1. Field of the Invention
This invention relates to agents and therapy to lessen morbidity and mortality by protecting against septic shock, Adult Respiratory Distress Syndrome (ARDS), and other inflammatory complications of shock. Particularly, this invention relates to the treatment of septic shock and the other complications resulting from septic shock by down-regulating the expression of certain cell-cell adhesion receptors or ligands involved in the inflammatory response during septic shock. More specifically, this invention relates to therapy with antisense oligonucleotides which reduce expression of adhesive proteins and protect against septic shock and reduce associated inflammatory damage (like ARDS). Particularly this invention relates to the use of antisense oligonucleotides complementary to human mRNAs or pre-mRNAs coding for VCAM-I to be used in a therapeutic treatment of sepsis (henceforth to include sepsis, the sepsis syndrome, septic shock and all other manifestations of the sepsis disease, including but not inclusive of, adult respiratory distress syndrome, multi-organ failure, or cardiovascular dysfunction). Mediators of sepsis produce endothelial dysfunctions that result in the development of an intravascular inflammatory response and subsequent damage to the endothelial cells with migration of leukocytes into the surrounding tissues. This invention also relates to the treatment of sepsis with antisense oligonucleotides targeted to cellular based receptors or their ligands where these receptors or ligands are involved in the inflammatory response during the development of sepsis. This invention further relates to the inhibition of the synthesis of VCAM-I with antisense oligonucleotides.
2. Description of the Prior Art
Septic shock is defined as a type of shock associated with overwhelming infection. Most commonly, the infection is produced by gram-negative bacteria although other bacteria, viruses, fungi and protozoa may also be causes. As summarized in Infectious Diseases and Medical Microbiology, 2nd edition, edited by Braude et al., Chapter 92, pages 700 et seq.
"Shock is a syndrome of generalized metabolic failure resulting from prolonged inadequacy of tissue perfusion. Its early clinical manifestations reflect malfunction of those organs most dependent on uninterrupted blood flow, particularly the brain, as well as compensatory adjustments designed to maintain adequate arterial pressure. As these adjustments fail, urinary output decreases and biochemical indices of distorted metabolism are detectable; specifically nonoxidative glycolysis with low yield of high energy chemical bonds testifies to the widespread nature of the disorder. In the end, it is the failure of energy production rather than damage to a particular organ that leads to death.
Other terms, such as `circulatory collapse,` `circulatory failure,` and `hypoperfusion,` have been substituted for `shock` in an attempt to pinpoint the specific nature of the derangement. When it occurs as a specific complication of infection, it is referred to as `infectious shock,` `septic shock,` `bacteremic shock,` and even `endotoxin shock.` The last three terms specifically implicate bacterial infection and are therefore too restrictive. Because `infectious shock` is sufficiently broad as well as concise, this term will be used in the present chapter.
Shock may occur in the course of almost any severe infection, but it is particularly characteristic of bacteremia due to gram-negative bacilli. The importance of endotoxin, the lipopolysaccharide (LPS) composing part of all gram-negative cell walls, is readily apparent because it produces a similar syndrome in experimental animals. Partly because of the extensive use of endotoxin as an investigative tool, endotoxin shock is commonly regarded as the prototype of infectious shock." The shock is believed to be caused by the action of endotoxins, other products of the infectious agent, or host mediators released in response to the infectious agent on the vascular system. Such action causes altered patterns of perfusion of tissues and large volumes of blood to be sequestered in the capillaries and veins.
Sepsis, the sepsis syndrome, and septic shock are not discrete entities, but rather terms that delineate increasingly severe stages of the same disease. Septic shock, a frequently fatal reaction following bacterial infection, has been estimated to occur at a rate of 175 per 100,000 people yearly in the general population and rises to 500 per 100,000 for those people admitted to hospitals (Johnston, J. (1991) J. NIH 3: 61-65). Estimates range up to 400,000 cases of sepsis, 200,000 bouts of septic shock, and 100,000 deaths annually in the United States due to the septic shock induced syndrome (Snell, J. and J. E. Parrillo (1991) Chest 99: 1000-1009). Up to 40-50% of patients who develop septic shock die. The manifestation of septic shock involves a severe decrease in systemic vascular resistance and maldistribution of blood flow. Septicemia, a systemic disease associated with the presence and persistence of pathogenic microorganisms or their toxins in the blood, is currently ranked as the thirteenth leading cause of death in the United States (Annual Summary of Births, Marriages, Divorces, and Deaths: United States, 1988. Hyattsville, Md.: U.S. Department Health and Human Services, Public Health Service, CDC, 1989: 7. Monthly vital statistics report. 1989: 37[13]). Reasons underlying this high incidence of death from septic shock involve increased usage of cytotoxic and immunosuppressive drug therapies which impairs host defense mechanisms or increased use of invasive diagnostic devices or increased patient age (Snell, J. and J. E. Parrillo (1991) Chest 99: 1000-1009). Further causes of impaired host defense mechanisms include diabetes, malignant neoplasms, cirrhosis or extensive burns. The rising rate of infections from organisms other than gram-negative bacteria also contribute to the rise in septic shock induced death. Any bacteria can cause septic shock, however, the gram-negative bacteria (E. coli, Pseudomonas sp. and Bacteroides sp.) in particular evoke septic shock due to the presence of lipopolysaccharide (LPS) in their cell walls. Bacterial LPS, also known as endotoxin, at concentrations as low as a few .mu.g/L can activate immune cells. The majority of damage induced from the presence of LPS is not due to the actual LPS itself, but is in fact a result of the body's complex reaction to the foreign LPS. This response is mediated by immune cell activation and the resultant damage that these activated cells cause to the host tissues.
Septicemia is difficult to reverse and the preferred treatment following the initial signs of hypoperfusion or shock include infusion of normal saline or lactated Ringer's solution. If shock persists then an aggressive fluid challenge is begun and the use of dopamine and/or norepinephrine is recommended. Cardiovascular insufficiency results from alterations to the myocardium and the vasculature and it is myocardial dysfunction that is responsible for hypotension or multiple organ system failure (Snell, J. and J. E. Parrillo (1991). Chest 99: 1000-1009). Unresponsive hypotension usually results from low systemic vascular resistance due to cardiovascular insufficiency which can not be corrected by any therapy. Multiple organ failure usually affects the lungs, kidneys, liver, heart, and central nervous system.
Treatment of septic shock is complex, requiring therapies directed at ameliorating the source of infection [antibiotics], blocking effects of products of the infectious agent and inflammatory mediators on tissues [anti-endotoxin (patent Young et al. U.S. Pat. No. 4,918,163) and anti-cytokine agents (patent Mandell et al. U.S. Pat. No.4,965,271)], and maintenance of cardiovascular function [volume expansion and pressor agents]. However, mortality still runs at about 100,000 patients per year (40 to 50% of those in shock) and no therapies are available to prevent vascular contractile defects.
Other current approaches to the treatment of sepsis or septic shock involve neutralization of LPS with specific monoclonal antibodies, interference of cytokine-mediated immune responses, or inactivation of cell adhesion proteins with monoclonal antibodies. Targeting of LPS mediated sepsis, however, will be effective only against gram-negative bacteria since LPS is only found in their cell walls. Monoclonal antibodies to the lipid A domain of LPS have had some success at intervention with LPS mediated septic shock from gram-negative bacteria, but not for non-gram-negative induced septic shock (Ziegler, E. J. et al. (1991) N. Eng. J. Med. 324: 429-436). Thus, while the gram-negative LPS may be the most potent inducer of sepsis, gram-positive bacterial infections occur in 60-70% of all cases. Intervention with cytokine mediated activation of the immune response as a means of preventing septic shock would not only interfere with gram-negative induced sepsis, but also shock caused by gram-positive bacterial infection or other agents. The development of an effective therapy to treat all bacterial induced septic shock would be of obvious benefit to patients who are at an increased risk of bacterial induced sepsis and provide increased survival from septic shock and the complications that arise during septic shock induced dysfunctions. Another approach would be interference with the cellular response to the various endogenous mediators (cytokines, PAF, arachidonic acid metabolites, histamine, endorphins, etc) responsible for vasculature effects. These approaches are not currently approved for therapy and are in clinical trials.
One of the major effects experienced by the vasculature is destruction of endothelial cells by leukocytes. Inflammation is characterized by the local accumulation of leukocytes, plasma proteins, and fluid usually at an extravascular site. Inflammatory processes are intrinsically destructive to the surrounding tissues and may, in certain circumstances such as allograft rejection or sepsis, be more harmful than beneficial. Thus, an appropriate strategy for the treatment or prevention of sepsis or septic shock would be down-regulation, but not total ablation, of the inflammatory response. Down-regulation of specific cell adhesion receptors and/or ligands to the receptors would be one approach to preventing, or lessening, the inflammatory mediated damage to endothelial cells in the vasculature.
The involvement of the immune response in the development of septic shock and its lethal consequences provides a target that is applicable to the use of antisense oligonucleotides. Antisense oligonucleotides can be used to inhibit expression of the key receptors and cellular ligands involved in the activation of the immune response. The migration of leukocytes into tissues is the central event in the immune or inflammation response. This migration to and subsequent emigration into the tissue is responsible for the successful host response to injury and infection. The leukocytes are also potentially harmful and contribute to the pathology of many inflammatory disorders. The precise mechanism of this injury is not known, but the generation of free oxygen radicals and release of proteolytic enzymes have been implicated and may act together in leukocyte induced endothelial cell damage (Varani, J. et al. (1989), Am. J. Path. 135: 429-436). Evidence for the leukocyte adhesion to endothelial cells has been attributed to specific surface proteins.
There are many lines of evidence that indicate that inflammatory reactions are modulated by the interaction of circulating leukocytes with adhesion molecules on the endothelial cells of the luminal surface of blood vessels. These vascular adhesion molecules arrest circulating leukocytes and, thus, perform the first step in recruitment of these cells to sites of inflammation. Two cytokine inducible adhesion molecules, ICAM-1 and ELAM-1, found on the surface of the leukocytes have been characterized as important to the recruitment of circulating leukocytes to the sites of inflammation (Simmons, D. et al. (1988) Nature 331: 624-627; Staunton, D. E. et al. (1988) Cell 52: 925-933; Staunton, D. E. et al. (1989) Nature 339: 61-64). However, neither ICAM-1 or ELAM-1 appear to be involved in the recruitment or adhesion of lymphocytes, B- or T-cells, to activated endothelial cells. ELAM-1 is selective for PMNs, and perhaps monocytes, but does not bind lymphocytes (Bevilacqua, M. P. (1987) Proc. Natl. Acad. Sci. USA 84: 9238-9242; Bevilacqua, M. P. (1989) Science 243: 1160-1165). ICAM-1 is the ligand for the leukocyte integrin receptor molecule, LFA-1, however, antibodies against LFA-1 do not block the adhesion of lymphocytic cells to activated endothelial cells (Haskard, D. et al. (1986) J. Immunol. 137: 2901-2906). The above results suggest another pathway for lymphocyte adhesion to activated endothelium.
A prominent feature of endothelial cell activation by cytokines or endotoxin is the alteration of their surface adhesive properties due to the induction of adhesion molecules expression (Pober, J. S. and R. S. Cotran (1990) Physiol. Rev. 70: 427-451). The induction of these molecules leads to the hyperadhesive surface changes observed in vivo during various physiological conditions. Vascular cell adhesion molecule 1 (VCAM-1) was identified in activated human umbilical endothelial cells by expression cloning (Osborne, L. et al. (1989) Cell 59: 1203-1211) and specific monoclonal antibodies (Rice, G. E. and M. P. Bevilacqua (1989) Science 246: 1303-1306). VCAM-1 is an inducible endothelial cell surface molecule which has been demonstrated to mediate intercellular adhesion via interaction with the integrin VLA-4, which is expressed on monocytes, lymphocytes, basophils, eosinophils, and certain tumor cells, but not neutrophils (Elices, M. J. et al. (1990) Cell 60: 577-584; Taichman, D. B. et al. (1991) Cell Regul. 2: 347-356; Bochner, B. S. et al. (1991) J. Exp. Med. 173: 1553-1556; Rice, G. E. et al. (1990) J. Exp. Med. 171: 1369-1374). VCAM-1 expression is inducible on vascular endothelium in pathological conditions, however, it is constitutively expressed on some nonvascular cells (Rice, G. E. et al. (1990) J. Exp. Med. 171: 1369-1374; Rice, G. E. et al. (1991) AM. J. Pathol. 138: 385-393). In the inflammatory process, VCAM-1 is upregulated at the level of translation on endothelial cells of the postcapillary venules (Briscoe, D. M. et al. (1991) Transplantation 51: 537-547).
VCAM-1 is a transmembrane protein and a member of the immunoglobin gene superfamily. Contained within the VCAM-1 molecule are six immunoglobin-like domains which interact with the VLA-4 receptor on lymphocytes. Several lines of evidence are consistent with an important role for VCAM-1 in lymphocyte recruitment in the inflammatory process: 1) VCAM-1 expression is rapidly induced by the cytokines interleukin-1 and tumor necrosis factor alpha and this induction is sustained for up to 72 hours. This time course of VCAM-1 induction parallels the sustained mononuclear lymphocytic infiltration that occurs in delayed hypertension reactions (Dvorak, H. F. et al. (1980) Int. Rev. Exp. Pathol. 21: 195-199). 2) Human umbilical vein endothelial cells (HUVEC) express ICAM-1 and ELAM-1 upon exposure to cytokines and their expression occurs at sites of cytokine injection in vivo (Cotran, R. S. and Pobar, J. S. (1988) In Endothelial Cell Biology, N. Simionescu and M. Simionescu, eds.(New York: Plenum Press), pp. 335-347). HUVEC also express VCAM-1 upon exposure to cytokines (Osborne, L. et al. (1989) Cell 59: 1203-1211). 3) Frozen sections of human synovium exhibit the capacity for binding of lymphocytes to inflamed vessels and not normal vessels, consistent with the presence of inducible VCAM-1 at these sites. Taken together, these results suggests that VCAM-1 may be the central mediator of lymphocyte recruitment to the sites of inflammation in vivo. While the role of VCAM-1 in the adherence of resting T-cells to interleukin-1 stimulated endothelial cells has been suggested, there is little evidence that VCAM-1 is involved in the binding of either activated T-cells or in the transendothelial migration of T-cells (Oppenheimer-Marks, N. et al. (1991) J. Immunol. 147: 2913-2921). Also, VCAM-1 does not appear to play a vital role in the adherence of activated T-cells to endothelial cells.
Based upon the above information, it may be possible to inhibit the binding of resting T-cells to either unstimulated or activated endothelium by disruption of the recognition that occurs between VCAM-1 and VLA-4. The use of monoclonal antibodies against either VLA-4 and/or VCAM-1 can be useful in preventing the recognition of the receptor/ligand pair for each other. Another possible approach for interfering with the recognition process is via a down-regulation of VCAM-1 synthesis and, thereby, a reduction in the presentation of this molecule to the surface of activated endothelial cells. Antisense oligonucleotides provide an attractive approach for inhibiting the presentation of VCAM-1 molecules to the surface of endothelial cells.
Involved in the activation of the inflammatory and immune response, as during the development of sepsis and septic shock, is the expression of many essential cell adhesion proteins and receptors. Adhesion molecules are activated by various cellular mediators, exogenous or endogenous to the host, and therefore, the logical approach is down-regulation of adhesion protein expression as opposed to treatments aimed at the multiple activators. Thus, the use of antisense oligonucleotides to specifically down-regulate adhesion protein expression would be of obvious advantage to most therapeutic approaches to septic shock.
Research by others into PKC inhibition and treatment of inflammatory responses have disclosed that endothelial cells express adhesive proteins in response to sepsis associated stimuli such as endotoxin or cytokines, such as interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF). Magnuson, D. K. et al. ((1989) Surgery 106: 216-223) and Lane, T. A. et al. ((1990) Biophys. Res. Comm. 172: 1273-1281) have shown that these adhesive proteins can be reduced on endothelial cell surfaces by inhibition of PKC with staurosporine or 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H7). Surface presentation of these adhesive proteins enhances white blood cell infiltration and activation which can result in tissue damage in inflammatory states like septic shock. In addition, PKC activation enhances endothelial cell permeability resulting in edema. This response to inflammatory agents was also abrogated by exposure of the cells to the PKC inhibitor H7 (Lynch, J. J. et al. (1990) J. Clin. Invest. 85: 1991-1998). Abnormal leukocyte accumulation is implicated in a variety of inflammatory states such as: reperfusion injury, autoimmune diseases, and acute respiratory distress syndrome (ARDS). The damage is thought to result from the release of toxic oxygen radicals and proteases that potentiate tissue damage. The use of anti-adhesive protein antibodies or adhesive like proteins was shown to reduce tissue damage in select models of reperfusion injury (Vealder, N. B. et al. (1988), J. Clin. Inv. 81: 939-944; Simpson, P. J. et al. (1988) J. Clin. Inv. 81: 624-629; Horgan, M. J. et al. (1989) Am. J. Physiol. 259: L315-L319; International patent application of Vadas, M., and M. Berndt (1991) Application #WO 91/07993) and endotoxin induced damage (Rosen, H. and S. Gordon (1989) Br. J. Exp. Path 70: 385-394). When antibodies are used as a treatment they do not control the levels of expression of these proteins and the antibodies typically have short half-lives in circulation. An additional complication of antibodies is the potential for immunogenic reactions to large foreign proteins. Compared to antibodies, smaller molecules like antisense oligonucleotides can overcome these disadvantages and also provide selective control of expression of a single cellular protein.
The mRNA coding for VCAM-I has been cloned and the nucleic acid sequence is available for selective targeting with antisense oligonucleotides (Osborn, L. (1989) Cell 59: 1203-1211).