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Use of gelsolin to treat infections

Use of gelsolin to treat infections description/claims

This application is a divisional application of U.S. non-provisional Application No. 11/129,670, filed May 12, 2005, currently pending, which claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 60/570,233, filed May 12, 2004, the entire contents of both of which are incorporated herein by reference.

The invention is directed to the use of gelsolin to treat infections and sepsis and to the use of gelsolin to monitor and evaluate treatments of infections. The invention also provides methods for up-regulating interleukin expression and methods for down-regulating pro-inflammatory cytokine expression.

Despite significant advances in diagnosis and therapy, infections and sepsis remain a major cause of morbidity and mortality throughout the world. Even with aggressive management, many patients with severe infections develop complications and some die. Sepsis claims more than 200,000 lives in the United States annually (Angus, D. C. & Wax, R. S. (2001) Crit Care Med 29, S109-16). An intense inflammatory response accompanies the early phase of sepsis with markedly increased plasma levels of pro-inflammatory cytokines (Riedemann, N. C., Guo, R. F. & Ward, P. A. (2003) Nat Med 29, 517-24), in addition to other biochemical abnormalities. Much concerted research has gone into attempts at inhibition of specific inflammatory mediators in hope of developing pharmacologic treatments for sepsis. Nevertheless, activated protein C (APC) is the only drug proven to reduce the mortality of severe sepsis with an absolute reduction of death by 6% (Bernard, G. R., Vincent, J. L., Laterre, P. F., LaRosa, S. P., Dhainaut, J. F., Lopez-Rodriguez, A., Steingrub, J. S., Garber, G. E., Helterbrand, J. D., Ely, E. W. & Fisher, C. J., Jr. (2001) N Engl J Med 344, 699-709).

The negative health effects of infections and sepsis provide a strong incentive to identify new treatments as well as improved tests and approaches to evaluate therapy of infections and sepsis.

This invention is based on the surprising discovery that gelsolin treats infections and protects against the toxic manifestations of infections. We have found that mice injected with gelsolin subsequent to the exposure to an infectious agent unexpectedly survived better than mice given saline. Thus, the invention involves in one aspect, the administration of gelsolin to a subject to treat an infection. The invention is also directed to methods of using gelsolin to treat the biological effects of infections.

Gelsolin (GSN), specifically cytoplasmic gelsolin (cGSN), first discovered as an intracellular actin-binding protein involved in cell motility (Yin, H. L. & Stossel, T. P. (1979) Nature 281, 583-6) is also an abundant secretory protein (Yin, H. L., Kwiatkowski, D. J., Mole, J. E. & Cole, F. S. (1984) J Biol Chem 259, 5271-6. The exported isoform of gelsolin, designated plasma gelsolin (pGSN), has 25 additional amino acids and originates from alternative splicing of a single gene (Kwiatkowski, D. J., Stossel, T. P., Orkin, S. H., Mole, J. E., Colten, H. R. & Yin, H. L. (1986) Nature 323, 455-8).

In each of the following aspects and embodiments of the invention, the use of pGSN is preferred.

According to one aspect of the invention, methods of treating an infection in a subject are provided. The methods include administering gelsolin to the subject having or at risk of having an infection in an effective amount to treat the infection. The infection may be caused by a gram-positive bacterium, an acid-fast bacillus, a spirochete, an actinomycete, a virus, a fungus, a parasite, Ureaplasma species including Ureaplasma urealyticum, Mycoplasma species including Mycoplasma pneumonia, Rickettsia species, Chlamydia species including Chlamydia psittaci, Chlamydia trachomatis and Chlamydia pneumoniae, and Pneumocystis species including Pneumocystis carinii. In some embodiments, the subject is otherwise free of indications calling for treatment with gelsolin. In some embodiments, the gelsolin is administered subsequent to exposure of the subject to the infection. In certain embodiments, the gelsolin is administered at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours subsequent to exposure of the subject to the infection. In some embodiments, the gelsolin is administered at least about 1, 2, 3, 4, 5, 6, 7, or more days subsequent to exposure of the subject to the infection.

According to yet another aspect of the invention, methods of treating a gram-negative bacterial infection in a subject are provided. The methods include administering gelsolin to the subject in an effective amount at a time subsequent to the exposure of the subject to a gram-negative bacterial infection. In some embodiments, the subject is otherwise free of indications calling for treatment with gelsolin. In some embodiments, the gelsolin is administered at least about one hour subsequent to exposure of the subject to the gram-negative bacterial infection. In certain embodiments, the gelsolin is administered at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours subsequent to after exposure of the subject to the gram-negative bacterial infection. In some embodiments, the gelsolin is administered at least about 1, 2, 3, 4, 5, 6, 7, or more days subsequent to exposure of the subject to the gram-negative bacterial infection. In some embodiments, gelsolin is administered prior to exposure of the subject to the gram-negative bacterial infection.

According to another aspect of the invention, methods of treating or preventing the effects of lipopolysaccharide endotoxin (LPS) in a subject are provided. The methods include administering to the subject an effective amount of gelsolin at a time subsequent to LPS exposure to protect the subject against the effects of LPS. In some embodiments, the subject is otherwise free of indications calling for treatment with gelsolin. In some embodiments, the gelsolin is administered at least about one hour subsequent to the exposure of the subject to the LPS. In certain embodiments, the gelsolin is administered at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 or more hours subsequent to exposure of the subject to LPS. In some embodiments, the gelsolin is administered at least about 1, 2, 3, 4, 5, 6, 7, or more days subsequent to exposure of the subject to LPS.

According to another aspect of the invention, methods of treating or preventing gram-negative bacterial septic shock in a subject exposed to LPS are provided. The methods include administering to the subject an effective amount of gelsolin at a time subsequent to LPS exposure to treat the gram-negative bacterial septic shock in the subject. In some embodiments, the subject is free of indications calling for treatment with gelsolin. In some embodiments, the gelsolin is administered at least about one hour subsequent to exposure of the subject to LPS. In certain embodiments, the gelsolin is administered at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours subsequent to exposure of the subject to LPS. In some embodiments, the gelsolin is administered at least about 1, 2, 3, 4, 5, 6, 7, or more days subsequent to exposure of the subject to LPS.

According to another aspect of the invention, a method for up-regulating interleukin (IL) expression in a subject is provided. The method involves administering gelsolin to the subject in an effective amount to up-regulate IL expression in the subject. In some embodiments, the subject is free of indications calling for treatment with gelsolin. Up-regulation of IL expression may be either due to increased expression of the IL or due to decreased degradation of the IL or a combination of an increased expression of the IL and a decreased degradation of the IL.

In some embodiments, the expression of IL is increased by at least approximately 25% relative to control. In other embodiments, the expression of IL is increased by at least approximately 50% or 75% relative to control. In some embodiments, the expression of IL is increased by at least approximately 2-fold relative to control. In some embodiments, the IL expression is increased at least approximately 3-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold relative to control. In general, the IL expression level in a control is the level of IL expression in a subject to whom gelsolin was not administered but is otherwise identical to the treated subject. Methods of measuring IL levels are known to those of ordinary skill in the art.

According to another aspect of the invention, a method for up-regulating IL expression in vitro is provided. The method involves contacting a cell capable of expressing IL with a sufficient amount of gelsolin to up-regulate the level of IL expression in the cell. In some embodiments, the expression of IL is increased by at least approximately 25% relative to control. In other embodiments, the expression of IL is increased by at least approximately 50% or 75% relative to control. In some embodiments, the expression of IL is increased by at least approximately 2-fold relative to control. In general, the level of IL expression in a control is that level of expression in a cell that is not contacted with gelsolin but is otherwise identically treated to the cell contacted with gelsolin. In some embodiments, the IL expression is increased at least approximately 3-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold relative to control. Methods of measuring IL levels are known to those of ordinary skill in the art.

The time period in which the IL expression is increased is, at least in part, a function of the cell type and on the specific culture vessel used. In general, this time period ranges from 2-3 hours (for short-term increase) to about 2-3 days (for medium-term increase) to several weeks (for long-term increase). Routine procedures known to those of ordinary skill in the art can be used to determine the level of IL expression as a function increasing doses of gelsolin or increasing incubation time or of the cells with gelsolin. One preferred IL is IL-10.

According to another aspect of the invention, a method for down-regulating pro-inflammatory cytokine expression in a subject is provided. The method involves administering to the subject gelsolin in an effective amount to down-regulate pro-inflammatory cytokine expression in the subject. In some embodiments, the subject is otherwise free of indications calling for treatment with gelsolin. Down-regulation of pro-inflammatory cytokine expression may be either due to a decreased expression of the cytokine or due to an increased degradation of the cytokine or a combination of a decreased expression of the cytokine and an increased degradation of the cytokine. In general, the expression of pro-inflammatory cytokine is decreased by at least approximately 10% relative to control. In some embodiments, the expression of pro-inflammatory cytokine is decreased by at least approximately 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% relative to control. In some embodiments the expression of pro-inflammatory cytokine is decreased by 100% relative to control. In general, the pro-inflammatory cytokine expression level in a control is the level of pro-inflammatory cytokine expression in a subject to whom gelsolin was not administered but is otherwise identical to the treated subject. Methods of measuring pro-inflammatory cytokine levels are known to those of ordinary skill in the art. One preferred pro-inflammatory cytokine is IL-1β. Another preferred pro-inflammatory cytokine is IFN-α.

According to one aspect of the invention, a method for down-regulating expression of pro-inflammatory cytokines in vitro is provided. The method involves contacting a cell capable of expressing a pro-inflammatory cytokine with a sufficient amount of gelsolin to down-regulate the level of pro-inflammatory cytokine expression in the cell. In general, the expression of a pro-inflammatory cytokine is decreased by at least approximately 10% relative to control. In general, the level of a pro-inflammatory cytokine expression in a control is that level of expression in a cell that is not contacted with gelsolin but is otherwise identically treated to the cell contacted with gelsolin. In some embodiments, the pro-inflammatory cytokine expression is decreased at least approximately 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% relative to control. In some embodiments the expression of pro-inflammatory cytokine is decreased by 100% relative to control. Methods of measuring pro-inflammatory cytokine levels are known to those of ordinary skill in the art.

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