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
1999-11-04
2002-12-31
Jones, Dameron L. (Department: 1619)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
Testing efficacy or toxicity of a compound or composition
C435S007100, C562S400000
Reexamination Certificate
active
06500405
ABSTRACT:
BACKGROUND OF THE INVENTION
Microtubules are intracellular filamentous structures present in all eukaryotic cells. As components of different organelles such as mitotic spindles, centrioles, basal bodies, cilia, flagella, axopodia and the cytoskeleton, microtubules are involved in many cellular functions including chromosome movement during mitosis, cell motility, organelle transport, cytokinesis, cell plate formation, maintenance of cell shape and orientation of cell microfibril deposition in developing plant cell walls. The major component of microtubules is tubulin, a protein composed of two subunits called alpha and beta. An important property of tubulin in cells is the ability to undergo polymerization to form microtubules or to depolymerize under appropriate conditions. This process can also occur in vitro using isolated tubulin.
Microtubules play a critical role in cell division as components of the mitotic spindle, an organelle which is involved in distributing chromosomes within the dividing cell precisely between the two daughter nuclei. Various drugs and pesticides prevent cell division by binding to tubulin or to microtubules. Anticancer drugs acting by this mechanism include the alkaloids vincristine and vinblastine, and the taxane-based compounds paclitaxel and docetaxel {see, for example, E. K. Rowinsky and R. C. Donehower,
Pharmacology and Therapeutics,
52, 35-84 (1991)}. Other antitubulin compounds active against mammalian cells include benzimidazoles such as nocodazole and natural products such as colchicine, podophyllotoxin and the combretastatins. Benzimidazole compounds which bind to tubulin are also widely used anthelmintics {McKellar, Q. A. and Scott, E. W.,
J. Vet. Pharmacol. Ther.,
13, 223-247 (1990)}. Anti-tubulin herbicides are described in “The Biochemical Mode of Action of Pesticides”, by J. R. Corbett, K. Wright and A. C. Baillie, pp. 202-223, and include dinitroanilines such as trifluralin, N-phenylcarbamates such as chlorpropham, amiprophos-methyl, and pronamide. Fungicides believed to act by binding to tubulin include zarilamide {Young, D. H. and Reitz, E. M.,
Proceedings of the
10
th International Symposium on Systemic Fungicides and Antifungal Compounds,
Reinhardsbrunn, ed by H. Lyr and C. Polter, 381-385, (1993)}, the benzimidazoles benomyl and carbendazim, and the N-phenylcarbamate diethofencarb {Davidse, L. C and Ishi, H. in “Modern Selective Fungicides”, ed. by H. Lyr, 305-322 (1995)}.
Due to the success of tubulin as a biochemical target for drugs and pesticides, there is considerable interest in discovering new compounds which bind to tubulin. Various cell-free methods are available for detecting such compounds. A common method involves measuring the ability of test compounds to inhibit the polymerization of isolated tubulin into microtubules in vitro {see for example, E. Hamel,
Medicinal Research Reviews,
16, 207-231 (1996)}. In a second method, interactions of test compounds with isolated tubulin can be detected in binding assays by measuring the ability of the test compound to influence binding of a second tubulin-binding ligand, used as a probe. (The term “test compound” means a compound which one wishes to evaluate, i.e. to test, for its ability to affect tubulin). Typically, the probe is radiolabeled to enable binding to be measured. A test compound which binds to tubulin may influence binding of the probe by binding to the same site on the tubulin protein as the probe, and thus reduce the amount of probe which binds. Alternatively, binding may be influenced by means of an “allosteric” interaction in which the test compound binds to a different site from that of the probe and induces a conformational change in the tubulin protein which affects the binding site of the probe. Such an allosteric interaction may either increase or decrease binding of the probe. A third approach involves measuring the effect of test compounds on tubulin-associated guanosine triphosphatase activity {Duanmu, C., Shahrik, L. K., Ho, H. H. and Hamel, E.,
Cancer Research,
49, 1344-1348 (1989)}.
To screen large numbers of compounds by any of these methods is feasible at present only using tubulin from mammalian brain tissue, since it has not been possible to isolate sufficiently large amounts of purified tubulin from other sources. This limits the usefulness of these methods since many anti-tubulin compounds show great specificity with respect to their effects on microtubules from different sources. For example, the herbicides oryzalin and amiprophosmethyl inhibit the polymerization of plant tubulin but not brain tubulin, whereas colchicine is more than 100-fold more effective as an inhibitor of brain tubulin polymerization than of plant tubulin polymerization {Morejohn, L. C. and Fosket, D. E., ‘Tubulin from Plants, Fungi, and Protists’, in “Cell and Molecular Biology of the Cytoskeleton”, ed. by J. W. Shay, 257-329 (1986)}.
The present invention relates to the use of certain amide derivatives, known to inhibit the growth of eukaryotic cells, including fungal and plant cells {see, for example, U.S. Pat. Nos. 3,661,991, 4,863,940 and 5,254,584}. Said amides have now been found useful as probes in binding assays to screen compounds for antitubulin activity, a use which U.S. Pat. Nos. 3,661,991, 4,863,940 and 5,254,584 neither disclose nor suggest. While radiolabeled probes such as colchicine {see for example, M. H. Zweig and C. F. Chignell,
Biochemical Pharmacology,
22, 2141-2150 (1973)} and vinblastine (see for example, R. Bai et al.,
Journal of Biological Chemistry,
265, 17141 (1990)} have been used extensively in binding assays using isolated tubulin, these compounds bind noncovalently to tubulin.
One advantage of the amide derivatives of this invention over existing antitubulin compounds in competitive binding assays results from their unique ability to bind covalently in a highly specific manner to tubulin, specifically to the beta-subunit of tubulin. (A covalent bond is a nonionic chemical bond characterized by the sharing of electrons by two atoms). In binding assays it is necessary to measure the amount of the probe which is bound to tubulin, and this generally involves separating the tubulin-bound probe from unbound probe. In the case of the amides, since binding is covalent, the tubulin-bound probe is chemically stable allowing easy separation from the unbound probe by methods such as filtration or centrifugation. This enables their use not only in assays using isolated tubulin but also in assays using whole cells, crude cell extracts, and partially purified tubulin preparations, thus obviating the need for isolated tubulin and enabling tubulin-binding assays to be carried out in many different types of cell or cell extract.
One aspect of the present invention involves use of amide probes in binding assays to screen large numbers of compounds in order to identify those compounds with antitubulin activity using whole cells, cell extracts or isolated tubulin. For example, test compounds which bind to plant or fungal tubulin may be detected in assays using plant or fungal cells, thus providing a means of detecting antitubulin compounds with herbicidal or fungicidal activity. Similarly, amide probes may be used to detect compounds which bind to tubulin in mammalian cells or cell extracts, thus providing a means of detecting antitubulin compounds with anticancer activity.
A second aspect of the current invention involves use of amide probes in binding assays to evaluate the sensitivity of a cell population to an antitubulin compound. For example, the current invention can be used to evaluate the sensitivity of a tumor cell population to an antitubulin drug such as paclitaxel, vincristine or vinblastine, thus providing a means of predicting drug sensitivity of a patient's tumor at the time of diagnosis or relapse using cells isolated by biopsy, and consequently guiding selection of the optimal chemotherapy regimen. Frequently, treatment of n
Carpenter Clark B.
Dow AgroSciences LLC
Jones Dameron L.
Ludwig Kenneth B.
LandOfFree
Use of certain amides as probes for detection of antitubulin... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Use of certain amides as probes for detection of antitubulin..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Use of certain amides as probes for detection of antitubulin... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2974178