Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1997-10-23
2001-04-03
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S004000, C435S021000, C435S029000, C435S015000, C435S024000, C435S023000, C435S069200, C435S068100, C435S173300, C536S001110, C536S024320, C536S024310
Reexamination Certificate
active
06210917
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to pharmaceutical agents and methods for use in the treatment of cancer. More specifically, the invention relates to treatment of cancer cells with L-alanosine to inhibit de novo adenosine 5′-triphosphate (ATP) synthesis in such cancer cells. In particular, the invention deprives cancer cells that have developed P-glycoprotein-mediated multiple drug resistance of the energy required to maintain such drug resistance.
HISTORY OF THE INVENTION
One of the greatest limitations on the efficacy of cancer chemotherapy is the tendency of cancer cells to develop broad-spectrum resistance to a host of anti-cancer and cytotoxic drugs. Such multiple drug resistance (MDR) is believed to occur to varying degrees in most cancers, either from the onset of the cancer or on recurrence following chemotherapy.
MDR is believed to be mediated by the activity of a cell surface phospho-glycoprotein, P-glycoprotein. Increased expression of the gene which encodes P-glycoprotein (mdr) is found in many malignant cells and may be upregulated by the onset of a malignancy and/or cellular contact with chemotherapeutic agetns. Once active, P-glycoprotein is believed to function as a “hydrophobic vacuum cleaner” which expels hydrophobic drugs from targeted cells. Such drugs include a host of anti-cancer drugs and cytotoxic agents, such as the Vinca alkaloids (e.g., vinblastine), the anthracyclines (e.g., doxorubicin), the epipodophyllotoxins (e.g., etoposide), the taxanes (e.g., taxol), antibiotics (e.g., actinomycin D), antimicrotubule drugs (e.g., colchicine), protein synthesis inhibitors (e.g., puromycin), toxic peptides (e.g., valinomycin), topoisomerase inhibitors (e.g., topotecan), DNA intercalators (e.g., ethidium bromide) and anti-mitotics.
MDR has been the subject of intense research for many years. Efforts to counter MDR have primarily involved the use of hydrophobic competitors for P-glycoprotein binding, such as calcium channel blockers, cephalosporins, steriods, immunosuppressants, antihypertensives, anti-arrhythmics, lipophilic cations, detergents and antidepressants. To varying degrees, most of these competitors eventually fail to sufficiently overcome MDR for reasons including their interference with chemotherapeutic drug uptake and unexpected toxicities.
The precise mechanism of MDR action for P-glycoprotein is not yet certain. However, it is clear that P-glycoprotein requires greatly increased stores of ATP (as compared to normal cells) to function. Thus, as mdr expression and P-glycoprotein activity increases in growing and/or metastasizing cancer cells, so too does synthesis and metabolic turnover of ATP by those cells. In order for cells to produce ATP, they must maintain a source of adenine, the metabolic substrate for synthesis of ATP. Failing an adequate natural source of adenine, cells can resort to an alternative pathway in which adenine is salvaged from metabolism of methylthioadenosine (MTA). Thus, de novo and salvage adenine metabolism is significantly increased in cells with active P-glycoprotein.
SUMMARY OF THE INVENTION
It has been discovered that MDR can be prevented or overcome in certain cancer cells by application of an inhibitor of de novo adenine synthesis to such cells. Cancer cells which are sensitive to the treatment of the invention are those which cannot salvage adenine by metabolism of MTA. In particular, cells from which the gene which encodes MTAse protein has been deleted are not able to metabolize MTA to adenine (“MTAse deficient cells”). In the presence of therapeutically effective dosages of a de novo AMP synthesis inhibitor, such cells become starved of adenine and cannot produce sufficient levels of ATP to maintain P-glycoprotein activity.
The invention provides a method for treating and preventing MDR in MTAse deficient cancers by contacting MTAse deficient cells with a therapeutically effective amount of a de novo purine synthesis inhibitor which inhibits de novo adenine synthesis. In one aspect of the inventive method, the purine synthesis inhibitor is L-alanosine.
A method is also provided for treating and limiting MDR in MTAse competent cancer cells. According to this aspect of the invention, MTAse competent cancers are treated with a combination of chemotherapeutic agents and purine synthesis inhibitors, thus limiting the acquisition and maintainance of MDR in such cells.
In another aspect, the invention provides a method for determining whether particular cancer cells are MTAse deficient (and therefore susceptible to treatment to prevent MDR) by providing assays to determine whether the cells lack MTAse protein. The preferred assay for use in this regard is one for detection of homozygous deletions from cells of the gene which encodes MTAse protein.
Also provided are kits for use in the methods of the invention which include reagents for use in performing the MTAse deficiency assay of the invention as well as pharmaceutical compositions of a purine synthesis inhibitor. In one aspect, the inhibitors provided are L-alanosine and/or its active metabolite, L-alanosinyl AICOR.
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Guicherit et
Carrera Carlos J.
Carson Dennis A.
Leary Louise N.
The Regents of the University of California
Townsend and Townsend and Crew
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