Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
1999-08-12
2002-07-02
Lankford, Jr., Leon B. (Department: 1651)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C424S094200, C424S094650, C435S212000, C435S220000, C435S221000, C435S222000
Reexamination Certificate
active
06413512
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to a method of treating human and animal disease utilizing proteolytic enzymes of plant or microbial origin (“proteases” or “proteinases”) and, more particularly, to a method of modifying the development or manifestation of conditions affected by the action of biologically-active molecules which may be specifically bound or stimulated by activated alpha-2-macroglobulins. Such biologically-active molecules include, but are not limited to, cytokines and other signaling molecules, such as tumor necrosis factor-alpha (TNF-&agr;), leptin, beta-amyloid (&bgr;A), and transforming growth factors (TGF).
BACKGROUND OF THE INVENTION
In many diseases and injuries there is a marked increase in proinflammatory cytokines in the blood. The increased cytokines are believed to contribute to the pathology of the condition. Infection, cancer and tissue injury trigger the production of cytokines. These hormone-like peptides can enter the bloodstream to alter the physiology of distant tissues, or they may behave as paracrine mediators that act only locally. In some disease or injury states, cytokines are beneficial to the host but, in others, cytokines cause the most striking manifestations of the disease (e.g., shock, tissue injury and weight loss). K. J. Tracey, et al.,
Tumor Necrosis Factor: A Pleiotropic Cytokine and Therapeutic Target
, Ann. Rev. Med. 45: 491-503 (1994). Proteases and cytokines are intimately interrelated in the body in that cytokines are involved in regulating the production of proteases (Westermarck, J. & Kahari, V. M.,
Regulation of Matrix Metalloproteinase Expression in Tumor Invasion
, Faseb J. 13: 781-92 (1999)), and proteases are frequently involved in the liberation of soluble cytokines. Excess circulating proteases also play an important role in the manifestation of disease or injury.
Normal human blood serum contains significant quantities of anti-proteinases, such as alpha-2-macrolobulin (&agr;2M), which function to inhibit excess activity of proteases in vivo. &agr;
2
M has a high molecular weight (Mr
human
=725,000), and is composed of two noncovalently bonded pairs of identical subunits joined by disulfide bonds. Feldman et. al.,
Model of &agr;
2
-
Macroglobulin Structure and Function
, Proc. Nat'l. Acad. Sci. USA, 82: 5700-5704 (September 1985). The major source of plasma &agr;
2
M is the liver (hepatocytes), although other cells including macrophages synthesize and secrete &agr;
2
M. This may explain the presence of &agr;
2
M in interstitial sites and malignant tissues. LaMarre et al.,
Cytokine Binding and Clearance Properties of Proteinase
-
Activated &agr;
2
-
Macroglobulins
, Lab. Investigation 65(1):3-14 (1991). &agr;
2
M inhibits proteinases of all four (4) catalytic classes. Barrett et al.,
The Interaction of &agr;
2
-
Macroglobulin With Proteinases: Characteristics and Specificity of the Reaction, and a Hypothesis Concerning its Molecular Mechanism
, Biochem. J. 133: 709-724 (1973); Harpel et al.,
Studies on Human Plasma &agr;
2
-
Macroglobulin-Enzyme Interactions
, J. Exp. Med. 138: 508-521 (1973); Salveson et al.,
Covalent Binding of Proteases in Their Reaction With &agr;
2
-
Macroglobulin
, Biochem. J. 187: 695-701 (1980). Proteinases inhibited by &agr;
2
M include the coagulation proteinases thrombin and Factor Xa, the fibrinolytic enzymes urokinase-type and tissue-type plasminogen activators as well as plasmin, kallikrein of the contact system, the neutrophilic proteinases elastase, cathepsin G, and collagenase, and several bacterial proteinases. DeBoer et al.,
Alpha-
2-
Macroglobulin Functions as an Inhibitor of Fibrinolytic, Clotting, and Neutrophilic Proteinases in Sepsis: Studies Using a Baboon Model
, Infection and Immunity 61(12): 5035-5043 (1993).
Because of the biological importance of activated &agr;
2
M (&agr;
2
M*) scavenging of proteinases, several laboratories have attempted to elucidate the structure and function of &agr;
2
M. The trap mechanism and structure identified by Feldman et al. has been generally accepted. According to the “trap” hypothesis, proteinases cleave specific peptide bonds within the “bait” amino acids sequence of the &agr;
2
M subunits and become entrapped within the interior of the &agr;
2
M as a result of its conformational change. Feldman et. al., supra at 5701. Methylamine mimics the proteinase-induced conformational changes by reacting with the &agr;
2
M thiol ester bonds, and has therefore been commonly used in studies of &agr;
2
M*.
A substantial body of research has established that &agr;
2
M* complexes acquire a high affinity for binding proinflammatory cytokines, resulting in their removal from the body along with the protease bound in the &agr;
2
M* complex. Activated &agr;
2
M has been shown to play a pivotal role in regulating inflammatory and homeostatic mechanisms of disease and injury by binding major mediators such as tumor necrosis factor-alpha (TNF-&agr;), transforming growth factor-&bgr;1 (TGF-&bgr;1), transforming growth factor-&bgr;2 (TGF-&bgr;2), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), nerve growth factor (NGF), interleukin-1&bgr;(IL- 1&bgr;) and interleukin-6 (IL-6). Wolleberg et.al.,
Binding of Tumor Necrosis Factor Alpha to Activated Forms of Human Plasma Alpha
2
Macroglobulin
, Amer. J. Path., 138 (2): 265-272 (1991). Recent research suggests that various proinflammatory cytokines, and especially TNF-&agr;, play a major role in rheumatoid arthritis. M. Feldmann, et at.,
Role of Cytokines in Rheumatoid Arthritis
, Ann. Rev. Immun., 14: 397-440 (1996).
One of the hallmarks of most disease processes is acute inflammation, which features the release of neutrophil-derived oxidants. It has recently been shown that “oxidation serves as a switch mechanism that down-regulates the progression of acute inflammation by sequestering TNF-alpha, IL-2, and IL-6, while up-regulating the development of tissue repair processes by releasing bFGF, beta-NGF, PDGF, and TGF-beta from binding to alpha2M.” Wu, Patel & Pizzo,
Oxidized Alpha
2-
Macroglobulin (alpha
2
M
)
Differentially Regulates Receptor Binding by Cytokines/Growth Factors: Iimplications for Tissue Injury and Repair Mechanisms in Inflammation
. J. Immunol. 161: 4356-65 (1998). The conformationally modified or “activated” &agr;
2
M is rapidly cleared from the circulation as it more readily binds to specific cell-surface receptors on hepatocytes, macrophages, and fibroblasts and undergoes receptor-mediated endocytosis. These cellular receptors rapidly clear &agr;
2
M-protease and &agr;
2
M-methylamine complexes from the systemic circulation, primarily in the liver. Native &agr;
2
M, on the other hand, is not receptor recognized and has a prolonged half-life in circulation.
Activated &agr;
2
M is believed to also play a role in the mediation and regulation of leptin. Leptin is a 16kDa polypeptide consisting of 167 amino acids that is expressed and secreted from adipose tissue under the control of the “obese gene.” Zhang Y, et al.,
Positional Cloning of the Mouse Obese Gene and its Human Homologue
, Nature, 372:425-432 (1994). Murine studies indicate that leptin acts on the CNS to regulate body weight through the control of appetite and energy expenditure. Pelleymounter M A., et al.,
Effects of the Obese Gene Product on Weight Regulation in ob/ob Mice
, Science, 269:540-43 (1995). Leptin has also been shown to affect sympathetic nerve activity, insulin resistance, and renal sodium excretion. Haynes W G, et al.,
Cardiovascular Consequences of Obesity: Role ofLeptin
, Clin. Exp. Pharm. Physiol., 25:65-69 (1998). Obesity is associated with increased activity of the sympathetic nervous system and, therefore, there appears to be a causal link between excess leptin and other systemic, and especially cardiovascular consequences of obesity. See id.
There are a variety of undesirable consequences of obesity, including insulin resistance, dyslipoproteinemia, and hypercoagulability, all of which are probably due to increases in circulati
Andrews Lary D.
Collier Anthony W.
Greaves Lynn S.
Houston Devin B.
Lankford , Jr. Leon B.
Mahoney Joseph A.
Mayer Brown Rowe & Maw
National Enzyme Company
Stiebel Thomas R.
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