Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Oxidoreductases
Reexamination Certificate
1998-05-08
2001-02-20
Prouty, Rebecca E. (Department: 1632)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Oxidoreductases
C435S006120, C435S189000
Reexamination Certificate
active
06190658
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally relates to a genetically modified superoxide dismutase enzyme and methods for treating oxidative damage by administration of such an enzyme.
BACKGROUND OF THE INVENTION
Active oxygen species are involved in the pathogenesis of a variety of diseases. Accumulation of active oxygen species, and oxygen-free radicals, as well as direct oxygen toxicity can cause oxidative damage to cells. Oxidative damage can occur in various lung diseases, cancer and inflammatory diseases, and in other conditions involving hypoxia or ischemia-reperfusion injury, such as organ transplantation.
In many laboratory models and in a few clinical trials, superoxide dismutase (SOD) has proven therapeutically useful in protecting injured tissues from one of these active oxygen species, the superoxide radical (McCord et al., Superoxide production and human disease, 1992
, Molecular Basis of Oxidative Damage by Leukocytes
, Boca Raton: CRC Press, pp. 225-239; Delanian et al., 1994
, Radiother. Oncol
. 32:12-20; Sanchiz et al., 1996
, Anticancer. Res
. 16:2025-2028). Other enzymes which are part of the cell's arsenal against these active oxygen species include catalase and glutathione peroxidase, which eliminate H
2
O
2
. The ability of SOD to protect tissues against any particular insult (ischemia, inflammation, hyperoxia, etc.), however, depends on several parameters, such as its rate of plasma clearance, its ability to equilibrate between extracellular fluid compartments, and its ability to closely approach negatively-charged cell surfaces.
In humans, three isozymes of SOD have been extensively characterized: the cytosolic cuprozinc SOD (Cu,ZnSOD), a 32 kDa dimer; the mitochondrial manganese SOD (MnSOD), an 89 kDa tetramer; and an extracellular SOD (ECSOD), a 135 kDa tetrameric glycoprotein which is also a cuprozinc enzyme. ECSOD is genetically related to the smaller cytosolic enzyme, Cu,ZnSOD, and is found in a number of tissues but at a much lower concentration than either of the other two enzymes. It is, however, the major SOD in extracellular fluids. Plasma membranes of endothelial and parenchymal cells of various tissues are often exposed to the superoxide radical generated by activated phagocytes, and are partially protected from this oxidative attack by an extracellular superoxide dismutase (ECSOD) electrostatically bound to their surfaces. ECSOD is found as three different forms: ECSOD-A with no heparin affinity, ECSOD-B with low heparin affinity, and ECSOD-C with high heparin affinity. Sandstrom et al. (1992
, J. Biol. Chem
. 267:18205-18209) have suggested that forms A and B are generated by proteolytic cleavage of a carboxyl-terminal “tail” found on ECSOD-C. The highly hydrophilic positively charged nature of this carboxyl-terminal “tail” imparts the high heparin affinity which allows the enzyme to be largely bound to heparan sulfate on endothelial surfaces.
Under normal physiological circumstances, intracellularly generated superoxide is efficiently handled by the cytosolic Cu,ZnSOD and mitochondrial MnSOD. However, under pathological conditions, large amounts of superoxide and its metabolites are produced extracellularly by activated neutrophils and other phagocytic cells. In this case, one major site of oxidant attack is endothelial cell surfaces, where membrane perturbation leading to cell death (possibly through apoptosis or programmed cell death) may be induced. Therefore, it seems clear that the plasma membranes of vascular endothelial cells and parenchymal cells should be protected when there is excessive production of these species. Surface-bound ECSOD-C normally protects endothelial cell surfaces from superoxide attack. However, proteases released by inflammatory cells may cleave the ECSOD “tail” allowing the enzyme to become soluble and rendering the endothelium susceptible to superoxide attack.
To protect the endothelium, most therapeutic efforts prior to the present invention have attempted to utilize the cytosolic Cu,ZnSOD, which unfortunately has undesirable pharmacological properties, including a short plasma half life following intravenous (i.v.) injection (6 to 15 minutes, depending on species), with rapid clearance by the kidneys, and a net negative charge at physiologic pH. This net negative charge precludes close contact with cellular surfaces and/or movement into interstitial spaces. In contrast, MnSOD is positively charged at physiologic pH and has a longer plasma half-life of about 4 hours. In an isolated perfused heart model, MnSOD equilibrates more quickly than the native Cu,ZnSOD and is more protective (Omar et al., 1991
, J. Mol. Cell Cardiol
., 23:149-159; incorporated by reference herein in its entirety). ECSOD, however, may have a substantial advantage over both Cu,ZnSOD and MnSOD because of its ability to bind to the endothelium. Recombinant ECSOD, however, has been produced only in a low-yield mammalian cell culture system (Tibell et al., 1987
, Proc. Natl. Acad. Sci. USA
, 84:6634-6638). All attempts to express this protein in a bacterial high-expression system have failed.
Moreover, while recombinant human ECSOD was found to be cardioprotective in the isolated rat heart subjected to ischemia and reperfusion, the effects noted by Sjöquist et al. “were essentially the same as those observed in hearts perfused with bovine Cu,ZnSOD” (Sjöquist et al., 1991
, J. Cardiovasc. Pharmacol
. 17:678-683), although Pisarenko et al. found ECSOD somewhat more than twice as effective as bovine Cu,ZnSOD (Pisarenko et al., 1994
, J.Cardiovasc.Pharmacol
. 24:655-663). In a similar study that used a spin-trapping agent to assess free radical production, the ECSOD was found to reduce free radical concentrations “at least to the same extent as Cu—Zn superoxide dismutase” (Johansson et al., 1990
, Cardiovasc.Res
. 24:500-503). Abrahamsson et al. (1992, Circ.Res. 70:264-271) used pyrogallol to generate superoxide radicals in a bath containing rabbit aorta rings. The free radical exposure inhibited the rings' ability to relax when acetylcholine was added to the bath, and this effect could be blocked by SOD added to the bath. Surprisingly, ECSOD produced a dose-response curve identical to that seen with Cu,ZnSOD. However, when the rings were pretreated with SOD, then washed, and then exposed to the radical-generating systeir. followed by acetylcholine, ECSOD showed its ability to “stick” and provide a degree of protection even after washing. The protection provided by Cu,ZnSOD was effectively all washed away. All in all, ECSOD has been rather disappointing in its ability to protect tissues from radical-mediated injury, being only marginally better than Cu,ZnSOD.
Inoue et al. (1990
, FEBS Lett
. 269:89-92) have attempted to improve the properties of Cu,ZnSOD by genetic engineering. They created a fusion gene consisting essentially of the cDNA encoding human Cu,ZnSOD fused to a synthetic oligonucleotide region that adds C-terminal amino acids of human ECSOD. This mutant is called HB-SOD, because it has heparin-binding properties similar to ECSOD. Functionally, the HB-SOD is superior to native Cu,ZnSOD in some models, but not in others. In the spontaneously hypertensive rat HB-SOD lowered the blood pressure, whereas native Cu,ZnSOD at the same dosage had no effect at all (Nakazono et al., 1991
, Proc. Natl. Acad. Sci. USA
88:10045-10048). In carrageenan-induced foot edema in the rat, however, HB-SOD and native Cu,ZnSOD had the same relative effect (Oyanagui et al., 1991
, Biochem.Pharmacol
. 42:991-995).
Therefore, although much work in the past three decades has attempted to utilize SOD as a therapeutic agent, with most of the effort being focused on the cytosolic SODs, while there have been successes under laboratory conditions where dosing and clearance may be carefully controlled, very little of this success has found its way into clinical application where the picture becomes more complex. Moreover, many of the recombinant SOD agents have been difficult to produce in useful quantities. Thus, there remains a need in the a
Flores Sonia C.
Gao Bifeng
McCord Joe M.
Monshipouri M
Prouty Rebecca E.
Sheridan & Ross P.C.
Webb-Waring Institute for Biomedical Research
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