Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1995-02-28
2003-07-01
Nolan, Partick J. (Department: 1644)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
Reexamination Certificate
active
06586222
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the area of immunology/biochemistry, and particularly concerns the development and production of compositions and methods for identifying inhibitors of protein hormone release, and prophylactic and therapeutic uses of the inhibitors for treating diseases associated with elevated levels of the hormones. More specifically, the invention facilitates the identification of compositions and methods for identifying inhibitors of a TNF&agr; convertase. These inhibitors may be used to treat a variety of diseases, particularly sepsis, rheumatoid arthritis, cachexia, AIDS and autoimmune diseases, and thus affords the physician alternate treatment regimes.
BACKGROUND OF THE INVENTION
In the United States alone nosocomial bacteremia develops in about 194,000 patients per year, and of these about 75,000 die. Maid, D. G., 1981
, Nosocomial Infect
., (Dikson, R. E., Ed.), page 183, Yrke Medical Books, U.S.A. Most of these deaths are attributable to six major gram-negative bacilli—
Pseudomonas aeruginosa, Escherichia coli
, Proteus, Klebsiella, Enterobacter and Serratia. The current treatment for bacteremia is the administration of antibiotics which have limited effectiveness in treatment of septic shock frequently associated with bacterial infection.
The precise pathology of the septic shock sometimes associated with bacteremia is not completely elucidated. Nevertheless, it is known that certain bacterial endotoxins called lipopolysaccharides (LPS), are the primary causative agents. LPS consists of at least three significant antigenic regions: lipid A; core polysaccharide; and O-specific polysaccharide. The latter is also referred to as O-specific chain or simply O-antigen. The O-specific chain is a long-chain polysaccharide built up from repeating polysaccharide units. The number of polysaccharide units differs among different bacterial species and may vary from one to as many as six or seven monosaccharide units. While the O-specific chain varies among different gram-negative bacteria, the lipid A and core polysaccharides are similar if not identical.
LPS initiates a cascade of biochemical events that can lead to the death of the patient. It is widely believed that an early result of exposure to LPS is the stimulation of macrophage cells and the production of tumor necrosis factor alpha (TNF&agr;). Based on this belief, considerable effort has been expended to produce neutralizing antibodies and other molecules that could inhibit the effects of TNF&agr; and which could serve as valuable clinical adjuncts to the standard antibiotic therapies used in the treatment of septic shock. Tracey et al.,
Nature
330:662 (1987).
While many cell types are capable of expressing TNF&agr;, including for example, T and B lymphocytes, fibroblasts, and endothelial cells, the principal source is macrophages. TNF&agr; has been reported to exist in both membrane-bound and soluble secreted forms. Decker et al.,
J. of Immunol
. 138:957 (1987); Kriegler et al.,
Cell
53:45 (1988). Human TNF&agr; has been cloned and consists of a 17 kD polypeptide, plus a 76-amino-acid pro sequence containing the residues that appear to be responsible for anchoring proTNF&agr; as a type II membrane protein. The 17 kD molecule is a key agent involved in initiating the biochemical cascade responsible for sepsis. TNF&agr; may exist as both a membrane-bound 26 kD form, and a soluble form corresponding to the 17 kD species. Kreigler et al.,
Cell
53:45 (1988). The 26 kD form is the precursor, or prohormone, of the mature 17 kD molecule. The two forms of TNF&agr; may have different biological effects, primarily as a result of differences in tissue distribution. Kriegler et al. supra
Because TNF&agr; plays a key role in the sequelae of sepsis and is believed to be an inflammatory agent in diseases, there is a need to identify and develop anti-TNF&agr; prophylactics/therapeutics. Anti-TNF&agr; antibody has shown promise in in studies employing a baboon model system. (Hinshaw et al.,
Circulatory Shock
30:279 (1989)). However, these studies involve non-human anti-TNF&agr; and non-human anti-TNF&agr; antibody. In addition, TNF&agr; is involved in inducing the expression of human immunodeficiency virus (HIV) in human cells that carry latent virus. Folks et al.,
PNAS
(
USA
) 86:2365 (1989).
TNF&agr; also plays a role in various autoimmune diseases, particularly rheumatoid arthritis. Duff et al.,
International Conference on Tumor Necrosis Factor and Related Cytotoxins
, 175:10 (1987). Compounds or methods for inhibiting TNF&agr; action will have considerable application for the treatment of a variety of diseases of immunologic origin. As described in Fiers et al.,
FEBS Lett
. 285:199 (1991), a variety of other serious human conditions including cerebral malaria, graft-versus-host disease and ischemia/reprefusion injury, are also associated with TNF&agr; biological activity.
In addition to anti-TNF&agr; antibodies, other molecules with TNF&agr; inhibitory activity are being sought. Non-antibody TNF&agr; inhibitors are described by Seckinger et al.,
Exp. H. Med
. 167:151 (1988), and Seckinger et al.,
Biol. Chem
. 264:11966 (1989), and in European Patent Application No. 88830365.8, inventors Wallach et al. The inhibitors are present in the urine of febrile patients, and are reported to have molecular weights of about 27,000-33,000. These inhibitors are reported to be soluble forms of the TNF&agr; receptor. Although these molecules exhibit TNF&agr;-inhibitory activity, neither of the inhibitors is shown to be effective in the treatment of septic shock in humans.
From the foregoing discussion it is apparent that there is a need to identify and develop additional modulators of TNF&agr; activity, both antibody-based or otherwise, that are efficacious in the treatment of TNF&agr;-mediated diseases.
SUMMARY OF THE INVENTION
An approach to modulating the activities of TNF&agr; according to the present invention involves the inhibition of TNF&agr; convertases, for example PR-3, which are capable of converting locally produced, membrane-bound proTNF&agr; to TNF&agr; which contributes significantly to the pathologic processes of diseases such as septic shock and such as preventing or inhibiting the formation of the 17 kD, or lower molecular weight forms of TNF&agr; might be a valuable prophylactic for the prevention of AIDS in HIV-positive patients by preventing the expression of virus that is latent in the patient and others described above.
In its most general form, the invention described herein presents methods and compositions for inhibiting the production of a mature form of TNF&agr;, from its prohormone precursor, proTNF&agr; in its 26 kD form or multimers thereof and its soluble 20 kD form or multimers thereof. These compositions are useful for preventing or treating diseases in patients associated with elevated levels of mature TNF&agr; including septic shock, AIDS, cerebral malaria, graft versus host disease, ischemia/reperfusion injury, rheumatoid arthritis, and cachexia. The invention also relates to methods (e.g. colorimetric and autoradiographic) for identifying molecules that inhibit the production of a mature form of TNF&agr;. Such inhibitors are distinguishable from anti-TNF&agr; antibody or soluble TNF&agr; receptor, which block TNF&agr; activity by binding to TNF&agr;.
This method may be used to identify medicaments such as prophylactics and/or therapeutics for the treatment of diseases associated with the production of mature TNF&agr; such as those discussed above. Medicaments identified by this method interfere with the cleavage of the 26 kD proTNF&agr; prohormone by enzymes termed convertases. Thus, these medicaments inhibit the production of lower molecular weight molecules (i.e., circulating mature forms of TNF&agr; having subunits of 17 kD molecular weight) which play a role in the induction of “septic shock” associated with sepsis and other diseases. Specifically, preferred inhibitors as described herein interfere with the activity of a TNF&agr; convertase to prevent removal of
Halenbeck Robert F.
Jewell David A.
Koths Kirston E.
Kriegler Michael
Perez Carl
Blackburn Robert P.
Chiron Corporation
Nolan Partick J.
Pochopien Donald J.
LandOfFree
Recombinant PR-3 and compositions thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Recombinant PR-3 and compositions thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Recombinant PR-3 and compositions thereof will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3002836