Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing – Testing efficacy or toxicity of a compound or composition
Reexamination Certificate
1998-01-21
2001-02-06
Saucier, Sandra E. (Department: 1651)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
Testing efficacy or toxicity of a compound or composition
C424S009100, C514S002600
Reexamination Certificate
active
06183723
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATION
Not applicable.
BACKGROUND
The plasma transport of Cobalamin (Cbl; Vitamin B
12
) to all tissues/cells occurs bound to a plasma transporter, transcobalamin II (TC II), by receptor-mediated endocytosis [1] via transcobalamin II-receptor (TC II-R). Recent studies [2] have shown that TC II-R is expressed as a non-covalent homodimer of molecular mass of 124 kDa in all human [2], rat [3] and rabbit [4] tissue plasma membranes. The plasma membrane expression of TC II-R is important for the tissue/cellular uptake of Cbl since its functional inactivation in vivo by its circulatory antiserum causes intracellular deficiency of Cbl which in turn results in the development of Cbl deficiency of the animal as a whole [4]. Although TC II-R is expressed in the plasma membrane of all cells, its polarity of expression in epithelial cells is not known.
Recent immunoblot studies [3] have revealed that in the rat kidney, TC II-R protein is expressed in both the isolated apical and basolateral membranes with an enrichment in the basolateral membranes by about
8
fold. However, how TC II-Cbl internalized via the TC II-R from either the apical or basolateral surface is processed is not known. Recent TC II-
57
[Co]Cbl uptake studies [4] using filter grown polarized Caco-2 cells have shown that
57
[Co]Cbl taken up from the basolateral side in these cells was utilized as Cbl coenzymes suggesting that these cells derive Cbl essential for their use from the basolateral side.
Despite these studies, the details of intracellular sorting of Cbl and TC II by a polarized epithelial cell are poorly understood. The present studies were undertaken to address the issues related to polarized expression and function of TC II-R in human intestinal derived Caco-2 cells, a well established cell model used extensively to study nutrient transport and general intestinal epithelial cell biology [5, 6].
Needed in the art of cell biology is a method by which cell surface receptors, such as TCII-R, that bind to polypeptide ligands can be used to transport various drugs, including Cbl, across epithelial cell barriers, such as the one that exists in the intestine.
SUMMARY OF THE INVENTION
The present invention is a method of treating a patient with a therapeutic drug, preferably a drug that cannot normally be administered orally because it is inactivated prior to absorption or is not absorbed. In one embodiment a hydrophobic drug, preferably a synthetic organic molecule, is conjugated to a cobalamin molecule. The cobalamin drug conjugate is then bound to transcobalamin II and an effective amount is orally delivered to the patient.
The cobalamin/drug conjugate bound to transcobalamin II will be transcytosed from the intestinal lumen to circulation as an intact complex. The complex present in the circulation will be taken up by all cells and following internalization be targeted to lysosomes, wherein the complex will be degraded and the drug liberated.
In another form of the present invention, physiological amounts of cobalamin are orally delivered to vitamin B
12
-deficient patients. An effective amount of the complex of transcobalamin II-cobalamin will be supplied and orally ingested by patients.
In another embodiment of the present invention, a drug is conjugated directly to a transcobalamin II molecule and an effective amount is orally delivered to the patient.
In another embodiment, the present invention is the transcobalamin II-cobalamin-therapeutic drug complex. In another embodiment, the present invention is the transcobalamin II-therapeutic drug complex. In another embodiment, the present invention is the transcobalamin II-cobalamin complex.
It is a feature of the present invention that one is able to orally deliver therapeutic drugs to the circulation of a patient. This feature is especially important when the drug selected is one that is destroyed by acid or proteases if it is administered orally.
It is another feature of the present invention that one is able to deliver a therapeutic drug across the gastrointestinal tract to the circulation of the patient through the mediation of the transcobalamin II receptor expressed in the gastrointestinal tract.
Other features, advantages and objects of the present invention will become apparent after review of the specification, claims and drawings.
REFERENCES:
Barshop et al., “Transcobalamin II deficiency presenting with methylmalonic aciduria and homocystinuria and abnormal absorption of cabalamin”, American Journal of Medical Genetics, 1990, vol. 35, pp. 222-228.
Pathare et al., “Synthesis of cobalamin-biotin conjugates that vary in the position of cabalamin coupling. Evaluation of cabalamin derivative binding to transcoabalamin II”, Bioconjugate Chem. , 1996, vol. 7, No. 2, pp. 217-232.
Li et al., Isolation and sequence analysis of various forms of human transcobalamin II, Biochimica et Buophysica Act, 1993, vol. 1172, pp. 21-30.
S. Bose, et al., “Membrane Expression and Interactions of Human Transcobalamin II Receptor,”J. Biol. Chem.270 (14):8152-8157, 1995.
S. Bose, et al., “Regulation of Expression of Transcobalamin II Receptor in the Rat,”Biochem. J.310:923-929, 1995.
S. Bose, et al., “Dimerization of Transcobalamin II Receptor,”J. Biol. Chem.271(20):11718-11725, 1996.
S. Bose, et al., “In vitro and in vivo Inactivation of Transcobalamin II Receptor by its Antiserum,”J. Biol. Chem.271(8):4195-4200, 1996.
S. Bose, et al., “Bipolar Functional Expression of Transcobalamin II Receptor in Human Intestinal Epithelial Caco-2 Cells,”J. Biol. Chem.272(77):3538-3543, 1997.
B.A. Cooper and D.S. Rosenblatt, “Inherited Defects of Vitamin B12Metabolism,”Ann. Rev. Nutr.7:291-320, 1987.
N. Dan and D.F. Cutler, “Transcytosis and Processing of Intrinsic Factor-Cobalamin in Caco-2 Cells,”J. Biol. Chem.269(29):18849-18855, 1994.
K.S. Ramanujam, et al., “Expression of Cobalamin Transport Proteins and Cobalamin Transcytosis by Colon Adenocarcinoma Cells,”Am. Physiol. Soc.pp. G416-G422, 1991.
K.S. Ramanujam, et al., “Leupeptin and Ammonium Chloride Inhibit Intrinsic Factor Mediated Transcytosis of [57Co] Cobalamin across Polarized Renal Epithelial Cells,”Biochem. Biophys. Res. Comm.182(2):439-446, 1992.
M. Ramasamy, et al., “Cobalamin Release from Intrinsic Factor and Transfer to Transcobalamin II within the Rat Enterocyte,”Am. Physiol. Soc., pp. G791-G797, 1989.
B. Seetharam, “Gastrointestinal Absorption and Transport of Cobalamin (Vitamin B12),” Physiology of the Gastrointestinal Tract, Third Edition, L.R. Johnson, Raven Press, New York, pp. 1997-2026, 1994.
Bose Santanu
Seetharam Bellur
Afremova Vera
MCW Research Foundation
Quarles & Brady LLP
Saucier Sandra E.
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