Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1997-04-11
1998-09-22
Tsang, Cecilia J.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
530350, 530402, 530410, 514 6, A61K 3514, C07K 100
Patent
active
058115218
DESCRIPTION:
BRIEF SUMMARY
This application claims benefit of USC Provisional Appln. No. 60/000,210 filed Jun. 16, 1995.
FIELD OF THE INVENTION
This invention relates to crosslinking reagents for proteins such as hemoglobin, and more particularly to chemical crosslinking reagents having a plurality of reactive sites, some of which react with globin chains of hemoglobin to effect crosslinking therebetween, and others of which remain available, after such crosslinking, for further reaction. From another aspect, the present invention relates to crosslinked hemoglobin conjugates, in which other groups or molecules, e.g. biomolecules, are chemically bonded to crosslinked hemoglobin.
BACKGROUND OF THE INVENTION AND PRIOR ART
Proposals have been made in the past to take advantage of the bioacceptability of hemoglobin, and its ability to circulate through the body in the bloodstream, as a drug delivery aid. Thus it has been proposed to prepare conjugates of hemoglobin with biologically active materials, and to inject such conjugates into the body, for therapeutic purposes. The biologically active material is assumed to remain biologically active when conjugated to the hemoglobin, or to be released from the hemoglobin in biologically active form after administration to and circulation through the body.
Site-directed cross-linking reagents for the specific modification of human hemoglobin have been developed and reported previously. The cross-linked protein can potentially be used as a red cell substitute and also as a carrier in bioconjugation. Cross-linkers with structurally defined bridging moieties and highly selective reaction sites can produce specifically defined linkages in the protein. This permits altered properties of the modified protein to be clearly related to its structure.
Thus U.S. Pat. No. 5,250,665 Kluger and Wodzinska, issued Oct. 5, 1993, discloses that crosslinking reagents such as trimesoyl tris(methyl phosphate) react with deoxy hemoglobin at the .alpha.-amino group of .beta.-val 1 and the .epsilon.-amino group of lys 82 of both .beta.-subunits, thereby utilizing all of the functional groups of the crosslinking reagent in hemoglobin crosslinking.
While common cross-linking reagents have two reaction sites, we have shown that a reagent with three reaction sites can cross-link a protein with great efficiency and novel utility. Once two sites on the cross-linker have reacted, the third site may react with a group on a protein or remain available for reaction with exogenous reagents.
Thus U.S. Pat. No. 5,399,671 Kluger and Song, issued Mar. 21, 1995, discloses that, in contrast with the teachings of Kluger and Wodzinska above, demonstrates that a reagent with three reaction sites can cross-link a protein with great efficiency and novel utility, but that once two sites on the trifunctional crosslinker have reacted, the third site may react with a group on a protein or remain available for reaction with exogenous reagents. The outcome depends on the functional group of the cross-linker. Reagents such as trimesoyl tris(3,5-dibromosalicylate) (TTDS) reacts only at .beta.-lys 82 of the two .beta. subunits, leaving the third ester group available reaction with added nucleophiles, and thereby providing a basis for bioconjugation and delivery of bioactive materials to the bloodstream of patients.
International Patent Application PCT/US92/09713 Somatogen Inc., published May 13, 1993, describes conjugates of human hemoglobin with various biological molecules, and proposes the use of such conjugates as drug carriers for drug delivery to the human body. The conjugates of this patent utilize cysteine residues, notably genetically engineered cysteine residues, on the globin chains of the hemoglobin, as coupling sites for the biological molecules to be carried, thereby effecting modification of the globin chains of the hemoglobin molecule.
The present invention has, as one objective, the provision of novel crosslinking agents for preparing crosslinked hemoglobin capable of conjugation to other biomolecules, and the provision of pro
REFERENCES:
patent: 5250665 (1993-10-01), Kluger et al.
patent: 5399671 (1995-03-01), Kluger et al.
Kluger et al, J. Am. Chem. Soc., vol. 114, No. 24, pp. 9275-9279, 1992.
Paal et al, J. Am. Chem. Soc., vol. 118, No. 43, pp. 10380-10383, 1996.
Kluger Ronald H.
Paal Krisztina
Mohamed Abdel A.
Tsang Cecilia J.
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