Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-04-01
2002-09-24
Caputa, Anthony C. (Department: 1642)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S350000, C530S387100, C435S007100
Reexamination Certificate
active
06455494
ABSTRACT:
FIELD OF THE INVENTION
TECHNICAL FIELD
The present invention relates to GPI-anchored p97, a secreted form of p97 and derivatives thereof; methods of using p97 in modulating iron transport, in the delivery of drugs, and in the treatment of conditions involving disturbances in iron metabolism; and methods of treating and diagnosing Alzheimer's Disease.
BACKGROUND OF THE INVENTION
Iron is a fundamental component required by all cells for growth and normal physiological processes (Crichton, R. R. and Charloteaux-Wauters, M. Eur. J. Biochem. 164:485-506 and Ponka, P. et al, Iron Transport and Storage, CRC Press, Boca Raton, Ann Arbor and Boston, 1990). Rapidly proliferating cells have a higher iron requirement than quiescent cells. In humans this iron requirement is thought to be provided by the binding of iron to the major serum iron-transporting protein, transferrin. Transferrin bound to iron can bind as a complex to the transferrin receptor expressed on the plasma membrane (Ponka, P. et al, Iron Transport and Storage, CRC Press, Boca Raton, Ann Arbor and Boston, 1990). After binding, the iron/transferrin/transferrin receptor complex remains membrane bound and is concentrated and then endocytosed via endocytotic vesicles. The endosomes become acidified and the iron is released from the complex within the cell and the apotransferrin remains bound to the receptor and is recycled to the surface where it is released to participate in the uptake of further iron into the cell (Kuhn L. C. et al., in Iron Transport and Storage, CRC Press, Boca Raton, Ann Arbor and Boston, 1990, p. 149).
Disruption of blood circulation deprives cells of oxygen and iron and may result in cell death. Deposition of iron from cell death, for example in ischemic injury may result in the generation of highly reactive and toxic superoxide or hydroxyl free radicals which can result in further tissue damage. Accordingly, the abundance of iron and its availability can greatly alter survival of damaged tissues. Rapidly proliferating cells, such as malignant cells, have an increased requirement for iron and must possess efficient mechanisms to obtain iron. Limiting the ability of malignant cells to acquire iron may provide a method of killing tumor cells or of modulating their uncontrolled cell growth.
Fe is rarely found in the blood plasma in the free state since it is highly toxic (Lauffer, R. B. (1992). Iron and Human Disease (Boca Ranton, Fla.: CRC Press)) and Tf serves mainly to mop up free Fe and to shuttle Fe, in a soluble non-toxic form, among the organs of the body. The established mechanism by which cells acquire Fe from Tf involves Tf binding to the transferrin receptor (TR) and Fe being internalized by the mechanism of receptor mediated endocytosis(RME) (Aisen, P. (1989). Iron carriers and iron proteins. In Iron carriers and iron proteins. T. M. Loehr, ed. (New York: VCH), pp. 353-372.; Thorstensen, K. and Romslo, I.
Biochem. J.,
271, 1-10, 1990). Since normal levels of serum Tf are high and about 99% of Fe in the plasma is bound to Tf (May, P. M. et al, (1980). Biological significance of low molecular weight iron(III) complexes. In Metal ions in biological systems. H. Sigel, ed. (New York: Marcel Dekker Inc.), pp. 29-76), Fe uptake is believed to be regulated by the level of TR expression (Thorstensen, K. and Romslo, I.
Biochem. J.,
271, 1-10, 1990; Young, S. P. and Aisen, P.
Hepatology,
1, 114-119, 1981; Brissot, P. et al.,
J.Clin.Invest.,
76, 1463-1470, 1985). Any free Fe generally circulates as low molecular weight complexes such as citrate (Grootveld, M. et al.,
J. Biol. Chem.,
264, 4417-4422, 1989) and certain amino acids or in association with other serum proteins such as albumin (May, P. M. et al, (1980). Biological significance of low molecular weight iron(III) complexes. In Metal ions in biological systems. H. Sigel, ed. (New York: Marcel Dekker Inc.), pp. 29-76). High levels of free Fe are usually only found in the plasma from dying cells or during iron overload disorders such as haemochromatosis (Smith, L., West.
J. Med.,
153, 296-308, 1990), thalassaemia (Modell, B. and Berdoukas, V. (1984). The clinical approach to thalessemia (New York: Grune and Stratton).) and atransferrinanemia (Kaplan, J. et al,
J. Biol. Chem.,
266, 2997-3004, 1991).
Based on studies where cells were grown in serum free, hence Tf-free, media and in cases of iron overload disorders it has become evident that some cells are able to obtain Fe independent of Tf and the RME pathway.
Although cellular iron uptake has been shown to be mediated mainly by the transferrin receptor (Doering, T. L. et al, J. Biol. Chem. 265:611-614, (1990), a non-transferrin-mediated pathway has been implicated for iron incorporation into cells, including leukemic cells (Basset, P. et al, Cancer Res. 46:1644-1647, 1986), HeLa cells (Sturrock, A. et al, J. Biol. Chem. 265:3139-3145, 1990), hepatocytes (Thorstensen, K., J. Biol. Chem. 263:16837-16841, 1988) and melanoma cells (Richardson, D. R. and Baker, E., Biochem. Biophys. Acta. 1053:1-12, 1990; Richardson, D. R. and Baker, E., Biochem. Biophys. Acta. 1091:294-302, 1991a and; Richardson, D. R. and Baker, E., Biochem. Biophys. Acta. 1093:20-28, 1991a).
p97, also known as melanotransferrin, a human melanoma-associated antigen, was one of the first cell surface markers associated with human skin cancer (Hellstrom, K. E. and Hellstrom, I. (1982) in Melanoma Antigens and Antibodies, Ed. Reisfield, R. and Ferrone, S., Plenum Press, New York, pp187-341). p97 is a monomeric membrane-associated protein with a molecular mass of 97,000 daltons (Brown, J. P. et al. J. Immunol. 127:539, 1981) and has been suggested as a melanoma specific marker (Estin, C. D. et al., Proc. Nat. Acad. Sci. U.S.A. 85:1052-1056, 1988). As well as being associated with the cell surface of melanomas and some other tumors and cell lines (Brown, J. P. et al., Proc. Nat. Acad. Sci. U.S.A. 78:539, 1981), p97 has also been found in certain fetal tissue (Woodbury, R. G. et al., Int. J. Cancer 27:145, 1981) and, more recently on endothelial cells of the human liver (Sciot, R., et al., Liver 9:110, 1989).
The primary structure of p97, deduced from its mRNA sequence indicates that it belongs to a group of closely related iron binding proteins found in vertebrates (Rose, T. M. et al., Proc. Nat. Acad. Sci. U.S.A. 83:1261, 1986). This family includes serum transferrin, lactoferrin and avian egg white ovotransferrin. Human p97 and lactoferrin share 40% sequence homology (Baker, E. N. et al., Trends Biochem. Sci. 12:350, 1987), however, in contrast to the other molecules of the transferrin family, p97 is the only one which is directly associated with the cell membrane. The deduced sequence of p97 has, in addition to a transferrin-like domain, a hydrophobic segment at its C terminal which was thought to allow the molecule to be inserted into the plasma membrane (Rose, T. M. et al., Proc. Nat. Acad. Sci. USA 77:6114, 1980).
Detergent-solubilized p97 has been reported to bind iron (Doering, T. L. et al., J. Biol. Chem. 265:611-614, 1990). However, the role of p97 in iron transport is far from clear. Iron binding to p97 at the plasma membrane has not been demonstrated and, despite numerous studies, no evidence of a role for p97 in iron mediated transport has been obtained to date. Recent studies have concluded that p97 does not play a role in iron transport (Richardson, D. R. and Baker, E. Biochem. Biophys. Acta. 1103:275-280, 1992; Richardson, D. R. and Baker, E. Biochem. Biophys. Acta. 1093:20-28, 1991 and; Richardson, D. R. and Baker, E. Biochem. Biophys. Acta. 1091:294-302, 1991). The physiological role of p97 in normal and malignant cells has not been determined.
Alzheimer's Disease has become a significant health care problem due to increases in number and longevity of the elderly. In the near future, it is predicted that a significant proportion of the elderly population may be affected. The incidence of Alzheimer's Disease increases sharply from 1% at age 65, to over 20% at age 80. After age 85, nearly half of
Food Michael R.
Jefferies Wilfred A.
Kennard Malcolm
McGeer Patrick L.
Rothenberger Sylvia
Bereskin & Parr
Caputa Anthony C.
Davis Minh-Tam
Gravelle Micheline
University of British Columbia
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