Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
2000-12-14
2002-06-25
Dees, Jose′ G. (Department: 1616)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
Reexamination Certificate
active
06410260
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention describes various bioadhesive sealants. One is a hemostatic agent commonly known as a “fibrin glue” and is a novel fibrin glue that lacks fibrinogen. There is also provided a biosealant composition. Methods of use and manufacture of the fibrin glue and biosealant are also provided. The fibrin glue and biosealant compositions and methods of the present invention are suitable for arresting blood flow, maintaining hemostasis, and for accelerating and ameliorating the healing process after various types of surgical and nonsurgical procedures or wound healing in mammals, including humans.
2. Description of the Related Art
The use of fibrin glue has been explored in various surgical disciplines as reported by Lemer, R. & Binur, N. S.,
J. Surg. Res
., 48:165-181 (1990); Gibble, J. W. & Ness, P. M.,
Transfusion
30:741-747 (1990); Sierra, D. H.,
J. Biometer. Applic
. 7:309-352 (1993); Brennan, M.,
Blood Reviews
, 5:240-244 (1991); Dresdale, A., et al.,
Surgery
97:750-755 (1985); Sponitz, W., et al.,
Amer. Surg
., 59:460-462 (1987); Schlag, G. & Redl, H. (Eds), GYNECOLOGY AND OBSTETRICS-UROLOGY (1986); FIBRIN SEALANT IN OPERATIVE MEDICINE, vol 3. Springer Verlag (Berlin); and Burnou Radosevich, M. et al.,
Vox Sang
, 58:77-84 (1990).
For example,; surgeons, dentists and hematologists have reported that fibrin glue is an effective bioadhesive. Experience in animals and humans suggests that: an advantage of using fibrin glue rather than synthetic plastics (e.g., cyanoacrylate) or sutures is that fibrin glue promotes local coagulation, thereby preventing bleeding even in hemophiliacs. Fibrin glue also appears to support regrowth of new tissue and the extracellular matrix.
In the past, fibrin glue has been formed by mixing two components, exogenous human fibrinogen (obtained from a source other than the patient being treated, such as a freeze-dried plasma protein concentrate of fibrinogen/Factor XIII/fibronectin) and an activating enzyme, such as thrombin. Prior to use, the plasma protein concentrates were conventionally solubilized in the presence of calcium chloride. Thrombin induced activation of fibrinogen resulted in the formation of fibrin. Factor XIII and calcium participated in the cross-linking and stabilization of the fibrin to produce a tight mesh of polymeric fibrin glue. Applied to tissue, the fibrin clot adhered to the site of application. The rate of coagulation and mechanical properties of the clot were dependent on the concentration of fibrinogen and thrombin.
Traditional fibrin glue preparations are described in International Applications No. W093/05067 to Baxter International, Inc.; W092/13495 to Fibratek, Inc.; and W091/09641 to Cryolife, Inc.
Thrombin is a common physiological instigator of clotting. Thrombin from a number of mammalian sources, most commonly bovine, is routinely used in commercially-available fibrin glues. Human thrombin can be employed in the formulation of fibrin glue, as can other appropriate catalyzing enzymes, such as reptilase or selected venoms (Fenton II, J. W. et al.,
J Biol. Chem
., 252:3587-3598 (1977); Gaffney P. J. et al., Thrombos. Haemostas., 67:424-427 (1992); European Patent Application No. EP 0 439 156 A1., Stocker K., et al.,
Toxicon
, 20:265-273 (1982); and Pirkle H. & Stocker K.,
Thrombos. Haemiostas
. 65:444-450 (1991)).
Fibrinogen may be in an intimate admixture with other proteins that are typically found in uncoagulated whole blood, in platelet-rich plasma, in plasma, in cryoprecipitate, or in precipitates of plasma obtained by a method such as Cohn precipitations of plasma. Such additional protein components may include, for example, fibronectin, immunoglobulin, particularly, IgG, and plasminogen.
Thrombin is derived from blood plasma by the fractionation of plasma. Comprehensive reviews on the preparative techniques of each have been published and are the basis for most commercial plasma fractionation procedures used by those skilled in the art (Fenton II, J. W. supra; Gaffney P. J. supra; EP 0 439 156 A1; and U.S. Pat. No. 5,143,838).
However, the prior art fibrin glue compositions required the mixture of thrombin with exogenous fibrinogen to form an unwanted, premature fibrin clot, prior to the application of the fibrin glue to the tissue being treated. For example, U.S. Pat. No. 5,607,694 issued to Marx on Mar. 4, 1997, discloses a fibrin glue composition in which exogenous fibrinogen and thrombin are mixed together prior to the application of the mixture to the tissue being treated. Marx further teaches the addition of liposomes to the fibrin glue mixture for the delivery of agents to the tissue being treated.
Similarly, U.S. Pat. No. 5,290,552 issued to Sierra et al. on Mar. 1, 1994, discloses a surgical adhesive material comprising a composition of fibrinogen, collagen, thrombin and calcium, in which the thrombin is mixed with exogenous fibrinogen prior to the application of the fibrin glue to the tissue.
Therefore, there exists a need for a fibrin glue that can be applied directly to the tissue being treated to form a clot on contact without the use of added exogenous fibrinogen and without pre-mixing the fibrinogen to the fibrin glue.
SUMMARY OF THE PRESENT INVENTION
The invention is a novel fibrin glue without fibrinogen that has thrombo plastin, thrombin, and calcium, together in a pharmaceutically acceptable carrier, such as water.
The fibrin glue without fibrinogen of the invention may compoise of 0.0001 to 99.99% thromboplastin, 0.00001 to 10,000 U/ml thrombin and about 0.015-0.025 M calcium, but preferably, there is about 0.5-1.5% or about 1% thromboplastin, about 250-1000 U/ml thrombin, and 0.02 M calcium.
The fibrin glue without fibrinogen of the invention may contain other clotting factors, growth factors, antibiotics, trace metals, etc, as long as it contains no significant amounts of fibrinogen which would cause the premature formation of a clot. In a preferred embodiment, the fibrin glue also contains Factor VII, Factor IX, and Factor X. There can be about 0.001-1000 U off each of these Factors.
In yet another embodiment, the fibrin glue without fibrinogen is formulated in a form selected from the group consisting of a bandage, surgical dressing, wound packing, swab, liquid, aerosol, paste, ointment, f
6
am, gel, emulsion, powder and moldable form, and the like.
Methods of manufacturing the novel glue are also provided. For example, one method is: isolating prothrombin from blood, converting the isolated prothrombin to thrombin, isolating. thromboplastin from blood. Next, the method involves blending the prothrombin and/or thrombin F and thromboplastin plus calcium in a pharmaceutically acceptable carrier to produce the fibrin glue without fibrinogen. The prothrombin may be converted to thrombin before blending or after blending with the other components of the glue. In one specific method of isolating prothrombin from blood described herein, the procedure also produces Factor VII, Factor IX and Factor X. Thus, the fibrin glue of the invention also comprises at least these additional Factors without requiring additional preparative techniques.
In another embodiment of the invention, the method of manufacturing a fibrin glue without fibrinogen involves blending thrombin, thromboplastin and calcium in a pharmaceutically acceptable carrier to form a fibrin glue without fibrinogen. The method may also involve blending Factor VII, Factor IX and Factor X. The thrombin, thromboplastin, Factor VII, Factor IX and Factor X may be produced through recombinant DNA techniques or may be purified from blood or other suitable tissue source.
The invention also teaches methods of using the fibrin glue without fibrinogen. Because F the novel glue does not pre-clot and does not require the mixing of components prior to or simultaneously with use, it is particularly suitable for use in surgical procedures where the glue can be applied to an internal portion of a patient via a tube, such as an endoscope, or via a syringe, and the like. Altern
Dees Jose′ G.
George Konata M.
Miller & Martin LLP
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