Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Transferases
Reexamination Certificate
1999-01-20
2001-07-31
Naff, David M. (Department: 1651)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Transferases
C424S059000, C424S094630, C424S401000, C435S016000, C435S177000, C435S193000, C514S002600, C530S402000, C530S812000
Reexamination Certificate
active
06267957
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the linkage of agents to tissue by transglutaminase and involves methods, products and kits relating thereto.
BACKGROUND OF THE INVENTION
Transglutaminases are a family of calcium-dependent enzymes mediating covalent cross-linking reactions between specific peptide bound (4-glutamyl residues and various primary amino groups of peptide-bound lysines or polyamines, acting as amine donor substrates (Davies, et al.,
Adv. Exp. Med. Biol.
250, 391-401, 1988). These enzymes stabilize biological structures via the formation of isopeptide cross-links. In mammals, at least five enzymatically active transglutaminases have been identified, cloned and sequenced. The number of proteins acting as glutaminyl substrates for transglutaminases is restricted, and no obvious consensus sequence around these substrates' glutamines has been found.
Three main lines of investigation have been conducted surrounding transglutaminases. These enzymes have been used to label membrane proteins and, in the absence of exogenous amines, to catalyze the formation of (4-glutamyl)-lysyl cross-links between them. The labeling is quite specific and can be carried out under mild (physiological) reaction conditions. Thus, for example, transglutaminases were used to study rhodopsin in the intact disc membrane, as only residues of rhodopsin located in the aqueous phase in the exposed side of the disc membranes were expected to be labeled. In these experiments, rhodopsin was labeled by transglutaminase using putrescine and dansylcadaverine as detectable substrates.
The role of transglutaminases in living cells also has been studied, for example, using the cell-penetrating labeled substrate fluoresceincadaverine for detecting amine acceptor protein substrates accessible to active transglutaminase in living cells. A similar strategy was employed using 5-(biotinamido)-pentylamine as a label. Such labeled substrates can be detected directly, for example by fluorescence, or can be detected indirectly, for example using antibodies, to identify native proteins to which the labeled substrate has been covalently attached by transglutaminase. See, Pober, J. S. et al.,
Biochemistry
, Vol. 17, No. 11:2163-2169 (1978); Lajemi, M. et al.,
Histochemical Journal
29:593-606 (1997).
More recently, an investigation was carried out to determine if polyglutamine is a transglutaminase substrate. It was determined that as long as polypeptides including stretches of polyglutamine are rendered sufficiently soluble by the flanking residues, all were excellent substrates of transglutaminase. Based upon these studies, it was speculated that certain diseases such as Spinocerebellar ataxia Type I, Machado-Joseph disease, and Dentato-Rubral pallidoluysian atrophy which are characterized by proteins having polyglutamine stretches, may arise as a result of aggregation of such proteins acted upon by a transglutaminase.
It also is described in U.S. Pat. No. 5,525,336 (the disclosure of which is incorporated herein by reference in its entirety) that transglutaminases and corneocyte proteins, the natural substrates of transglutaminases, can be used together as cosmetic treatments to cross-link preparations of corneocyte proteins to the outer layer of skin, hair or nails to form a protective layer on the skin, hair or nails.
U.S. Pat. No. 5,490,980 describes selecting agents having or modifying agents to have an aliphatic amine, and then attaching those agents to skin, hair or nails using transglutaminase. While the idea was sound in principle, in practice the '980 applicants achieved results that were barely above background. (See Example Section of '980 patent). An aliphatic amine was applied in the examples as a single linking molecule or prophetically in clusters (according to a formula in the '980 patent). In selecting the amine moiety of the pair of known transglutaminase substrate moieties, the '980 patent taught away from using the carboxamide substrate moiety.
SUMMARY OF THE INVENTION
It has been discovered, surprisingly, that certain substrates of transglutaminase are particularly desirable for use as linking molecules to attach agents to proteinaceous material such as body tissue. It also has been discovered that molecules, including native peptides and conjugates according to the invention, can be screened to determine those that can be substrates of transglutaminases, and then such molecules can be attached to body tissue. Method of attaching agents to body tissue and methods of screening molecules using transglutaminase are provided. In addition, compositions of matter suitable as substrates for transglutaminase and kits containing such molecules together with transglutaminase are provided.
According to one aspect of the invention, a method is provided for attaching a non-corneocyte protein, non-labeling agent to a body tissue. A conjugate of the agent and a linking molecule having a carboxamide, the linking molecule being a carboxamide-bearing substrate of transglutaninase, is applied to the body tissue. Transglutaminase also is applied to the body tissue, in an amount effective for cross-linking the conjugate to the body tissue via the linking molecule. The cross-linking then is allowed to occur. In certain embodiments the agent is not fibronectin (i.e., a nonfibronectin agent). In certain embodiments the agent is not an extracellular matrix protein (i.e. a non-extracellular protein agent). Preferably the linking molecule comprises a polymer of at least 3, 4 or 5 linked units, each unit being a carboxamide substrate of transglutaminase.
According to another aspect of the invention, a method is provided for attaching a non-corneocyte protein, non-labeling agent to a body tissue. The method involves selecting a non-corneocyte protein, non-labeling agent that is a carboxamide substrate for transglutaminase. The agent, in an isolated form, then is applied to the body tissue in the presence of a sufficient amount of transglutaminase to cross-link the isolated agent to the body tissue. The cross-linking then is allowed to occur. In this embodiment, the agent can be a conjugate of a native, non-corneocyte, non-labeling active agent and a linking molecule not native to the agent. It also is the case that the agent can be a native agent free of conjugation with groups not native to the agent. The agent in certain embodiments is a non-extracellular matrix protein agent.
In either of the foregoing embodiments, the linking molecule can be any number of a variety of molecules. In some embodiments, the linking molecule is at least one glutamine. The linking molecule, likewise, can be one bearing multiple reactive carboxamides, such as two or more contiguous linked L or D glutamines. D glutamines have the advantage of being physiologically more stable than L glutamines. In a preferred embodiment, the linking molecule is a polymer rich in carboxamides that are substrates of transglutaminase, such as a polymer rich in glutamine. The linking molecule also can be a polymer rich in both carboxamides and aliphatic amines, such as one rich in both glutamine and lysine. A polymer rich in glutamine, lysine, or glutamine and lysine is a molecule wherein at least 20% of the units of the polymer are glutamine, lysine or glutamine and lysine, respectively or wherein the molecule includes at least three, preferably four and most preferably five contiguous, linked transglutamines substrates, preferably linked by peptide bonds. A polymer rich in glutamines, lysines or glutamines and lysines, can be a polymer that contains at least 30% glutamines, lysines or glutamines and lysines, at least 40% glutamines, lysines or glutamines and lysines, or even 50% or more glutamines, or glutamines and lysines.
In certain preferred embodiments, the methods described above involve first preparing the body tissue for the attachment of the agent to the body tissue. In one important embodiment, a separate “complementary” linking molecule that is attachable to the linking molecule by transglutaminase is first att
Compton Bruce J.
Corey George D.
Dijan Philippe
Green Howard
Naff David M.
Wolf Greenfield & Sacks P.C.
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