Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-01-23
2002-10-22
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C536S023100
Reexamination Certificate
active
06468757
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods of screening for drug binding to serum proteins using spectrofluorimetry. Kits useful for performing fluorimetric screening of drug binding to serum proteins are also disclosed.
BACKGROUND OF THE INVENTION
Potent, pharmacologically active new drug candidates can be effective in vivo only if they are able to achieve and maintain therapeutic concentrations at the site of action. Pharmaceutical properties such as solubility, partition coefficient, permeability, and protein binding contribute to in vivo disposition and, frequently, these properties are important determinants of clinical outcome. The recent successes of combinatorial chemistry in accelerating drug discovery have also increased the interest in rapid, resource-sparing approaches to determining pharmaceutical properties.
The binding of drugs to serum proteins is particularly important, because it affects both the activity of drugs and their disposition (Huang et al., “Effect of Altered Disopyramide Binding on its Pharmacologic Response in Rabbits,”
Journal of Pharmacology & Experimental Therapeutics
, 223:469-71 (1982); Qin et al., “Decreased Elimination of Drug in the Presence of Alpha-1-acid Glycoprotein is Related to a Reduced Hepatocyte Uptake,”
Journal of Pharmacology & Experimental Therapeutics
, 269:1176-81 (1994)). According to the “free drug” hypothesis, only unbound drug exerts pharmacological activity (Recant et al., “Thyroid Function in Nephrosis,”
Journal of Clinical Investigation
, 31:789 (1952)) and disposition is often altered by drug binding (Shand et al., “Perfusion-Limited of Plasma Drug Binding on Hepatic Drug Extraction,”
Life Sciences
, 19:125-30 (1976); Jansen, “Influence of Plasma Protein Binding Kinetics on Hepatic Clearance Assessed from a “Tube” Model and a “Well-stirred” Model,”
Journal of Pharmacokinetics & Biopharmaceutics
, 9:15-26 (1981)). Consequently, it is important to know the affinity of a drug for serum proteins.
A variety of techniques have been proposed for protein binding measurements including dialysis, ultrafiltration (Huang, “Errors in Estimating the Unbound Fraction of Drugs Due to the Volume Shift in Equilibrium Dialysis,”
Journal of Pharmaceutical Sciences
, 72:1368-9 (1983)), circular dichroism (Ascoli et al., “Stereospecific and Competitive Binding of Drugs to Human Serum Albumin: A Difference Circular Dichroism Approach,”
Journal of Pharmaceutical Sciences
, 84:737-41 (1995)), and extrinsic fluorescence (Sudlow et al., “Spectroscopic Techniques in the Study of Protein Binding: The Use of 1-Anilino-8-Naphthalenesulphonate as a Fluorescent Probe for the Study of the Binding of lophenoxic and lopanoic Acids to Human Serum Albumin,”
Molecular Pharmacology
, 9:649-57 (1973); Sudlow et al., “The Characterization of Two Specific Drug Binding Sites on Human Serum Albumin,”
Molecular Pharmacology,
11
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(1975); Epps et al., “A General, Wide-range Spectrofluorometric Method for Measuring the Site-Specific Affinities of Drugs Toward Human Serum Albumin,”
Analytical Biochemistry
, 227:342-50 (1995); Suarez Varela et al., “Spectrofluorimetric Study of the Binding of 1-Anilnonaphthalene-8-Sulfonate to Bovine Serum Albumin,”
Journal of Pharmaceutical Sciences
, 81:843-4 (1992)). Despite the fact that the displacement of extrinsic fluorophores such as warfarin and dansylglycine has been proposed as the basis for a rapid protein binding assay (Epps et al., “A General, Wide-range Spectrofluorometric Method for Measuring the Site-Specific Affinities of Drugs Toward Human Serum Albumin,”
Analytical Biochemistry
, 227:342-50 (1995)) and the fact that such assays are drug nonspecific and rapid, they are indirect because they utilize the interaction between two drugs to produce an extrinsic signal.
The present invention is directed to overcoming these deficiencies in the art.
SUMMARY OF THE INVENTION
The present invention relates to a method of screening for drug binding to serum proteins. This method includes preparing at least two solutions, each of the at least two solutions containing a concentration of a serum protein characterized by broad specificity in binding to xenobiotics and a concentration of a candidate drug, wherein the concentration of the candidate drug is different for each of the at least two solutions and, optionally, one of the at least two solutions is a control solution characterized by a candidate drug concentration of zero; exposing each of the at least two solutions to a light source; measuring fluorescent emission by the serum protein or a serum protein-candidate drug complex for each of the at least two solutions upon the exposing; and determining whether a change in fluorescence emission is measured for an increased concentration of the candidate drug, wherein the change in fluorescence emission indicates binding of the candidate drug to the serum protein.
The present invention also relates to a method of screening for drug binding to serum proteins, where a dissociation constant (K
d
) for the candidate drug and the serum protein can be calculated based on the measured fluorescence emissions. This method includes preparing at least two solutions, each of the at least two solutions containing a concentration of a serum protein characterized by broad specificity in binding to xenobiotics and a concentration of a candidate drug, wherein the concentration of the candidate drug is different for each of the at least two solutions and, optionally, one of the at least two solutions is a control solution characterized by a candidate drug concentration of zero; exposing each of the at least two solutions to a light source; measuring fluorescent emission by the serum protein or a serum protein-candidate drug complex for each of the at least two solutions upon the exposing; and calculating a dissociation constant (K
d
) for the candidate drug and the serum protein based on the measured fluorescence emissions.
Another aspect of the present invention relates to a kit useful for performing a fluorimetric screening of drug binding to serum proteins. The kit includes a plurality of detection cells compatible for use with a fluorimetric device, one or more solutions each having a predetermined concentration of a serum protein characterized by broad specificity in binding to xenobiotics, and instructions for combining a volume of the one or more solutions with a quantity of a drug in the detection cells, exposing the detection cells to the fluorimetric device, and analyzing fluorimetric emission data.
The present invention uses spectrofluorimetry, a technique that has been widely used to study biomolecular interactions and which has many advantages over other techniques such as dialysis and ultrafiltration. The advantages arise primarily because fluorescence data are obtained without separating the bound and unbound species, which reduces the time required for the experiment and eliminates the need for a size-selective membrane. The dialysis and ultrafiltration methods require analysis of free and total drug concentration which can be resource and time consuming. Additionally, these methods cannot be used with drugs that bind extensively to the membrane (MacKichan, “Influence of Protein Binding and Use of Unbound (Free) Drug Concentrations” in
Applied Pharmacokinetics: Principles of Therapeutic Drug Monitoring
, pp 5.1-5.48, Evans et al. (eds.), Applied Therapeutics, Vancouver, Wash., (1992), which is hereby incorporated by reference in its entirety); this is often a serious problem with highly hydrophobic drugs. Although the displacement of extrinsic fluorophores such as warfarin and dansylglycine has been proposed as the basis for a rapid protein binding assay (Epps et al., “A General, Wide-range Spectrofluorometric Method for Measuring the Site-Specific Affinities of Drugs Toward Human Serum Albumin,”
Analytical Biochemistry
, 227:342-50 (1995), which is hereby incorporated by reference in its entirety), intrinsic fluorescence offers advantages over extrinsic fluorescence and has the potenti
Morris Marilyn E.
Ramanathan Murali
Nixon & Peabody LLP
The Research Foundation of State University of New York
Wang Andrew
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