylamine resin. Matched. The peptide resin was treated with CH2 (J2 twice, then CH,CAl2/1 Washed once with 50% TFA in % dimethyl sulfide and washed once with 50% TFA in CHzC4/1% dimethyl sulfide. 50% TFA in sulfide for 20 min or CH2CI2/1% dimethyl sulfide The t-Boa group was removed by treatment with 25% TFA in Fido for 30 min. . Next, the peptide resin was washed 5 times with 5X CH2Cl2; CH2Cl! During ~ Neutralize by washing twice with 10% diisopropylethylamine (0% diisopropylethylamine); C14 Washed 5 times with 2CA'2. In the presence of 1.5 equivalents of DIEA, 3 equivalents of no symmetry water (see below) for 45 minutes or 4 equivalents of active ester (see below) for 2 hours. processing to attach the next amino acid. The peptide resin was then diluted with CH2Cl2. Times: CH, Cl, 2 times with 33% ethanol in medium: then washed twice with CH2C/2 Purified.

6.1 equivalents of amino acids were treated with 3 equivalents of DEC in CHClt for 20 minutes on ice. Symmetric anhydride-activated amino acids were prepared by. 30 minutes on ice 4 equivalents each of the amino acids, HOBt and D, in dimethylformamide (DMF) Active esters of hydroxybenzotriazole (HOBt) were prepared from IC. Ta. CH, (J!, medium, 4 equivalents each of amino acids, EACNOx) on ice for 30 min. and ethyl hydroxyiminocyanoacetate (EACNOx) from DIC. Active esters were prepared. Kaiser Ninhydrin Test (Kaiser er) et al., Anal.

Biochem, 34:, 595.1970) Confirmed completion.

Incomplete bonds are repeated once or twice, and if still incomplete, the synthesis continues. The peptide resin was acetylated with acetic anhydride before washing. Deprotect the peptides and H F processing (Immunodynamics), Sandier (performed according to the indicators set by the State of California) or by Yajima (Yajima). ajima) et al. (Journal of Chemical Society (Chemical ・Cominike Noyons XJChem, S oc, , Chem, Cai m, ), pp. 107-108, 1974), 1:101.05 trifle. Oromethanesulfonic acid: TFA: For treatment with thioanisole, nitane dithiol Therefore, it was cut from the resin. The crude deprotected peptides were eluted with 5% acetic acid. Desalt on a column of Adex G-25 or 10-100 volumes of ethyl Precipitated twice from TFA solution with ether. Next, the peptides were added to Bi-Dac ( Vydac) C+a column and 0% to 90% CH3C containing 01% TFA Purified by reverse phase HPLC using a gradient of N/H20. purified pepti The solutions of the compounds were evaporated, redissolved in water, and lyophilized to dryness. High-speed atomic bombardment material The identity of the peptides was confirmed by quantitative spectroscopic (F, ABMS) analysis. .

Shih et al. (Brondinges of National Academy) Of Science! -- Nisuei (Proc, Natl, Acad, Sc i, US, A), 871436.1990, incorporated herein by reference). The synthetic peptides 5P-6, 5P-6ASSP-8, 5P-8A, 5P-12A.

and 5P-14A were radiolabeled. Some experiments require radioactively labeled LDL. Ta. In these cases, the above-mentioned Lees et al. (Proceedings of ・National Academy of Sciences, 80:5 098.1983, incorporated herein by reference). McF　Arlane) LDL can be converted to 125 iodine by a modification of the iodine-chloride method. Labeled with plain. Sephadex G-25 gel filtration "desalting" column or equivalent Radioactively labeled riboproteins or synthetic peptides are removed by passage through the The bound radioisotope was separated with 125I using chloramine-T as follows: Synthetic hebutides 5P-15a, 5P-17, 5P-19aSSP-21aSSP -34a, 5P-2 translations 5P29 and 5P30 were radiolabeled.

2.51M sodium phosphate/37.51M NaCl buffer (pH 7,4) 2 Dissolve peptide (400Ig) in 00μA and mix with 1mci (3μI) + 251. It matched. Talolamine-T (30 μ7.8 u/l/in H20) was added to the mixture. After 35 minutes, the reaction was quenched with sodium bisulfite (6 μI!s 8 rich 9/rich 1). Quenched by addition. Radiolabeled peptides were added to Blo-Ge1 P-2 ( Bio-Rad, Bercules, CA) Kara Gel filtration was performed on a vacuum filter (ICix 30 cm). Elute with 0.1% BSA in IM acetic acid. did. A 5-rich liter top fraction was collected, then 0.45 ml of both fractions were collected. oh Iodinated peptides eluted in other fractions 9 to 12 were pooled and treated with INNaOH. The pH was adjusted to 5 and then 7.5 with 1M NaHCO3.

The actual procedure was the same as above, except that the reaction mixture was adjusted to a final concentration of 50% ethanol. Iodination of 5P-17 was performed. After iodination, Uno serum aliquots up to a final concentration of 10% Lubumin was added to precipitate the radiolabeled peptide, and the precipitate was transferred to a 2000 rpm layer. The samples were collected by centrifugation for 15 minutes at . The pellets were then washed 4 times with 1 part water each. After the final wash, the precipitate was dissolved in 10% BSA 5m/ml. alternatively , 20% BSA to a final concentration of 10% and a volume not exceeding 51N Peptide (in 50% ethanol) was added. Then, in the same manner as above, the solution was Passed through a BioGelP 2 (10cm x 1.5cm) column and 0. Eluted with 1% BSA in acetic acid. Remove excess buffer using nitrogen pressure , the column was washed with 5 mA of 11% B5A 10.1 M acetic acid, and the most radioactive fractions were pooled for injection.

Alternatively, synthetic peptides can be directly treated with technetium (Tc) or It is known to chelate various metals, including radioactive isotopes such as indium. The combination of chelating groups such as diethylenetriaminepentaacetic acid (DTPA), which is Label indirectly via a bond.

Direct binding to semi is performed as follows. 5C1+Ci' in aqueous solution Q, 5d "Tc (in the form of 9@"Tc O4-) in a 0.2M 1i sodium carbonate solution (pH 8,0) 1 to 611 g of synthetic peptide in 0.5 ml, preferably added to 2 irises 9 Add and mix thoroughly for 10 minutes at room temperature.

Raise the pH to 8.0-9.0 with 0.25M sodium hydroxide if desired. next Then, to the mixture was added reduced sodium dithionite (freshly dissolved in 0.5 d of distilled water). 57°5 mmol) 10119 is added. The mixture was slowly heated at room temperature for 30 minutes. and stir.

Unbound technetium and natate by molecular tube chromatography Separate the radiolabeled synthetic peptide fraction from rhium dithionite. EDTA-heavy Carbonate mild solution (0.2M sodium bicarbonate IJ sea urchin, pH 8.0, O, OOIM E A 1×5 Qcm column of Sephadex G-25 equilibrated with DTA) is suitable for the separation. are doing.

The column was standardized with blue dextran and potassium iodide, respectively, to Measure medium volume and column capacity. The reaction mixture was applied to the column to remove bicarbonate. Elute column fractions using salt-EDTA buffer. Special for synthetic peptides Macromolecule radioactive peaks eluting at characteristic positions are isolated and prepared for use.

Indirect coupling to Shiwa Tc is performed as follows. Chelating ligands, e.g. DTP A (Hnatowich et al., Science ), 220 + 613.1983) or bromoacetyl para-aminobenzene Njiru EDTA (BABE).

Meares et al. 1. Analyt, B iochem, 1 42:142.1984) at the N-terminus or C-terminus of the peptide. Join. These documents are cited herein. Next, the synthetic peptide Technetium can be added to DTPA-synthetic peptides or by the methods described above for direct labeling. chelates to BABE-synthetic peptide. Technechi in the form of “’Tc04-” of the DTPA-synthetic peptide and added reduced sodium dithionite to the mixture. Add a solution (pH 8.0-9.0). Column chromatography (above and Similarly, the TPA peptide was converted into unconjugated 99'Tc and Tel[assembly] by and sodium dithionite. The preparation was then prepared according to Mears et al. by silica gel chromatography substantially as described in 1), or Characterized by HPLC. The 9°Te1lt recognition peptide was added to a pharmaceutically acceptable The drug may be administered in a carrier solution or bound to a lipid emulsion.

structure The molecular composition or structure of synthetic apoB peptides is similar to that of the apoB portion of LDL. Polyclonal antisera were raised to each peptide to determine similarity. Its ability to bind LDL was tested. As a control, antiserum raised to human LDL was used as a control. used.

Specific anti-LDL antisera are available from many sources (e.g., Hoechst Pharmacy). Cal Incoholated (Hoechst Pharmaceutical) , Inc, ), Cincinnati, OH and Marble-Courern (M arburg-Lahn), West Germany, and Highland Laboratories ・Incoholated (Hyland Laboratories).

Inc., 4501 Colorado Blvd., Los Angeles, Calif. It can be purchased from (A). Alternatively, the antisera can be prepared using a number of professional methods known to those skilled in the art. It can be prepared by any of the following methods: For the assays described herein Anti-LDL antiserum was prepared as follows. saline or barbital buffer In 1iuF, F. Ischman et al. (Atherosclerosis Arteriosclerosis, 7: 361.1987) 5 to 201 Ig of LDL prepared according to (Difco Laboratories), Det. (Royt, Michigan). This is a 5d loop fitted with a 20 gauge needle. Lipoprotein and adjuvant in a Luer-Lock syringe separately and through a 1-inch piece of 0.030-inch inside diameter polyethylene tubing. This was most easily done by connecting two needles. Then connect the two needles and The contents of the syringe are effectively transferred from one syringe to another via the tube. discharge.

Prepare a stable, ream-like emulsion, which is finally poured completely into one of the syringes. and remove the connecting pipe. The emulsified antigen was injected subcutaneously into the back of experimental rabbits. number If multiple rabbits must be injected with the same antigen, a larger syringe and Using a larger amount of material, give each rabbit 1.5% of the initial antigen solution. emal equivalent to zl John 2*I was injected. An alternative method for preparing large quantities of emulsions is (Mickle) Grinder (Mickle Company) ), Hampton, Middlesex, UK, Brinkman Instruments Brinkmann I instruments, INC.), West Burley, New York] in one tube. Add the stock solution and adjuvant, cap it, and remove the steel cap of this magnetic shaker. place it on one of the card receptacles. Replace the second sample or water balance with another sample. Place the lead on the lead, turn on the machine, and apply the lead to maximum excursion for approximately 10 minutes. Obtained The emulsion was drawn into a syringe through a round 18-gauge needle and a 20-gauge Inject subcutaneously through a needle.

For the preparation of high antibody titer antisera, the animals were incubated for exactly 3 injections for the first injection. ~Get a “boost J” every 5 weeks. Usually after 2 injections Good antiserum can be obtained. Animals treated in this way remain immune for long periods of time. will yield a very large amount of antiserum. The amount of antiserum must be in the range of 11 or more. If required, human immunization may be performed in the same manner, except that 2-3 times the amount of immunizing antigen is required. You can use azalea. Animals are bled 6-10 days after each booster injection. desired A small test bleed may be performed to test antibody levels and purity.

The blood is allowed to clot at room temperature for several hours and then left in the refrigerator overnight. Store the sample in a cold place. Centrifuge, remove the clot with a cotton swab (applicator, 5 ticks), and remove any remaining clots. Centrifuge the remaining blood cells into the pellet and decant the serum. 1g/ml as a preservative Add sodium azide. Antisera that are used regularly should be kept in the refrigerator or Or it can be stored at -15' to -20°C.

To prepare anti-synthetic peptide antiserum, 1 zq/ml of truncated synthetic peptide It was dissolved in PBS (pH 7,4) at a concentration of ml. Equal volume of peptide solution Mix with complete adjuvant and vortex thoroughly until a viscous emulsion is obtained. . New Sealant White Rabbit (Millbrook Farms) Four dorsal quarters of F art Is), Amnost, Massachusetts) A total of 0.5 tq of synthetic peptide was injected subcutaneously into the circle. After 2-3 weeks, the Rabbits were boosted with peptide 0.519 emulsified in incomplete Freund's adjuvant. (injected at the same site). 8-10 days after the first boost, the The animals were given a second identical boost and 8-10 days later, 30 liters of blood was drawn.

To test for immunological cross-reactivity, Peptide in 501M bicarbonate (pH 9.6) was tested for immunological cross-reactivity. 1100n/well by incubating overnight at 4°C. The synthesized peptide or LDL was prepared using a microtiter plate (Imuran・■・Dynachi). Labda (Ima+ulon II Dynatech Labs), Cha in phosphate-buffered saline (pH 7.40). PBS) and 1% serum albumin (BSA) for further incubation overnight. and blocked for non-specific binding. Control wells were coated with BSA only. I clothed it. After washing twice with PBS, the wells were prepared for synthetic peptides. Serial dilutions of rabbit polyclonal antibodies (1:1 made in PBS, 3% BSA) 0-1:100.000) and incubated for 45 minutes at room temperature.

After thorough washing (3x with PBS, O11% BSA), the wells were washed with goat antiseptic solution. IgG Horseradish Peroxidase Conjugate (Atlantic) Tsuku Antibodies (Atlantfc Antfbodies), Scarepo Lowe, ME) at a 1:2000 dilution. After the final wash, the wells 3.3'-5,5'-tetramethylbenzidine (TMB) Oxidase substrate (Kirkegaar & Berry Labda) d and PerryL abs), Gaithersburg, Maryland). ELISA-5 automated plate meter (Physica) , New York, NY) at 650 stops every 2 minutes. The result is based on It is expressed as the initial rate of transformation (ODsse/hour change), which is Determined by linear regression of 15 data points.

Each data point is a collection of three measured data points from the same plate taken at the same time. It represents the average of the total.

Apply LDL to ELI SA plate and apply 5P-4 (PCT/US891018 54) was treated with antiserum. Anti-5P-4 antiserum binds LDL on the plate. Immunological evidence shows that 5P-4 and LDL have structural similarities. confirmed. In similar experiments, anti-5P-4 antisera were used to test 5P-4 and 5P-4A and conservatively substituted peptides 5P-6, 5P-6A, 5P-8, 5P-8A.

It was found that these peptides bind 5P-42A and AP-14A. has structural similarity to 5P-4 and therefore to LDL. It was shown that

animal model・ The ability of the peptides described herein to target sites of vascular injury as follows: was assayed for. Stingray male new sealant white rabbits (2~3 & 9 each) ・ARI Breeding Lab sX West Bridgewater, MA). blood vessels To induce damage, the Baumgartner method (Fishman et al., Atherosclerosis (Art. eriosclerosis), 7:361.1987). The endothelium of the aorta was removed. That is, each animal was treated with ketamine and ether. , or alternatively, quinolazine (20u/xi) and Keta lar) (50+g/m/), the left femoral artery was isolated; A 4F Fuogatay embolectomy catheter (model 12-040- 4F.

Edwards Laboratories, Inc. aboratories I incorporated), Lang Ante, Cali Fornia) was introduced and advanced under fluoroscopic visualization to the level of the diaphragm.

The catheter was treated with a radiographic contrast agent (Conray, Marine). Clots, St. Fleiss, Missouri) approximately 3 psi below the balloon inflation pressure. Inflated to high pressure. Removal of the catheter, ligation of the femoral artery, and closure of the wound Previously, a dilatation catheter was passed through the abdominal aorta three times to remove the aortic endothelium. . The animals were cured 4-5 weeks prior to injection of labeled synthetic peptides.

Watanabe hereditary hyperlipidemia (WHHL) rabbits were also used as an animal model. that The National Heart Lung and Blood Institute ( National Heart Lung and Blood I n5ti Approximately 3 from the WHHL rabbit program of tuteX Bethesda, Maryland One month old and weighing 1.54 g was obtained. The animal was kept until it reached a weight of 3-4 kq. It was bred in At this weight they exhibit significant aortic atherosclerosis. did.

Each labeled synthetic peptide preparation in column elution buffer (e.g., 150-400 μC (containing 1251-labeled peptide of i or more) was expanded and cured. were injected into the peripheral ear vessels of white or WHHL rabbits. Between 0 and 24 hours Serial blood samples were obtained from the contralateral ear and analyzed for radioactivity. After the injection, the first The labeled peptide concentration in blood samples collected over 10 minutes was supplemented until 0:00. Radioactivity at time 0 was determined by calculation of mean plasma radioactivity. Has a half-life of approximately 1 minute or less Peptides 5P15a, 5P17.5P19a, 5P21a, SP2g from plasma After 71 hours of rapid withdrawal of 5P34a and 5P30, plasma levels were was less than 10% of the administered dose and decreased by a further 1% over the next 3 hours. Pe Ptyids 5P15a, 5P17.5P21a, 5P28.5P34a and 5P 30 drops to 3-6% plasma levels and peptide 5P19a is removed more quickly The concentration decreased to 0.3% (at 4 hours).

One to 24 hours after injection of the labeled synthetic peptide preparation, each animal was given a region of deendothelialized aorta. Evans Blue Dye (Allied Chemical Company (A)) llied Chemical Co+tpany), National Aniline Intravenous injection of 4 ml of 05% solution of Divisi B, New York, NY) did. Thirty minutes later, the animals were killed by lethal injection of bendoparvicur. kill After that, the aorta was completely removed, washed with physiological saline, and soaked in 10% trichloroacetic acid. It solidified.

Ventral lavaged and solidified aortas from animals injected with radiolabeled synthetic peptides. opened along the surface. These pieces were then pinned together and treated with 10% trichloroacetic acid. I let it harden for 2 hours and took a photo. Then solidify and open blood vessels with more covered with Tic (Saran) wrap and high speed X-ray film (Kodak). ・Place on Kodak Orthofilm 0H-1), Kodak "X-OMAT" at -70°C before development in X-OMAT for 90 seconds. 3 in “Set” (“X-〇maticCassette”) (24 x 30cx) It was stored for 4 weeks.

Representative results are shown in FIGS. 8 to 13. The tax endothelial artery wall is stained blue (see above) Evans Blue dye), which appears as dark areas in the photograph (Figures 3 to 8, A), radiolabel accumulation is indicated by dark areas in the autoradiograph (second Figures 3 to 8, B). All peptides are found in endothelial tissue in a pattern characteristic of LDL. Accumulated intensively at the leading edge to play. In each case, autoradio The graphs (Figures 3-8, B) show that the aortic lesion caused by previous injury is heated. The clear cleavage position of the synthetic peptide is shown for imaging at the ring (re-endothelialized) edge. This lesion is characterized by a number of important features, including lipoprotein accumulation and other pathological changes. The synthetic pigment is known to resemble human atherosclerosis. The localization of putide at the site of injury and its ability to image are useful for imaging vascular disease. shows the utility of the present invention.

These and other results are shown in Tables 3 and 4. Control peptides used is 5P-2 (part of the heparin and LDL receptor binding site of apolipoprotein E). ) and the receptor and heparin-binding domain of apolipoprotein B, SP -1, 1, A. 12SI-labeled synthetic peptides in the aorta and adrenal glands Correction for differences in mean plasma concentrations of labeled compounds to compare relative accumulation of was necessary. The average concentration of synthetic peptide-related +251 radioactivity was determined by the plasma decay curve. was calculated by numerical integration of and division by the time from isotope injection.

Table 3 B59 51)6. A"+MV" 312 '251 24? 1:B127 5p6Ax 350 I25I 4 ↓1:B122 5p6A” 348I 251 4 +1! B139 sp6. A engineering 552”I 4 +j■ 1B146 5p6A" 550.4"I5:i'JL-45p6A" 282'2'I 5.

Table 3 (continued) 1B115 5p5K 418”’I 4 +j□ 1B115-1 ap6” 398°9 125i 4 + IIBIII sp 8”-4-MVtx419 1231 4 +1iB103 sp8” 424 "I 5 = -1■ 1B 124 sp 8・4662...I 4+1MV=Cholestero Low ester microvesicles = radioactive ヰ=cold jl! Table 4 All peptides have plasma lifetimes of approximately 1 minute. 0.2~l, QmCi throw Radiolabeled peptides were injected intravenously in given doses.

FIG. 1 FIG. 2 −−1−→ $+J voice-paji Summary book Synthetic peptides that have affinity for and accumulate at sites of vascular injury or administering a peptide analog to a patient and then administering the peptide analog within the patient's vasculature. Asymptomatic atherosclerosis by detecting the location of peptide or peptide analogues Vascular diseases including arteriosclerosis can be diagnosed. The synthetic peptide or peptide The analogue is apolipoprotein B1, apolipoprotein A-1, or elastin protein. may contain amino acid sequences that overlap sufficiently with those of the region so that the peptide tide or peptide analogues accumulate at sites of vascular injury.

international search report """"-"'Q--,, PCT/US91103 [+265erial N o, PC/L1391/Q:13j6^rt ur+it La9B X, C1ass sleeping 1-9. drawn t6 @ flrst prod uct, a061Looor6tel*-9pepticn*, C1ass 5ri starvation tubc↓ass. 5ri.

C15i+ss 4, 41 Satoshi nd 47-62. drawr+ta a fir tm・thod lowe use, for5 dsect4an 61　1n)ury,　C4asg　514. mubc↓111 hot.

11 Claims 1 of 1 * drawI'I t-C3-geeond product, apoAipoprot*1n-Apept4de snow, C 1ass 5W, subclaim 359°=1111 f! ! C1m1s s 14-211. drswn to a third product, e lsgtLn pepttр■{+.

C111C11l 5 pieces (a, mubeLmm* 53゜

Claims (1)

[Claims] 1. Peptides or peptide analogs [have sequences]: [I have an array]: [I have an array]: [I have an array]: [I have an array]: [I have an array]: [There is an array]; and [There is an array] A vascular injury site characterized by consisting of an amino acid sequence selected from the group consisting of Peptides or peptides that have affinity for the site and the property of accumulating at the site analog. 2. The amino acid sequence [There is an array] The peptide or peptide analog according to claim 1, consisting of: 3. The amino acid sequence [There is an array] The peptide or peptide analog according to claim 1, consisting of: 4. The amino acid sequence [There is an array] The peptide or peptide analog according to claim 1, consisting of: 5. The amino acid sequence [There is an array] The peptide or peptide analog according to claim 1, consisting of: 6. The amino acid sequence [There is an array] The peptide or peptide analog according to claim 1, consisting of: 7. The amino acid sequence [There is an array] The peptide or peptide analog according to claim 1, consisting of: 8. The amino acid sequence [There is an array] The peptide or peptide analog according to claim 1, consisting of: 9. The amino acid sequence [There is an array] The peptide or peptide analog according to claim 1, consisting of: 10. The peptide or peptide analog is an amphipathic domain of apolipoprotein A-I. and has a net charge of −2 or more, thereby making the peptide or or peptide analogs accumulate at the site of vascular injury. Peptides or peptides that have affinity for the site and the property of accumulating at the site analog. 11. The peptide or peptide according to claim 10, wherein the amphipathic domain further comprises an α-helix. or peptide analogs. 12. - at least a net charge of 2 or more and an amphipathic domain of apoA-I; It has an amino acid sequence that sufficiently overlaps with a portion of the amino acid sequence, thereby 11. The peptide of claim 10, wherein the tide or peptide analog accumulates at sites of vascular damage. or peptide analog. 13. Peptide or peptide analog [has sequence] The peptide or peptide analog according to claim 10, having an amino acid sequence consisting of Gu. 14. The peptide or peptide analog consists of a hydrophobic domain and -2 or more the peptide or peptide analog has a net charge of It has an affinity for and accumulates at the site of vascular damage. Peptides or peptide analogs that have the property of multiplying 15. Claim 1: The peptide or peptide analog is derived from a blood vessel-related protein. 4. The peptide or peptide analog according to 4. 16. The peptide or peptide according to claim 15, wherein the blood vessel-related protein is elastin. analog. 17. The peptide or peptide analog has an affinity for blood vessel wall components. 15. The peptide or peptide analog according to claim 14. 18. The peptide or peptide according to claim 17, wherein the blood vessel wall component is collagen. analog. 19. 18. The peptide or peptide according to claim 17, wherein the blood vessel wall component is a proteoglycan. Petit analog. 20. The peptide or peptide according to claim 17, wherein the blood vessel wall component is elastin. analog. 21. The peptide or peptide analog has a dissociation constant of 10-6 or less and elastin 21. The peptide or peptide analog of claim 20, which binds. 22. 15. The peptide or peptide according to claim 14, wherein the hydrophobic domain consists of a β-chain. analog. 23. - a net charge of 2 or more and an amino acid sequence of at least a portion of elastin; has an amino acid sequence that overlaps sufficiently with that of the peptide or peptide 15. The peptide or peptide analog of claim 14, wherein the analog accumulates at said site. 24. Peptide analog [There is an array] (wherein X is at least 1) The peptide or peptide analog according to claim 16, having an amino acid sequence consisting of Gu. 25. 25. The peptide or peptide analog according to claim 24, wherein X is 3. 26. The amino acid sequence [There is an array] (wherein X is at least 1) 17. The peptide or peptide analog according to claim 16, consisting of: 27. 27. The peptide or peptide analog according to claim 26, wherein X is 4. 28. 27. The peptide or peptide analog according to claim 26, wherein X is 3. 29. A claim in which the amino terminus of the peptide or peptide analog is acetylated. 1, 10 or 14. 30. Claims that the carboxy terminus of the peptide or peptide analog is amidated Item 1, 10 or 14. The peptide or peptide analog according to Item 1, 10 or 14. 31. Claim 1, 10 or 1, wherein the peptide or peptide analog is water soluble. 4. The peptide or peptide analog according to 4. 32. Claims 1, 10 wherein the peptide or peptide analog is dissolved in a physiological fluid. or the peptide or peptide analog described in 14. 33. 33. The peptide or peptide according to claim 32, wherein the physiological fluid has a physiological pH. analog. 34. 34. The peptide or peptide analog according to claim 33, wherein the physiological fluid is plasma. Gu. 35. Claim 1, 10 or 1 further comprising a detectable label attached thereto. 4. The peptide or peptide analog according to 4. 36. 36. The peptide or peptide analog of claim 35, wherein the label is radioactive. 37. The radioactive labels are 131I, 125I, 123I, 111In, 99mTc, selected from the group consisting of 203Pb, 198Hg, 97Ru and 201Tl 37. A peptide or peptide analog according to claim 36. 38. 38. The peptide or peptide according to claim 37, wherein the radioactive label is 99mTc. analog. 39. 36. The peptide or peptide agent according to claim 35, wherein the label comprises a paramagnetic contrast agent. Nalog. 40. (a) The peptide or peptide analog according to claim 1, 10 or 14 introduced into the subject, (b) circulating the introduced peptide or peptide analog within the vascular system; accumulating at least a portion of the peptide or peptide analog at the site; (c) the location in the vasculature of the peptide or peptide analog accumulated at the site; The process of detecting A method for detecting damage in a subject's vascular system, comprising: 41. 41. The vascular damage in the vasculature comprises atherosclerosis. Method. 42. The peptide or peptide analog is an amphipathic domain of apolipoprotein A-I. and has a net charge of −2 or more, thereby 41. The method of claim 40, wherein the tide analog accumulates at the site of vascular injury. 43. 43. The method of claim 42, wherein the amphipathic domain further comprises an alpha helix. 44. The peptide or peptide analog consists of a hydrophobic domain and -2 or more peptides or peptide anacogs, thereby causing vascular damage. 41. The method of claim 40, wherein the method accumulates at the site. 45. Claim 4: The peptide or peptide analog is derived from a blood vessel-related protein. The method described in 4. 46. 46. The method according to claim 45, wherein the blood vessel-related protein is elastin. 47. Peptides or peptides in which the introduction step (a) has an acetylated amino terminus 41. The method of claim 40, comprising introducing the analog into the subject. 48. The introduction step (a) is a peptide or peptide having an activated carboxy terminus. 41. The method of claim 40, comprising introducing the analog into the subject. 49. Introducing step (a) comprises a peptide having a detectable label attached to it or 41. The method of claim 40, comprising administering a peptide analog. 50. 50. The method of claim 49, wherein the detectable label is a paramagnetic contrast agent. 51. 50. The method of claim 49, wherein the detectable label is a radioactive label. 52. The radioactive labels are 131I, 125I, 123I, 111In, 99mTc, selected from the group consisting of 203Pb, 198Hg, 97Ru and 201Tl 52. The method of claim 51. 53. The introduction step (a) is administration of the peptide or peptide analog by intraarterial injection. 41. The method of claim 40. 54. The introduction step (a) is administration of the peptide or peptide analog by intravenous injection. 41. The method of claim 40. 55. The introduction step (a) may consist of oral administration of the peptide or peptide analogue. The method according to claim 40. 56. The introduction step (a) may consist of nasal administration of the peptide or peptide analogue. The method according to claim 40. 57. The detection step (c) further detects blood vessels in which the peptide or peptide analog has accumulated. 41. The method of claim 40, comprising imaging a region of the system. 58. Furthermore, a step of quantifying the detected amount of labeled peptide or peptide analog 41. The method of claim 40. 59. The introduction step (a) has an affinity for the arterial injury site and a tendency to accumulate at the site. claim consisting of administering a second peptide or peptide analog having the quality The method according to item 40. 60. The detection step (c) further includes detecting the labeled peptide or peptide analog in vitro. 41. The method of claim 40, comprising monitoring. 61. The detection step (c) is the detection of radioactive labels using a gamma scintillation camera. 52. The method of claim 51, comprising in vitro monitoring. 62. Detection step (c) is in vitro monitoring of paramagnetic contrast agent using a magnetic resonance imaging system. 51. The method of claim 50, comprising a ring. 63. Treatment with a peptide or peptide analog according to claim 1, 10 or 14. administration of low-density lipoproteins to a blood vessel wall of a subject, comprising administering to the subject an amount effective to How to suppress white binding.