Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-11-22
2003-03-18
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S024000, C435S213000, C514S002600, C514S018700, C514S019300, C514S012200, C530S330000, C530S331000, C530S350000, C560S034000, C560S035000, C560S036000
Reexamination Certificate
active
06534283
ABSTRACT:
FIELD OF THE INVENTION
The present invention is a method for the prevention and treatment physiologic shock involving the inhibition or removal of proteases in the small intestine and in circulation to prevent the generation of the mediators of shock. It is also a method for the identification of the proteases involved in shock to allow for the development of protease inhibitors for use in the treatment of shock.
BACKGROUND OF THE INVENTION
Shock is a life-threatening complication in situations associated with trauma including burns, surgery, ischemia, sepsis, and other critical care applications. Shock is a broad term that describes a group of circulatory syndromes, all of which result in general cellular hypoxia. This leads to a depletion of the adenosine triphosphate (ATP), the failure of the sodium-potassium pump, mitochondrial dysfunction, and ultimately the release of a variety of toxic substances, including superoxides. Superoxides are toxic to essentially all tissues. They react with proteins and cause unfolding and are able to induce DNA damage. Additionally, cellular activation in the circulation can be detected by leukocytes or endothelial cells resulting in superoxide production, pseudopod projections, enzyme release, cytokine release, and expression of membrane adhesion molecules. Cell activation fundamentally alters the biomechanics of microvascular blood flow by a shift in rheological, adhesive, and cytotoxic cell properties. Eventually these stress responses give rise to irreversible cardiovascular collapse because of their combined effects on the microcirculation.
The interaction between activated leukocytes, both neutrophils and monocytes, and endothelial cells leads to accumulation of leukocytes in various organs, leading to cytotoxicity and cell death. Although such processes are mediated by humoral activators in the plasma of systemic circulation, an inflammation in organs throughout the body may eventually lead to multi-organ failure. When leukocytes are activated, neutrophil pseudopod formation is upregulated and several membrane adhesion molecules are expressed. This process lowers cell deformability and leads to accumulation of neutrophils in microcirculation. Not only may leukocytes start inflammation, but the abnormal cellular entrapment in the microcirculation also leads to immune suppression because of the reduced numbers of circulating cells.
Shock is a multifaceted systemic response to any of a number of stress inducing stimuli that results in cellular activation and release of a number of interacting response mediators, including cytokines, inflammatory and immune mediators, and nitric oxide (NO). During an immune response, oxygen free radicals and superoxides are generated to kill pathogens. However, oxygen free radicals and superoxides are also damaging to the host cells, resulting in oxidation of lipids, proteins and nucleic acids. The mediators of shock orchestrate complex biological interactions and amplification of signals that result in a systemic response to a localized insult.
Due to the multifaceted nature of factors inducing shock, development of therapeutics has been difficult. Most therapies have focused on the modulation of a single factor (e.g. cytokines, NO, endotoxin) to mitigate the effects of shock. Unfortunately, inhibition of any one of these pleiotropic factors is ineffective. Organ specific therapies can support life, but are not an ideal option as they often sacrifice remote organ function.
One potential therapeutic molecule that has been suggested for use in shock is bacterial/permeability-increasing protein (BPI), a protein involved in the immune response (Ammons, U.S. Pat. No. 6,017,881). Intestinal ischemia, frequently associated with shock, results in the breakdown of the intestinal mucosal permeability barrier allowing for the translocation of bacteria and/or endotoxin from the intestinal lumen to the vascular system. During shock, endotoxin has been detected in the portal vein, but its role in shock has not been clearly defined. BPI is a protein isolated from granules of mammalian polymorphonuclear cells (PMNs). PMNs are blood cells involved in the defense of the body against invading microorganisms. BPI is highly specific for gram negative bacteria and seems to have no deleterious effects on other pathogens or host cells. Administration of BPI to rats results in a decrease in the adverse physiological effects of intestinal ischemia which may catalyze the other symptoms of shock. However BPI only effects one of the pathways that are activated in shock, so it is of limited use. Additionally, BPI acts by attacking the endotoxin and bacteria afterthey have been released from the intestine into the bloodstream; therefore, it can not be used to prevent the occurrence shock.
There are no satisfactory drugs, treatment methods, or interventions available for the prevention of shock. All currently available methods for the treatment of shock deal with the symptoms, rather than the cause. For this reason, current clinical approaches are limited in their efficacy and can only prevent further damage from occurring.
SUMMARY OF THE INVENTION
Until the initiators, rather than the downstream mediators, of shock are identified, it will not be possible to develop satisfactory methods to prevent or treat shock. The present invention is the discovery of a role for proteases, from the pancreas, circulating cells, and other tissues, as the initiators of shock. During a normal immune response, proteases are generated by a number of circulating cells with no systemic ill effects. However during shock, there is a n overactivation of the immune system resulting in an overproduction of proteases. Similarly under normal circumstances after eating, pancreatic digestive enzymes are released into the small intestine after eating with no adverse effects. However, during shock, the intestinal permeability barrier of the small intestine is compromised, revealing protease susceptible sites not present under normal conditions. This “self-digestion” process produces a variety of protease digestion products that are responsible for shock. Such digestion products may include lipid, carbohydrate or other components from post-translational modifications. Therefore, shock is most effectively treated by the inhibition or elimination of the proteases that generate the activators of shock. Ideally by preventing the activation of the proteases by inhibiting the conversion of the proenzyme to the enzyme.
The present invention is also a method for the prevention and treatment of shock involving the inhibition or elimination of proteases present in the lumen of the small intestine and in circulation during shock. Protease inhibition can be achieved prophylactically by administration of protease inhibitors to the subject before a known potential shock inducing event (e.g. cardiac surgery). Altematively, in trauma situations, protease inhibitors can be introduced directly into the small intestine by lavage, inserted either endoscopically or directly into the intestine, to flush the proteases out of the intestine. Additionally, protease inhibitors could be administered intravenously in both situations to inhibit proteases in circulating cells and throughout the body. Any of a number of protease inhibitors could be used for the purpose of inhibiting or treating shock including any of the known plasma protease inhibitors (e.g. anti-trypsin, anti-chymotripsin, C1 inhibitor, antithrombin III, alpha-2-macroglobulin), non-toxic amino acid/peptide substrate analog inhibitors, and non-amino/peptide chemical analogs that bind to the active site of the protease and block function. Inhibitor selection is based on the desired route of administration, desired pharmacokinetic properties, interaction with other patient medications, and other issues known to one skilled in the art.
The present invention is also a method for identifying the proteases that are involved in shock to allow for the development of protease inhibitors that are specific to those activated in shock. An idea
Hugli Tony
Mitsuoka Hiroshi
Schmid-Schoenbein Geert
Brown Martin Haller & McClain LLP
Carlson Karen Cochrane
Kam Chih-Min
The Regents of the University of California
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