Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1999-12-17
2002-09-24
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S013000
Reexamination Certificate
active
06455269
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the therapeutic administration of fibrinolytic metalloproteinases, and more specifically to a method for administering such agents in vivo via localized delivery to vascular thrombi in order to effect clot lysis.
BACKGROUND OF THE INVENTION
Vascular occlusions caused by blood clots such as thrombi and embolisms are serious medical maladies that can become limb or life threatening if not timely treated. Devices and methods have been developed for the treatment and removal of vascular blood clots. By way of illustration, see U.S. Pat. No. 4,447,236 (Quinn), issued May 8, 1984; U.S. Pat. No. 4,692,139 (Stiles), issued Sep. 8, 1987; U.S. Pat. No. 4,755,167 (Thistle et al.), issued Jul. 5, 1988; U.S. Pat. No. 5,167,628 (Boyles), issued Dec. 1, 1992; U.S. Pat. No. 5,222,941 (Don Michael). issued Jun. 29, 1993; U.S. Pat. No. 5,250,034 (Appling et al.), issued Oct. 5, 1993: U.S. Pat. No.5,370,653 (Cragg), issued Dec. 6, 1994; U.S. Pat. No.5,380,273 (Dubrul et al.), issued Jan. 10, 1995; U.S. Pat. No. 5,498,236 (Dubrul et al.), issued Mar. 12, 1996; U.S. Pat. No. 5,626,564 (Zhan et al.), issued May 6, 1997; U.S. Pat. No. 5,709,676 (Alt), issued Jan. 20, 1998; U.S. Pat. No. 5,865,178 (Yock), issued Feb. 2, 1999, and WO 90/07352 (published Jul. 12, 1990). Such methods and devices include infusion catheters for delivering thrombolytic or fibrinolytic agents to the blood clot and dissolving it. Infusion catheters are typically used in conjunction with enzymatically active agents that are capable of degrading the fibrin in the clot and thus effectively dissolving the clot. Such enzymes are typically referred to as “thrombolytic” or “fibrinolytic” agents.
Fibrolase is a known fibrinolytic zinc metalloproteinase that was first isolated from the venom of the southern copperhead snake (
Agkistrodon contortrix contortrix
). See Guan et al., Archives of Biochemistry and Biophysics, Volume 289, Number 2, pages 197-207 (1991); Randolph et al., Protein Science, Cambridge University Press (1992), pages 590-600; European Patent Application No. 0 323 722 (Valenzuela et al.), published Jul. 12, 1989; and U.S. Pat. No. 4,610,879 (Markland et al.), issued Sep. 9, 1986. Fibrolase has been shown to be fibrinolytic, and this metalloproteinase has been documented to have proteolytic activity against the fibrinogen A&agr;-chain, with reduced proteolytic cleavage of the B&bgr;-chain and no activity against the &ggr;-chain of fibrinogen; Ahmed et al., Haemostasis, Volume 20, pages 147-154 (1990). Because fibrin is a principal component of blood clots, the fibrinolytic properties of fibrolase point to its potential as a clot dissolving agent for in vivo thrombolytic use; see Markland et al., Circulation, Volume 9, Number 5, pages 2448-2456 (1994), and Ahmed et al., above.
Novel Acting Thrombolytic (NAT) is a modified form of fibrolase that differs from fibrolase in that NAT contains 201 amino acids with an N-terminal sequence of SFPQR, whereas the N-terminal sequence of native fibrolase begins with EQRFPQR and is 203 amino acids in length. The amino-terminal modification was designed to prevent chemical reactions at amino acid residues that were capable of forming a variable quantity of cyclized glutamine (pyroglutamic acid) which have the potential to create lot-to-lot variations in quality and uniformity of the product. Thus, NAT can be viewed as a more stable molecule.
Despite these structural differences, NAT and fibrolase are similar with respect to enzymatic (fibrinolytic) activity. This similarity in biological activity is consistent with data indicating that the active site of the fibrolase molecule spans amino acids 139-159, as described by Manning in Toxicon, Volume 33, pages 1189-1200 (1995), and its predicted location in three-dimensional space is distant from the amino-terminus. The active site of the fibrolase and NAT molecules contains a zinc atom which is complexed by three histidine residues.
Published literature on venom-derived fibrolase has demonstrated its proteolytic activity against fibrinogen at the Lys
413
-Leu
414
site and against the oxidized &bgr;-chain of insulin at the Ala
14
-Leu
15
site; Retzios and Markland, Thrombosis Research, Volume 74, pages 355-367 (1994); Pretzer et al., Pharmaceutical Research, Volume 8, pages 1103-1112 (1991), and Pretzer et al., Pharmaceutical Research, Volume 9, pages 870-877 (1992). NAT has also been determined to have proteolytic activity on these substrates at the same cleavage sites.
In contrast to fibrinolytic metallo-proteinases such as fibrolase and NAT, clot lysing agents such as streptokinase, urokinase and tissue-type plasminogen activator (tPA) are plasminogen activators which promote thrombolysis by activation of the endogenous fibrinolytic system. More specifically, plasminogen activators catalyze the conversion of plasminogen into plasmin, a serine protease. Plasmin is capable of cleaving fibrinogen and fibrin at arginyl-lysyl bonds, and it is through the generation of plasmin that the plasminogen activators ultimately effect fibrin degradation and clot lysis. Current commercially available thrombolytic agents are plasminogen activators, such as urokinase, streptokinase or tPA.
Unlike the plasminogen activator class of thrombolytic drugs, fibrinolytic metalloproteinases, such as fibrolase and NAT, do not rely on the endogenous fibrinolytic system (conversion of plasminogen to plasmin). Hence, this class of clot lysing agents can be distinguished from the plasminogen activators by their unique mode of action and are defined as “direct” fibrinolytic agents.
Alpha
2
-macroglobulin is a prevalent proteinase inhibitor present in mammalian serum and is one of the largest of the serum proteins (having a molecular weight of 725 kilodaltons). The specificity of &agr;
2
-macroglobulin for proteinases is broad, encompassing serine, cysteine, aspartic and metalloproteinase classes. The &agr;
2
-macroglobulin molecule is a tetramer of identical subunits that are disulfide bonded in pairs with a non-covalent association of the half molecules. Thus, under reducing conditions, native &agr;
2
-macroglobulin can be dissociated into its four monomeric subunits.
Each subunit of &agr;
2
-macroglobulin possesses a region that is very susceptible to proteolytic cleavage (the “bait” region). Proteolysis of the bait region induces a conformational change in &agr;
2
-macroglobulin, which entraps the proteinase within the &agr;
2
-macroglobulin molecular structure. This process is described in the literature as a “venus fly-trap” interaction. Once the proteinase is entrapped, it is sterically hindered and therefore cannot access its macromolecular substrate.
In addition, a covalent bond can form between &agr;
2
-macroglobulin and many of the proteinases that it entraps. As mentioned, entrapment of a proteinase induces a conformational change in the &agr;
2
-macroglobulin molecule. It is presumed that upon this conformational change, a thioester bond on the interior of the &agr;
2
-macroglobulin molecule becomes reactive and can form a covalent bond with nucleophilic residues (such as lysine) of the entrapped proteinase. Thus, within the general circulation, &agr;
2
-macroglobulin can effectively neutralize a variety of proteinases.
Moreover, the conformational change in &agr;
2
-macroglobulin brought about by the entrapment of a proteinase results in a form that is recognized by the reticuloendothelial system. Clearance of &agr;
2
-macroglobulin-entrapped proteinases is generally described with half-life values in minutes and is believed to occur through the low-density lipoprotein (LDL)-receptor related protein expressed on macrophages, hepatocytes and fibroblasts. For more on &agr;
2
-macroglobulin, see Methods in Enzymology, edited by A. J. Barrett, Academic Press, Inc., Philadelphia, (1981), pages 737-754.
Alpha
2
-macroglobulin is capable of forming a macromolecular complex with fibrolase, NAT and other proteinases. Unlike some proteinases that can form a dissociable complex with &agr;
2
-macroglobulin, fibrola
Amgen Inc.
Gitomer Ralph
Levy Ron K.
Nicastro Karen L.
Odre Steven M.
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