Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1998-12-24
2001-05-15
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S334000, C435S343100, C435S343200, C435S354000
Reexamination Certificate
active
06232088
ABSTRACT:
The present invention relates to a treatments to prevent immune rejection reactions, such as graft vs. host disease (GVHD), with a hydrolase effective to remove cell surface adhesion molecules involved in triggering such immune reactions. One very particular embodiment uses a krill-derived multifunctional enzyme and a family of crustacean and fish derived enzymes having substantial structural or functional similarity to the multifunctional enzyme derived from antarctic krill.
The aquatic or other enzymes that are substantially or functional structurally similar to the krill-derived multifunctional enzyme have the same utility as the krill enzyme. In particular, these enzymes are useful for treating viral infections such as and other disorders, as outlined for example in U.S. patent application Ser. No. 08/600,273, now U.S. Pat. No. 5,958,406, 08/486,820, U.S. patent application Ser. No. 08/338,501 (filed Nov. 22, 1994) and U.S. patent application Ser. No. 08/385,540, now U.S. Pat. No. 5,945,102.
SUMMARY OF THE INVENTION
The invention further provides (a) methods relating to certain conditions using effective amounts of hydrolase, (b) compositions for use in such methods, (c) pharmaceutical compositions containing effective amounts of hydrolase for use in such methods, and (d) uses of the hydrolase composition for manufacturing a medicament for use in such methods. The methods are include:
treating a tissue, body fluid or composition of cells to remove or inactivate a cell adhesion component comprising, wherein the enzyme is administered to the tissue, body fluid or composition of cells, preferably a cell-adhesion component removing or inactivating effective amount or an immune rejection inhibiting amount of the enzyme is administered, wherein preferably the tissue, body fluid or composition of cells is treated extra-corporeally, although they may also be treated in situ in an animal; or
treating or prophylactically preventing HIV infection, preferably administering an HIV infection treating or preventing effective amount of the enzyme
The method comprises administering a composition comprising a hydrolase described above.
The invention further provides (a) methods for treating or prophylactically preventing a cell-cell or cell-virus adhesion syndrome comprising administering an anti-adhesion effective amount of a hydrolase effective to remove or inactivate a cellular or viral acceptor or receptor adhesion component that is involved in the cell-cell or cell-virus adhesion, (b) compositions or substances for use in such methods, (c) pharmaceutical compositions containing effective amounts of enzyme for use in such methods, and (d) uses of the enzyme composition for manufacturing a medicament for use in such methods. Preferably, the syndrome comprises inflammation, shock, tumor metastases, autoimmune disease, transplantation rejection reactions or microbial infections. Preferably, (a) the syndrome is selected from the group consisting of graft versus host disease, organ or tissue transplantation rejection, autoimmune disease and associated conditions, microbial infection, immune disorder, cystic fibrosis, COPD, atherosclerosis, cancer, asthma, septic shock, toxic shock syndrome, conjunctivitis, reperfusion injury and pain, and (b) a cell surface receptor, associated with the cell-cell or cell-virus adhesion syndrome, is removed or inactivated by the administered hydrolase, where the cell surface receptor can be selected from the group consisting of ICAM-1, ICAM-2, VCAM-1, CD3, CD4, CD8, CD11, CD18, CD28, CD29D, CD31, CD44, CD49, CD62L, CD102, GP39, integrins and asialo GM1 ceramide.
The invention further provides a pharmaceutical composition for removing or inactivating a cell-surface adhesion molecule comprising a cell-surface adhesion molecule removing or inactivating effective amount of a hydrolase. Such hydrolases include a number of enzymes such as cod trypsin and other hydrolases, including, as one specific example, proteases with multiple classes of proteolytic activity such as the multifunctional enzyme having: activity comprising at least one of a chymotrypsin, trypsin, collagenase, elastase or exo peptidase activity; a molecular weight between about 20 kd and about 40 kd as determined by SDS PAGE; and substantial homology to the krill-derived multifunctional hydrolase. Such compositions typically include a pharmaceutically acceptable diluent or carrier.
The invention still further provides a pharmaceutical composition for treating or prophylactically preventing a cell-cell or cell-virus adhesion syndrome comprising a cell-cell or cell-virus adhesion syndrome treating or preventing effective amount of a composition comprising a hydrolase. For example, in some embodiments the hydrolase is multifunctional enzyme having: activity comprising at least one of a chymotrypsin, trypsin, collagenase, elastase or exo peptidase activity; a molecular weight between about 20 kd and about 40 kd as determined by SDS PAGE; and substantial homology to the krill-derived multifunctional hydrolase. Such compositions typically include a pharmaceutically acceptable diluent or carrier.
In a preferred embodiment, HIV-infected patients are treated to slow the progression of the associated diseases by the process of (1) isolating T-cells from the patient, (2) treating the T-cells with a hydrolase effective to remove CD4, and (3) injecting the T-cells into the patient.
In one aspect, the method of extra-corporeally treating a tissue, body fluid or composition of cells to remove cell adhesion components reduces the immune rejection of a tissue, body fluid or composition of cells that is transplanted from one individual to another. In another aspect, such treatments remove or inactivate the cell adhesion components found in the treated tissue, body fluid or composition of cells involved in a microbial infection.
In some specific embodiments, the invention relates to a hydrolase having multifunctional activity comprising at least one of a chymotrypsin, trypsin, collagenase, elastase or exo peptidase activity, a molecular weight between about 20 kd and about 40 kd as determined by SDS PAGE, and substantial homology to krill-derived multifunctional hydrolase. Preferably, the enzyme has a molecular weight of from about 26 kd to about 32 kd as determined by SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis (“PAGE”), more preferably about 29 kd. Preferably, the enzyme has an N-terminal sequence comprising: I-V-G-G-X-E/D-B-X-X-X-X-Z/B′-P-Z/H-Q-B-X-B′/Z, wherein X is any amino acid, Z is an aromatic amino acid, B is an amino acid having a C1 to C6 alkyl side chain, and B′ is leucine or isoleucine. More preferably, all amino acids represented by X, Z or B are natural amino acids. Preferably, the enzyme has an N-terminal sequence comprising: I-V-G-G-X-E/D-B wherein X is any amino acid, B is an amino acid having a C1 to C6 alkyl side chain. Preferably, the enzyme is the krill-derived multifunctional hydrolase. Thus, in one embodiment, the N-terminal sequence is I-V-G-G-X-E-V-T-P-H-A-Y-P-W-Q-V-G-L-F-I-D-D-M-Y-F (SEQ ID NO. 20). Preferably, the enzyme has the N-terminal sequence: I-V-G-G-N/M-E-V-T-P-H-A-Y-P-W-Q-V-G-L-F-I-D-D-M-Y-F (SEQ ID NO. 1).
In these specific embodiments, preferably, the multifunctional enzyme of the invention has at least two of the identified proteolytic activities, more preferably at least three, still more preferably at least four. Yet more preferably, the enzyme has all of the identified proteolytic activities. Preferably, the multifunctional enzyme has substantial anti cell-cell and cell-virus adhesion activity. Preferably, the multifunctional enzyme has substantial homology with the krill-derived multifunctional hydrolase.
In another aspect of this specific embodiment, the multifunctional enzyme shall include an amino acid sequence having at least about 70% identity with a “reference sequence” described below, more preferably at least about 80% identity, still more preferably at least about 90% identity, yet still more preferably at least about 95% ide
Franklin Richard L.
St. Pierre Yves
Dechert
Park Hankyel T.
Phairson Medical Inc.
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