Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-07-17
2003-04-08
Eyler, Yvonne (Department: 1646)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C424S094100, C530S350000
Reexamination Certificate
active
06544947
ABSTRACT:
TECHNICAL FIELD
This application relates to a novel use of cancer markers such as kallikreins, including prostate-specific antigen (PSA), as inhibitors of angiogenesis useful for treating angiogenesis-related diseases such as angiogenesis-dependent cancer. The invention further relates to novel compositions and methods for curing angiogenesis-dependent cancer. In addition, the present invention relates to molecular probes for monitoring biosynthesis, to antibodies that are specific for serine proteases including kallikreins, to the development of peptide agonists and antagonists to kallikrein receptors, and to cytotoxic agents linked to kallikrein peptides.
BACKGROUND OF THE INVENTION
Cancer Markers
The discovery of cancer markers and tumor markers has significantly enhanced not only diagnosis of cancer but has also contributed to the monitoring of cancer patients for assessing disease progression. A rise in cancer markers is a yardstick with which benign diseases can be distinguished from metastatic disease and also used to evaluate the efficacy of treatments. A decline in cancer markers is often a predictor of possible residual disease if the timing of blood sampling is soon after therapy. Numerous cancer markers are known in the art and are utilized in detection assays such as immunoassays depending upon the intrinsic characteristics of each marker (antigen specificity, molecular heterogeneity) and individual factors (nonspecific increases and renal and hepatic diseases).
Kallikrein
Kallikrein and kallikrein-like enzymes belong to a multigene family of serine proteases present in tissues and body fluids of numerous animals such as mammals and reptiles (i.e. snake venom). Included in the kallikrein family is hk1, a pancreatic/renal kallikrein; hk2, a human glandular kallikrein present in seminal fluid, a protease that activates urokinase type plasminogen activator; and prostate-specific antigen (hk3), a single-chain glycoprotein found in prostate tissue. Pre-kallikrein is converted by limited proteolysis into an active serine protease, and is one of the five major proteins involved in the activation and inhibition of surface mediated pathways in blood clotting. Pre-kallikrein is an important component of the biochemical junctures of intrinsic coagulation with other plasma proteolytic pathways required in the initiation, amplification, and propagation of surface-mediated defense reactions wherein various proteins such as bradykinin are involved. Thus, the molecular events of the contact phase of coagulation activation and inhibition involve pre-kallikrein and the plasma biochemical systems. (Colman et al. 1987).
Plasma kallikrein circulates in the blood as the precursor “pre-kallikrein.” Plasma pre-kallikrein is synthesized in the liver and secreted into plasma. However, only 25% of the protein exists as free pre-kallikrein and approximately 75% circulates bound to high molecular weight kininogen (HMWK). The molecular weight of human plasma pre-kallikrein, as assessed by gel filtration, is approximately 100,000 Daltons. By SDS polyacrylamide gel electrophoresis, plasma pre-kallikrein consists of two components having molecular weight 85,000 Daltons and 88,000 Daltons, depending whether the sample has undergone reduction. In plasma, the concentration of pre-kallikrein is estimated to be 35 &mgr;g to 50 &mgr;g/ml.
Following proteolysis, pre-kallikrein is activated to kallikrein and current studies do not demonstrate any clear cut difference in physiochemical or immunochemical properties of zymogen pre-kallikrein, and active enzyme kallikrein in the absence of reduction. Hageman factor (also known as Factor XII
a
), and Hageman factor fragment (also known as Factor XII
f
), are both able to convert pre-kallikrein to kallikrein. Unlike pre-kallikrein on reduced SDS gel electrophoresis, kallikrein has two types of subunits: a heavy chain with a molecular weight of approximately 52,000 Daltons, and two light chain variants with a molecular weight of approximately 36,000 Daltons and 33,000 Daltons. Pre-kallikrein circulates mostly complexed to high molecular weight kininogen HMWK, and it is thought that this complex may have protective functions for the pre-kallikrein. Following activation from pre-kallikrein to kallikrein, HMWK is cleaved to release bradykinin. Bradykinin is one of the most potent vasodilators known. (Colman et al. p.254).
The gene for plasma pre-kallikrein has not been isolated or characterized thus far. The messenger RNA for plasma pre-kallikrein, however has been characterized as a cDNA and shown to be approximately 2,300 nucleotides in length. It codes for a leader sequence of 19 amino acids and a mature polypeptide chain of 619 amino acids. The latter peptide in plasma pre-kallikrein is one amino acid longer than that in Factor XI. The activation reaction of pre-kallikrein to kallikrein is due to the cleavage of the peptide bond following arginine 371. Plasma kallikrein is generated as an enzyme composed of a heavy chain (371 amino acids) and a light chain (248 amino acids), held together by a disulfide bond. The catalytic domain or light chain of plasma kallikrein, contains three important amino acids (His-44, Asp-93 and Ser-188) that are directly involved in catalysis. In addition, plasma kallikrein contains 5 N-linked carbohydrate chains as established by amino acid sequence analysis.
The proteins and enzymes of the clotting cascade may perform multiple functions, for example, Factor XII
a
may cleave pre- kallikrein to kallikrein, and Factor XI to XI
a
. Kallikrein can initiate reciprocal activation, generating additional Factor XII
a
from Factor XII. Plasma kallikrein leads to the conversion of plasminogen to plasmin and Factor XII
a
also converts plasminogen to plasmin. Kallikrein cleavage of HMWK results in the release bradykinin and may also elevate blood pressure by directly converting pro-renin to renin.
Alteration of any of the components of the vascular system, namely vessel cell wall, plasma proteins and platelets can result in an angiogenic disorder. There appear to be two major mechanisms under which the multiple inciting etilogies can be catagorized: endothelial injury and tissue injury. Endothelial injury relates to disease states such as infections that specifically injure the endothelium, with resultant kallikrein-kinin activation.
Injury to the vascular endothelium, such as occurs in endotoxemia, exposes basement membrane. Consequently collagen, along or in combination with proteoglycans or other components, activates Factor XII. Following Factor XII activation, intrinsic coagulation, activation of fibrinolysis and kinin formation occur. (Colman et al. p. 976).
Patients with bacterial infections, especially those caused by gram negative bacteria, may have elevated levels of plasma kallikrein. The hypotensive effect of kallikrein may contribute to the development of disseminated intravascular coagulation by reducing blood flow to reticuloendothelial organs thereby impairing clearance of activated coagulation factors.
Prostate-Specific Antigen
One important member of the kallikrein family is prostate-specific antigen. (Riegman et al.) The prostate-specific antigen (PSA) molecule is a single-chain glycoprotein consisting of approximately 237 amino acids and has a molecular weight of 28,430 daltons as determined by ion-spray mass spectroscopy. (Sokoll et al. 1997). The gene for PSA is located on the long arm of chromosome 19 and is approximately 6 kilobases in size, consisting of 4 introns and 5 exons. The PSA gene is under androgen regulation as evidenced by an androgen-responsive element in the promoter region. PSA is thought to be translated as a 261 amino acid prepropeptide. Although not isolated, the 244 propeptide zymogen form of PSA results after cleavage of the leader peptide during translation. The 237 amino acid active enzyme then is surmised to result from subsequent cleavage with as yet unidentified proteases. Structurally, the molecule is thought to possess five disulfide bonds owing to the presence of 10 cysteine residues with
Fortier Anne H.
Holaday John W.
Andres Janet L.
EntreMed, Inc.
Eyler Yvonne
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