Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-12-10
2004-03-23
Borin, Michael (Department: 1631)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S324000, C536S023100
Reexamination Certificate
active
06710030
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of biochemistry and medicine and in particular to the cloning, sequencing, and production of contortrostatin and its precursor.
BACKGROUND OF THE INVENTION
Breast cancer is one of the leading causes of death among non-smoking women and the spread of the disease from the breast to distant sites is a major cause of death in breast cancer patients. At the time of diagnosis over 60% of breast cancer patients will have disease that has spread from the primary site in the breast to some distant site. Spread of cancer to remove sites, e.g. bone, lungs, liver, brain, a process called metastasis, is a characteristic of malignancy and often leads to inoperable disease. Metastasis is the most common factor leading to death from breast cancer. Control of metastasis offers an important avenue for breast cancer treatment. Cancer cells metastasize through the blood or lymph vessels. The first step of metastasis involves the attachment of cancer cells to tissues around the primary site, i.e., to the extracellular matrix (ECM) via cell surface integrins and other adhesion receptors. Integrins mediate cell—cell and cell-substratum interactions and are involved in bi-directional signaling that links the ECM with cytoskeletal proteins. Integrins play an important role in the interaction of mammary carcinoma cells with the ECM. In the second step, cancer cells secrete digestive enzymes that degrade the surrounding tissues allowing the tumor cells to invade these tissues. Eventually, the tumor cells enter the blood or lymphatic system where they repeat the adhesion and invasion steps at a distant (metastatic) site. At this remote site, tumor cells induce the formation of new blood vessels (a process called neovascularization), in and around the growing tumor. These new blood vessels supply nutrients to the metastatic tumor and allow it to grow. Treatments that block any of these steps should act to inhibit metastasis.
Integrins on cancer cells play important roles in tumor invasion and spread. They are a family of proteins found on the cell surface of many cell types that mediate interactions between cells, and between cells and their surroundings. Integrins are heterodimers, composed of &agr; and &bgr; subunits involved in cell—cell and cell-substratum interactions. Integrins serve as receptors for extracellular matrix proteins such as fibronectin, fibrinogen, vitronectin, collagen and laminen. Some of these interactions have been shown to be mediated via an Arg-Gly-Asp (RGD) sequence present in the matrix proteins. Both the &agr; and &bgr; subunits are required for fibrinogen binding. For example, one of the members of the superfamily of integrin cell surface receptors is the platelet membrane glycoprotein (GP)IIb/IIIa which interacts with plasma fibrinogen in platelet aggregation.
CN binds to a specific integrin on the surface of blood platelets, and blocks the ability of platelets to adhere to one another (a process called platelet aggregation). Platelets are small fragments of bone marrow cells that are found in the blood stream. They have both beneficial and harmful activities. Their useful action is to stop bleeding following injury by facilitating the formation of a blood clot. But, under certain conditions they are involved in blocking arteries that supply nourishment to the heart—an action that can lead to a heart attack.
Integrin cell surface receptors have been investigated in the role of platelets in mediating coronary artery thrombosis and rethrombosis in the genesis of acute myocardial infarction [Zucker, M. B.,
Sci. American
242:86 (1990)]. For platelet aggregation an RGD sequence present in fibrinogen is essential for the interaction with (GP)IIb/IIIa [Ginsberg, M. H. et al.,
Thrombos. Haemostas
. 59:1 (1988)]. Because of its inhibition of platelet aggregation, snake venom has been the subject of various investigations.
A number of proteins purified from venom of snakes of the Crotalidae and Viperidae families have been found to inhibit glycoprotein (GP)IIb/IIIa mediated platelet aggregation [see, e.g., Huang, T. F. et al.,
J. Biol. Chem
. 262:16157 (1987); Gan, Z. R. et al.,
J. Biol. Chem
. 263:19827 (1988); Yasuda, T. et al.,
J. Am. Coll. Cardiol
. 16:714 (1990); Trikha, M. et al.,
Fibrinolysis
4 (Suppl. 1):105 (1990); Trikha, M. et al.,
Blood
76 (Suppl. 1):479a (1990); Holahan, M. A. et al.,
Pharmacology
42:340 (1991); Shebuski, R. J. et al.,
Circulation
82:169 (1990); Yasuda, T. et al.,
Circulation
83:1038 (1991)]. These proteins, classified as disintegrins, are typically disulfide rich. Moreover, all disintegrins isolated thus far, with the exception of barbourin [Scarborough, R. M. et al.,
J. Biol. Chem
. 266:9359 (1991)] contain an RGD (Arg-Gly-Asp) sequence that has been implicated as being involved in the inhibition of integrin-mediated interactions. In particular, the RGD sequence of the disintegrins may compete for fibrinogen binding sites on the platelet membrane, thereby inhibiting platelet aggregation induced by ADP or other agents.
Nonetheless, there appears to be increasing evidence that disintegrins may have unique surface geometry which facilitates interactions with integrins by mechanisms other than those based solely upon the RGD site. For example, the finding that a mutated, chemically synthesized derivative of echistatin (in which alanine was substituted for arginine in the RGD sequence) still possessed some biological activity, suggests that other regions in the protein may be involved in binding and that there may be some flexibility in the RGD binding site [Connolly, T. M. et al.,
Circulation
82 (Suppl. III):660 (1990)]. Synthetic RGD peptides, due to their small size, generally do not possess the molecular topography of the disintegrins and therefore cannot interact via the multiplicity of mechanisms likely to be involved in disintegrin binding.
In other investigations, prevention of reocclusion following thrombolysis using tissue-type plasminogen activator in a canine model system has been reported using 30 &mgr;g/kg plus 3 &mgr;g/kg/min bitistatin, an 83 amino acid disintegrin derived from the venom of
Bitis arietans
[Shebuski et al., supra], or 15 &mgr;g/kg/min i.v. echistatin, a 49 amino acid disintegrin derived from the venom of
Echis carinatus
[Holahan et al., supra]. In the reported methods, an initial bolus of heparin (100 U/kg i.v.) and subsequent hourly boluses of 50 U/kg were used to increase activated partial thromboplastin times at least 1.5-fold over the control. Whereas it had previously been observed that heparin in combination with tissue-type plasminogen activator (tPA) did not affect the incidence of acute reocclusion in this model system, the addition of echistatin or bitistatin lead to dramatic reductions in the incidence of acute thrombotic reocclusion. The administration of heparin was, however, apparently necessary for prevention of acute thrombotic reocclusion.
Similarly, kistrin (a 68 amino acid disintegrin derived from the venom of
Agkistrodon rhodostoma
) was evaluated in conjunction with recombinant tissue-type plasminogen activator in a canine model of coronary artery thrombosis with superimposed high grade stenosis [Yasuda et al. (1991), supra]. An effective dose of 4 &mgr;g/kg/min was determined to be sufficient to prevent reocclusion. Simultaneous systemic therapeutic heparin anticoagulation was used; the dose of heparin was selected to maintain the activated partial thromboplastin time more than two-fold throughout the experimental observation period.
U.S. Pat. No. 5,066,592 to Huang et al. describes the use of trigramin, a 72 amino acid disintegrin isolated from the venom of
Trimeresurus gramineus
, to inhibit fibrinogen binding to human platelets and thereby to inhibit fibrinogen-induced aggregation of human platelets. Trigramin is also reported to inhibit binding of von Willebrand factor to platelets. Trigramin is reported to inhibit
125
I-
Markland, Jr. Francis S.
Zhou Qing
Borin Michael
Fulbright & Jaworski L.L.P.
University of Southern California
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