USOO6267957B1 (12) United States Patent (10) Patent No.: US 6,267,957 B1 Green et al. (45) Date of Patent: Jul. 31, 2001

(54) ATTACHINGAGENTS TO TISSUE WITH 61-172807 8/1986 (JP). TRANSGLUTAMINASE AND A 02169511 6/1990 (JP). TRANSGLUTAMINASE SUBSTRATE 2-204407 8/1990 (JP). O3038511 2/1991 (JP). 03083908 4/1991 (JP). (76) Inventors: Howard Green, 82 Williston St., 5-56785 3/1993 (JP). Brookline, MA (US) 02146; George D. 05085924 4/1993 (JP). Corey, 65 Harding St., Newton, MA WO95/24929 9/1955 (WO). (US) 02165; Bruce J. Compton, 30 WO92/12238 7/1992 (WO). Cottage St., Lexington, MA (US) WO 94/18945 9/1994 (WO). 02173; Philippe Dijan, 170, rue de la WO94/23738 10/1994 (WO). Convention, 75015 Paris (FR) WOUS96/ 11990 4/1996 (WO). Notice: Subject to any disclaimer, the term of this WO 98/13381 4/1998 (WO). patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. OTHER PUBLICATIONS Rice, “The Cornified Envelope of Terminally Differentiated (21) Appl. No.: 09/234,358 Human Epidermal Keratinocytes Consists of Cross-Linked Protein", Cell, 11:417-422 (1977). (22) Filed: Jan. 20, 1999 Simon, “Enzymatic Cross-Linking of Involucrin and Other Proteins by Keratinocyte Particulates in Vitro,” Cell, Related U.S. Application Data 40:677–683 (1985). (60) Provisional application No. 60/071,908, filed on Jan. 20, Steven, “Biosynthetic Pathways of Filaggrin and Loricrin 1998. Two Major Proteins Expressed by Terminally Differentiated (51) Int. Cl." ...... A61K 38/45; A61K 38/48; Epidermal Keratinocytes,” J. of Structural Biology, A61K 38/00; C12N 11/02; CO7K 17/02 104:150–162 (1990). (52) U.S. Cl...... 424/94.5; 424/59; 424/94.63; Highley, “The Epidermal Keratinization Process,” Cosmet 424/401; 435/16; 435/177; 435/193; 514/2; ics & Toiletries, 99:57-62 (1984). 530/402; 530/812 Rialdi et al., 1“Filaggrin Overview; Functions and Cosmetic (58) Field of Search ...... 435/16, 174, 177, Aim,” Cosmetics & Toiletries, 103:89–94 (1988). 435/193; 424/94.63, 94.5, 401, 59; 514/2; 530/402,812 (List continued on next page.) Primary Examiner-David M. Naff (56) References Cited (74) Attorney, Agent, or Firm Wolf, Greenfield & Sacks, P.C. U.S. PATENT DOCUMENTS 4,279,996 7/1981 Yoshioka et al...... 435/69 (57) ABSTRACT 4,338,214 7/1982 Fischer et al...... 252/545 4,369,037 1/1983 Matsunaga et al. ... 8/127.51 Methods, products and kits are provided for attaching agents 4,517,175 5/1985 Iwabuchi et al...... 424/70 to tissue with a linking molecule in the presence of trans 4,699,778 10/1987 Marty ...... 424/59 glutaminase. The linking molecule and/or agent is a Sub 4,705,682 11/1987 Moeller et al...... 525/70 Strate of transglutaminase. The agent can be a nonprotein or 4,726,942 2/1988 Lang et al...... 424/47 an enzyme Such as cholinesterase or phosphodiesterase. The 4,832,946 5/1989 Green ...... 424/70 transglutaminase may be exogenously added or be endog 4,839,168 6/1989 Abe et al...... 424/74 enous in tissue. In Specific embodiments, the linking mol 4,879,116 11/1989 Fox et al...... 424/682 ecule contains at least two contiguous linked glutamines or 4.885,169 12/1989 Gazzani ...... 424/104 at least three contiguous linked lysines. A conjugate of the 4,973,473 11/1990 Schneider et al. . - - - - - 424/63 agent and the linking molecule may be applied to tissue, and 5,075,019 12/1991 Evans et al...... 252/34 5,080,888 1/1992 Grollier et al...... 424/61 in the presence of transglutaminase covalently bonded to the 5,091,173 2/1992 Buultjens et al...... 424/70 tissue via the linking molecule. A complementary linking 5,100,956 3/1992 O'Lenick, Jr. . ... 514/54.1 molecule rich in lysines may be first attached to the tissue in 5,135,913 8/1992 Pickart ...... 424/16 the presence of transglutaminase, and then covalently 5,156,956 10/1992 Motoki et al...... 425/68.1 bonded to a glutamine-containing linking molecule of the 5,490,980 2/1996 Richardson et al...... 424/94.6 conjugate in the presence of transglutaminase. In another 5,525,336 6/1996 Green et al...... 424/94.5 embodiment, a linking molecule containing multiple 5,773,577 6/1998 Cappello ...... 530/350 glutamines is covalently bonded to tissue in the presence of transglutaminase, and an agent containing multiple lysines is FOREIGN PATENT DOCUMENTS covalently bonded to the linking molecule in the presence of 13742 9/1988 (AU). transglutaminase. Alternatively, the linking molecule con 1964.7863A 5/1998 (DE). tains multiple lysines and the agent contains multiple 0285 474 10/1988 (EP). glutamines. Two tissues can be sealed together by holding 0481504 10/1991 (EP). 0511 116 10/1992 (EP). the tissues in contact with each other in the presence of O704221A 3/1996 (EP). transglutaminase. 0615 745 5/1997 (EP). 2659352 9/1991 (FR). 48 Claims, 2 Drawing Sheets US 6,267,957 B1 Page 2

OTHER PUBLICATIONS KVedar, “Characterization of Sciellin, a Precursor to the Cornified Envelope of Human Keratinocytes,” Differentia (Abstract) Women's Wear Daily, p. 6 (Oct. 9, 1992). Banks-Schlegal, “Involucrin Synthesis and Tissue Assem tion, 49:195-204 (1992). bly by Keratinocytes in Natural and Cultured Human Epi Markova, Profilaggrin Isa Major Epiderman Calcium-Bind thelia,” J. of Cell Biology, 90:732, 737 (1981). ing Protein, Molecular and Cellular Biology, 13:613-625 Eckert, "Structure and Evolution of the Human Involucrin (1993). Gene,” Cell, 46:583–589 (1986). Marvin, “Cornifin, a Cross-Linked Envelope Precursor in Etoh, “Involucrin Acts as a Transglutaminase Substrate at Keratinocytes that Is Down-Regulated by Retinoids,” Bio Multiple Sites,” Biochemical and Biophysical Research chemistry, 89:11029–11030 (1992). Communications, 136:51-56 (1986). Fietz, “The cDNA-deduced Amino Acid Sequence for Tri Mehrel, “Identification of a Major Keratinocyte Cell Enve chohyalin, A Differentiation Marker in the Hair Follicle, lope Protein, Loricrin.” Cell, 61:1103–1112 (1990). Contains a 23 Amino Acid Repeat,” J. of Cell Biology, Phillips, “Primary Structure of Keratinocyte Transglutami 110:427–436 (1990). nase,” Biochemistry, 87.9333–9337 (1990). Steven, M. et al., “Protein Composition of Cornified Cell Rice, “Presence in Human Epidermal Cells of a Soluble Envelopes of Epidermal Keratinocytes,” J. Cell Science, Protein Precursor of the Cross-Linked Envelope: Activation 107:693–700 (1994). of the Cross-Linking by Calcium Ions,” Cell, 18:681-694 Folk, “Transglutaminase (Guinea Pig Liver).” in Methods in (1979). Enzymology, vol. 17A, 1970, p. 889-894, Tabor H. and C. Tabor, Eds. International Search Report: PCT/US99/01193 (related PCT Green, “Terminal Differentiation of Cultured Human Epi Case-WO). dermal Cells,” Cell, 11:405-416 (1977). Davies, et al., Adv. Exp. Med. Biol. 250, 391-401 (1988). Greenberg, “Transglutaminases: Multifunctional Pober, J.S., et al., Biochemistry, vol. 17, No. 11:2163–2169 Cross-linking Enzymes that Stabilize Tissues.” FASEB J., (1978). 5:3071-3077 (1991). Hohl, “Cornified Cell Envelope, Dermatologica, Lajemi, M., et al., Histochemical Journal 29:593-606 180:201–211 (1990). (1997). Hohl, “Characterization of Human Loricrin,” J. of Biologi Kahlem, et al., Proc. Natl. Acad. Sci., USA, Vol. 93, cal Chemistry, 266:6626–6636 (1991). pp. 14580–14585 (Dec., 1996)(Appendix A). U.S. Patent Jul. 31, 2001 Sheet 1 of 2 US 6,267,957 B1

Fig. 1 U.S. Patent Jul. 31, 2001 Sheet 2 of 2 US 6,267,957 B1

US 6,267,957 B1 1 2 ATTACHINGAGENTS TO TISSUE WITH natural Substrates of transglutaminases, can be used together TRANSGLUTAMINASE AND A as cosmetic treatments to cross-link preparations of corneo TRANSGLUTAMINASE SUBSTRATE cyte proteins to the outer layer of skin, hair or nails to form a protective layer on the Skin, hair or nails. This application claim benefit to provisional application U.S. Pat. No. 5,490,980 describes selecting agents having Ser. No. 60/071,908 Jan. 20, 1998. or modifying agents to have an aliphatic amine, and then attaching those agents to skin, hair or nails using trans FIELD OF THE INVENTION glutaminase. While the idea was Sound in principle, in This invention relates to the linkage of agents to tissue by practice the 980 applicants achieved results that were barely transglutaminase and involves methods, products and kits above background. (See Example Section of 980 patent). relating thereto. An aliphatic amine was applied in the examples as a single linking molecule or prophetically in clusters (according to a BACKGROUND OF THE INVENTION formula in the 980 patent). In selecting the amine moiety of Transglutaminases are a family of calcium-dependent the pair of known transglutaminase Substrate moieties, the enzymes mediating covalent cross-linking reactions 15 980 patent taught away from using the carboxamide Sub between specific peptide bound (4-glutamyl residues and Strate moiety. various primary amino groups of peptide-bound lysines or polyamines, acting as amine donor Substrates (Davies, et al., SUMMARY OF THE INVENTION Adv. Exp. Med. Biol. 250, 391-401, 1988). These enzymes It has been discovered, Surprisingly, that certain Substrates Stabilize biological Structures via the formation of isopeptide of transglutaminase are particularly desirable for use as croSS-links. In mammals, at least five enzymatically active linking molecules to attach agents to proteinaceous material transglutaminases have been identified, cloned and Such as body tissue. It also has been discovered that Sequenced. The number of proteins acting as glutaminyl molecules, including native peptides and conjugates accord Substrates for transglutaminases is restricted, and no obvious ing to the invention, can be Screened to determine those that consensus Sequence around these Substrates glutamines has 25 can be Substrates of transglutaminases, and then Such mol been found. ecules can be attached to body tissue. Method of attaching Three main lines of investigation have been conducted agents to body tissue and methods of Screening molecules Surrounding transglutaminases. These enzymes have been using transglutaminase are provided. In addition, composi used to label membrane proteins and, in the absence of tions of matter Suitable as Substrates for transglutaminase exogenous amines, to catalyze the formation of and kits containing Such molecules together with trans (4-glutamyl)-lysyl cross-links between them. The labeling is glutaminase are provided. quite Specific and can be carried out under mild According to one aspect of the invention, a method is (physiological) reaction conditions. Thus, for example, provided for attaching a non-corneocyte protein, non transglutaminases were used to Study rhodopsin in the intact 35 labeling agent to a body tissue. A conjugate of the agent and disc membrane, as only residues of rhodopsin located in the a linking molecule having a carboxamide, the linking mol aqueous phase in the exposed side of the disc membranes ecule being a carboxamide-bearing Substrate of were expected to be labeled. In these experiments, rhodopsin transglutaninase, is applied to the body tissue. Transglutami was labeled by transglutaminase using putrescine and dan nase also is applied to the body tissue, in an amount effective Sylcadaverine as detectable Substrates. 40 for cross-linking the conjugate to the body tissue via the The role of transglutaminases in living cells also has been linking molecule. The croSS-linking then is allowed to occur. Studied, for example, using the cell-penetrating labeled In certain embodiments the agent is not fibronectin (i.e., a Substrate fluoresceincadaverine for detecting amine acceptor nonfibronectin agent). In certain embodiments the agent is protein Substrates accessible to active transglutaminase in not an extracellular matrix protein (i.e. a non-extracellular living cells. A similar Strategy was employed using 45 protein agent). Preferably the linking molecule comprises a 5-(biotinamido)-pentylamine as a label. Such labeled Sub polymer of at least 3, 4 or 5 linked units, each unit being a Strates can be detected directly, for example by fluorescence, carboxamide Substrate of transglutaminase. or can be detected indirectly, for example using antibodies, According to another aspect of the invention, a method is to identify native proteins to which the labeled substrate has provided for attaching a non-corneocyte protein, non been covalently attached by transglutaminase. See, Pober, J. 50 labeling agent to a body tissue. The method involves Select S. et al., Biochemistry, Vol. 17, No. 11:2163–2169 (1978); ing a non-corneocyte protein, non-labeling agent that is a Lajemi, M. et al., Histochemical Journal 29:593-606 carboxamide Substrate for transglutaminase. The agent, in an (1997). isolated form, then is applied to the body tissue in the More recently, an investigation was carried out to deter presence of a Sufficient amount of transglutaminase to mine if polyglutamine is a transglutaminase Substrate. It was 55 croSS-link the isolated agent to the body tissue. The croSS determined that as long as polypeptides including Stretches linking then is allowed to occur. In this embodiment, the of polyglutamine are rendered sufficiently soluble by the agent can be a conjugate of a native, non-corneocyte, flanking residues, all were excellent Substrates of trans non-labeling active agent and a linking molecule not native glutaminase. Based upon these Studies, it was speculated to the agent. It also is the case that the agent can be a native that certain diseaseS Such as Spinocerebellar ataxia Type I, 60 agent free of conjugation with groups not native to the agent. Machado-Joseph disease, and Dentato-Rubral pallidoluy The agent in certain embodiments is a non-extracellular sian atrophy which are characterized by proteins having matrix protein agent. polyglutamine Stretches, may arise as a result of aggregation In either of the foregoing embodiments, the linking mol of Such proteins acted upon by a transglutaminase. ecule can be any number of a variety of molecules. In Some It also is described in U.S. Pat. No. 5,525,336 (the 65 embodiments, the linking molecule is at least one glutamine. disclosure of which is incorporated herein by reference in its The linking molecule, likewise, can be one bearing multiple entirety) that transglutaminases and corneocyte proteins, the reactive carboxamides, Such as two or more contiguous US 6,267,957 B1 3 4 linked L or D glutamines. D glutamines have the advantage integument, a wound bed, internal organs or internal tissue of being physiologically more Stable than L. glutamines. In of a living Subject. a preferred embodiment, the linking molecule is a polymer According to the foregoing methods, the agent can be any rich in carboxamides that are Substrates of transglutaminase, variety of agents, including cosmetics Such as bulking Such as a polymer rich in glutamine. The linking molecule agents and coloring agents, Sunscreen agents, enzymes also can be a polymer rich in both carboxamides and including cholinesterase and phosphodiesterase, pharmaceu aliphatic amines, Such as one rich in both glutamine and tical agents, ligands of ligand-receptor complexes, receptors lysine. A polymer rich in glutamine, lysine, or glutamine and of ligand-receptor complexes and the like. In one important lysine is a molecule wherein at least 20% of the units of the embodiment, the agent is a member of a noncovalent cou polymer are glutamine, lysine or glutamine and lysine, pling pair, Such as biotin and avidin, to provide a universal respectively or wherein the molecule includes at least three, linker as discussed in greater detail below. In certain preferably four and most preferably five contiguous, linked embodiments, particularly those employing pharmaceutical transglutamines Substrates, preferably linked by peptide agents, the bond between the agent and the linking molecule bonds. A polymer rich in glutamines, lysines or glutamines can be a bond which cleaves under normal physiological and lysines, can be a polymer that contains at least 30% 15 conditions or which can be caused to cleave specifically, for glutamines, lysines or glutamines and lysines, at least 40% example, by light. In many instances where the agent is not glutamines, lysines or glutamines and lysines, or even 50% itself a Substrate of transglutaminase, the agent is a non or more glutamines, or glutamines and lysines. protein. In certain preferred embodiments, the methods described According to another aspect of the invention, a method is above involve first preparing the body tissue for the attach provided for attaching an agent to a body tissue. A linking ment of the agent to the body tissue. In one important molecule, that is covalently bonded to the agent in the embodiment, a separate “complementary' linking molecule presence of transglutaminase, is attached to the body tissue. that is attachable to the linking molecule by transglutami Then, an agent that is a Substrate of transglutaminase is nase is first attached to the body tissue to provide multiple, applied to the body tissue. Transglutaminase also is applied accessible linking Sites for the attachment of the linking 25 to the body tissue, in an amount effective to croSS-link the molecule to the body tissue. AS used herein a pair of agent to the linking molecule. CroSS-linking then is allowed molecules which are covalently joined by transglutaminase to occur. The linking molecule can be attached to the body are Said to be complementary molecules. The complemen tissue by any Suitable means, but preferably is itself a tary linking molecule can be attached to the body tissue by Substrate of transglutaminase and preferably is attached to any Suitable means, but preferably is attached by applying the body tissue by applying the linking molecule to the body the complementary linking molecule to the body tissue, and tissue together with transglutaminase, the transglutarminase applying transglutaminase to the body tissue in an amount being present in an amount effective to croSS-link the linking effective for cross-linking the complementary linking mol molecule to the body tissue. Preferred agents and linkers are ecule to the body tissue. Cross-linking then is allowed to as discussed above. Most preferred linking molecules are occur. Preferably, the complementary linking molecule is a 35 glutamine, lysine and polymers of glutamine and/or lysine polymer rich in lysine, glutamine, or both glutamine and or polymers that are rich in glutarnine, or lysine, or both lysine. glutamine and lysine. Layers of Such linking molecules can be attached to body In this embodiment, the agent can be any Substance tissue. To exemplify, polyglutamine could first be attached to including those listed above (with or without conjugated the Surface of a body tissue using transglutaminase. Then, 40 complementary linking molecules depending on whether the polylysine could be attached to the polyglutamine using agent is itself a Substrate of transglutaminase) but also transglutaminase. Subsequently polyglutamine could be including visible labels, extracellular matrix proteins and attached to the polylysine by transglutaminase, and So forth, corneocyte proteins. Preferred body tissues are as described to create by amplification alternating layers of Such mol above. The transglutaminase may be endogenous trans ecules on the body tissues, for example, for bulking pur 45 glutaminase. poses or to provide an even, continuous bed of reactive According to another aspect of the invention, a method is groups for linking an active agent to the body tissue. provided for attaching an agent to a body tissue. The method For example, polymers comprising polyglutamine may involves first attaching to the body tissue a linking molecule first be attached to a body tissue as primary linking mol 50 which is covalently bondable to the agent in the presence of ecules. Then, polymers comprising the complementary transglutaminase. Then, the method involves applying to the linker (e.g. polylysine) can be attached to the body tissue via body tissue having the linking molecule attached thereto an the polymers comprising polyglutamine. Finally, agents agent that is a Substrate of transglutaminase and that is conjugated with polyglutamine then may be applied to the covalently bondable to the linking molecule in the presence coated body Surface and easily attached to the exposed 55 of transglutaminases, the applying carried out in the pres amines of the polylysines. ence of the Sufficient amount of transglutaminase effective to In important embodiments, the native agent is not itself a croSS-link the agent to the cross-linking molecule. CroSS Substrate of transglutaminase. Thus, it is required that the linking then is allowed to occur. Preferred agents, linking agent be conjugated to a Substrate of transglutaminase molecules and body tissues are as described above. whereby the agent may be attached to the body tissue by 60 According to another aspect of the invention, a method for Such a Substrate which acts as the linking group. It also is attaching a noneXtracellular matrix protein, preferably possible to modify peptide agents by adding a side group, nonlabeling, agent to a body tissue is provided. The method whereby the agent which itself is not a Substrate of trans involves applying to the body tissue a conjugate of the agent glutaminase is converted to a Substrate of transglutaminase. and a linking molecule, the linking molecule being a poly According to the foregoing methods, the agents and 65 mer carrying at least 3 aliphatic amines Spaced along the conjugates are attached to proteinaceous material. The pre polymer, applying to the body tissue transglutaminase in an ferred proteinaceous material is body tissue, including the amount effective for crosslinking the linking molecule to the US 6,267,957 B1 S 6 body tissue, and allowing crosslinking to occur. The ali The invention also involves the use of transglutaminase to phatic amines can be the Side chain of L or D lysines. D glue two tissues together. Two tissues are held under force lysines have the advantage of being physiologically more in contact with one another in the presence of an effective Stable than Llysines. Most preferably, the linking molecule amount of transglutaminase, whereby the transglutaminase is Selected from the group consisting of at least 3, at least 4 causes the cross-linking of the tissue to occur. Preferably, the and at least 5 contiguous lysines attached to one another Surfaces of the tissues to be glued to one another are treated directly by peptide bonds. The polymer also can be one rich with a Substrate of transglutaminase Such as polymers rich in in aliphatic amines Such as one rich in lysines, as described glutamine, lysine or both glutamine and lysine to create above. Preferred agents and body tissues are as described highly reactive Surfaces in the presence of transglutaminase. above. These highly reactive Surfaces are bonded to one another. According to another aspect of the invention, composi Even more preferably, the Surfaces of the tissue are first tions of matter are provided. The compositions include treated with a linking molecule to crosslink the linking conjugates of a non-corneocyte, non-labeling agent and a molecule to the Surfaces, then a linking molecule comple linking molecule having a carboxamide, the linking mol mentary to the first is applied to crosslink the linking ecule being a carboxamide bearing Substrate of 15 molecules to one another and glue the tissue. The trans transglutaminase, wherein the agent is Selected from the glutaminase may be exogenously Supplied. The tissue may group consisting of a SunScreen agent, a cosmetic, an be held together by any conventional means, Such as Sutures, enzyme, a coloring agent, a pharmaceutical agent, a member tape, Stapes and the like. of a ligand/receptor pair, a component of a high-affinity The agent also can be in a vehicle Such as a microparticle, non-covalent coupling pair, a tissue Sealant, an insecticide, e.g. a microSphere or a nanosphere, the microSphere or an insect repellant, a bactericide, a fungicide, and the like. nanosphere being rich in carboxamide or aliphatic amine The linking group is not native to the agent. Preferred Substrates of transglutaminase, Such as glutamines, lysines, linking molecules are as described above. In certain or glutamines and lysines, whereby the microSphere or embodiments, particularly those involving the pharmaceu nanosphere can be attached to a body tissue. tical agents, the bond between the agent and the linking 25 group or molecule is a hydrolyzable bond or light cleavable According to Still another aspect of the invention, a bond. In certain important embodiments, the agent is a composition of matter is provided comprising a conjugate of non-protein. In other important embodiments, the agent is an a linking molecule that is a Substrate of transglutaminase and active agent. In other important embodiments, the agent, in an agent that is Selected from the group consisting of a its native form free of conjugation to the linking molecule, component of a ligand-receptor pair, a component of a is not itself a Substrate of transglutaminase. high-affinity noncovalent binding pair and a microparticle. In this embodiment the linking molecule can be a car Another composition is as described above, except that boxyamine Substrate of transglutaminase or a hydrophobic the linking molecule is one bearing multiple, spaced ali terminal amine Substrate of transglutaminase, Such as lysine phatic amines. Such linking molecules carry at least three, or known hydrophobic amine Substrates. preferably at least 4 and more preferably, at least 5 aliphatic 35 amines that are Substrates of transglutaminase, attached to These and other aspects of the invention are described in the backbone of the linking molecule and Separated from further detail below. one another and Spaced at discrete intervals. The linking molecule can be a polymer, and, in one important embodi BRIEF DESCRIPTION OF THE DRAWING ment has at least 3, 4 or 5 contiguous lysines attached 40 FIG. 1 depicts a kit according to the invention. directly to one another by peptide bonds. In another embodi FIG.2 depicts the skin of a mouse treated according to the ment the polymer is rich in aliphatic amines. invention. According to other aspects of the invention, kits are provided. One Such kit includes a package housing a first FIG. 3 depicts the mouse of FIG. 2 after 10 days. container containing a composition of matter as described 45 DETAILED DESCRIPTION above and a Second container containing transglutaminase. The kit can further comprise a third container housed by the The invention is based in part on the discovery that package, the third container containing a linking molecule polymers bearing multiple reactive (with transglutaminase) that is a Substrate of transglutaminase and that is covalently carboxamides or multiple reactive aliphatic amines are par bondable, in the presence of transglutaminase, to the com 50 ticularly useful linking molecules for attaching agents to position contained in the first container. The various con protinaceous material Such as skin and hair. The closest prior tainers also can contain vehicles, preservatives, buffers, art teaches away from using carboxamides and also from calcium chelators and calcium (which is necessary for the using polymers bearing multiple reactive aliphatic amines as activity of transglutaminase). defined herein, for Such a purpose as described in greater detail below. AS mentioned above, the tissue can be pretreated to make 55 it more receptive to the action of transglutaminase. In one In general, the agents are chemical agents and include embodiment described above, this is accomplished by pharmaceutical agents, enzymes, cosmetics, Sunscreen attaching polymers rich in glutamine, lysine or both agents, ligands of ligand-receptor pairs, receptors of ligand glutamine and lysine to the body tissue. In other receptor pairs, components of high affinity noncovalent embodiments, the tissue is treated to expose reactive 60 bonding pairs, insecticides and repellants, bactericides, glutamines and/or lysines by Washing, chemical treatment, fungicides, tissue Sealants, labels, Structural proteins, chelat etc. Detergents and lipases can be used to remove fatty acids ing agents, microparticles and the like. Examples are listed and oils. Roughening agents Such as pumice, Silica and below. Sandpaper can be employed to remove dead tissue and other In certain embodiments the agent is a noncorneocyte, obstructions, and chemical agents Such as Sodium hydroxide 65 nonlabeling active agent. Thus, Specifically excluded in can be used to expose reactive groups. Combinations of the these embodiments is corneocyte proteins. Corneocyte pro foregoing are contemplated. teins have been shown in the prior art to be among the US 6,267,957 B1 7 8 natural Substrates of transglutaminase. In certain embodi incorporated herein by reference. Unlike the 980 patent, ments the agent also is a non-extracellular matrix protein however, which depicts Single aliphatic amine moieties and agent. A non-extracellular matrix protein agent is one that is plural Such moieties as independent Substituents in certain not an extracellular matrix protein. Fibronectin, an extracel circumstances, the present invention involves in one aspect lular matrix protein, also has been shown in the prior art to using a plurality of aliphatic amines Spaced apart at discrete be a Substrate of transglutaminase. A nonlabeling active intervals, preferably along the length of a branched or agent is one that is not simply a passive label with no unbranched polymer. It has been discovered, Surprisingly, function, when applied to a body tissue, other than being a that the Spacing of the reactive moieties can be important to label. Specifically excluded are labeled corneocyte proteins, achieving the results of the present invention. One embodiment involves linking molecules that are labeled fibronectin, labeled extracellular matrix proteins, polymers having multiple units, which units each bear an putrescine, dansylcadaverine, 5-(biotinamido)-pentylamine, aliphatic amine Substrate of transglutaminase. The polymer fluoresceincadaverine and the like. Such compounds have can be a homopolymer or a heteropolymer. AS used herein been used in the prior art to detect on cells or cell extracts, in connection with linking molecules, a polyaliphatic amine Substrates of transglutaminase. Substrate of transglutaminase is a linking molecule with at By active agent it is meant that the agent, once coupled to 15 least three aliphatic amines Spaced apart from one another at a biological tissue in Vivo or in vitro, has, maintains or can discrete intervals along the backbone of the linking be released to have a desired activity Such as a desired molecule, Separated by one or more backbone atoms. This is physiological activity or therapeutic activity. Examples of most easily envisioned, for example, with polymers rich in active agents are pharmaceutical agents, SunScreen agents, lysine, whereby discrete units of the polymer carry the insecticides, bactericides, fungicides, etc. AS used herein, an aliphatic amine, each being Separately a Substrate for trans active agent is not a cosmetic agent and is not a labeling glutaminase. The linking molecule itself may be a polymer agent including diagnostic agents. of contiguous lysines, preferably at least 3, at least 4 and at The agents are linked to proteinaceous material. When least 5 Such contiguous lysines. Polymers of contiguous used in Vivo, the agents are attached to a body tissue. units, each carrying an aliphatic amine, are preferred. 25 The most preferred linking molecules are polymers rich in Particularly important body tissueS as Sites of attachment are a carboxamide moiety or an aliphatic amine moiety, Such as the integument (including specifically skin, nails, hair, glutamine, lysine or both glutamine and lysine. A polymer mucous membranes and the Surface of the eye), internal rich in glutamine or lysine is a molecule wherein at least organs, internal tissue and wound beds. In in vitro 20% of the units of the polymer carry a carboxamide, an applications, the tissue may be a body tissue, a tissue or cell aliphatic amine, or both, Such as glutamine, lysine or isolate, isolated proteins, Synthetic proteins, cell cultures and glutamine and lysine, or wherein the molecule includes at the like for use, for example, in assay Systems according to least 3, preferably 4 and most preferably 5 Separate and the invention. discretely spaced by a regular distance carboxamides or In certain embodiments, conjugates of agents and linking aliphatic amines, Such as occurs with contiguous, linked molecules are applied, for example, to body tissue and 35 glutamines or lysines. It should be understood, however, that covalently linked to that tissue using transglutaminase. a chain of as few as two glutamines or lysines can be AS used herein, a conjugate means two entities Stably attached to or tethered to an agent to render the agent a bound to one another by any physiochemical means. It is Substrate of transglutaminase. important that the nature of the attachment be of Such a AS noted above, the invention in one aspect involves nature that it does not impair Substantially the effectiveness 40 attaching active agents to proteinaceous materials using of the agent or the Substrate binding ability of the linking transglutaminase, wherein the native agent itself is a Sub molecule. Keeping these parameters in mind, any linkage Strate of transglutaninase. Such agents typically will be known to those of ordinary skill in the art may be employed, polypeptides or proteins and most typically will contain covalent or noncovalent. Covalent is preferred. Such means reactive glutamines, lysines or both. To determine whether and methods of attachment are well known to those of 45 an agent itself is a Substrate of transglutaminase (or a ordinary skill in the art. modified agent, or a covalent conjugate), a simple Screening Typically the agents used according to the invention are method is employed. not themselves, in their native form, Substrates for trans The Screening method involves Selecting a noneXtracel glutaminase. Such agents, however, can be modified accord lular matrix protein, nonlabeling agent, preferably an active ing to the invention to render the agent a Substrate of 50 agent, that is a Substrate for transglutaminase. The agent is transglutaminase. This may be accomplished for example by applied, in an isolated form, to a proteinaceous material Such adding a carboxamide side group(s) to an appropriate moiety as a body tissue, a body tissue isolate, or more preferably, a of the agent (i.e. a modified agent) or by covalently coupling polymer rich in glutamine, a polymer rich in lysine or a glutamine, lysine or both glutamine and lysine to the agent polymer rich in glutamine and lysine. Then, transglutami to form a conjugate that is a Substrate of transglutaminase. 55 nase is applied to the proteinaceous material in an amount The most preferred method is to couple polyglutamine, Sufficient and under appropriate conditions to cross-link the polylysine, a mixed polymer of glutamine and lysine, agent to the proteinaceous material if the agent is a Substrate involucrin (a natural Substrate of transglutaminase) or a of transglutaminase. Then it is determined whether the agent fragment of involucrin to the agent to form an appropriate covalently binds to the proteinaceous material. The amounts conjugate. 60 of materials and conditions employed for these assays are Preferred linking molecules are polymers bearing mul derivable from the examples below and, in general, can be tiple reactive carboxamides and/or aliphatic amines that are derived by those of ordinary skill in the art without undue Substrates of transglutaminase. Carboxamides that are Sub experimentation from, for example, the publication by Strates of transglutaminase are well known and include Kahlem, et al., Proc. Natl. Acad. Sci., USA, Vol. 93, pp. glutamines. Aliphatic amines that are Substrates of trans 65 14580-14585, December, 1996. glutaminase also are well known and are exemplified in, for In constructing conjugates, it may be desirable to vary not example, U.S. Pat. No. 5,490,980, the disclosure of which is only the number of glutamines and/or lysines in the linking US 6,267,957 B1 9 10 molecule, but it also may be desirable to tether the linking dimethylaminopropyl-carbodiimide hydrochloride. Hetero molecule to the active agent via a Spacer. This can remove, bifunctional cross-linkers that react with carbohydrates and for example, any problems that might arise from Steric sulfhydryls include 4-N-maleimidomethyl-cyclohexane-1- hindrance, wherein acceSS by transglutaminase to the reac carboxylhydrazide.2 HCl, 4-(4-N-maleimidophenyl)- tive moiety of the linking molecule is hindered. These butyric acid hydrazide.2 HCl, and 3-2-pyridyldithio Spacers can be any of a variety of molecules, preferably propionyl hydrazide. The cross-inkers are bis-S-4- nonactive, Such as Straight or even branched carbon chains azidosalicylamido)ethyldisulfide and glutaraldehyde. of C-C, Saturated or unsaturated, phospholipids, amino Amine or thiol groups may be added at any nucleotide of a acids, and in particular glycine, and the like, naturally Synthetic nucleic acid So as to provide a point of attachment occurring or Synthetic. Additional Spacers include alkyl and for a bifunctional croSS-linker molecule. The nucleic acid alkenyl carbonates, carbamates, and carbamides. These are may be Synthesized incorporating conjugation-competent all related and may add polar functionality to the Spacers reagents such as Uni-Link AminoModifier, 3'-DMT-C6 Such as the C-C previously mentioned. Amine-ON CPG, AminoModifier II, N-TFA-C6 The conjugations or modifications described herein AminoModifier, C6-ThiolModifier, C6-Disulfide Phos employ routine chemistry, which chemistry does not form a 15 phoramidite and C6-Disulfide CPG (Clontech, Palo Alto, part of the invention and which chemistry is well known to Calif.). those skilled in the art of chemistry. The use of protecting In constructing conjugates, it also may be desirable to groupS and known linkerS Such as mono and heterobifunc attach the agent to the linking molecule by a bond that tional linkers are well documented in the literature and will cleaves under normal physiological conditions or that can be not be repeated here. caused to cleave specifically upon application of a Stimulus Attachment according to the invention thus need not be Such as light, whereby the agent can be released. In certain directed attachment. The components of the compositions of instances, the agent may be inactive in its conjugated form the invention may be provided with functionalized groups to and activated only when released. In other instances, the facilitate their attachment and/or linker groups may be agent would be released to exert an activity remote from its interposed between the components of these compositions to 25 point of attachment to the body tissue. In still other facilitate their attachment. In addition, the components of instances, the agent would be released in a Sustained fashion, the compositions of the present invention may be Synthe to prolong the release of the agent versus an agent applied sized in a single process, whereby the components could be to tissue but not covalently coupled to the tissue. Readily regarded as one and the same entity. For example, a protein cleavable bonds include readily hydrolyzable bonds, for agent may be Synthesized recombinantly to include a poly example, ester bonds, amide bonds and Schiff's base-type glutamine at one end for linking the polypeptide via trans bonds. Bonds which are cleavable by light are well known. glutaminase. Noncovalent methods of conjugation may also be used. Specific examples of covalent bonds include those Noncovalent conjugation includes hydrophobic interactions, wherein bifunctional croSS-linker molecules are used. The ionic interactions, high affinity interactions Such as biotin cross-linker molecules may be homobifunctional or 35 avidin and biotin-Streptavidin complexation and other affin heterobifunctional, depending upon the nature of the mol ity interactions. In one embodiment, a molecule Such as ecules to be conjugated. Homobilfinctional cross-linkers avidin is attached to a linking molecule Such as poly have two identical reactive groups. Heterobifunctional glutamine. This conjugate, once attached to tissue according croSS-linkers are defined as having two different reactive to the invention, then becomes a universal linking moiety for groups that allow for Sequential conjugation reaction. Vari 40 any agent attached to a biotin molecule. ous types of commercially available croSS-linkers are reac AS mentioned above, the linking molecules may be part of tive with one or more of the following groups: primary a microparticle Such as a microSphere or a nanosphere and amines, Secondary amines, Sulphydryls, carboxyls, carbon the agent may be contained in the microparticle, either yls and carbohydrates. Examples of amine-Specific croSS physically entrapped therein, covalently bonded thereto or linkers are bis(Sulfo Succinimidyl) Suberate, bis(2- 45 otherwise physiochemically attached to the microparticle. In (Succinimidooxycarbonyloxy)ethylsulfone, disuccinimidyl preferred embodiments, the microSpheres or nanospheres Suberate, disuccinimidyl tartarate, dimethyl adipimate.2 carry, at least on their Surface, polymers rich in glutamine, HCl, dimethyl pimelimidate.2 HCl, dimethyl suberimidate.2 lysine, or both glutamine and lysine. The methods for HCl, and ethylene glycolbis-Succinimidyl-Succinate. manufacturing microparticles according to the prior art are Cross-linkers reactive with Sulfhydryl groups include 50 well documented and do not form a basis for the present bismale imidohexane, 1,4-di-3'-(2-pyridyldithio)- invention. The present invention differs from those of the propion amido)butane, 1-p-azidosalicylamido)-4- prior art only in that either the polymers of the microSpar iodoacetamidobutane, and N-4-(p-azidosalicylamido) ticle Structure themselves contain or are derivatized to butyl-3'-2'-pyridyldithiopropionamide. Cross-linkers contain glutamines and/or lysines, or polymers of glutamine, preferentially reactive with carbohydrates include azidoben 55 lysine or glutamine and lysine are included within the Zoyl hydrazine. Cross-linkers preferentially reactive with mixture of polymers forming the matrix, whereby Such carboxyl groups include 4-p-azido Salicylamido polymers are entrapped throughout and at the Surface of the butylamine. Heterobifunctional cross-linkers that react with microparticles. Examples of microSpheres and nanospheres amines and sulfhydryls include N-succinimidyl-3-2- and their method of manufacture may be found in U.S. Pat. pyridyldithiopropionate, Succinimidyl4-iodoacetyl 60 No. 5,075,019, PCT WO95/24929, PCT WO94/23738 and aminobenzoate, Succinimidyl 4-N-maleimidomethyl PCT/US96/11990, the disclosures of which are incorporated cyclohexane-1-carboxylate, m-maleimidobenzoyl-N- herein by reference. hydroxysuccinimide ester, SulfoSuccinimidyl 6-3-2- Agents in an isolated form are Sometimes applied accord pyridyldithiopropionamidohexanoate, and SulfoSuccinim ing to the invention. Isolated as used herein will depend idyl 4-N-maleimidomethylcyclohexane-1-carboxylate. 65 upon the agent employed. In general, isolated as used herein Heterobifunctional cross-linkers that react with carboxyl means that the material is essentially free of other Substances and a mine groups include 1 - ethyl-3-3- to an extent practical and appropriate for the intended use of US 6,267,957 B1 11 12 the material. In the case of pharmaceuticals and cosmetics, The invention also involves kits. Referring to FIG. 1, the the materials are likely to be Substantially pure. In the case kit is a package 10 comprising a housing 12 holding a first of proteins, the proteins are Sufficiently pure and Sufficiently container 14, a Second container 16 and a third container 18. free from other biological constituents of the host cells from The first container can contain any of the agents or conju which the proteins are derived so as to be useful in the gates that are Substrates of transglutaminase, as described methods according to the invention. Typically, Such agents above. The Second container can contain transglutaminase. will be at least 95% or more pure. The third container can contain, for example, a linking molecule for preparing the Surface of the body tissue for Agents are Sometimes described as native agents herein. application of the agents and conjugates of the invention. A native agent is one as it occurs in nature (isolated or The transglutaminase preferably is Stored in the presence of Synthesized to duplicate a naturally occurring molecule), a chelating agent Such as EDTA, and either one of the first without modification or conjugation as described herein. or third containers contains calcium for activating the trans AS mentioned above, the body tissue, to which the agents glutaminase when applied to the tissue. The various con and conjugates of the invention are to be applied, may be tainers may also contain preservatives, buffers, vehicles, and pretreated to facilitate the reaction with transglutaminase. the like, as is conventional. The package also may house Such treatments include Washings, abrasive treatments 15 instructions for using the materials according to the inven including physical agents Such as pumice, Silica and tion. oatmeal, enzymes Such as papain, bromelins and the like and The conjugates and agents of the invention are applied in chemical agents Such as alpha hydroxy acids and glycolic effective amounts. An effective amount, in general, means acids. The main object is to treat the body tissue So as to that amount necessary to achieve the purpose for which the expose or create reactive glutamines and/or lysines. agent is applied. If the agent is a pharmaceutical agent, then Likewise, as mentioned above, the body tissue may be the amount is that amount necessary to delay the onset of, pretreated by putting down a layer or reactive groups, Such Slow the progression of, halt altogether the onset or pro as by applying to the body tissue polymers rich in lysine, gression of or diagnose a particular condition being treated. glutamine or both lysine and glutamine. These materials In the case of a cosmetic agent, the effective amount will be 25 that amount necessary to achieve the desired cosmetic result. may be attached to the body tissue by any conventional In the case of a Sunscreen agent, an effective amount will be means, but, according to the invention, also may be attached that amount necessary to achieve Suitable protection from using transglutaminase. the Sun as is conventional. Effective amounts will, of course, It should be noted that glutamine, lysine, and polymers of depend on the particular condition being treated, Severity of glutamine and lysine are described above. AS used herein, the condition, the needs of the patient, individual patient Such terms embrace nonpeptidic multimers of glutamine and parameters including age, physical condition, Size and lysine whereby amino acid analogs are used to replace these weight, concurrent treatment, frequency of treatment and amino acids in the polyglutamine or polylysine Substrates. mode of treatment. These factors are well-known to those of Some well known classes of peptide mimetics and ordinary skill in the art and can be addressed with no more pseudopeptides are: azabicycloalkane amino acids; thiazabi 35 than routine experimentation. The mode of delivery typi cycloalkane amino acids, oxazabicycloalkane amino acids, cally will be topical. Other modes of delivery are, diazabicycloalkane amino acids. D-amino acids are an nonetheless, appropriate depending on the condition being important embodiment. treated. AeroSols are an example of an appropriate mode of The transglutaminase may be exogenously added trans delivery. glutaminase or may be endogenous transglutaminase present 40 The agent may be a Sunscreen agent. Examples of Sun at the tissue. Screen agents include: p-aminobenzoate analogS Such as In one embodiment transglutaminase is used to glue two 2-ethylhexyl-4-dimethylaminobenzoate (Padimate O); tissues to one another. This can be accomplished in a variety p-methoxy-2-ethyl-hexyl-cinnamate (Parsol 1789); oxyben of wayS. Transglutaminase, a Substrate of transglutaminase, Zone (benzophenone-3); ethylhexylsalicylate; diphenylacry or both can be supplied to the surfaces of two tissues which 45 late polyisobutylene; alkyl-S,S-diphenylacrylate and then are held in contact with one another for a period of time "-cyano-S,S-diphenylacrylate, 1-(4-aminophenyl)-2- Sufficient to permit transglutaminase to crosslink the tissues morpholinylethanone; (1-(4-methoxylphenyl)-3-(4-tert to one another. In one circumstance, exogenously Supplied butyl-phenyl)propan-1-3-dione; methyl anthranilate; Octoc transglutaminase is applied to the Surfaces of the tissues to rylene; Tretinoin "-hydroxyacid, diphenylacrylate crosslink Substrates of transglutaminase to one another, 50 polyisobutyle ne; 1-(4-amin op he nyl)-2- which Substrates are present and are endogenous on the morpholinylethanone; diphenylacrylate polyisobutylene; Surfaces of the tissue. In another circumstance, exogenously digalloyl trioleate; glyceryl p-aminobenzoate, 4-(omega Supplied Substrates of transglutaminase are applied to the -dialkylaminoalkoxy)phenylmethylene)-1,3,3-trimethyl-2- Surfaces of the tissues and are acted upon by endogenous oXabicyclo(2.2.2)octan-6-ones, 5-(arylmethylene)-1,3,3- transglutaminase to crosslink the tissue Surfaces to one 55 trimethyl-2-oxabicyclo(2.2.2)octan-6-ones, melanin. another. In another circumstance both transglutaminase and The agent may also be a cosmetic agent. Examples of Substrate of transglutaminase are applied to the Surfaces of cosmetic components include: Vitamin C, Alpha-tocopherol the tissue to crosslink the Surfaces to one another. In this (Vit. E analog); Ammonium lauryl Sulfate; Cocamidopropyl Situation, a Single Substrate Such as polyglutamine could be Betaine; Lauramide DEA, Cocamide DEA; Methyl paraben; applied, one end attaching to one Surface and the other end 60 Propyl paraben; Butyl paraben; Salicylic acid; Propylene attaching to the opposing Surface of the tissues to be glycol; EDTA; BHT; BHA, TBHQ; DMDM hydantoin; crosslinked to one another. Alternatively, a first Substrate (a Imidazolidinyl urea; Potassium Sorbate; Sodium Benzoate; linking molecule Such as polyglutamine) could be applied to phenoxyethanol; Polysorbate 20 and 80; Sodium laurylether create first reactive Surfaces and a second Substrate (a sulfate; Oleyl betaine; Tego betaine; Sorbitol; Glycerin complementary linking molecule Such as polylysine) could 65 monolaurate; Glycerol Stearate. be applied to crosslink the primary linking molecules on A preferred cosmetic agent is any of the known bulking opposing Surfaces to one another. agents which can be added to the hair or nails to provide US 6,267,957 B1 13 14 body and Strength. Cationic Surfactant/polymers, fatty Diflunisal; Dihydrocodeine Bitartrate; Dimefadane; alcohols (non-ionic Surfactant), waxes or esters, non-ionic Dipyrone; Doxpicomine Hydrochloride; Drinidene; Ena polymers (e.g. polyglycols) for thickening, and insoluble doline Hydrochloride; Epirizole; Ergotamine Tartrate; Silicone. The preferred bulking agent is the cationic Ethoxazene Hydrochloride; Etofenamate; Eugenol; Feno Surfactant, which places a dispersive charge on the hair. profen; Fenoprofen Calcium; Fentanyl Citrate; Floctafe Additional bulking agents can be Solutions of proteins, nine; Flufenisal; Flunixin; Flunixin Meglumine; Flupir peptides, and polynucleotides or combinations thereof. Par tine Maleate; FluproduaZone; Fluradoline Hydrochloride; ticular bulking agents include collagen, keratins, plant Struc Flurbiprofen; Hydromorphone Hydrochloride; Ibufenac; Indoprofen; Ketazocine; Ketorfanol; Ketorolac tural proteins, Silk, fibrin, mucopolysaccharide and elastin. Tromethamine; Letimide Hydrochloride; Levomethadyl Bulking agents are well known to those of ordinary skill in 1O Acetate; Levomethadyl Acetate Hydrochloride; Levo the art. nantradol Hydrochloride; Levorphanol Tartrate; Lofemi The agent also can be a tissue Sealant. TiSSue Sealants are Zole Hydrochloride; Lofentanil Oxalate; Lorcinadol; Lor those used in wound healing to mechanically Seal wounds. noxicam, Magnesium Salicylate; Mefenamic Acid, The use of transglutaminase to covalently attach Such mate Menabitan Hydrochloride; Meperidine Hydrochloride; rials would add mechanical and adhesive Strength to this 15 Meptazinol Hydrochloride; Methadone Hydrochloride; Sealant. Such tissue Sealants are composed typically of Methadyl Acetate; Methopholine; Methotrimeprazine; fibrinogen, collagen, hyaluronic acid, Synthetic peptides and Metkephamid Acetate; Mimbane Hydrochloride; Mirfen the like. They also can be polyglutamines, polylysines, or tanil Hydrochloride; Molinazone; Morphine Sulfate; polymers of both glutamine and lysine, corneocyte proteins Moxazocine; Nabitan Hydrochloride; Nalbuphine Hydro and the like. chloride; Nalmexone Hydrochloride; Namoxyrate; Nant The agents also can be insect repellants. A widely used radol Hydrochloride; Naproxen; Naproxen Sodium; insect repellant is N-N-diethyl-3-methylbenzamide. The Naproxol; Nefopam Hydrochloride; Nexeridine Hydro agent also may be cultured cells and cultured body tissues chloride; Noracymethadol Hydrochloride; Ocfentanil used for wound healing, cartilage replacement, corneal Hydrochloride; Octazamide; Olvanil; Oxetorone Fuma 25 rate; Oxycodone; Oxycodone Hydrochloride; Oxycodone replacements and other like Surgical procedures. Terephthalate; Oxymorphone Hydrochloride; Pemedolac, AS mentioned above, the agent may be a pharmaceutical Pentamorphone; Pentazocine; Pentazocine Hydrochlo agent. ride; Pentazocine Lactate; Phenazopyridine Hydrochlo When administered the pharmaceutical agents of the ride; Phenyramidol Hydrochloride; Picenadol Hydrochlo invention are applied in pharmaceutically acceptable ride; Pinadoline; Pirfenidone; PiroXicam Olamine; amounts and in pharmaceutically acceptable compositions. Pravadoline Maleate; Prodilidine Hydrochloride; Profa Such preparations may routinely contain Salts, buffering dol Hydrochloride; Propiram Fumarate; Propoxyphene agents, preservatives, compatible carriers and optionally Hydrochloride; Propoxyphene Napsylate; Proxazole; other therapeutic or nontherapeutic ingredients. When used Proxazole Citrate; Proxorphan Tartrate; Pyrroliphene in medicine, the Salts should be pharmaceutically 35 Hydrochloride; Remifentanil Hydrochloride; Salcolex; acceptable, but nonpharmaceutically acceptable Salts may Salethamide Maleate; Salicylamide; Salicylate Meglu conveniently be used to prepare pharmaceutically accept mine; Salsalate; Sodium Salicylate; Spiradoline Mesylate; able salts thereof and are not excluded from the scope of the Sufentanil; Sufentanil Citrate; Talmetacin; Talniflumate; invention. Talosalate; Tazadolene Succinate; Tebufelone; Tetry Examples of categories of pharmaceutical agents include: 40 damine; Tifurac Sodium; Tilidine Hydrochloride; Tiopi analgesic; amino acid; antagonist, anti-acne agent, anti nac; Tonazocine Mesylate; Tramadol Hydrochloride; allergic, anti-asthmatic; antibacterial; anticholinergic, anti Trefentanil Hydrochloride; Trolamine; Veradoline Hydro fungal; antiglaucoma agent; antihistamine; anti-infective; chloride; Verilopam Hydrochloride; Volazocine; Xorpha anti-infective, topical; anti-inflammatory; antikeratinizing nol Mesylate; Xylazine Hydrochloride; Zenazocine agent; antimicrobial; antimycotic; antineoplastic, 45 MeSylate, Zomepirac Sodium Zucapsaicin. antineutropenic, antiproliferative; antipruritic, antisebor Antiacne: Adapalene; Erytiromycin Salnacedin; Inocoterone rheic, carbonic anhydrase inhibitor; cholinergic, cholinergic Acetate. agonist, diagnostic aids, ectoparasiticide; fluorescent agent, Antiallergic: Amlexanox; Astemizole, AZelastine Hydro glucocorticoid; hair growth Stimulant; histamine H2 recep chloride; Eclazolast; Minocromil; Nedocromil; Nedocro tor antagonists, immunizing agent; immunomodulator; 50 mil Calcium; Nedocromil Sodium; Nivinmedone Sodium; immunoregulator; immunostimulant; immunosuppreSSant; Pemirolast Potassium; Pentigetide; Pirquinozol; Poi keratolytic, mucosal protective agent, radio; wound healing Sonoak Extract; Probicromil Calcium; ProXicromil, Repi agent. rinast; Tetrazolast Meglumine; Thiazinamium Chloride; Analgesic: Acetaminophen; Alfentanil Hydrochloride; Ami Tiacrilast; Tiacrilast Sodium; Tiprinast Meglumine; Tix nobenzoate Potassium; Aminobenzoate Sodium; Ani 55 OX. doxime; Anileridine; Anileridine Hydrochloride; Anilo Antiasthmatic: Ablukast, Ablukast Sodium; AZelastine pam Hydrochloride; Anirolac, Antipyrine, Aspirin; Hydrochloride; Bunaprolast; Cinalukast; Cromitrile Benoxaprofen; Benzydamine Hydrochloride; Biciifadine Sodium; Cromolyn Sodium; Enofelast; Isamoxole; Keto Hydrochloride; Brifentanil Hydrochloride; Bromadoline tifen Fumarate; Levcromakalim, Lodoxamide Ethyl, Maleate; Bromfenac Sodium; Buprenorphine Hydrochlo 60 Lodoxamide Tromethamine; Montelukast Sodium, Onta ride; Butacetin; Butixirate; Butorphanol; Butorphanol Zolast; Oxarbazole; Oxatomide; Piriprost; Piriprost Potas Tartrate; Carbamazepine; Carbaspirin Calcium, Carbi sium; Pirolate; Pobilukast Edamine; Quazolast; Repiri phene Hydrochloride; Carfentanil Citrate; Ciprefadol nast; Ritolukast, Sulukast, TetraZolast Meglumine; Succinate; Ciramadol; Ciramadol Hydrochloride; Clonix Tiaramide Hydrochloride; Tibenelast Sodium; Tome eril; Clonixin; Codeine; Codeine Phosphate; Codeine 65 lukast; Tranilast; Verlukast; Verofylline; Zarirlukast. Sulfate; Conorphone Hydrochloride; Cyclazocine; Dex Antibacterial: AcedapSone; AcetoSulfone Sodium; Alame oxadrol Hydrochloride; Dexpemedolac, Dezocine; cin; Alexidine, Amdinocillin; Amdinocillin Pivoxil, Ami US 6,267,957 B1 15 16 cycline; Amifloxacin; Amifloxacin MeSylate; Amikacin; Phosphate; Mequidox; Meropenem; Methacycline; Meth Amikacin Sulfate; AminoSalicylic acid; Aminosalicylate acycline Hydrochloride; Methenamine; Methenamine Sodium; Amoxicillin; Amphomycin; Ampicillin; Ampicil Hippurate; Methenamine Mandelate; Methicillin Sodium; lin Sodium; Apalcillin Sodium; Apramycin; ASpartocin, Metioprim; Metronidazole Hydrochloride; Metronidazole Astromicin Sulfate, Avilamycin; AVoparcin, Azithromy Phosphate; Mezlocillin; Mezlocillin Sodium; Minocy cin; Azlocillin; AZlocillin Sodium; Bacampicillin Hydro cline; Minocycline Hydrochloride; Mirincamycin Hydro chloride; Bacitracin; Bacitracin Methylene Disalicylate; chloride; Monensin; Monensin Sodium; Nafcillin Bacitracin Zinc, Bambermycins, Benzoylpas Calcium; Sodium; Nalidixate Sodium; Nalidixic Acid; Natamycin; Berythromycin; Betamic in Sulfate; Biapenem; Nebramycin; Neomycin Palmitate; Neomycin Sulfate; Biniramycin, Biphenamine Hydrochloride; Bispyrithione Neomycin Undecylenate; Netilmicin Sulfate; Neutramy Magsulfex; Butikacin; Butirosin Sulfate; Capreomycin cin; Nifuradene; Nifuraldezone; Nifuratel; Nifuratrone; Sulfate; Carbadox; Carbenicillin Disodium; Carbenicillin Nifurdazil; Nifurimide; Nifurpirinol; Nifurcquinazol; Nifi Indanyl Sodiumn; Carbenicillin Phenyl Sodium; Carbe urthiazole; Nitrocycline; Nitrofurantoin; Nitromide; Nor nicillin Potassium; Caru monam Sodium; Cefaclor; floxacin; Novobiocin Sodium; Ofloxacin; Onnetoprim; Cefadroxil, Cefamandole; Cefamandole Nafate; Cefa 15 Oxacillin Sodium; OXimonam, OXimonam Sodium; OXo mandole Sodium; Cefaparole; Cefatrizine; Cefazaflur linic Acid, Oxytetracycline, Oxytetracycline Calcium; Sodium; Cefazolin; Cefazolin Sodium; Cefbuperazone; Oxytetracycline Hydrochloride; Paldimycin; Parachlo Cefdinir, Cefepime; Cefepime Hydrochloride; Cefetecol; rophenol; Paulomycin; Pefloxacin; Pefloxacin Mesylate; Cefixime; Cefinenoxime Hydrochloride; Cefinetazole; Penamecillin; Penicillin G BenZathine; Penicillin G Cefinetazole Sodium; Cefonicid Monosodium; Cefonicid Potassium; Penicillin G Procaine; Penicillin G Sodium; Sodium; Cefoperazone Sodium; Ceforanide; Cefotaxime Penicillin V; Penicillin V BenZathine; Penicillin V Hydra Sodium; Cefotetan, Cefotetan Disodium; Cefotiam bamine; Penicillin V Potassium; Pentizidone Sodium; Hydrochloride; Cefoxitin; Cefoxitin Sodium; Cefpimi Phenyl Aminosalicylate; Piperacillin Sodium; Pirbenicil zole; Cefpimizole Sodium; Ce?piramide; Ce?piramide lin Sodium; Piridicillin Sodium; Pirlimycin Hydrochlo Sodium; Cefpirome Sulfate; Cefpodoxime Proxetil, Cef 25 ride; Pivampicillin Hydrochloride; Pivampicillin Pamo prozil; Cefroxadine; Cefsulodin Sodium; Ceftazidime; ate; Pivampicillin Probenate; Polymyxin B Sulfate; Ceftibuten; Ceftizoxime Sodium; Ceftriaxone Sodium; Porfiromycin; Propikacin; Pyrazinamide; Pyrithione Cefuroxime; Cefuroxime Axetil, Cefuroxime Pivoxetil, Zinc, Quindecamine Acetate, Quinupristin, Racepheni Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; col; Ramoplanin; Ranimycin; Relomycin; Repromicin; Cephalexin Hydrochloride; Cephaloglycin; Cephalori Rifabutin; Rifametane; Rifamexil; Rifamide; Rifampin; dine; Cephalothin Sodium; Cephapirin Sodium; Cephra Rifapentine; Rifaximin; Rolitetracycline; Rolitetracycline dine; Cetocycline Hydrochloride; Cetophenicol; Nitrate; Rosaramicin; Rosaramicin Butyrate; Rosarami Chloramphenicol; Chloramphenicol Palmitate; Chloram cin Propionate; Rosaramicin Sodium Phosphate; Rosa phenicol Pantothenate Complex; Chloramphenicol ramicin Stearate; RoSoxacin; RoxarSone; Roxithromycin; Sodium Succinate; Chlorhexidine Phosphanilate; 35 Sancycline; Sanfeltrinem Sodium; Sarmoxicillin; Sarpicil Chloroxylenol; Chlortetracycline Bisulfate; Chlortetracy lin; Scopafungin; Sisomicin; Sisomicin Sulfate, Spar cline Hydrochloride; Cinoxacin; Ciprofloxacin; Ciprof floxacin; Spectinomycin Hydrochloride, Spiramycin; loxacin Hydrochloride; Cirolemycin; Clarithromycin; Stallimycin Hydrochloride; Steffimycin; Streptomycin Clinafloxacin Hydrochloride; Clindamycin; Clindamycin Sulfate; Streptonicozid; Sulfabenz, Sulfabenzamide; Sul Hydrochloride; Clindamycin Palmitate Hydrochloride; 40 facetamide; Sulfacetamide Sodium; Sulfacytine; Sulfadi Clindamycin Phosphate; Clofazimine; Cloxacillin Benza azine, Sulfadiazine Sodium; Sulfadoxine, Sulfalene; Sul thine; Cloxacillin Sodium; Cloxyquin; Colistimethate famerazine; Sulfameter, Sulfamethazine; Sulfamethizole; Sodium; Colistin Sulfate; Coumermycin; Coumermycin Sulfamethoxazole; Sulfamonomethoxine, Sulfamoxole; Sodium; Cyclacillin, CycloSerine; Dalfopristin; DapSone; Sulfanilate Zinc, Sulfanitran; SulfaSalazine, Sulfasomi Daptomycin; Demeclocycline, Demeclocycline Hydro 45 zole; Sulfathiazole; Sulfazamet, Sulfisoxazole; Sulfisox chloride; Demecycline; Denofungin; Diaveridine, azole Acetyl; Sulfisoxazole Diolamine; Sulfomyxin; Dicloxacillin; Dicloxacillin Sodium; Dihydrostreptomy Sulopenem; Sultamicillin; Suncillin Sodium; Talampicil cin Sulfate; Dipyrithione; Dirithromycin; Doxycycline; lin Hydrochloride; Teicoplanin; Temafloxacin Hydrochlo Doxycycline Calcium; Doxycycline Fosfatex; Doxycy ride; Temocillin; Tetracycline; Tetracycline Hydrochlo cline Hyclate; Droxacin Sodium, Enoxacin; Epicillin; 50 ride; Tetracycline Phosphate Complex; Tetroxoprim; Epitetracycline Hydrochloride; Erythromycin; Erythro Thiamphenicol; Thiphencillin Potassium; Ticarcillin mycin Acistrate; Erythromycin Estolate; Erythromycin Cresyl Sodium; Ticarcillin Disodium; Ticarcillin Mono Ethylsuccinate; Erythromycin Gluceptate; Erythromycin Sodium; Ticlatone; Tiodonium Chloride; Tobramycin; Lactobionate; Erythromycin Propionate; Erythromycin Tobramycin Sulfate; Tosufloxacin; Trimethoprim; Trime Stearate; Ethambutol Hydrochloride; Ethionamide; 55 thoprim Sulfate; Trisulfapyrimidines; Troleandomycin; Fleroxacin; Floxacillin; Fludalanine; Flumequine; Fosfo Trospectomycin Sulfate; Tyrothricin; Vancomycin; Van mycin; Fosfomycin Tromethamine; Fumoxicillin; Fura comycin Hydrochloride, Virginiamycin; Zorbamycin. Zolium Chloride; Furazolium Tartrate; Fusidate Sodium; Anticholinergic: Alverinc Citrate; Anisotropine Methylbro Fusidic Acid, Gentamicin Sulfate; Gloximonam; Grami mide; Atropine; Atropine Oxide Hydrochloride; Atropine cidin; Haloprogin; Hetacillin; Hetacillin Potassium; 60 Sulfate; Belladonna; Benapryzine Hydrochloride; Ben Hexedine, Ibafloxacin; Imipenem; Isoconazole, Isepami Zetimide Hydrochloride; Benzilonium Bromide; cin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamy Biperiden; Biperiden Hydrochloride; Biperiden Lactate; cin; Levofuraltadone; Levopropylcillin Potassium; Clidinium Bromide; Cyclopentolate Hydrochloride; Dex Lexithromycin; Lincomycin; Lincomycin Hydrochloride; etimide; Dicyclomine Hydrochloride; Dihexyverine Lomefloxacin; Lomefloxacin Hydrochloride; Lomefloxa 65 Hydrochloride; Domazoline Fumarate; Elantrine; cin Mesylate; Loracarbef; Mafenide; Meclocycline; Elucaine; Ethybenztropine, Eucatropine Hydrochloride; Meclocycline Sulfosalicylate; Megalomicin Potassium Glycopyrrolate; Heteronium Bromide; Homatropine US 6,267,957 B1 17 18 Hydrobromide; Homatropine Methylbromide; Hyos mchlorenone; Carbamide Peroxide; Cetalkonium Chlo cyamine, Hyoscyamine Hydrobromide, Hyoscyamine ride; Cetylpyridinium Chloride: Chlorhexidine Hydro Sulfate; Isopropamide Iodide; Mepenzolate Bromide; chloride; Clioquinol; Domiphen Bromide; Fenticlor; Methylatropine Nitrate; Metoquizine; Oxybutynin Chlo Fludazonium Chloride; Fuchsin, Basic; Furazolidone; ride; Parapenzolate Bromide; Pentapiperium Methylsul Gentian Violet; Halquinols; Hexachlorophene: Hydrogen fate; Phencarbamide; Poldine Methylsulfate; Proglumide; Peroxide; Ichthammol; Imidecyl Iodine; Iodine; Isopropyl Propantheline Bromide; Propenzolate Hydrochloride; Alcohol; Mafenide Acetate; Meralein Sodium; Mer Scopolamine Hydrobromide; Tematropium Methylsul cufenol Chloride; Mercury, Ammoniated; Methylbenze fate; Tiquinamide Hydrochloride; Tofenacin Hydrochlo thonium Chloride; Nitrofurazone; NitromerSol; Octeni ride; Toquizine; Triampyzine Sulfate; Trihexyphenidyl dine Hydrochloride; Oxychlorosene; Oxychlorosene Hydrochloride; Tropicamide. Sodium; Parachlorophenol, Camphorated; Potassium Per Antifungal: Acrisorcin; Ambruticin; Amphotericin B; AZa manganate; PoVidone-Iodine, Sepazonium Chloride; Sil conazole, AZaserine; Basifungin; Bifonazole; Biphe ver Nitrate; Sulfadiazine, Silver; Symclosene; Thimer namine Hydrochloride; Bispyrithione Magsulfex; Buto fonate Sodium; Thimerosal: Troclosene Potassium. conazole Nitrate, Calcium Undecylenate; Candicidin; 15 Anti-inflammatory: Alclofenac; Alclometasone Dipropi Carbol-Fuchsin; Chlordantoin; Ciclopirox, Ciclopirox onate, AlgeStone Acetonide; Alpha Amylase, Amcinafal; Olamine, Cilofimgin; Cisconazole; Clotrimazole; Amcinafide; Amfenac Sodium; Amiprilose Hydrochlo Cuprimyxin; Denofungin; Dipyrithione; Doconazole; ride; Anakinra, Anirolac, AnitraZafen; ApaZone, Bal Econazole; Econazole Nitrate; Enilconazole; Ethonam Salazide Disodium; Bendazac, Benoxaprofen, Benzy Nitrate; Fenticonazole Nitrate; Filipin; Fluconazole; damine Hydrochloride; Bromelains; Broperamole; Flucytosine; Fungimycin, Griseofulvin; Hamycin; ISO BudeSonide; Carprofen, Cicloprofen, CintaZone; Clipro conazole, Itraconazole; Kalafungin; Ketoconazole; fen; Clobetasol Propionate; Clobetasone Butyrate; Clopi Lomofungin; Lydimycin; Mepartricin; Miconazole; rac; Cloticasone Propionate; Cormethasone Acetate; Miconazole Nitrate; Monensin; Monensin Sodium; Naf Cortodoxone, Deflazacort, DeSonide, DeSoximetasone; tifine Hydrochloride; Neomycin Undecylenate; Nifuratel; 25 Dexamethasone Dipropionate; Diclofenac Potassium; Nifurmerone; Nitralamine Hydrochloride; Nystatin; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Octanoic Acid, Orconazole Nitrate; Oxiconazole Nitrate; Sodium; Diflunisal; Difluprednate; Diftalone; Dimethyl Oxifimgin Hydrochloride; Parconazole Hydrochloride; Sulfoxide; Drocinonide; Endrysone; Enlimomab, Enoli Partricin; Potassium Iodide; Proclonol; Pyrithione Zinc; cam Sodium; Epirizole, Etodolac, Etofenamate, Felbinac, Pyrrolnitrin; Rutamycin; Sanguinarium Chloride; Saper Fenamole; Fenbufen, Fenclofenac, Fenclorac, Fendosal; conazole, Scopafungin; Selenium Sulfide, Sinefungin; Fenpipalone, Fentiazac, Flazalone, Fluazacort, Flufe Sulconazole Nitrate; Terbinafine; Terconazole; Thiram; namic Acid, Flumizole; Flunisolide Acetate; Flumixin; Ticlatone; Tioconazole, Tolciclate; Tolindate, Tolnaftate; Flunixin Meglumine; Fluocortin Butyl, Fluorometholone Triacetin; Triafungin; Undecylenic Acid; Viridofulvin; Acetate; FluguaZone; Flurbiprofen; Fluretofen; Flutica Zinc Undecylenate; Zinoconazole Hydrochloride. 35 sone Propionate; Furaprofen; Furobufen; Halcinonide; Antiglaucoma agent: AlprenoXime Hydrochloride; Col Halobetasol Propionate; Halopredone Acetate; Ibufenac; forsin; Dapiprazole Hydrochloride; Dipivefrin Hydro Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol; chloride; Naboctate Hydrochloride; Pilocarpine; Pirnab Ilonidap, Indomethacin; Indomethacin Sodium; Indopro C. fen, Indoxole, Intrazole; Isoflupredone Acetate; ISOXepac, Antihistaminic: Acrivastine; Antazoline Phosphate, Astemi 40 Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lor Zole, AZatadine Maleate; Barmastine; Bromodiphenhy noxicam, Lote prednol Etabonate, Meclofenamate dramine Hydrochloride; Brompheniramine Maleate; Sodium; Meclofenamic Acid; Meclorisone Dibutyrate; Carbinoxamine Maleate; Cetirizine Hydrochloride; Chlo Mefenamic Acid; Mesalamine; Meseclazone; Methyl rpheniramine Maleate; Chlorpheniramine Polistirex; Cin prednisolone Suleptanate; Morniflumate; Nabumetone; narizine; Clemastine; Clemastine Fumarate; Closiramine 45 Naproxen Naproxen Sodium; Naproxol; Nimazone; Aceturate; Cycliramine Maleate; Cyclizine, Cyprohepta Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin, dine Hydrochloride; Dexbrompheniramine Maleate; Dex Oxyphenbutazone; Paranyline Hydrochloride; Pentosan chlorpheniramine Maleate; Dimethindene Maleate; Polysulfate Sodium; Phenbutazone Sodium Glycerate; Diphenhydramine Citrate; Diphenhydramine Hydrochlo Pirfenidone; Piroxicam, Piroxicam Cinnamate; Piroxicam ride; Dorastine Hydrochloride; Doxylamine Succinate; 50 Olamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Ebastine; Levocabastine Hydrochloride; Loratadine; ProquaZone; Proxazole; Proxazole Citrate; Rimexolone; Mianserin Hydrochloride; Noberastine; Orphenadrine RomaZarit; Salcolex, Salnacedin; Salsalate, Sangui Citrate; Pyrabrom; Pyrilamine Maleate; Pyroxamine narium Chloride; SeclaZone ; Sermetacin, Sudoxicam, Maleate; Rocastine Hydrochloride; Rotoxamine; Tazifyl Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; line Hydrochloride; Temelastine; Terfenadine; Tripelen 55 Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesi namine Citrate; Tripelennamine Hydrochloride; Triproli cam; Tesimide; Tetrydamine; Tiopinac; Tixocortol Piv dine Hydrochloride; Zolamine Hydrochloride. alate; Tolimetin; Tolimetin Sodium; Triclonide; Triflumi Anti-infective: Difloxacin Hydrochloride; Lauryl Isoquino date, Zidometacin, Zomepirac Sodium linium Bromide; Moxalactam Disodium; Ornidazole; Antikeratinizing agent: Doretinel; Linaroteine, Pelretin. Pentisomicin; Sarafloxacin Hydrochloride; Protease 60 Antimicrobial: Aztreonam; Chlorhexidine Gluconate, Imi inhibitors of HIV and other retroviruses; Integrase Inhibi durea; Lycetamine; Nibroxane, PiraZmonam Sodium; tors of HIV and other retroviruses; Cefaclor (Ceclor); Propionic Acid; Pyrithione Sodium; Sanguinarium Chlo Acyclovir (Zovirax); Norfloxacin (Noroxin); Cefoxitin ride; Tigemonam Dicholine. (Mefoxin); Cefuroxime axetil (Ceftin); Ciprofloxacin Antimycotic: Amorolfine. (Cipro). 65 Antineoplastic: Acivicin, Aclarubicin; Acodazole Hydro Anti-infective, topical: Alcohol; Aminacrine Hydrochloride; chloride; Acronine; Adozelesin; Aldesleukin; Altre Benzethonium Chloride: Bithionolate Sodium; Bro tamine, Ambomycin; Ametantrone Acetate; Aminoglute US 6,267,957 B1 19 20 thimide, AmSacrine; Anastro Zole; Anthramycin; inhibitors, antagonist D; antagonist G, antarelix; anti Asparaginase; ASperlin, AZacitidine, AZetepa, AZOtomy dorsalizing morphogenetic protein-1, antiandrogen, pros cin; Batimastat; BenZOdepa, Bicalutamide, Bisantrene tatic carcinoma, antiestrogen; antineoplaston; antisense oli Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleo gonucleotides, aphidicolin glycinate, apoptosis gene mycin Sulfate; Brequinar Sodium; Bropirimine; Busul modulators, apoptosis regulators, apurinic acid, ara-CDP fan; Cactinomycin; Calusterone; Caracemide, Carbe DL-PTBA, arginine deaminase, asulacrine, atameStane; atri timer; Carbo platin; Car mustine: Carubic in mustine; axinastatin 1; axinastatin 2; axinastatin 3; aza Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Setron; azatoxin; azatyrosine; baccatin III derivatives, Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; balanol; batimastat; BCR/ABL antagonists; benzochlorins; Cyclophosphamide, Cytarabine; Dacarbazine; Dactino benzoylstauroSporine; beta lactam derivatives, beta mycin; Daunorubicin Hydrochloride; Decitabine; Dexor alethine; betaclamycin B; betulinic acid; bFGF inhibitor; maplatin, DeZaguanine, DeZaguanine MeSylate; Diazi bicalutamide, bisantrene, bisaZiridinylspermine; bisnafide; quone; Docetaxel, Doxorubicin; Doxorubicin bistratene A, bizelesin; breflate; bropirimine; budotitane; Hydrochloride; Droloxifene; Droloxifene Citrate; Dro buthionine Sulfoximine, calcipotriol, calphostin C; camp moStanolone Propionate, DuaZomycin; Edatrexate, Eflo 15 tothecin derivatives, canarypox IL-2, capecitabine; rnithine Hydrochloride; Elsamitrucin; Enloplatin; Enpro carboxamide-amino-triazole; carboxyamidotriazole, CaRest mate; Epipropidine, Epirubicin Hydrochloride; M3; CARN 700; cartilage derived inhibitor; carzelesin; Erbulozole; Esorubicin Hydrochloride; Estramustine; casein kinase inhibitors (ICOS), castanospermine; cecropin Estramustine Phosphate Sodium; Etanidazole; Ethiodized B; cetrorelix; chlorins, chloroquinoxaline Sulfonamide, cica Oil I 131; Etoposide; Etoposide Phosphate; Etoprine; prost, cis-porphyrin, cladribine; clomifene analogues, clot Fadrozole Hydrochloride; Fazarabine; Fenretinide; rimazole; collismycin A, collismycin B; combretastatin A4, Floxuridine; Fludarabine Phosphate; Fluorouracil; combretastatin analogue, conagenin, crambescidin 816; cri Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcit Snatol; cryptophycin 8; cryptophycin A derivatives, curacin abine; Gemcitabine Hydrochloride; Gold Au 198; A., cyclopentanthraquinones, cycloplatam, cypemycin; cyt Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; 25 arabine ocfoSfate; cytolytic factor; cytostatin, dacliximab, Ilmofosine; Interferon Alfa-2a, Interferon Alfa-2b; Inter decitabine; dehydrodidemnin B; deslorelin; dexifosfamide; feron Alfa-n1;Interferon Alfa-n3; Interferon Beta-Ia; deXraZOxane, deXVerapamil, diaziquone; didemnin B; didox; Interferon Gamma-Ib, Iproplatin; Irinotecan Hydrochlo diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, ride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; 9-, dioxamycin; diphenyl Spiromustine, docosanol; dolas Liarozole Hydrochloride; Lometrexol Sodium; Lomus etron, doxifluridine, droloxifene, dronabinol; duocarmycin tine; Losoxantrone Hydrochloride; Masoprocol; May SA, ebSelen; ecomustine, edelfosine; edrecolomab, eflorni tansine; Mechlorethamine Hydrochloride; Megestrol thine; elemene; emitefur, epirubicin, epristeride, estramus Acetate; Melengestrol Acetate; Melphalan; Menogaril; tine analogue; estrogen agonists, estrogen antagonists, Mercaptopurine; Methotrexate; Methotrexate Sodium; etanidazole, etoposide phosphate; exemestane, fadrozole; Metoprine; Meturedepa; Mitindomide; Mitocarcin; 35 fazarabine; fenretinide; filgrastim; finasteride, flavopiridol; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mito fleZelastine; fluiasterone, fludarabine; fluorodaunorunicin sper; Mitotane; Mitoxantrone Hydrochloride; Mycophe hydrochloride; forfenimex, formeStane; fostriecin, fotemus nolic Acid, Nocodazole; Nogalamycin; Ornaplatin; Oxi tine, gadolinium teXaphyrin, gallium nitrate; galocitabine; Suran, Paclitaxel, Pegaspargase, Peliomycin; ganirelix; gelatinase inhibitors, gemcitabine; glutathione Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipo 40 inhibitors, hepSulfam; heregulin; hexamethylene bisaceta broman; Piposulfan; Piroxantrone Hydrochloride; Plica mide; hypericin; ibandronic acid; idarubicin; idoxifene, mycin; Plomestane; Porfimer Sodium; Porfiromycin; idramantone, ilmofoSine, illomastat; imidazoacridones, imi Prednimustine; Procarbazine Hydrochloride; Puromycin; quimod, immunostimulant peptides; insulin-like growth Puromycin Hydrochloride; Pyrazofurin; Riboprine; factor-1 receptor inhibitor; interferon agonists, interferons, Rogletimide; Safingol; Safingol Hydrochloride; Semus 45 interleukins, iobenguane; iododoxorubicin; ipomeanol, 4-, tine, SimtraZene, Sparfosate Sodium, SparSomycin; irinotecan; iroplact, irSogladine, isobengaZole, isohomohali Spirogennanium Hydrochloride, Spiromustine, Spiropl condrin B; itasetron, jasplakinolide; kahalalide F; atin; Streptonigrin; Streptozocin; Strontium Chloride Sr lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan lentinan Sulfate; leptolstatin; letrozole; leukemia inhibiting Sodium; Tegafur; Teloxantrone Hydrochloride; Temopor 50 factor; leukocyte alpha interferon; leuprolide--estrogen fin, TenipoSide; Teroxirone; Testolactone; Thiamiprine; progesterone; leuprorelin; levamisole; liarozole; linear Thioguanine; Thiotepa; Tiazofurin, TirapaZamine; Topo polyamine analogue, lipophilic disaccharide peptide; lipo tecan Hydrochloride; Toremifene Citrate; Trestolone philic platinum compounds, lisSoclinamide 7; lobaplatin; Acetate; Triciribine Phosphate; Trimetrexate; Trimetrex lombricine, lometrexol; lonidamine; loSOXantrone; lovasta ate Glucuronate; Triptorelin; Tubulozole Hydrochloride; 55 tin, loxoribine; lurtotecan; lutetium teXaphyrin, lySofylline; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vin lytic peptides, maitansine; mannostatin A, marimastat; blastine Sulfate; Vincristine Sulfate; Vindesline; Vindesline masoprocol; maspin; matrilysin inhibitors, matrix metallo Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleu proteinase inhibitors, menogaril; merbarone; meterelin; rosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; methioninase; metoclopramide; MIF inhibitor; mifepris Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; 60 tone, miltefosine, mirimoStim, mismatched double Stranded Zorubicin Hydrochloride. RNA, mitoguaZone, mitolactol, mitomycin analogues, Other anti-neoplastic compounds include: 20-epi-1,25 mitonafide; mitotoxin fibroblast growth factor-Saporin; dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclaru mitoxantrone; mofaroteine; molgramoStim; monoclonal bicin; acylfulvene, adecypenol; adoZelesin; aldesleukin; antibody, human chorionic gonadotrophin, monophosphoryl ALL-TK antagonists, altretamine; ambamustine, amidox; 65 lipid A+myobacterium cell wall Sk, mopidamol, multiple amifostine; aminolevulinic acid; amrubicin; amsacrine; drug resistance gene inhibitor, multiple tumor Suppressor anagrelide, anastrozole; andrographolide, angiogenesis 1-based therapy; mustard anticancer agent; my caperoxide B; US 6,267,957 B1 21 22 mycobacterial cell wall eXtract; myriap or one; pertensive drugs (e.g., reserpine); Thiol depleters (e.g., N-acetyldinaline; N-substituted benzamides; nafarelin; buthionine and sulfoximine) and Multiple Drug Resistance nagreStip; naloxone-pentazocine, napavin, naphterpin, nar reducing agents Such as Cremaphor EL. The compounds of tograstim; nedaplatin; nemorubicin; neridronic acid; neutral the invention also can be administered with cytokines Such endopeptidase, nilutamide, nisamycin; nitric oxide modula as granulocyte colony Stimulating factor. tors, nitroxide antioxidant; nitrullyn; O6-benzylguanine; Antineutropenic: Filgrastim; Lenograstim; MolgramoStim; octreotide, okicenone, oligonucleotides, onapristone; RegramoStim; SargramoStim. Antiproliferative agent: Piritrexim Isethionate. Ondansetron; ondansetron; oracin, oral cytokine inducer, Antiprotozoal: Amodiaquine, AZanidazole; Bamnidazole; ormaplatin, OSaterone, Oxaliplatin, oxaunomycin; paclitaxel Camidazole; Chlortetracycline Bisulfate; Chlortetracy analogues, paclitaxel derivatives, palauamine, palmitoyl cline Hydrochloride; Flubendazole; Flunidazole; rhizoxin; pamidronic acid; panaxytriol; panomifene, para Halofuginone Hydrobromide; Imidocarb Hydrochloride; bactin; paZelliptine, pegaspargase; peldesine, pentosan Ipronidazole; Metronidazole; Misonidazole; Moxinida polysulfate Sodium; pentostatin; pentrozole, perflubron; per zole; NitarSone; Partricin; Puromycin; Puromycin Hydro fosfamide; perillyl alcohol; phenaZinomycin; phenylacetate; chloride; Ronidazole; Sulnidazole; Tinidazole. phosphatase inhibitors, picibanil; pilocarpine hydrochloride; 15 Antipruritic: Cyproheptadine Hydrochloride; Methdilazine; pirarubicin, piritrexim; placetin A; placetin B; plasminogen Methdilazine Hydrochloride; Trimeprazine Tartrate. activator inhibitor; platinum complex, platinum compounds, Antipsoriatic: Acitretin; Anthralin, AZaribine, Calcipotriene; platinum-triamine complex, porfimer Sodium; porfiromycin; Cycloheximide; Enazadrem Phosphate; Etretinate; Liaro propyl bis-acridone; prostaglandin J2, proteasome inhibi Zole Fumarate, Lonapalene; Tepoxalin. tors, protein A-based immune modulator, protein kinase C Carbonic anhydrase inhibitor: Acetazolamide; Acetazola inhibitor, protein kinase C inhibitors, microalgal; protein mide Sodium; Dichlorphenamide; Dorzolamide Hydro tyrosine phosphatase inhibitors, purine nucleoside phospho chloride; Methazolamide; Sezolamide Hydrochloride. rylase inhibitors, purpurins, pyrazoloacridine, pyridoxylated Cholinergic: Aceclidine; Bethanechol Chloride; Carbachol; hemoglobin polyoxyethylene conjugate, raf antagonists, Demecarium Bromide; Dexpanthenol; Echothiophate raltitrexed; ramosetron; ras farnesyl protein transferase 25 Iodide; Isoflurophate; Methacholine Chloride; Neostig inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine mine Bromide; Neostigmine Methylsulfate; Physostig demethylated; rhenium Re 186 etidronate; rhizoxin; mine; Physostigmine Salicylate; Physostigmine Sulfate; ribozymes, RII retinamide, rogle timide, rohitukine; Pilocarpine; Pilocarpine Hydrochloride; Pilocarpine romurtide, roquinimex, rubiginone B1, rubOXyl; Safingol; Nitrate; Pyridostigmine Bromide. Saintopin; SarCNU; Sarcophytol A, SargramoStim; Sidi 1. Diagnostic aid: Aminohippurate Sodium; Anazolene mimetics, Semustine, Senescence derived inhibitor 1; Sense Sodium; Arclofenin, Arginine, Bentiromide, Benzylpeni oligonucleotides, Signal transduction inhibitors, Signal trans cilloyl Polylysine; Butedronate Tetrasodium; Butilfenin; duction modulators, Single chain antigen binding protein; Coccidioidin; Corticorelin Ovine Triflutate; sizofiran; Sobu ZOxane, Sodium borocaptate, Sodium pheny Corticotropin, Repository; Corticotropin Zinc Hydroxide; lacetate, Solverol, Somatomedin binding protein; Sonermin; 35 Diatrizoate Meglumine; Diatrizoate Sodium; Diatrizoic Sparfosic acid; Spicamycin D; Spiromustine; Splenopentin; Acid; Diphtheria Toxin for Schick Test; Disofenin; Edro Spongistatin 1; Squalamine; Stem cell inhibitor, Stem-cell phonium Chloride; Ethiodized Oil, Etifenin; Exametaz division inhibitors; stipiamide; stromelysin inhibitors; sulfi ime, Ferristenc, Ferumoxides, FerumoxSil; Fluorescein, nosine; Superactive vasoactive intestinal peptide antagonist; Fluorescein Sodium; Gadobenate Dimeglumine; Gadot Suradista; Suramin; Swainsonine, Synthetic glycosaminogly 40 eridol, Gadodiamide, Gadopentetate Dimeglumine; cans, tallimustine, tamoxifen methiodide, tauromustine; taZ Gadoversetamide; Histoplasmin; Impromidine Hydro arotene, tecogalan Sodium, tegafur, tellurapyrylium; telom chloride; Indigotindisulfonate Sodium; Indocyanine erase inhibitors, temoporfin, temozolomide; teniposide; Green; Iobenguane Sulfate I 123, Iobenzamic Acid; Iocar tetrachlorodecaoxide, tetraZomine; thaliblastine; thalido mate Meglumine, Iocarmic Acid, Iocetamic Acid, Ioda mide; thiocoraline; thrombopoietin; thrombopoietin 45 mide, Iodamide Megilumine, Iodipamide Meglumine; mimetic; thymalfasin; thymopoietin receptor agonist, thy Iodixanol, Iodoxamate Meglumine, Iodoxamic Acid; motrinan; thyroid Stimulating hormone, tin ethyl etiopurpu Ioglicic Acid, Ioglucol, Ioglucomide, Ioglycamic Acid; rin; tirapazamine, titanocene dichloride; topotecan; topsen Iogulamide, Iohexol, Iomeprol, Iopamidol, Iopanoic tin, toremifene; totipotent Stem cell factor; translation Acid, Iopentol; Iophendylate; profenin, Iopronic Acid; inhibitors, tretinoin, triacetyluridine, triciribine, trimetrex 50 Ioprocemic Acid, Iopydol, Iopy done, Iosefamic Acid; ate, triptorelin; tropisetron; turosteride, tyrosine kinase IoSeric Acid, IoSulamide Meglumine, IoSumetic Acid; inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogeni Iotasul; Iotetric Acid; Iothalamate Meglumine; Iothala tal Sinus-derived growth inhibitory factor; urokinase recep mate Sodium; Iothalamic Acid; Iotrolan; IotroXic Acid; tor antagonists, vapreotide; variolin B; Vector System, eryth IoverSol, Ioxaglate Meglumine, Ioxagiate Sodium, Ioxa rocyte gene therapy, VelareSol, Veramine, Verdins, 55 glic Acid, Ioxilan, Ioxotrizoic Acid, Ipodate Calcium; verteporfin, Vinorelbine; VinXaltine, Vitaxin; Vorozole; Zan Ipodate Sodium; ISOSulfan Blue; Leukocyte Typing oterone; Zeniplatin, Zilascorb, Zinostatin stimalamer. Serum; Lidofenin; Mebrofenin; Meglumine; Metriza Anti-cancer Supplementary Potentiating Agents: Tricy mide; Metrizoate Sodium; Metyrapone; Metyrapone Tar clic anti-depressant drugs (e.g., imipramine, desipramine, trate; Mumps Skin Test Antigen; Pentetic Acid; Propyli amitryptyline, clomipramine, trimipramine, doxepin, 60 odone; Quinaldine Blue, Schick Test Control; Sermorelin nortriptyline, protriptyline, amoxapine and maprotiline); Acetate; Sodium Iodide I 123; Sprodiamide; Stannous non-tricyclic anti-depressant drugs (e.g., Sertraline, traz Pyrophosphate; Stannous Sulfur Colloid; Succimer; Teri odone and citalopram); Ca' antagonists (e.g., Verapamil, paratide Acetate; Tetrofosmin; Tolbutamide Sodium; nifedipine, nitrendipine and caroverine); Calmodulin inhibi Tuberculin; Tyropanoate Sodium; Xylose. tors (e.g., prenylamine, trifluoropera Zine and 65 Ectoparasiticide: Nifluridide; Permethrin. clomipramine); Amphotericin B; Triparanol analogues (e.g., Glucocorticoid: Amcinonide; Beclomethasone Dipropi tamoxifen); antiarrhythmic drugs (e.g., quinidine); antihy onate, Betame thaSone; Betame thaSone Acetate; US 6,267,957 B1 23 24 Betamethasone Benzoate, Betamethasone Dipropionate; Hydrochloride I 123; Iomethin I 125; Iomethin I 131; Betamethasone Sodium Phosphate; Betamethasone Val Iothalamate Sodium I 125; Iothalamate Sodium I 131; erate; Carbenoxolone Sodium; Clocortolone Acetate; Clo Iotyrosine 1131; Liothyronine I 125; Liothyronine I 131; cortolone Pivalate; Clop rednol; Corticotropin; Merisoprol Acetate Hg 197; Merisoprol Acetate Hg 203; Corticotropin, Repository; Corticotropin Zinc Hydroxide; Merisoprol Hg 197; Selenomethionine Se 75; Technetium Cortisone Acetate; CortivaZol, Descinolone Acetonide; Tc 99 m Antimony Trisulfide Colloid; Technetium Tc 99 Dexamethasone; Dexamethasone Sodium Phosphate; m Bicisate; Technetium Tc 99 m Disofenin; Technetium Diflucortolone; Diflucortolone Pivalate; Flucloronide; Tc 99 m Etidronate; Technetium Tc 99 m. Exametazime; Technetium Tc 99 m Furifosmin; Technetium Tc 99 m Flumethasone; Flumethasone Pivalate; Flunisolide; Fluo Gluceptate; Technetium Tc 99 m Lidofenin; Technetium cinolone Acetonide; Fluocinonide; Fluocortolone; Fluo Tc 99 m Mebrofenin; Technetium Tc 99 m Medronate; cortolone Caproate, Fluorometholone, Fluperolone Technetium Tc 99 m Medronate Disodium; Technetium Acetate; Fluprednisolone; Fluprednisolone Valerate; Flu Tc 99 m Mertiatide; Technetium Tc 99 m Oxidronate; randrenolide; Formocortal; Hydrocortisone; Hydrocorti Technetium Tc 99 m Pentetate; Technetium Tc 99 m Some Acetate, Hydrocortisone Buteprate; Hydrocortisone Pentetate Calcium Trisodium; Technetium Tc 99 m Ses Butyrate; Hydrocortisone Sodium Phosphate; Hydrocor 15 tamibi; Technetium Tc 99 m Siboroxime; Technetium Tc tisone Sodium Succinate; Hydrocortisone Valerate; 99 m Succimer; Technetium Tc 99 m Sulfur Colloid; Medrysone; Methylprednisolone; Methylprednisolone Technetium Tc 99 m Teboroxime; Technetium Tc 99 m Acetate; Methylprednisolone Sodium Phosphate; Meth Tetrofosmin; Technetium Tc 99 m. Tiatide; Thyroxine 1 ylprednisolone Sodium Succinate; Nivazol; Parametha 125; Thyroxine 1131; Tolpovidone 1131; Triolein 1125; Some Acetate; Prednicarbate; Prednisolone; Prednisolone Triolein 1131. Acetate; Prednisolone Hemisuccinate; Prednisolone Wound healing agent: ErSofermin. Sodium Phosphate; Prednisolone Sodium Succinate; The invention thus may be used, inter alia, to localize Prednisolone Tebutate; Prednisone; Prednival; Ticabe drugs to a tissue Such as a wound bed or for localized Some Propionate; Tralonide; Triamcinolone; Triamcino delivery to a tissue, to hold a drug, insect repellant, bacte lone Acetonide; Triamcinolone Acetonide Sodium; Tri 25 ricide fungicide, growth factors, cytokine, and the like at a amcinolone Diacetate; Triamcinolone Hexacetonide. particular location to prevent the drug from being flushed Hair growth stimulant: Minoxidil. away to other body Sites where it is not needed, to apply Histamine H2 receptor antagonists: Ranitidine (Zantac); bulking agents and other cosmetic agents to the integuments, Famotidine (Pepcid); Cimetidine (Tagamet); Nizatidine Such as the skin, hair and nails, to hold Sunscreen agents at (Axid). the Surface of the skin for longer periods of time, to hold Immunizing agent: Antirabies Serum; Antive nin anti-nerve gas enzymes at the Surface of the skin whereby (Latrodectus mactans); Antivenin (Micrurus Fulvius); nerve gas can be deactivated, to hold or link chemical agents Antivenin (Crotalidae) Polyvalent; BCG Vaccine; Botu to the skin which can in turn act as binding sites for other lism Antitoxin; Cholera Vaccine; Diphtheria Antitoxin; agent or alternatively, as reactive sites for catalytic buildup Diphtheria Toxoid; Diphtheria Toxoid Adsorbed; 35 of multiple alternating layers, to link hydrophobic com Globulin, Immune; Hepatitis B Immune Globulin; Hepa pounds to the Skin, thereby making the Skin hydrophobic, to titis B Virus Vaccine Inactivated; Influenza Virus Vaccine; link conditioners to the hair, thereby giving hair the appear Measles Virus Vaccine Live; Meningococcal Polysaccha ance of greater bulk and to link agents to organs or tissues ride Vaccine Group A; Meningococcal Polysaccharide which are to be transplanted. Vaccine Group C; Mumps Virus Vaccine Live; Pertussis 40 Immune Globulin; Pertussis Vaccine; Pertussis Vaccine EXAMPLES Adsorbed; Plague Vaccine; Poliovirus Vaccine Inacti Example 1 vated; Poliovirus Vaccine Live Oral; Rabies Immune Durable Suntan Preparation and Kit. Globulin; Rabies Vaccine, Rh(D) Immune Globulin; A kit is provided for producing a durable Sunscreen. The Rubella Virus Vaccine Live; Smallpox Vaccine; Tetanus 45 kit includes as a first component a conjugate of a low Antitoxin; Tetanus Immune Globulin; Tetanus Toxoid; molecular weight Sunscreen agent and a linking agent. This Tetanus Toxoid Adsorbed; Typhoid Vaccine; Yellow component is an aqueous solution, pH 6.4 of 50 mM Fever vaccine; Vaccinia Immune Globulin; Varicella polylysyl-methoxy-2-ethylhexyl-cinnamate, 0.1 V (% propy Zoster Immune Globulin. lene glycol, 0.5 mM, EDTA, 0.1 wt % BHT, 0.1 wt % Immunomodulator: Dimepranol Acedoben; Imiquimod; 50 potassium Sorbate, 0.05 wt % polysorbate 20 and 80 and 1 Interferon Beta-1b; Lisofylline; Mycophenolate Mofetil, mM sodium laurylether sulfate. Component 2 of the kit is a Prczatide Copper Acetate. calcium chloride activator Solution. This is an aqueous Immunoregulator: AZarole, Fanetizole MeSylate; Frentizole; solution at about 25 mM calcium chloride. Component 3 of Oxamisole Hydrochloride; Ristianol Phosphate; Thymo the kit is lyophilized transglutaminase. The lyophilized pentin; Tilomisole. 55 preparation can contain 10 mg of recombinant tissue trans Immunostimulant: Loxoribine; Teceleukin. glutaminase in 2% sucrose, 0.1 mM EDTA, and 5 mM Immunosuppressant: AZathioprine, AZathioprine Sodium; glycine buffer, pH 7.2. Cyclosporine; Daltroban; Gusperimus Trihydrochloride; Three Vials containing the three kit components are Sirolimus; Tacrolimus. Mucolytic: Acetylcysteine; Car opened. About 10 mL of component 1 is added to 10 mg of bocysteine; Domiodol. 60 component 3, and the combination is mixed by Swirling. Mucosal Protective agents: Misoprostol (Cytotec). Then this combination is added to about 90 mL of compo Radioactive agent: Fibrinogen 1125; Fludeoxyglucose F 18; nent 1. Finally, about 10 mL of component 2 is added to the Fluorodopa F 18; Insulin I 125; Insulin I 131; Iobenguane mixture, with this final combination mixed by gentle Swirl I 123; Iodipamide Sodium I 131; Iodoantipyrine I 131; ing. The mixture then is applied to a washed and Scraped Iodocholesterol I 131; Iodohippurate Sodium I 123; 65 skin Surface. The mixture is uniformly spread on the skin Iodohippurate Sodium I 125; Iodohippurate Sodium I and allowed to remain for ten minutes. The exceSS Solution 131; Iodopyracet I 125; Iodopyriacet I 131; Iofetamine is removed by Washing. US 6,267,957 B1 25 26 Example 2 A. Mouse was epilated. Seven days later, a concentrated Durable Topical Antifungal Preparation and Kit. reaction Solution containing guine a pig A kit is provided for producing durable antifungal pro transglutaminase, dansyl labeled polyglutamine and tection. The kit contains three components. Component 1 is Ca" at 10 mM was applied to the left side (FIG.2). The a conjugate of an antifungal agent and a linking agent. This control (right side) was pretreated for 10 mins with 100 component is an aqueous Solution, pH 6.4, containing 0.01 mM cyStamine, the excess liquid was drained and the wt % polylysyl-amphotericin B conjugate, 10 V % ethanol, Same reaction Solution containing 25 mM cyStamine 0.1 v 9% propylene glycol, 0.5 mM EDTA, 0.1 wt % BHT. Component 2 is a calcium chloride activator Solution as was applied. After 30 minutes, both sites were washed described for Example 1. Component 3 is a lyophilized with a solution of 1% SDS. The mouse was then transglutaminase preparation as described in Example 1. photographed under UV illumination (312 nm). The The three containers containing components 1, 2 and 3 are left Side shows Strong fluorescence of dansyl poly opened. Ten mL of component 1 is added to component 3, glutamine whereas the right Side shows very weak and they are mixed by Swirling. The mixture then is added fluorescence (FIG. 2). to about 90 mL of component 1. To this mixture is added B. Same mouse was photographed again five days later. component 2. This final combination is mixed by gentle 15 There is still considerable fluorescence at the site of Swirling. After this, the material is applied to the Surface of enzymatic coupling (left, FIG. 3), but the control fluo skin as described in Example 1. rescence (right, FIG. 3) has virtually disappeared. Example 3 Reaction Solution Long-Term Protective Preparation for Anticholinesterase 10 ul buffer containing 100 uM Tris pH8.2, 10 MNCaCl, Nerve Gas and Kit. and 10 mM DTT A kit for providing long-term protection from anticho 3 ul dansylated polyglutamine (5 uM) linesterase nerve gas is provided. Component 1 of the kit 3 ul (13.3 mU/ul) partially purified guinea pig trans includes recombinant cholinesterase coupled to biotin (e.g., glutaminase by reaction in the presence of N.N. Succinimide). Compo We claim: nent 2 is polyglutamine coupled to avidin. Component 2 is 25 1. A method for attaching a non-corneocyte protein, applied to the Surface of the Skin in the presence of nonlabeling agent to a body tissue comprising: transglutaminase, as described above in connection with applying to the body tissue a conjugate of the agent and Examples 1 and 2. After the avidin is coupled to the skin via a carboxamide-containing linking molecule having at the polyglutamine, then component 1 is added to bind the least two contiguous linked glutamines and being a biotin to the avidin, thereby coupling the cholinesterase to Substrate of transglutaminase, the skin. Said applying to the body tissue being carried out in the presence of transglutaminase in an amount effective to Example 4 covalently crosslink the conjugate to the body tissue via A Mousse for Thickening Hair. the linking molecule, and A dispensing can with three reservoirs (a calcium ion 35 Solution, a transglutaminase Solution and a hair bulking or allowing Said crosslinking to occur. thickening agent Such as a mucopolysaccharide linked to 2. The method of claim 1, wherein the linking molecule polyglutamine) is provided. The three Solutions are mixed, is Selected from the group consisting molecules having: as is conventional with Such dispensing cans, as they are (a) at least three contiguous linked glutamines, being applied onto tissue Such as hair. The mousse can be 40 (b) at least four contiguous linked glutamines, and combed through the hair, left on the hair for at least ten (c) at least five contiguous linked glutamines. minutes, and then rinsed. 3. The method of claim 1, wherein the linking molecule comprises 5 or more contiguous glutamines attached directly Example 5 to one another by peptide bonds. It has been shown in previous Studies that polyglutamine 45 4. The method of claim 1, wherein the linking molecule attached to other peptides remains an excellent Substrate of comprises a polymer of amino acids and wherein at least transglutaminase. Under optimal conditions, virtually all of 20% of the amino acids are glutamines. the glutamine residues acted as amine acceptors in the 5. The method of claim 4, wherein at least 30% of the reaction with an aliphatic amine, and lengthening the amino acids are glutamines. Sequence of polyglutamine increases the reactivity of each 50 6. The method of claim 4, wherein at least 40% of the glutamine residue. In the presence of transglutaminase, amino acids are glutamines. peptides containing polyglutamine become croSS-linked to 7. The method of claim 1, further comprising first attach polylysine. The details of the reaction conditions and the ing to the body tissue a complementary linking molecule manner of applying labels whereby the reaction may be bearing multiple reactive aliphatic amines, the complemen visualized under UV light are described in detail in Kahlem 55 tary linking molecule being an aliphatic amine Substrate of et al., Proc. Natl. Acad. Sci. USA, 1996 93:14580-14585 transglutaminase, wherein the conjugate is crosslinked to the (Appendix A). The same polyglutamines, but attached to body tissue by crosslinking the aliphatic amines of the agents as described herein, and, in general, the same con complementary linking molecule and the carboxamide of ditions as described in Kahlem et al. may be applied in the linking molecule to one another by Said transglutaminase. above-described examples and, in general, in the-practice of 60 8. The method of claim 7, wherein the complementary the present invention. The disclosure of this reference, as linking molecule is attached to the body tissue by well as any other reference mentioned herein, is incorpo applying to the body tissue the complementary linking rated by reference in its entirety. molecule, Example 6 applying to the body tissue an amount of transglutaminase Polyglutamine Containing a Fluorescent Marker is 65 effective for crosslinking the complementary linking Covalently Attached to the Surface of the Skin Through the molecule to the body tissue, and Action of Transglutaminase allowing Said crosslinking to occur. US 6,267,957 B1 27 28 9. The method of claim 8, wherein a polymer rich in 26. The method of any one of claim 20, wherein the body lysine is the complementary linking molecule. tissue is Selected from the group consisting of skin, hair, 10. The method of claim 9, wherein the linking molecule nails, wound bed tissue and internal body tissue. is a polymer having 4 or more contiguous glutamines 27. The method of claim 20, wherein the agent in a directly attached to one another by peptide bonds. nonprotein. 11. The method of claim 9, wherein the polymer rich in 28. The method of claim 20, wherein the agent is an lysine has 4 or more contiguous lysines directly attached to enzyme. one another by peptide bonds. 29. The method of claim 28, wherein the enzyme is 12. The method of claim 1, wherein the agent is not itself Selected from the group consisting of a cholinesterase and a a Substrate of transglutaminase. 1O phosphodiesterase. 13. The method of claim 1, wherein the body tissue is Selected from the group consisting of skin, hair, nails, wound 30. A method for attaching an agent to a body tissue bed tissue and internal body tissue. comprising 14. The method of claim 1, wherein the body tissue is first attaching to the body tissue a carboxamide Selected from the group consisting of skin, hair and nails, containing linking molecule having at least two con and wherein the agent is Selected from the group consisting 15 tiguous linked glutamines that is covalently bondable of a cosmetic agent, a bulking agent, a SunScreen agent, a to the agent in the presence of transglutaminase, then coloring agent, a pharmaceutical agent, a ligand-receptor complex and a receptor of a ligand-receptor complex. applying to the body tissue having the linking molecule 15. The method of claim 1, wherein the agent is an attached thereto an agent that is a Substrate of trans enzyme. glutaminase and that is covalently bonded to the linking 16. The method of claim 15, wherein the agent is selected molecule in the presence of transglutaminase, Said from the group consisting of a cholinesterase and a phos applying to the body tissue being carried out in the phodiesterase. presence of a Sufficient amount of transglutaminase 17. The method of claim 14, wherein crosslinking forms effective to covalently crosslink the agent to the linking a bond between the agent and the linking molecule that is 25 molecule, and hydrolyzable under normal physiological conditions. allowing the crosslinking to occur. 18. The method of claim 1, wherein the agent is a 31. The method of claim 30, wherein the linking molecule nonprotein. is a polymer having multiple units that is a Substrate of 19. The method of claim 18, wherein the agent is not itself transglutaminase. a Substrate for transglutaminase. 32. The method of claim 31, wherein the agent comprises 20. A method for attaching an agent to a body tissue a polymer having multiple units which carry an aliphatic comprising amine that is a Substrate of transglutaminase. first attaching to the body-tissue a carboxamide 33. The method of claim 31, wherein the agent comprises containing linking molecule having at least two con 35 a polymer rich in lysine. tiguous linked glutamines that is covalently bondable 34. The method of claim 30, wherein the agent is selected to the agent in the presence of transglutaminase, then from the group consisting of a visible label, a component of applying to the body tissue having the linking molecule a high affinity noncovalent coupling pair, a receptor or a attached thereto an agent that is a Substrate of trans ligand of a receptor/ligand pair, a pharmaceutical agent, a glutaminase and that is covalently bondable to the 40 cosmetic agent and a SunScreen agent. linking molecule in the presence of transglutaminase, 35. The method of claim 30, wherein the body tissue is Selected from the group consisting of, Skin, hair, nails, applying to the body tissue transglutaminase in an amount wound bed tissue and internal body tissue. effective in crosslinking the agent to the linking 36. The method of claim 30, wherein the agent is a molecule, and 45 nonprotein. allowing Said croSSlinking to occur. 37. The method of claim 30, wherein the agent is an 21. The method of claim 20, wherein the linking molecule enzyme. is a Substrate of transglutaminase and wherein the linking 38. The method of claim 37, wherein the enzyme is molecule is attached to the body tissue by Selected from the group consisting of a cholinesterase and a applying to the body tissue the linking molecule, 50 phosphodiesterase. applying to the body tissue transglutaminase in an amount 39. A method for attaching a nonextracellular matrix effective to crosslinking the linking molecule to the protein agent to a body tissue comprising: body tissue, and applying to the body tissue a conjugate of the agent and allowing Said croSSlinking to occur. a linking molecule having at least three contiguous 22. The method of claim 20, wherein the linking molecule 55 lysines attached directly to one another by peptide is a polymer having multiple units that is a Substrate of bonds, transglutaminase. Said applying to the body tissue being carried out in the 23. The method of claim 22, wherein the agent comprises presence of transglutaminase in an amount effect to a polymer having multiple units which carry an aliphatic covalently crosslink the linking molecule to the body amine that is a Substrate of transglutaminase. 60 tissue, and 24. The method of claim 23, wherein the agent comprises allowing the crosslinking to occur. a polymer rich in lysine. 40. The method of claim 39, wherein the linking molecule 25. The method of claim 20, wherein the agent is selected is Selected from the group consisting of molecules having at from the group consisting of a visible label of a high affinity least 4 and at least 5 contiguous lysines attached directly to noncovalent coupling pair, a pharmaceutical agent, a recep 65 one another by peptide bonds. tor or a ligand of a receptor/ligand pair, a cosmetic, and a 41. The method of claim 39, wherein the linking molecule SunScreen agent. is a polymer Selected from the group consisting of polymers US 6,267,957 B1 29 30 containing at least 20% lysines, at least 30% lysines, and at 45. The method of claim 39, wherein the agent is a least 40% lysines. nonprotein. 42. The method of claim 39, wherein the agent is selected 46. The method of claim 39, wherein the agent is an from the group consisting of a component of a high affinity noncovalent coupling pair, a receptor or a ligand of a enzyme. receptor/ligand pair, a pharmaceutical agent, a cosmetic 47. The method of claim 46, wherein the enzyme is agent and a Sunscreen agent. Selected from the group consisting of a cholinesterase and a 43. The method of claim 39, wherein the body tissue is phosphodiesterase. Selected from the group consisting of skin, hair, nails, wound 48. The method of claim 1 or 39, wherein the trans bed tissue and internal body tissue. 10 glutaminase is exogenously added transglutaminase. 44. The method of claim 39, wherein the agent is not itself a Substrate of transglutaminase. k k k k k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 6,267,957 B1 Page 1 of 1 DATED : July 31, 2001 INVENTOR(S) : Howard Green et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 27 Line 52, delete the first occurrence of “to' and insert therefor -- in --. Line 64, delete the Second occurrence of “of” and insert therefor --, --. Column 28 Line 1, delete “any one of.

Signed and Sealed this Thirteenth Day of August, 2002

Attest.

JAMES E. ROGAN Attesting Officer Director of the United States Patent and Trademark Office UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 6,267,957 B1 Page 1 of 1 DATED : July 31, 2001 INVENTOR(S) : Howard Green et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Title page, Item 76, Inventors, please delete “Dijan' and insert therefor -- Djian --.

Signed and Sealed this Eighth Day of April, 2003

JAMES E ROGAN Director of the United States Patent and Trademark Office