Drug – bio-affecting and body treating compositions – Extract – body fluid – or cellular material of undetermined... – Blood
Reexamination Certificate
2000-02-10
2003-06-17
Meller, Michael V. (Department: 1651)
Drug, bio-affecting and body treating compositions
Extract, body fluid, or cellular material of undetermined...
Blood
C435S212000, C435S183000, C435S214000, C424S094640
Reexamination Certificate
active
06579537
ABSTRACT:
CROSS REFERENCES TO RELATED APPLICATIONS
The present application claims priority to Austrian application number A 206/99, filed Feb. 12, 1999.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable
BACKGROUND OF THE INVENTION
The invention relates to a method for producing protein compositions comprising fibronectin and fibrinogen and, optionally, further ingredients as well as to protein compositions obtainable according to this method.
Tissue adhesives based on fibrinogen (“fibrin adhesives”) have been known for a long time. They serve for a seamless or suture-supporting connection of human or animal tissues or organ parts, for sealing wounds, haemostasis and assisting wound healing.
Their mode of action is based on an imitation of the final phase of blood coagulation.
By the action of thrombin, (soluble) fibrinogen at first is converted into fibrin monomers which aggregate spontaneously and form a sticky mass, a so-called fibrin clot. Simultaneously, factor XIII (F XIII) present is activated by thrombin in the presence of calcium ions to factor XIIIa. By the latter, the aggregated fibrin monomers and also fibronectin possibly present are cross-linked to a high polymer by new peptide bonds forming. By this cross-linking reaction, the strength of the clot formed is substantially increased. Generally, the clot adheres well to wound and tissue surfaces, which i.a. leads to the adhesive and haemostatic effect.
Therefore, fibrin adhesives frequently are used as two-component adhesives which comprise the fibrinogen component together with a thrombin solution which additionally contains calcium ions.
A particular advantage of a fibrin adhesive consists in that the latter does not remain at its site of application as a foreign body, but is completely resorbed just as in natural wound healing, and is replaced by newly formed tissue. Various cells, such as, e.g., macrophages and, subsequently, fibroblasts migrate into the clot, lyse and resorb the clot material and form new tissue.
Although the complicated procedures of wound healing so far by no means have become completely clear, it is considered to be certain that the presence of fibronectin in the clot is of crucial importance for the growing in of cells and thus for wound healing.
A fibrin clot of optimum composition threrefore should also comprise a content of fibronectin in addition to its main component, fibrinogen.
Although the mode of action of fibrin adhesives substantially corresponds to the natural processes of blood coagulation, for a sufficient efficacy (adhesive strength, haemostatic effect), a substantially higher concentration of the active components (in particular, of fibrinogen) is necessary than is present in blood, the fibrinogen concentration of human blood amounting to approximately 2.5-3 mg/ml.
It has been reported that by means of PEG precipitation a fibrinogen solution could be obtained with which a satisfactory adhesive strength could be attained already at fibrinogen concentrations of below 30 mg/ml in an artificial test system (WO 92/13495), yet with this, for many purposes of tissue adhesion a high density of the fibrin network (and the latter is substantially only attainable by high fibrinogen concentrations in the tissue adhesive) cannot be ensured,
To ensure an optimum efficacy, therefore, the fibrinogen content in a fibrin adhesive should be at least 70 mg/ml. The production of such concentrated ready-to-use fibrinogen or fibrin adhesive solutions, respectively, does, however, involve some difficulties:
Since the ready-to-use solutions are not storage-stable over longer periods of time, they have to be prepared upon demand either by reconstitution from lyophilized preparations or by thawing of liquid-deep frozen solutions.
Because of the relatively poor solubility of fibrinogen and the simultaneously required high fibrinogen concentration in an effective fibrin adhesive, in general this is still more cumbersome and time-consuming than desired by users thereof, despite diverse proposals for improvements. It is understandable that particularly in the field of emergency surgery, a particularly rapid and simple availability of a fibrin adhesive is required.
Moreover, concentrated fibrin adhesive solutions generally are highly viscous due to their high fibrinogen concentrations. A relatively low viscosity, however, is desirable not only for easier handling, but also for specific modes of application of a fibrin adhesive, e.g. when applying it by means of spray devices (such as, e.g., Duploject® with the associated spray set), or by means of a catheter.
Both requirements, i.e. rapid availability and low viscosity of the ready-to-use fibrin adhesive solutions, are even more difficult to meet if preparation thereof (dissolution or thawing, respectively) is to be effected without further auxiliary means, such as heating and/or stirring equipment, at room temperature, and if the fibrin adhesive preparations additionally contain high-molecular substances, in particular fibronectin. For also fibronectin—particularly in combination with fibrinogen—is relatively difficult to dissolve and generally leads to an even poorer solubility and an increased viscosity of fibrin adhesives.
Methods for producing fibrinogen-containing preparations which can be used as tissue adhesives comprise i.a. their production from cryoprecipitate, optionally with further washing and precipitation steps with ethanol, ammonium sulphate, polyethylene glycol, glycine or &bgr;-alanine, and their production from plasma within the scope of the known plasma fractionation methods, respectively (cf., e.g., “Methods of plasma protein fractionation”, 1980, ed.: Curling, Academic Press, pp. 3-15, 33-36 and 57-74, or Blomb{overscore (a)}ck B. and M., “Purification of human and bovine fibrinogen”, Arkiv Kemi 10, 1959, p. 415 f.).
In the prior art, also various suggestions have been made to reduce the viscosity of highly concentrated fibrinogen solutions. Thus, e.g., the addition of solubilizers, such as substances containing urea or guanidine residue, e.g. arginine (cf. DE 3203775-A1), or the addition of unphysiologically high salt concentrations has been known. However, it has been shown that such tissue adhesives have cytotoxic and proliferation-inhibiting properties, respectively (Redl et al., Med. Welt 36, 1985, pp. 769-776)
According to EP 0 804 933, the addition of substances improving the solubility of fibrinogen has been suggested. Such substances are, e.g., vitamins, aromatic compounds, such as compounds derived from benzene or phenol, or those derived from heterocyclic compounds, such as piperidine, pyridine or pyrimidine.
Thus, it is an object of the present invention to overcome the drawbacks of known preparations, and to further improve the known preparations, respectively, and to provide protein compositions comprising a high fibrinogen content and an easily adjustable ratio of fibrinogen to fibronectin as well as, optionally, further ingredients, which are, e.g., suitable for an improved preparation of ready-to-use tissue adhesives, while particularly maintaining properties, such as a good cell compatibility, or the formation of a physiological fibrin structure after mixing with a thrombin solution. At the same time, also the viscosity properties of such protein compositions or pharmaceutical preparations are to be improved.
A further object of the present invention is to provide a simplified and more rapid method of producing such protein compositions. In particular, it shall also be possible to easily carry out the method on an industrial scale.
SUMMARY OF THE INVENTION
According to the invention, these objects are achieved by a method for producing protein compositions which comprise fibrinogen and fibronectin, which method is characterized in that a starting solution comprising fibrinogen and fibronectin is treated with a precipitating composition which comprises two different components that modify the solubility of fibrinogen and/or fibronectin, so that in a single-step precipitation a fibrinogen and fibro
Broermann Ralf
Seelich Thomas
Baxter Aktiengesellschaft
Guthrie Janice
Meller Michael V.
Parent Annette S.
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