Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1995-05-30
2001-09-18
Sayala, Chhaya D. (Department: 1761)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S021800, C530S395000
Reexamination Certificate
active
06291427
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to an anticoagulant combination of LACI and sulfated polysaccharides and, more particularly, to a combination of LACI and heparin or similar such anticoagulant sulfated polysaccharides which exerts a synergistic anticoagulant action in whole plasma.
Blood clotting can be activated via the intrinsic or the extrinsic pathways. The intrinsic pathway begins with the contact phase which involves the interaction of factor XII, kallikrein, high molecular weight kininogen, a foreign surface and factor XI. The product of this reaction, factor XI
a
, converts factor IX to factor IX
a
, which subsequently hydrolyzes factor X to factor X
a
in the presence of activated factor VIII, phospholipid, and calcium. Alternatively, the extrinsic pathway is initiated when plasma factor VII/VII
a
, binds to tissue factor (TF; thromboplastin) to form a complex which proteolytically activates factors IX and X. Once factor X
a
is formed, either via the intrinsic or the extrinsic pathway, it can bind factor V
a
, phospholipid, and calcium to form the prothrombinase complex which converts prothrombin to thrombin. Ultimately, thrombin causes the fibrin clot to form.
Heparin has been widely used as an anticoagulant in clinical conditions. The anticoagulant effect of heparin is to a large extent a direct consequence of its catalytic action on the inhibition of thrombin by antithrombin III, and to a lesser extent its catalytic action on the inhibition by antithrombin III of other coagulation proteases including factors XII
a
, XI
a
, IX
a
, X
a
and kallikrein (1-4). In the absence of heparin, antithrombin III does not inhibit factor VII
a
(5-8). In the presence of heparin, factor VII, was reported to be resistant to inhibition (6) or inhibited 50% by antithrombin III in 11 min (7), 75-90 min (8) or 6 hours (5). Thus the rate of factor VII, inhibition by antithrombin III is so slow that antithrombin III is unlikely a physiological regulator of the TF/factor VII pathway in the presence or the absence of heparin (9). In addition to the antithrombin III-dependent inhibition of proteases of the intrinsic pathway, heparin can also exert anticoagulant action by displacing factor X
a
and prothrombin from the prothrombinase complex in an antithrombin III-independent fashion (10, 11).
In the past few years evidence has accumulated that regulation of the extrinsic pathway may primarily involve a plasma-derived protein called lipoprotein-associated coagulation inhibitor (LACI) (12). This protein also has been referred to as extrinsic pathway inhibitor (EPI) (13), or tissue factor inhibitor (TFI) (14). The inhibitor is capable of complexing with factor X
a
directly, and inhibits TF activity by formation of an inert TF/factor VII
a
/factor X
a
/Ca
2+
/inhibitor complex (12). Following the purification of apparently related inhibitor from Hep G2 hepatoma (14), the cDNA coding for the protein was subsequently cloned (15). Recently, expression of recombinant protein has generated large quantity of protein for in vitro and in vivo use.
The isolation of LACI from the conditioned media of Hep G2 cells, SK-Hep-1 cells, and Chang liver cells also is disclosed in co-pending application Ser. No. 07/77,366, filed Jul. 23, 1987, and the cloning of the cDNA coding for the LACI protein also is disclosed in co-pending application Ser. No. 07/123,753, filed Nov. 23, 1987, now allowed.
References cited herein by numbers in parentheses are listed hereinbelow.
BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to a novel anticoagulant combination of lipoprotein-associated coagulation inhibitor (LACI) and sulfated polysaccharides. It has been surprisingly found that this combination exerts a synergistic anticoagulant action in whole plasma.
In a preferred embodiment of the invention, LACI and heparin cause a greatly enhanced anticoagulation compared to either LACI or heparin alone. Many related sulfated polysaccharides having known anticoagulant activity were also found to enhance the LACI-dependent inhibition of TF-induced clotting. By weight, the relative potencies of these compounds are in the following order: low molecular weight heparin (mean M
r
=5,100)>unfractionated heparin>low molecular weight heparin (mean M
r
=3,700)>pentosan polysulfate>dermatan sulfate>dextran sulfate>heparan sulfate.
Because of the unique mechanism and ability of LACI in the inhibition of TF-induced coagulation, LACI has been described heretofor as a potential therapeutic protein for the treatment/prevention of thrombotic diseases. The synergistic use of heparin and LACI in combination as described herein for therapeutic applications thus is highly attractive for the following reasons: first, heparin is widely available and may reduce the amount of LACI required for treatment by potentiating the LACI function; second, heparin and LACI in combination inhibit both the intrinsic and extrinsic pathways of coagulation; and third, the combination may be effective in clinical conditions where heparin alone is not sufficient, e.g. disseminated intravascular coagulation where TF may be generated in large amounts.
The dosages of the LACI and sulfated polysaccharides used for inhibiting coagulation preferably are small but effective amounts for producing a synergistic anticoagulation result. Use of from about 0.1 to about 4 units of heparin per ml of plasma in combination with from about 0.1 &mgr;g to about 5 &mgr;g of LACI per ml of plasma is preferred for the synergistic anticoagulant activity. Other sulfated polysaccharides can also be used in various amounts and proportions with LACI to produce synergistic anticoagulant effects. Use of the following amounts, respectively, of these other sulfated polysacharides with from about 0.1 to about 5 &mgr;g of LACI are preferred for synergistic anticoagulant activity:
0.2-2 &mgr;g/ml low molecular weight heparin (mean M
r
=5,100)
1-10 &mgr;g/ml low molecular weight heparin (mean M
r
=3,700),
4.5-45 &mgr;g/ml pentosan polysulfate,
34-340 &mgr;g/ml dermatan sulfate,
50-500 &mgr;g/ml dextran sulfate (mean M
r
=6,000-8,000), and
100-1,000 &mgr;g/ml heparan sulfate.
As used herein, LACI is defined to mean lipoprotein-associated coagulation inhibitor as described by Wun et al.,
J. Biol. Chem
. 263, 6001-6004 (1988). LACI can be isolated from various known sources, e.g., the conditioned media of cultured liver cells such as Hep G2 cells, SK hepatoma cells and Chang liver cells, or produced by recombinant DNA procedures. Although specific methods of isolation or production of LACI are described herein, it will be understood that the invention is not limited to any particular source of the LACI.
As used herein, one unit of heparin is defined to mean one U.S.P. (United States Pharmacopoeia) unit. The U.S.P. unit of heparin is that quantity which will prevent 1.0 ml of citrated sheep plasma from clotting for one hour after the addition of 0.2 ml of a 1:1000 CaCl
2
solution. Heparin is generally obtained by isolation from mammalian tissues containing mast cells such as the liver and lung. As used herein, the term “heparin” also is meant to include the pharmaceutically acceptable water soluble salts thereof, e.g., the sodium salt. Suitable examples of commercially available heparin sodium products are Lipo-Hepins® (Riker Laboratories), Liquaemin® Sodium (Organon), and Panheprin® (Abbott Laboratories).
REFERENCES:
patent: 4379142 (1983-04-01), Port et al.
patent: 91/19514 (1991-12-01), None
Wun, J. Biol. Chem. 263, 6001-6004 (1988).
Broze et al., Blood 71, 335-343 (988).
Broze & Miletich, Proc. Natl. Acad. Sci. 84, 1886-1890 (1987).
Rapapport, Blood 73, 359-365 (1989).
Goodman and Gilman's “The Pharmaceutical Bases of Therapeutics,” Ch. 58, pp 1348-1353, 6th ed. 1980, section on “Heparin”.
The Merck Index, No. 4571, 11th ed. 1989.
Girard et al., Science 248, 1421-1424 (1990).
G.D. Searle & Co.
Meyer Scott J.
Sayala Chhaya D.
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