Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-02-02
2001-12-25
Kaufman, Claire M. (Department: 1646)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600
Reexamination Certificate
active
06333311
ABSTRACT:
BACKGROUND OF THE INVENTION
Lactoferrin (LF) is a metal binding glycoprotein of M
r
77,000 found in milk, tears, saliva, bronchial, intestinal, vaginal and other secretions. LF is also present in the secondary granules of neutrophils. Lactoferrin plays an important role in numerous inflammatory and immune response functions such as regulation of monocyte colony stimulating factor synthesis, regulation of interleukin synthesis, activation of natural killer cell activity, inhibition of metastasis, and maturation of T-cells.
The amino acid sequence of LF has been determined by protein-sequencing and sequencing of cDNA clones. Human LF (hLF) consist of a polypeptide chain of 692 amino acids. The amino terminal region of hLF contains two clusters of basic residues, RRRR (SEQ ID NO:1) (residues 2-5) and RNMRKVR (SEQ ID NO:2) (residues 25-31). The LF polypeptide is folded into two globular lobes, each of which contains an iron-binding cleft. The high affinity of LF for iron confers to the protein certain antibacterial properties and, in addition, may play a role in the absorption of dietary iron by the small intestine.
Some of the biological activities of LF do not arise from the binding of iron but from its capacity to bind to other molecules. Direct intermolecular interactions between hLF and human lysozyme (hLZ) may explain the synergy between the antibacterial action of these two proteins. Interaction of hLF with bacterial outer membrane components such as lipopolysaccharide (LPS) and porins presumably plays an important role in the antimicrobial activity of hLF. Binding of hLF to the lipid A portion of LPS inhibits the LPS priming of neutrophils for enhanced fMLP-triggered superoxide release. Interaction of LF with heparin may account for the neutralization of the anticoagulant activity of heparin.
Some biological activities of LF arise from interactions between LF and cells via membrane bound receptors. For example, LF binding to specific receptors on monocytes, macrophages and activated lymphocytes results in inhibition of cytokine production. Cells that exhibit specific binding to hLF include liver cells, intestinal cells, mammary gland epithelial cells, monocytic cell lines, activated lymphocytes, and platelets.
BRIEF SUMMARY OF THE INVENTION
The invention provides compositions containing human lactoferrin, or lactoferrin variants deleted for one or more arginine residues in the amino-terminal region of the protein (i.e., in the first basic cluster), and uses of the compositions. In one aspect the invention is a composition containing a human lactoferrin variant deleted for one or more arginine residues in first basic cluster. The invention is particularly directed to the human lactoferrin variants hLF
−2N
, hLF
−3N
, hLF
−4N
, and hLF
−5N
. These binding properties of these variants differ in advantageous ways from those of natural lactoferrin. In one aspect, the composition is a pharmaceutical composition, optionally comprising bovine milk. In some embodiments, the human lactoferrin or lactoferrin variant is saturated with iron, typically at least 95% saturated.
The invention also relates to the uses of human lactoferrin and lactoferrin arginine-deletion variants. In one aspect, the invention provides methods for activating a lactoferrin receptor, for example the 105 kd lactoferrin receptor, by administering hLF or an hLF variant.
In another aspect, the invention provides a method for reducing or inhibiting release of a cytokine, such as IL-1, IL-2 or TNF&agr;, from lactoferrin-receptor bearing cells in a patient, by administering lactoferrin or a lactoferrin variant.
In other aspects, the invention provides methods in which human lactoferrin or a lactoferrin variant is administered to a patient to inhibit myelopoieses, for treatment of a chronic inflammatory bowel disease, or to reduce TNF&agr;-mediated neutrophil degranulation in a patient.
In another aspect, the invention provides a method for delivering iron to a lactoferrin-receptor-bearing cell in a patient by administering to the patient a composition of human lactoferrin or a lactoferrin variant which is at least about 95% saturated with iron. Administration of these compounds are beneficial, for example, in treatment of anemia or iron storage diseases.
In another aspect, the invention provides methods in which human lactoferrin or a lactoferrin variant is administered to a patient to reduce inflammation. Administration of hLF and hLF variants is useful for reducing reperfusion injury in a patient after myocardial infarction.
In other aspects, the invention provides methods in which human lactoferrin or a lactoferrin variant is administered to a patient to inhibit growth of a solid tumor in a patient and for stimulating natural killer (NK) cells in a patient.
The invention also provides methods for inhibiting entry into a cell of viruses, for example, CMV, HIV or HSV1 viruses, for which viral entry is mediated by an interaction between the virus and a cell surface proteoglycan.
In a related aspect, the invention is a composition containing human lactoferrin in which the first basic cluster of the lactoferrin is neutralized, for example, by the binding of an anti-lactoferrin monoclonal antibody or heparin, such that the lactoferrin binds a Jurkat cell 105 kD lactoferrin receptor with higher affinity than does natural lactoferrin.
In another aspect, the invention provides pharmaceutical compositions comprising a lactoferrin variant containing the first basic cluster, but not containing the second basic cluster.
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Elass-Rochard, E. et al., “Lactoferrin-Lipopolysaccharide interaction: involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding toEscherichia coli055B5 lipopolysaccharide”Biochem. J. 312:839-845 (1995).
Hutchens, T.W. et al., “Structurally intact (78-kDa) forms of maternal lactoferrin purified from urine of preterm infants fed human milk: Identification of a trypsin-like proteolytic cleavage event in vivo that does not result in fragment dissociation”Proc. Natl. Acad. Sci. USA 88:2994-2998 (Apr. 1991).
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van Berkel, P.H.C. et al., “Glycosylated and unglycosylated human lactoferrins both bind iron and show identical affinities toward human lysozyme and bacterial lipopolysaccharide, but differ in their susceptibilities towards tryptic proteolysis”Biochem. J. 312:107-114 (1995).
Wu, H.-f. et al., “Characterization of the Glycosaminoglycan-Binding Region of Lactoferrin”Arch. Biochem. Biophys. 317(1):85-92 (Feb. 20, 1995).
Ziere, G.J. et al., “Removal of 14 N-terminal Amino Acids of Lactoferrin Enhances its Affinity for Parenchymal Liver Cells and Potentiates the Inhibition of &bgr;-Very Low Density Lipoprotein Binding”J. Biol. Chem. 268(36):27069-27075 (Dec. 25, 1993).
Legrand, et al., “The N-Terminal Arg2, Arg3and Arg4of Human Lactoferrin Interact with Sulphated Molecules But Not With the Receptor Present on Jurkat Human Lymphoblastic T-Cells”,Biochem J.,327(Pt. 3):841-846 (1997).
van Berkel et al., “N-Terminal Stretch Arg2, Arg3, Arg4and Arg5of Human Lactoferrin is Essential for Binding to Heparin, Bacterial Lipopolysaccharide, Human Lysozyme and DNA”,Biochem. J.,328(Pt. 1):145
Nuijens Jan
van Berkel Patrick
Kaufman Claire M.
Pharming
Townsend and Townsend / and Crew LLP
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