Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-05-16
2002-12-03
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007210, C435S287100, C435S288400
Reexamination Certificate
active
06489094
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to an improved in vitro method for determining the potential for drug-drug interaction involving cytochrome P450s (CYP) with new chemical entities. This invention further relates to the in vitro determination testing of in vivo drug-drug interactions, particularly as they affect liver metabolism, as an initial or primary screen for compounds as drug candidates.
BACKGROUND OF THE INVENTION
Unfavorable drug-drug interactions (DDI) are responsible for approximately 1-2% of clinically relevant DDI, which while a relatively small number, are nevertheless an important factor in determining whether a new chemical entity will successfully make it beyond a drug discovery program to development. In addition, the late discovery of a clinically significant drug-drug interaction (which would likely eliminate a drug from use) for an otherwise promising candidate could result in the significant economic waste of testing resources already expended on a project.
It is therefore important to screen for potential interactions early on, as well as to select the most appropriate in vivo studies. In this regard, drug interactions with cytochrome P450s (CYPs) are particularly important. CYP1A2, CYP2C, CYP2D6 and CYP3A4 represent greater than 90% of total hepatic P450 and nearly 80% of therapeutic drugs are metabolized by these same enzymes. Interaction with one or more of these enzymes in vivo would pose a potentially relevant event in the clinic. Recently, it has been established that in vitro systems have proven capable of predicting the likelihood of DDI as they allow identification of the CYPs responsible for metabolism as well as determination of the relative contribution to overall elimination of the inhibited pathways.
Since the number of molecules synthesized by pharmaceutical companies has dramatically increased with the utilization of combinatorial chemistry, there is now a shift in emphasis towards earlier implementation of higher throughput in vitro studies for metabolism or lead optimization. The prediction of drug-drug interactions of new chemical entities (NCEs) using in vitro methods, such as human microsomes (HLMs), hepatocytes or individual expressed CYPs has escalated both in importance and scale of use, as one way to reliably avoid potential interactions in vivo.
Actual DDI testing, such as determination of IC
50
or Ki values, is relatively efficient and rapid. It is however, the preparation of the samples, to provide valid in vitro results that is the most exacting portion of the testing protocol and it is such preparation, within rigid short time limit constraints (as a function of testing material degradation), available personnel, equipment, and limited amount of material (microsome, compound samples, etc.), that has limited increased scale-up of initial evaluations and screening of compounds.
Current methodologies involving manual test sample preparation have proven inadequate with respect to increased throughput. A major factor for such inadequacy is the instability of the testing materials, particularly the microsomes which tend to decrease in activity by approximately one hour after being thawed and incubated from freezer storage. Unless the preparation, timing and testing are carefully controlled and coordinated, inaccurate results are likely to be obtained from materials that have not been prepared and tested within the available time window before significant loss of enzyme activity occurs.
Furthermore, due to the vast number of compounds to be tested for initial screening, it is viable to synthesize only small quantities of the compounds for such initial testing. In addition, there is the availability of only limited amounts of microsomes for the testing of the vastly increased number of compounds as would be required for the increased through-put. Accordingly, in addition to the time coordination required for scaled up testing, there is a need for adequately obtaining accurate test results with limited availability of testing materials.
Currently, manual preparations for determinations of DDI in vitro, are gaining in speed and efficiency through the use of 96 and 384 well plates and multi-well pipetting of test sample material. However, other operations of thawing of samples, incubation, plate transport, mixing, etc. are still done manually, with attendant problems of timing and coordination which limit the number of testing samples which can be reliably prepared.
At least four test plates are used in a single testing procedure or run (for compound/CYP enzyme substrate, reaction, filtration and collection) that involve the steps of thawing, preparation, incubation, mixing, and processing of inhibitor compounds being tested (NCEs) with a particular probe (cytochrome P450-specific substrates) and thawed microsomes which are buffered and provided with cofactor. Coordination and efficiency of handling of the various plates during processing is exponentially more difficult with the increased number of compounds being tested and the short time frame allowed for processing which does not change with the increase of tests for which the compounds are prepared.
SUMMARY OF THE INVENTION
It is accordingly an object of the present invention to provide an efficient time-coordinated automated system for preparing a large number of drug candidate compounds for drug-drug interactions testing as an initial screen for viability, within the time constraints of material degradation. Preparation includes the operations of material thawing and incubation, test well filling, mixing (with attendant reactions), and vacuum filtration and coordinated test plate transport to operational stations.
It is a further object of the present invention to provide a system which automatically effects the continuous requisite steps of preparation and test well filling with HLM (after thawing, buffering and addition of co-factor), addition of CYP-specific probe substrates and inhibitor compounds (NCEs) to be tested and controls; incubation, mixing, test tray or plate handling and transport to preparation, operation and testing stations, all within a time window needed, prior to actual testing, before the onset of material degradation.
It is still yet another object of the present invention to effectively minimize test sample amounts, particularly with availability of only small amounts of synthesized inhibitor compound samples and microsomes, by enhancing testing intra and inter reproducibility of standards and particularly with automated single concentration testing of compounds and enzymes.
Co-pending provisional application no. 60/193,717, filed Mar. 31, 2000, describes the use of single point concentrations for effective DDI determinations. The disclosure thereof is included herein in its entirety by reference thereto, which disclosure already minimizes the need for materials with attendant speed in obtaining results without any significant loss of accuracy in obtaining viable results and which utilization is further enhanced by the automation thereof.
It is another object of the present invention to provide a fully operational test preparation system which is self operable to the limits of its storage capacity of processed components and materials, without manual intervention.
Generally the present invention comprises a synchronized, time coordinated method and device for the fully automated multiple-plate in vitro preparation of compounds which are candidates for drugs (NCEs), for drug-drug interaction testing, particularly for testing involving cytochrome P450 (CYP), CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, as a step screening for viability in mammals and particularly humans. To facilitate the time coordinated preparation, non-degrading elements involved in the preparation are made ready prior to an actual test run with degrading materials such as microsomes. Thus, generally stable candidate compounds (NCEs) are preferably pre-mixed in multi-well plates with the CYP-specific probe substrates in the requisite permutations and combinations, for use dur
Ekins Sean
Johnson Diane Lynn
Kelly Kevin George
Ginsburg Paul H.
Nissenbaum Israel
Pfizer Inc.
Richardson Peter C.
Tate Christopher R.
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