A fibrin/fibrinogen binding conjugate for forming a depot for the sustained release of a pharmaceutically active substance from a fibrin clot. The conjugate comprises a fibrin/fibrinogen binding moiety bound to a pharmaceutically active substance either directly or via an intervening substance capturing moiety such as an antibody. The conjugate can also be a recombinant fusion protein comprising a fibrin/fibrinogen binding moiety such as VEGF.sub.165 C-terminal domain fused to a wound-healing substance such as leptin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a biomatrix containing a conjugate which is able to bind to a fibrin gel and preferably to fibrinogen as well. The conjugate's binding affinity to fibrinogen is transferred to the binding of fibrin after cleavage of the fibrinogen to fibrin.

One conjugate according to the present invention comprises: a binding moiety which binds to fibrin/fibrinogen, a substance capturing moiety capable of reversibly binding to a pharmaceutically active substance, and the pharmaceutically active substance. According to the present invention the fibrin/fibrinogen-binding moiety is bound to the substance capturing moiety, preferably covalently. For example a fibrin/fibrinogen-binding protein or a part thereof which binds to fibrin/fibrinogen may be bound or coupled to a substance capturing moiety. This coupling may be accomplished by chemical linkers, by recombinant DNA technology, by peptide synthesis or combinations of these techniques.

Another conjugate of the invention comprises a fibrin/fibrinogen binding moiety covalently linked directly to a pharmaceutically active substance, without an intervening substance capturing moiety. A depot formed with this conjugate provides sustained release of the pharmaceutically active substance based on the release kinetics of the fibrin/fibrinogen binding moiety from fibrin, as well as the natural dissolution of the fibrin clot over time. In this instance, the pharmaceutically active substance retains its activity even when it remains covalently linked to the fibrin/fibrinogen binding moiety.

The fibrin/fibrinogen-binding moiety may be derived from naturally occurring (e.g. physiological) binding proteins, such as thrombin, fibronectin, bacterial fibrinogen binding proteins, basic fibroblast growth factor, integrins, tissue-type plasminogen activator, VEGF.sub.165, and similar proteins exhibiting at least one fibrin/fibrinogen-binding moiety.

In a further embodiment a nucleic acid, particularly DNA may be used as a fibrin/fibrinogen-binding moiety. This DNA need not have a coding function and therefore can even be of random sequence although care has to be taken to avoid a possibly inflammatory motif, especially a CpG-motif, within the sequence. For the purposes of the present invention DNA is used in any form, which means single- or double stranded DNA, linear or circular DNA, as a fibrin/fibrinogen-binding moiety.

For the present conjugate, proteins may either be used in their physiological form or in a processed form. For example, such physiological binding partners may be processed by known biochemical techniques, in order to provide at least the fibrin/fibrinogen-binding parts of these proteins. Alternatively, the parts known to bind to fibrin/fibrinogen can also be provided by recombinant DNA technology. For many fibrin/fibrinogen-binding proteins a three-dimensional structure has been described or proposed, enabling one of skill in the art to select those parts of these proteins which are relevant for fibrin/fibrinogen-binding for use in the present invention. Other substances with binding affinity for fibrin/fibrinogen may be analyzed for their putative fibrin/fibrinogen-binding sites based on known three-dimensional models of the above mentioned proteins, e.g. by sequence analysis, if such substances are proteins or protein derivatives or selection by phage display.

The choice of the substance capturing moiety or the directly bound pharmaceutically active substance is essentially dependent on the pharmaceutically active substance to be administered by the fibrin depot. Suitable pairs of substance capturing moieties and pharmaceutically active substances are known in the art.

For example a substance-capturing moiety may be an antibody, a receptor or a part thereof, which specifically recognizes and reversibly binds the pharmaceutically active substance of interest (e.g. as an antigen or ligand). Herein, the term "antibody" includes a complete antibody of any class, comprising the constant domain as well as the variable antigen binding domain, as well as parts of antibodies or antibody derived molecules, e.g. fragments or recombinant constructs. Indeed, most of the parts of such "classical" antibodies may be omitted as long as the essential moiety, namely the variable binding region, which allows the binding of the pharmaceutically active substance, is present.

A further example of a substance capturing moiety may be the group of antibody binding molecules, e.g. bacterial proteins like protein A or protein G or Fc-receptor of macrophages, as well as fragments or recombinant constructs thereof.

According to a preferred-embodiment of the present invention monoclonal antibodies or the antigen binding regions of monoclonal antibodies are used as substance capturing moieties. Further, coupling of such a monoclonal antibody or parts thereof to a fibrin/fibrinogen-binding moiety, especially a fibrin binding protein, may be established by classical protein chemistry.

The present invention may be adapted for all pharmaceutically active substances possible, especially for those for which a suitable binding partner is already known (e.g. antigen/antibody, receptor/ligand, complex partners). The binding partner to be applied as a drug is bound to the conjugate only via its individual corresponding binding partner, the latter being covalently coupled to the fibrin/fibrinogen-binding moiety.

Herein the term "reversible binding" refers to non-covalent binding based on electrostatic forces which confer an affinity between the substance capturing moiety and the pharmaceutically active substance, whereby the pharmaceutically active substance is released over time to diffuse from the fibrin clot.

Preferred pharmaceutically active substances to be used in the present conjugate are antibiotics, growth factors, receptors for tissue components, tissue adhesive substances, anti-tumor agents, cell adhesive substances, nucleic acids, plasma proteins, anti-proteases, fibrinolysis-inhibitors, hormones, heparinoids, wound-healing substances and mixtures thereof. When the pharmaceutically active substance is a fibrinolysis inhibitor such as aprotinin, as part of the inventive conjugate, the clot to which the conjugate is bound will last longer than a clot which merely contains free aprotinin, which would readily diffuse out of the clot.

These substances may either be directly pharmaceutically active or allow an improved action of another pharmaceutically active substance, which may be applied simultaneously or separately with the present drug depot. For example, receptors for tissue components or tissue adhesive substances may be applied which allow an improved performance of a tissue adhesive based on fibrinogen. Other examples which change the adhesive properties of a tissue adhesive are substances which may be provided with the present conjugate. If applied together with a "classical" tissue adhesive, the presence of such pharmaceutically active substances which have an influence on the adhesion properties may influence the adhesive or non-adhesive capacity of the fibrinogen tissue adhesive to specific tissues or cells. Other substances, such as nucleic acids or anti-tumor agents may also be applied together with a specific fibrin/fibrinogen basis to form a depot for these substances at the site necessary for a desired effect. Also substances useful for image based diagnostic methods e.g. for X-rays or magnetic resonance induction (MRI) or colors may be used according to the present invention.

According to a preferred embodiment of the present invention the conjugate or the bifunctional molecule is designed for the incorporation in a "classical" tissue adhesive system. Such a system usually comprises a fibrinogen and a thrombin containing preparation similar to a "one-" or "two component" glue resulting in fibrin formation at the site of application or a preformed fibrin preparation, e.g. a fibrin fleece. The formed fibrin clot or the fibrin fleece allows e.g. wound closure or tissue adhesion. Further ingredients in this system are e.g. Factor XIII (as a cross-linker), fibrinolysis-inhibitors, etc (see e.g. Fibrin Sealing in Surgical and Non-Surgical Fields, Schlag G., Redl H. editors, Vols. 1 9).

The fibrin/fibrinogen-binding moiety and the substance-capturing moiety are preferably covalently bound by a linker substance, especially linker substances which are used and have proven to be successfully applied in protein chemistry. This preferred embodiment is especially suited if enhanced flexibility of the moieties is desired.

Although the pharmaceutically active form of the conjugate according to the present invention comprises the pharmaceutically active substance, the present invention also relates to the conjugate without the drug. Such a "naked" conjugate may be easily transformed into a pharmaceutically active form by "loading" the conjugate comprising the fibrin/fibrinogen-binding moiety and the substance-capturing moiety with the individual drug wherefore the substance-capturing moiety has been designed.

A specific embodiment of the present invention relates to a conjugate wherein the drug to be applied has been designed to carry a fibrin/fibrinogen containing moiety. According to this aspect of the present invention, the substance-capturing moiety may be omitted. Also this conjugate may be designed by protein chemistry, peptide synthesis and/or recombinant technology by combining a fibrin/fibrinogen-binding moiety with the pharmaceutically active substance, e.g. by direct covalent binding or by binding with suitable linker substances. Also these conjugates, which do not need a separate "loading" with the pharmaceutically active substance, may be used in a common tissue adhesive system as described above.

A preferred fibrin/fibrinogen binding conjugate is based on the present inventors' discovery that the C-terminal domain of VEGF.sub.165 is responsible for the fibrin-binding capacity of that molecule. Herein, the term "C-terminal domain of VEGF.sub.165" refers to amino acid residues C104-R165 as depicted in U.S. Pat. No. 5,332,671, FIGS. 10a and 10b (Ferrara and Leung). VEGF.sub.165 is further described in Tischer et al, J. Biol. Chem. (1991) 266:11947 11954; Sahni et al, Blood (2000) 96:3772 3778; and Houck et al, J. Biol.Chem. (1992) 267:26031 26037). The fibrin-binding domain of this conjugate is not limited to the natural amino acid sequence coded for by exons 5, 7, and 8, as depicted in FIG. 10 of the present document. The fibrin-binding properties of the resulting fusion protein can be altered in order to increase or decrease the release kinetics from the fibrin depot by adding, deleting, or mutating specific amino acid residues in the VEGF.sub.165 C-terminal domain. VEGF.sub.165 also contains a natural plasmin cleavage site at the beginning of its C-terminal domain. Thus the C-terminal domain of VEGF.sub.165 can be used to form a fusion protein with a pharmaceutically active substance, without the need for a "substance-binding moiety" between the fibrin binding moiety and the pharmaceutically active substance. When the conjugate is incorporated into a fibrin clot at a wound site, the natural plasminogen in the patient's plasma entering the wound is converted to plasmin, which in turn cleaves the fusion protein to release the pharmaceutically active substance, allowing its diffusion from the clot. Another mechanism for release is based on the dissociation rate of the VEGF.sub.165 C-terminal domain from fibrin; according to this mechanism, the entire conjugate is released and diffuses from the clot, allowing the pharmaceutical substance to act at the wound site, outside the clot. The following are examples of pharmaceutically active substances which can be fused to the C-terminal moiety of VEGF.sub.165: cytokines, growth factors, and wound-healing substances such as leptin (Frank et al, J. Clin. Invest. (2000) 106:501 509; Sierra-Honigmann et al., Science (1998) 281:1683 1686), IL-8, MCP-1, and PF-4. antibiotic peptides such as magainins, defensins, and granulysin. fibrinolysis inhibitors such as aprotinin and Kunitz domains of human lipoprotein-associated coagulation inhibitor (LACI-D1; Markland et al., Biochemistry (1996) 35:8045 8057).

The pharmaceutically active substance thus released can serve to direct the growth, migration, and differentiation of specific cell types, thus enhancing wound healing and neovascularization during tissue repair.

According. to another aspect the present invention relates to a kit for forming a depot for a pharmaceutically active substance comprising a tissue adhesive based on fibrinogen and a conjugate according to the present invention. The conjugate may be provided in a separate form ready to be mixed before medical use. The "ready to use" mixture of the tissue adhesive based on fibrinogen and the conjugate according to the present invention may be applied with means and methods as already. known in the art for "classical" tissue adhesives, especially with the fibrinogen component of such adhesives. This fibrinogen component may be mixed in a known way with a component containing an activity for processing fibrinogen to fibrin, preferably a thrombin preparation.

A kit according to the present invention may therefore also contain suitable devices for administering the tissue adhesive and the conjugate and optionally the fibrinogen to fibrin processing activity. Examples for such devices are described in EP 0 037 393 A, EP 0 315 222 A, EP 0 156 098 A, EP 0 210 160 A and EP 0 292 472 A, which are incorporated herein by reference.

According to another aspect the present invention relates to a method for producing a depot of a pharmaceutically active substance comprising providing a conjugate according to the present invention, administering this conjugate at a depot site together with a fibrinogen preparation, allowing processing of said fibrinogen to fibrin whereby a fibrin clot is formed, and allowing binding of the conjugate to said fibrinogen or the fibrin clot formed.

Processing of the fibrinogen to fibrin may either be performed by thrombin already being present at the site of administration or by an exogenously added fibrinogen processing activity. Apart from thrombin or thrombin derived proteases, other proteases such as streptylase, protease III and venom proteases like e.g. baxotropin, may be used for cleaving the fibrinogen molecule. The binding of the conjugate to fibrin/fibrinogen may take place after forming of the fibrin clot. However, it is preferred to allow this binding process at an earlier stage, e.g. during the fibrinogen processing step or (most preferred) even before, so that binding of the conjugate according to the present invention takes place at the fibrinogen level. This results in a fibrin depot which has a homogeneous distribution of conjugate throughout the whole depot. On the other hand, if the conjugate is intended to be located mainly on the surface of the fibrin depot, binding of the conjugate should be allowed after forming of the fibrin clot.

Another aspect of the present invention relates to a depot for a pharmaceutically active substance, comprising a conjugate according to the present invention and fibrin (e.g. a suitable fibrin matrix). Such a depot is e.g. obtainable by administration of a conjugate according to the present invention to a fibrin network base.

Yet still another object of the present invention is drawn to a method for treating a patient suffering from a pathological state, said pathological state being treatable with a pharmaceutically active substance, comprising administering to this patient an effective amount of a tissue adhesive based on fibrinogen and a conjugate according to the present invention.

Thereby a depot of the pharmaceutically active substance with suitable releasing properties is provided which allows a suitable treatment of the patient with the pharmaceutical substance without the need of continuously and separately providing this substance.
 

Claim 1 of 13 Claims

1. Fibrin/fibrinogen-binding conjugate comprising a fibrin/fibrinogen-binding moiety that is a nucleic acid, a substance capturing moiety capable of reversibly binding to a pharmaceutically active substance, wherein said fibrin/fibrinogen-binding moiety is bound to said substance capturing moiety.

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