Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1998-11-24
2001-04-10
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S021000, C435S015000, C435S024000, C435S023000, C435S004000, C435S069200, C435S068100
Reexamination Certificate
active
06214571
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to pharmaceutical agents and methods for use in chemotherapeutic treatment of cancer. More specifically, the invention relates to the identification of cancer cells which cannot metabolize methylthioadenosine phosphorylase to adenine for the salvage synthesis of adenine nucleotides, and the use of L-alanosine to inhibit de novo adenosine 5′-monophosphate (AMP) synthesis in such cancer cells.
HISTORY OF THE INVENTION
Methylthioadenosine (MTA) is cleaved in healthy mammalian cells by methylthioadenosine phophorylase (MTAse) into adenine and methylthioribose-1-P, the latter of which is a substrate for metabolic synthesis of methionine. Adenine is salvaged into a cellular pool of adenosine 5′-monophosphate (AMP), from which cells derive adenosine 5′-triphosphate (ATP) for metabolic energy and 2′-deoxyadenosine-5′-triphosphate (DATP) for DNA synthesis.
Based on early in vitro studies, the L isomer of a bacterial antibiotic alanosine (obtained from
Streptomyces alanosinicus;
hereafter, “L-alanosine”) appeared to have promise for use as an anti-viral and anti-tumor agent. In particular, it is believed that L-alanosine inhibits adenylosuccinate synthetase (ASS) conversion of inosine 5′-monophosphate (IMP) to AMP, thus depleting target cells of AMP and ATP (in the absence of adenine). However, clinical studies of the therapeutic efficacy of L-alanosine in human cancer patients have been disappointing (see, e.g., data collected in Tyagi and Cooney,
Adv. Pharmacol. Chemotherapy,
20:69-120, 1984 [results then to date offered “little encouragement” regarding L-alanosine's efficacy for treatment of human cancers]; Creagan, et al.,
Cancer,
52:615-618, 1993 [Phase II studies had overall response rate of only 4%]; Creagan, et al.,
Am.J.Clin.Oncol.,
7:543-544, 1984 [Phase II study in melanoma patients; little therapeutic response observed]; VonHoff, et al.,
Invest. New Drugs,
9:87-88, 1991 [no objective responses observed in breast cancer patients]). Eventually, all clinical trials of L-alanosine for use in treatment of cancer were abandoned.
Another known inhibitor of ASS activity is hadacidin. However, hadacidin is believed to be more toxic than L-alanosine in humans. Further, the activity of other inhibitors of de novo purine synthesis (such as methotrexate, 6-mercaptopurine, 6-thioguanine and dideazatetrahydrofolate) which block IMP synthesis (and therefore theoretically eliminate IMP as a source for AMP production) has been circumvented in vivo by salvage of hypoxanthine, which is abundant in plasma, for use as a substrate for IMP production. Hence, to date the in vivo performance of agents which block the adenine metabolic pathway for intracellular AMP production has been frustratingly poor.
However, with the development of an assay of sufficient sensitivity to identify homozygous deletions of the gene encoding MTAse in certain human cancer cells (see, commonly assigned parent U.S. patent application Ser. No. 08/176,855), it now appears that the tumors treated in the clinical trials of L-alanosine produced MTAse and were therefore able to provide sufficient adenine to maintain a pool of AMP despite inhibition of AMP production from IMP. The present invention therefore provides a method for identifying cells which lack MTAse and for treating such cells by depleting them of AMP.
SUMMARY OF THE INVENTION
It has been discovered that cells from which the gene which encodes MTAse protein has been deleted and are therefore not able to metabolize MTA to adenine (“MTAse deficient cells”) are selectively killed in vivo on contact with therapeutically effective dosages of a de novo AMP synthesis inhibitor such as L-alanosine. Thus, while L-alanosine is not therapeutically effective against all cancer cells, it is therapeutically effective against MTAse deficient cells.
In one aspect, the invention provides a method for determining whether particular cancer cells are MTAse deficient 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.
In another aspect, the invention provides a method for treating MTAse deficient cancers by contacting MTAse deficient cells with a therapeutically effective amount of a de novo purine synthesis inhibitor which inhibits the activity of ASS, preferably L-alanosine. The ASS inhibitory agents of the invention may be administered by any clinically acceptable means, but are preferably administered by continuous infusion at concentrations below the maximally tolerated dose to prolong the desired inhibitory activity and minimize toxicity to host tissues.
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 an ASS inhibitor, preferably L-alanosine and/or its active metabolite, L-alanosinyl AICOR.
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Carrera Carlos J.
Carson Dennis A.
Cottam Howard B.
Nobori Tsutomu
Leary Louise N.
The Regents of the University of California
Townsend & Townsend & Crew LLP
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