WO1988003413A1 - Detection de maladies vasculaires - Google Patents

Detection de maladies vasculaires Download PDF

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Publication number
WO1988003413A1
WO1988003413A1 PCT/US1987/002939 US8702939W WO8803413A1 WO 1988003413 A1 WO1988003413 A1 WO 1988003413A1 US 8702939 W US8702939 W US 8702939W WO 8803413 A1 WO8803413 A1 WO 8803413A1
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WO
WIPO (PCT)
Prior art keywords
biologically active
conjugate
reagent
antibodies
active molecule
Prior art date
Application number
PCT/US1987/002939
Other languages
English (en)
Inventor
Robert S. Lees
Original Assignee
New England Deaconess Hospital Corporation
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by New England Deaconess Hospital Corporation filed Critical New England Deaconess Hospital Corporation
Publication of WO1988003413A1 publication Critical patent/WO1988003413A1/fr

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Classifications

    • 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
    • A61K49/16—Antibodies; Immunoglobulins; Fragments thereof
    • 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
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1018—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo

Definitions

  • the technical field of this invention concerns methods and means for detecting vascular diseases, such as atherosclerotic lesions, and in particular, methods and means employing labeled, target-seeking, biologically active molecules to detect abnormal arterial structures or compositions.
  • Atherosclerosis is a disease which causes the thickening and hardening of the arteries, particularly the larger artery walls. It is characterized by lesions of raised fibrous plaque 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 bulge into the lumen.
  • a lesion typically will consist of a central core of lipid and necrotic cell debris, capped by a collagen fibromuscular layer. Complicated lesions will also include calcified deposits and exhibit various degrees of necrosis, thrombosis and ulceration.
  • Atherosclerosis and related vascular diseases are most often clinically silent. Since lifestyle changes, drug therapy and other means exist for delaying or reducing vascular occlusion or the stresses on various body organs which result from atherosclerotic lesions, the early detection of atheromatous plaque in the vascular system would be of considerable value in permitting preventive intervention at a time when it can be most effective.
  • LDLs low density lipoproteins
  • the labeled LDLs can be imaged with a gamma camera or other radiation detector to provide information on the location and extent of plaque in the vascular system.
  • labeled LDL's image acute, rapidly growing atherosclerosic lesions, as reported by the present inventor and colleagues, Lees et al., Journal of Nuclear Medicine, Vol. 24, pp. 154-156, 1983, and herein incorporated by reference.
  • Vascular diseases including asymptomatic atherosclerosis, can be diagnosed by administering a conjugate diagnostic reagent to a patient and then detecting the location and concentration of the conjugate reagent within the patient's vascular system.
  • the conjugate reagents have a target-seeking, biologically active molecule as one component and a labeling means capable of extracorporeal detection as the other component.
  • the biologically active molecules are chosen to have affinity for structural elements of the arterial wall which are either not present in the normal wall but are found in atherosclerotic lesions, or which are not available to the blood-borne reagent within normal arterial walls, but are exposed to the reagent in atherosclerotic lesions.
  • the invention encompasses conjugates of radionuclides with various antibodies or fragments thereof which have affinity for certain arterial wall components which are exposed to the blood in atherosclerosis.
  • Two conjugated reagents having particularly useful BAMs are disclosed.
  • the BAMs have high affinity for elastin and chondroitin sulfate proteoglycans (CSPG) , respectively.
  • the conjugates of the present invention can be administered individually or in conjunction with each other.
  • Suitable radionuclides include Co-57, Cu-67, Ga-67, Ga-68, Ru-97, Tc-99m, In-111, In-113m, 1-123, 1-125, 1-131, Hg-197, Au-198, and Pb-203.
  • the BAMs and radionuclides can be linked by direct labeling (e.g.,- by acidic buffered reactions or oxidative procedures) or by ligand exchange or chelation.
  • the radionuclides are preferably imaged with a radiation detection means capable of detecting gamma radiation, such as a gamma camera or the like.
  • radiation imaging cameras employ a conversion medium (wherein the high energy gamma ray is absorbed, displacing an electron which emits a photon upon its return to the orbital state) , photoelectric detectors arranged in a spatial detection chamber (to determine the position of the emitted photons), and circuitry to analyze the photons detected in the chamber and produce an image.
  • a conversion medium wherein the high energy gamma ray is absorbed, displacing an electron which emits a photon upon its return to the orbital state
  • photoelectric detectors arranged in a spatial detection chamber (to determine the position of the emitted photons)
  • circuitry to analyze the photons detected in the chamber and produce an image.
  • the invention can also be practiced with non-radioactive labeling means, such as magnetic contrast agents capable of detection in magnetic resonance imaging (MRI) systems.
  • MRI magnetic resonance imaging
  • a strong magnetic field is used to align the nuclear spin vectors of the atoms in a patient's body. The field is then disturbed and an image of the patient is read as the nuclei return to their equilibrium alignments.
  • target-seeking BAMs can be linked to diamagnetic contrast agents, such as gadolinium, cobalt, nickel, manganese or copper complexes, to form conjugate diagnostic reagents that are imaged extracorporeally with an MRI system.
  • determinants can be used to identify atherosclerotic plaque.
  • CSPG chondroitin sulfate proteogylcans
  • determinants can also be used as targets against which BAMs can be raised and used in conjugate reagents.
  • Such determinants may include, for examples, particular species of collagen and glycoproteins, such as HLA-DR or similar proteins, which have been reported to be present on the surface of atherosclerotic smooth muscle cells.
  • the elastin present in the arterial wall becomes enzymatically degraded. This fragmented elastin is chemically different from native elastin. Additionally, the composition of the proteoglycans which coat the arterial wall changes markedly; the content of other proteoglycans decreases and that of chondroitin sulfate proteoglycan increases. These two chemical changes are exploited in the present invention.
  • antibodies are raised against elastin and chondroitin sulf te proteoglycans (CSPG) . These antibodies or fragments derived therefrom are then used to create conjugated diagnostic reagents.
  • CSPG chondroitin sulf te proteoglycans
  • Human elastin was used to image balloon angioplasty-induced lesions in rabbit aorta.
  • the particular antibodies were polyclonal sheep anti-human lung amorphorous elastin antibodies obtained from Dr. J. Rosenbloom of the University of Pennsylvania.
  • Such antibodies can be generated independently by known techniques.
  • human elastin can be isolated from a lung autopsy specimen, emulsified with Freund's complete adjuvant and then injected subcutaneously at multiple sites into a sheep (e.g., 10 - 20 milligrams). One month later, the animal can be boosted (e.g., with 5 milligrams of the same antigenic preparation), and one week after that, the animal can be bled.
  • the immunoglobulin s fraction can be recovered from the whole blood serum by passage through a Protein A-Sepharose affinity chromotography column.
  • the anti-elastin antibodies were labeled with 1-125 (i.e., sodium iodide) as described by McFarlane in Vol. 182, Nature, p. 53 (1958). Excess 1-125 can be dialyzed off against physiological saline solution at a pH of about 8. The 1-125 labeled antibody was then injected intravenously (approximately 300 millicuries per animal) in each of three rabbits which had four weeks previously undergone balloon diendothelialization of the abdominal aorta. Twenty-four to forty-eight hours later, the rabbits were sacrificed, the aortas removed, washed with normal saline, cut open lenghtwise, and covered with polyester wrap. The aortas were then carefully placed on a sheet of high speed X-ray film, and the audioradiograph was allowed to develop for four weeks,
  • the audioradiograph showed clear-cut localization of the elastin on the image at the healing (reendothelization) edge of the aortic lesions produced by the previous trauma. Since this lesion is known to resemble human arteriosclerosis in many important respects, including accumulation of lipoproteins and other pathological changes, the ability of the antiserum to localize at the trauma site and permit the imaging thereof demonstrates the utility of the present invention in imaging vascular disease.
  • antibodies to human chondroitin sulfate proteoglycan were similarly used to image the abdominal aortas of rabbits which had been previously balloon diendothelialized.
  • the particular antibodies were monoclonal anti-CSPG antibodies obtained from Drs. M. Lark and T. Wight of the University of Washington.
  • Such antibodies can be generated independently by known techniques.
  • monoclonal antibodies or active fragments can be obtained by applying generally known cell fusion techniques (cf. G. Kohler, C. Milstein, Eur. J. Immunol. 6 511-519 (1976) and M.
  • Monoclonal antibodies are prepared by obtaining mammalian lymphocytes (preferably spleen cells), committing the lymphocytes to produce antibodies (e.g., by immunizing the mammal with the particular antigenic determinant of interest beforehand), fusing the lymphocytes with myeloma (or other immortal) cells to form hybrid cells, and then culturing a selected hybrid cell colony in' vivo or in vitro to yield antibodies Identical ih structure and reactivity.
  • mammalian lymphocytes preferably spleen cells
  • committing the lymphocytes to produce antibodies e.g., by immunizing the mammal with the particular antigenic determinant of interest beforehand
  • myeloma or other immortal
  • chondrotin-sulfate proteoglycans can be prepared by isolating CSPG from smooth muscle cells in tissue culture.
  • the CSPG can be extracted from the culture medium and purified by ion exchange chromotography.
  • Mice or other animals can be challenged with a solution of the above-derived CSPG in Freund's adjuvant (i.e., 1 - 5 milligram CSPG per milliliter of medium emulsified with one milliliter of adjuvant) injected into the peritoneal cavities of the animals. Six weeks later a similar injection
  • Freund's adjuvant i.e., 1 - 5 milligram CSPG per milliliter of medium emulsified with one milliliter of adjuvant
  • mice can be administered as a booster.
  • mice are killed and their spleens homogenized.
  • the spleen cells are hybridized with mouse myeloma cells by the above-referenced procedure of Kohler and Milstein.
  • the hybridomas so produced are screened to select a cell line producing antibodies which react with human aortic chondroitin sulphate proteoglycan.
  • Large scale antibody production can be obtained from such anti-CSPG producing cell lines by various techniques, including the Induction of ascites tumors (i.e., after priming with pristane) and the purification of such antibodies from the ascites fluid by Protein A-Sepharose affinity chromotography.
  • the pure IgM antibody was labeled with 1-125 by the same method as described in Example 1. and injected into each of three rabbits in a similar manner. Audioradiographs produced as described above clearly showed that the labeled anti-CSPG antibodies imaged the bare, injured areas of the arterial wall and did not image either healthy arterial wall regions or those areas where rapid reendothelialization was occurring. Thus, this antibody can image aspects of vascular disease which are distinct from those detected by the anti-elastin antibodies discussed in Example 1.
  • Active fragments can be derived from the monoclonal antibodies disclosed herein by a number of techniques. For example, purified monoclonal antibodies can be treated in a buffer solution with an enzyme, such as pepsin and subjected to HPLC gel filtration. The appropriate fraction containing Fab can then be collected and concentrated by membrane filtration or the like.
  • an enzyme such as pepsin
  • HPLC gel filtration The appropriate fraction containing Fab can then be collected and concentrated by membrane filtration or the like.
  • the antibodies and fragments used herein can be labeled preferably with radioactive labels, by a variety of techniques other than the above-described McFarland technique.
  • the biologically active molecules can also be labeled with a radionuclide via conjugation with the cyclic anhydride of diethylenetriamine penta-acetic acid (DTPA) or bromoacetyl aminobenzyl ethylamine diamine tetra-acidic acid (BABE) .
  • DTPA diethylenetriamine penta-acetic acid
  • BABE bromoacetyl aminobenzyl ethylamine diamine tetra-acidic acid

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Physics & Mathematics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Radiology & Medical Imaging (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Les maladies vasculaires y compris l'athérosclérose asymptomatique peuvent être diagnostiquées en administrant un réactif conjugué diagnostic à un patient et en détectant la position et la concentration du réactif conjugué dans le système vasculaire du patient. Les réactifs conjugués ont une molécule biologiquement active de ciblage qui constitue un composant et des moyens d'étiquetage pouvant effectuer une détection extracorporelle et constituant l'autre composant. Les molécules biologiquement actives (BAMs) sont choisies pour avoir une affinité pour des éléments structuraux de la paroi artérielle qui, soit ne sont par présents dans la paroi normale mais se trouvent dans des lésions athérosclérotiques ou artéritiques, soit ne sont pas disponibles pour le réactif transporté par le sang dans des parois artérielles normales mais sont exposées au réactif dans des lésions athérosclérotiques ou artéritiques.
PCT/US1987/002939 1986-11-10 1987-11-09 Detection de maladies vasculaires WO1988003413A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US92901286A 1986-11-10 1986-11-10
US929,012 1986-11-10

Publications (1)

Publication Number Publication Date
WO1988003413A1 true WO1988003413A1 (fr) 1988-05-19

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PCT/US1987/002939 WO1988003413A1 (fr) 1986-11-10 1987-11-09 Detection de maladies vasculaires

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EP (1) EP0288558A4 (fr)
JP (1) JPH01501796A (fr)
AU (1) AU601126B2 (fr)
CA (1) CA1285223C (fr)
WO (1) WO1988003413A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2091570A1 (fr) * 2006-12-20 2009-08-26 GE Healthcare AS Agents de contraste

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK408289D0 (da) * 1989-08-18 1989-08-18 Novo Nordisk As Diagnostisk reagens
GB201102189D0 (en) * 2011-02-08 2011-03-23 King S College London Materials and methods relating to cardiovascular imaging

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4577636A (en) * 1982-11-23 1986-03-25 The Beth Israel Hospital Association Method for diagnosis of atherosclerosis
US4647445A (en) * 1984-03-28 1987-03-03 Massachusetts Institute Of Technology Radiolabelled lipoproteins and method for making same
US4660563A (en) * 1984-12-31 1987-04-28 Massachusetts Institute Of Technology Method and means for detection of arterial lesions

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4577636A (en) * 1982-11-23 1986-03-25 The Beth Israel Hospital Association Method for diagnosis of atherosclerosis
US4647445A (en) * 1984-03-28 1987-03-03 Massachusetts Institute Of Technology Radiolabelled lipoproteins and method for making same
US4660563A (en) * 1984-12-31 1987-04-28 Massachusetts Institute Of Technology Method and means for detection of arterial lesions

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2091570A1 (fr) * 2006-12-20 2009-08-26 GE Healthcare AS Agents de contraste

Also Published As

Publication number Publication date
CA1285223C (fr) 1991-06-25
AU601126B2 (en) 1990-08-30
AU8322687A (en) 1988-06-01
EP0288558A1 (fr) 1988-11-02
EP0288558A4 (fr) 1990-01-08
JPH01501796A (ja) 1989-06-22

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