Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1999-12-02
2002-09-24
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S007100, C435S284100, C435S288300, C435S297200, C436S063000, C530S802000, C422S082010, C422S105000, C204S400000, C204S403060
Reexamination Certificate
active
06455303
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method and an apparatus for detection of endogenous and synthetic receptor antagonists and receptor modulators, such as e.g. drugs and pharmaceutically active substances. More specifically, the present invention relates to a method and an apparatus based on the use of a miniaturized liquid-based separation technique coupled to a biosensor activated by a receptor agonist and thus giving rise to a measurable response that is affected in a measurable way by the receptor antagonist to be detected.
BACKGROUND OF THE INVENTION
Biologically active compounds generally lack features enabling sensitive detection thereof by conventional techniques, and their roles in biochemical and physiological processes are therefore often difficult to elucidate.
The detection of biologically active compounds is of particular interest in the pharmaceutical field, e.g. during development of new drugs. Since many native and synthetic substrates constituting commercial drugs act as inhibitors of dysfunctional events in the human body, it is of importance to find systems that enable screening or detection of molecules with that mode of action.
In recent years, there has been an exponential increase in the number of compounds which are interesting for screening. Synthetic libraries from drug companies and natural products have been some of the sources of these compounds. The compounds origin from a broad spectrum of different organisms, such as bacteria, insects, plants and marine organisms. This, together with the introduction of combinatorial libraries for the manufacturing of several thousands of compounds have led to a great demand for new screening techniques which are faster and more selective than the ones used today. Known methods used for drug screening are generally based on pure chemical binding between compounds extracted from, for example, natural products and target molecules, such as receptors, enzymes or nucleic acids. The target molecules can also be included in biological systems, such as living cells, where the merits of chemical recognition and biological amplification are combined.
The use of specific target molecules for the evaluation of a compound's biological potential is based on the creation of systems of biological relevance for the analyzed compound. Strategies in this field often include expression of cloned cDNA in different cell systems for the production of a functional target molecule in its natural environment.
There are also examples of screening systems which are based on cell effects where the response cannot be traced to a single target molecule.
Several different techniques are presently used for biological screening and characterization of potential drugs, and some examples of these techniques are given below.
Microphysiometry
During the growth of a typical biological cell, carbon-containing nutrients such as glucose are taken up and acidic metabolic products such as lactic acid are released. In microphysiometry these changes in metabolic rate are recorded as changes in the rate of acidification of the medium surrounding the cells (see e.g. Raley-Susman, K. M., et al., J. Neurosci. 12:773, 1992; Baxter, G. T., et al., Biochemistry 31:10950, 1992; Bouvier, C., et al., J. Recept. Res. 13:559, 1993; and McConnell, H. M., et al., Science 257:1906, 1992). Virtually any molecule that affects the cell can be detected by this method. Such molecules include neurotransmitters, growth factors, cytokines and so forth. The microphysiometry is unable to distinguish between different antagonists acting on the same receptor system and can therefore not be used for binary or more complex solutions of such agents. Other drawbacks of this system are the low-level detection, measuring changes in pH is far less selective than measuring responses on the receptor level, and slow recovery rates.
Immunoassays
This group of techniques is based on in vitro procedures for screening of specific antigens (see e.g. Tu, J., et al., Clin. Chem. 44:232, 1998; Pinilla, C., et al., Biomed. Pept. Proteins Nucleic Acids 1:199, 1995; Tawfik, D. S., et al., Proc. Natl. Acad. Sci. USA 90:373, 1993; and Houghten, R. A., et al., Biotechniques 13:412, 1992). Antibodies, often immobilized, are used as targets for antigens. The antigen-antibody interaction is detected by a second antibody, which is labeled by, e.g., a radioactive isotope. The problems with these immuno-based techniques are related to the difficulties in raising specific antibodies for small molecules that are identical or resemble endogenous compounds. Another problem is related to the handling of radioactive substances.
Use of Combinatorial Libraries
Synthetic combinatorial libraries have proven to be a valuable source of diverse structures useful for large-scale biochemical screening (see e.g. Sastry, L., et al., Ciba Found Symp. 159:145, 1991; Huse, W., Ciba Found Symp. 159:91, 1991; Persson, M. A., et al., Proc. Natl. Acad. Sci. USA 88:2432, 1991; Kang, A. S., et al., Proc. Natl. Acad. Sci. USA 88:4363, 1991; Houghten, R. A., et al., Nature 354:84, 1991; Clackson, T., et al, Nature 352:624, 1991; and Ostresh, J. M., et al., Proc. Natl. Acad. Sci. 91:11138, 1991). The libraries are generated by a combination of solution and solid-phase chemistries and are cleaved off the solid-support for screening. When mixtures of compounds are screened, however, the possibility exists that the most active compound will not be identified.
Separation Techniques Coupled to Mass Spectrometry
Separation techniques such as liquid chromatography, gas chromatography and capillary electrophoresis coupled to mass spectrometry or tandem-mass spectrometry create analytical systems available for structure evaluation (see e.g. Hsieh, S., et al., Anal. Chem. 70:1847, 1998; Tretyakova, N. Y., et al., J. Mass. Spectrom. 33:363, 1998; Bonnichsen, R., et al., Zacchia 6:371, 1970; Taylor, G. W., et al., Br. J. Clin. Pharmacol. 42:119, 1996; and McComb, M. E., et al., J. Chromatogr. A 800:1, 1998). Mass spectrometry gives information about the molecular weight of the analyzed molecule. With refined and controlled fragmentation of large molecules it is also possible to extract information about the sequence.
Enzyme Assays Using Proteases
Many proteases have become targets for drug discovery (see e.g. Carroll, C. D., et al., Adv. Exp. Med. Biol. 436:375, 1998; Ferry, G., et al., Mol. Divers. 2:135, 1997; and Jiracek, J., et al., J. Biol. Chem. 270:21701, 1995), from viral proteases required for the generation of active viral proteins to mammalian proteases that process pro-hormones to their active mature forms. Assays have been developed in bacterial systems to screen for compounds that inhibit protease activity. Most of these involve the co-expression of both the protease and a target reporter gene (the gene that encoded the protein which creates a measurable effect) in the same cell. A number of in vitro biochemical assays have also been developed. In most of these cases, a peptide containing the protease cleavage site is labeled at one end using either a radioactive or a fluorescent tag. The other end of the peptide molecule is adhered to a plate or a bead. In the presence of an active protease, the peptide is cleaved and the labeled end is released. The loss of signal from the labeled end of the peptide molecule after washing reflects the activity of the protease and can be easily monitored. For detection of protease inhibitor the grade of maintenance of the signal can instead be measured. These assays are, however, often time consuming since they involve genetic engineering.
A significant limitation of the above mentioned methods is the capacity; the number of compounds that can be rapidly evaluated is extremely low. Alternative methods for high through-put screens are needed.
Another major disadvantage of these known biological screening systems is that they involve extensive multistep purification and isolation of the compounds which are to be tested.
Capillary-based separation methods for identifying bioact
Jardemark Kent
Orwar Owe
Cellectricon AB
Conlin David G.
Edwards & Angell LLP
Nguyen Bao-Thuy L.
Padmanabhan Kartic
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