Chemistry: analytical and immunological testing – Including chromatography
Reexamination Certificate
2000-05-12
2003-06-17
Ludlow, Jan (Department: 1743)
Chemistry: analytical and immunological testing
Including chromatography
C435S007100, C436S086000, C436S164000, C436S171000, C436S173000
Reexamination Certificate
active
06579720
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for analyzing complex mixtures of compounds. More particularly, the present invention is directed to a high efficiency method for screening a complex mixture of compounds for a predetermined characteristic, such as a chemical or biological property, and identifying the compounds in the mixture exhibiting said characteristic.
BACKGROUND AND SUMMARY OF THE INVENTION
The emergence of automated chemical synthesis platforms coupled with combinatorial techniques as a routine tool in the pharmaceutical industry that has enabled the synthesis of large numbers of compounds in a relatively short time. Millions of potential new drug candidates are synthesized every year, and both pharmaceutical and biotechnology industries have embraced the challenge in recent years of developing new, faster and more efficient ways to screen pharmaceutical compounds in order to rapidly identify “hits” and develop them into promising lead candidates.
The development of a chemical lead into an ideal marketable medicine requires a balance in potency, safety, and pharmacokinetics. The overall cost of bringing a new medicine to the market place is very high: recent surveys indicate that the average new chemical entity taken to market in the United States requires 10 to 15 years of research and costs more than $300 million. The major reasons for failure in development often involve clinically unacceptable kinetics, bioavailability or toxicity and the need for uncovering this information at earlier (less costly) stages of the drug discovery process is evident. Thus, there is a significant need to develop methods for rapidly evaluating these properties, as well as the bioactivity potential, for the ever-increasing number of compounds generated by combinatorial chemistry. Analytical researchers have turned their attention to development of high-throughput analytical approaches. Chromatographic systems have been developed and specifically designed for automated high-throughput identification, purity assessment, purification and biological screening of combinatorial libraries. In parallel to this, a great deal of research effort has been devoted to the optimization of screening assays to meet the requirements of high throughput screening. In theory, any assay performed on the bench top can be applied in HTS (High Throughput Screening), but the adaptation of such assays to an automated format may not be straightforward and often requires modifications of the assay to circumvent constraints imposed by HTS. The ideal assay is one that can be performed in a single well with no other manipulation other than the addition of the sample to be tested. A number of assay formats have been developed or modified over the past few years to conform to the constraints imposed by HTS, particularly emphasizing automation, and miniaturization.
The technological advances directed toward the implementation of fast and high volume chromatographic systems have rapidly converged toward automated systems for accommodating the large number of compounds typically produced in parallel syntheses. Despite recent developments of rapid and efficient methods for high throughput screening, intense efforts are ongoing to create new, more economical and more efficient methods for carrying out screening processes.
A few methods addressing the need for more efficient screening techniques have emerged over the past few years. Recently, a chemical-screening scouting technique for preliminary chemical characterization for natural product extracts was used for the dereplication and prioritization of HIV-inhibitory aqueous natural product extracts by Boyd M. R. et al., “A Chemical Screening Strategy for the Dereplication (elimination from further consideration) and Prioritization of HIV-Inhibitory Aqueous Natural Products Extracts”,
J. Nat. Prod.,
1993, 56, No 7, pp 1123-1129). The method is based on a preliminary chemical characterization, or “chemical screening”, of natural product extracts by performing a series of chromatographic separations on different columns addressing distinct chemical/physicochemical properties of the solutes. For example, Sephadex G-25 cartridges were utilized first to provide information about molecular size and weight. Bonded-phase cartridge, C
4
wide pore (300 Å) and C
18
narrow pore (60 Å), were utilized to determine the relative polarity of active constituents. Four fractions were collected from each cartridge; they were tested side by side with the parent supernatant for anti-HIV activity. Thus, a distinctive or characteristic chromatographic profile of the active constituents was obtained, along with information about the recovery of activity and, by inference, the stability of the active compounds. This chemical-screening approach was first validated with a number of HIV-inhibitory standards, e.g., AZT, Dextrin sulfate, Cyclosporin, and Oxathiin carboxanilide. As illustrated in
FIG. 1
, different patterns of elution were observed for each compound tested. The recurring patterns of bioactivity elution could be readily discerned from the matrix form shown. Similarly, several sponge extracts were evaluated for the elution pattern in the chemical screen (FIG.
2
).
The chemical-screening approach was used to gain insight into the general chemical nature of potential new anti-HIV lead compounds, to identify and dereplicate additional recurring classes of antiviral compounds, and to select chromatographic procedures for initial fractionation of natural product extracts.
FIG. 3
illustrates the overall dereplication and chemical screening strategy.
Julian et al at Eli Lilly and Company have developed a system that delivers data with reasonable throughput (Julian, R. K. Jr.; Higgis, R. E.; Gygi, J. D.; Hilton, M. D., “A Method for Quantitatively Differentiating Crude Natural Extracts Using High-Performance Liquid Chromatography-Electrospray Mass Spectrometry”,
Anal. Chem.,
1998, 70, pp 3249-3254). The system comprises three components: (a) HPLC separation using standard reversed-phase C
18
gradient separation on the crude extract. (b) ESI-MS detection of effluent analytes, and (c) a computational image analysis techniques, the data are reduced to a list containing the m/z value and retention time of each ion. The ion lists are then compared in a pairwise fashion to compute a sample similarity index between two samples to allow effective comparison of the large data sets that are generated by the analysis.
Identifying the activity of compounds in complex mixtures, and isolating the active compounds, has been a method in the field of drug discovery for more than a century. Interestingly, many complex mixtures for use in humans contain only partially characterized complex mixtures, as found for example in herbal medicines. The chromatography of complex mixtures, particularly those mixtures which contain compounds with similar physical chemical properties, routinely generates chromatograms of co-eluting compounds. Examples include the chromatography of natural and synthetic chemical libraries using mass spectrometry to detect the compounds. Overlapping peaks is an unavoidable modern experimental problem in modern drug discovery whereby chromatography of complex mixtures is used as a chemical-source for identifying active compounds.
Methods to identify and isolate the activity of compounds comprising complex mixtures frequently will involve an initial test for activity in a crude sample preparation. If there is no activity, then there is usually no reason for further testing. However, if there is one or more activities in the crude sample preparation then the task of identifying the compounds containing the one or more activities is performed. The crude mixture may in fact be not only a natural product extract, but also pooled fractions from any chemical or biological source. The conventional methods routinely used to identify the active compounds in complex mixtures include the following steps.
Step 1) Prepare the crude sample, or aliquot of the crude sam
Pidgeon Charles
Rooke Nadege M.
Ruell Jeffrey A.
Barnes & Thornburg
BDC Pharma LLC
Ludlow Jan
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