Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-06-22
2002-10-22
Redding, David A. (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S030000, C435S286400, C435S287300, C435S288400, C435S288700, C356S904000
Reexamination Certificate
active
06468736
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to a system which dramatically increases the speed and efficiency by which substances can be tested for their effects upon a myriad of biochemical processes, for example in living cells. The system can be applied to many fields including application in high throughput drug screening. When applied to the field of high throughput drug screening the system only requires a fraction of the cells currently needed for such tests, enables microminiturization of the process, and reduces the cost of drug screening by reducing the amount of reagents, cells, and disposable materials utilized in the screening process.
2. Discussion of the Background
Scientific research in general, and medical research as a specific example, often requires the evaluation of certain compositions relative to other compositions, plant cells, animal cells, etc. A common example of such research would be in the discovery and development of new drugs.
The discovery and development of a new drug occurs via two main stages. An initial discovery stage aims to the identification and optimization of chemical lead structures among the numerous compounds synthesized to interact with a molecular target putatively involved in the pathophysiology of a human disease. A development stage then follows that assesses the pharmacokinetics, safety and efficacy properties of those drugs found to be potential candidate in humans. Recent advances in drug discovery include the synergistic development of two new technologies in biomedical research known as Combinatorial Chemistry (CC) and High Throughput Screening (HTS). CC, via computer-aided drug design and automated organic synthesis, allows thousands of compounds (a library) of systematic variants of a parent chemical structure to be produced in parallel. Pharmaceutical researchers can now create in a relatively short time millions of new compounds designed to target a specific cellular substrate such as receptors, enzymes, structural proteins and DNA, thus increasing the need for rapid and broadly applicable methods to screen these compounds. While it is important to screen compounds for the targets they were designed for, it is also important to be able to screen compounds for their unintended targets to anticipate potential side effects of selected candidate drugs and to find new uses for these substances if the side effect turns out to be a desired property. The development of HTS has been making it feasible, through automation and miniaturization techniques, to screen upwards to millions of drug candidates a year with robotic workstations running continuously 24 hours a day, 7 days a week. Billions of animal cells expressing the molecular target against which a library is made are grown in 96, 384, or 1536 micro-well plates and, via automated drug and liquid delivery and computerized read-out devices, are tested for a biological response to the drugs.
In conventional HTS systems, animal cells are placed in each of the individual wells of the micro-well plates and are subject to many different processes to test for a response to applied drug candidates. However, an extremely large number of novel drug candidates can now be made available by CC. The conventional approach in HTS systems has been to increase the number of individual wells in the micro-well plates to increase the number of drug candidates that can be screened at one time.
The Scintillation Proximity Assay by Amersham, as disclosed in U.S. Pat. No. 4,271,139 and U.S. Pat. No. 4,382,074 as examples, is a one-step radioisotope-based assay that can be easily automated for HTS. However, the advantages of this sensitive and simple technique are challenged by increasing constrains on the use as well as the cost of disposal of radioactive materials. Thus, new nonradioisotope based screening alternatives have been sought. The development of fluorescent probes able to penetrate living cells, or be biochemically synthesized by cells, such as with chimeric constructs of green fluorescent proteins (GFP), and target protein receptors and enzymes in combination with improved optical instrumentation and means of delivering light and detecting signals has made fluorescence based technique the preferred alternative for many research applications. Fluorimetric Imaging Plate Reader (FLIPR) is a recently developed technique which permits kinetic measurements of intracellular fluorescence on cells labeled with an indicator whose fluorescence properties change upon binding to a cellular substrate targeted by a given drug. FLIPR allows for simultaneous and real time measurements of 96 (and recently 384) samples every second and finds an ideal application in HTS for candidate drugs targeting cell membrane receptors or channels whose activation leads to intracellular ion fluxes in a matter of seconds as in the case of the internal release or influx of calcium ions. In the pharmaceutical industry, HTS is currently performed on commercially available cell lines established from a variety of embryonic and adult animal tissues both normal and pathological. To create cell lines, cells are made immortal via exposure to defined agents such as viruses or chemicals thus acquiring the ability to continuously grow and divide in culture. However, it is generally recognized that, as a result of the immortalization procedure, changes in the expression of certain genes can randomly occur leading to a cell phenotype which might deviate from that of the parental tissue. For example, immortalized liver cells might have lost the ability to express a certain receptor, or to express it in the correct form or cellular compartment as the parental liver cells. Consequently, upon establishment, cell lines are tested for the expression of specific markers, receptors, enzymes, etc. and categorized accordingly.
In contrast to immortal cell lines, primary cell cultures derive from cells freshly isolated from a given organ or tissue. No viral or chemical intervention are used to pressure the cell division cycle and, thus, the cells will survive in vitro for only a short period of time, generally 10-15 days, and need to be re-established quite frequently during a research project. Primary cells are obtainable from a variety of animal models as well as human tissues surgically removed mainly for pathological reasons. Because of their short life span, primary cells maintain the biological stigmata of the original tissue virtually unchanged and, thus, are the research model considered closest to the in vivo environment. Therefore, drug screening on primary cells is highly desirable because it both decreases the chances to miss a valuable lead and increases the physiological relevance of the data collected. However, the dependence of conventional HTS on a tremendously high volume of biological substrate—billions of cells grown and processed in 96-, 384-, or 1536-micro-well plates—has prevented the application of widespread drug screening to primary cells because they are only available in limited quantities. Thus, cell lines exhibiting the biological target against which a drug library has been made are the unique and invaluable source of biological substrate fitting the needs of HTS currently available in drug discovery.
In many of the currently available HTS methodologies—e.g. fluorescence imaging based—the vast majority of cells grown are wasted because, among all the cells present in a given well and exposed to a drug candidate, only those occupying a microscopic field are ultimately monitored for their response. Along with the cells, precious chemical compounds and expensive reagents and supplies are dissipated making the process wasteful and time-consuming, thus reducing the overall afforded by HTS. As discussed above, conventional HTS systems provide individual cells in individual wells of micro-well plates. FIG.
1
(
a
) shows a standard 96-well micro-well plate
100
including 96 individual wells
110
, and an individual well
110
is shown in FIG.
1
(
b
). Each micro-well
11
Atto Instruments, LLC.
Redding David A.
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