Protamine fragment compositions and methods of use

Details Protamine fragment compositions and methods of use Protamine fragment compositions and methods of use

2000-11-16

2003-09-23

Low, Christopher S. F. (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S002600, C530S350000

Reexamination Certificate

active

06624141

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of protein biochemistry and medicine. More particularly, it concerns bioactive, low-toxicity fragments of protamine, and a number of different uses of such protamine fragment compositions. Provided are protamine fragments, compositions, combinations and kits and various methods and uses of such fragments, e.g., in the neutralization of heparin and for association with a variety of therapeutic proteins, including insulin.
2. Description of Related Art
Heparin has become the clinical anticoagulant drug of choice, and is used universally for: prophylaxis of postoperative thromboembolism, in patients with stroke, during various surgical situations, and particularly in all procedures involving extracorporeal blood circulation (ECBC) (Jaques, 1980; Majerus et al., 1996). Extracorporeal blood circulation is employed in numerous clinical situations such as kidney dialysis, open-heart operations, cardiac catheterizations, blood oxygenation, plasmapheresis, organ transplantation, and the implantation of artificial organs. In the United States alone, approximately 15 million ECBC procedures are performed annually (Ma et al., 1994). Approximately 33 metric tons of heparin, representing 500 million doses, are used worldwide each year (Linhardt, 1991). The production figures obtained from the pharmaceutical industry suggest that clinical use of heparin continues to grow rapidly (Linhardt, 1991).
Heparin consists of a heterogeneous mixture of sulfated glycosaminoglycans with a molecular weight ranging from 3,000 to 40,000 daltons. It is made of a repeating unit of D-glucuronic acid and N-acetyl-D-glucosamine residues (Bourin and Lindahl, 1993). The anticoagulant function of heparin was discovered over 70 years ago (Howell, 1922). Heparin exerts its anticoagulant activity primarily via interaction with antithrombin III (Rosenberg, 1987).
Antithrombin III (ATIII) is a circulating inhibitor of the serine proteases in the coagulation cascade, acting more particularly on thrombin and factor Xa but also on factors IXa, XIa, and XIIa. It possesses an arginine center that binds to the active serine site of thrombin (and also the other coagulation factors) to form a covalent bond (Griffith, 1983). Normally this reaction proceeds rather slowly. Binding of heparin to ATIII, however, induces a conformational change of ATIII, rendering the arginine center more accessible to thrombin interaction, and producing a 1000-fold acceleration of the inhibitory effect (Rosenberg, 1987). The binding of heparin to ATIII involves a unique pentasaccharide sequence containing a 3-O-sulfated glucosamine residue (Choay et al., 1981), and entails interaction between specific lysine residues on ATIII and sulfate and carboxylate groups in heparin (Choay et al., 1981; Rosenberg et al., 1979).
Heparin also acts as a template that helps bring thrombin in close proximity to ATIII. Thrombin then cleaves the reactive site bond of ATIII, to which it becomes covalently bound and is irreversibly inhibited. The released heparin can then act on other ATIII molecules. Heparin, however, does not act as a template for the interaction of antithrombin III and factor Xa (Casu et al., 1981). Thus smaller heparin fragments, such as the low molecular weight heparin possessing the ATIII-binding sequence, are able to inhibit factor Xa but not thrombin (Verstraete, 1990).
Systemic heparinization, however, results in a high incidence of bleeding complications (Hirsh, 1984; Kelton and Hirsh, 1984). Major bleeding occurs in 8% to 33% of patients who receive various forms of heparin therapy (Levin and Hirsh, 1986). Nearly 25% of all patients suffering from acute renal failure are subject to increased bleeding risk during and immediately following dialysis (Swartz and Port, 1979). The incidence of bleeding increase with elderly or diabetic patients, patients with ulcers or other multiple traumata, and patients with current cardiac or vascular surgery.
Aside from hemorrhage, there are also other complications associated with the use of heparin, particularly when the drug is administered over a long period. These added complications include thrombocytopenia, alopecia, arterial embolus, and interference with bone repair and maintenance (Hirchboeck et al., 1954). In fact, heparin has been cited as “the drug responsible for the most deaths in patients who are reasonably healthy” (Porter and Jick, 1977).
Low molecular weight heparin (LMWH) was derived from native heparin in an attempt to abate the induced bleeding risk (Verstraete, 1990; Holmer et al., 1986). It contains the specific pentasaccharide sequence in heparin that is required for ATIII binding, and thus fully retains the antithrombotic effect of heparin through inhibition of factor Xa by ATIII. However, it is of insufficient length to bind thrombin and catalyze the inhibition of thrombin by ATIII. LMWH is more effective than regular heparin in preventing deep vein thrombosis and pulmonary embolism after orthopedic surgery but has similar incidence of bleeding (Jensen and Ens, 1993). In a large randomized study in which a continuous intravenous infusion of heparin was compared with a fixed subcutaneous dose of LMWH in patients with venous thrombosis, the incidence of major bleeding was only marginally lower with LMWH (Hull et al., 1992). Enoxparin (Clexane), Kabi-2165 (Fragmin), CY-216 (Fraxiparine), and Novo LHN-1 (Logiparin) are a few commercial LMWH products approved for clinical use.
A major drawback of LMWH lies in the absence of an appropriate clinical antidote to combat the potential risk of induced bleeding. Neither protamine nor platelet factor 4 (PF4, a naturally occurring protein from platelet that is under extensive investigation as a potential replacement of protamine as the anti-heparin agent (Cook et al., 1992) can fully neutralize the anticoagulant effects of LMWH (Lechner et al., 1995; Ryn-McKenna et al., 1990).
To reduce post-operative bleeding, protamine, a clinical heparin antagonist, is routinely administered after cardiac and vascular surgery to reverse the anticoagulant activity of heparin (Jaques, 1973). Protamine consists of a group of heterogeneous polycationic peptides with an average molecular weight of about 4500 daltons. It is generally obtained from fish (Ando et al., 1973). Nearly 67% of the amino acid composition of protamine is arginine (Ando et al., 1973). The polycationic protamine combines electrostatically with the polyanionic heparin to form a stable complex that is devoid of anticoagulant activity. Each milligram of protamine neutralizes approximately 90 units of heparin derived from bovine lung tissue or 115 units of heparin derived from porcine intestinal mucosa. Protamine, however, cannot completely neutralize the anticoagulant activities of low molecular weight heparins (Lechner et al., 1995; Ryn-McKenna et al., 1990; Harenberg et al., 1985; Diness and Ostergaard, 1986; Wakefield et al., 1994), apparently due to an insufficient binding affinity between protamine and LMWH.
In addition to its function as a heparin antagonist, protamine also finds another major pharmacological application. It prolongs the adsorption of insulin, and is therefore combined with insulin to formulate protamine zinc insulin (PZI) and neutral protamine Hagedorn (NPH) insulin. Such formulations allow insulin-dependent diabetic patients to achieve euglycemia with less frequent insulin injections.
However, despite its nearly universal use in clinical practice, current formulations of protamine are nevertheless toxic. Protamine toxicity ranges from mild hypotension (Katz et al., 1987; Ovrum et al., 1992; Kirklin et al., 1986), to severe systemic vascular collapse requiring prompt intervention (Lowenstein et al., 1983; Just-Viera et al., 1984; Hurby et al., 1995), or idiosyncratic fatal cardiac arrest (Olinger et al., 1980; Cobb and Fung, 1982; Sharath et al., 1985; Neidhart et al., 1992).
The protamine toxicity is mediated by several pathways: (i) non-immunological pathway; (ii)

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