Antisense compounds to CD14

Details Antisense compounds to CD14 Antisense compounds to CD14

1998-11-06

2001-06-26

Wang, Andrew (Department: 1635)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C435S006120, C536S023100, C536S024300, C536S024500

Reexamination Certificate

active

06251873

ABSTRACT:

TECHNICAL FIELD
The present invention relates to an oligonucleotide comprising a sequence complementary to a part of a gene encoding human CD14. Further, it relates to a pharmaceutical composition comprising said nucleotide and a pharmacologically acceptable carrier.
BACKGROUND TECHNOLOGY
500,000 people in the United States suffer from sepsis caused by bacterial infection and 175,000 people die. The disease is highly lethal and effective therapeutic method is not established (Science, Volume 264, page 365, 1994). The cause has been considered to be a direct effect of lipopolysuccharide (hereinafter designated as “LPS”, which is almost synonymic for endotoxin). In 1985, Beutler et al. reported that anti-TNF antibody-administered mouse exhibits resistance to a lethal amount of endotoxin (Science, Volume 229, page 869, 1995). In the meanwhile, Tracy et al. discovered that endotoxin-analogous shock and organic impairment occur in recombinant TNF&agr;-administered animal (Science, Volume 234, page 470, 1996), whereby it was found that the septic shock is caused not by direct effect of LPS, but by excess cytokine production from a macrophage activated by stimulation of LPS, namely hyper-cytokinemia. This discovery was an opportunity to try a therapeutic method targeting TNF&agr; produced in an excess amount by stimulation of LPS. However, the clinical test targeting the TNF&agr; conducted in the beginning of 1990's ended up with disappointing results, wherein good result was not obtained in indexes, e.g. a survival rate of 28 days after (Nature Medicine, Volume 3, page 1193, 1997).
At present, antibiotics are employed for the purpose preventing bacterial infection at present, whereas it is reported that these antibiotics destroy bacterial bodies and a large amount of LPS is released into blood (Scand. J. Infect. Dis., Volume 101, page 3, 1996). This means that the use of antibiotics may cause septic shock or endotoxic shock. Accordingly, in order to prevent the shock, it is important to block the stimulation of LPS simultaneously with the administration of antibiotics.
CD14 is a glycosyl phosphatidylinositol-linked glycoprotein with a molecular weight of 55 kd which is expressed with differentiation and maturation of bone marrow cell. Todd et al. reported the CD14 as surface antigen of human peripheral blood monocytes (New York, Springer-Verlag, pages 424 to 433, 1984). It has been clarified that CD14 is present on membrane of macrophage, monocyte, Kupffer cells, and neutrophil.
Goyert et al, reported the DNA sequence of human CD14 in 1988 (Nucleic Acid Research, Volume 16, No. 9, page 4173, 1988), and Yamamoto et al. reported the DNA sequence of mouse CD14 in 1988 (Somat. Cell Mol. Genet., Volume 14, page 427, 1988). It has been suggested that the CD14 gene is present in a gene cluster of fifth chromosome, where a hematopoietiesis differentiating proliferating factor group such as IL-3 or GM-CSF, G-CSF, etc. are present, and that CD14 gene is involved in the differentiation and maturation of hematopoietiesis tissue. However, detailed function thereof has been unknown.
In 1990, Wright et al. reported that the CD14 is a receptor of LPS of Gram-negative Bacillus endotoxin (Wright et al., Science, Volume 249, page 1431, 1990). Further, recent study discovered that the CD14 binds not only to LPS but also to proteoglycan (Gupta et al., J. Biol. Chem, Volume 271, No. 38, page 23310, 1996). It is also reported that the ingredients of Gram-negative bacteria and Gram-positive bacteria activate the cells through CD14 (Jerome et al., Immunity Volume 1, page 509, 1994). In other words, it is estimated that when organisms are infected with a bacteria, CD14 binds to the bacterial ingredients, whereby macrophage and monocyte expressing the CD14 are activated and various inflammatory factors (inflammatory cytokine, e.g. TNF&agr;, IL-1, IL-6, IL-8, PAI-2, MCP-1, etc., arachidonic metabolites, PAF and nitrogen monoxide, etc.) are released and induced, whereby it contributes to the prevention of the bacterial infection in the early phase of infection (Matthew et al., J. Biol. Chem., Volume 60, page 728, 1996). On the other hand, it is also estimated that under disease conditions, such as sepsis, activation of macrophage due to a large quantity of LPS from bacteria leads to release of a large amount of TNF&agr; into blood, and causes shock (Fearn. S et al., J. Exp. Med., Volume 181, page 857, 1995).
At present, the cytokine production mechanism by LPS via CD14 is estimated as described below. In short, aggregated LPS originated from bacterium together with LPS-binding protein (LBP) forms a complex in blood, and consequently, the LPS monomer becomes capable of efficiently binding to CD14 molecules on the macrophage in a proportion of 1:1. Signal of the LPS bound to the surface of the cells is transmitted into the cell through an unknown route analogous to ceramide; NF&kgr;B which is a transcription factor is activated in the cell; and the production of various cytokines including TNF&agr; is induced (Ulevith et al., Annual Review of Immunology, 13, 437, 1995). These facts indicate that primary response of the host in the case of bacterial infection initiates from the response of the CD14 on monocyte/macrophage to LPS or Gram-positive bacterium ingredients.
By the way, there are two forms of the CD14 molecule, i.e. membrane-binding form and soluble-form. The production of the soluble CD14 is assumed that the membrane-binding CD14 is cleaved by protease to become soluble CD14 (Philip et al., Eur. J. Immunol., Volume 25, page 604, 1995).
It is reported that the soluble CD14 binds to LPS molecule in the blood and transports it to HDL, so that the soluble CD14 serves for the clearance of the LPS (Wurfel et al., J. Exp. Med., Volume 186, page 1743, 1995). On the other hand, it is assumed that the membrane CD14 binds to LPS, allows to transmit the signal into cells to induce inflammatory cytokine. In short, the CD14 possesses functions contrary to each other, i.e. the effect of removing LPS and the effect of inducing inflammatory factors.
JP-A 5-501399 discloses a method for treating sepsis by employing anti-CD14 antibody. The anti-CD14 antibody inhibits the binding between CD14 and LPS, to enable blockage of the signal via CD14 to thereby suppress the expression of the inflammatory cytokine. The sepsis is thereby treated. WO93/19772 and WO96/2057 disclose the treatment of sepsis by employing soluble CD14.
Nevertheless, when high mortality and numbers of patients of septic shock are taken into consideration, provision of more effective medicines is required.
DISCLOSURE OF THE INVENTION
The present inventors have investigated in order to provide more effective medicines against septic shock. The inventors have predicted that the inflammatory cytokine produced from Kupffer cells in liver by LPS stimulation plays an important role, and have assumed that specific blocking of the binding between the LPS and the CD14 on the Kupffer cell in a way of not affecting the soluble CD14 contributing the removal of LPS, or the CD14 on aveolar macrophage or peritoneal macrophage, or on other macrophages contributing for bacterial infection prevention on each site would be clinically effective. The inventors have assumed that the use of an antisense oligonucleotide accumulative in liver should act with the CD14 on the liver Kupffer cells at a high selectivity.
It is known that CD14 is very weakly expressed on mouse Kupffer cell in normal state whereas CD14 is strongly expressed when the cell is stimulated by LPS. On the other hand, it has been known that the liver is an organ most susceptible to shock, and that the reduction of liver function considerably affects constitutional symptom. The present inventors provide a medicine effective to sepsis or septic shock based on a new view of selectively inhibiting the CD14 on Kupffer cell whose expression is induced by LPS stimulation, and mainly inhibiting the production of inflammatory cytokine from Kupffer cells. In other words, the present inventors provide an antisense oli

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