Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
1998-07-28
2001-06-26
Higel, Floyd D. (Department: 1613)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C548S338100, C530S362000, C530S403000, C530S404000, C530S405000, C530S406000, C530S387100
Reexamination Certificate
active
06252087
ABSTRACT:
FIELD OF THE INVENTION
This invention generally relates to a class of nitroaromatic compounds that, when activated by reductive metabolism, bind to hypoxic cells. This reductive metabolism and binding increase as the oxygen concentration of cells decreases, which enables these compounds to be used as indicators of hypoxia. The present invention presents novel nitroaromatic compounds; immunogenic conjugates comprising the novel nitroaromatic compounds and proteins; and monoclonal antibodies specific for the novel nitroaromatic compounds of the invention, their protein conjugates, their reductive byproducts, and adducts formed between mammalian hypoxic cells and the compounds of the invention. The invention is further directed to methods for detecting levels of low oxygen in tissue. Detection may be done directly using methods such as imaging techniques involving specific isotopes attached to the nitroaromatic drug, or indirectly using the monoclonal antibodies (mAbs) in immunohistological assays. Still further, the present invention is directed to kits for performing the methods of the invention.
BACKGROUND OF THE INVENTION
One of the most important goals in oncology is the identification and elimination of treatment resistant cells; hypoxic cells are the most familiar examples of this type of cell. Kennedy, el al.,
Biochem. Pharm
. 1980, 29, 1; Moulder, et al.,
Int. J. Radioat. Oncol. Biol. Phys
. 1984, 10, 695; Adams,
Cancer
, 1981, 48, 696. Hypoxic cells are seldom found in normal tissues, and are generally found only in conjunction with certain tumors, vascular diseases, or after a stroke.
As certain tumors enlarge, the tissue often outgrows its oxygen and nutrient supply because of an inadequate network of functioning blood vessels and capillaries. Although the cells deprived of oxygen and nutrients may ultimately die, at any given time a tumor may produce viable hypoxic cells. These hypoxic cells, although alive, have very low oxygen concentrations because of their remoteness from the blood vessels.
The level of molecular oxygen has important implications in disease diagnosis and prognosis. In medical oncology, for example, hypoxic cells in solid tumors may be highly resistant to killing by some forms of chemotherapy. When chemotherapeutic agents are administered to patients, the agents are carried through the functioning blood vessels and capillaries to the target tissue. Because hypoxic tissue lacks a fully functioning blood supply network, the chemotherapeutic drugs may never reach the hypoxic cells; instead, intervening cells scavenge the drug. The result is that the hypoxic cells survive and recurrence of the tumor is possible. Kennedy, et al., supra.
Tissue hypoxia also hinders the effectiveness of radiation therapy, especially of neoplasms. Radiation treatment is most effective in destroying oxygen containing cells because oxygen is an excellent radiation sensitizer. The presence of hypoxic cells impedes this treatment because their low oxygen concentration renders the ionizing radiation relatively ineffective in killing the cancerous cells. Therefore, hypoxic cells are more likely to survive radiation therapy and eventually lead to the reappearance of the tumor. The importance of hypoxic cells in limiting radiation responsiveness in animal tumors is well known, Adams, supra; Moulder, et al., supra; Chapman, et al., “
The Fraction of Hypoxic Clonogenic Cells in Tumor Populations,” in Biological Bases and Clinical Implications of Tumor Radioresistance
61, G. H. Fletcher, C. Nevil, & H. R. Withers, eds., 1983. Studies have revealed that such resistant cells greatly affect the ability of radiation and chemotherapy to successfully sterilize tumors in animals. Substantial work since that time has shown similar problems in human tumors. Despite the progress in animal studies regarding the identification of hypoxic cells, limited success has been achieved in humans. One reason for this disparity may relate to differences in tumor growth and other host related factors, but in addition, there has been no suitably accurate method to assess tissue oxygen at a sufficiently fine resolution.
Venous oxygen pressure is generally ~35 Torr, an oxygen level providing nearly full radiation sensitivity. As the oxygen level decreases below 35 Torr, radiation resistance gradually increases, with half-maximal resistance at about 3.5 Torr, and full resistance at about 0.35 Torr. Therefore, it is necessary to measure much lower oxygen levels than are usually encountered in normal tissue. Current technology does not meet this need. Oxygen partial pressure measured using current techniques often yields an average value for large numbers of neighboring cells. This is a severe impediment for detection and diagnosis because histological evaluation of solid tumors suggest that important changes in cellular oxygen can occur over dimensions of even a few cell diameters. Urtasun, el al., Br.
J. Cancer
, 1986, 54, 453. Nitroheterocyclic drugs have been under extensive investigation as oxygen indicators. It is known that this class of compounds has the potential for resolution at the cellular level and can provide sufficient sensitivity to monitor the low oxygen partial pressures described above. This technique involves the administration of nitroaromatic drugs to the tissue of interest. The drugs undergo bioreductive metabolism at a rate which increases substantially as the tissue's oxygen partial pressure decreases. The result of this bioreductive metabolism is that reactive drug products are formed which combine chemically to form adducts with predominantly cellular proteins. Because the metabolic binding of these compounds to cellular macromolecules is inhibited by oxygen, these compounds bind to hypoxic cells in preference to normal, healthy, oxygen-rich tissue. This preferential metabolic binding, or adduct formation, provides a measure of the degree of hypoxia. Koch, et al.,
Int. J. Radiation Oncology Biol. Phys
., 1984,10, 1327.
Misonidazole (MISO) 3-methoxy-1-(2-nitroimidazol-1-yl)-2-propanol, and certain of its derivatives have been under extensive investigation as indicators of hypoxia in mammalian tissue. Chapman, et al.,
Int. J. Radiat. Oncol. Biol. Phys
., 1989,16, 911; Taylor, et al.,
Cancer Res
., 1978, 38, 2745; Varghese, et al.,
Cancer Res
., 1980, 40, 2165. The ability of certain misonidazole derivatives to form adducts with cellular macromolecules, referred to as binding throughout this application, has formed the basis of various detection methods.
For example,
3
H or
14
C labeled misonidazole has been used in vitro and in vivo, with binding analyzed by liquid scintillation counting or autoradiography. Chapman, 1984 supra; Urtasun, 1986, supra; Franko, et al.,
Cancer Res
., 1987, 47, 5367. A monofluorinated derivative of misonidazole has utilized the positron emitting isotope F18 for imaging bound drug in vivo, Rasey, et al.,
Radiat. Res
., 1987, 111,292. The method of the preparation of the PET derivative of ethanidazole was described in Tewson T. J. Synthesis of [
18
F] Fluoroetanidazole: a potential new tracer for imaging hypoxia.
Nuclear Medicine & Biology
, 24(8):755-60, 1997.
A hexafluorinated derivative of misonidazole (1-(2-hydroxy-3-hexafluoro-isopropoxy-propyl)-2-nitroimidazole has been assayed directly (no radioactive isotopes) via nuclear magnetic resonance spectroscopy (NMR or MRI) techniques. Raleigh, et al.,
Int. J. Radiat. Oncol. Biol. Phys
., 1984, 10, 1337. Polyclonal antibodies to this same derivative have allowed immunohistochemical identification of drug adducts. Raleigh, et al.,
Br. J. Cancer
, 1987, 56, 395. An iodine isotope has been incorporated into another azomycin derivative, azomycin arabinoside, allowing radiology techniques of detection. Parliament, et al.,
Br. J. Cancer
, 1992, 65, 90.
A fluorescence immunohistochemical assay for detecting hypoxia is described in the literature. Raleigh, et al., 1987, supra. A method for preparing immunogenic conjugates for use in such assays is broadly disclosed in U.S. Pat. No. 5,0
Baird Ian R.
Dolbier, Jr. William R.
Evans Sydney M.
James Brian R.
Kachur Alexander V.
Higel Floyd D.
The Trustees of the University of Pennsylvania
Woodcock Washburn Kurtz Mackiewicz & Norris LLP
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