EP0600869A1 - Synthetische peptide zur abbildung von arterien - Google Patents
Synthetische peptide zur abbildung von arterienInfo
- Publication number
- EP0600869A1 EP0600869A1 EP91910995A EP91910995A EP0600869A1 EP 0600869 A1 EP0600869 A1 EP 0600869A1 EP 91910995 A EP91910995 A EP 91910995A EP 91910995 A EP91910995 A EP 91910995A EP 0600869 A1 EP0600869 A1 EP 0600869A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- peptide
- ala
- leu
- analog
- peptide analog
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/14—Peptides, e.g. proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/775—Apolipopeptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2123/00—Preparations for testing in vivo
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- 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 fibrous plaques which form within the arterial lumen. The plaques are most prevalent in the abdominal aorta, coronary arteries, or carotid arteries, and they increase progressively with age. They commonly present dome-shaped, opaque, glistening surfaces 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,
- the injury at, or deformities of, the arterial lumen presented by the plaque and associated deposits result in occluded blood flow, and ultimately in angina, cerebral ischemia, renal hypertension, ischemic heart disease, stroke, and diseases of other organs, if untreated.
- 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.
- 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, perforation of the artery, arrhythmia, stroke, infarction, and even death can occur. Because of the risks involved, arteriograms typically are reserved for individuals with advanced or acute atherosclerotic disease.
- LDLs low density lipoproteint
- LDLs circulating in the blood are known to bind to atherosclerotic plaques (Lees et al., J. Nucl. Med. 24:154, 1983). This binding most likely occurs via apolipoprotein B-100 (apo B-100), the protein moiety of the LDL 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.
- LDLs can be labelled with a non-radioactive, paramagnetic contrast agent capable of detection in magnetic resonance imaging (MRI) systems.
- MRI magnetic resonance imaging
- Yet another object of the present invention is to provide a method, which is non-invasive, of detecting and mapping vascular injury.
- the invention features a peptide or peptide analog having an affinity for, and propensity to accumulate at, a site of vascular injury, whereby the peptide or peptide analog includes an amino acid sequence selected from the group including:
- peptide is meant any chain of 30 amino acids or less.
- peptide analog is meant a peptide which differs in amino acid sequence from the native peptide only by conservative amino acid substitutions, for example,
- 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
- a peptide analog may also 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.
- the invention features a peptide or peptide analog having an affinity for, and a propensity to accumulate at, a site of vascular injury; the peptide or peptide analog is derived from an amphiphilic domain, preferably including an ⁇ -helix, of apolipoprotein A-I
- 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.
- derived from is meant having an amino acid sequence identical or substantially identical to the sequence of, as used herein, apolipoprotein A-I.
- 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).
- 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:
- 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 -2 or greater, such that the peptide or peptide analog accumulates at the site of injury.
- the peptide or peptiade analog is derived from a vascular-associated protein, for example, elastin.
- vascular-associated protein By “derived from” is meant having an amino acid sequence identical to or substantially identical to (as defined above) the sequence of, as used herein, a vascularassociated protein.
- 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
- the peptide or peptide analog has an affinity for a vascular wall
- the peptide or peptide analog binds elastin with a dissociation constant of 10 -6 or less (i.e., or with greater affinity, as measured in vitro by the method of Podet et al., Arteriosclerosis and Thrombosis 11:116, 1991); the hydrophobic domain of the peptide or peptide analog includes a ⁇ -strand.
- the peptide or peptide analog includes a ⁇ -strand.
- 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 peptide or peptide analog may include the amino acid
- Tyr-(Val-Gly-Val-Ala-Pro-Gly) x wherein x is at least 1 and, preferably, 3; or the peptide or peptide analog may include the amino acid sequence: Tyr- (Val-Pro-Gly-Val-Gly) x , wherein x is at least 1 and, preferably, 3 or, more
- the peptide or peptide analog has an acetylated amino terminus and/or an amidated carboxy terminus.
- peptide or peptide analogues include: H 2 N-Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asp- Ala-Glu-Gly-Ala-Lys-CONH 2 ;
- the synthetic peptide or peptide analogues are useful for detecting and imaging injury in the vascular 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.
- the peptide or peptide analog is water soluble; or is soluble in a physiological fluid, preferably, one which is at
- peptide or peptide analog is linked to a detectable label to enable its monitoring within the subject.
- detectable labels include a radioisotope, e.g., 131 I, 125 I, 123 I, 111 ln, 99m Tc, 203 Pb, 198 Hg, Ru 97 , or 201 T1; or a paramagnetic contrast agent.
- radioisotope e.g., 131 I, 125 I, 123 I, 111 ln, 99m Tc, 203 Pb, 198 Hg, Ru 97 , or 201 T1
- paramagnetic contrast agent e.g., a paramagnetic contrast agent.
- vascular system of the subject with, for example, a gamma scintillation camera or an MRI system.
- 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 above.
- the peptide or peptide analog to be introduced may be administrated by arterial or venous injection.
- non-hydrolyzable derivative may be any non-hydrolyzable derivative.
- 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.
- the invention includes a method for inhibiting the binding of low density lipoprotein to the vascular wall(s) of a subject involving administering to the subject a therapeutically-effective amount of a peptide or peptide analog of the forms set forth above.
- vascular diseases including asymptomatic atherosclerosis
- vascular diseases 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 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 inexpensively, quickly, and in large quantity (e.g., by recombinant DNA technology).
- the peptides of the invention may be used for the prevention or alleviation of vascular diseases 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.
- FIG. 1 shows a schematic model of the apo B-100 configuration, when included in the LDL molecule, and surface-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
- FIG. 4 shows a photograph (A) and an onlay
- FIG. 5 shows a photograph (A) and an onlay
- FIG. 6 shows a photograph (A) and an onlay
- FIG. 7 shows a photograph (A) and an onlay
- FIG. 8 shows a photograph (A) and an onlay
- 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
- Such synthetic peptides having these characteristics may have an amino acid sequence that is analogous to portions of known polypeptides which have an affinity for a site of vascular injury, i.e., have a
- lipoprotein or elastin which is responsible for its
- the synthetic peptides of the present invention may be homologous with portions of the apo B-100 moiety of LDL, the apo A-I moiety of HDL, or elastin.
- Peptides useful in the invention are those which successfully target vascular lesions. Thus, it is
- 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.
- fragments are of low net charge (i.e., between -2 and +2), allowing an interaction, e.g., with the highly negatively-charged vascular wall.
- 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
- hydrophilic domain of either positive charge or low negative charge (i.e., -2 or greater; i.e., or more positive) which facilitates solubility.
- Preferred peptides of class I i.e., those peptides which include an amphiphilic domain (i.e., a domain which has both a hydrophobic and a hydrophilic surface) are identified, e.g., as described in Kaiser and Kezdy (Ann .
- 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.
- Preferred peptides of class II i.e., those peptides which include both (a) a hydrophobic domain which
- vascular cell surface or a hydrophobic vascular-associated component e.g., elastin
- a positively-charged or slightly negatively-charged domain that facilitates solubility are identified using e.g., the methods for predicting hydrophobicity and
- peptides of this class 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
- secondary structure i.e., the presence of an ⁇ -helix or ⁇ -strand
- amphiphilic ⁇ -helix one may construct an amphiphilic ⁇ -helix
- 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
- amphiphilicity may be derived from a calculation of the "hydrophobic moment", i.e., the measure of the
- peptide analogues having different amino acid sequences, provided that the local charge distribution (and overall net charge) and secondary structure, and hence the biological activity (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
- peptides may be designed which include a region(s) of amphiphilic, hydrophobic, hydrophilic and/or secondary structure embedded within a longer amino acid stretch.
- 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 vascularassociated protein molecule) because such regions are most likely to interact with the vascular wall or with a vascularassociated protein domains (i.e., regions of the protein which are present on the external surface of a protein molecule, preferably a vascularassociated 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 may 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. Biochem . 47:251, 1978); extracellular domains are generally hydrophilic or
- amphiphilic in character such domains are frequently surrounded by hydrophobic stretches which correspond to transmembrane domains.
- the peptides can be synthesized by any of a number of established procedures, including, e.g., the expression of a recombinant DNA encoding that peptide in an appropriate host cell.
- these peptides can be produced by the established procedure of solid phase 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.
- 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.
- Peptides may be tested for their ability to effectively target vascular lesions using an in 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 (Roberts et al., J. Lipid Res . 24:1160, 1983; Lees et al., J. Nuclear Med. 24:154, 1983). Accordingly, a rabbit whose abdominal aorta has been balloon de-endothelialized
- This strain of rabbit develops spontaneous atherosclerosis at about 2 months of age, and they often die of heart attacks.
- each labelled synthetic peptide may 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.
- 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
- the peptides of the invention may be used to diagnose vascular injury or, alternatively, to inhibit binding of LDL to vascular walls.
- the peptide is first administered to a subject, e.g., a patient. Administration may be accomplished by arterial or venous injection. 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.
- a non-hydrolyzable derivative of the peptide e.g., a keto methylene derivative
- administration may be accomplished nasally.
- labelled synthetic peptide is suspended in a pharmaceuticallyacceptable 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
- MV microemulsion
- the labelled peptide is administered in an amount sufficient for later detection (generally, 0.5-1 mg intravenously or 5-100 mg orally).
- the peptide is labelled with, e.g., a radioisotope such as 123 I, 125 I or 9 9m Tc, 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.
- 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.
- paramagnetic contrast agents such as gadolinium, cobalt, nickel, manganese or iron complexes
- the peptide is administered in a therapeutically-effective dose, generally 5-100 mg intravenously or intramuscularly. Treatment may be
- Apoliprotein B is the protein moiety of low density lipoprotein.
- the primary structure of apo B-100 has become available by virtue of its cloning (see e.g., Knott et al., Nature 323:734-742, 1986; Yang et al., Nature
- SP-12 is a truncated form of SP-4 in which the last five amino acid residues were replaced with a single leucine (Leu) 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
- 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 SP15a, SP17, SP19a, and SP21a are summarized in Table 2. Helical wheel diagrams demonstrating the
- FIG. 2 amphiphilic and ⁇ -helical nature of these peptides are shown 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,
- amphiphilic peptide i.e., SP34a
- APOA-I CONSENSUS apo A-I consensus peptide
- APOA-I CONSENSUS an apo A-I consensus peptide
- 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).
- Table 2 A helical wheel diagram of SP-34a is shown in FIG. 2 (described above).
- aM olecular weight (calculated/observed); expressed as the parent ion (M+H) + , as determined by Fast Atom Bombardment Mass Spectrometry.
- cCharge is expressed as the difference between positively and negatively charged groups on the peptide at neutral pH.
- Elastin is a major component of skin, arteries, lung, and other tissues (Rosenbloom; Robert and Robert,
- elastin proteins are generally composed of a number of repeated units. Two such repeated units are the
- VPGVG Val-Pro-Gly-Val-Gly
- VGVAPG Val-Gly-Val-ala-Pro-Gly
- 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.
- a hydrophobic extracellular vascular wall component e.g., elastin
- the peptides preferably include a hydrophilic domain or a net positive or weak negative charge.
- SP-28 includes three repeats of the elastin
- 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.
- 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 peptides 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
- 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, Biology, Academic Press, New York, 1980; Stewart and Young, Solid-Phase Peptide Synthesis , 2nd ed., Pierce Chemical Co., Rockford, IL, 1984).
- the peptide resin was next washed five times with 5X CH 2 Cl 2 ; neutralised with two washes of lot diisopropylethylamina (DIEA) in CH 2 Cl 2 ; and washed five times with CH 2 Cl 2 .
- the next amino acid was coupled by treatment with either three equivalents of symmetrical anhydride (see below) for 45 minutes or four equivalents of aotive eater (see below) for 2 hours, in the presence of 1.5 equivalents of DIEA.
- the peptide resin was then washed four times with CH 2 Cl 2 ; twice with 33% ethanol in CH 2 Ol 2 ; and twice with CH 2 Cl 2 .
- Symmetrical anhydride-activated amino acids ware prepared by treating 6.1 equivalents of amino acid with three equivalents of Die in CH 2 Cl 2 for 20 minutes, on ice.
- Active esters of hydroxybenzotriaasole (HOBt) ware prepared from four equivalents each of amino acid, HOBt, and Die in dimethyl formamide (DKF) for 30 minutes on ice.
- Active asters of ethylhydroxyiminocyanoacetate (EACNOx) were
- 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
- the synthetic peptides SP-6, SP-6A, SP-8, SP-8A, SP- 12A, and SP-14A were radiolabelled by the chloramine T method as described in Shih et al. (Proc . Natl . Acad. Sci . USA 87:1436, 1990, herein incorporated by reference).
- LDL was labelled with 125 -iodine by a previously described modification of the McFarlane 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
- the peptide (400 ⁇ g) was dissolved in 200 ⁇ l of 2.5 mM sodium phosphate/37.5 mM NaCl buffer, pH 7.4 and mixed with 1 mCi (3 ⁇ l) 125 I. Chloramine-T (30 ⁇ l, 8 mg/ml in H 2 O) was added to the mixture and, after 35 seconds, the reaction was quenched by the addition of sodium bisulfite (60 ⁇ l, 8 mg/ml). Radiolabeled peptides were gel-filtered on a BioGel P-2 (Bio-Rad, Hercules, CA) column (1 cm X 30 cm) and eluted with 0.1% BSA in 0.1M 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 IN NaOH and then to 7.5 with 1M NaHCO 3 .
- the radiolabeled peptide was precipitated by addition of 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) of water and, after the final wash, the precipitate was dissolved in 5 ml . of 10% BSA.
- 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 known to chelate a variety of metals including radioisotopes such as 111 -indium.
- Tc technetium
- DTPA diethylenetriamine pentaacetic acid
- Direct coupling to 99m Tc is carried out as follows. 50 mCi 99m Tc (in the form of 99a TcO 4 -), 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 10 minutes at room temperature. The pH is raised to 8.0 - 9.0 if
- the radiolabelled synthetic peptide fraction is separated from uncoupled technetium and sodium dithionite by molecular sieve chromatography.
- the column is standardized with blue dextran and potassium iodide to determine 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.
- Indirect coupling to 99m Tc is carried out as follows.
- a chelating ligand e.g., DTPA (as per Hnatowich et al., Science 220: 613, 1983) or bromoacetylparaaminobenzyl EDTA (BABE; as per Meares et al., Analyt . Biochem . 142: 142, 1984) is covalently bound to the N- or C-terminus of the peptide.
- DTPA as per Hnatowich et al., Science 220: 613, 1983
- BABE bromoacetylparaaminobenzyl EDTA
- 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 99m TcO 4 - is added to the DTPA-synthetic peptide, and to the mixture is
- 99m Tc-labelled synthetic-DTPA peptide is separated from uncomplexed 99m 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 99m Tc-labelled peptide is
- lipid emulsion administered either in a pharmaceutically-acceptable carrier solution or bound to a lipid emulsion.
- Specific anti-LDL antisera may be purchased from a number of sources (e.g., Hoechst Pharmaceutical, Inc.,
- antisera may be prepared by any number of protocols known to those skilled in the art.
- 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 ml of saline or barbital buffer, was emulsified with an equal volume of Freund's complete adjuvant (Difco).
- the animal 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 immune state and will 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 is allowed to clot at room temperature for several hours and is then placed overnight in the
- 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
- the peptide solution was mixed with an equal volume of complete Freund's adjuvant 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 four 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.
- microtiter plates (Immulon II Dynatech Labs, Chantilly, VA) were coated with the purified synthetic peptide or LDL by an overnight incubation at 4°C with 100 ng peptide per well in 50 mM carbonate, pH 9.6, and blocked for nonspecific binding with an additional overnight incubation with phosphatebuffered saline, pH 7.4 (PBS), 1% bovine serum albumin
- BSA Bovine serum albumin
- Control wells were coated with BSA alone. After washing twice with PBS, the wells were filled with serial dilutions (1:10 to 1:100,000 made in PBS, 3% BSA) of a rabbit polyclonal antibody generated against the synthetic peptide (s) and incubated for 45 minutes at room temperature. Following thorough washing (3X with PBS, 0.1% BSA), the wells were filled with a 1:2000 dilution of goat anti-rabbit IgG-horseradish peroxidase conjugate (Atlantic Antibodies,
- Results were expressed as the initial velocity of substrate conversion (change in OD 650 /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 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 LDL on the plates, providing immunological confirmation that SP-4 and LDL have structural similarities.
- anti-SP-4 antiserum was shown to bind SP-4 and SP-4A as well as the conservatively
- 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).
- the catheter was inflated to a pressure of about 3 psi above the balloon inflation pressure with radiographic contrast medium (Conray, Mallinkrodt, St. Louis, MO). 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 weeks before injection of the labelled synthetic peptides.
- radiographic contrast medium Conray, Mallinkrodt, St. Louis, MO
- 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 1.5 kg. The animals were raised until they were 3-4 kg in weight. At this weight, they exhibited marked aortic atherosclerosis. Each labelled synthetic peptide preparation
- Peptides SP15a, 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).
- Saran high speed x-ray film
- FIGS. 8-13 Representative results are shown in FIGS. 8-13.
- Deendothelialized 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.
- the autoradiograph (FIG 3-8, B) demonstrates clear-cut localization of the synthetic peptide on the image at the healing (reendothelizing) edge of the aortic lesions produced by the previous trauma.
- Control peptides used were SP-2 (part of the heparin and LDL receptor binding site of apolipoprotein E) and SP-11A which is a receptor and heparin binding domain of apolipoprotein B.
- the mean concentration of synthetic peptide-asso ⁇ iated 125 I 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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US51814290A | 1990-05-03 | 1990-05-03 | |
US51821590A | 1990-05-03 | 1990-05-03 | |
US518142 | 1990-05-03 | ||
PCT/US1991/003026 WO1991016919A1 (en) | 1990-05-03 | 1991-05-02 | Synthetic peptides for arterial imaging |
US518215 | 1995-08-23 |
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EP0600869A4 EP0600869A4 (de) | 1993-03-22 |
EP0600869A1 true EP0600869A1 (de) | 1994-06-15 |
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JP (1) | JPH05507276A (de) |
AU (1) | AU663291B2 (de) |
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WO (1) | WO1991016919A1 (de) |
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US5972890A (en) * | 1988-05-02 | 1999-10-26 | New England Deaconess Hospital Corporation | Synthetic peptides for arterial imaging |
US5726153A (en) * | 1988-05-02 | 1998-03-10 | New England Deaconess Hospital Corporation | Synthetic peptides for arterial imaging |
US5997844A (en) * | 1991-02-08 | 1999-12-07 | Diatide, Inc. | Technetium-99m labeled peptides for imaging |
US5965107A (en) * | 1992-03-13 | 1999-10-12 | Diatide, Inc. | Technetium-99m labeled peptides for imaging |
US5989519A (en) * | 1992-03-13 | 1999-11-23 | Diatide, Inc. | Technetium-99m labeled peptides for imaging inflammation |
CA2131816C (en) * | 1992-03-13 | 2001-08-28 | Richard T. Dean | Technetium-99m labeled peptides for imaging inflammation |
WO1994004563A1 (en) * | 1992-08-14 | 1994-03-03 | Shino-Test Corporation | PEPTIDES CONTAINING RESPECTIVE AMINO ACID SEQUENCES SELECTED FROM AMONG THOSE OF LIPOPROTEIN(a) AND APOLIPOPROTEIN(a), ANTIBODIES RESPECTIVELY RECOGNIZING THESE AMINO ACID SEQUENCES, AND METHOD OF ASSAYING WITH THESE ANTIBODIES |
US5413778A (en) * | 1992-10-05 | 1995-05-09 | The Regents Of The University Of Michigan | Labelled monocyte chemoattractant protein material and medical uses thereof |
US5605671A (en) * | 1992-10-05 | 1997-02-25 | The Regents Of The University Of Michigan | Radiolabeled neutrophil activating peptides for imaging |
AU5537994A (en) * | 1992-10-22 | 1994-05-09 | Mallinckrodt Medical, Inc. | Therapeutic treatment for inhibiting vascular restenosis |
DE4301871A1 (de) * | 1993-01-13 | 1994-07-14 | Diagnostikforschung Inst | Neue Mittel zur Diagnose von Gefäßerkrankungen |
DE4425778A1 (de) * | 1994-07-13 | 1996-01-18 | Diagnostikforschung Inst | Komplexverbindungen zur Diagnose von Gefäßerkrankungen |
US6489446B1 (en) | 1996-08-07 | 2002-12-03 | Hsc Research And Development Limited Partnership | Self-aligning peptides modeled on human elastin and other fibrous proteins |
JP2001505539A (ja) * | 1996-08-07 | 2001-04-24 | プロテイン・スペシャルティーズ,リミテッド | エラスチン及び他の線維状タンパク質由来の自己整列ペプチド |
DE69735243T2 (de) * | 1996-11-27 | 2006-11-02 | Boston Heart Foundation, Inc., Cambridge | Neue low density lipoprotein bindende proteine und deren verwendung in der diagnostik und behandlung von atherosklerose |
US6605588B1 (en) | 1996-11-27 | 2003-08-12 | Boston Heart Foundation, Inc. | Low density lipoprotein binding proteins and their use in diagnosing and treating atherosclerosis |
US6632923B1 (en) | 1996-11-27 | 2003-10-14 | Boston Heart Foundation, Inc. | Low density lipoprotein binding proteins and their use in diagnosing and treating atherosclerosis |
US7368099B2 (en) * | 2003-02-27 | 2008-05-06 | Kyushu University, National University Corporation | MRI contrast agents |
AU2008251900A1 (en) | 2007-05-10 | 2008-11-20 | Elastin Specialties, Inc. | Synthetic peptide materials for joint reconstruction, repair and cushioning |
GB201102189D0 (en) * | 2011-02-08 | 2011-03-23 | King S College London | Materials and methods relating to cardiovascular imaging |
WO2023086771A2 (en) * | 2021-11-05 | 2023-05-19 | Trustees Of Tufts College | Materials for valuable metal recovery |
Citations (4)
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EP0162414A2 (de) * | 1984-05-15 | 1985-11-27 | Research Corporation | Amphipatische Peptide und deren pharmazeutische Zusammensetzung |
WO1986004144A1 (en) * | 1984-12-31 | 1986-07-17 | International Genetic Engineering, Inc. | Peptide fragments of human apolipoprotein, type-specific antibodies and methods of use |
WO1989010098A1 (en) * | 1988-04-21 | 1989-11-02 | Uab Research Foundation | Stimulation of chemotaxis by chemotactic peptides |
WO1989010760A1 (en) * | 1988-05-02 | 1989-11-16 | New England Deaconess Hospital Corporation | Synthetic peptides for arterial imaging |
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CA1242643A (en) * | 1983-08-12 | 1988-10-04 | Eric T. Fossel | Nmr imaging utilizing chemical shift reagents |
US4660563A (en) * | 1984-12-31 | 1987-04-28 | Massachusetts Institute Of Technology | Method and means for detection of arterial lesions |
-
1991
- 1991-05-02 CA CA002080493A patent/CA2080493A1/en not_active Abandoned
- 1991-05-02 AU AU79928/91A patent/AU663291B2/en not_active Ceased
- 1991-05-02 EP EP91910995A patent/EP0600869A1/de not_active Withdrawn
- 1991-05-02 WO PCT/US1991/003026 patent/WO1991016919A1/en not_active Application Discontinuation
- 1991-05-02 JP JP91510596A patent/JPH05507276A/ja active Pending
Patent Citations (4)
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EP0162414A2 (de) * | 1984-05-15 | 1985-11-27 | Research Corporation | Amphipatische Peptide und deren pharmazeutische Zusammensetzung |
WO1986004144A1 (en) * | 1984-12-31 | 1986-07-17 | International Genetic Engineering, Inc. | Peptide fragments of human apolipoprotein, type-specific antibodies and methods of use |
WO1989010098A1 (en) * | 1988-04-21 | 1989-11-02 | Uab Research Foundation | Stimulation of chemotaxis by chemotactic peptides |
WO1989010760A1 (en) * | 1988-05-02 | 1989-11-16 | New England Deaconess Hospital Corporation | Synthetic peptides for arterial imaging |
Non-Patent Citations (3)
Title |
---|
JOURNAL OF CELL BIOLOGY vol. 107, no. 5, November 1988, NEW YORK,USA pages 1987 - 1993 BLOOD ET AL 'IDENTIFICATION OF A TUMOR CELL RECEPTOR FOR VGVAPG,AN ELASTIN-DERIVED CHEMOTACTIC PEPTIDE' * |
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES,USA vol. 87, February 1990, WASHINGTON D.C.,USA pages 1436 - 1440 SHIH ET AL 'FOCAL ACCUMULATION OF AN APOLIPOPROTEIN B-BASED SYNTHETIC OLIGOPEPTIDE IN THE HEALING RABBIT ARTERIAL WALL' * |
See also references of WO9116919A1 * |
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