Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1998-10-15
2001-03-13
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S025000, C435S184000
Reexamination Certificate
active
06200771
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to a novel cGMP-specific phosphodiesterase that provides a method for identifying compounds potentially useful for the treatment and prevention of pre-cancerous and cancerous lesions in mammals.
Cancer and precancer research is replete with publications that describe various biochemical molecules that are over-expressed in neoplastic tissue, leading one research group after another to research whether specfic over-expressed molecules are responsible for the disease, and whether, if the over-expression were inhibited, neoplasia could be alleviated. For example, in familial adenomatous polyposis (“FAP”), Waddell in 1983 (Waddell, W. R. et al., “Sulindac for Polyposis of the Colon,”
Journal of Surgical Oncology,
24:83-87, 1983) hypothesized that since prostaglandins were over-expressed in such polyps, non-steroidal anti-inflammatory drugs (“NSAIDs”) should alleviate the condition because NSAIDs inhibited prostaglandin synthetase (PGE
2
) activity. Thus, he administered the nonsteroidal anti-inflammatory drug (“NSAID”) sulindac (an inhibitor of PGE
2
) to several FAP patients. Waddel discovered that polyps regressed and did not recur upon such therapy. PGE
2
inhibition results from the inhibition of cyclooxygenase (COX) caused by NSAIDs. The success by Waddell and the PGE
2
/COX relationship seemingly confirmed the role of two other biochemical targets—PGE
2
and COX—in carcinogenesis, and the subsequent literature reinforced these views.
Sulindac and other NSAIDs when chronically administered, aggravate the digestive tract where PGE
2
plays a protective role. In addition, they exhibit side effects involving the kidney and interference with normal blood clotting. Thus for neoplasia patients, such drugs are not a practical chronic treatment for FAP. These side effects also prohibit NSAIDs' use for any other neoplasia indication requiring long-term drug administration.
Recent discoveries have lead scientists away from the COX/PEG
2
targets, since those targets do not appear to be the primary (or perhaps even secondary targets) to treat neoplasia patients successfully. Pamukcu et al., in U.S. Pat. No. 5,401,774, disclosed that sulfonyl compounds, that have been reported to be practically devoid of PGE2 and COX inhibition (and therefore not NSAIDs or anti-inflammatory compounds) unexpectedly inhibited the growth of a variety of neoplastic cells, including colon polyp cells. These sulfonyl derivatives have proven effective in rat models of colon carcinogenesis, and one variant (now referred to as exisulind) has proven effective in human clinical trials with FAP patients.
Thus like so many other proteins over-expressed in neoplasias, PGE
2
/COX over-expression is not a cause of neoplasia, rather a consequence. But the discoveries by Pamucku et al., however, have raised the question about how their compounds act —what do such compounds do to neoplastic cells?
Piazza, et al. (in U.S. Pat. No. 5,858,694) discovered that compounds (such as the sulfonyl compounds above) inhibited cyclic GMP, phosphodiesterase, namely, PDE5 and that other such compounds could be screened using that enzyme, which could lead to the discovery of still other pharmaceutical compositions that are anti-neoplastic, and that can be practically devoid of COX or PGE2 inhibition. In addition, anti-neoplastic PDE5-inhibiting compounds can induce apoptosis (a form of programmed cell death or suicide) in neoplastic cells, but not in normal cells. Thus, the conventional wisdom that chemotherapeutics cannot be effective without also killing normal cells is being reversed by such discoveries.
Further, new research presented below has shown that not all compounds exhibiting PDE5 inhibitions induce apoptosis in neoplastic cells. For example, the well-known PDE5 inhibitors, zaprinast and sildenafil, do not induce apoptosis, or even inhibit cell growth in neoplastic cells in our hands, as explained below. However, because pro-apoptotic PDE5 inhibitors induced apoptosis selectively (i.e., in neoplastic but not in normal cells), and many did so without substantial COX inhibition, the usefulness of PDE5 as a screening tool for desirable anti-neoplastic compounds is unquestioned.
However, an even more accurate and selective screening tool than PDE5 to find anti-neoplastic, pro-apoptotic but safe compounds is desirable.
SUMMARY OF THE INVENTION
In the course of researching why some PDE5 inhibitors induced apoptosis while others did not, we uncovered a form of cyclic GMP-specific phosphodiesterase, not previously described. This new phosphodiesterase activity was previously uncharacterized, possibly because it is expressed only in neoplastic tissue, or perhaps because it is a mutation of a known/characterized PDE. This new PDE is useful in screening pharmaceutical compounds for desirable anti-neoplastic properties.
In its broadest aspects, this new PDE is characterized by having:
(a) cGMP specificity over cAMP
(b) positive cooperative kinetic behavior in the presence of cGMP substrate;
(c) submicromolar affinity for cGMP; and
(d) insensitivity to incubation with purified cGMP-dependent protein kinase
Other characteristics of this novel PDE include: it has a reduced sensitivity to inhibition by zaprinast and E4021, it can be separated from classical PDE5 activity by anion-exchange chromatography, it is not activated by calcium/calmodulin, and it is insensitive to rolipram, vinpocetine and indolodan.
This invention relates to a novel in vitro method for screening test compounds for their ability to treat and prevent neoplasia, especially pre-cancerous lesions, safely. In particular, the present invention provides a method for identifying test compounds that can be used to treat and prevent neoplasia, including precancerous lesions. The compounds so identified can have minimal side effects attributable to COX inhibition and other non-specific interactions. The compounds of interest can be tested by exposing the novel PDE described above to the compounds, and if a compound inhibits this novel PDE, the compound is then further evaluated for its anti-neoplastic properties.
One aspect of this invention, therefore, involves a screening method to identify a compound effective for treating neoplasia that includes ascertaining the compound's inhibition of this novel PDE and its inhibition of COX. Preferably, the screening method of this invention further includes determining whether the compound inhibits the growth of tumor cells in a cell culture.
In another aspect, the screening method of this invention involves determining the COX inhibition activity of the compound, determining the inhibition activity of the compound against this novel PDE, and determining whether the compound induces apoptosis in tumor cells.
By screening compounds in this fashion, potentially beneficial and improved compounds for treating neoplasia can be identified more rapidly and with greater precision than possible in the past. Further benefits will be apparent from the detailed description that follows.
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Li Han
Liu Li
Pamukcu Rifat
Piazza Gary A.
Thompson W. Joseph
Cell Pathways Inc.
Gitomer Ralph
Stevenson Robert W.
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