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  Linkage of agents using microparticlesRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Enzyme Or Coenzyme Containing, Transferases (2. ), Lyase (4.), Isomerase (5.), Ligase (6.)The Patent Description & Claims data below is from USPTO Patent Application 20060110379. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application Ser. No. 09/620,783, filed Jul. 21, 2000, entitled "Linkage of Agents to Body Tissue Using Microparticles and Transglutaminase" (allowed) which is a continuation-in-part of U.S. Patent application Ser. No. 09/359,920, filed Jul. 22, 1999, entitled "Conjugates of Agents and Transglutaminase Substrate Linking Molecules" (allowed) which is a continuation-in-part of U.S. patent application Ser. No. 09/234,358, filed Jan. 20, 1999, entitled "Attaching Agents to Tissue with Transglutaminase and a Transglutaminase Substrate," now U.S. Pat. No. 6,267,957, which claims benefit to provisional application No. 60/071,908, filed Jan. 20, 1998, each of which is incorporated herein in its entirety. FIELD OF THE INVENTION [0002] This invention relates to the delivery of agents to tissue using microparticles and involves methods, products and kits relating thereto. BACKGROUND OF THE INVENTION [0003] Transglutaminases are a family of calcium-dependent enzymes mediating covalent cross-linking reactions between specific peptide bound .gamma.-glutamyl residues and various primary amino groups of aliphatic amines, 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. [0004] 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 (.gamma.-glutamine)-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. [0005] 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). [0006] 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. [0007] 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 transglutaminase 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. [0008] 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 [0009] The invention relates to microparticles and their use in delivering agents to tissues, preferably external surfaces such as skin, nails and hair. More specifically, the invention provides compositions, kits and methods for delivery of a variety of agents using microparticles which can be covalently linked to such tissues. [0010] In one aspect, the invention provides a method of treating a subject to attach microparticles to a skin surface of the subject. The method involves contacting the skin surface with microparticles having surface available transglutaminase substrate reactive groups (i.e., carboxamide-bearing substrates or transglutaminase or aliphatic amine-bearing substrates of transglutaminase that are available (for reaction) at the surface of a microparticle) in an amount sufficient to attach the microparticles to the skin surface in the presence of endogenous transglutaminase, and allowing the microparticles to remain in contact with the skin surface for a time sufficient to permit a layer of microparticles to covalently attach to the skin surface. [0011] In a related aspect, the invention provides a method similar to the preceding method except in the use of exogenous transglutaminase rather than endogenous transglutaminase. Thus, the method involves contacting the skin surface with microparticles having surface available transglutaminase substrate reactive groups in an amount sufficient to attach the microparticles to the skin surface in the presence of exogenous transglutaminase, applying exogenous transglutaminase to the skin surface, and allowing the microparticles and exogenous transglutaminase to remain in contact with the skin surface for a time sufficient to permit a layer of microparticles to covalently attach to the skin surface. [0012] In one embodiment, the surface available transglutaminase substrate reactive groups comprise aliphatic amines. In a related embodiment, the surface available transglutaminase reactive groups are lysines. In another embodiment, the surface available transglutaminase substrate reactive groups comprise carboxamides. In a related embodiment, the surface available transglutaminase substrate reactive groups are glutamines. The layer of microparticles may be planar or non-planar. [0013] In one embodiment, the microparticles further comprise an active agent. The active agent may be selected from the group consisting of a cosmetic agent, a bulking agent, a hair conditioning agent, a hair fixative, a sunscreen agent, a moisturizing agent, a depilatory agent, an anti-nerve gas agent, a film forming agent, a vitamin, an insect repellant, a coloring agent, a pharmaceutical agent, a ligand-receptor complex and a receptor of a ligand-receptor complex. In another embodiment, the active agent is not itself a substrate of transglutaminase. The active agent may be a non-nucleic acid active agent or it may be a non-protein active agent, but it is not so limited. [0014] In yet another embodiment, the microparticles may further comprise a synthetic polymer. In one embodiment, the synthetic polymer is latex. In another embodiment, it is polystyrene. In one instance, the microparticle is porous, while in another it is hollow (i.e., a microsphere or a microcapsule). In certain embodiments, the size of the microparticle is less than 5 .mu.m, or less than 1 .mu.m, or 100 nm to 500 nm, or less than 100 .mu.m, or 20 nm to 90 nm, or 20 nm to 35 nm, or less than 20 nm, or 1 nm to 10 nm, or 5 nm to 10 nm. In some embodiments, the microparticles enter the cornified layer of the skin but not the layer of living cells. However, in these latter embodiments, the agent contained within the microparticle may be able to enter the layer of living cells. [0015] In important embodiments, the microparticles are non-biodegradable. The microparticles preferably are water insoluble and more preferably, are detergent insoluble. [0016] In one embodiment, the transglutaminase substrate reactive groups are part of a polymer. The polymer may be a polymer of amino acid residues, non-amino acid residues, or a mixture of amino acid and non-amino acid residues, but it need not be so limited. In one embodiment, at least 50% of the residues in the polymer possess aliphatic amines or at least 50% possess carboxamides. In a related embodiment, at least 50% of the residues in the polymer are lysines or at least 50% are glutamines. The polymer may be covalently attached to the microparticle, but need not be. In one embodiment, the polymer is rich in aliphatic amines or rich in carboxamides, or rich in both, preferably at a surface available terminus or at a surface available loop. In another embodiment, the polymer is lysine-rich or glutamine-rich, or both, at a surface available terminus or at a surface available loop. In certain embodiments, the polymer comprises a polymer selected from the group consisting of at least two contiguous linked aliphatic amines or carboxamides, at least three contiguous linked aliphatic amines or carboxamides, at least four contiguous linked aliphatic amines or carboxamides, at least five contiguous linked aliphatic amines or carboxamides. In related embodiments, the polymer comprises a polymer selected from the group consisting of at least two contiguous linked lysines or glutamines, at least three contiguous linked lysines or glutamines, at least four contiguous linked lysines or glutamines, and at least five contiguous linked lysines or glutamines. In other embodiments, the polymer comprises a polymer selected from the group consisting of at least five contiguous linked aliphatic amines or carboxamides, at least ten contiguous linked aliphatic amines or carboxamides, at least fifteen contiguous linked aliphatic amines or carboxamides, at least twenty contiguous linked aliphatic amines or carboxamides, at least thirty, at least forty, at least fifty, at least 60 and at least 70 contiguous linked aliphatic amines or carboxamides. In other embodiments, the polymer comprises a polymer selected from the group consisting of at least five contiguous linked lysines or glutamines, at least ten contiguous linked lysines or glutamines, at least fifteen contiguous linked lysines or glutamines, and at least twenty contiguous linked lysines or glutamines. Many of the embodiments provided herein which relate to lysine or lysine-rich polymers apply equally to aliphatic amines and polymers rich in aliphatic amines, respectively. Similarly, many of the embodiments provided herein which relate to glutamine or glutamine-rich polymers apply equally to carboxamides and polymers rich in carboxamides. [0017] In yet another aspect of the invention, a composition is provided that is a microparticle, preferably non-biodegradable, comprising an active agent and a polymer rich in aliphatic amine transglutaminase substrates that are preferably surface available. In an important embodiment, the microparticle comprises an active agent and a lysine-rich polymer having transglutaminase substrate reactive groups, wherein the microparticle is non-biodegradable, and the transglutaminase substrate reactive groups are surface available. [0018] The invention further provides a composition comprising a microparticle that comprises an active agent and either a polymer rich in carboxamide transglutaminase substrates or a polymer rich in aliphatic amine transglutaminase substrates, wherein the transglutaminase substrates are preferably surface available. In one important embodiment, the microparticle comprises an active agent and a glutamine-rich polymer having transglutaminase substrate reactive groups, wherein the transglutaminase substrate reactive groups are surface available. In one embodiment, the microparticle comprises an active agent and a polymer rich in both aliphatic amines and carboxamides. Pharmaceutical preparations comprising a microparticle composition and a pharmaceutically acceptable carrier are also provided. [0019] In one embodiment, the transglutaminase substrate reactive groups are surface available in an amount sufficient to attach the microparticle to a skin surface in the presence of endogenous transglutaminase. In another embodiment, the transglutaminase substrate reactive groups are surface available in an amount sufficient to attach the microparticle to a skin surface in the presence of exogenous transglutaminase. [0020] In another embodiment, the microparticle is non-biodegradable. Preferably, the microparticle is water insoluble and, even more preferably, it is detergent insoluble. In certain embodiments, the microparticle is less than 5 .mu.m, or less than 1 .mu.m, or 100 nm to 500 nm, or less than 100 nm, or 20 nm to 90 nm, or 20 nm to 35 nm, or less than 20 nm, or 1 nm to 10 nm, or 5 nm to 10 nm. The microparticle may be porous or it may be hollow, but is not so limited. Continue reading... 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