Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-04-27
2003-02-11
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007210, C435S023000, C435S024000, C435S029000, C435S032000, C435S375000, C435S377000, C436S063000
Reexamination Certificate
active
06518032
ABSTRACT:
BACKGROUND OF TEE INVENTION
This invention relates to the biological process of programmed cell death and, more specifically to rapid methods of measuring apoptotic activity and of screening for compounds which modulate apoptosis.
Cells can die by at least two fundamentally different biological processes, One process, termed necrosis, refers to cell or tissue death which usually occurs as a result of massive physical or chemical insult. Necrosis is characterized, in part, by cell swelling organelle disintegration and the leakage of cell cytoplasm into the extracellular space and is generally considered to be a passive process.
The alternative process of cell death is known as apoptosis or programmed cell death. This mechanism of cell death in mammalian cells is characterized by a set of morphological and biochemical changes that reflect an active cell suicide. Apoptotic changes include cell shrinkage, nuclear chromatin condensation and margination, and DNA fragmentation. Biochemical events include the externalization of phosphatidyl serine and the activation of aspartate-specific cysteine proteases.
In regard to the latter biochemical event, proteases within the cysteine aspartic acid protease family, also known as the ICE/CED-3 family, are critical for effecting the process of apoptosis. These enzymes are cysteine proteases and exhibit substrate specificity for cleavage after an aspartic acid residue. Due to these characteristics, these enzymes are now referred to by the above term “cysteine aspartic acid proteases” or “caspases”. During the apoptotic process, caspase activity is generated in cells and known inhibitors of the caspase family of proteases inhibit apoptosis.
Apoptosis is important clinically for several reasons. In the field of oncology, many of the clinically useful drugs kill tumor cells by inducing apoptosis. For example, cancer chemotherapeutic agents such as cisplatin, etoposide and taxol all induce apoptosis in target cells. In addition, a variety of pathological disease states can result from the failure of cells to undergo proper regulated apoptosis. For example, the failure to undergo apoptosis can lead to the pathological accumulation of self-reactive lymphocytes such as that occurring in many autoimmune diseases, and can also lead to the accumulation of virally infected cells and to the accumulation of hyperproliferative cells such as neoplastic or tumor cells. The development of efficacious compounds which are capable of specifically inducing apoptosis would therefore be of therapeutic value in the treatment of these pathological diseases states.
In contrast, the inhibition of apoptosis is also of clinical importance. For example, cells are thought to die by apoptosis in the brain and heart following stroke and myocardial infarction, respectively. Moreover, the inappropriate activation of apoptosis can also contribute to a variety of other pathological disease states including, for example, acquired immunodeficiency syndrome (AIDS), neurodegenerative diseases and ischemic injuries other than these listed above. As apoptotic inducers are of benefit in the previously mentioned disease states, specific inhibitors of apoptosis would similarly be of therapeutic value in the treatment of these latter pathological disease states.
Drug discovery benefits from the use of efficient high throughput methods which can rapidly identify specific molecules that interact with the target of interest. The identification of compounds which specifically modulate the apoptotic pathway so far has been hindered by the lack of such methods. Available methods are either limited by the lack of specificity and/or efficiency. For example, most anti-cancer drugs are screened for their ability to kill cells and therefore will identify compounds that induce both necrosis or apoptosis. Moreover, many of these methods are often cumbersome in that they require assessment of cell viability and take days to perform.
Attempts have been made to create methods that are specific for apoptosis. For example, the amount of DNA degradation induced in a cell population has been used for a measurement of apoptosis. However, DNA degradation is a relatively late step in the apoptotic process. Moreover, DNA degradation is not truly specific for apoptosis since DNA eventually becomes degraded in necrotic cells too.
Other methods currently employed use metabolic determinations as an attempt to measure apoptosis in a relatively shorter time frame. For example, cells undergoing apoptosis show impaired mitochondrial function which can be measured using dyes such as alamar blue or by colourimetric assays such as reduction of MTT (3-(4.5-dimethyl) thiazol-2-yl-2,5-diphenyl tetrazolium bromide) to formazan. However, impaired mitochondrial function is not specific for apoptosis as it is also a characteristic exhibited by necrotic cells.
There has been one method described where apoptotic measurements appear specific and are conducted in relatively short time periods. For example, the successful measurement of caspase activity by measuring the fluorescent cleavage product of the CPP32 substrate analog DEVD-AMC has been reported (Armstrong et al.
J. Biol. Chem
. 271:16850-16855 (1996)). However, this method required separate preparations of the cell lysate and reaction mixture as well as additional manipulations, including sample washing in the assay procedure. In addition to the extra time required to perform these additional manipulations, this method could not be performed in a single step due to the requirement for separate preparation of lysate and reaction mixture. Therefore, regarding high throughput assays for apoptosis, what benefit might have been gained in specificity was lost due the inefficiencies incurred in order to measure the caspase activity.
Another method specific for apoptosis has been reported where separate preparations of cell lysate and reaction mixture has not been required (Los et al.
Nature
375:81-83 (1995)). This method similarly determined the caspase activity following induction of apoptosis by measuring the cleavage product of an ICE substrate analogue. Separate preparation of samples and reaction was avoided due to the use of a detergent which does not completely lyse the cells. However, the incomplete solubilization of cellular components can result in decreased sensitivity of the method. Moreover, the detection of substrate cleavage was performed by a separate procedure and, as with the method of Armstrong et al. above, similarly required additional manipulations and steps which lengthened the time period for the procedure.
Thus, there exists a need for rapid and efficient methods to identify compounds which can specifically modulate the apoptotic pathway for the therapeutic treatment of human diseases. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The invention provides a single-well, microscale method of determining the specific apoptotic activity of a cell. The method consists of contacting a cell population of about 1×10
5
cells for a time period of between about 30 minutes and 4 hours with a sufficient volume of medium containing an apoptotis-specific diagnostic reagent and a diagnostic accessory reagent so as to cover the cell population, and determining the activity of the apoptotis-specific diagnostic reagent. The invention also provides a method of identifying a compound which induces apoptosis. The method consists of (a) providing a cell over-expressing a cell survival polypeptide at a level which is sufficient to prevent the induction of apoptosis; (b) treating the cell over-expressing the cell survival polypeptide with a direct stimulus of the cell death pathway; (c) adding a compound to be tested for apoptotic inducing activity, and (d) determining cellular apoptotic activity, the presence of which is indicative of the compound being an apoptotic inducer. The invention further provides a rapid method of identifying a compound which inhibits apoptosis. The method consists of (a) separately co
Armstrong Robert C.
Diaz Jose-Luis
Fritz Lawrence C.
Tomaselli Kevin J.
IDUN Pharmaceuticals Inc.
Saunders David
Seed Intellectual Property Law Group PLLC
LandOfFree
Rapid methods for identifying modifiers of cellular... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Rapid methods for identifying modifiers of cellular..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Rapid methods for identifying modifiers of cellular... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3148273